m. pharm. (pharmaceutical analysis & quality assurance...

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Chapter 5 Valacyclovir - Introduction

5.1 Introduction

Valacyclovir is an antiviral drug. The chemical formula of Valacyclovir

hydrochloride is L-valine, 2-[(2-amino-1,6-dihydro-6oxo-9H-purin-9-yl) methoxy]

ethyl ester, mono hydrochloride with the molecular formula C13H20N6O4.HCl and a

molecular weight of 360.80 (Figure 5.1).

Figure 5.1 Chemical structures of Valacyclovir (A), Valacyclovir-D8 (B)

Valacyclovir hydrochloride is rapidly absorbed from the gastrointestinal tract and

nearly completely converted to acyclovir and L-valine by first-pass intestinal and

hepatic metabolism by enzymatic hydrolysis. Acyclovir is converted to a small extent

to inactive metabolites by aldehyde oxidase and by alcohol and aldehyde

dehydrogenase. Neither Valacyclovir nor acyclovir is metabolized by cytochrome P450

enzymes. Plasma concentrations of unconverted Valacyclovir are low and transient,

generally becoming non-quantifiable by 3 hours after administration. Peak plasma

concentrations of Valacyclovir are generally less than 0.5 µg/mL at all doses. The

absolute bioavailability of acyclovir after oral administration is 54.5% ± 9.1%. The

binding of Valacyclovir to human plasma proteins ranges from 13.5% to 17.9%.

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The binding of acyclovir to human plasma proteins ranges from 9% to 33% 51

Several techniques such as HPLC and LC-MS/MS methods have been

reported in the literature for the quantitative estimation of Valacyclovir and Acyclovir

in biological fluids 52-58

and pharmaceutical dosage forms59-61

,A number of methods

were developed in animals such as rabbit55

rat56

and horse57

plasma for quantification

of Valacyclovir and Acyclovir by LC-MS/MS. Only a few methods were reported in

human plasma for quantification of Valacyclovir and Acyclovir 52-54

by LC-MS/MS.

Among all Yadav M, Upadhyav V et al 52

achieved best results. They developed the

method with linearity between the concentration range of 5-1075 ng/mL for

Valacyclovir, 47.6-10225 ng/ mL for Acyclovir using the mobile phase ratio of 0.1%

formic acid: methanol (30:70) on Gemini C18 column . They compared the drug with

fluconazole as an internal standard. They have not achieved sensitive less than

5 ng/mL.52

In bioanalytical method development, usage of deuterated internal standard is

very helpful to find the exact matrix effect at analyte and internal standard retention

times. Till now, as of our knowledge, there is no method reported for comparision of

Valacyclovir with its deuterated internal standard.

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5.2 Experimental Investigations

5.2.1 Materials and reagents

Valacyclovir and Valacyclovir-D8 TFA salt, Acyclovir were obtained from

Synfine Research Canada. Formic acid, Acetic acid, Ammonium hydroxide, NH4OH,

Ammonium formate were obtained from Merck Mumbai. Methanol, Acetonitrile

(HPLC grade) were Obtained from J.T.Baker, Mumbai. Ammonium formate,

Ammonia solution (NH4OH, 25%, Reagent grade), Formic acid, Glacial acetic acid

(CH3COOH, reagent grade) were Obtained from sd.Fine chemicals Mumbai.

Dichloromethane, were purchased from Merck Speciality Chemicals Ltd, Mumbai,

India. Human plasma was procured from Navazeevan Blood bank, Hyderabad.

Millipore water was used from Milli-Q system.

5.2.2 Instrumentation and equipment

Refer Chapter - 3.2.2

5.2.3 Preparation of Reagents and Solvents

Table 5.1 Preparation of Reagents and Solvents

Reagents and Solvents preparation

0.1% Formic acid Dilute 1 mL of formic acid to 1000 mL with water.

30% Methanol in 0.1% formic acid Mix 300 mL of methanol with 700 mL of 0.1% formic acid.

1N Acetic acid Dilute 60 mL of glacial acetic acid to 1000 mL with water (prepare

daily).

30% Formic acid Dilute 30 mL of formic acid to 100 mL with water.

2.5% NH4OH in methanol Mix 10 mL of ammonia solution with 90 mL of methanol (prepare

daily).

10mM Ammonium formate, PH 5.0

Dissolve 1.26 g of ammonium formate into 2 L of water. Adjust PH to

5.0 ± 0.05 with formic acid.

10mM Ammonium formate, PH 3.1

Dissolve 1.26 g of ammonium formate into 2 L of water. Adjust PH to

3.1 ± 0.05 with formic acid.

50% Methanol Mix 500 mL of methanol with 500 mL of water.

Reconstitution solution Mix 800 mL of 10mM ammonium formate, P

H 3.1 with 200 mL of

methanol.

20% Methanol

(Auto sampler wash) Mix 200 mL of methanol with 800 mL of water.

Acidified plasma Add approximately 5 mL of 30% formic acid to 100 mL of plasma.

Mobile phase

Mix 10mM Ammonium formate PH 5.0 : Methanol in the ratio of 80:20

and

Filter through 0.45 m filter

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5.2.4 Preparation of Stock solutions

Table 5.2 Preparation of Stock solutions

Name of the solution Concentration Volume (mL) Diluent

Valacyclovir stock solution 100.o µg/mL 100 mL Methanol

Valacyclovir- D8 stock solution 100.0 µg/mL 100 mL Methanol

Acyclovir stock solution 1000.0 µg/mL 10 mL Methanol

5.2.5 Preparation of standards and quality control (QC) Samples

Standard stock solution of Valacyclovir (100 g/mL) was prepared in

50% methanol. From this stock, analytical standards were prepared at concentration

levels of 0.5, 1.0, 5.0, 35.0, 70.0, 140.0, 280.0, 420.0, 560.0 and 700.0 ng/mL by

appropriate dilution with human plasma.

From the standard stock solution, Quality control samples were prepared separately

at LLOQ (0.5 ng/mL) low (1.5ng/mL) medium, (210.0 ng/mL) and high

(490.0 ng/mL) concentrations. All the samples were stored in -80°C freezer until

analysis.

5.3 Method Development

The goal of this research is to develop and validate a simple, selective,

sensitive, rapid, rugged and reproducible assay method for the quantitative

determination of Valacyclovir from plasma samples. In the way to develop a simple

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and easy applicable method for Valacyclovir assay in human plasma for

pharmacokinetic study, HPLC with MS/MS detection was selected as the method of

choice.

Mass parameter Optimization,Chromatographic Optimization and Extraction

optimization to be optimized carefully to achieve the best results.

The MS optimization was performed by direct infusion of solutions of both

Valacyclovir and Valacyclovir-D8 into the ESI source of the mass spectrometer. Other

parameters, such as the nebulizer and the heater gases and Declustering potential(DP),

Entrance potential(EP),Collision energy(CE) was optimized to obtain a better spray

shape, resulting in better ionization and droplet drying to form the protonated ionic

Valacyclovir and Valacyclovir- D8 molecules.

A CAD product ion spectrum for Valacyclovir and Valacyclovir- D8 yielded

high-abundance fragment ions of m/z (amu) 152.0 and m/z (amu) 152.0 respectively

Shown in Figure 5.2 and Figure 5.3.

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Figure 5.2 Parent and Product ion mass spectra of Valacyclovir

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Figure 5.3 Parent and Product ion mass spectra of Valacyclovir-D8

Chromatographic conditions, especially, selection of column, composition and

nature of the mobile phase were optimized through several trials to achieve best

resolution and increase the signal of Valacyclovir and Valacyclovir- D8. Separation

was tried using various combinations of mobile phase with variety of columns like

YMC Pack pro C18, RP-Amide, Ascentis Express RP-amide, X-Bridge, Discovery

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Cyano, Kromasil 100- 5CN. After the MRM channels were tuned, the mobile phase

was changed from more aqueous phase to organic phase to obtain a fast and selective

LC method. A good separation and elution were achieved using 10 mM ammonium

formate (pH 5.0): methanol (80:20 v/v) as the mobile phase, at a flow-rate of

0.25 mL/ min and injection volume of 5 µL. Chromatographic analysis of the analyte

and IS was initiated under isocratic conditions with an aim to develop a simple

separation process with a short run time.

Extraction was performed by different extraction techniques like SPE, LLE,

Precipitation methods. Finally a simple SPE technique was selected in the extraction

of Valacyclovir and Valacyclovir - D8 from the plasma samples.

Chromatographic conditions

Chromatographic separation was carried out on a reversed phase Zorbax, SB

C18, 4.6 x 75mm, 3.5 m column using a mixture of 10mM ammonium formate buffer

(PH 5) and methanol (80:20 v/v) as mobile phase with a flow-rate of 0.25 mL/min.

The column temperature was set at 45°C. Retention time of Valacyclovir and

Valacyclovir-D8 was found to be approximately 4.4 ± 0.2 min for both drug and IS.

Sample preparation

A Solid phase extraction procedure was used for extraction of drug and IS

from the plasma samples. For this purpose, 50µL of Valacyclovir- D8 (200ng/mL),

200 µL plasma ( respective concentration of plasma sample) was added into ria vials

then vortexed for 30 seconds followed by 200 µL of acetic acid solution was added

and vortexed briefly. SPE cartridges (Water Oasis, MCX LP, 3 cc, 60 mg) were

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conditioned with 2 mL of dichloromethane, 1.5 mL of 2.5% NH4OH in methanol.

After that, samples from ria vials were loaded the onto SPE cartridge. Wash the

cartridges with 1.5 mL of water followed by 1.5 mL of methanol. Allowed to dry the

cartridge then eluted cartridges with 1.5 mL of 2.5% NH4OH in methanol into

prelabled ria vials. These samples were evaporated to dryness under the nitrogen

stream at 40°C. Finally, the residue was reconstituted with 400 µL of reconstitution

solution (10mM ammonium formate (PH 3.1): methanol 80:20) and vortexed briefly.

Then the samples were transferred into auto sampler vials injected into the

LC-MS/MS system.

Calibration curve parameters and regression model

The analytical curves of Valacyclovir were constructed in the concentrations

ranging from 0.5- 700.0 ng/mL in human plasma. Calibration curves were obtained by

weighted linear regression (weighing factor: 1/x2). The ratio of Valacyclovir peak area

to Valacyclovir-D8 peak area was plotted against the ratio of Valacyclovir

concentration in ng/mL. The fitness of calibration curve was confirmed by

back-calculating the concentrations of calibration standards.

Method Development Conclusion

The developed method is suitable for estimation of plasma concentrations for

Valacyclovir as a single analytical run, in clinical samples from Pharmacokinetic

studies. This was followed by method validation.

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5.4 Method Validation

The objective of the work is to validate specific HPLC- MS method for the

determination of Valacyclovir in human plasma for clinical / Pharmacokinetic study.

Chromatography

Representative chromatograms of Plasma blank, blank +IS, LOQ, ULOQ,

LLOQC, LQC, MQC, HQC, Calibration curve are shown in Figure 5.6 to 5.14.

Figure 5.4 MRM Chromatogram of Blank Human Plasma Sample

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Figure 5.5 Chromatogram of Blank + IS

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Figure 5.6 Chromatogram of LOQ Sample (Valacyclovir & IS)

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Figure 5.7 Chromatogram of ULOQ Sample (Valacyclovir & IS)

135


Figure 5.8 Chromatogram of LLOQ Sample (Valacyclovir & IS)

136


Figure 5.9 Chromatogram of LQC Sample (Valacyclovir & IS)

137


Figure 5.10 Chromatogram of MQC Sample (Valacyclovir & IS)

138


Figure 5.11 Chromatogram of HQC Sample (Valacyclovir & IS)

Figure 5.12 Calibration Curve of Valacyclovir

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Blank Matrix Screening

During validation, blank plasma samples from 10 different lots were processed

according to the extraction procedure and evaluate the interference at the retention

times of analyte and internal standard. The 6 free interference lots were selected from

the 10 lots. Results are presented in Table 5.3.

Table 5.3 Screening of Different batches of blank matrix

(Human K2EDTA Plasma) for interference free Valacyclovir blank plasma

Matrix identification

Blank plasma Area

Analyte

(Valacyclovir)

RT

Internal standard

RT

AP/3451/09/10 0 0

AP/3452/09/10 0 0

AP/3453/09/10 27 0

AP/3454/09/10 0 0

AP/3455/09/10 35 0

AP/3456/09/10 0 0

AP/3457/09/10 28 0

AP/3458/09/10 0 0

AP/3459/09/10 0 0

AP/3460/09/10 0 0

Blank +IS with AP/3451/09/10 0 557914

LOQ with AP/3451/09/10 4932 551115

Blank Matrix Specificity and Limit of Quantification

During specificity run, the LLOQ standard was prepared in one of the

screened blank plasma including the spiking of working range of internal standard.

Blank plasma samples from 10 different lots, 6 LLOQ standards were processed

according to the extraction procedure. The responses for the blank plasma from 10

different lots were compared to the LLOQ standard of the analyte and internal

140


standard. No significant response (≤ 20% for the analyte response and ≤ 5% of the

internal standard response) was observed at the retention times of the analyte or the

internal standard in blank plasma as compared to the LLOQ standard. Results are

presented in Table 5.5

The specificity experiment shall be considered for calculation of LOQ

experiment. Results are presented in Table 5.4

Table 5.4 Specificity of Different batches of blank matrix

(Human K2EDTA Plasma) for Valacyclovir

Matrix

Identification

LLOQ

Area

Internal

standard

(IS) area

Interference with

Analyte(% of

LLOQ Response)

Interference

with IS(% of IS

Response)

AP/3451/09/10 4930 551118 0 0

AP/3452/09/10 4876 568732 0 0

AP/3454/09/10 4798 563455 0 0

AP/3456/09/10 4689 558974 0 0

AP/3458/09/10 4768 557687 0 0

AP/3459/09/10 4812 568794 0 0

Acceptance criteria:

1. Analyte response should be ≤ 20% of LOQ Response in at least 75% of the

blank.

2. Internal standard response should be ≤ 5% of mean internal standard response

in at least 75% of the blank.

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Table 5.5 Limit of Quantitation for analyte (Valacyclovir)

Matrix identification Blank plasma area at

Analyte RT

LLOQ

response

LLOQ S/N

RATIO

AP/3451/09/10

0 4932 17.4

0 4836 15.9

0 4860 15.2

0 4734 17.8

0 4677 17.4

0 4505 13.6

N 6 6 6

Mean 0 4757 16.2

LLOQ was spiked in -AP/3451/09/10


1. Mean S/N ratio of LLOQ should be ≥ 5.

2. S/N ratio is analyst software generated data.

Intra Batch Accuracy and precision

Intra batch accuracy and precision evaluation were assessed by analyzing

1 calibration curve and 6 replicate each of the LLOQ, LQC, MQC, HQC, from

precision and accuracy batch-1.

The Intra batch percentage of nominal concentrations for Valacyclovir was

ranged between 94.43% and 97.86%.

The Intra batch percentage of coefficient of variation is 0.74% to 4.09% for

Valacyclovir.

Results are presented in Table 5.6

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Table 5.6 Intra batch (Within-Batch) Accuracy and Precision for determination

of Valacyclovir levels in human plasma

Analytical

Run ID

LLOQ

0.50 ng/mL

Low QC

1.50 ng/mL

Mid QC

210.00 ng/mL

High QC

490.00 ng/mL

Conc.

found

%

nominal

Conc.

found

%

nominal

Conc.

found

%

nominal

Conc.

found

%

nominal

P&A Batch 1

0.49 97.00 1.43 95.33 206.00 98.10 478.00 97.55 0.47 94.20 1.43 95.33 204.00 97.14 482.00 98.37 0.47 94.80 1.55 103.33 208.00 99.05 476.00 97.14 0.48 96.00 1.42 94.67 206.00 98.10 472.00 96.33 0.49 97.60 1.44 96.00 204.00 97.14 486.00 99.18 0.44 87.00 1.44 96.00 205.00 97.62 478.00 97.55

N 6

6

6

6

Mean 0.47 1.45 205.50 478.67

SD (±) 0.02 0.05 1.52 4.84

CV (%) 4.09 3.36 0.74 1.01

%Accuracy 94.43

96.78

97.86

97.69


1. % CV ≤ 15 % except LLOQ for which it is ≤ 20%.

2. Mean % Nominal (100 ±15% and for LLOQ 100±20%).

Inter Batch Accuracy and Precision

Inter batch accuracy and precision evaluation were assessed by analyzing 5

sets of calibration curves for Valacyclovir and 5 sets of QC samples, 6 replicates each

of the LLOQ, LQC, MQC and HQC.

The inter batch percentage of nominal concentrations for Valacyclovir was

ranged between 95.07% and 102.15%.

The Inter batch percentage of coefficient of variation is 3.29% to 7.80% for

Valacyclovir.


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Table 5.7 Inter batch (Between-Batch) Accuracy and Precision for determination

of Valacyclovir levels in human plasma

Analytical

Run ID

LLOQ

0.50 ng/mL

Low QC

1.50 ng/mL

Mid QC

210.00 ng/mL

High QC

490.00 ng/mL

Conc.

found

%

nominal

Conc.

found

%

nominal

Conc.

found

%

nominal

Conc.

found

%

nominal

P&A

Batch 1

0.49 97.00 1.43 95.33 206.00 98.10 478.00 97.55

0.47 94.20 1.43 95.33 204.00 97.14 482.00 98.37

0.47 94.80 1.55 103.33 208.00 99.05 476.00 97.14

0.48 96.00 1.42 94.67 206.00 98.10 472.00 96.33

0.49 97.60 1.44 96.00 204.00 97.14 486.00 99.18

0.44 87.00 1.44 96.00 205.00 97.62 478.00 97.55

P&A

Batch 2

0.47 94.40 1.40 93.33 199.00 94.76 458.00 93.47

0.49 97.00 1.41 94.00 185.00 88.10 426.00 86.94

0.47 93.60 1.45 96.67 192.00 91.43 462.00 94.29

0.46 92.60 1.33 88.67 202.00 96.19 452.00 92.24

0.47 94.20 1.37 91.33 199.00 94.76 424.00 86.53

0.47 93.80 1.49 99.33 203.00 96.67 428.00 87.35

P&A

Batch 3

0.54 107.20 1.66 110.67 216.00 102.86 497.00 101.43

0.56 111.60 1.53 102.00 201.00 95.71 458.00 93.47

0.56 111.60 1.49 99.33 203.00 96.67 456.00 93.06

0.55 109.40 1.49 99.33 202.00 96.19 476.00 97.14

0.53 105.60 1.48 98.67 210.00 100.00 470.00 95.92

0.58 116.60 1.46 97.33 203.00 96.67 470.00 95.92

P&A

Batch 4

0.50 100.20 1.47 98.00 189.00 90.00 429.00 87.55

0.49 98.60 1.38 92.00 199.00 94.76 418.00 85.31

0.53 105.40 1.46 97.33 202.00 96.19 457.00 93.27

0.53 106.40 1.55 103.33 212.00 100.95 469.00 95.71

0.53 106.00 1.51 100.67 212.00 100.95 482.00 98.37

0.52 103.40 1.51 100.67 199.00 94.76 466.00 95.10

P&A

Batch 5

0.54 107.60 1.55 103.33 211.00 100.48 504.00 102.86

0.50 99.00 1.38 92.00 202.00 96.19 486.00 99.18

0.59 118.40 1.40 93.33 196.00 93.33 464.00 94.69

0.50 100.00 1.43 95.33 201.00 95.71 478.00 97.55

0.56 112.40 1.42 94.67 202.00 96.19 481.00 98.16

0.56 112.80 1.40 93.33 197.00 93.81 493.00 100.61

N 30

30

30

30

Mean 0.51 1.46 202.33 465.87 SD(±) 0.04 0.07 6.65 22.27 % CV 7.80 4.68 3.29 4.78

%Nominal 102.15

97.18

96.35

95.07

Acceptance criteria: Same as presented in Table 5.6

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Calibration Curve

Calibration curves are found to be consistently accurate and precise for

Valacyclovir over 0.50 -700.00 ng/mL for calibration range. The correlation

coefficient is greater than 0.9988 for Valacyclovir. Back calculations were made from

the calibration curves to determine Valacyclovir concentrations of each calibration

standard.

Results are presented in Tables 5.8 & 5.9

Table 5.8 Summary of calibration curve parameters for Valacyclovir in human

plasma

Analytical Run ID A B C

Coefficient of

regression

(r2)

P&A Batch-1 -0.000001537 0.02206 0.0004753 0.9980

P&A Batch-2 -0.000004956 0.02394 -0.0003083 0.9994

P&A Batch-3 -0.000002361 0.02194 -0.0009788 0.9994

P&A Batch-4 -0.000002326 0.02453 -0.0002957 0.9987

P&A Batch-5 -0.000001149 0.02193 0.00008181 0.9989

N 5 5 5

Mean -000002.4658 0.02288 -0.00021 0.9988

SD (±) 000001.48562 0.001255 0.000539 0.00058

CV (%) -60.24 5.48 -262.82 0.05

Regression model y = ax2 +bx+c

where:

y = peak area ratio (PAR) of valacyclovir to internal standard.

x = concentration (ng/mL) of valacyclovir in plasma.


1. Coefficient of regression (r) ≥ 0.9980.

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Table 5.9 Back-calculated standard concentrations from each calibration curve

for Valacyclovir in human plasma.

Analytical Run

ID

Nominal Concentration (ng/mL)

CS1 CS2 CS3 CS4 CS5

0.50

ng/mL

1.00

ng/mL

5.00

ng/mL

35.00

ng/mL

70.00

ng/mL

P&A Batch-1 0.52 0.91 4.81 37.30 70.70

P&A Batch-2 0.51 0.99 4.77 36.50 70.90

P&A Batch-3 0.51 0.96 4.90 35.30 72.40

P&A Batch-4 0.51 0.98 4.75 35.40 72.20

P&A Batch-5 0.50 1.02 4.74 37.10 69.80

N 5 5 5 5 5

Mean 0.51 0.97 4.79 36.32 71.20

SD(±) 0.01 0.04 0.07 0.93 1.09

CV% 1.87 4.05 1.36 2.57 1.53

%Nominal 101.72 97.26 95.88 103.77 101.71

Analytical run

ID

Nominal Concentration (ng/mL)

CS6 CS7 CS8 CS9 CS10

140.00

ng/mL

280.00

ng/mL

420.00

ng/mL

560.00

ng/mL

700.00

ng/mL

P&A Batch-1 141.00 282.00 419.00 556.00 700.00

P&A Batch-2 139.00 284.00 416.00 556.00 702.00

P&A Batch-3 138.00 285.00 426.00 555.00 693.00

P&A Batch-4 147.00 280.00 395.00 561.00 715.00

P&A Batch-5 141.00 269.00 414.00 571.00 704.00

N 5 5 5 5 5

Mean 141.20 280.00 414.00 559.80 702.80

SD(±) 3.49 6.44 11.55 6.69 7.98

CV% 2.47 2.30 2.79 1.19 1.14

%Nominal 100.86 100.00 98.57 99.96 100.40

Acceptance criteria

1. Mean % Nominal (100±15%) except lowest calibration standard.

2. Mean % Nominal (100±20%) for lowest calibration standard (CS1).

3. % CV ≤ 15% except lowest calibration standard (CS1) for which it is ≤ 20%.

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Recovery

The percentage recovery of Valacyclovir was determined by comparing the

mean peak area of Valacyclovir in extracted LQC, MQC, HQC samples with freshly

prepared unextracted LQC, MQC, HQC samples respectively.

The mean % recovery for LQC, MQC, HQC samples of Valacyclovir were

88.48%, 105.88% and 103.15% respectively.

The mean recovery of Valacyclovir across QC levels is 99.17%.

The mean recovery of % CV recovery of Valacyclovir across QC levels is

10.9%.

For the internal standard, mean peak area of 18 extracted samples was

compared to the mean peak area of 18 unextracted IS solution. The mean %

recovery is 110.84%.

The % CV recovery of IS Valacyclovir- D8 for extracted is 7.9%.


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Table 5.10 Recovery of Analyte Valacyclovir and Valacyclovir-D8 from human

plasma

Standard

Extracted peak

response

Unextracted peak

response

Drug IS Drug IS

Low QC: 1.50 ng/mL

21804 614170 27316 729049

19882 608307 23875 655807

22415 708869 22084 599935

24321 769709 24652 657996

18076 575189 22374 628841

20168 651502 22846 630215

N 6 6 6 6

Mean 88.48

SD (±) 9.193

%CV 10.4

Medium QC: 210.00 ng/mL

3082852 666064 3005197 619888

2873999 667969 2830170 580764

2700670 619936 2357070 480503

3164543 729545 3024239 607338

2813385 623809 2504035 506765

2500981 575139 2460142 505789

N 6 6 6 6

Mean 105.88

SD(±) 9.071

%CV 8.6

High QC: 490.00 ng/mL

6632999 643479 6561725 603186

6093578 637962 5900211 535684

6016152 632263 5769571 514291

6211171 626532 6337750 572815

6070756 620561 5607332 504914

5914901 604012 5636899 509386

N 6 6 6 6

Mean 103.15

SD(±) 4.225

%CV 4.1

Drug IS

Mean recovery of across QC levels 99.17 110.84

Mean SD(±) of across QC levels 10.77 8.742

The Mean % CV across QC levels 10.9 7.9


1. The coefficient of variation for mean recovery across LQC, MQC and HQC

shall not exceed 25%.

2. The coefficient of variation for mean recovery of IS shall not exceed 25%.

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Matrix Effect

Samples were prepared at LQC & HQC level in triplicate in each of 6 different

lots of human plasma. A calibration curve and 6 replicates of LQC & HQC samples in

triplicate for each matrix were freshly prepared and analyzed in single run.

No significant matrix effect found in different sources of human plasma tested

for Valacyclovir, Valacyclovir- D8.

Results are presented in Tables 5.11 and 5.12.

Table 5.11 Assessment of Matrix Effect on determination of Valacyclovir

at LQC levels in human plasma

Identification of

matrix

Drug response

in Matrix at

LQC Level

Internal

standard

response

Matrix factor

AP/3451/09/10 4866 404298 0.011 AP/3451/09/10 4917 407166 0.012

AP/3451/09/10 5614 446360 0.0121 AP/3452/09/10 6080 457956 0.0126 AP/3452/09/10 6903 541129 0.0133

AP/3452/09/10 5836 428604 0.0128 AP/3453/09/10 6100 434136 0.0136 AP/3453/09/10 6634 450758 0.0141

AP/3453/09/10 6793 483352 0.0147 AP/3454/09/10 6848 454347 0.0141

AP/3454/09/10 6771 449004 0.0151 AP/3454/09/10 6781 442115 0.0151 AP/3455/09/10 6061 425255 0.0153 AP/3455/09/10 6255 415656 0.0143

AP/3455/09/10 6465 423037 0.015 AP/3456/09/10 5907 406537 0.0153

AP/3456/09/10 6285 425181 0.0145 AP/3456/09/10 4866 404298 0.0148

N 18 18 18

Grand Mean 0.013872 SD(±) 0.0013 % CV 9.36


1. Mean % Nominal 100±15% of nominal value.

2. % CV ≤ 15%.

149


Table 5.12 Assessment of Matrix Effect on determination of Valacyclovir

at HQC levels in human plasma

Identification of matrix Drug response

in Matrix at

HQC Level

Internal

standard

response

Matrix factor

AP/3451/09/10 4785575 368086 13.0012

AP/3451/09/10 4841203 364895 13.2674

AP/3451/09/10 4935020 373153 13.2252

AP/3452/09/10 5219207 393230 13.2727

AP/3452/09/10 5629933 415291 13.5566

AP/3452/09/10 5456931 404696 13.484

AP/3453/09/10 5464913 403621 13.5397

AP/3453/09/10 5544012 401873 13.7954

AP/3453/09/10 5191929 383555 13.5363

AP/3454/09/10 5464629 396315 13.7886

AP/3454/09/10 5427333 397085 13.6679

AP/3454/09/10 5489749 400656 13.7019

AP/3455/09/10 5231784 384228 13.6164

AP/3455/09/10 4965675 369029 13.456

AP/3455/09/10 5052930 371975 13.5841

AP/3456/09/10 5468764 392570 13.9307

AP/3456/09/10 5360425 391526 13.6911

AP/3456/09/10 5422345 390640 13.8807

N 18 18 18

Grand Mean 13.55533 SD(±) 0.2438 % CV 1.80


1. Mean % Nominal 100 ±15% of nominal value.

2. % CV ≤ 15%.

150


Dilution Integrity

Dilution integrity experiment was carried out at six replicate of two times

diluted (1 in 2 dilution) and four times diluted of approx 1.5 × ULOQ (1 in 4 dilution)

samples were prepared and concentrations were calculated including the dilution

factor against the freshly prepared calibration curve.

The % accuracy of Valacyclovir nominal concentrations ranged between

90.50% and 92.43% for 1 in 2 dilutions and 1 in 4 dilutions respectively.

The % CV is 4.29% to 4.59%.

Results are presented in Table 5.13.

Table 5.13 Assessment of Dilution integrity for Valacyclovir

at DQC Conc (ng/mL)

DQC

Dilution factor: ½

Nominal conc: 1125.00 ng/mL

DQC

Dilution factor: ¼

Nominal conc: 1125.00 ng/mL

Conc. Found % Nominal Conc. Found %Nominal

1090.00 96.89 1090.00 96.89

986.00 87.64 1050.00 93.33

1040.00 92.44 1040.00 92.44

976.00 86.76 1050.00 93.33

1030.00 91.56 949.00 84.36

987.00 87.73 1060.00 94.22

N 6

6

Mean

%Nominal 90.50 92.43

SD (±) 43.73 47.71

CV (%) 4.29 4.59


1. % CV ≤ 15%.

2. Mean % Nominal (100 ±15%).

151


Whole Batch Reinjection Reproducibility

To evaluate the whole batch reinjection reproducibility experiment, samples of

P & A batch-2 were kept at auto sampler temperature for approx. 26 hrs after the

initial analysis and were re-injected again after approx. 26 hrs. Concentrations were

calculated to determine precision and accuracy after reinjection.

The Accuracy of Valacyclovir QC samples in reinjection was between

100.23% and 106.84%.

The Precision (% CV) of Valacyclovir QC samples in reinjection was between

3.27 % and 5.47%.

Valacyclovir was found to be stable at autosampler temperature post

extraction (in reconstitution solution) for approx. 26 hrs and reproducible after

reinjection.


Table 5.14 Assessment of Whole Batch Re-injection Reproducibility during

estimation of Valacyclovir in human plasma

Analytical

Run ID

Low QC 1.50 ng/mL High QC 490.0 ng/mL

Comp sample Reinjection

sample Comp sample

Reinjection

sample

1.47 1.53 429 516 1.38 1.45 418 486 1.46 1.55 457 479 1.55 1.43 469 464 1.51 1.45 482 488 1.51 1.49 466 474

N 6 6 6 6

Mean 1.48 1.48 453.50 484.50 SD(±) 0.06 0.05 24.83 17.71 %CV 3.96 3.27 5.47 3.65

%NOM 100.23 106.84


1. % CV≤ 15% Except LLOQ for which it is ≤ 20%.

2. Mean % Nominal (100 ±15% and for LLOQ 100 ±20%).

3. 67% 0f the re-injected QCs at each level shall be within ± 20% of their previous

concentration.

152


Ruggedness-Different Analyst

To evaluate ruggedness experiment with different analysts, one P&A batch

(P&A-3) was processed by different analyst. The run consisted of a calibration curve

standards and 6 replicates of each LLOQ, LQC, MQC, HQC samples.

The Accuracy of Valacyclovir QC samples within the range of 98.43% to

116.90%.

The Precision of Valacyclovir QC samples within the range of 0.74% to

4.81%.

These results indicated that the method is rugged and reproducible by different

analyst.


Table 5.15 Ruggedness of the method for estimation of Valacyclovir Plasma

levels in human plasma with different Analyst.

Analytical

Run ID

LLOQ

0.50 ng/mL

Low QC

1.50 ng/mL

Mid QC

210.00 ng/mL

High QC

490.00 ng/mL

Analyst

ID 1

Analyst

ID 2

Analyst

ID 1

Analyst

ID 2

Analyst

ID 1

Analyst

ID 2

Analyst

ID 1

Analyst

ID 2

P&A Batch

3

0.49 0.54 1.43 1.66 206.00 216.00 478.00 497.00

0.47 0.56 1.43 1.53 204.00 201.00 482.00 458.00

0.47 0.56 1.55 1.49 208.00 203.00 476.00 456.00

0.48 0.55 1.42 1.49 206.00 202.00 472.00 476.00

0.49 0.53 1.44 1.48 204.00 210.00 486.00 470.00

0.44 0.58 1.44 1.46 205.00 203.00 478.00 470.00

N 6 6 6 6 6 6 6 6

Mean 0.47 0.55 1.45 1.52 205.50 205.83 478.67 471.17

SD (±) 0.02 0.02 0.05 0.07 1.52 5.91 4.84 14.81

CV (%) 3.93 3.16 3.36 4.81 0.74 2.87 1.01 3.14

%Accuracy 116.90

104.59

100.16

98.43



2. Mean % Nominal (100±15% & for LLOQ 100 ±20%).

153


Ruggedness-Different Column

To evaluate ruggedness experiment with different column, samples of P&A

batch-5 were reinjected on different columns with same and specifications,

Concentrations were calculated to determine precision and accuracy.

The Accuracy of Valacyclovir QC samples within the range of 98.05% to

114.44%.

The Precision of Valacyclovir QC samples within the range of 0.74% to

6.65%.

These results indicated that the method is rugged and reproducible by different

analyst.


Table 5.16 Ruggedness of the method for estimation of Valacyclovir Plasma

levels in human plasma with different Analytical column

Analytical

Run ID

LLOQ

0.50 ng/mL

Low QC

1.50 ng/mL

Mid QC

210.00 ng/mL

High QC

490.00 ng/mL

Column

ID

LC/312

Column

ID

LC/345

Column

ID

LC/312

Column

ID

LC/345

Column

ID

LC/312

Column

ID

LC/345

Column

ID

LC/312

Column

ID

LC/345

P&A Batch

5

0.49 0.54 1.43 1.55 206.00 211.00 478.00 504.00

0.47 0.50 1.43 1.38 204.00 202.00 482.00 486.00

0.47 0.59 1.55 1.40 208.00 196.00 476.00 464.00

0.48 0.50 1.42 1.43 206.00 201.00 472.00 478.00

0.49 0.56 1.44 1.42 204.00 202.00 486.00 481.00

0.44 0.56 1.44 1.40 205.00 197.00 478.00 493.00

N 6 6 6 6 6 6 6 6

Mean 0.47 0.54 1.45 1.43 205.50 201.50 478.67 484.33

SD(±) 0.02 0.04 0.05 0.06 1.52 5.32 4.84 13.63

CV (%) 3.93 6.65 3.36 4.29 0.74 2.64 1.01 2.81

%Accuracy 114.44

98.51

98.05

101.18



2. Mean % Nominal (100±15% & for LLOQ 100 ±20%).

154


Bench Top Stability (at room temp for 41.0 hrs)

Spiked LQC and HQC samples were retrieved from deep freezer and were kept at

room temperature for 41.0 hrs and were processed and analyzed along with freshly prepared

calibration standards, comparison LQC and HQC samples. Concentrations were calculated to

determine mean % change during stability period.

The mean Accuracy for LQC & HQC samples of Valacyclovir from

comparison samples were 96.55% and 97.48% respectively.

The plasma samples of Valacyclovir were found to be stable for

approximately 41.0 hrs min at room temperature.

Results are present in Table 5.17

Table 5.17 Assessment of stability of Analyte (Valacyclovir) in Biological matrix

at Room temperature


Comparison

samples

(0.00 hr)

Stability samples

(41.0 hrs)

Comparison

samples

(0.00 hr)

Stability samples

(41.0 hrs)

Conc.

found

%

nominal

Conc.

found

%

nominal

Conc.

found

%

nominal

Conc.

found

%

nominal

1.49 99.33 1.46 97.33 521.00 106.33 485.00 98.98 1.36 90.67 1.34 89.33 501.00 102.24 479.00 97.76 1.44 96.00 1.58 105.33 490.00 100.00 451.00 92.04 1.43 95.33 1.45 96.67 511.00 104.29 476.00 97.14 1.47 98.00 1.43 95.33 512.00 104.49 483.00 98.57 1.38 92.00 1.43 95.33 504.00 102.86 492.00 100.41

N 6

6

6

6

Mean 1.43 1.45 506.50 477.67 SD(±) 0.05 0.08 10.67 14.17

CV (%) 3.53 5.34 2.11 2.97 %Accuracy 95.22 96.55 103.37 97.48


1. % Ratio (stability/comparison) should be within 85-115 %.

2. %CV ≤ 15%.

3. Mean % Nominal (100 ±15%).

155


Freeze and Thaw Stability (after 3rd

cycle at -30°C)

Samples were prepared at LQC and HQC levels, aliquoted and frozen at -

30±5°C six samples from each concentration were subjected to three freeze and thaw

cycles (stability samples). These samples were processed and analyzed along with

freshly prepared calibration standards, LQC and HQC samples (comparison samples).

Concentrations were calculated to determine mean % change after 3 cycles.

The mean Accuracy for LQC & HQC samples of Valacyclovir from


The plasma samples of Valacyclovir were found to be stable after 3 cycles at -

30 ±5°C.


Table 5.18 Assessment of Freeze-Thaw stability of Analyte (Valacyclovir)

at -30±5°C


Comparison

samples

Stability sample

at 4th

cycle

Comparison

samples

Stability sample at

4th

cycle

Conc.

found

%

nominal

Conc.

found

%

nominal

Conc.

found

%

nominal

Conc.

found

%

nominal

1.49 99.33 1.45 96.67 521 106.33 486 99.18 1.36 90.67 1.36 90.67 501 102.24 470 95.92 1.44 96.00 1.4 93.33 490 100.00 467 95.31 1.43 95.33 1.49 99.33 511 104.29 475 96.94 1.47 98.00 1.48 98.67 512 104.49 499 101.84 1.38 92.00 1.39 92.67 504 102.86 495 101.02

N 6

6

6

6

Mean 1.43 1.43 506.50 482.00 SD(±) 0.05 0.05 10.67 13.36

CV (%) 3.53 3.69 2.11 2.77 %Accuracy

95.22 95.22 103.37 98.37

Acceptance criteria

Same as presented in Table 5.17

156


Autosampler stability at 2-8°C in autosampler

LQC and HQC samples were prepared and processed. These processed

samples were analyzed and kept in auto sampler for 79 hrs at 2-8°C and analyzed

along with freshly prepared calibration standard samples. Concentrations were

calculated to determine mean % change during stability period.

The mean Accuracy change for LQC & HQC samples of Valacyclovir from


Valacyclovir samples were stable for 79 hrs at 2-8°C in autosampler.


Table 5.19 Assessment of Autosampler stability of Analyte (Valacyclovir)

at 2-8°C


Comparison

samples (0.0 hr)

Stability samples

(79 hr)

Comparison

samples (0.0 hr)

Stability samples

(79 hr)

Conc.

found

%

nominal

Conc.

found

%

nominal

Conc.

found

%

nominal

Conc.

found

%

nominal

1.49 99.33 1.47 98.00 521 106.33 517 105.51 1.36 90.67 1.43 95.33 501 102.24 480 97.96 1.44 96.00 1.47 98.00 490 100.00 463 94.49 1.43 95.33 1.48 98.67 511 104.29 472 96.33 1.47 98.00 1.46 97.33 512 104.49 487 99.39 1.38 92.00 1.41 94.00 504 102.86 494 100.82

N 6

6

6

6

Mean 1.43 1.45 506.50 485.50

SD(±) 0.05 0.03 10.67 18.90

CV (%) 3.53 1.88 2.11 3.89

%Accuracy 95.22 96.89 103.37 99.08


157


Long-term stability (at -30°C temp for 55 days)

Spiked LQC and HQC samples were retrieved from deep freezer after 34 days

and were processed and analyzed along with freshly prepared calibration standards,

comparison LQC and HQC samples. Concentrations were calculated to determine

mean % change during stability period.

The mean Accuracy for LQC and HQC samples of Valacyclovir from


The plasma samples of Valacyclovir were found to be stable for

approximately 34 days at -30°C temp.


Table 5.20 Assessment of Long term plasma stability of Analyte

(Valacyclovir) at -30°C.

Low QC 1.50 ng/ml High QC 490.0 ng/ml Comparison

samples (0.0 hr)

Stability samples

(34 days)

Comparison samples

(0.0 hr)

Stability samples

(34 days)

Conc.

found

%

nominal

Conc.

found

%

nominal

Conc.

found

%

nominal

Conc.

found

%

nominal

1.49 99.33 1.5 100.00 521 106.33 506 103.27 1.36 90.67 1.38 92.00 501 102.24 496 101.22 1.44 96.00 1.41 94.00 490 100.00 481 98.16 1.43 95.33 1.38 92.00 511 104.29 498 101.63 1.47 98.00 1.42 94.67 512 104.49 504 102.86 1.38 92.00 1.33 88.67 504 102.86 500 102.04

N 6

6

6

6

Mean 1.43 1.40 506.50 497.50 SD(±) 0.05 0.06 10.67 8.89 CV (%) 3.53 4.05 2.11 1.79 %Accuracy 95.22 93.56 103.37 101.53


158


Short Term Stock Solution Stability of Valacyclovir, and Valacyclovir- D8 at

Room Temperature

Stock solution stability was determined by comparing the peak areas of freshly

prepared stock solutions (comparison samples) with stability stock solutions. Main

Stock solutions of Valacyclovir and Valacyclovir-D8 were freshly prepared and

aliquots of stocks were kept at room temperature for 9.0 hr (stability samples).

Aqueous equivalent highest calibration standard of Valacyclovir and solution of

Valacyclovir- D8 were prepared from the stability samples and analyzed. Areas of

stability samples and freshly prepared samples were compared to determine mean %

change during stability period.

The % CV for of Valacyclovir stock solution from comparison samples was

0.86% and % Ratio (stability/comparison) was 101.25

The % CV for of Valacyclovir- D8 stock solution from comparison samples

was 1.06% and % Ratio (stability/comparison) was 100.08

The % CV for Valacyclovir- D8 working solution (Internal standard spiking

solution) from comparison samples was 1.86% and % Ratio (stability/

comparison) was 101.57

Valacyclovir, Valacyclovir- D8 stock solutions and Valacyclovir- D8 spiking

solutions were found to be stable at room temperature for 9.0 hr.

Results are present in Tables 5.21 and 5.22

159


Table 5.21 Assessment of Short term stock solution stability of Analyte

(Valacyclovir) and Internal standard (Valacyclovir- D8) at Room temperature

Analyte Internal standard Comparison

Standard stock

solution response

(0.0 hr)

Stability stock

solution response

(9.0 hr)

Comparison stock

solution response

(0.0 hr)

Stability

Standard stock

Response

(9.0 hr)

1125523 1164383 247074 242627 1148859 1161817 238708 245090 1140661 1158323 244735 249679 1157040 1136488 243203 243594 1112872 1152873 240936 243848 1160514 1157245 240507 243193

N 6 6 6 6

Mean 1140911.50 1155188.17 242527.17 244671.83

SD (±) 18604.47 9975.97 3072.58 2586.71

CV (%) 1.63 0.86 1.27 1.06

%

Ratio

101.25

100.88


1. % change should be ± 15 %

Table 5.22 Assessment of Short term solution stability of internal standard

spiking solution (Valacyclovir- D8) at refrigerated conditions

Comparison solution (Internal

standard Spiking solution)

Response (0.0 hr)

Stability solution (Internal

standard spiking solution)

Response (9.0 hr)

240177 242981 238872 248870 232906 236707 241784 241009 231518 239699 238928 237213

N 6 6

Mean 237364.17 241079.83

SD (±) 4152.57 4478.55

CV (%) 1.75 1.86

% Ratio 101.57


1. % change should be ± 5%

160


Method validation Conclusion

As all the values obtained were within the Acceptance criteria. The method

stands validated and is suitable for estimation of plasma concentrations of

Valacyclovir in a single analytical run. The rugged, efficient Solid phase extraction

method provides exceptional sample clean up and constant recoveries using 200µl of

plasma. The high extraction efficiency, low limit of quantification, and wide linear

dynamic range make this a suitable method for use in clinical samples from

Pharmacokinetic studies following oral administration of Valacyclovir fixed dose

(1000/1000 mg) tablets in healthy human subjects.

5.5 Application

In order to evaluate the practical applicability of the developed method, we used it for

drug analysis throughout a project designed for bioequivalence analysis of Valacyclovir

generic product (Test tablet) with the innovator product. For this purpose, twenty healthy,

non-alcoholic, non-smoking, male volunteers were enrolled in this study. These volunteers

were, contracted in APL Research Pvt.Lt.D, Hyderabad, India. The clinical protocol was

approved by the IEC (Institutional Ethics Committee) as per ICMR (Indian council for

medical research) guidelines. The volunteers gave written informed consent after they had

received detailed instructions about the aims, restrictions and possible adverse effect, which

could be experienced as a result of taking the drug. Volunteers were healthy and had no

history of kidneys and metabolic diseases. Also they had a routine physical examination and

the routine laboratory tests found them to be normal. Subjects did not receive any medication

during the 2 weeks period prior to the start and also were not undergoing any pharmacological

treatment during the study period. The study was an open, randomized, two-period, two-

161


group crossover design over a 7days washout period between doses. The tablets were

administered to the volunteers in the next morning after an overnight fast, with 200 ml of

water. No other food was permitted for consumption during the sampling period. Blood

samples (2 ml) were collected via the catheter into K2EDTA anticoagulant containing tubes at

0, 0.33, 0.5, 0.67, 0.83, 1.0, 1.25, 1.5, 1.75, 2.0, 2.33, 2.67, 3.0, 3.33, 3.67, 4.0, 4.5, 5.0 and

6.0 h post-dosing. The blood samples were centrifuged at 4000 rpm for 10 min and the

plasma was separated stored at -80 °C until assayed for Valacyclovir content 40,41

The Mean Plasma concentration data for 20 volunteers is represented in Table

5.23 with respective concentration-time curve is shown in Figure 5.13.

Table 5.23. Valacyclovir Mean concentration (ng/mL) data for the subject samples obtained from

the LC-MS/MS

Time in hours Mean Plasma Concentration data

Test Reference

0 0 0

0.33 73.38014 81.51986

0.5 104.2249 98.44586

0.67 111.6632 105.0226

0.83 119.4778 105.2104

1 115.1178 114.2283

1.25 131.4125 143.7394

1.5 129.6616 157.2031

1.75 115.9391 139.2379

2 98.02067 125.6938

2.33 77.84638 93.85057

2.67 40.61176 55.72605

3 22.69424 29.17157

3.33 14.01033 16.90043

3.67 10.23967 11.39776

4 7.706762 8.646

4.5 5.779857 5.691524

5 4.134095 4.206143

6 2.475619

2.457048

162


Figure 5.13 Mean plasma Concentration Vs Time curve for Valacyclovir

5.6 Pharmacokinetic Studies

Pharmacokinetic parameters from the human plasma concentration samples

were calculated by a non compartmental statistics model using WinNon-Lin5.0

software (Pharsight, USA). Blood samples were taken for a period of 3 to 5 times of

the terminal elimination half-life (t1/2) and it was considered as area under the

concentration time curve (AUC) ratio higher than 80% as per FDA guidelines8-10

.

Plasma Valacyclovir concentration-time profiles were visually inspected Cmax and

Tmax values were determined. The AUC0–t was obtained by trapezoidal method.

AUC0-∞ was calculated up to the last measureable concentration and extrapolations

were obtained using the last measureable concentration and the terminal elimination

rate constant (Kel). The terminal elimination rate constant (Kel), was estimated from

the slope of the terminal exponential phase of the plasma of Valacyclovir

163


concentration-time curve by means of the linear regression method. The terminal

elimination half-life, t1/2, was then calculated as 0.693/Kel. Regarding AUC0–t, AUC0-∞

and Cmax bioequivalence was assessed by means of analysis of variance (ANOVA)

and calculating the standard 90% confidence intervals (90% CIs) of the ratios

test/reference (logarithmically transformed data). The bioequivalence was considered

when the ratio of averages of log-transformed data was within 80 to 125% for AUC0–t,

AUC0-∞ and Cmax. Pharmacokinetic data is shown in Table 5.24 and Table 5.25.

Table 5.24 Valacyclovir Pharmacokinetic data

Valacyclovir Pharmacokinetic data

Pharmacokinetic

Parameter

Test Reference

Mean±SD Mean±SD

Cmax

(ng/mL ) 157.20 ± 56.09 131.41 ± 51.74

AUC 0-t

(ng h/mL) 322.32 ± 10.85 286.78 ± 12.78

AUC 0-inf

(ng h/mL) 325.78± 8.44 290.89± 10.21

Tmax(hr) 1.5 1.25

t1/2 0.9760 1.125

Table 5.25 Valacyclovir Pharmacokinetic data (Test/Reference)

Pharmacokinetic

Parameter Cmax AUC 0-t

AUC 0-∞

Test/Reference 88.97 89.29

83.59

164


Pharmacokinetic Studies Conclusion

The present study provides firm evidence to support that the in house

Valacyclovir 1000 mg was bioequivalent with Valtrex Tablets (GSK, Australia) 1000

mg tablet under fasting conditions.

In vivo data was predicted by using Solid Phase Extraction procedure and

concentrations were found through Liquid Chromatography Tandem Mass

Spectroscopy detection. The Pharmacokinetic parameters assessed were AUC0-t,

AUC0-, Cmax, Tmax, t1/2. The bioequivalence criteria are based on the 90% confidence

intervals whose acceptance range is in between 80% -125%.

The results obtained for Valacyclovir was within the acceptance range.

Therefore, it can be concluded that the two Valacyclovir formulations (reference and

test) analyzed were bioequivalent in terms of rate and extent of absorption.

m. pharm. (pharmaceutical analysis & quality assurance...

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