development and validation of a combined method...

DEVELOPMENT AND VALIDATION OF A COMBINED METHOD FOR QUANTITATIVE DETERMINATION OF DISSOLUTION, ASSAY, AND CONTENT

UNIFORMITY IN BISOPROLOL FUMARATE/ HYDROCHLOROTHIAZIDE TABLETS USING HPLC AND UPLC

Niralee Thakar

A Thesis Submitted to the University of North Carolina Wilmington in Partial Fulfillment

of the Requirements for the Degree of Masters of Science

Department of Chemistry and Biochemistry

University of North Carolina Wilmington

2011

Approve By

Advisory committee

Jeremy Morgan Nadeem Zia

John Tyrell Chair

Accepted by

Dean, Graduate School

ii

TABLE OF CONTENTS

ABSTRACT ...................................................................................................................... iv

ACKNOWLEDGEMENTS ................................................................................................v

DEDICATION .................................................................................................................. vi

LIST OF TABLES ........................................................................................................... vii

LIST OF FIGURES ............................................................................................................x

INTRODUCTION AND BACKGROUND ...................................................................... 1

EXPERIMENTAL ...............................................................................................................7

Materials and Reagents ........................................................................................... 7

Equipment .............................................................................................................. 7

HPLC Instrumentations and Conditions ................................................................ 8

UPLC Instrumentations and Conditions ................................................................ 9

Preparation of the Solution for Dissolution Testing ............................................ 10

Preparation of the Solution for Assay and CU Testing ........................................ 12

RESULTS AND DISCUSSIONS .................................................................................... 15

Method Development ........................................................................................... 15

UPLC Method Transfer ........................................................................................ 18

Assay/CU Validation ........................................................................................... 19

Linearity/Range ........................................................................................ 20

Recovery/Accuracy .................................................................................. 21

Precision ................................................................................................... 22

Specificity ................................................................................................ 26

iii

Robustness of Extraction Procedure ........................................................ 27

Filter Study ............................................................................................... 28

Robustness of Chromatographic Parameter ............................................. 30

Stability of Sample Solution .................................................................... 34

Forced Degradation .................................................................................. 36

Method Comparison ................................................................................. 41

Dissolution Method Validation ............................................................................ 44

Linearity/Range ........................................................................................ 45

Recovery/Accuracy .................................................................................. 46

Precision ................................................................................................... 47

Specificity ................................................................................................ 49

Robustness of Degassing Procedure ........................................................ 50

Robustness of Sampling Procedure (Manual vs. Automated) ................. 53

Filter Study ............................................................................................... 54

Robustness of Chromatographic Parameters ........................................... 57

Stability of Sample Solution .................................................................... 59

Method Comparison ................................................................................. 62

Teflon Paddles vs. Non Teflon Paddles ................................................... 65

CONCLUSION ................................................................................................................ 67

REFFERENCES ...............................................................................................................69

APPENDIX .......................................................................................................................73

iv

ABSTRACT

In pharmaceutical and biomedical analysis, the main objective is to reduce

analysis time and maintain good efficiency; therefore, there has been substantial focus on

high speed chromatography separation. Recently, one of the most promising

developments in the area of fast chromatography separation is ultra performance liquid

chromatography (UPLC). A precise, accurate, and simple method was developed and

validated for bisoprolol Fumarate (BF) and hydrochlorothiazide (HCTZ). The two

combined active pharmaceutical ingredients (API) are in a single dosage form. The

method was developed and validated for dissolution, assay, and content uniformity (CU)

testing using high performance liquid chromatography (HPLC) and UPLC. The method

was validated according to ICH guidelines and current Sandoz Standard Operation

Procedure (SOP), for accuracy, precision, intermediate precision, linearity, range,

specificity, extraction study, filter study, system suitability, stability of solutions, force

degradation, and robustness.1 Also the method was compared with current United States

Pharmacopeias (USP) 20112 method for Bisoprolol Fumarate and Hydrochlorothiazide

Tablets. The results were compared between two different systems, traditional HPLC and

new UPLC. Also the advantages and disadvantage of UPLC and HPLC usage will be

discussed.

v

ACKNOWLEDGEMENTS

I would like to thank the following people who were instrumental in the completion of this thesis:

Dr. Nadeem B. Zia (Manager QA/QC Compliance, Sandoz)

Dr. John A. Tyrell (Department of Chemistry and Biochemistry, UNCW)

Dr. Jeremy Morgan (Department of Chemistry and Biochemistry, UNCW)

John Bredin (Manager, QC, Sandoz)

Richard Uveges (Director, QC, Sandoz)

Jason Miller (Chemist IV, QC, Sandoz)

Tenika Smith (Chemist II, QC, Sandoz)

Kathy Jones (Chemist II, QC, Sandoz)

Amber Vogt (Chemist II, QC, Sandoz)

Sandoz Inc. Wilson, NC for the financial support and for the opportunity

UNCW Faculty and Staff

vi

DEDICATION

I would like to dedicate this to my parents and my husband. My parents have

always encouraged me to study further. My husband has supported me in all manners.

vii

LISTS OF TABLES

Table Page

1. Retention time using HPLC ..................................................................................................... 8

2. HPLC gradient ......................................................................................................................... 9

3. Retention time using UPLC ................................................................................................... 10

4. UPLC gradient ....................................................................................................................... 10

ASSAY CU METHOD VALIDATION

5. Calculated linearity results for BF and HCTZ using HPLC and UPLC ................................ 21

6. Assay/CU accuracy/recovery results for BF and HCTZ using HPLC and UPLC ................. 22

7. Assay precision and intermediate precision results for BF using HPLC and UPLC ............. 24

8. Assay precision and intermediate precision results for HCTZ using HPLC and UPLC ........ 24

9. CU precision and intermediate precision results for BF using HPLC and UPLC ................. 25

10. CU precision and intermediate precision results for HCTZ using HPLC and UPLC ............ 26

11. Robustness of Extraction procedure results for BF and HCTZ using HPLC and UPLC ....... 28

12. Filter study results for BF and HCTZ using HPLC and UPLC ............................................. 29

13. Filter study’s percent difference results for BF and HCTZ using HPLC and UPLC ............. 30

14. Robustness Parameter for HPLC ........................................................................................... 30

15. Robustness Parameter for UPLC ........................................................................................... 31

16. Robustness system suitability results for BF and HCTZ using HPLC .................................. 32

17. Robustness system suitability results for BF and HCTZ using UPLC .................................. 33

18. Solution stability results for standard preparation using HPLC and UPLC ........................... 35

19. Solution stability results for sample preparation using HPLC and UPLC ............................. 36

20. Forced degradation results for BF in blend sample using HPLC ........................................... 39

21. Forced degradation results for BF in blend sample using UPLC ........................................... 39

22. Forced degradation results for HCTZ in blend sample using HPLC ..................................... 40

23. Forced degradation results for HCTZ in blend sample using UPLC ..................................... 40

viii

24. Method comparison results for BF using USP method .......................................................... 42

25. Method comparison results for HCTZ using USP method .................................................... 42

26. Method comparison results for HCTZ using current Sandoz method ................................... 43

27. Method comparison results for BF using current Sandoz method ......................................... 44

DISSOLUTION METHOD VALIDATION RESULTS

28. Linearity results for BF and HCTZ using HPLC and UPLC ................................................. 46

29. Recovery/Accuracy results for BF and HCTZ using HPLC and UPLC ................................ 47

30. Precision and intermediate precision results for BF and HCTZ using HPLC ....................... 48

31. Precision and intermediate precision results for BF and HCTZ using UPLC ....................... 49

32. Robustness of degassing procedure results for BF using HPLC ............................................ 51

33. Robustness of degassing procedure results for BF using UPLC ............................................ 51

34. Robustness of degassing procedure results for HCTZ using HPLC ...................................... 52

35. Robustness of degassing procedure results for HCTZ using UPLC ...................................... 52

36. Robustness of sampling procedure results for BF and HPLC using HPLC ........................... 54

37. Robustness of sampling procedure results for BF and HPLC using UPLC ........................... 54

38. Filter study result for BF and HCTZ using HPLC and UPLC ............................................... 56

39. Filter study’s percent difference result for BF and HCTZ using HPLC and UPLC .............. 56

40. Robustness parameter for HPLC ........................................................................................... 57

41. Robustness parameter for UPLC ........................................................................................... 57

42. Robustness system suitability results for BF and HCTZ using HPLC .................................. 58

43. Robustness system suitability results for BF and HCTZ using UPLC .................................. 59

44. Solution stability results for BF standard preparation using HPLC and UPLC ..................... 60

45. Solution stability results for BF sample preparation using HPLC and UPLC ....................... 61

46. Solution stability results for HCTZ standard preparation using HPLC and UPLC ............... 61

47. Solution stability results for HCTZ sample preparation using HPLC and UPLC ................. 61

48. Method comparison results for BF using USP method .......................................................... 63

ix

49. Method comparison results for HCTZ using USP method .................................................... 63

50. Method comparison results for BF using current Sandoz method ........................................ 64

51. Method comparison results for HCTZ using current Sandoz method ................................... 64

52. Teflon paddles vs. non Teflon paddles results for BF and HCTZ using HPLC .................... 65

53. Teflon paddles vs. non Teflon paddles results for BF and HCTZ using UPLC .................... 66

x

LISTS OF FIGURES

Figure Page

1. Structure of BF and HCTZ ........................................................................................... 1

2. Dissolution sample using HPLC chromatography ...................................................... 11

3. Dissolution sample using UPLC chromatography ..................................................... 11

4. Assay sample using HPLC chromatography .............................................................. 13

5. Assay sample using UPLC chromatography ............................................................. 13

6. Resolution ID using HPLC chromatography ............................................................. 14

7. Resolution ID using UPLC chromatography ............................................................. 14

INTRODUCTION AND BACKGROUND

Pharmaceutical dosage formulations containing the beta-blocker bisoprolol

fumarate (BF) and the diuretic hydrochlorothiazide (HCTZ) are used for the treatment of

high blood pressure.3 The combined mixture of BF, 1-[4-[[2-(1-methylethoxy)

ethoxy]methyl]-phenoxy]-3-[(1-methylethyl)amino] -2-propanolethylene-1,2-

dicarboxylic acid and HCTZ, 6-chloro-3,4-dihydro-2H-1,2,4-benzothiazine-7

sulfonamide-1,1-dioxide is a widely used as antihypertensive drug (Figure 1).4

Bisoprolol Fumarate HCTZ

Figure 1: Structure of BF and HCTZ

The HCTZ is a thiazide diuretic and is used for treatment of hypertension and oedema.5

HCTZ is one of the oldest thiazides used as a diuretic and is often prescribed in

combination with other drugs such as beta-blockers, ACE inhibitors, or angiotensin II

receptor blockers.6 The bisoprolol fumarate (BF)/ hydrochlorothiazide (HCTZ) tablets,

2.5 mg/6.25 mg, 5 mg/6.25 mg, and 10 mg/6.25 mg have two active pharmaceutical

ingredients (APIs).

2

Literature studies show that various analytical methods were reported for the

estimation of HCTZ in biological fluids. Several methods were reported for

quantification of BF and HCTZ separately. Very few methods were reported for

simultaneous estimation of BF and HCTZ using HPLC. There is no method reported for

simultaneous estimation of BF and HCTZ using UPLC. There is one method available in

2011 USP for the simultaneous analysis of both APIs using HPLC; but the dissolution

and Assay/content uniformity (CU) methods have different HPLC parameters. The

retention times and run time have not been mentioned for dissolution tests. The 2011

USP method for dissolution requires a dual wavelength detector (Photo Diode Array,

PDA) to quantitate BF and HCTZ, which is restricted to the use of a dual wavelength

detector HPLC instrument. The 2011 USP illustrates an HPLC method that uses for the

dissolution of BF /HCTZ in tablets L11 packing and aqueous 0.2% triethylamine solution

and acetonitrile (4:1 v/v) as the mobile phase in the isocratic mode. The method for assay

and CU tests requires the use of gradient mode and the total gradient run time is twelve

minutes. The 2011 USP method for assay has a different sample preparation for BF and

for HCTZ; therefore each solution needs to be injected separately in order to quantitate

each active. The USP illustrates an HPLC method for the assay/CU test of BF/HCTZ

tablets that uses L11 packing and aqueous dibutyl ammonium phosphate and acetonitrile

as the mobile phase in the gradient mode.2

Currently Sandoz has a method for quantitating BF and HCTZ in assay, CU, and

dissolution test solutions. The disadvantage of the current Sandoz method is that a

different set of HPLC parameters are required for each active, BF and HCTZ, in

assay/CU, and dissolution tests. This method uses a Phenomenex Prodegy C18, 3.9 mm x

3

300 mm, 10 μm analytical column and a mobile phase consistent of methanol, buffer

solution (pentanesulfonic acid and sodium acetate), and acetic acid (470:530:0.5) in an

isocratic mode for the quantitation of BF in assay, CU and dissolution test solutions.

Under the above conditions, BF elutes at 12.5 minutes with a total run time of 20 minutes

for each injection. Sandoz’s current HPLC method for HCTZ quantitation in assay, CU,

and dissolution tests uses Phenomenex Prodegy ODS(2), 4.6 mm x 100 mm, 5 μm

analytical column and buffer solution (potassium phosphate) and acetonitrile (mobile

phase A 90:10 and mobile phase B 10:90) as the mobile phase in the gradient mode.

Using this column and mobile phases, HCTZ elutes at 4 minutes with a total gradient

time of 20 minutes per injection. The assay/CU sample preparations are also different for

BF and HCTZ; therefore, multiple instruments and multiple days are needed to analyze

one tablet. Dissolution testing of BF and HCTZ also require different HPLC parameters

for each BF and HCTZ because pull time for BF is 20 minutes and HCTZ is 30 minutes.

During a literature search, one assay combined method3 was found for BF and

HCTZ. The method was developed for a different formulation and for different strengths

of HCTZ. The dissolution method and CU test methods are not addressed in this article.

This article describes an HPLC method for the assay of BF and HCTZ tablet using

sperisorb, 4.6 mm x 250 mm, 5 μm, cyano analytical column and 0.1 M aqueous

potassium dihydrogen phosphate buffer, acetonitrile, and tetrahydrofuran as the mobile

phase in the isocratic mode.3 Another combined method was found during literature

search for the assay of BF and HCTZ. In this method 0.1 M aqueous potassium

dihydrogen phosphate buffer and acetonitrile in the ratio of 70:30 v/v is used as a mobile

phase. Inertsil ODS 3V (250 mm x 4.6 mm, 5 μm) analytical column was used as a

4

stationary phase. This article does not address dissolution or CU method either.6 Both of

these methods, Sandoz’s current method, and the 2011 USP method use an HPLC

instrument to quantitate BF and HCTZ.

In order to make analysis user friendly, efficient, and cost effective, a new method

needs to be developed to analyze both APIs (BF and HCTZ product range) in one HPLC

and UPLC chromatographic run for dissolution, assay and CU. Multiple analysts can

prepare assay, CU, and dissolution solutions; and all those solutions can be analyzed

using one HPLC or UPLC instrument. In order to avoid the disadvantage of having two

different methods for dissolution and assay/CU, a study was conducted. The first part of

the present study was to develop and validate a simple, precise, specific, accurate, and

robust HPLC method for the simultaneous determination of BF and HCTZ in final

dosage form. The second part of the study was to transfer the HPLC method to UPLC

and validate the UPLC method. The important reasons for developing a UPLC method

are to make the laboratory more environmentally green, efficient, and cost effective than

when using HPLC. UPLC typically saves about 80 to 95 percent of mobile phase in

isocratic and gradient mode compared with HPLC. UPLC provides higher resolution,

speed, and potentially higher sensitivity compared to HPLC.7

For the past 30 plus years, HPLC has proven to be the predominant technology

used in pharmaceutical laboratories. Scientists have been searching for a “fast LC” as a

way to speed up analyses. In early 2004, the first commercially available UPLC was

invented by the Milford, Massachusetts based company, Waters Corporation. The Waters

UPLC is called the ACQUITY UPLCTM System.8 The fundamental principles of this

development are governed by the van Deemter equation (H = A + B/μ + Cμ) where: H is

5

the column’s plate height, and μ is the mobile phase linear flow rate. A, B, and C are

constants. The van Deemter equation describes the relationship between linear velocity

(flow rate) and plate height (HETP or column efficiency).9

Particle size is one of the variables that can be used to investigate

chromatographic performance.10 Chromatographic resolution can be improved by either

increasing the column length or reducing the particle size. Reducing the particle size is

far more effective than increasing the column length.11 The efficiency of the earlier

HPLCs increased by decreasing particle size in column packing from 10 μm in the 1970s

to 3.5 μm in the 1990s.12 The UPLC system is used with specially designed Acquity

UPLC columns containing a particle size of only 1.7 μm.8 As particle size gets smaller,

the column back pressure gets higher because the pressure required to pump mobile

phase through the column is inversely proportional to the square of the particle diameter.

The back pressure required for use of these small particle columns becomes high and this

presents a challenge to the pressure limitations of a conventional HPLC system.9 UPLC is

capable of having about 15000 psi back pressure compared to HPLC, which can have

about 4000 psi.7 Efficiency is three times greater with 1.7 μm particles compared to 5 μm

particles and two times greater compared to 3.5 μm particles. The resolution is 70 percent

higher with 1.7 μm particles than with 5 μm particles. The resolution is 40 percent higher

with 1.7 μm particles than with 3.5 μm particles. High speed is obtained because a

column length with 1.7 μm particles can be reduced by a factor of 3 compared to 5 μm

particles for the same efficiency, and flow rate can be three times higher. Therefore, the

separation in the UPLC can be nine times faster with equal resolution. The UPLC also

6

has a greater increase in sensitivity due to less band spreading during migration through a

column with smaller particles.12

Today’s pharmaceutical industry is looking for new ways to cut cost and shorten

time for the development of drugs while at the same time improving the quality of their

products. Faster separation can lead to higher output and less time consumption while

running multiple samples. The speed allows a greater number of analyses to be performed

in a shorter amount of time, thereby increasing sample output and lab productivity. The

transfer of the HPLC method to the UPLC method can be accomplished by simply

applying a scaling factor to the mobile phase flow rate and the sample injection volume.

The scaling factor was derived from the ratio of the column cross sectional area in order

to retain the mobile phase linear velocity.8 The use of the UPLC reduces the amount of

waste, mobile phase consumption, cost of analysis, and provides many of the benefits of

green chemistry.13 UPLC also offers significant theoretical advantages in resolution,

speed, sensitivity for analytical determination, and is capable of high speed acquisitions

for sampling rate.14

7

EXPERIMENTAL

Materials and Reagents:

BF Reference Standard, HCTZ, Reference Standard, 4-Amino (4-Amino-6-

chloro-1,3-benzenedisulfonamide), Reference Standard, CTZ (Chlorothiazide) Reference

Standard, and fumaric acid were supplied by the USP. Dimer ([6-chloro-N-[(6-chloro-7-

sulfamoyl-2,3-dihydro-4H-1,2,4-benzothiadiazine-4-yl 1,1-dioxide)methyl]3,4-dihydro-

2H-1,2,4-benzothiazine-7-sulfonamide 1,1-dioxide]) was purchased from Gyma and

manufacturer was Cambrex. 4-Amino is HCTZ’s degraded impurity. CTZ and Dimer are

HCTZ’s process related impurities. Bisoprolol impurity A (1-(4-hydroxymethyl)-

phenoxy-3-isopropylamino propan-2-ol) was purchased from Gyma and manufacturer

was Corden. Bisoprolol impurity A is Bisoprolol’s degraded impurity. Trifluoroacetic

Acid (TFA) was spectrophotometric grade and it was purchased from Aldrich Chemical

Company. Acetonitrile was ACS grade and was purchased from Fisher Brand.

Concentrated hydrochloric acid (12N) was purchased from Fisher brand. USP purified

water was used for making solutions.

Equipment:

Agilents 1100, Agilents 1260, and Waters 2695 equipped with gradient pump,

auto sampler, temperature controlled compartment, dual wavelength detector, and Photo

Diode Array detector (PDA) were used for all HPLC experiments. Waters Acquity

equipped with gradient pump, auto sampler, temperature controlled compartment, dual

wavelength detectors, and PDA was used for all UPLC experiments. Empower 1 and

Empower 2 were used as the data acquisition program to collect chromatograms for each

8

injection and to measure the retention times and resolutions. Microsoft Excel, Empower1

and Empower2 were used for calculations.

HPLC Instrumentations and Conditions:

A qualified HPLC systems equipped with an electronic injector were used for

analysis. The chromatographic separations were performed using Water Symmetry C18, 5

µm, 4.6 mm x 150 mm column, eluted with mobile phase at the flow rate of 1.0 mL/min.

Column temperature was at 35 °C. Measurement was performed with injection volume of

20 µL for dissolution testing and 10 µl for Assay/CU testing. UV detection was at 224

nm wavelength. Mobile Phase A was prepared by pipetting 1.0 mL of Trifluoroacetic

acid (TFA) into 1800 mL water and 200 mL acetonitrile. Mobile Phase B was prepared

by pipetting 1.0 mL of TFA into 1800 mL acetonitrile and 200 mL water. Table 1 shows

the retention time for each active. HPLC Gradient was followed according to table 2 with

a run time of 11 minutes.

Table 1: Retention Time using HPLC

(Note: The related retention times for 4-Amino, CTZ, and dimer are based on the HCTZ peak, and Fumaric acid and Bisoprolol impurity A are based on the Bisoprolol peak)

Active/Impurities

name Retention Time

(min) RRT

Fumaric acid 1.9 0.35 Bisoprolol imp A 3.2 0.59

4-Amino 3.8 0.88 CTZ 4.1 0.95

HCTZ 4.3 1.00 Bisoprolol 5.4 1.00

Dimer 6.1 1.40

9

Table 2: HPLC Gradient

Time (min) Mobile phase A% Mobile phase B% Curve

0 100 0 --- 6 40 60 6 7 40 60 6

7.5 100 0 6 11 100 0 6

UPLC Instrumentations and Conditions:

Qualified UPLC systems equipped with an electronic injector were used for

analysis. The chromatographic separations were performed using Water Acquity BEH

C18, 1.7 µm, 2.1 mm x 100 mm column, and eluted with mobile phase at the flow rate of

0.3 mL/min. Column temperature was at 35 °C. Measurements were performed with

injection volume of 10 µL for dissolution testing and 2 µL for Assay/CU testing. UV

detection was set at 224 nm wavelength. Mobile Phase A was prepared by pipetting 1.0

mL of TFA into 1800 mL water and 200 mL acetonitrile. Mobile Phase B was prepared

by pipetting 1.0 mL of TFA into 1800 mL acetonitrile and 200 mL water. Mobile phases

were the same for HPLC and UPLC instruments. The same mobile phases were used for

assay, CU, and dissolution testing for BF and HCTZ. Table 3 shows the retention time for

each active. UPLC Gradient was followed according to table 4 with a run time of 7

minutes.

10

Table 3: Retention Times using UPLC

(Note: The related retention times for 4-Amino, CTZ, and dimer are based on the HCTZ peak, and Fumaric acid and Bisoprolol impurity A are based on the Bisoprolol peak)

Table 4: UPLC Gradient

Time (min) Mobile phase A% Mobile phase B% Curve

0 95 5 --- 2 85 15 6 5 50 50 6

5.5 95 5 6

7 95 5 6

Preparation of the Solution for Dissolution Testing:

USP apparatus # 2, paddles were used for dissolution testing. Dissolution medium

was 0.1N hydrochloric acid. Dissolution volume was 900 mL. Speed for the paddle was

75 rpm. Sampling time for BF was 20 minutes and for HCTZ was 30 minutes. The final

solutions of the standards and samples contained about 2.8 µg/mL concentrations for 2.5

mg of BF label claim, 5.6 µg/mL concentrations for 5 mg of BF label claim, 11.2 µg/mL

concentrations for 10 mg of BF label claim, and 6.9 µg/mL concentrations for 6.25 mg of

HCTZ. Final sample solutions were filtered through a 0.45 µm PVDF Millex filter or

Active/Impurities name Time Relative

Retention Time (RRT)

Fumaric acid 0.9 0.20 Bisoprolol imp A 1.5 0.34

4-Amino 1.7 0.85 CTZ 1.8 0.90

HCTZ 2.0 1.00 Dimer 4.2 2.10

Bisoprolol 4.4 1.00

11

equivalent, discarding the first 3 mL of the filtrate. HPLC and UPLC chromatograms for

dissolution sample are shown in figure 2 and figure 3.

SampleName Bis HCTZ Bis MK061822 P D-1; Vial 11; Injection 1; Result Id 10452; Date Acquired Tuesday, February 08, 2011 8:58:59 PM EST

HC

TZ

bis

AU

0.00

0.10

0.20

0.30

Minutes0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00

Figure 2: Dissolution sample chromatogram using HPLC

SampleName Bis HCTZ bis MK061822 P D-1; Vial 1:B,3; Injection 1; Result Id 10336; Date Acquired Tuesday, February 08, 2011 8:04:03 PM EST

HC

TZ

bis

AU

0.00

0.20

0.40

0.60

0.80

Minutes0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00

Figure 3: Dissolution sample chromatogram using UPLC

12

Preparation of the Solution for Assay and CU Tests:

Diluent for Assay and CU was prepared by pipetting 1.0 mL of TFA into 1600

mL water and 400 mL acetonitrile. For assay samples, 20 tablets were ground and the

weight equivalent to 12.5 mg of HCTZ added to a 500 mL volumetric flask. For CU

samples, 10 tablets were weighed and each tablet was transferred into a separate 250 mL

volumetric flask. The flask was filled with diluent to half their volume, sonicated for 15

minutes with frequent swirling, and then shaken on a mechanical shaker for 15 minutes.

Samples were diluted to volume with diluent. Final sample solutions for assay and CU

were filtered through a 0.45 µm Whatman glass microfiber filter (GMF), discarding the

first 5 mL of the filtrate. The final solution of the standards and samples contained about

10 µg/mL concentration for 2.5 mg of BF label claim, 20 µg/mL concentration for 5 mg

of BF label claim, 40 µg/mL concentrations for 10 mg of BF label claim, and 25 µg/mL

concentration for 6.25 mg of HCTZ. Resolution ID was prepared to contain about 10

µg/mL final concentrations for fumaric acid and bisoprolol impurity A, 6.25 µg/mL final

concentrations for CTZ, 4-amino, and dimer, 1000 µg/mL final concentration for BF, and

625 µg/mL final concentration for HCTZ. HPLC and UPLC chromatograms for assay

sample are shown in figure 4 and figure 5. HPLC and UPLC chromatograms for

resolution ID having all impurities are shown in figure 6 and figure 7.

13

SampleName BisHCTZ_IntPrecision_MK061822_Assay1; Vial 1:A,8; Injection 1; Result Id 18105; Date Acquired Tuesday, February 15, 2011 7:28:15 PM EST

HC

TZ

Bis

op

rolo

lAU

0.00

0.10

0.20

0.30

0.40

Minutes0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00

Figure 4: Assay sample chromatogram using HPLC

SampleName BisHCTZ_IntPrecision_MK061822_Assay1; Vial 1:A,8; Injection 1; Result Id 20475; Date Acquired Tuesday, February 15, 2011 5:57:31 PM EST

HC

TZ

bis

op

rolo

lAU

0.00

0.20

0.40

0.60

Minutes0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00

Figure 5: Assay sample chromatogram using UPLC

14

Channel 2998; Processed Channel: PDA 224.0 nm; Result Id: 29684; Processing Method: bis trc md nnt LC02 RC

Bis

A -

3.2

28

4-a

min

o -

3.8

24

CT

Z -

4.0

58

HC

TZ

- 4

.31

3

bis

- 5

.36

8

Dim

er

- 6

.07

7

AU

0.00

1.00

2.00

3.00

Minutes0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00

Channel 2998; Processed Channel: PDA 224.0 nm; Result Id: 29684; Processing Method: bis trc md nnt LC02 RC

Bis

A -

3.2

28

4-a

min

o -

3.8

24

CT

Z -

4.0

58

HC

TZ

- 4

.31

3

bis

- 5

.36

8

Dim

er

- 6

.07

7

AU

0.00

0.10

Minutes0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00

Figure 6: Resolution ID chromatogram using HPLC

Channel PDA Spectrum; Processed Channel: PDA 224.0 nm; Result Id: 27175; Processing Method: bis trc md nnt up LC04 id

bis

A -

1.4

57

4 a

min

o -

1.6

73

CT

Z -

1.8

25

hct

z -

2.0

35

Dim

er

- 4

.24

0b

is -

4.3

93

AU

0.00

1.00

2.00

3.00

Minutes0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00

Channel PDA Spectrum; Processed Channel: PDA 224.0 nm; Result Id: 27175; Processing Method: bis trc md nnt up LC04 id

bis

A -

1.4

57

4 a

min

o -

1.6

73

CT

Z -

1.8

25

hct

z -

2.0

35

Dim

er

- 4

.24

0b

is -

4.3

93

AU

0.00

0.05

0.10

0.15

Minutes0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00

Figure 7: Resolution ID chromatogram using UPLC

15

RESULTS AND DISCUSSIONS

Method Development:

An isocratic method on HPLC and UPLC is generally simpler to follow compared

to a gradient method.15 The isocratic method using 0.1 M potassium phosphate buffer pH

4.5, acetonitrile and tetrahydrofuran (85:10:5 v/v/v) as a mobile phase did not give good

separation between 4-amino, CTZ, and HCTZ with Waters Spherisorb cyano column (4.6

mm x 250 mm, 5 μm, CNRC). Using this mobile phase and column, HCTZ eluted at 4.8

minutes and BF eluted at 5.9 minutes. Using the same column and 0.1 M potassium

phosphate buffer pH 4.5 and acetonitrile (85:15 v/v) as a mobile phase did not give good

resolution between CTZ and HCTZ. Bisoprolol impurity A and 4-amino impurity co-

elute. Analysis time was increased because tetrahydrofuran was not part of the mobile

phase. HCTZ eluted at 6.2 minutes and BF eluted at 18.7 minutes. Thus, adequate

resolution was not achieved by the isocratic mode, and a gradient method was developed.

There are many advantages of gradient methods. Gradient methods can improve

separation and shorten the run time16.

Waters symmetry C18 column was the perfect fit for the BF and HCTZ method

because it gives good reproducibility and has pH range from 2–817. Acetonitrile was used

in the mobile phase because acetonitrile is miscible with water in all proportions. Starting

with pure water as the mobile phase and adding acetonitrile to the water progressively

makes the mobile phase dispersive in character and gradually elutes more dispersive

substances. Acetonitrile does not associate strongly with water and thus, as opposed to

methanol, acetonitrile-water mixtures remain binary in character. Methanol also causes a

greater back pressure on the system due to the higher viscosity of methanol/buffer

16

compare to acetonitrile/buffer. TFA was used in a mobile phase to make a low pH buffer.

There are other acids that could be used to make a low pH buffer, but TFA has some

advantages as opposed to other acids. The main advantage is TFA helps to improve

resolution and sharpens the peaks. TFA is also volatile and easily removed. TFA has low

absorption within detection wavelengths; therefore TFA was used in a mobile phase to

make pH 2.2 buffer. BF has a pKa value of 9.5, and HCTZ has pKa value of 7.9 and 9.2.

For basic solutes, the retention normally decreases with decreasing pH of the mobile

phase as long as the pH does not exceed the log of the ionization constant for a base in

reverse phase HPLC. Generally, mobile phase and diluent are similar; they both have

similar pH and organic level. pH of the mobile phase is low (pH 2.2) in order to keep a

short analysis time, because BF and HCTZ are basic solutes.18,19 Maximum wavelength

for the analysis was determined by using a photodiode array (PDA) detector. A PDA

detects the absorption in UV to VIS region. A UV-VIS detector has only one sample-

side light-receiving section, but a PDA detector has multiple photodiode arrays to obtain

information over a wide range of wavelengths at one time. The idea is that spectra are

measured at intervals of one second or less during separation by HPLC with continuous

eluant delivery.20 The 224 nm was the maximum wavelength for BF absorbance. The 272

was the maximum wavelength absorbance for HCTZ. HCTZ had second highest

maximum wavelength absorbance at 225 nm. HCTZ gave a higher response compared to

BF; therefore, samples were analyzed at 224 nm wavelength.

The new developed method for assay, CU, and dissolution using HPLC and

UPLC met all acceptance criteria in the preliminary validation for filter study, extraction

study, linearity, specificity, and recovery were performed. In the assay/CU method, there

17

was a higher concentration for HCTZ, and linearity for HCTZ did not meet acceptance

criteria. The method was changed to the lower concentration for BF and HCTZ. Assay

linearity had a range of 5 μg/mL to 80 μg/mL for BF and a range of 12.5 μg/mL to 50

μg/mL for HCTZ. Both BF and HCTZ linearity had a correlation coefficient > 0.999 and

Y intercept < 3%.

The recovery for assay and dissolution was also performed. The dissolution

recovery met the acceptance criteria, but the assay recovery did not. Diluent for the assay

was changed from 80:20 (water:acetonitrile) diluent to a low pH by adding 1 mL of TFA

into 2 L of 80:20 water: acetonitrile diluent. The diluent with low pH helped to elute a

sharper peak shape for Bisoprolol impurity A, but did not help with recovery. Originally,

the 0.45 μm PVDF Millex filter was used for the assay and CU analysis. Because the

assay recovery was giving low results using 0.45 μm PVDF Millex filter, the filter was

changed to the 0.45 μm Whatman GMF/ with GMF filter. The 0.45 μm Whatman GMF/

with GMF filter gave good recovery results for the assay. Assay recovery was performed

with a range of 5 μg/mL to 80 μg/mL for BF and a range of 12.5 μg/mL to 50 μg/mL for

HCTZ (range from 50 % of lowest label claim to 200 % of highest label claim

concentration). Each individual assay recovery value was between 97.0–103.0 % for BF

and HCTZ. Dissolution recovery was performed with a range of 1.4 μg/mL to 22.2

μg/mL for BF and a range of 3.47 μg/mL to 13.9 μg/mL for HCTZ (range from 50 % of

lowest label claim to 200 % of highest label claim concentration). Each individual

dissolution recovery value was between 95.0–105.0% for BF and HCTZ.

For assay/CU analysis using UPLC, a 2 µL injection volume was used. With an

increased injection volume using UPLC, the HCTZ area got higher and failed to meet

18

acceptance criteria for linearity. For 2.5 mg of BF, assay sample concentration was 10

μg/mL. Dissolution sample concentration for 2.5 mg of BF was 2.8 μg/mL, which is

about four times lower than the assay sample concentration. Therefore, the injection

volume for dissolution was increased to 10 μL to bring the absorbance equivalent to

assay concentration, which is about five times more injection volume than the assay

sample injection volume. Linearity for dissolution met acceptance criteria for BF and

HCTZ with 10 μL injection volume.

The 2011 USP and current Sandoz method for BF/HCTZ tablets have the same

dissolution instrument parameters. Both methods have 0.1 N hydrochloric acid as the

dissolution medium, paddles, speed of 75 RPM and the temperature of the medium is set

at the temperature of the body, 37° C. The sample pull time is 20 minutes for BF and 30

minutes for HCTZ.

UPLC Method Transfer

The transfer of the HPLC method to UPLC method can be accomplished by

scaling factor to the mobile phase’s flow rate and the sample injection volume. HPLC

columns and UPLC columns are not perfectly comparable; therefore, a UPLC method

developed using a scaling factor does not give the same chromatography as HPLC

chromatography.21 BF and HCTZ have five impurities; in order to separate all impurities

and get better resolution, a 100 mm length column was a better choice. Water Acquity

BEH C18, 1.7 μm, 2.1 mm x 100 mm column is one of the most comparable columns to

the Water Symmetry C18, 5 μm, 4.6 mm x 150 mm. For injection volume scaling factor

was follow as equation 1.

19

Target Volume on UPLC (1)

= Original injection volume on HPLC x (Target Column Volume/ Original column Volume)

= 10 μL x (3.14 x 1.12 mm x 100 mm) / (3.14 x 2.32 mm x 150 mm)

= 1.52 μL

Initially flow rate of the mobile phase was calculated based on the scaling factor and

mathematical equation 2 was used.22

Target Flow rate on UPLC (2)

= Original flow rate on HPLC X d2 of target/ d2 of original

= 1.0 mL/min x 2.12 mm / 4.62 mm

= 0.21 mL/min

All other parameters such as column temperature, UV detection, and mobile

phase were the same as HPLC. For the sample injection volume, 1.5 μL did not give

good response of BF. More than 2.0 μL injection volume was overloading the column

with HCTZ and linearity did not meet acceptance criteria. Injection volume of 3.0 μL

gave 0.9998 correlation factor and 3.8 percent y-intercept for HCTZ. The % y-intercept

value should be no more than 3.0 percent of the target value. In order to make UPLC

analysis faster, the flow rate was increased from 0.2 mL/min to 0.3 mL/min.

Assay/CU Validation:

The assay/CU chromatographic method and sample preparation method were

validated by evaluating linearity, range, accuracy, precision, intermediate precision

(ruggedness), specificity, extraction study, filter study, robustness of chromatographic

20

parameters, system suitability, solution stability, force degradation, and analytical method

comparison according to the ICH guidelines Q2 (R1) and current Sandoz SOP.

Linearity/Range:

The linearity of an analytical procedure is its ability (within a given range) to

obtain test results which are directly proportional to the concentration (amount) of analyte

in the sample. Range is normally derived from linearity studies. Range is established by

confirming that the analytical procedure provides an acceptable degree of linearity,

accuracy and precision when applied to samples containing amounts of analyte within or

at the extremes of the specified range of the analytical procedure. According to the ICH

guideline, for the establishment of linearity, a minimum of five concentrations is

recommended.1 In this study, ten concentrations were used to established linearity.

According to the ICH guidelines, minimum specified ranges for the assay of an active

substance or a finished product is normally from 80 to 120 percent of the test

concentration, and for content uniformity, covering a minimum of 70 to 130 percent of

the test concentration1. In this linearity study, the range for BF was a 2 to 400 percent,

based on 2.5 mg of BF concentration. The range for HCTZ was 1 to 200 percent, based

on 6.25 mg of HCTZ concentration. Standard stock solutions of the BF and HCTZ were

diluted to prepare linearity of standard solution in the concentration range of 0.4–80 μg

mL-1 BF and 0.25–50 μg mL-1 HCTZ. Each active was analyzed to plot the linearity

curve. The slope, intercept, correlation coefficient, and residual sums of square were

determined. Linearity acceptance criteria according to the current Sandoz SOP are the

correlation coefficient for linearity cannot be less than 0.999 and the %y-intercept value

21

cannot be more than 3.0% of the target value. Assay CU Linearity results are shown in

table 5. Linearity plots are shown in Appendix A (1–4).

Table 5: Calculated linearity results for BF and HCTZ using HPLC and UPLC. Excel data analysis regression was used for calculation.

BF using HPLC

BF using UPLC

HCTZ using HPLC

HCTZ using UPLC

Linear Regression equation

(y = ax + b)

y= 16548.4x- 1159.2

y= 11466.2x- 625.6

y= 66231x+ 4348.5

y= 46362.8x+ 2129.9

Correlation Coefficient

0.9999 0.9999 0.9999 0.9999

Residual Sum of Square

28674424.6 6938606.2 336256842.1 78539392.4

y-intercept Value 0.69% 0.55% 0.26% 0.19%

Recovery/Accuracy:

The accuracy expresses the closeness of test results obtained by that procedure to

the true value. Recovery or accuracy is sometimes termed trueness. 1 Recovery of the

drug using this new method was determined by adding specific amount of APIs and

placebo to make marketed sample. In the recovery study, the range for BF was 50 to 800

percent (concentration range of 5–80 μg mL-1 BF) based on 2.5 mg of BF concentration.

The range for HCTZ was 50 to 200 percent (concentration range of 12.5–50 μg mL-1

HCTZ) based on 6.25 mg of HCTZ concentration. The recovery was performed in

triplicate for three concentrations. Two 100 percent API (with no placebo) samples were

also prepared for potency determination. Based of the APIs potency, percent recovery

was calculated using Excel. The recovery acceptance criteria according to the current

Sandoz SOP are that each individual recovery value should be between 97.0–103.0

22

percent and the mean recovery value at each level should be between 98.0–102.0 percent.

Assay/CU Accuracy/Recovery results are shown in table 6.

Table 6: Assay/CU Accuracy/Recovery results for BF and HCTZ using HPLC and UPLC

BF % Level BF using HPLC BF using UPLC HCTZ % Level HCTZ using HPLC HCTZ using UPLC

50% - 1 100.4 99.0 50% - 1 99.9 99.8

50% - 2 102.0 99.9 50% - 2 99.5 99.8

50% - 3 101.2 98.3 50% - 3 99.3 99.3

Average of 50% 101.2 99.1 Average of 50% 99.6 99.6

100% - 1 100.3 102.1 100% - 1 99.5 100.0

100% - 2 100.0 102.0 100% - 2 100.1 100.6

100% - 3 100.0 101.9 100% - 3 100.3 100.5


800% - 1 99.6 102.2 200% - 1 99.1 100.0

800% - 2 99.5 102.4 200% - 2 99.2 99.5

800% - 3 100.0 101.5 200% - 3 98.9 100.1


Precision:

The precision expresses the closeness of agreement between a series of

measurements obtained from multiple sampling of the same homogeneous sample under

the prescribed conditions. Precision was performed by preparing six assay preparations

and ten CU preparations at 100 percent concentration for each APIs according to the new

developed method. Intermediate precision was carried out by analyzing the sample by a

different analyst, using different instruments, and on different dates. The assay precision

acceptance criteria according to the current Sandoz SOP are that the percent RSD for

each of the six assay sample preparation should be no more than 2.0 percent. The assay

23

intermediate precision acceptance criteria are that the percent RSD for each of the six

assay sample preparations should be no more than 2.0 percent and the difference in

absolute mean/average difference between analyst one and analyst two should be no more

than 2.0 percent. The CU precision acceptance criteria according to the current Sandoz

SOP are that the acceptance value (AV) for each of the ten CU sample preparations

should be no more than 15.0 percent. The CU intermediate precision acceptance criteria

are that the acceptance value for each of the ten assay sample preparations should be no

more than 15.0 percent and the difference in mean between analyst one and analyst two

should be no more than 5.0 percent. The assay precision and intermediate precision

results for BF are shown in table 7. The assay precision and intermediate precision results

for HCTZ are shown in table 8. The CU precision and intermediate precision results for

BF are shown in table 9. CU precision and intermediate precision results for HCTZ are

shown in table 10.

24

Table 7: Assay precision and intermediate precision results for BF using HPLC and UPLC

Table 8: Assay precision and intermediate precision results for HCTZ using HPLC and UPLC

# of Assay preparations Analyst 1

HCTZ using HPLC

Analyst 2

HCTZ using HPLC

Analyst 1

HCTZ using UPLC

Analyst 2

HCTZ using UPLC

1 98.6 99.1 98.3 99.5

2 99.1 98.6 99.2 98.9

3 98.2 98.6 98.2 98.7

4 98.1 98.7 98.0 98.8

5 99.7 98.5 99.3 98.6

6 97.1 98.5 97.3 98.1

% Average 98.5 98.7 98.4 98.8

% RSD 0.9 0.2 0.8 0.4

Absolute % Difference

in Averages N/A 0.2 N/A 0.4

# of Assay preparations Analyst 1

BF using HPLC

Analyst 2

BF using HPLC

Analyst 1

BF using UPLC

Analyst 2

BF using UPLC

1 90.6 90.1 91.3 90.0

2 94.0 92.2 95.1 91.9

3 95.9 93.8 96.4 93.5

4 91.9 92.1 92.5 92.1

5 93.3 93.6 93.5 93.6

6 94.1 92.5 95.0 91.8

% Average 93.3 92.4 94.0 92.2

% RSD 2.0 1.5 2.0 1.4



25

Table 9: CU precision and intermediate precision results for BF using HPLC and UPLC

# of CU preparations Analyst 1

BF using HPLC

Analyst 2

BF using HPLC

Analyst 1

BF using UPLC

Analyst 2

BF using UPLC

1 92.8 93.2 93.4 93.7

2 89.5 90.6 90.1 91.0

3 97.8 86.8 98.4 86.9

4 89.1 89.5 89.7 89.6

5 90.5 91.3 90.7 92.0

6 91.4 91.0 91.9 91.4

7 93.5 91.0 94.3 91.0

8 93.9 87.5 94.1 87.4

9 93.9 97.6 94.4 98.3

10 90.6 92.5 91.4 92.7

% Average 92.3 91.1 92.8 91.4

AV Value 12.5 14.7 12.0 14.9

% RSD 2.9 3.4 2.8 3.5



26

Table 10: CU precision and intermediate precision results for HCTZ using HPLC and UPLC

# of CU preparations Analyst 1

HCTZ using HPLC

Analyst 2

HCTZ using HPLC

Analyst 1

HCTZ using UPLC

Analyst 2

HCTZ using UPLC

1 98.9 100.0 99.1 99.9

2 98.7 95.5 98.7 95.3

3 96.2 96.8 95.8 96.5

4 96.9 97.3 96.9 96.7

5 100.0 98.4 99.6 98.4

6 100.1 97.6 100.1 97.4

7 102.8 97.7 102.8 97.1

8 100.2 94.8 99.9 94.1

9 97.8 96.1 97.6 96.0

10 96.8 98.5 96.6 98.2

% Average 98.8 97.3 98.7 97.0

AV Value 4.8 4.9 4.9 5.5

% RSD 2.0 1.3 2.1 1.7



Specificity:

The ICH documents define specificity as the ability to assess unequivocally the

analyte in the presence of components that may be expected to be present, such as

impurities, degradation products, and matrix components. Specificity is also known as

selectivity. In this study, placebo and diluent was analyzed under HPLC and UPLC

conditions. The assay specificity chromatogram for the blank and placebo are shown in

Appendix B (1–4). The placebo peak (orange dye, which is used only in 5 mg BF/ 6.25

mg HCTZ tablets for coating) eluted at about 4.0 minutes using HPLC, and using UPLC

27

the placebo peak eluted at 1.4 minutes. The HCTZ peak eluted at about 4.3 minutes using

HPLC. The resolution between the placebo peak and HCTZ peak was 2.5 using HPLC.

The peak purity for BF and HCTZ met acceptance criteria using HPLC and UPLC, which

proved there was no other component that eluted at the same time as the HCTZ peak and

BF peak. The acceptance criteria for peak purity are that purity angle for the peak should

be less than the threshold angle. The peak purity is based on the difference in the purity

angle and the threshold angle expressed in degree. Both the purity angle and threshold

angle were measured by the use of complex algorithms, based on the conversion of the

peak spectra to vectors in a multi-dimensional space. For the determination of purity

angle, the peak apex is used as the reference spectrum; all the other spectral data

contained within that peak are compared to the spectra of the apex. The noise (threshold

angle) calculation is based on a segment of the baseline within the chromatographic run23.

Robustness of Extraction Procedure:

The robustness of extraction procedure was performed to determine whether the

sample preparation procedure has sufficient time for full extraction of the active

ingredient from the sample matrix. In this method, sonication and shaking were specified

for 15 minutes each. The study was conducted with the sonication and shaking of the

assay sample for 10, 15, and 30 minutes and at each time point samples were analyzed

using HPLC and UPLC. The acceptance criteria for the extraction procedure are that the

percent difference (absolute) should be no more than 2.0 percent from the method

suggested time point for the extraction to be considered robust. The robustness of

extraction procedure results are shown in Table 11.

28

Table 11: Robustness of extraction procedure results for BF and HCTZ using HPLC and UPLC

Extraction Time Points BF using

HPLC

BF Using

UPLC

HCTZ using

HPLC

HCTZ using

UPLC

10 min Sonication 92.5 93.7 98.0 98.1

15 min Sonication 93.1 94.6 98.8 99.0

30 min Sonication 92.9 94.7 98.8 99.2

Sonication maximum absolute %

difference in average 0.6 0.9 0.8 0.9

10 min Shaking 91.1 91.8 96.6 97.8

15 min Shaking 92.0 92.6 98.1 98.6

30 min Shaking 91.5 92.7 97.7 98.6

Shaking maximum absolute %

difference in average 0.9 0.8 1.5 0.8

Filter Study:

For the filter study, one assay sample and one assay standard were prepared as per

the new developed method. The standard and sample were filtered through a 0.45 μm

Whatman GMF/with GMF filter and a 0.45 μm Millex-HV PDVF filter. The first 3 mL

(3 mL total), the next 2 mL (5 mL total), the next 2 mL (7 mL total), and the last 3 mL

(10 mL total) were collected from both filters. A portion of the sample was also

centrifuged at 2000 RPM for 10 minutes. The results of the filtered samples were

compared with the results of the centrifuged sample. The filter study results are shown in

table 12. The filter is considered acceptable when the percent difference (absolute) varies

no more than 2.0 percent between collections for the standard. The filtered sample is

considered acceptable if the percent difference (absolute) is no more than 2.0 percent

different from the value obtained from the centrifuged sample. Percent difference results

29

are shown in table 13. In the percent difference results table for sample, only 7 ml

collected results are shown because in the developed method the first 5 ml of filtrate are

discarded. A 0.45 μm Millex-HV PVDF filter gave low results compared to the

centrifuged sample, because BF binds with a filter’s particles. HCTZ and BF results

using 0.45 μm Whatman GMF/with GMF filter are comparable to the centrifuged results;

therefore, the new developed method indicates to use 0.45 μm Whatman GMF/with GMF

filter for assay/CU.

Table 12: Filter study results for BF and HCTZ using HPLC and UPLC

Filter Sample BF using

HPLC

BF Using

UPLC

HCTZ using

HPLC

HCTZ using

UPLC

Centrifuge 93.6 94.5 98.6 99.1

Whatman

3 mL sample 93.9 94.5 99.0 98.9

5 mL sample 93.7 94.7 99.0 99.3

7 mL sample 93.6 94.7 98.7 99.2

10 mL sample 93.7 94.4 98.7 98.8

Millex

3 mL sample 57.7 57.1 98.8 98.3

5 mL sample 74.4 75.0 98.9 98.6

7 mL sample 79.7 79.9 98.8 98.9

10 mL sample 85.8 86.6 98.7 98.9

Whatman

3 mL standard 97.6 98.6 99.2 99.6

5 mL standard 99.0 99.6 99.5 99.9

7 mL standard 99.1 99.8 99.5 100.0

10 mL standard 99.0 99.8 99.4 100.1

Millex

3 mL standard 54.2 54.3 98.9 99.3

5 mL standard 79.0 79.6 99.4 100.1

7 mL standard 87.0 87.4 99.3 99.7

10 mL standard 92.6 93.0 99.4 100.0

30

Table 13: Filter study’s percent difference results for BF and HCTZ using HPLC and UPLC

Filter Sample

Absolute %

Difference BF

using HPLC

Absolute %

Difference BF

Using UPLC

Absolute %

Difference HCTZ

using HPLC

Absolute %

Difference HCTZ

using UPLC

Whatman 7 mL sample 0.0 0.2 0.1 0.1

Millex 7 mL sample 13.9 14.6 0.2 0.2

Whatman Standard 1.5 1.2 0.3 0.5

Millex Standard 38.4 38.7 0.5 0.8

Robustness of Chromatographic Parameter:

The robustness is a measure the capacity to remain unaffected by small deliberate

variations in procedural parameters listed in the procedure documentation and provides

an indication of its suitability during normal usage.1 According to the current Sandoz

SOP, robustness is considered established, once the system suitability meets the

acceptance criteria. If the system suitability criteria are not met under any of the altered

conditions the range may be narrowed accordingly until the robustness is established.

One assay sample was analyzed using the altered chromatographic conditions as per table

14 and 15.

Table 14: Robustness Parameter for HPLC Parameter Variation

Column Equivalency Waters Sunfire C18 4.6 mm x 150 mm 5 µm column

Flow Rate ± 0.2 mL/min

%ACN in Mobile Phase A ± 3 %

Temperature ± 5 °C

Wavelength ± 4 nm

31

Table 15: Robustness Parameter for UPLC

Parameter Variation

Column Equivalency Waters Aquity BEH HSS 2.1 mm x 50 mm 1.8 µm column




Wavelength ± 4 nm

For each condition, the system suitability was evaluated and peak purity was measured

for each API. The robustness system suitability results for BF and HCTZ using HPLC are

shown in table 16, and the robustness system suitability results for BF and HCTZ using

UPLC are shown in table 17.

32

Table 16: Robustness system suitability results for BF and HCTZ using HPLC

Initial Colu

mn

40 °C

(+5

°C)

30 °C

(-5

°C)

87%

A :

13%B

93%

A:

7%B

0.8

mL/min

flow

1.2

mL/min

flow

220

nm

228

nm

BF Retention Time 5.4 5.6 5.4 5.4 5.1 5.6 6.1 4.9 5.4 5.4

HCTZ Retention Time 4.3 4.8 4.1 4.5 3.9 4.5 5.1 3.7 4.3 4.3

% RSD for BF standard 0.1 0.4 0.2 0.1 0.3 0.0 0.1 0.2 0.1 0.1

% RSD for HCTZ Standard 0.2 0.4 0.1 0.1 0.1 0.1 0.1 0.1 0.2 0.1

BF Tailing 1.2 1.2 1.2 1.2 1.2 1.2 1.2 1.1 1.2 1.2

HCTZ Tailing 1.1 1.1 1.1 1.1 1.1 1.1 1.2 1.1 1.1 1.1

BF Plate Counts 96641 104217 102515 91030 83173 105348 95685 92787 98276 98361

HCTZ Plate Counts 43530 52545 40735 41998 30166 47818 47823 37296 43972 43685

BF Purity Angle 0.977 0.305 0.920 0.440 0.291 0.604 0.715 0.597 0.384 0.427

HCTZ Purity Angle 0.168 0.195 0.183 0.179 0.163 0.192 0.190 0.120 0.169 0.169

BF Threshold Angle 1.249 1.120 1.208 1.232 1.254 1.194 1.133 1.616 1.230 1.230

HCTZ Threshold Angle 1.015 1.011 1.015 1.016 1.017 1.014 1.011 1.020 1.015 1.015

BF %Result 94.8 95.2 95.1 95.3 93.8 95.1 95.0 94.9 94.6 94.7

Absolute %Difference of BF N/A 0.4 0.3 0.5 1.0 0.3 0.2 0.1 0.2 0.1

HCTZ % Result 97.8 97.8 97.8 97.8 96.5 97.8 97.5 97.7 96.0 97.8

Absolute % Difference of HCTZ N/A 0.0 0.0 0.0 1.3 0.0 0.3 0.1 1.8 0.0

Resolution Bis A to 4-amino 6.7 9.6 4.9 8.5 12.3 6.6 6.8 6.3 6.7 6.8

Resolution 4-amino to CTZ 2.7 2.7 2.7 2.5 2.3 2.8 2.4 2.8 2.7 2.7

Resolution CTZ to HCTZ 2.5 2.5 2.5 2.5 2.5 2.5 2.4 2.6 0.5 2.6

Resolution HCTZ to BF 10.1 7.9 12.0 8.1 10.3 9.8 8.2 11.6 2.1 10.4

Resolution BF to Dimer 8.0 11.3 6.0 10 8.1 8.1 8.0 7.9 8.2 8.1

33

Table 17: Robustness system suitability results for BF and HCTZ using UPLC

Initial Colu

mn

40 °C

(+5

°C)

33 °C

(-3

°C)

45 °C

(+10

°C)

87%

A:

13%B

93%A:

7%B

0.36

mL/min

flow

0.24

mL/min

flow

220

nm

228

nm

BF Retention Time 4.5 4.4 4.5 4.5 4.5 4.2 4.7 4.2 4.9 4.5 4.5

HCTZ Retention Time 2.0 2.1 1.9 2.1 1.9 1.8 2.3 1.7 2.5 2.0 2.0

% RSD for BF standard 0.1 0.2 0.2 0.3 0.2 0.2 0.5 0.3 0.3 0.1 0.2

% RSD for HCTZ Standard 0.2 0.2 0.2 0.3 0.3 0.3 0.3 0.2 0.3 0.2 0.2

BF Tailing 1.3 1.3 1.3 1.3 1.3 1.3 1.3 1.3 1.2 1.3 1.3

HCTZ Tailing 1.2 1.3 1.2 1.2 1.2 1.2 1.2 1.2 1.2 1.2 1.2

BF Plate Counts 214961 151678 208481 211839 211113 239623 210967 79010 216866 214902 214924

HCTZ Plate Counts 21338 19437 19048 22216 18258 28299 20636 10206 23484 21352 21302

BF Purity Angle 0.702 0.612 0.743 1.009 1.042 1.056 1.028 7.126 0.930 0.711 0.706

HCTZ Purity Angle 0.074 0.060 0.086 0.082 0.093 0.092 0.102 0.149 0.083 1.574 0.076

BF Threshold Angle 1.546 1.267 1.514 1.178 1.689 1.782 1.640 7.804 1.272 0.073 1.556

HCTZ Threshold Angle 1.078 1.014 1.086 1.022 1.086 1.092 1.097 1.197 1.029 1.077 1.079

BF %Result 94.1 90.7* 93.6 93.6 93.7 94.0 94.7 95.1 94.3 94.7 94.5

Absolute %Difference of BF N/A 0.6 0.5 0.5 0.4 0.1 0.6 1.0 0.2 0.6 0.4

HCTZ % Result 96.6 99.1* 96.4 96.3 95.9 96.8 97.6 97.6 97.6 96.7 96.8

Absolute % Difference of HCTZ N/A 0.4 0.2 0.3 0.7 0.2 1.0 1.0 1.0 0.1 0.2

Resolution Bis A to 4-amino 2.7 3.5 2.1 3.2 1.5 3.8 1.5 2.6 2.2 2.7 2.7

Resolution 4-amino to CTZ 2.3 2.2 2.0 2.4 1.8 1.6 3.0 2.2 2.2 2.3 2.3

Resolution CTZ to HCTZ 2.7 2.9 2.5 2.8 2.4 2.3 3.1 2.7 2.7 2.7 2.8

Resolution HCTZ to Dimer 31.1 29.3 28.9 31.9 27.0 28.7 31.9 31.7 28.9 31.4 31.8

Resolution Dimer to BF 1.5 0.8 2.7 0.9 3.8 1.6 1.3 2.0 1.0 1.5 1.5

* Initial condition results for column robustness study were 91.3% for BF and

99.5% for HCTZ using UPLC. The Initial condition results for column robustness were

different because column robustness was performed with a different sample on later day.

The system suitability criteria were met according to the ICH guidelines after

altering the chromatographic conditions for HPLC. HPLC and UPLC did not have all of

34

the impurities elute in the same order. The dimer eluted after BF using HPLC, but using

UPLC, the dimer eluted before BF because the HPLC column and UPLC column are not

perfectly comparable. In UPLC, low temperature caused the dimer to elute later and the

resolution between the dimer and BF failed to meet system suitability criteria at 30 °C

and at 33 °C column temperature. Because low temperature did not pass the resolution

criteria, the samples were analyzed with a 45 °C column temperature. The increase in

column temperature helps to get better resolution between the dimer and BF peaks using

UPLC.

Stability of Sample Solution:

The stability of the analytical solutions was studied by analyzing the standard and

sample solution immediately, after day 1, day 2, and day 6. The stability was assessed by

comparing the area response for standard preparation at different time intervals with the

freshly prepared standard and for standard and sample preparations, which were stored in

amber and clear volumetric flasks as well as at room temperature and cold refrigerator

temperature (4 °C–10 °C). The solution stability acceptance criteria according to the

current Sandoz SOP, are that the standard solution percent change when compared to the

initial value should be no more than 2.0 percent and the sample solution percent change

with respect to initial should be no more than 2.0 percent to be considering solution to be

stable. The solution stability results for the standard preparation using HPLC and UPLC

are shown in table 18. The solution stability results for the sample preparation using

HPLC and UPLC are shown in table 19. The purity angles for all stability of sample

solution were less than the threshold angle for BF and HCTZ. BF’s and HCTZ’s standard

35

and sample were stable in clear and amber flasks and in cold and room temperature at

least for six days.

Table 18: Solution stability results for standard preparation using HPLC and UPLC

BF using HPLC BF using UPLC HCTZ using HPLC HCTZ using UPLC

%

Result

Absolute %

Difference

% Result

Absolute %

Difference

% Result

Absolute %

Difference

% Result

Absolute %

Difference Initial day 99.6 N/A 100.0 N/A 99.8 N/A 100.1 N/A

Day 1

Clear Room Temp

100.7 1.1 100.7 0.7 100.5 0.7 100.4 0.3

Clear Cold Temp

100.4 0.8 100.2 0.2 100.3 0.5 100.2 0.1

Amber Room Temp

100.5 0.9 100.1 0.1 100.3 0.5 100.2 0.1

Amber Cold Temp

100.5 0.9 100.1 0.1 100.5 0.7 100.1 0.0

Day 2

Clear Room Temp

100.0 0.4 99.8 0.2 100.1 0.3 99.8 0.3

Clear Cold Temp

100.4 0.8 99.9 0.1 100.4 0.6 100.1 0.0

Amber Room Temp

99.9 0.3 99.8 0.2 100.1 0.3 100.0 0.1

Amber Cold Temp

100.2 0.6 100.3 0.3 100.3 0.5 100.3 0.2

Day 6

Clear Room Temp

99.7 0.1 99.9 0.1 99.7 0.1 100.1 0.0

Clear Cold Temp

100.0 0.4 100.1 0.1 100.0 0.2 100.1 0.0

Amber Room Temp

99.5 0.1 99.9 0.1 99.9 0.1 100.3 0.2

Amber Cold Temp

100.2 0.6 100.2 0.2 100.2 0.4 100.1 0.0

36

Table 19: Solution stability results for sample preparation using HPLC and UPLC

BF using HPLC BF using UPLC HCTZ using HPLC HCTZ using UPLC %

Result Absolute

% Difference

% Result

Absolute %

Difference

% Result

Absolute%

Difference

% Result

Absolute%

Difference Initial day 93.7 N/A 94.9 N/A 98.7 N/A 99.4 N/A

Day 1

Clear Room Temp

94.7 1.0 94.7 0.2 99.5 0.8 98.8 0.4

Clear Cold Temp

94.6 0.9 95.0 0.1 99.5 0.8 99.4 0.0

Amber Room Temp

95.0 1.3 94.7 0.2 99.7 1.0 99.0 0.4

Amber Cold Temp

94.8 1.1 95.4 0.5 99.6 0.9 99.6 0.2

Day 2

Clear Room Temp

94.8 1.1 94.7 0.2 99.1 0.4 98.9 0.5

Clear Cold Temp

94.5 0.8 94.9 0.0 99.5 0.8 99.5 0.1

Amber Room Temp

94.6 0.9 95.6 0.7 99.6 0.9 100.1 0.7

Amber Cold Temp

94.6 0.9 95.2 0.3 99.6 0.9 99.7 0.3

Day 6

Clear Room Temp

93.7 0.0 94.4 0.5 97.9 0.8 97.9 1.5

Clear Cold Temp

94.3 0.6 95.0 0.1 99.2 0.5 99.2 0.2

Amber Room Temp

94.1 0.4 94.7 0.2 99.4 0.7 99.3 0.1

Amber Cold Temp

94.4 0.7 95.0 0.1 99.4 0.7 99.3 0.1

Forced Degradation:

Forced degradation or stress testing is undertaken to demonstrate specificity when

developing stability-indicating methods. These studies also provide information about the

degradation pathways and degradation products that could form during storage. Forced

degradation studies may help facilitate pharmaceutical development as well in areas such

as formulation development, manufacturing, and packaging. Overstressing a molecule

can lead to degradation profiles that are not representative of real storage conditions and

37

perhaps not relevant to method development. Therefore, stress-testing conditions should

be realistic and not excessive. In this regard, it is the amount of stress that is important

and not necessarily the extent of degradation. Indeed, some compounds may not degrade

significantly after considerable exposure to stress conditions. The forced degradation

study was conducted on samples containing placebo alone, BF raw material alone, HCTZ

raw material alone and blend for BF/HCTZ tablets (BF raw material + HCTZ raw

material + placebo) for each condition. Intentional degradation was carried out by

exposing each sample to 20 mL of 1N hydrochloric acid for four hours at room

temperature, 20 mL of 1N sodium hydroxide for one hour at 65 °C using a water bath,

and 20 mL of 10% hydrogen peroxide for ninety minutes at 65 °C using a water bath. For

the thermal stress study, the BF raw material, placebo, and blend were kept as a dry

powder and placed in oven at 105 °C for two hours. The HCTZ raw material, placebo,

and blend were kept in a dry oven at 105 °C for five days. The photolytic stress study was

performed on dry powder and on sample solution. The dry powder samples were placed

under UV light at the short and long wavelengths for five days and then the samples were

prepared according to the developed method. For sample solution, each sample was

prepared according to the method and then the sample solutions were placed under UV

light at short and long wavelengths. Based on previous forced degradation studies the

HCTZ degrades quickly under UV light; therefore, HCTZ solutions were analyzed after

three hours under UV light. BF solutions were placed under UV light and were analyzed

every day up to five days. For the humidity stress study, dry powder samples were placed

in a desiccator with water on the bottom and tightly covered. The desiccator was placed

at room temperature for five days and then the samples were prepared according to the

38

method. Moisture saturated headspace (MSH) studies were carried out on the dry powder

for each sample. The samples were placed in a desiccator with water on the bottom and

tightly covered. The desiccator was placed in an oven maintained at 90 °C for forty eight

hours. After 48 hours, the samples were prepared according to the new developed

method. The control samples were prepared with no extra stress to compare with forced

degradation samples. All forced degradation samples were analyzed using a PDA for the

peak purity evaluation. The acceptance criteria of the degradation for each condition

should be no more than 15 percent. Acceptance of a higher amount of degradation can be

assessed on a case-by-case basis. The purity angle should be less than threshold angle for

BF and HCTZ. These methods were compare entire UV spectra recorded at various

points across the LC peak with the spectrum recorded at the apex of the peak, which can

reveal details of the retention time as well as the UV spectrum of the co eluting

impurity24. In each of the cases, it was observed that the purity angle was less than the

threshold angle indicating that the BF and HCTZ peaks observed were pure. The forced

degradation results for BF in blend sample using HPLC are shown in table 20. The forced

degradation results for BF in blend sample using UPLC are shown in table 21. The forced

degradation results for HCTZ in blend sample using HPLC are shown in table 22. The

forced degradation results for HCTZ in blend sample using UPLC are shown in table 23.

39

Table 20: Forced degradation results for BF in Blend sample using HPLC

Condition Time % BF Absolute %

Difference Purity Angle Threshold Angle

Control N/A 98.6 N/A 0.244 1.058

UV (dry) 5 days 100.0 1.4 0.200 1.047

UV (solution) 5 days 85.9 12.7 0.194 1.051

Thermal (105 °C) 2 hrs 91.1 7.5 0.480 1.067

Humidity 5 days 101.5 2.9 0.227 1.051

MSH (90 °C) 48 hrs 94.4 4.2 0.585 1.057

Acid (1N HCl at RT) 4 hrs 95.8 2.8 0.236 1.058

Base (1N NaOH 65 °C) 1 hr 84.9 13.7 0.228 1.070

Peroxide (10% H2O2 at 65 °C) 90 min 85.6 13.0 0.890 1.020

Table 21: Forced degradation results for BF in Blend sample using UPLC

Condition Time % BF Absolute %


Control N/A 98.0 N/A 0.452 1.302

UV (dry) 5 days 99.2 1.2 0.234 1.336

UV (solution) 5 days 85.5 12.5 1.479 2.490

Thermal (105 °C) 2 hrs 91.7 6.3 0.461 1.278

Humidity 5 days 100.9 2.9 0.123 1.160

MSH (90 °C) 48 hrs 95.3 2.7 0.389 1.268


Base (1N NaOH 65 °C) 1 hr 84.3 13.7 1.175 1.931


40

Table 22: Forced degradation results for HCTZ in Blend sample using HPLC

Condition Time % HCZAbsolute %


Control N/A 97.7 N/A 0.166 1.015

UV (dry) 5 days 98.5 0.8 0.186 1.015

UV (solution) 3 hrs 87.2 10.5 0.176 1.019

Thermal (105 °C) 5 days 91.7 6.0 0.187 1.016

Humidity 5 days 99.0 1.3 0.190 1.016

MSH (90 °C) 48 hrs 82.1 15.6 0.178 1.019


Base (1N NaOH 65 °C) 1 hr 81.7 16.0 0.163 1.018


Table 23: Forced degradation results for HCTZ in Blend sample using UPLC

Condition Time % HCZAbsolute %


Control N/A 97.8 N/A 0.114 1.123

UV (dry) 5 days 99.0 1.2 0.057 1.066

UV (solution) 3 hrs 88.0 9.8 0.170 1.179

Thermal (105 °C) 5 days 92.3 5.5 0.079 1.082

Humidity (RT) 5 days 99.5 1.7 0.054 1.048

MSH (90 °C) 48 hrs 83.8 14.0 0.211 1.123


Base (1N NaOH 65 °C) 1 hr 81.7 16.1 0.095 1.131


BF degraded most at the high temperature environment, base, and peroxide. BF in

solution degraded under UV light after 5 days. HCTZ degraded most in the humid

41

environment, base, and peroxide. HCTZ also degraded a lot under UV light. HCTZ had

solution stability for six days at room temperature under room (fluorescent) light.

Humans perceive white as a combination of several frequencies of light, which can be

simplified to just a few primary colors, such as the red, green and blue used in television.

A blend of phosphors is used to generate what is considered to be a white light. A

phosphor wavelength commonly used in lamps for red is 611 nm to 658 nm, for green is

491 nm to 575 nm, and for blue is 424 nm to 491 nm. For forced degradation study,

samples were placed under UV at 254 nm and 360 nm. HCTZ sample degraded at low

wavelength. In fluorescent light wavelengths are more than 400 nm and therefore,

samples were stable for six days under fluorescent light.

Method Comparison:

Currently a method is in place at Sandoz, which is approved and used for the

determination of the assay for BF and HCTZ in dosage form. A USP method is also

available. Method comparison was executed to demonstrate the equivalency of the

methods. Six assay samples were prepared according to the USP method, Sandoz current

method, and the new developed method. The acceptance criteria are the difference in the

mean value of the six sample preparations between each method and should be no more

than 2.0 percent absolute. The method comparison results for BF using the USP method

are shown in table 24. The method comparison results for HCTZ using the USP method

are shown in table 25. The method comparison results for BF using the current Sandoz

method are shown in table 26. The method comparison results for HCTZ using the

current Sandoz method are shown in table 27.

42

Table 24: Method comparison results for BF using USP method.

# of Prep % BF

(USP Method)

% BF (New

HPLC method)

% BF (New

UPLC method)

1 92.4 90.6 91.3

2 92.7 94.0 95.1

3 94.4 95.9 96.4

4 92.0 91.9 92.5

5 92.8 93.3 93.5

6 92.5 94.1 95.0

Mean 92.8 93.3 94.0

% RSD 0.9 2.0 2.0

Absolute % Difference in average between new HPLC method 0.5 N/A N/A

Absolute % Difference in average between new UPLC method 1.2 N/A N/A

Table 25: Method comparison results for HCTZ using USP method.

# of Prep % HCTZ

(USP Method)

% HCTZ (New

HPLC method)

% HCTZ (New

UPLC method)

1 99.9 98.6 98.3

2 98.8 99.1 99.2

3 98.7 98.2 98.2

4 97.9 98.1 98.0

5 101.1 99.7 99.3

6 101.7 97.1 97.3

Mean 99.7 98.5 98.4

% RSD 1.5 0.9 0.8

Absolute % Difference in average between new HPLC method 1.2 N/A N/A

Absolute % Difference in average between new UPLC method 1.1 N/A N/A

43

Table 26: Method comparison results for BF using current Sandoz method.

# of Prep % BF(Current

Sandoz Method)

% BF (New

HPLC method)

% BF (New

UPLC method)

1 93.2 90.6 91.3

2 93.8 94.0 95.1

3 93.1 95.9 96.4

4 94.0 91.9 92.5

5 93.4 93.3 93.5

6 93.4 94.1 95.0

Mean 93.5 93.3 94.0

% RSD 0.4 2.0 2.0

Absolute % Difference in average

between new HPLC method 0.2 N/A N/A


between new UPLC method 0.5 N/A N/A

44

Table 27: Method comparison results for HCTZ using current Sandoz method.

# of Prep % HCTZ

(Current Sandoz Method)

% HCTZ (New

HPLC method)

% HCTZ (New

UPLC method)

1 98.3 98.6 98.3

2 99.8 99.1 99.2

3 98.9 98.2 98.2

4 98.7 98.1 98.0

5 98.1 99.7 99.3

6 98.5 97.1 97.3

Mean 98.7 98.5 98.4

% RSD 0.6 0.9 0.8


between new HPLC method 0.2 N/A N/A


between new UPLC method 0.3 N/A N/A

Dissolution Method Validation:

Dissolution profiles are often performed to demonstrate how a product may

behave in the human body over time. Normally, the dissolution medium is 0.1N

hydrochloric acid to mimic the aqueous environment of the stomach. The temperature of

the medium was set at the body temperature, 37 °C. The dissolution parameters were kept

the same as the USP method and the current Sandoz method. The current Sandoz method

required using Teflon paddles, but the USP method does not specify for any specific type

of paddles. Therefore, Teflon paddles vs. non Teflon paddles study was conducted during

method validation. The dissolution chromatographic conditions and the sample

preparation method were validated by evaluating accuracy, precision, intermediate

precision, linearity, range, specificity, filter study, robustness of degassing procedure,

45

robustness of sampling procedure, system suitability, solution stability, robustness of

chromatographic parameters, method comparison, and Teflon paddles vs. non Teflon

paddles according to the ICH guidelines Q2 (R1) and current Sandoz SOP.

Linearity/Range:

Range is established by confirming that the analytical procedure provides an

acceptable degree of linearity, accuracy, and precision when applied to samples

containing amounts of analyte within or at the extremes of the specified range of the

analytical procedure. In this study, seven concentrations were used to established

linearity. According to the ICH guidelines, the minimum specified ranges for the

dissolution of an active substance or a finished product is normally from 80 to 120

percent of the test concentration.1 In this linearity study, the range for BF was 10 to 400

percent, based on 2.5 mg of BF sample concentration. The range for HCTZ was 20 to 200

percent, based on 6.25 mg of HCTZ sample concentration. The standard stock solution of

the BF and HCTZ was diluted to prepare the linearity of the standard solution in the

concentration range of 0.56–22.4 μg mL-1 BF and range of 1.4–13.0 μg mL-1 HCTZ. Each

active was analyzed to plot the linearity curve. The slope, intercept, correlation

coefficient, and residual sums of square were determined. The linearity acceptance

criteria according to the current Sandoz SOP are that the correlation coefficient for

cannot be less than 0.99 and the %y-intercept value cannot be more than 5.0 percent of

the target value. The dissolution linearity results for BF and HCTZ using HPLC and

UPLC are shown in table 28. The dissolution linearity plots are shown in Appendix E (1–

4)

46

Table 28: Linearity results for BF and HCTZ using HPLC and UPLC.

BF using HPLC

BF using UPLC

HCTZ using HPLC

HCTZ using UPLC

Linear Regression equation

(y = ax + b)

y= 33902.0x+ 2163.9

y= 73377.3x+ 368.9

y= 137000.2x+ 4743.8

Y= 293342.5x+ 22453.5

Correlation Coefficient

0.999 0.999 0.999 0.999

Residual Sum of Square

3318369 1352032 39004807 1931642671

y-intercept Value 2.23 0.18 0.50 1.09

Recovery/Accuracy:

Recovery of the drug using this new method was determined by spiking a specific

amount of API and placebo to make marketed sample. In this recovery study, the range

for BF was 50 to 200 percent (concentration range of 1.4–22 μg mL-1 BF) based on 2.5

mg of BF in the sample concentration. The range for HCTZ was 50 to 150 percent

(concentration range of 3.5–13.9 μg mL-1 HCTZ) based on 6.25 mg of HCTZ in the

sample concentration. Recovery was performed in triplicate for three concentrations. Two

100 percent API (with no placebo) samples were also prepared for potency

determination. Based from the APIs’ potency, percent recovery was calculated. The

recovery acceptance criteria according to the current Sandoz SOP are that the mean

recovery value should be between 95 to 105 percent. The dissolution recovery/accuracy

results for BF and HCTZ using HPLC and UPLC are shown in table 29.

47

Table 29: Recovery/Accuracy results for BF and HCTZ using HPLC and UPLC.

BF % Level BF using

HPLC

BF using

UPLC HCTZ % Level

HCTZ using

HPLC

HCTZ using

UPLC

50% - 1 102 102 50% - 1 101 101

50% - 2 101 101 50% - 2 101 101

50% - 3 101 102 50% - 3 101 101

Average of 50% 101 102 Average of 50% 101 101

100% - 1 100 100 100% - 1 100 100

100% - 2 99 100 100% - 2 100 100

100% - 3 100 100 100% - 3 100 100


800% - 1 99 100 150% - 1 100 99

800% - 2 99 100 150% - 2 100 99

800% - 3 99 100 150% - 3 100 99


Precision:

The precision study was performed by doing a dissolution using six tablets. A

different analyst performed the intermediate precision testing on a different instrument

and date and the results compared with the original precision study. The acceptance

criteria according to the current Sandoz SOP are that the percent RSD for each of the six

sample preparation should be no more than 5.0 percent for dissolution precision. The

dissolution intermediate precision acceptance criteria are that the percent RSD for each of

the six sample preparations should be no more than 5.0 percent and the difference in the

mean between analyst one and analyst two should be no more than 5.0 percent. The

precision and intermediate precision for BF and HCTZ using HPLC results are shown in

48

table 30. The precision and intermediate precision for BF and HCTZ using UPLC results

are shown in table 31.

Table 30: Precision and intermediate precision results for BF and HCTZ using HPLC

Sample # Analyst 1

BF using HPLC

Analyst 2

BF using HPLC

Analyst 1

HCTZ using HPLC

Analyst 2

HCTZ using HPLC

1 93 86 98 94

2 88 85 96 95

3 89 90 99 97

4 90 91 95 100

5 88 92 98 96

6 93 87 98 97

% Average 90 88 97 96

% RSD 2.5 3.1 1.7 2.2


in Averages N/A 2 N/A 1

49

Table 31: Precision and intermediate precision results for BF and HCTZ using UPLC

Sample # Analyst 1

BF using UPLC

Analyst 2

BF using UPLC

Analyst 1

HCTZ using UPLC

Analyst 2

HCTZ using UPLC

1 93 87 98 94

2 88 86 96 94

3 90 90 99 96

4 90 91 95 100

5 88 93 99 96

6 93 88 98 96

% Average 90 89 97 96

% RSD 2.5 3.2 1.7 2.3


in Averages N/A 1 N/A 1

Specificity:

In the specificity study, a placebo was prepared according to the dissolution

parameters. The placebo sample was pulled from a dissolution vessel after 20 minutes

and 30 minutes as per dissolution parameter. The placebo solution and dissolution

medium were analyzed under HPLC and UPLC conditions. No interfering peaks were

above the limit of quantitation in the blank or the placebo injection. The dissolution

specificity chromatogram for blank and placebo are shown in Appendix D (1–6). The

placebo peak (orange dye which is used only in 5 mg BF/ 6.25 mg HCTZ tablets for

coating) elutes at about 4.0 minutes using HPLC and at about 1.4 minutes using UPLC.

The HCTZ peak elutes at about 4.3 minutes using HPLC. The resolution between the

placebo peak and HCTZ peak is 2.5 using HPLC. The peak purity for BF and HCTZ met

50

acceptance criteria using HPLC and UPLC, which proved there were no other

components eluted at the same time as the HCTZ peak and as the BF peak.

Robustness of Degassing Procedure:

The robustness of degassing procedure was performed to determine whether the

degassing procedure for the dissolution medium affects the rate of dissolution release for

each API. In this method, the degassing method was not specified. The study was

conducted to prove the degassing of the dissolution medium does not affect the rate of

dissolution release. The dissolution robustness of the degassing procedure was

performed using six tablets with non-degassed dissolution medium, vacuum-degassed

(for 15 minutes) dissolution medium, and helium sparged (for 15 minutes) dissolution

medium. The amount of dissolved oxygen in the dissolution medium was measured prior

to dispensing the dissolution medium to the dissolution vessels. The acceptance criteria

of the degassing procedure may be considered un-biased on the rate of dissolution if the

mean percent dissolved for all six tablets between each bath is within ± 5 percent

absolute of one another. The robustness of degassing procedure results for BF using

HPLC are shown in table 32. The robustness of degassing procedure results for BF using

UPLC are shown in table 33. The robustness of degassing procedure results for HCTZ

using HPLC are shown in table 34. The robustness of degassing procedure results for

HCTZ using UPLC are shown in table 35.

51

Table 32: Robustness of degassing procedure results for BF using HPLC

# of Tablets Non Degassed Vacuum Degassed Helium Sparged

1 88 83 93

2 91 83 88

3 89 85 90

4 83 95 89

5 91 93 88

6 86 88 93

% Average 88 88 90

Absolute % difference between

highest mean to lowest mean 2

Table 33: Robustness of degassing procedure results for BF using UPLC


1 89 84 93

2 91 84 88

3 91 86 90

4 84 96 90

5 92 94 88

6 87 89 93

% Average 89 89 90



52

Table 34: Robustness of degassing procedure results for HCTZ using HPLC


1 99 95 98

2 96 97 96

3 94 97 99

4 91 98 95

5 100 99 98

6 96 97 98

% Average 96 97 97



Table 35: Robustness of degassing procedure results for HCTZ using UPLC


1 99 95 98

2 96 98 96

3 92 97 99

4 89 98 95

5 100 99 99

6 96 97 98

% Average 95 98 97



In the non-degassed dissolution medium, 84.4 percent of dissolved oxygen

was measured prior to dispensing the medium. In the vacuum-degassed dissolution

medium, 71.2 percent of dissolved oxygen was measured prior to dispensing the medium.

In the helium sparged dissolution medium, 30.8 percent of dissolved oxygen was

measured prior to dispensing the medium. All results are within 5 percent, therefore the

53

degassing procedure of the dissolution medium does not affect the rate of dissolution

release.

Robustness of the Sampling Procedure (Manual vs. Automated):

The robustness of the sampling procedure was performed to prove that neither

manual nor automated pulling the sample from the dissolution vessels affected the rate of

the dissolution. In this method, the sampling procedure was not specified. The dissolution

robustness of the sampling procedure using six tablets was performed by pulling a sample

manually from each of the dissolution vessels and by pulling a sample automatically

using on automated instrument from each of the dissolution vessels. The acceptance

criteria of the sampling procedure may be considered unbiased on the rate of dissolution

if the mean percent dissolved for all six tablets between each bath is within ± 5 percent

absolute of one another. The robustness of sampling procedure results for BF and HCTZ

using HPLC are shown in table 36. The robustness of sampling procedure results for BF

and HCTZ using UPLC are shown in table 37.

54

Table 36: Robustness of sampling procedure results for BF and HCTZ using HPLC

Tablet # % BF

(manual)

% BF

(automated)

% HCTZ

(manual)

% HCTZ

(automated)

1 93 88 98 95

2 88 98 96 99

3 90 77 99 87

4 89 71 95 82

5 88 94 98 100

6 93 92 98 100

% Average 90 86 97 94

Absolute % Difference in Averages N/A 4 N/A 3

Table 37: Robustness of sampling procedure results for BF and HCTZ using UPLC

Tablet # % BF

(manual)

% BF

(automated)

% HCTZ

(manual)

% HCTZ

(automated)

1 93 89 98 96

2 88 99 96 100

3 90 81 99 86

4 90 75 95 80

5 88 95 95 100

6 93 93 98 101

% Average 90 89 97 97

Absolute % Difference in Averages N/A 1 N/A 0

Filter Study:

In the filter study, the dissolution of one tablet was performed and one standard

was prepared according to the new developed method. The standard and sample were

filtered through a 0.45 μm Whatman GMF/with GMF filter and a 0.45 μm Millex-HV

55

polyvinylidenefluoride (PVDF) filter. The first 3 mL (3 mL total), the next 2 mL (5 mL

total), the next 2 mL (7 mL total), and the last 3 mL (10 mL total) were collected from

both filters. A portion of the sample was also centrifuged at 2000 RPM for 10 minutes.

The filtered samples were compared with the centrifuged sample. The dissolution filter

study data is shown in table 38 for BF and HCTZ using HPLC and UPLC. The filter is

considered acceptable when the percent difference (absolute) varies no more than 2.0

percent between collections for the standard. The filtered sample is also considered

acceptable if the percent difference (absolute) is no more than 2.0 percent different from

the value obtained from the centrifuged sample. The percent difference data for filter

study is shown in table 39 for BF and HCTZ using HPLC and UPLC. In the percent

difference results table for the samples, only 5 ml collected results are shown because in

the developed method it is mentioned to discard the first 3 ml of filtrate. HCTZ and BF

results using 0.45 μm Millex-HV PVDF and 0.45 μm Whatman GMF/with GMF filter

are comparable to the centrifuged results.

56

Table 38: Filter study results for BF and HCTZ using HPLC and UPLC

Filter Sample BF using

HPLC

BF Using

UPLC

HCTZ using

HPLC

HCTZ using

UPLC

Centrifuge 93.5 92.0 98.2 98.5

Whatman

3 mL sample 91.9 88.8 98.6 98.4

5 mL sample 94.2 91.7 98.5 98.5

7 mL sample 93.5 91.7 98.2 98.7

10 mL sample 93.8 91.8 97.8 98.5

Millex

3 mL sample 91.7 91.0 98.6 96.4

5 mL sample 92.2 91.8 98.6 98.5

7 mL sample 91.5 91.8 98.5 98.8

10 mL sample 91.6 91.9 98.5 98.4

Whatman

3 mL standard 98.7 95.8 99.9 99.9

5 mL standard 99.7 99.4 99.8 100.1

7 mL standard 99.4 99.5 99.7 100.1

10 mL standard 99.3 99.6 99.7 1002

Millex

3 mL standard 100.7 98.3 99.9 98.6

5 mL standard 99.0 99.7 99.8 100.2

7 mL standard 99.7 99.5 100.0 100.0

10 mL standard 100.1 99.8 100.0 100.0

Table 39: Filter study’s percent difference results for BF and HCTZ using HPLC and UPLC

Filter Sample

Absolute %

Difference BF

using HPLC

Absolute %

Difference BF

Using UPLC

Absolute %

Difference HCTZ

using HPLC

Absolute %

Difference HCTZ

using UPLC

Whatman 5 mL sample 0.7 0.3 0.3 0.0

Millex 5 mL sample 1.3 0.2 0.4 0.0

Whatman Standard 1.0 3.8 0.2 0.3

Millex Standard 1.7 1.5 0.2 1.6

57

Robustness of Chromatographic Parameter:

In the robustness of the chromatographic parameter study, the dissolution of one

tablet was performed and one standard was prepared according to the new developed

method. The dissolution sample and standard were analyzed using the altered

chromatographic conditions as per table 40 for HPLC parameters and 41 for UPLC

parameters. According to the current Sandoz SOP, robustness has to be established; if the

system suitability criteria do not meet under any of the altered conditions, the range may

be narrowed accordingly until robustness is established.

Table 40: Robustness Parameter for HPLC Parameter Variation

Column Equivalency Waters Sunfire C18 4.6 mm x 150 mm 5 µm column




Wavelength ± 4 nm

Table 41: Robustness Parameters for UPLC

Parameter Variation

Column Equivalency Waters Aquity BEH Shield 2.1 mm x 50 mm 1.7 µm column




Wavelength ± 4 nm

58

For each condition, the system suitability was evaluated for each BF and HCTZ. The

robustness system suitability results for BF and HCTZ using HPLC are shown in table

42, and the robustness system suitability results for BF and HCTZ using UPLC are

shown in table 43.

Table 42: Robustness system suitability results for BF and HCTZ using HPLC

Initial Colu

mn

40 °C

(+5

°C)

30 °C

(-5

°C)

93%

A:

7%B

87%

A:

13%B

0.8

mL/min

flow

1.2

mL/min

flow

220

nm

228

nm





BF Tailing 1.2 1.2 1.1 1.1 1.2 1.1 1.2 1.1 1.1 1.2

HCTZ Tailing 1.1 1.1 1.1 1.1 1.1 1.1 1.1 1.1 1.1 1.1

BF Plate Counts 97382 103393 102357 93892 113544 95410 98025 98025 97493 97257


BF %Result 93 93 93 93 93 93 94 93 93 93

Absolute % Difference of BF N/A 0 0 0 0 0 1 0 0 0

HCTZ % Result 100 100 100 100 100 100 100 100 100 100

Absolute % Difference of HCTZ N/A 0 0 0 0 0 0 0 0 0


59

Table 43: Robustness system suitability results for BF and HCTZ using UPLC

Initial Colu

mn

40 °C

(+5

°C)

30 °C

(-5

°C)

87%

A:

13%B

93%

A:

7%B

0.36

mL/min

flow

0.24

mL/min

flow

220

nm

228

nm





BF Tailing 1.3 1.3 1.3 1.3 1.3 1.3 1.3 1.3 1.3 1.3

HCTZ Tailing 1.3 1.3 1.4 1.3 1.4 1.3 1.3 1.4 1.3 1.3

BF Plate Counts 70273 59913 71356 71638 60758 93092 65553 76964 70072 70332


BF %Result 94 95 94 93 95 94 94 95 94 94

Absolute % Difference of BF N/A 1 0 1 1 0 0 1 0 0

HCTZ % Result 100 100 100 100 99 100 100 100 100 100

Absolute % Difference of HCTZ N/A 0 0 0 1 0 0 0 0 0


The system suitability criteria were met according to the ICH guidelines even

after altering the chromatographic conditions for HPLC and UPLC.

Stability of Sample Solution:

The stability of the analytical solutions of the method was studied by analyzing

the standard and sample solution immediately after day 1, day 2, and day 3. The stability

was assessed by comparing the area response for the standard preparation at different

time intervals with the freshly prepared standard and for sample and standard

preparations. The standard and sample were stored in amber and clear volumetric flasks

for HCTZ and BF at room temperature and at cold refrigerator temperature (4 °C–10 °C).

The solution stability acceptance criteria according to the current Sandoz SOP are that the

60

standard solution percent change when compared to the initial value should be no more

than 2 percent and the sample solution percent change with respect to initial should be no

more than 2 percent to be considered stable. The dissolution solution stability results for

the BF standard using HPLC and UPLC are shown in table 44. The solution stability

results for the BF sample using HPLC and UPLC are shown in table 45. The solution

stability results for the HCTZ standard using HPLC and UPLC are shown in table 46.

The solution stability results for the HCTZ sample using HPLC and UPLC are shown in

table 47

Table 44: Solution stability results for BF standard using HPLC and UPLC

BF using HPLC BF using UPLC

% Result Absolute %

Difference % Result Absolute %

Difference Initial day 99.0 N/A 99.4 N/A

12 hrs

Clear Room Temp 98.5 0.5 98.9 0.5 Clear Cold Temp 100.1 1.1 99.6 0.2

Amber Room Temp 98.0 1.0 99.5 0.1

Day 1 (24hr)


Amber Room Temp 96.4 2.6 96.7 2.7

Day 2 Clear Room Temp 95.8 3.2 95.0 4.4 Clear Cold Temp 99.6 0.6 100.6 1.2

61

Table 45: Solution stability results for BF sample using HPLC and UPLC

BF using HPLC BF using UPLC

% Result Absolute %



12 hrs Clear Room Temp 84.6 1.8 85.8 1.4 Clear Cold Temp 88.0 1.6 88.7 1.5

Amber Room Temp 87.0 0.6 87.9 0.7

Day 1 (24hr)


Amber Room Temp 85.9 0.5 86.2 1.0 Day 2 Clear Cold Temp 87.9 1.5 88.6 1.4

Table 46: Solution stability results for HCTZ standard using HPLC and UPLC

HCTZ using HPLC HCTZ using UPLC % Result Absolute %



Day 1 Clear Room Temp 99.8 0.4 99.7 0.3

Amber Room Temp 99.7 0.5 99.7 0.3


Amber Room Temp 98.9 1.3 99.1 0.9


Amber Room Temp 98.5 1.7 98.7 1.3


Amber Room Temp 99.6 0.6 98.7 1.3

Table 47: Solution stability results for HCTZ sample using HPLC and UPLC

HCTZ using HPLC HCTZ using UPLC % Result Absolute %




Amber Room Temp 98.1 0.5 97.9 0.5


Amber Room Temp 96.9 2.0 97.3 1.1


Amber Room Temp 95.8 2.8 95.9 2.5

62

BF’s standard and sample were stable in cold temperature for at least 2 days and

were stable at room temperate for 12 hrs. HCTZ’s standard was stable in clear and amber

flask for at least for four days and HCTZ’s sample was stable in amber flask for at least

two days.

Method Comparison:

Currently there is a method in place at Sandoz which is approved and used to

determine the dissolution rate. A USP method is also available for the determination of

dissolution rate. Therefore, a method comparison study was executed to demonstrate the

equivalency of this method. A dissolution using six tablets was performed using the USP

method, Sandoz current method, and this new developed method. The acceptance criteria

are that the difference in the mean value of the six tablets preparation between each

method should be no more than 5.0 percent absolute. The method comparison results for

BF using the USP method are shown in table 48. The method comparison results for

HCTZ using the USP method are shown in table 49. The method comparison results for

BF using the current Sandoz method are shown in table 50. The method comparison

results for HCTZ using the current Sandoz method are shown in table 51.

63

Table 48: Method comparison results for BF using USP method

Tablet # % BF

(USP Method)

% BF (New

HPLC method)

% BF (New

UPLC method)

1 92 93 93

2 92 88 88

3 100 90 90

4 89 89 90

5 88 88 88

6 93 93 93

Mean 92 90 90

% RSD 4.5 2.5 2.5

Absolute % Difference between new HPLC method 2 N/A N/A

Absolute % Difference between new UPLC method 2 N/A N/A

Table 49: Method comparison results for HCTZ using USP method

Tablet # % HCTZ

(USP Method)

% HCTZ (New

HPLC method)

% HCTZ (New

UPLC method)

1 97 98 98

2 100 96 96

3 97 99 99

4 97 95 95

5 98 98 95

6 99 98 98

Mean 98 97 97

% RSD 1.3 1.7 1.7



64

Table 50: Method comparison results for BF using current Sandoz method

Tablet # % BF (Current

Sandoz Method)

% BF (New

HPLC method)

% BF (New

UPLC method)

1 80 93 93

2 92 88 88

3 97 90 90

4 90 89 90

5 88 88 88

6 93 93 93

Mean 90 90 90

% RSD 6.7 2.5 2.5



Table 51: Method comparison results for HCTZ using current Sandoz method

Tablet # % HCTZ(Current

Sandoz Method)

% HCTZ (New

HPLC method)

% HCTZ (New

UPLC method)

1 97 98 98

2 100 96 96

3 98 99 99

4 97 95 95

5 98 98 95

6 100 98 98

Mean 98 97 97

% RSD 1.2 1.7 1.7



65

Teflon Paddles vs. Non Teflon Paddles:

Currently there is a method in place at Sandoz which is approved and used to

determine the rate of dissolution. The current Sandoz method required the use of Teflon

paddles, but the USP method does not specify the types of paddles required for the

dissolution. Therefore, dissolution was performed using Teflon paddles and non-Teflon

paddles. The acceptance criteria are that the difference in the mean value of the six tablets

preparations between each type of paddles should be no more than 5.0% absolute. Teflon

paddles vs. non-Teflon paddles results for BF and HCTZ using HPLC are shown in table

52. Teflon paddles vs. non-Teflon paddles results for BF and HCTZ using UPLC are

shown in table 53.

Table 52: Teflon paddles vs. non Teflon paddles results for BF and HCTZ using HPLC

Tablet # % BF (Non

Teflon)

% BF

(Teflon)

% HCTZ

(Non Teflon)

% HCTZ

(Teflon)

1 93 89 98 97

2 88 92 96 100

3 90 100 99 97

4 89 89 95 97

5 88 88 98 98

6 93 93 98 99

% Average 90 92 97 98

Absolute % Difference in Averages 2 N/A 1 N/A

66

Table 53: Teflon paddles vs. non Teflon paddles results for BF and HCTZ using UPLC

Tablet # % BF (Non

Teflon)

% BF

(Teflon)

% HCTZ (Non

Teflon)

% HCTZ

(Teflon)

1 93 91 98 97

2 88 90 96 100

3 90 90 99 98

4 90 89 95 97

5 88 89 95 99

6 93 94 98 100

% Average 90 91 97 98

Absolute % Difference in Averages 1 N/A 1 N/A

67

CONCLUSION

BF is a cardio selective β1-adrenergic blocker. HCTZ is thiazide diuretic and

administered orally in the treatment of hypertension and oedema. Fixed dose combination

containing BF and HCTZ is available in tablet dosage form in the market. The present

paper describes a precise, accurate, specific, and sensitive HPLC and UPLC methods for

simultaneous estimation of BF and HCTZ in tablet dosage forms. The protocol was

written for assay, CU, and dissolution testing, and then a formal method validation was

performed. The formal method validation included specificity, linearity, range, accuracy,

precision, intermediate precision, robustness of extraction procedure, filter study, method

comparison, force degradation, solution stability, and robustness testing for assay, CU,

and dissolution.15 The robustness testing included alterations in wavelength, pH of buffer,

mobile phase ratio, flow rate, and column temperature.2 After the method validation

testing was successfully completed with all of the results meeting the acceptance criteria

for assay, CU, and dissolution testing using HPLC, the method validation was performed

using UPLC according to ICH Guidelines and current Sandoz SOP. Method comparison

was conducted by preparing six assay preparations and one dissolution using developed

method for HPLC and UPLC, the current Sandoz method, and the USP method. All

method comparison results for BF and HCTZ are within 2 percent absolute difference,

which proves that the developed method is competitively as good as the current Sandoz

and USP methods. The newly developed method has a much faster analysis time. Also

the developed method is simple because mobile phase and chromatographic parameters

are the same for assay, CU, and dissolution testing. For assay and CU testing, HCTZ and

68

BF can be analyzed from the same test solutions. The new method for BF and HCTZ has

numerous advantages over the current Sandoz and USP methods.

69

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73

APPENDIX

Appendix A-1: Assay linearity data for BF using HPLC

HPLC Bisoprolol

%level conc area8% 80.2592 13259036 60.1944 9996364 40.1296 6673383 30.0972 5006652 20.0648 3331121 10.0324 168400

0.5 5.0162 835850.2 2.0065 335040.1 1.0032 166780.04 0.4013 6703

0SUMMARY OUTPUT

Regression StatisticsMultiple R 0.999992417 Y-intercept Value: Y-intercept/100% area * 100R Square 0.999984835 1159.227554/168400*100Adjusted R Square 0.999982939 0.688% ~ 0.7% ~1%Standard Error 1893.225574Observations 10

ANOVAdf SS MS F Significance F

Regression 1 1.89077E+12 1.89E+12 527512.9 1.44631E-20Residual 8 28674424.59 3584303Total 9 1.89079E+12

Coefficients Standard Error t Stat P-value Lower 95% Upper 95% Lower 95.0% Upper 95.0%Intercept 1159.227554 825.1220966 1.404916 0.197665 -743.508643 3061.963751 -743.508643 3061.963751conc 16548.37112 22.78445896 726.3008 1.45E-20 16495.83003 16600.91221 16495.83003 16600.91221

RESIDUAL OUTPUT

Observation Predicted area Residuals1 1329317.593 -3414.5929192 997278.0016 2357.9984223 665238.4102 2099.5897634 499218.6146 1446.3854345 333198.8189 -86.818895426 167179.0232 1220.9767757 84169.12539 -584.12538958 34363.18669 -859.18668839 17761.20712 -1083.207121

10 7800.019381 -1097.0193811159.227554

Residuals Plot for Bisoprolol using HPLC

-4000

-3000

-2000

-1000

0

1000

2000

3000

0 200000 400000 600000 800000 1000000 1200000 1400000

Residuals

Linearity Plot for Bisoprolol using HPLC

0

200000

400000

600000

800000

1000000

1200000

1400000

0.0000 10.0000 20.0000 30.0000 40.0000 50.0000 60.0000 70.0000 80.0000 90.0000

conc (µg/ml)

area

area

Predicted area

Y-intercept: 1159.227554Slope: 16548.37112Correlation Coefficient: 0.999992417

74

Appendix A-2: Assay linearity data for BF using UPLC

%level conc (µg/ml) area8% 80.2592 9197616 60.1944 6909904 40.1296 4578493 30.0972 3442932 20.0648 2282741 10.0324 114290

0.5 5.0162 569900.2 2.0065 228640.1 1.0032 11350

0.04 0.4013 45180

SUMMARY OUTPUT

Regression StatisticsMultiple R 0.999996178R Square 0.999992356 Y-intercept Value: Y-intercept/100% area * 100Adjusted R Square 0.999991401 625.6115106/114290*100Standard Error 931.3032643 0.547% ~ 0.5% ~1%Observations 10


Regression 1 9.07753E+11 9.08E+11 1046611 9.33393E-22Residual 8 6938606.161 867325.8Total 9 9.0776E+11

Coefficients Standard Error t Stat P-value Lower 95% Upper 95% Lower 95.0% Upper 95.0%Intercept -625.6115106 405.8887185 -1.54134 0.161804 -1561.593179 310.3702 -1561.59318 310.370158conc (µg/ml) 11466.21717 11.2079835 1023.04 9.33E-22 11440.37149 11492.06 11440.37149 11492.0628

RESIDUAL OUTPUT

Observation Predicted area Residuals1 919643.3467 117.65334442 689576.1071 1413.8928863 459508.8676 -1659.8675724 344475.2478 -182.24780175 229441.628 -1167.6280316 114408.0083 -118.00826027 56891.19837 98.801625218 22381.11244 482.88755649 10877.75047 472.2495335

10 3975.73328 542.2667198-625.6115106

Linearity Plot for Bisoprolol using UPLC

y -intercept: -625.6115106Correlation coefficient: 0.999996178

Slope: 1146.21717

-200000

0

200000

400000

600000

800000

1000000

0.0000 10.0000

20.0000

30.0000

40.0000

50.0000

60.0000

70.0000

80.0000

90.0000

conc (µg/ml)

are

aarea

Predicted area

Residuals Plot for Bisoprolol using UPLC

-2000

-1500

-1000

-500

0

500

1000

1500

2000

0 200000 400000 600000 800000 1000000

Residuals

75

Appendix A-3: Assay linearity data for HCTZ using HPLC

%level conc (µg/ml) area

8% 49.3914 32639216 37.0435 24649484 24.6957 16478373 18.5372 12373172 12.3478 8232431 6.1739 416777

0.5 3.0870 2067160.2 1.2348 831320.1 0.6174 411790.04 0.2470 16702

0SUMMARY OUTPUT

Regression StatisticsMultiple R 0.999985343R Square 0.999970686Adjusted R Square 0.999967022 Y-intercept Value: Y-intercept/100% area * 100Standard Error 6483.2172 4348.506558/1647837*100Observations 10 0.264% ~ 0.3% ~0%



Coefficients Standard Error t Stat P-value Lower 95% Upper 95% Lower 95.0% Upper 95.0%Intercept 4348.506558 2825.681582 1.538923 0.162385 -2167.53107 10864.54 -2167.53107 10864.5442conc (µg/ml) 66231.42149 126.7833456 522.3984 2.02E-19 65939.05838 66523.78 65939.0584 66523.7846

RESIDUAL OUTPUT

Observation Predicted area Residuals1 3275609.813 -11688.813452 2457794.487 7153.5132763 1639979.16 7857.8399984 1232092.073 5224.9268275 822163.8333 1079.166726 413256.1699 3520.8300817 208802.3382 -2086.3382398 86130.03923 -2998.0392319 45239.27289 -4060.272895

10 20704.81309 -4002.8130934348.506558

Linearity Plot for HCTZ using HPLC

correlation coefficient: 0.999985343y-intercept: 4348.506558

Slope: 66231.42148

0

500000

1000000

1500000

2000000

2500000

3000000

3500000

0.0000 10.0000 20.0000 30.0000 40.0000 50.0000 60.0000

conc (µg/ml)a

rea

area

Predicted area

Residuals Plot for HCTZ using HPLC

-15000

-10000

-5000

0

5000

10000

0 500000 1000000 1500000 2000000 2500000 3000000 3500000

Residuals

76

Appendix A-4: Assay linearity data for HCTZ using UPLC

%level conc (µg/ml) area

8% 49.3914 22863076 37.0435 17238144 24.6957 11489483 18.5372 8653572 12.3478 5758561 6.1739 288507

0.5 3.0870 1444720.2 1.2348 578550.1 0.6174 290650.04 0.2470 12043

0SUMMARY OUTPUT

Regression StatisticsMultiple R 0.999993014R Square 0.999986027Adjusted R Square 0.999984281 Y-intercept Value: Y-intercept/100% area * 100Standard Error 3133.276887 2129.914173/1148948*100Observations 10 0.185% ~ 0.2% ~0%



Coefficients Standard Error t Stat P-value Lower 95% Upper 95% Lower 95.0% Upper 95.0%Intercept 2129.914173 1365.624892 1.559663 0.157459 -1019.22451 5279.0529 -1019.22451 5279.05286conc (µg/ml) 46362.80354 61.27317877 756.6574 1.04E-20 46221.50725 46504.1 46221.50725 46504.0998

RESIDUAL OUTPUT

Observation Predicted area Residuals1 2292052.762 -5745.7619142 1719572.05 4241.9500213 1147091.338 1856.6619564 861565.3981 3791.6018935 574610.6261 1245.3738916 288370.2701 136.7298597 145250.0922 -778.09215728 59377.98537 -1522.9853679 30753.94977 -1688.94977

10 13579.52841 -1536.5284122129.914173

Linearity Plot for HCTZ using UPLCy-intercept:2129.91417

Correlation coefficient:0.999993014Slope: 46362.8035

0

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1000000

1500000

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0.0000

10.0000

20.0000

30.0000

40.0000

50.0000

60.0000

conc (µg/ml)a

rea

area

Predicted area

Residuals Plot for HCTZ using HPLC

-8000

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0

2000

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0 500000 1000000 1500000 2000000 2500000

Residuals

77

Appendix B-1: Assay blank specificity chromatograph using HPLC

Channel 2998; Processed Channel: PDA 224.0 nm; Result Id: 32421; Processing Method: biso assay nnt 122210

1.9

53

AU

-0.015

-0.010

-0.005

0.000

0.005

0.010

0.015

Minutes0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00

Appendix B-2: Assay placebo specificity chromatograph using HPLC

Channel 2998; Processed Channel: PDA 224.0 nm; Result Id: 32423; Processing Method: biso assay nnt 122210

1.9

56

4.0

01

AU

-0.010

0.000

0.010

0.020

Minutes0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00

1

2

3

4

5

6

7

8

9

Peak Name RT Area USP Plate Count USP Tailing

fumaric acid

biso A

4-amino

CTZ

HCTZ

bisoprolol

Dimer

1.810

1.956

3.150

3.800

4.001

4.045

4.300

5.400

6.100

50579

43569

392

27517

0.8

2.7

Processed Channel Descr.: PDA 224.0 nm

78

Appendix B-3: Assay blank specificity chromatograph using UPLC

AU

-0.015

-0.010

-0.005

0.000

0.005

0.010

Minutes

0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00

Channel PDA Spectrum; Processed Channel: PDA 224.0 nm; Result Id: 26405; Processing Method: biso assay nnt 122210 uplc

Processed ChannelDescr.: PDA 224.0 nm

Appendix B-4: Assay placebo specificity chromatograph using UPLC

1.37

1

AU

-0.015

-0.010

-0.005

0.000

0.005

0.010

Minutes

0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00

Channel PDA Spectrum; Processed Channel: PDA 224.0 nm; Result Id: 26407; Processing Method: biso assay nnt 122210 uplc

79

Appendix C-1: Dissolution linearity data for BF using HPLC

% conc (μg/ml) area Bisoprolol HPLC20 0.5545 1980450 1.3862 50060

100 2.7724 96876200 5.5449 190210400 11.0898 377409600 16.6347 565981800 22.1796 754422

0SUMMARY OUTPUT

Regression StatisticsMultiple R 0.999996523R Square 0.999993047Adjusted R Square 0.999991656Standard Error 814.6618222Observations 7


Regression 1 4.77235E+11 4.77E+11 719080.2 4.32867E-14Residual 5 3318369.423 663673.9Total 6 4.77238E+11

Coefficients Standard Error t Stat P-value Lower 95% Upper 95% Lower 95.0% Upper 95.0%Intercept 2163.855972 461.3850863 4.689913 0.005386 977.8297881 3349.882156 977.8297881 3349.882156conc 33902.04939 39.97949386 847.986 4.33E-14 33799.279 34004.81978 33799.279 34004.81978

RESIDUAL OUTPUT

Observation Predicted area Residuals1 20962.16266 -1157.8626562 49159.62268 900.02731823 96155.38939 721.10060874 190146.9228 63.557189595 378129.9896 -721.10264866 566113.0565 -131.75648687 754096.1233 326.036675

2163.855972

Line Fit Plot for Bisoprolol using HPLC

0

100000

200000

300000

400000

500000

600000

700000

800000

900000

0.0000 5.0000 10.0000 15.0000 20.0000 25.0000conc (μg/ml)

are

aarea

Predicted area

Y-intercept: 2163.855972Slop: 33902. 04939Correlation Coefecient: 0.999996523

Residuals plot for BF using HPLC

-1500

-1000

-500

0

500

1000

1500

0 100000 200000 300000 400000 500000 600000 700000 800000

Residuals

80

Appendix C-2: Dissolution linearity data for BF using UPLC

% conc (μg/ml) area Bisoprolol UPLC20 0.5545 4110150 1.3862 101282

100 2.7724 203950200 5.5449 407653400 11.0898 814748600 16.6347 1220780800 22.1796 1627600

0SUMMARY OUTPUT





RESIDUAL OUTPUT

Observation Predicted area Residuals1 41055.8257 44.774298672 102086.198 -804.29804753 203803.4853 146.01470894 407238.0598 415.41722175 814107.2088 640.46624726 1220976.358 -196.73772727 1627845.507 -245.6367017

368.9108039

0

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400000

600000

800000

1000000

1200000

1400000

1600000

1800000

0.0000 5.0000 10.0000 15.0000 20.0000 25.0000conc (μg/ml)

are

a

area

Predicted area

Line Fit Plot for Bisoprolol using UPLC

Correlation Coefficient:0.999999698Y-intercept:368.9108039Slop:73377.34305

Residuals plot for BF using UPLC

-1000

-800

-600

-400

-200

0

200

400

600

800

0 500000 1000000 1500000 2000000

Residuals

81

Appendix C-3: Dissolution linearity data for HCTZ using HPLC

% conc (μg/ml) area HCTZ HPLC20 1.3830 19058650 3.4576 47916075 5.1864 715969100 6.9152 955815133 9.2202 1268421150 10.3728 1426946200 13.8304 1896402

0SUMMARY OUTPUT





RESIDUAL OUTPUT

Observation Predicted area Residuals1 194220.382 -3634.2520352 478435.2916 724.61635563 715281.0497 688.34634814 952126.8077 3688.4323415 1267908 513.02035476 1425818.324 1127.6863267 1899509.84 -3107.849689

4743.775629

Line Fit Plot for HCTZ using HPLC

0

500000

1000000

1500000

2000000

2500000

0.0000 2.0000 4.0000 6.0000 8.0000 10.0000 12.0000 14.0000 16.0000

conc (μg/ml)

area

area

Predicted area

Correlation Coefficient:0.999990562Y-intercept: 4743.775629Slop:137000.2499

Residuals plot for HCTZ using HPLC

-5000

-4000

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-1000

0

1000

2000

3000

4000

5000

0 500000 1000000 1500000 2000000

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82

Appendix C-4: Dissolution linearity data for HCTZ using UPLC

% conc (μg/ml) area HCTZ UPLC20 1.3830 41158550 3.4576 103221875 5.1864 1546985100 6.9152 2063130133 9.2202 2744672150 10.3728 3082812200 13.8304 4050133

0SUMMARY OUTPUT

Regression StatisticsMultiple R 0.999898061R Square 0.999796132Adjusted R Squa 0.999755359Standard Error 19655.2419Observations 7


Regression 1 9.47304E+12 9.473E+12 24520.687 2.015E-10Residual 5 1931642671 386328534Total 6 9.47497E+12


RESIDUAL OUTPUT

Observation Predicted area Residuals1 428157.5467 -16572.656722 1036713.61 -4495.7802333 1543843.663 3141.6868424 2050973.716 12156.038925 2727118.959 17552.800916 3065233.822 17578.388077 4079493.928 -29360.47778

22453.50438

Line Fit Plot for HCTZ using UPLC

0

500000

1000000

1500000

2000000

2500000

3000000

3500000

4000000

4500000

0.0000 2.0000 4.0000 6.0000 8.0000 10.0000 12.0000 14.0000 16.0000conc (μg/ml)

are

a

area

Predicted area

Correlation Coefficient:0.999898061Y-intercept: 22453.50438Slop: 293342.5727

Residuals plot for HCTZ using UPLC

-40000

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0

10000

20000

0 500000

1E+06 2E+06 2E+06 3E+06 3E+06 4E+06 4E+06 5E+06

Residuals

83

Appendix D-1: Dissolution blank specificity chromatograph using HPLC

SampleName blank spl S D; Vial 4; Injection 1; Result Id 8521; Date Acquired Monday, February 07, 2011 6:06:24 PM EST

AU

-0.010

0.000

0.010

0.020

0.030

Minutes0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00

Appendix D-2: Dissolution BF placebo specificity chromatograph using HPLC

SampleName Bis S D; Vial 6; Injection 1; Result Id 8523; Date Acquired Monday, February 07, 2011 6:30:02 PM EST

AU

-0.010

0.000

0.010

0.020

0.030

Minutes0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00

Appendix D-3: Dissolution HCTZ placebo specificity chromatograph using HPLC ( g )

SampleName HCTZ S D; Vial 7; Injection 1; Result Id 8524; Date Acquired Monday, February 07, 2011 6:41:51 PM EST

AU

-0.010

0.000

0.010

0.020

0.030

Minutes0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00

84

Appendix D-4: Dissolution blank specificity chromatograph using UPLC

SampleName blank spl S D; Vial 1:A,4; Injection 1; Result Id 8454; Date Acquired Monday, February 07, 2011 5:37:22 PM EST

AU

0.00

0.10

0.20

Minutes0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00

Appendix D-5: Dissolution BF placebo specificity chromatograph using UPLC

SampleName Bis S D; Vial 1:A,6; Injection 1; Result Id 8456; Date Acquired Monday, February 07, 2011 5:53:19 PM EST

AU

0.00

0.10

0.20

Minutes0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00

Appendix D-6: Dissolution HCTZ placebo specificity chromatograph using UPLC

SampleName HCTZ S D; Vial 1:A,7; Injection 1; Result Id 8457; Date Acquired Monday, February 07, 2011 6:01:18 PM EST

AU

0.00

0.10

0.20

Minutes0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00

85

Appendix E: Method Validation Protocol for Assay and CU Testing

Analytical Method Validation Protocol for the

Quantitative Determination of Bisoprolol

Fumarate/ Hydrochlorothiazide 2.5 mg/6.25 mg, 5

mg/6.25 mg, and 10 mg/6.25 mg Tablets and All

Related Compounds in the Finished Product

Dosage Form

Protocol #: MVP00126v1

Author’s Signature: ____________________________________ Date: ______________ Name/Department/ Title: Niralee Thakar /QC/Chemist II

Reviewed By: Date:

Name/Department/ Title: Jason Miller/QC/Chemist IV

Reviewed By: Date:

Name/Department/ Title: John Bredin/QC/Manager

Approved By: Date:

Name/Department/ Title: Richard Uveges/QC/Director

86

1.0 OBJECTIVE The purpose of this protocol is to describe the procedure to validate a new method for the simultaneous determination of Bisoprolol and Hydrochlorothiazide and their related compounds in Bisoprolol Fumarate/Hydrochlorothiazide Tablets. This new method will be used for the determination of assay, content uniformity, and related compound analysis. These studies will be conducted to demonstrate that each method is suitable for its intended use.

2.0 INTRODUCTION Currently, at Sandoz, Inc in Wilson, NC there are Bisoprolol Fumarate/Hydrochlorothiazide Tablets, 2.5/6.25 mg, 5/6.25 mg, 10/6.25 mg products being manufactured using various analytical methods. The product contains its own monograph for the separate testing of assay, content uniformity, and related compounds for each Active Pharmaceutical Ingredient (API). The analytical method has also been previously validated by Sandoz, Inc at Wilson, NC. However, recently a new method was developed that can be used as an alternative to combine many of the testing procedures into one united analytical method. The purpose of this protocol is to demonstrate that the new analytical methods using HPLC and UPLC are suitable for the simultaneous determination of Bisoprolol and HCTZ assay, content uniformity, and related compounds in the Bisoprolol Fumarate/Hydrochlorothiazide Tablets, 2.5/6.25 mg, 5/6.25 mg, 10/6.25 mg. This validation will also demonstrate that this new method has shown equivalency with the existing analytical methods at Sandoz, Inc as well as the USP method and is, therefore, considered to be an acceptable alternative.

3.0 REGULATORY REQUIREMENTS 3.1 Related Compounds

3.1.1 Bisoprolol Fumarate Related Compound Bisoprolol A Impurity, NMT 1.0% Any other individual related compound, NMT 0.5% Total other related compound (excluding Bisoprolol A Impurity), NMT 1.5%

3.1.2 HCTZ 4-Amino Impurity, NMT 1.0%

Chlorothiazide Impurity, NMT 0.5% (QC Release), NMT 1.0% (Stability)

Individual Related Compound, NMT 0.5% Total related compound (excluding 4-Amino Impurity), NMT 1.0%

3.2 Assay

3.2.1 The tablets contain not less than 90.0% and not more than 110.0% of the labeled amount of Active Pharmaceutical Ingredient (API).

87

3.3 Content Uniformity

3.3.1 Level 1: The acceptance value (AV) for 10 units is not more than 15.0.

3.3.2 Level 2: If the acceptance value (AV) for Level 1 is over 15.0, test an additional 20 units. The requirements are met if the acceptance value for the 30 units is not more than 15.0 and each unit contains not less than 0.75M or not more than 1.25M.

4.0 STANDARDS 4.1 Bisoprolol Fumarate Reference Standard: Use the current USP lot or

certified In-House Standard. 4.2 Hydrochlorothiazide (HCTZ) Reference Standard: Use the current USP

lot or certified In-House Standard. 4.3 4-Amino Reference Standard (4-Amino-6-chloro-1,3-

benzenedisulfonaminde): Use the current USP lot or equivalent. 4.4 CTZ (2H-1,2,4-benzothiadiazine-7-sulfonamide,6-chloro-1,1-dioxide):

Use the current USP lot or equivalent. 4.5 Dimer ([6-chloro-N-[(6-chloro-7-sulfamoyl-2,3-dihydro-4H-1,2,4-

benzothiadiazine-4-yl 1,1-dioxide)methyl]3,4-dihydro-2H-1,2,4-benzothiazine-7-sulfonamide 1,1-dioxide]): Use the Manufacturer’s reference standard or equivalent.

4.6 Bisoprolol Base Compound A ((+/-)-1-[4-(Hydroxymethyl)-phenoxy]-3-[(1-methylethyl)-amino]-2-propanol): Use the manufacturer’s reference standard or equivalent.

4.7 Fumaric Acid: Use USP Reference Standard or 99+%, Aldrich Chemical Company, or equivalent.

5.0 REAGENTS

5.1 Trifluoroacetic Acid: 99+%, Aldrich Chemical Company or equivalent for spectrophotometric grade.

5.2 Acetonitrile: Fisher Brand or equivalent for ACS grade. 5.3 Water: USP purified water or equivalent.

6.0 INSTRUMENTATION/EQUIPMENT 6.1 Use a qualified HPLC system equipped with an electronic injector. 6.2 Use a qualified UPLC system equipped with an electronic injector.

7.0 ANALYTICAL METHODS

7.1 Solution Preparations Diluent: Pipet 1.0 ml of Trifluoroacetic acid into 1600ml of water and 400ml of acetonitrile and mix.

88

Mobile Phase A: Pipet 1.0 ml of Trifluoroacetic acid into 1800ml of water and 200ml of acetonitrile and mix. Mobile Phase B: Pipet 1.0 ml of Trifluoroacetic acid into 1800ml of acetonitrile and 200ml of water and mix. Standard Stock Solution: For 2.5mg Bisoprolol/ 6.25HCTZ Weigh 20mg of Bisoprolol Fumarate and 50mg of HCTZ standards into same 200ml volumetric flask. Add 10.0 ml of Acetonitrile and sonicate for about 5 minutes. Add half volume of diluent and sonicate to dissolve, dilute to volume with diluent. This solution may contain about 100µg/ml of Bisoprolol and 250µg/ml of HCTZ. For 5mg Bisoprolol/ 6.25HCTZ Weigh 20mg of Bisoprolol Fumarate and 25mg of HCTZ standards into same 100ml volumetric flask. Add 5.0 ml of Acetonitrile and sonicate for about 5 minutes. Add half volume of diluent and sonicate to dissolve, dilute to volume with diluent. This solution may contain about 200µg/ml of Bisoprolol and 250µg/ml of HCTZ. For 10mg Bisoprolol/ 6.25HCTZ Weigh 40mg of Bisoprolol Fumarate and 25mg of HCTZ standards into same 100ml volumetric flask. Add 5.0 ml of Acetonitrile and sonicate for about 5 minutes. Add half volume of diluent and sonicate to dissolve, dilute to volume with diluent. This solution may contain about 400µg/ml of Bisoprolol and 250µg/ml of HCTZ. Working Standard Preparation: Pipette 5.0ml of standard stock into 50ml volumetric flask and dilute to volume with diluent and mix well for all strengths. This solution may contain the concentration for each component as outlined in Table 1. Table 1: Assay Standard Preparation Concentrations

7.2 Sample Preparations Assay Preparation (Prepare in duplicate) Weigh NLT 20 tablets and determine the average tablet weight. Grind the tablets to a fine powder using a mortar and pestle. Weigh a portion equivalent to about 12.5 mg of Hydrochlorothiazide into a 500-mL volumetric flask. (Weigh about 330mg of powder) Add to ½volume with

Conc. Table 2.5mg/6.25mg 5mg/6.25mg 10mg/6.25mg HCTZ 25µg/ml 25µg/ml 25µg/ml

Bisoprolol 10µg/ml 20µg/ml 40µg/ml

89

diluent and sonicate for 15 minutes with frequent shaking. Shake the solution on a mechanical shaker for 15 minutes. Allow the solution to cool to room temperature. Dilute to volume with diluent and mix well. Filter a portion of the sample through a 0.45μm Whatman GMF filter, discarding the first 5ml of the filtrate. This solution will contain the concentrations for each component as outlined in Table 2. Table 2: Assay Sample Preparation Concentrations

Content Uniformity Sample Preparation Randomly select and weigh 10 tablets individually. Transfer each tablet individually into separate 250-mL volumetric flasks. Add diluent to ½ volume and sonicate for 15 minutes with frequent shaking. Shake the flasks on a mechanical shaker for 15 minutes. Allow the solutions to cool to room temperature. Dilute to volume with diluent and mix well. Filter a portion of the sample through a 0.45μm Whatman GMF filter, discarding the first 5ml of the filtrate. This solution will contain the concentrations for each component as outlined in Table 3. Table 3: Content Uniformity Sample Preparation Concentrations

Bisoprolol Fumarate/ HCTZ Tablets

Conc. (µg/mL)

2.5 mg/ 6.25 mg 10 / 25 5 mg/ 6.25 mg 20 / 25 10 mg/ 6.25 mg 40 / 25

Related Compound Sample Preparation Weigh 10 tablets and transfer them into a 100-mL volumetric flask. Add to ½ volume with diluent and sonicate for 15 minutes with frequent shaking. Shake the solution on a mechanical shaker for 15 minutes. Allow the solution to cool to room temperature. Dilute to volume with diluent and mix well. Filter a portion of the sample through a 0.45μm Whatman GMF filter, discarding the first 5ml of the filtrate. This solution will contain the concentration for each component as outlined in Table 4.

Bisoprolol Fumarate/ HCTZ Tablets

Conc. (µg/mL) mg of Powder (validation)

2.5 mg/ 6.25 mg 10 / 25 330mg 5 mg/ 6.25 mg 20 / 25 330mg 10 mg/ 6.25 mg 40 / 25 330mg

90

Table 4: Related Compound Sample Preparation Concentrations.

Bisoprolol Fumarate / HCTZ Tablets

Conc. (µg/mL)

2.5 mg/ 6.25mg 250 / 625 5 mg/ 6.25mg 500 / 625 10 mg/ 6.25mg 1000 / 625

The need for Resolution/Identification Solutions will be assessed as part of this validation.

7.3 Chromatographic Conditions for HPLC

Apparatus: HPLC equipped with an electronic injector. Detector: Ultraviolet set at 224-nm. Column: Waters Symmetry C18 4.6mm x 150mm 5 µm Flow Rate: 1.0mL/min Injection Volume: 10 µL Column Temperature: 35oC Gradient: Retention Times: (Note: The relative retention times for 4-Amino, CTZ, and Dimer are based on the HCTZ peak, and Fumaric acid and Bisoprolol A are based on the Bisoprolol peak)

Component Retention Time

(min) RRT

Fumaric acid 1.9 0.35 Bisoprolol A imp 3.3 0.61

4-Amino 3.9 0.89 CTZ 4.1 0.93

HCTZ 4.4 1.00 Bisoprolol 5.4 1.00

Dimer 6.1 1.39

Time % A % B Curve 0 100 0 --- 6 40 60 6

7 40 60 6

7.5 100 0 6 11 100 0 6

91

7.4 Chromatographic Conditions for UPLC Apparatus: UPLC equipped with an electronic injector. Detector: Ultraviolet set at 224-nm. Column: Waters Acquity BEH C18 2.1mm x 100mm 1.7 µm Column Temperature: 35oC Flow Rate: 0.30 mL/min Injection Volume: 2.0 µL Sampling Rate: 10 points/second Injection Mode: Partial Loop with Needle Overfill Gradient:

Time % A % B Curve 0 95 5 --- 2 85 15 6 5 50 50 6

5.5 95 5 6 7 95 5 6

Retention Times: (Note: The relative retention times for 4-Amino, CTZ, and Dimer are based on the HCTZ peak, and Bisoprolol A imp and Fumaric acid are based on that of the Bisoprolol peak)

Component Retention Time

(min) RRT

Fumaric acid 0.9 0.20 Bisoprolol A imp 1.5 0.34

4-Amino 1.7 0.85 CTZ 1.8 0.90

HCTZ 2.0 1.00 Dimer 4.2 2.10

Bisoprolol 4.4 1.00

7.5 Calculations Assay

......

100.....%

CLwtSampleDilStdAreaPkStd

ATWDilSplWtStdAreaPkSplActive

For Content Uniformity

.....

100.....%

CLDilStdAreaPkStd

DilSplWtStdAreaPkSplActive

92

For Related Compounds

ZCLofTabletsDilStdAreaPkStd

DilSplWtStdAreaPkIRCIRC

..#...

100.....%

Total Related Compounds

IRCTRC %% (excluding 4-Amino for HCTZ determination)

Where, Spl.Pk.Area is the Sample Peak Area. Std.Pk.Area is the Standard Peak Area. Std.Wt. is the Standard Weight. Std.Dil is the Standard Dilution. Spl.Wt. is the Sample Weight. ATW is the Average Tablet Weight. L.C. is the Label Claim. # of Tablets is number of tablets are used in sample preparation. IRC.Pk.Area is the Individual Related Compound Peak Area TRC is the Total Related Compounds Z is the Response Factor The Response Factors for each related compound of interest will be determined as part of this method validation.

8.0 VALIDATION PARAMETERS

8.1 Placebo Preparation for Bisoprolol Fumarate/HCTZ Tablets 2.5/6.25mg, 5/6.25mg, and 10/6.25mg.

Table 5: Placebo Preparation for Method Validation Studies.

EDP # Ingredients Variance

Placebo Preparation

(mg/unit)

E280 Bisoprolol Fumarate 2.5/5.0/10.0 --- F109 Hydrochlorothiazide 6.25 ---

E370 Dicalcium Phosphate

Anhydrous 80 – 140 140

G623 Corn Starch NF 12 – 22 22 E188 Microcrystalline Cellulose 10 - 25 25 E352 Colloidal Silicone Dioxide 0.25 – 2 2 F550 Magnesium Stearate 0.5 – 2 2 H915 Opadry White YS-1-7003

2-8 8

H929 Opadry Orange YS-1-13174 8 H927 Opadry Orange YS-1-13148 8

Placebo Total Weight (mg) per Unit 215

93

8.2 Resolution/Identification Solutions

Resolution/Identification Solutions will be prepared as part of this method validation. The need for these solutions in further laboratory testing will be assessed based on sound scientific judgment and the results of these validation tests. 4-Amino Stock Preparation: Accurately weigh about 3.13 mg of 4-Amino into a 25-mL volumetric flask. Add about 3.0 mL of Acetonitrile to volumetric flask and sonicate for about 5 minutes. Add about 10 mL of diluent and sonicate to dissolve, dilute to volume with diluent, and mix well. This solution should contain about 125.2 µg/mL of the 4-Amino HCTZ impurity. CTZ Stock Preparation: Accurately weigh about 3.13 mg of CTZ into a 25-mL volumetric flask. Add about 3.0 mL of Acetonitrile to volumetric flask and sonicate for about 5 minutes. Add about 10 mL of diluent and sonicate to dissolve, dilute to volume with diluent, and mix well. This solution should contain about 125.2 µg/mL of the CTZ HCTZ impurity. Dimer Stock Preparation: Accurately weigh about 39 mg of Dimer into a 25-mL volumetric flask. Add about 3.0 mL of Acetonitrile to volumetric flask and sonicate for about 5 minutes. Add about 10 mL of diluent and sonicate to dissolve, dilute to volume with diluent, and mix well. This solution should contain about 124.8 µg/mL of the Dimer HCTZ impurity. Note: The Dimer is present as a mix with HCTZ at 8%, therefore the weight was adjusted to reach a target concentration of 124.8 µg/mL. Bisoprolol A Impurity Stock Preparation: Accurately weigh about 5 mg of Bisoprolol A imp into a 25-mL volumetric flask. Add about 3.0 mL of Acetonitrile to volumetric flask and sonicate for about 5 minutes. Add about 10 mL of diluent and sonicate to dissolve, dilute to volume with diluent, and mix well. This solution should contain about 200 µg/mL of the Bisoprolol A imp.

Fumaric Acid Stock Preparation: Accurately weigh about 5 mg of Fumaric acid into a 25-mL volumetric flask. Add about 3.0 mL of Acetonitrile to volumetric flask and sonicate for about 5 minutes. Add about 10 mL of diluent and sonicate to dissolve, dilute to volume with diluent, and mix well. This solution should contain about 200 µg/mL of the Fumaric acid. (This is a process related impurity and will be used for resolution determination only)

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Bisoprolol Fumarate/ HCTZ Resolution/Identification Solution Preparation: Weigh 100 mg of Bisoprolol Fumarate Raw Material or reference standard and 62.5 mg of HCTZ Raw Material or reference standard into same 100 mL volumetric flask. Add 10ml of Acetonitrile to volumetric flask and sonicate for about 5 minutes. Pipet 5.0 mL of each impurity stock into same 100 mL volumetric flask. Add 30 mL of diluent and sonicate to dissolve. Dilute to volume with diluent. This solution contains about 10 µg/ml of Fumaric acid and Bisoprolol imp A, 6.24 µg/ml, 6.26 µg/ml of CTZ and 4-amino, 1000 µg/ml of Bisoprolol Fumarate, and 625 µg/ml of HCTZ.

8.3 Linearity The linearity will be performed as outlined below. The range for assay linearity will cover from 200% of highest label claim of the assay concentration down to the LOQ lever for each active ingredient (Bisoprolol Fumarate and HCTZ). For related compound linearity, the range for each related compound will cover from 2 % of highest label claim of the related compound concentration down to the LOQ level.

Table 6: Linear Ranges for Each Active

Component Concentration Range

(µg/mL) %Level for samples

Bisoprolol Fumarate

80 - 0.4

200 - 1 (10mg/6.25mg) 400 – 2 (5mg/6.25mg)

800 – 4 (2.5mg/6.25mg)

HCTZ 50 – 0.25 200 – 1

Bisoprolol A Imp 20 - 0.4

2 – 0.04 (10mg/6.25mg)

4 – 0.08 (5mg/6.25mg)

8 – 0.016 (2.5mg/6.25mg)

4-Amino 12.5 – 0.25 2 – 0.04

CTZ 12.5 – 0.25 2 – 0.04

Dimer 12.5 – 0.25 2 – 0.04

Linearity Solution A: Weigh about 40 mg of Bisoprolol Fumarate Reference Standard and 25 mg of HCTZ Reference Standard into the same 100-mL volumetric flask. Add 5 mL of Acetonitrile and sonicate for 5 minutes. Add diluent to ½ volume and sonicate to dissolve. Dilute to volume with diluent and mix well. This solution contains about 400 µg/mL of Bisoprolol Fumarate and 250 µg/mL of HCTZ.

Linearity Solution B: Weigh about 10 mg of Bisoprolol A Impurity, 6.25 mg of 4-Amino, and 6.25 mg of CTZ into 50-mL volumetric flask. Add 5 mL of Acetonitrile and sonicate for 5 minutes. Pipet 25.0 mL of Linearity Solution A into the same 50-mL volumetric flask and sonicate to dissolve.

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Dilute to volume with diluent and mix well. This solution contains about 200 µg/mL of Bisoprolol Fumarate, 200 µg/mL of Bisoprolol A Impurity, 125 µg/mL of 4-amino, 125 µg/mL of CTZ, and 125 µg/mL of HCTZ. Linearity Solution C: Weigh about 19.5 mg of dimmer into 25-mL volumetric flask. Add 3 mL of Acetonitrile and sonicate for 5 minutes. Add diluent to ½ volume and sonicate to dissolve. Dilute to volume with diluent and mix well. This solution contains about 62.4 µg/mL of Dimer.

Table 7: Linearity Solution Preparations for Assay and Related Compounds

% Level Bisoprolol (Based on

1000 µg/mL)

% Level HCTZ

(Based on 625 µg/mL)

Amount of Solution VF

(mL)

Conc. For Bisoprolol (µg/mL)

Conc. For

HCTZ (µg/mL)

8.00 8.00 Pipet 5.0 mL of Linearity Solution A 25 80.0 50.00 6.00 6.00 Pipet 15.0 mL of Linearity Solution A 100 60.0 37.50 4.00 4.00 Pipet 5.0 mL of Linearity Solution A 50 40.0 25.00 3.00 3.00 Pipet 15.0 mL of Linearity Solution A 200 30.0 18.75 2.00 2.00 Pipet 5.0 mL of Linearity Solution B 50 20.0 12.50 1.00 1.00 Pipet 5.0 mL of Linearity Solution B 100 10.0 6.25 0.50 0.50 Pipet 2.5 mL of Linearity Solution B 100 5.0 3.125 0.20 0.20 Pipet 2.5 mL of Linearity Solution B 250 2.0 1.25 0.10 0.10 Pipet 1.0 mL of Linearity Solution B 200 1.0 0.625 0.04 0.04 Pipet 1.0 mL of Linearity Solution B 500 0.4 0.25

Table 8: Related Compound Linearity Solution Preparations for HCTZ Dimer

% Level Bisoprolol (Based on

1000 µg/mL)

% Level HCTZ

(Based on 625 µg/mL)

Amount of Solution VF

(mL) Conc.

(µg/mL)

2.00 2.00 Pipet 5.0 mL of Linearity Solution C 25 12.50 1.00 1.00 Pipet 5.0 mL of Linearity Solution C 50 6.25 0.50 0.50 Pipet 2.5 mL of Linearity Solution C 50 3.125 0.20 0.20 Pipet 4.0 mL of Linearity Solution C 200 1.25 0.10 0.10 Pipet 2.0 mL of Linearity Solution C 200 0.625 0.04 0.04 Pipet 1.0 mL of Linearity Solution C 250 0.25

Report the slope, y-intercept, and the residual sum of squares. Acceptance Criteria (Assay): The correlation coefficient (R) for each API should be NLT 0.999. The % y-intercept value should be NMT 3.0% of the target value. Acceptance Criteria (Related Compounds): The correlation coefficient (R) for each related compound should be NLT 0.99. The % y-intercept value should be NMT 25% based on the specification value.

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8.4 Response Factors The response factors will be based on the slope of the linearity for each related compound relative to that of their respective API. The calculation will be performed as follows:

CompoundlatedofSlope

APIofSlopeFactorsponse

ReRe

8.5 Limit of Quantitation (LOQ) The Limit of Quantitation will be validated by the preparation of a solution for each respective sample matrix spiked with each applicable compound at the respective limit of quantitation. Prepare solution as outlined below. Inject LOQ solution six times into the chromatographic system. Calculate the %RSD for the replicate injections. Bisoprolol Fumarate/ HCTZ Tablets LOQ Solution: Accurately weigh ~4300mg of the Bisoprolol Fumarate/ HCTZ Tablets placebo preparation into a 200-mL volumetric flask. Pipet 4.0 mL of 2% Linearity Solution into the same 200 mL volumetric flask. Dilute to volume with diluent and mix well. This solution should contain about 0.4 µg/mL of Bisprolol A Impurity, 0.4 µg/mL of Bisoprolol, 0.25 µg/mL of 4-Amino, 0.25 µg/mL of CTZ, and 0.25 µg/mL HCTZ. Bisoprolol Fumarate/ HCTZ Tablets Dimer LOQ Solution: Accurately weigh ~4300mg of the Bisoprolol Fumarate/ HCTZ Tablets placebo preparation into a 200-mL volumetric flask. Pipet 4.0 mL of 2% Linearity Solution for Dimer into the same 200 mL volumetric flask. Dilute to volume with diluent and mix well. This solution should contain about 0.25 µg/mL Dimer. Note1: Due to this run being a gradient with many related compounds peak being present in the solution, finding an acceptable interval for the signal-to-noise may be difficult. In the even that this occurs the LOQ study may be repeated spiking sample containing the placebo matrix with one related compound at a time. Therefore, each sample preparation would contain only one related compound of interest for the LOQ determination. Note2: Should the acceptance criteria not be met new solutions at a higher concentration may be prepared. However, the concentration must be ≤ than that of the specification concentration. Acceptance Criteria: The %RSD must be NMT 10.0% for each related compound. The average signal-to-noise ratio must be NLT 10.

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8.6 Limit of Detection (LOD) The limit of detection for each compound will be extrapolated from the limit of quantitation results. The following equation should be used for this determination.

NSmLOQ

ConcLOD

/

3.

Where, Conc is the concentration of the limit of quantitation solution. mLOQ S/N is the mean signal to noise ratio observed of the six LOQ sample injections. 3 is the target signal to noise at the limit of detection. Acceptance Criteria: Report the extrapolated limit of detection for each component. Include the concentration and the % level based on the related compound sample preparation.

8.7 Accuracy Studies for Assay The accuracy will be performed to cover a range of 200% of the highest label claim of the assay concentration down to 50% of the lowest label claim. Each solution is to be prepared as outlined in Table 9 and Table 10. Two 100% raw material samples will be prepared for potency determination.

Table 9: Accuracy Determination for Bisoprolol Fumarate/HCTZ Tablets – Assay (Bisoprolol Fumarate)

% Level Bis/HCTZ (based on 5/6.25mg

LC)

Amount of

Bisoprolol Fumarate

(mg)

Amount of

HCTZ (mg)

Amount of

Placebo (mg)

VF (mL)

# of Preparations

Conc. of Bisoprolol Fumarate (µg/mL)

Conc. of HCTZ

(µg/mL)

100% 10 12.5 0 500 2 20 25 25% 2.5 12.5 430 500 3 5 25

100% 10 12.5 430 500 3 20 25400% 40 12.5 430 500 3 80 25

Table 10: Accuracy Determination for Bisoprolol Fumarate/HCTZ Tablets – Assay (HCTZ)

% Level Bis/HCTZ (based on 5/6.25mg

LC)

Amount of

Bisoprolol Fumarate

(mg)

Amount of

HCTZ (mg)

Amount of

Placebo (mg)

VF (mL)

# of Preparations


Conc. of HCTZ

(µg/mL)

100% 20 12.5 0 500 2 40 25 50% 20 6.25 430 500 3 40 12.5

100% 20 12.5 430 500 3 40 25 200% 20 25 430 500 3 40 50

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Note: The amount of Bisoprolol Fumarate will be added at the highest strength (10mg/6.25mg) as this is worst case.

Acceptance Criteria: Each individual recovery value should be between 97.0 – 103.0%. The mean recovery value at each level should be between 98.0 – 102.0%.

8.8 Accuracy Studies for Related Compounds

The accuracy studies for related compounds determination will be performed for finished product formulation. All solutions are to be prepared as outlined in Table 11 and 12. Two un-spiked samples will be prepared and used as controls. All sample preparations will be prepared as instructed for the related compounds sample preparation or as instructed below. The Impurity Stock Solutions prepared in Section 8.2 can be used for the accuracy studies. (At least need 100 mL of impurity stock solution to perform Accuracy testing) Accuracy Solution A: Pipet 6.0 mL of Bisoprolol A Impurity stock from Related Compound Stock Solutions prepared in Section 8.2 into 25 mL volumetric flask. Dilute to volume with diluent. This solution should contain about 48 µg/mL of Bisprolol A Impurity.

Table 11: Accuracy Determination for Bisoprolol Fumarate/ HCTZ Tablets

% Level (Bisoprolol A Impurity,

4-amino)

% Level (CTZ

)

Amount of

Bisoprolol

Fumarate (mg)

Amount of HCT

Z (mg)

Amount of

Placebo (g)

Amount of RC Solution (mL)

VF (mL

)

# of Preparatio

ns

Conc. of Related

Compounds (µg/mL)

0% 0% 100 62.5 ~2.15 NA 100 2 ---

0.2%1 0.2% 100 62.5 ~2.15

1.0 mL of accuracy solution A

1.0 mL of 4-Amino 1.0 mL of CTZ

100 3

0.48 (Bisoprolol A)

1.25 (4-Amino, CTZ)

1.0%2 0.5% 100 62.5 ~2.15

2.5 mL of Bisoprolol A imp


100 6

5.0 (Bisoprolol A)

6.25 (4-Amino) 3.13 (CTZ)

2.0%3 1.0% 100 62.5 ~2.15

10.0 mL of Bisoprolol A imp 10.0 mL of 4-Amino

5.0 mL of CTZ 100 3

20.0 (Bisoprolol A)

12.5 (4-Amino) 6.25 (CTZ)

Note1: The %Level for the Bisoprolol is based on the 2.5/6.26mg strength as this is worst case. Note2: The %Level for the Bisoprolol is based on the 5/6.26mg strength as this is for middle concentration. Note3: The %Level for the Bisoprolol is based on the 10/6.25 mg strength as this is worst case.

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Table 12: Accuracy Determination for Dimer in Bisoprolol Fumarate/ HCTZ Tablets

% Level (Dimer)

Amount of

Bisoprolol

Fumarate (mg)

Amount of HCT

Z (mg)

Amount of

Placebo (g)

Amount of RC Solution (mL)

VF (mL

)

# of Preparatio

ns

Conc. of Related

Compounds (µg/mL)

0% 50 31.25 ~1.08 NA 50 2 --- 0.2% 50 31.25 ~1.08 0.5 mL of Dimer 50 3 1.25 1.0% 50 31.25 ~1.08 2.5 mL of Dimer 50 6 6.25 2.0% 50 31.25 ~1.08 5.0 mL of 4-Amino 50 3 12.5 Note: Due to limited availability of the Dimer the accuracy study had to be scaled accordingly.

Acceptance Criteria: The mean recovery at each level must be within the range outlined in Table 13. Due to the low levels of some of the impurities a wider range may be acceptable. The acceptance of a wider range will be based on sound scientific judgment and approved by management.

Table 13: Acceptance Criteria for Related Compound Accuracy % Level Range < 0.5% 80 – 120%

0.5% - 5% 90 – 110%

8.9 Method Precision for Assay Precision studies for finished product will be conducted by the analysis of six sample preparations of the lowest strength dosage as this is the worst case for all. Samples will be prepared as directed in the assay procedure.

Expired lots for Bisoprolol Fumarate/HCTZ 2.5/6.25 mg Tablets will be used for the assay precision: Acceptance Criteria: The %RSD for each of the six sample preparations should be NMT 2.0%.

8.10 Intermediate Precision for Assay

The Intermediate Precision will be performed as described in Section 8.9. All samples will be prepared by a different chemist, on a different day, using different mobile phase, sample and standard preparations, column and a different chromatographic system. Acceptance Criteria: The %RSD for each of the six sample preparations should be NMT 2.0%. The difference in mean between chemist 1 and chemist 2 should be NMT 2.0%.

8.11 Ruggedness for Content Uniformity – Day 1

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Content Uniformity ruggedness studies for finished product will be conducted by the analysis of ten (10) individual sample preparations of the lowest strength dosage as this is the worst case for all. As the content uniformity sample preparation utilizes whole intact tablets and the assay procedure uses a sample grind the ruggedness of this procedure will need to be evaluated. Samples will be prepared as directed in the content uniformity procedure. Expired lots for Bisoprolol Fumarate/HCTZ 2.5/6.25 mg Tablets will be used for the content uniformity: Acceptance Criteria: The acceptance value (AV) for 10 units is not more than 15.0. Report the mean and the range.

8.12 Ruggedness for Content Uniformity – Day 2

The Day 2 ruggedness for content uniformity will be performed as described in Section 8.11. All samples will be prepared by a different chemist, on a different day, using different mobile phase, sample and standard preparations, column and a different chromatographic system. Acceptance Criteria: The acceptance value (AV) for 10 units is not more than 15.0. Report the mean and the range. The % difference in mean between Day 1 and Day 2 should be NMT 5.0%.

8.13 Method Precision for Related Compounds Precision studies will be executed by spiking six representative samples with each known related compound. These samples will be prepared as directed in the related compounds method each in a 100-mL volumetric flask. Also prepare in duplicate for each precision analysis a placebo sample spiked with raw material at 100% to use as a control. Table 14: Method Precision Sample Preparations

Method Amount of Bisoprolol

Fumarate (mg)

Amount of HCTZ

(mg)

Amount Placebo (g)

Amount of RC Stock Solution

VF (mL)

Bisoprolol Fumarate/HCTZ

Tablets 100 62.5 ~2.15

2.5 mL of Bisoprolol A imp


100

Table 15: Method Precision Sample Preparations for Dimer

Method Amount of Bisoprolol

Fumarate (mg)

Amount of HCTZ

(mg)

Amount Placebo (g)

Amount of RC Stock Solution

VF (mL)

Bisoprolol Fumarate/HCTZ

Tablets 50 31.25 ~1.08

2.5 mL of Dimer imp

50

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Note1: Due to limited availability of the Dimer the method Precision study had to be scaled accordingly. Note2: The six middle level accuracy preparations will be used for method precision determination as well. Acceptance Criteria: The %RSD for each related compound in the six sample preparations must be as specified in Table 16.

Table 16: Acceptance Criteria for Method Precision % Level %RSD < 0.2% RSD ≤ 20%

0.2 – 0.5% RSD ≤ 10% 0.5 – 5% RSD ≤ 5%

8.14 Intermediate Precision for Related Compounds

A different chemist on a different day should prepare samples as directed in Section 8.13 using different columns, mobile phase, chromatographic systems, and standard preparations.

Acceptance Criteria: All acceptance criteria for method precision must be met for intermediate precision. The difference in mean between day 1 and day 2 values should be as outlined in Table 17.

Table 17: Acceptance Criteria for Intermediate Precision

% Level % Difference (Absolute)NMT 0.1% ∆ ≤ 0.05% 0.2 – 0.3% ∆ ≤ 0.10% 0.4 – 0.5% ∆ ≤ 0.15% 0.6 – 0.8% ∆ ≤ 0.20% 0.9 – 3.0% ∆ ≤ 0.3%

Limit > 3.0% ∆ ≤ 10% relative 8.15 Specificity

The specificity will be assessed by analysis using a Photo Diode Array (PDA) Detector. Solutions of each individual related compound, the blanks, and placebo solution will be analyzed. Related Compound ID Solutions: Inject each of the individually prepared Related Compound Stock Solutions from Section 8.2. Blank: Inject the diluent into the chromatographic system. Blank: Pipet 5.0mL of Acetonitrile into a 100mL volumetric flask. Dilute to volume with diluent and mix well.

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Placebo Preparation: Prepare a placebo sample preparation for assay and related compounds for each finished product. The sample should be extracted as outlined in the analytical method. Acceptance Criteria: Report the retention time for each related compound relative to that of its respective API in the standard preparation. There should be no interfering peak above the limit of quantitation in the blank or the placebo preparation.

8.16 Robustness of Extraction Procedure for Assay

A study will be performed to determine if the sample preparation procedure is sufficient for full extraction of the active ingredient from the sample matrix. Expired lots for Bisoprolol Fumarate/HCTZ 2.5/6.25 mg Tablets will be used for the extraction study. 8.16.1 Sonication Study Randomly select and weigh 20 tablets. Grind the tablets to a fine powder using a dry mortar and pestle. Transfer an accurately weighed portion of the powder equivalent to 12.5 mg of HCTZ into a 500-mL volumetric flask. Add diluent to ½ volume and sonicate for 10 minutes with frequent shaking. Remove the flask from the sonication bath. Dilute to volume with diluent and mix well. Remove NLT 20 mL from flask. Replace the flask and sonicate for another 5 minutes with frequent shaking (15 minutes total). Remove NLT 20 mL from the flask. Sonicate the flask for another 15 minutes (30 minutes total) with frequent shaking. Remove NLT 20 mL from the flask. Filter a portion of the sample through a 0.45μm Whatman GMF filter, discarding the first 5ml of the filtrate. Analyze all samples as described in the analytical method.

8.16.2 Shaking Study Using the same sample grind from the sonication study weigh an accurately weighed portion of the powder equivalent to 12.5 mg of HCTZ into a 500-mL volumetric flask. Add diluent to ½ volume and sonicate for 15 minutes with frequent shaking. Shake the flask on a mechanical shaker for 10 minutes. Remove the flask and dilute to volume with diluent. Remove NLT 20 mL from flask. Replace the flask and shake for another 5 minutes (15 minutes total). Remove NLT 20 mL from the flask. Shake the flask for another 15 minutes (30 minutes total). Remove NLT 20 mL from the flask. Filter a portion of the sample through a 0.45μm Whatman GMF filter, discarding the first 5ml of the filtrate. Analyze all samples as described in the analytical method.

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Acceptance Criteria: The % difference (absolute) should be NMT 2.0% from the method specified time point for the extraction to be considered robust.

8.17 Robustness of Extraction Procedure for Related Compounds

A study will be performed to determine if the sample preparation procedure is free of bias on the related compounds analysis. Expired lots for Bisoprolol Fumarate/HCTZ 2.5/6.25 mg Tablets will be used for the extraction study. 8.17.1 Sonication Study Randomly select and weigh 10 tablets into a 100-mL volumetric flask. Add diluent to ½ volume and sonicate for 10 minutes with frequent shaking. Remove the flask from the sonication bath. Dilute to volume with diluent and mix well. Remove NLT 20 mL from flask. Replace the flask and sonicate for another 5 minutes with frequent shaking (15 minutes total). Remove NLT 20 mL from the flask. Sonicate the flask for another 15 minutes (30 minutes total) with frequent shaking. Remove NLT 20 mL from the flask. Filter a portion of the sample through a 0.45μm Whatman GMF filter, discarding the first 5ml of the filtrate. Analyze all samples as described in the analytical method.

8.17.2 Shaking Study Randomly select and weigh 10 tablets into a 100-mL volumetric flask. Add diluent to ½ volume and sonicate for 15 minutes with frequent shaking. Shake the flask on a mechanical shaker for 10 minutes. Remove the flask and dilute to volume with diluent. Remove NLT 20 mL from flask. Replace the flask and shake for another 5 minutes (15 minutes total). Remove NLT 20 mL from the flask. Shake the flask for another 15 minutes (30 minutes total). Remove NLT 20 mL from the flask. Filter a portion of the sample through a 0.45μm Whatman GMF filter, discarding the first 5ml of the filtrate. Analyze all samples as described in the analytical method. Acceptance Criteria: The extraction time is free of bias for related compounds if the absolute difference between each individual impurity for each time point is as outlined in Table 18, and no additional peak above reporting level is found between each time point.

Table 18: Acceptance Criteria for Related Compounds Extraction Study

%Level Absolute Difference ≤ 0.5% NMT 0.1%

0.5% - 5.0% NMT 10% (relative)

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8.18 Filter Study for Assay and Related Compounds Expired lots for Bisoprolol Fumarate/HCTZ 2.5/6.25 mg Tablets will be used for the filter study. Assay: Prepare a sample as outlined in the assay portion of the analytical method described in this protocol. Using the assay sample preparation and the working standard preparation filter a portion through a 0.45µm Millex-HV PDVF filter and a 0.45µm Whatman GMF with GMF filter. Collect the first 3 mL (3 mL total), the next 2 mL (5 mL total), the next 2 mL (7 mL total) and the last 3 mL (10 mL total) when 10 mL of the solution is passed through the same filter. Centrifuge a portion of the sample at 2000rpm for 10 minutes. Use the centrifuged assay sample for comparison purposes.

Acceptance Criteria (Assay): The filter is considered acceptable when the % difference (absolute) varies NMT 2.0% between collections for the standard. The filtered sample is considered acceptable if the % difference (absolute) is NMT 2.0% different from the value obtained from the centrifuged sample. Related Compounds: Prepare a sample preparation as outlined for related compounds as described in the analytical method section of this protocol. The sample should be assessed by the collection of the first 3 mL (3 mL total), the next 2 mL (5 mL total), the next 2 mL (7 mL total), and the last 3 mL (10 mL total) when 10 mL of solution is passed through the same filter. Use a 0.45-µm Millex-HV PDVF and a 0.45-µm Whatman GMF with GMF filter. Centrifuge a portion of the sample at 2000rpm for 10 minutes. A portion of the centrifuged should be injected for comparison purposes. Acceptance Criteria: The filter is free from bias if the absolute difference of individual impurity between the filtered sample and the centrifuged samples (i.e. unfiltered) is NMT 0.1% for the observed level 0.1% to less than 0.5%, NMT 10% (relative) for the observed level 0.5 to 5.0% and no additional peak above the reporting level is found.

8.19 Robustness (Assay and Related Compounds) of Chromatographic Parameters

Prepare one sample as described in Method Precision for Assay Section 8.9 and Method Precision for Related Compounds Section 8.13 and analyze using the chromatographic conditions outlined in this protocol. Analyze both the related compounds and assay sample preparations using the altered chromatographic conditions as per Tables 19 and 20. The assay robustness analysis should be injected on a chromatographic system

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equipped with a Photodiode Array Detector (PDA) and analyzed for peak purity. Note: A method precision sample (Section 8.9 and Section 8.13) may be used for robustness studies for related compounds and assay.

Table 19: Robustness Parameters for HPLC

Parameter Variation Column Equivalency Waters Sunfire C18 4.6mm x 150mm 5µm column

Flow Rate ± 0.2 mL/min %ACN in Mobile Phase A ± 3%

Temperature ± 5°C Wavelength ± 4 nm


Parameter Variation

Column Equivalency Waters Aquity BEH Shield RP18 2.1mm x 100mm 1.7µm

column Flow Rate ± 0.06 mL/min

%ACN in Mobile Phase A ± 3% Temperature ± 5°C Wavelength ± 4 nm

Evaluate the system suitability. If the system suitability criteria are not met under any of the altered conditions the range may be narrowed accordingly until robustness is established. Acceptance Criteria: The purity angle for the Bisoprolol Fumarate and HCTZ peaks should be less than the threshold angle for the assay sample preparation. Report the results of system suitability. Report the results of the samples for both assay and related compounds. Report the relative retention times of each related compounds for all robustness conditions.

8.20 Stability of Sample Solutions (Assay and Related Compounds) The stability of the sample solutions will be evaluated for each solution that could be used with this method. Prepare one sample for assay, related compounds, and each working standard following the analytical procedure described in this protocol. Analyze the assay, related compound, and standards on the day of preparation. Pour a portion of the sample and standard preparations into both clear and amber flasks to be stored on the bench top at room temperature and under refrigeration. Analyze the standards and samples against freshly prepared standards for not less than 3 Days with one day following Day 3. The stability of the Resolution/Identification solutions

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will be assessed over an extended time and may be added to the report later as an addendum. The assay samples should be analyzed using a PDA detector for peak purity. Acceptance Criteria (Assay): The standard solution % change when compared to the initial value should be NMT 2.0% for acceptability in assay analysis. The sample solution % change with respect to initial should be NMT 2.0% to be considered standard. The purity angle should be less than that of the threshold angle for the peak to be considered pure.

Acceptance Criteria (Related Compounds): The standard solution % change when compared to the initial value should be NMT 10%. The sample solution % change should be as follows in Table 21 with respect to the initial.

Table 21: Acceptance Criteria for Related Compound Solution Stability

Observed Level <0.5% ± 0.1% absolute Observed Level 0.5% to <0.5% ≤ 10% relative

Observed Level >0.5% ≤ 5% relative For test solution, no additional peak ± 0.1% with respect to initial should be observed

8.21 Forced Degradation for Bisoprolol Fumarate/HCTZ Tablets

The forced degradation study will be performed using samples consisting of placebo for Bisoprolol Fumarate/HCTZ Tablets, Bisoprolol Fumarate Raw Material and HCTZ Raw Material only, and the Finished Product Sample Blend for Bisoprolol Fumarate /HCTZ Tablets (Bisoprolol Fumarate + HCTZ + placebo) for each condition. There will be 3 samples total for each condition tested. As the purpose of stability indicating studies is to determine the routes of degradation for each component the sample will be prepared at the related compound sample preparation and diluted appropriately to the assay concentration for recovery analysis. Should any condition prove to be too harsh with degradation more than 15% then the experiment may be repeated using different parameters. All forced degradation studies should be analyzed using a Photo-Diode Array Detector (PDA) and analyzed for both the assay and related compound analysis. Refer to Table 22 for guidance. Table 22: Samples for Bisoprolol Fumarate /HCTZ Forced Degradation

Sample Bisoprolol Fumarate

HCTZ Placebo (Bisoprolol Fumarate /HCTZ

Tablets) Bisoprolol Fumarate/HCTZ Placebo NA NA Bisoprolol Fumarate and HCTZ Raw

Material NA

Bisoprolol Fumarate /HCTZ Blend

Control Sample Preparation: Prepare one of each sample listed above and transfer the equivalent amount of 100 mg of Bisoprolol Fumarate,

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62.5mg of HCTZ, and 4300mg of placebo into the 100-mL volumetric flask. Prepare the samples as outlined in the analytical procedure for related compounds and analyze. Pipet 4.0 mL of the stock solution into a 100-mL volumetric flask. Dilute to volume with diluent and mix. Filter a portion of the sample through a 0.45μm Whatman GMF filter, discarding the first 5ml of the filtrate. Analyze the sample for assay. Photolytic Degradation of Dry Powders: Prepare one of each sample listed above and transfer the equivalent amount of 100 mg of Bisoprolol Fumarate, 62.5mg of HCTZ, and 4300mg of placebo into the 100-mL volumetric flask. Place the flasks under UV light at the short and long wavelengths for 5 days. After 5 days remove the flasks and prepare the samples as directed in Control Sample Preparation for assay and related compounds. Photolytic Degradation of Sample Solutions: Prepare one of each sample listed above and transfer the equivalent amount of 100 mg of Bisoprolol Fumarate, 62.5mg of HCTZ, and 4300mg of placebo into the 100-mL volumetric flask. Prepare the samples as directed in the Control Sample Preparation for assay and related compounds. Place the flasks under UV light at the short and long wavelengths. Analyze the samples up to 5 days until NMT 15% degradation is observed. Note1: Portions taken from the control sample preparation may be used for this study. Note2: Based on previous forced degradation studies the HCTZ may degrade quickly under UV light. Therefore, inject the solution after 3 hours under UV light. Thermal Degradation of Dry Powders: Prepare one of each sample listed above and transfer the equivalent amount of 100 mg of Bisoprolol Fumarate, 62.5mg of HCTZ, and 4300mg of placebo into the 100-mL volumetric flask. Place the flasks in a calibrated oven maintained at 105°C for 2 hours for Bisoprolol Fumarate and 5 days for HCTZ. After 2 hours and 5 days remove the appropriate flasks (one set of each samples at 2 hours and another set of each samples at 5 hours) and prepare the samples as directed in the Control Sample Preparation for assay and related compounds. Dry Powders Under Room Temperature Humidity: Prepare one of each sample listed above and transfer the equivalent amount of 100 mg of Bisoprolol Fumarate, 62.5mg of HCTZ, and 4300mg of placebo into the small glass beakers. Place the beakers in a desiccator with water on the bottom and cover tightly. Leave the unit undisturbed on the bench top at room temperature for 5 days. After 5 days remove the beakers and

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transfer the contents into a 100-mL volumetric flask. Prepare the samples as directed in the Control Sample Preparation for assay and related compounds.

Dry Powders Under Moisture Saturated Headspace (MSH): Prepare one of each sample listed above and transfer the equivalent amount of 100 mg of Bisoprolol Fumarate, 62.5mg of HCTZ, and 4300mg of placebo into the small glass beakers. Place the beakers in a dessicator with water on the bottom and cover tightly. Leave the entire unit in a calibrated oven maintained at 90°C for 48 hours. After 48 hours remove the beakers, allow them to cool to room temperature, and transfer the contents into a 100-mL volumetric flask. Prepare the samples as directed in the Control Sample Preparation for assay and related compounds. Acid Hydrolysis: Prepare one of each sample listed above and transfer the equivalent amount of 200 mg of Bisoprolol Fumarate, 125mg of HCTZ, and 8600mg of placebo into the 200-mL volumetric flask. Pipet 10.0 mL of 1N HCl into each flask and store the flasks on bench top at room temperature for 4 hours. After 4 hours add 10.0 mL of 1N NaOH to neutralize the reaction. Pipet 8.0 mL of ACN into the same volumetric flasks. Add diluent to ½ volume and extract according to the Control Sample Preparation for assay and related compounds.

Alkaline Hydrolysis: Prepare one of each sample listed above and transfer the equivalent amount of 200 mg of Bisoprolol Fumarate, 125mg of HCTZ, and 8600mg of placebo into the 200-mL volumetric flask. Pipet 10.0 mL of 1N NaOH into each flask and heat the flasks in a water bath maintained at 65°C for 1 hour. After 1 hour remove the flasks from the bath and add 10.0 mL of 1N HCl to neutralize the reaction. Pipet 8.0 mL of ACN into the same volumetric flasks. Add diluent to ½ volume and extract according to the Control Sample Preparation for assay and related compounds. Chemical Oxidation: Prepare one of each sample listed above and transfer the equivalent amount of 200 mg of Bisoprolol Fumarate, 125mg of HCTZ, and 8600mg of placebo into the 200-mL volumetric flask. Pipet 10.0 mL of 10% H2O2 into each flask and heat the flasks in a water bath maintained at 65°C for 90 minutes. After 90 minutes remove the flasks from the bath and allow them to cool to room temperature. Pipet 4.0 mL of ACN into the same volumetric flasks. Add diluent to ½ volume and extract according to the Control Sample Preparation for assay and related compounds. Acceptance Criteria: Degradation for each condition of NMT 15% should be observed. Acceptance of a higher amount of degradation will be assessed on a case-by-case basis. All samples should be analyzed using

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a PDA. The purity angle should be less than the threshold angle for both Bisoprolol Fumarate and HCTZ in the assay sample preparations.

8.22 Analytical Method Comparison Study – Assay and Related

Compounds There is currently a method in place (as listed below) at Sandoz, Inc at Wilson, NC that is approved and used to determine the assay and related compounds for the product being tested in these validation studies. Therefore, a method comparison should be executed to demonstrate the equivalency of this method. Upon establishment of equivalency this new analytical method may be adopted as an alternative to the current method being use for this product. Six samples will be prepared for assay and six samples will be prepared for related compounds. New samples will be prepared again and tested according to the new proposed method outlined in this protocol. The results of these two studies will then be compared to determine the equivalency of the analytical method. There is also a USP method. This method will be compared to USP method as well, even though we do not use USP method for testing Bisoprolol Fumarate/HCTZ tablets at Sandoz, Inc at Wilson, NC. Note: The Method Precision for Assay may be used for this study. Table 23: Expired lots for following will be used for the equivalency study:

Product Sandoz

Monograph New Proposed

Method # of Assay

Preparations # of RC

Preparations Bisoprolol Fumarate /HCTZ

Tablets B002QC MVP000126v1 6 6

Bisoprolol Fumarate /HCTZ Tablets

USP 33 MVP000126v1 6 6

An expired lot for following strengths should be used for this study as this is worse case:

Bisoprolol Fumarate /HCTZ 2.5/6.25 mg Tablets Acceptance Criteria (Assay): The difference in the mean values of the six sample preparations between each method should be NMT 2.0% absolute. Acceptance Criteria (Related Compounds): Calculate the individual and total related compounds found in the sample preparations. The number and amount of the related compounds peaks should be comparable between two methods in each sample preparation. Calculate the individual and total related compounds found in the sample preparations. The absolute difference between the propose method and the current method is NMT 0.25% for the average of total related compounds. The absolute

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difference between individual related compounds with specification values should be NMT 0.1%. Should the values be outside the acceptance criteria the level of equivalency will be decided on and approved by management within Sandoz, Inc at Wilson, NC and discussed within the validation report.

9.0 SYSTEM SUITABILITY The system suitability parameters will be assessed and determined following the successful completion of this method validation protocol.

10.0 DATA ANALYSIS AND REPORT Upon completion of the study, all raw data will be reported in Sandoz technical laboratory notebooks. All data generated and calculated from this validation study will be evaluated and reviewed for accuracy. An Analytical Method Validation Report (MVR) will be generated by the analyst performing the validation.

11.0 REFERENCES 11.1 B002QC – Bisoprolol Fumarate and Hydrochlorothiazide Tablets,

2.5mg/6.25mg, 5mg/6.25mg, and 10mg/6.25mg 11.2 SOP QC-117 – Analytical Method Validation

12.0 ATTACHMENTS 12.1 NA

13.0 REVISION HISTORY Version Number

Type of Change Justification for Change Date Initiated

1 New New 10/09/2010 (NNT)

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Appendix F: Method Validation Protocol for Dissolution Testing

Analytical Method Validation Protocol

For the Quantitative Determination of the Dissolution Rate for

Bisoprolol Fumarate/Hydrochlorothiazide Tablets,

2.5mg/6.25mg, 5mg/6.25mg, 10mg/6.25mg

Protocol #: MVP00127v1

Author’s Signature: ____________________________________ Date: ______________ Name/Department/ Title: Niralee Thakar/QC/Chemist II

Reviewed By: Date:

Name/Department/ Title: Jason Miller/QC/Chemist IV

Reviewed By: Date:

Name/Department/ Title: John Bredin/QC/Manager

Approved By: Date:

Name/Department/ Title: Richard Uveges/QC/Director

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14.0 OBJECTIVE

The purpose of this protocol is to describe the procedure to validate a new method for the simultaneous determination of the dissolution rate of Bisoprolol and Hydrochlorothiazide in Bisoprolol Fumarate/Hydrochlorothiazide Tablets, 2.5mg/6.25mg, 5mg/6.25mg, and 10mg/6.25mg. These studies will be conducted to demonstrate that this method is suitable for its intended use.

15.0 INTRODUCTION Currently, at Sandoz, Inc in Wilson, NC the dissolution for Bisoprolol and HCTZ in Fumarate/Hydrochlorothiazide Tablets, 2.5mg/6.25mg, 5mg/6.25mg, and 10mg/6.25mg are analyzed using two different chromatographic systems. Each analytical method has also been previously validated by Sandoz, Inc at Wilson, NC. However, recently new methods for HPLC and UPLC were developed that can be used as an alternative to combine the analysis of Bisoprolol Fumarate and Hydrochlorothiazide.

The purpose of this protocol is to demonstrate that the new analytical methods using HPLC and UPLC are suitable for the simultaneous determination of the dissolution rate in Bisoprolol Fumarate/Hydrochlorothiazide Tablets, 2.5mg/6.25mg, 5mg/6.25mg, and 10mg/6.25mg. This validation will also demonstrate that this new method has shown equivalency with the existing analytical methods at Sandoz, Inc and is therefore, considered to be an acceptable alternative. Also this validation for new method will compare to USP method.

16.0 REGULATORY REQUIREMENTS Specifications Dissolution (Stage 1): Not less than 85% of the labeled amount of Bisoprolol Fumarate dissolved in 20 minutes and Hydrochlorothiazide in 30 minutes for each of the 6 tablets tasted. Q = 80%. Dissolution (Stage 2): If the requirements of Stage 1 are not met, test another 6 tablets. The requirements are met if the average dissolution for all 12 tablets tested is not less than 80% and no tablet is less than 65% of the labeled amount of Bisoprolol Fumarate in 20 minutes and Hydrochlorothiazide in 30 minutes. Dissolution (Stage 3): If the requirements of Stage 2 are not met, test another 12 tablets. The requirements are met if the average dissolution of the all 24 tablets tested is not less than 80% and not more than 2 tablets are less than 65% of the labeled amount of Bisoprolol Fumarate in 20 minutes and Hydrochlorothiazide in 30 minutes. No tablet is less than 55% of the labeled amount of Bisoprolol Fumarate in 20 minutes and Hydrochlorothiazide in 30 minutes.

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17.0 STANDARDS 17.1 Bisoprolol Fumarate Reference Standard: Use the current USP lot or

certified In-House Standard. 17.2 Hydrochlorothiazide (HCTZ) Reference Standard: Use the current USP

lot or certified In-House Standard.

18.0 REAGENTS 18.1 Water: Use purified water or equivalent. 18.2 Acetonitrile: Fisher Scientific or equivalent. 18.3 Hydrochloric acid: Use concentrated hydrochloric acid (12N). Fisher

brand or equivalent. 18.4 Trifluoroacetic acid: 99+%, Aldrich Chemical Company or equivalent for

spectrophotometric grade.

19.0 INSTRUMENTATION/EQUIPMENT 19.1 Use a qualified HPLC system equipped with an electronic injector. 19.2 Use a qualified UPLC system equipped with an electronic injector.

20.0 ANALYTICAL METHODS

20.1 Solution Preparations

Dissolution Medium (0.1N HCl): Add 50 mL of Hydrochloric acid into about 6000 mL of water. Heat to 37°C ± 0.5°C and thoroughly degas. Mobile Phase A: Pipet 1.0mL of Trifluoroacetic acid into 1800mL of water. Add 200mL of acetonitrile and mix. Mobile Phase B: : Pipet 1.0mL of Trifluoroacetic acid into 1800mL of acetonitrile. Add 200mL of water and mix. Bisoprolol Standard Stock Solution: Weigh about 28 mg of Bisoprolol Fumarate Reference standard into 200 mL volumetric flask. Add ½ volume of dissolution medium to the flask and sonicate to dissolve. Dilute to volume with dissolution medium and mix well. This solution may contain about 140 µg/mL of Bisoprolol Fumarate.

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Hydrochlorothiazide Standard Stock Solution: Weigh about 23 mg of Hydrochlorothiazide Reference standard into a 200-mL volumetric flask. Add 5 mL of acetonitrile into the flask and sonicate for about 5 minutes. Add ½ volume of dissolution medium to the flask and sonicate to dissolve. Dilute to volume with dissolution medium and mix well. This solution may contain about 115 µg/mL of Hydrochlorothiazide. Working Standard Preparation: For all strengths Follow Table 1 below and pipette appropriate amount of standard stock into the appropriate volumetric flask. Dilute to volume with dissolution medium. Table 1: Working standard preparation

20.2 Dissolution Apparatus Parameters

Medium: 0.1N HCl Temperature: 37.0°C ± 0.5°C Volume: 900 mL Apparatus: Paddle (USP #2) Speed: 75 RPM Time Point: 20 minutes (for Bisoprolol), 30 minutes (HCTZ)

20.3 Sample Preparations

Fill each of the six dissolution vessels in a water bath with 900 mL of degassed dissolution medium. Allow to thermally equilibrate to temperature of 37.0°C ± 0.5°C. Weigh six individual tablets and place one tablet into each of the six dissolution vessels. Carry out the dissolution procedure according to the parameters outlined above. At 20 minutes, collect a portion for Bisoprolol (about 5 mL). At 30 minutes, collect a portion for HCTZ (about 10 mL). Filter each of the samples through a 0.45µm Millex HV PVDF filter or equivalent, discarding the first 3 mL of the filtrate. NOTE 1: The dissolution apparatus parameters and dissolution procedure for both dissolutions are the same except that Bisoprolol Fumarate is sampled at 20 minutes and Hydrochlorothiazide is sampled at 30 minutes.

Label Claim (Bisoprolol/

HCTZ)

Amount pipette from Bisoprolol standard

stock (mL)

Amount pipette from

HCTZ standard

stock (mL)

Volumetric Flask Size

(mL)

Bisoprolol Conc.

(µg/ml)

HCTZ Conc.

(µg/ml)

2.5mg/6.25mg 4.0 12.0 200 2.8 6.9 5mg/6.25mg 4.0 6.0 100 5.6 6.9 10mg/6.25mg 4.0 3.0 50 11.1 6.9

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Both dissolutions can be done at the same time provided that samples are taken at 20 and 30 minutes. Since 5 mL is withdrawn after 20 minutes, no correction is needed since the maximum error is 0.6%. Note that the USP general chapter <711> Dissolution states that the volume of dissolution medium may vary by ± 1% from the stated volume. NOTE 2: The dissolution apparatus must conform to the specifications of USP general chapter <711>. Check the shafts for wobble (follow the specifications as per Sandoz SOP QC-020).

20.4 Chromatographic Conditions for HPLC

Apparatus: HPLC equipped with an electronic injector. Detector: Ultraviolet set at 224-nm. Column: Waters Symmetry C18, 5µm, 4.6mm x 150mm Column Temperature: 35oC Flow Rate: 1.0mL/min Injection Volume: 20 µL Run Time: 10 minutes Linear Gradient:

Time % A % B Curve

0 100 0 --- 6 40 60 6 7 40 60 6

7.5 100 0 6 11 100 0 6

Retention Times: HCTZ at about 4.4 minutes Bisoprolol Fumarate about 5.4 minutes Column Wash: The column should be washed using (50:50) Water:ACN

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20.5 Chromatographic Conditions for UPLC Apparatus: UPLC equipped with an electronic injector. Detector: Ultraviolet set at 224-nm. Column: Waters Acquity BEH C18 2.1mm x 100mm 1.7 µm Column Temperature: 35oC Flow Rate: 0.300 mL/min Injection Volume: 10 µL Sampling Rate: 5 points/second Injection Mode: Full Loop Linear Gradient:

Time % A % B Curve 0.0 95 5 --- 2.0 85 15 6 5.0 50 50 6 5.5 95 5 6 7 95 5 6

Retention Times: HCTZ is about 2.0 minutes Bisoprolol Fumarate is about 4.4 minutes Weak Column Wash: (50:50) Water:MeOH Strong Column Wash: (50:50) Water:ACN Column Wash: The column should be washed with (50:50) Water:ACN and stored in 100% ACN.

20.6 Calculation

% Dissolution

TabletCLDilStdAreaPkStd

VolSplPotWtStdAreaPkSplActive

1......

100......%

Where, Spl.Pk.Area is the Sample Peak Area. Std.Pk.Area is the Standard Peak Area. Std.Wt. is the Standard Weight. Std.Dil is the Standard Dilution. Pot is the Potency of the Reference Standard Spl. Vol. is the Sample Volume (900 mL) L.C. is the Label Claim.

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21.0 VALIDATION PARAMETERS

21.1 Placebo Preparation

Table 2: Placebo Preparation for Method Validation Studies.

EDP # Ingredients Variance

Placebo Preparation

(mg/unit)

E280 Bisoprolol Fumarate 2.5/5.0/10.0 --- F109 Hydrochlorothiazide 6.25 ---

E370 Dicalcium Phosphate

Anhydrous 80 – 140 140

G623 Corn Starch NF 12 – 22 22 E188 Microcrystalline Cellulose 10 - 25 25 E352 Colloidal Silicone Dioxide 0.25 – 2 2 F550 Magnesium Stearate 0.5 – 2 2 H915 Opadry White YS-1-7003

2-8 8

H929 Opadry Orange YS-1-13174 8 Opadry Orange YS-1-13148 8

Placebo Total Weight (mg) per Unit 215 Note: The lowest strength of Bisoprolol Fumarate/HCTZ Tablets (2.5 mg/6.25 mg) is shown to have the highest ratio of placebo to active ingredient in the tablets and is therefore considered to be the worst case.

21.2 Linearity

The linearity will be performed as outlined below and cover the specified ranges for each product. The range will cover from 200% of the highest target dissolution concentration down to the 20% level for lowest dissolution concentration.

Table 3: Linear Ranges for Each Compound

% Level Concentration Range

(µg/mL) Bisoprolol Fumarate 2.5 mg (800% - 20%) 5 mg (400% - 10%) 10 mg (200% - 5%)

22.2 – 0.6

HCTZ 6.25 mg (200% - 20%)

13.9 – 1.4

Linearity Solution A: Weigh about 28 mg of Bisoprolol Fumarate into a 200-mL volumetric flask. Add dissolution medium to ½ volume and sonicate to dissolve. Dilute to volume with dissolution medium and mix well. This solution should contain about 140 µg/mL of Bisoprolol Fumarate.

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Linearity Solution B: Weigh about 23.2 mg of HCTZ into a 200-mL volumetric flask. Add about 5.0 mL of acetonitrile into the flask and sonicate for 5 minutes. Add dissolution medium to ½ volume and sonicate to dissolve. Dilute to volume with dissolution medium and mix well. This solution should contain about 116 µg/mL of HCTZ. Table 4: Linearity Solution Preparations for Dissolution

% Level Bisoprolol

(Based on 2.5 mg)

% Level HCTZ (Based on 6.25

mg) Amount of Solution

VF (mL)

Bis/HCTZ Conc.

(µg/mL)

800 200 8 mL of A & 6 mL of B 50 22.4/13.9 600 150 12 mL of A & 9 mL of B 100 16.8/10.44 400 133 4 mL of A & 4 mL of B 50 11.2/9.28 200 100 4 mL of A & 6 mL of B 100 5.6/6.96 100 75 4 mL of A & 9 mL of B 200 2.8/5.22 50 50 2 mL of A & 6 mL of B 200 1.4/3.5 20 20 2 mL of A & 6 mL of B 500 0.56/1.4

Acceptance Criteria: The correlation coefficient (R) for each API should be NLT 0.99. The % y-intercept value should be NMT 5% of the target value.

21.3 Accuracy Studies for Dissolution The accuracy for each product will be performed to cover a range of 200% of the highest sample concentration to 50% of the lowest sample concentration. Each solution is to be prepared as outlined in the Tables 5 and 6. Two 100% raw material samples will be prepared for potency determination. Accuracy Solution A: Weigh about 278 mg of Bisoprolol Fumarate into a500-mL volumetric flask. Add dissolution medium to ½ volume and sonicate to dissolve. Dilute to volume with dissolution medium and mix well. This solution should contain about 556 µg/mL of Bisoprolol Fumarate.

Accuracy Solution B: Weigh about 173.5 mg of HCTZ raw material into a 500-mL volumetric flask. Add about 5.0 mL of acetonitrile into the flask and sonicate for 5 minutes. Add dissolution medium to ½ volume and sonicate to dissolve. Dilute to volume with dissolution medium and mix well. This solution should contain about 347 µg/mL of HCTZ. Accuracy Sample Preparation: Pipet indicated amount of accuracy solution and weigh placebo into separate 1000-mL volumetric flasks. Dilute to volume with Dissolution medium. Add a stir bar to each flask and stir the solution for 20 minutes for Bisoprolol Fumarate accuracy (Table 5) and 30 minutes for HCTZ accuracy (Table 6). The stir plate may be kept at very low heat to maintain the dissolution temperature.

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Table 5: Accuracy Determination for Bisoprolol Fumarate - Dissolution

% Level

(Based on

2.5mg)

Pipet from Accuracy solution A

(mL)

Pipet from Accuracy solution B

(mL)

Amount of

Placebo (mg)

VF (mL)

# of Preparations


200% 10 0 0 1000 2 5.6 50% 2.5 20 239 1000 3 1.4

200% 10 20 239 1000 3 5.6 800% 40 20 239 1000 3 22.2

Table 6: Accuracy Determination for HCTZ - Dissolution

% Level

(Based on

10mg)

Pipet from Accuracy solution A

(mL)

Pipet from Accuracy solution B

(mL)

Amount of

Placebo (mg)

VF (mL)

# of Preparations

Conc. of HCTZ (µg/mL)

100% 0 20 0 1000 2 6.9 50% 20 10 239 1000 3 3.5

100% 20 20 239 1000 3 6.9 200% 20 30 239 1000 3 13.9 Note: The amount of Bisoprolol Fumarate will be added at the highest strength (10mg/6.25mg) as this is worst case.

Acceptance Criteria: The % Mean recovery must be between 95 - 105%.

21.4 Method Precision for Dissolution Precision studies for each finished product will be conducted by the analysis of six tablets of the lowest strength dosage form as this is the worst case. Sample will be prepared as directed in the dissolution procedure.

Expired lots for Bisoprolol Fumarate/HCTZ 2.5/6.25 mg Tablets will be used for the dissolution precision. Acceptance Criteria: The RSD for n=6 must be ≤ 5.0% at Q time point.

21.5 Intermediate Precision for Dissolution The Intermediate Precision will be performed as described in Section 8.4. All samples will be prepared by a different chemist, on a different day, using different mobile phase, different dissolution baths, sample and standard preparations, column and a different chromatographic system. Acceptance Criteria: The RSD limits for intermediate precision mentioned as per method precision must be met. The difference in mean values between chemist 1 and chemist 2 must be < 5.0% absolute at the prescribed time point.

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21.6 Specificity Bisoprolol Fumarate/HCTZ Tablets Placebo Solution Preparation: Weigh about 215 mg of the tablet placebo into a dissolution vessel containing 900 mL of dissolution medium. Perform the dissolution as described in the analytical method. At 20 minutes, collect a portion for Bisoprolol (about 5 mL). At 30 minutes, collect a portion for HCTZ (about 10 mL). Filter each of the samples through a 0.45µm Millex HV PVDF filter or equivalent, discarding the first 3 mL of the filtrate. Blank 1: Pipet 5.0 mL of acetonitrile into a 200-mL volumetric flask. Dilute to volume with dissolution medium and mix well. Pipet 3.0 mL of this blank solution into a 50-mL volumetric flask. Dilute to volume with water and mix well. Inject this solution into the chromatographic system. Blank 2: Inject the dissolution medium (0.1N HCl). Acceptance Criteria: There should be no interfering peak above 1% at the same time as the API in the chromatogram of the standard preparation.

21.7 Robustness Degassing Procedure

Dissolution Medium Preparation 1 (Non-Degassed): Mix 50ml of Hydrochloride acid into 6L of DI water. Heat the media to 37°C ± 0.5°C. Measure the amount of dissolved oxygen prior to dispensing. Dissolution Medium Preparation 1 (Vacuum Degassed): Mix 50ml of Hydrochloride acid into 6L of DI water. Degas the media for 15 minutes using a vacuum degasser. Heat the media to 37°C ± 0.5°C. Measure the amount of dissolved oxygen prior to dispensing. Dissolution Medium Preparation 1 (Helium Sparge): Mix 50ml of Hydrochloride acid into 6L of DI water. Helium sparge the media for NLT 12 minutes. Heat the media to 37°C ± 0.5°C. Measure the amount of dissolved oxygen prior to dispensing. Perform the dissolution as outlined in the analytical method. Use the lowest strength for the product. Expired lots for Bisoprolol Fumarate/HCTZ 2.5/6.25 mg Tablets will be used for the dissolution. Acceptance Criteria: The degassing procedure may be considered un-biased on the rate of dissolution if the mean %dissolved for all six tablets between each bath is within ± 5% absolute of one another.

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21.8 Robustness of Sampling Procedure (Manual vs. Automated) Prepare the dissolution media as described by the analytical method. Perform the dissolution using six tablets of the lowest strength lots. At 20 minutes, collect a portion manually for Bisoprolol (about 5 mL). At 30 minutes, collect a portion manually for HCTZ (about 10 mL) from each vessel, while simultaneously using an automated sampling apparatus to pull about 5 mL samples at 20 and 30 minutes. Filter each of the samples through a 0.45-µm Millex-HV PVDF filter or equivalent, discarding the first 3 mL of the filtrate. Automated Sampling Apparatus Parameter Change following parameter from the default All Pump flow rates: 15.0 mL/minutes Flush: 3.0 mL Offset: 3.0 mL Expired lots for Bisoprolol Fumarate/HCTZ 2.5/6.25 mg Tablets will be used for the dissolution. Acceptance Criteria: The sampling procedure may be considered un-biased on the results if the mean %dissolved for all six tablets between each sampling procedure is within ± 5% absolute of one another.

21.9 Robustness of Sample Preparation (Filter Study) Prepare the dissolution media as described by the analytical method. Perform the dissolution using one tablet of the lowest strength lots. After 20 minutes and 30 minutes manually pull about 60 mL of samples from each vessel. Prepare two separate filter study solutions, one for bisoprolol and one for HCTZ. Filter a portion of the sample and standard preparation using a 0.45-µm Millex-HV PVDF filter collecting the first 3 mL of filtrate, the next 2 mL of filtrate (5 mL total), the next 2 mL of filtrate (7 mL total), and the next 3 mL of filtrate (10 mL total). Perform the filter study again using a 0.45-µm Whatman GMF filter. Centrifuge the sample preparation for 10 minutes at 2000 rpm and compare all results to the centrifuged sample. Expired lots for Bisoprolol Fumarate/HCTZ 2.5/6.25 mg Tablets will be used for the dissolution. Acceptance Criteria: The sample preparation procedure may be considered un-biased on the results if the %dissolved for all sample collections is within ± 2% absolute of one another.

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21.10 Robustness of Chromatographic Parameters The robustness of the chromatographic parameters will be assessed by the injection of six samples prepared as directed in the analytical method. The mean of each bath will then be compared to determine the robustness of the chromatographic parameters. Six samples from the lowest strength for each product will be used for this study. Expired lots for Bisoprolol Fumarate/HCTZ 2.5/6.25 mg Tablets will be used for the dissolution.

Table 8: Robustness Parameters for HPLC

Parameter Variation Column Equivalency Water Sunfire C18 4.6mm x 150mm 5µm Column

Flow Rate ± 0.2 mL/min %ACN in Mobile Phase A ± 3%


Note: The method precision samples may be used for this study if injected within 48 hours from the day of preparation.


Parameter Variation

Column Equivalency Waters Aquity BEH Shield RP18 2.1mm x 100mm 1.7µm

column Flow Rate ± 0.06 mL/min

%ACN in Mobile Phase A

± 3%


Note: The method precision samples may be used for this study if injected within 48 hours from the day of preparation. Evaluate the system suitability. If the system suitability criteria are not met under any of the altered conditions the range may be narrowed accordingly until robustness is established. Should the robustness study exceed 48 hours from preparation new samples must be prepared and injected with new initial conditions for comparison purposes. Acceptance Criteria: Report the results of the mean of the six samples for each chromatographic condition. Report the results of system suitability (i.e. standard peak tailing and plate counts). The mean results must be between ± 2% absolute when compared to the mean if the initial sample injections for the chromatographic condition to be considered robust.

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21.11 Stability of Solutions Prepare one sample and standard following the analytical procedure described in this protocol. Analyze the sample and standard on the day of preparation. Pour a portion of the sample and standard preparations into both clear and amber flasks to be stored on the bench top. Analyze the standards and samples against freshly prepared standards for not less than 3 Days with one day following Day 3. The samples should be analyzed using a PDA detector for peak purity. Acceptance Criteria: The standard solution % change when compared to the initial value should be NMT 2% for acceptability. The sample solution % change with respect to initial should be NMT 2% to be considered stable. The purity angle should be less than that of the threshold angle for the peak to be considered pure.

21.12 Analytical Method Comparison Study There is currently a method in place (as listed below) at Sandoz, Inc at Wilson, NC that is approved and used to determine the rate of dissolution for the product being tested in these validation studies. Therefore, a method comparison should be executed to demonstrate the equivalency of these methods. Upon establishment of equivalency this new analytical method may be adopted as an alternative to the current method being use for this product. Six samples will be prepared for the products listed below in Table 10. New samples will be prepared again and tested according to the new proposed method outlined in this protocol. The results of these two studies will then be compared to determine the equivalency of the analytical methods. Note: The Method Precision for may be used for this study as the proposed method portion. Table 10: Expired lots for the following will be used for the equivalency study:

Product Sandoz

Monograph New Proposed

Method # of

Tablets Bisoprolol Fumarate/HCTZ

Tablets B002QC MVP0012v1 6

Bisoprolol Fumarate/HCTZ Tablets

USP 33 MVP0012v1 6

Expired lots for Bisoprolol Fumarate/HCTZ 2.5/6.25 mg Tablets will be used for the dissolution.

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Acceptance Criteria: The absolute difference between the mean of the six tablets tested using the proposed method and the current method is NMT 5%. Should the values be outside the acceptance criteria the level of equivalency will be decided on and approved by management within Sandoz, Inc at Wilson, NC and discussed within the validation report.

22.0 DATA ANALYSIS AND REPORT

Upon completion of the study, all raw data will be reported in Sandoz technical laboratory notebooks. All data generated and calculated from this validation study will be evaluated and reviewed for accuracy. An Analytical Method Validation Report (MVR) will be generated by the analyst performing the validation.

23.0 REFERENCES 23.1 B002QC – Bisoprolol Fumarate/Hydrochlorothiazide Tablets

2.5mg/6.25mg, 5mg/6.25mg, and 10mg/6.25mg 23.2 Sandoz SOP QC-117 “Analytical Method Validation”

24.0 ATTACHMENTS 24.1 None

25.0 REVISION HISTORY Version Number

Type of Change Justification for Change Date Initiated

1 New New 10/29/2010 (NNT)

development and validation of a combined method...

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