simultaneous determination of sitagliptin phosphate...

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CHAPTER-3

Simultaneous determination of Sitagliptinphosphate monohydrate and Metforminhydrochloride in tablets by validated ultraperformance liquid ChromatographyMethod

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3.1 Introduction

Sitagliptin phosphate monohydrate (SP) chemically, (7-[(3R)-3-amino-

1-oxo-4-(2,4,5-trifluorophenyl)butyl]-5,6,7,8-tetrahydro-3-(trifluoro meth-

yl)-1,2,4-triazolo[4,3-a] pyrazine phosphate monohydrate) [1] is an oral

anti-diabetic, which is available in 25 mg, 50 mg and 100 mg tablets for

oral administration. SP is indicated for the improvement of glycemic

control in patients with type II diabetes mellitus as monotherapy or

combination therapy with metformin or a peroxisome proliferator-

activated receptor gamma (PPAR) agonist (e.g., thiazolidinediones) when

the single agent does not provide adequate glycemic control. Sitagliptin

phosphate monohydrate is a white to off white crystalline powder and

soluble in water, slightly soluble in methanol, very slightly soluble in

ethanol, acetone, acetonitrile, insoluble in isopropanol and isoproprl

acetate. The empirical formula of sitagliptin phosphate monohydrate is

C16H15F6N5O.H3PO4.H2O. The molecular weight of sitagliptin phosphate

monohydrate is 523.33.

Metformin hydrochloride (MH) chemically, N,N-dimethylimidodi-

carbonimidic diamide hydrochloride [2] is an antidiabetic agent [3]. It is

first line drug for choice for the treatment of type II diabetes, particularly

in over weight and obese people and those with normal kidney function.

It works by lowering the blood sugar and helping the body to use insulin

more efficiently. It is available in 500 mg, 850 mg and 1000 mg tablets

(immediate release) and in 500 mg, 750 mg (slow release) for oral

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administration. It is a white to off-white crystalline powder and freely

soluble in water and is practically insoluble in acetone, ether, and

chloroform. The empirical formula of metformin hydrochloride is

C4H11N5.HCl. The molecular weight of metformin hydrochloride is 165.63.

Merck and Co. market sitagliptin in combination with metformin in a

single dosage form as JANUMET TM [4]. JANUMET is available for oral

administration as tablets containing 64.25 mg sitagliptin phosphate

monohydrate and metformin hydrochloride equivalent to 50 mg

sitagliptin as free base and 500 mg metformin hydrochloride (JANUMET

50 mg/500 mg) or 1000 mg metformin hydrochloride (JANUMET 50

mg/1000 mg). Each film-coated tablet of JANUMET contains the

following inactive ingredients: microcrystalline cellulose,

polyvinylpyrrolidone, sodium lauryl sulfate, and sodium stearyl

fumarate. In addition, the film coating contains the following inactive

ingredients: polyvinyl alcohol, polyethylene glycol, talc, titanium dioxide,

red iron oxide, and black iron oxide.

Metformin works by decreasing glucose (sugar) production in the liver

and decreasing absorption of glucose by the intestines. Sitagliptin works

by regulating the levels of insulin your body produces after eating.

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Fig. 3.1.F1: Chemical structures of Sitagliptin phosphate andMetformin hydrochloride

(a) Sitagliptin phosphateF

F

F

N

NN

N

CF3

. H3PO4

H NH2O

Molecular formula: C16H15F6N5O.H3PO4

Molecular weight: 505.3

(b) Metformin hydrochloride

Molecular formula: C4H11N5.HCl

Molecular weight: 165.63

The literature reveals that there are some of the methods have been

reported for metformin. Few UV spectrophotometric methods [5-6], HPLC

[7-11] and ion-pair HPLC [12] method have been reported for the

estimation of metformin. SP is not yet official in any of the

pharmacopoeia but MH is official in IP [13], BP [14] and USP NF [15].

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Literature survey reveals that only LC‐MS [16-19] methods were reported

for the determination of sitagliptin phosphate in plasma and urine of

humans, rats and dogs. Literature indicated spectroflourometric and

spectrophotometric methods for the determination of sitagliptin in

pharmaceutical dosage forms [20]. Described the mass spectrometry

method for simultaneous quantitation of metformin and sitagliptin from

mouse and human dried blood spots using laser diode thermal

desorption tandem mass spectrometry [21].

The review of the literature revealed that ultra performance liquid

chromatographic (UPLC) method is not reported for the simultaneous

estimation of the SP and MH in combined pharmaceutical dosage form.

Therefore, it was thought worthwhile to develop a simple, precise,

accurate reverse phase Ultra performance liquid chromatographic

method for the simultaneous estimation of SP and MH in combined

tablet dosage form.

Forced degradation or stress studies of drug substance and products

play an integral role in the development of pharmaceuticals. The results

of degradation studies facilitate the stability indicating method

development. The current ICH guidelines requires that the analysis of

stability samples should be done by using stability-indicating assay

methods (SIAM’s), developed and validated after stress testing on drug

under variety of conditions, including hydrolysis, oxidation, photolysis

and thermal degradation.

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The target is to develop a suitable stability-indicating LC assay

method for simultaneous determination of sitagliptin phosphate and

metformin hydrochloride in combined dosage forms. In this chapter

described a stability indicating UPLC method which can separate

metformin and sitagliptin impurities along with separation of sitagliptin

and metformin.

3.2 Experimental

3.2.1 Materials:

Bulk sample of sitagliptin phosphate monohydrate and metformin

hydrochloride were received from research development department of

Dr. Reddy’s laboratories limited, Hyderabad, India. Commercially

available Janumet [4] tablets manufactured by Merck & Co., Inc., NJ,

USA. Hexane-1- sulfonic acid sodium salt and acetonitrile (HPLC grade)

were purchased from Merck specialties private limited, India. Water was

de-ionized and purified on a Milli-Qwater purification system (Millipore,

Bedford, MA, USA) and used to prepare all solutions.

3.2.2 Instrumentation and Chromatographic Conditions:

The UPLC system was Waters alliance 2695 including pump, auto

sampler and PDA detector 2996. Aquity UPLC BEH C8 (100 x 2.1 mm,

1.7 µm) was used as stationary phase. The mobile phase composition

used was the buffer 10mM potassium dihydrogen phosphate buffer and

2mM hexane-1- sulfonic acid sodium salt (pH adjusted to 5.5 with

diluted phosphoric acid) and acetonitrile with gradient program

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(Time(min)/% acetonitrile): 0/8, 2/8, 4/45, 6/45, 8/8, 10/8). Prior to

use, the mobile phase was filtered by using 0.2 µm filter and degassed.

The mobile phase pumped at 0.2 mL min-1 and water was used as

sample diluent. The column temperature was 27°C (ambient) and eluants

were monitored at 210 nm. The injection volume for samples and

standards was 0.5 µL. The total analysis run time was 10 min.

3.2.3 Standard preparation:

A standard solution containing 50 µg mL-1 of sitagliptin phosphate

and 500 µg mL-1 of metformin hydrochloride were prepared by dissolving

in diluent.

3.2.4 Sample Preparation:

One tablet containing 50 mg of SP and 500 mg of MH was transferred

to a 1000 mL standard volumetric flask and dissolved in1000 mL of

diluent. The solution was filtered through 0.2 µm Millipore PVDF filter.

Then 0.5 µL of these solutions were injected in the column. Prepared

impurity blend solution of metformin impurity1 & 2 and sitagliptin

impurity with 0.10 mg mL-1 concentration in diluent.

3.2.5 Generation of stress samples:

One lot of Junumet tablets were selected for stress testing. From the

ICH Stability guideline: “stress testing is likely to be carried out on a

single batch”. Different kinds of stress degradation conditions (like heat,

humidity, acid, base, oxidative and light) were performed on one lot of

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sitagliptin phosphate and metformin hydrochloride tablets based on the

guidance available from ICH Stability Guideline (Q1AR2). The details of

the stress conditions applied are as follows:

a) Acid hydrolysis: Drug solution in 0.1N HCl at 70° C for 48 h.

b) Base hydrolysis: Drug solution in 0.1N NaOH at 70°C for 48 h.

c) Study in Neutral solution: Drug product dissolved in water was heated

at 70°C for 48 h.

d) Oxidative stress: Drug solution in 3% hydrogen peroxide at room

temperature for 4 h.

e) Thermal stress: Tablets were subjected to dry heat at 105°C for 10 d.

f) Photolytic degradation (254 nm): for 10 days.

3.3 Method development and optimization of chromatographic

conditions:

Forced degradation studies were performed to develop a stability

indicating UPLC method for the simultaneous quantitative determination

of sitagliptin phosphate and metformin hydrochloride in combined

pharmaceutical dosage forms.

Sitagliptin phosphate (pKa = 7.7) and Metformin hydrochloride (pKa =

12.4) are basic in nature. The main target of the chromatographic

method is to get the separation of metformin impurities (met impurity-1

and met impurity-2) from metformin and sitaglipin impurity from

sitagliptin. The impurities blend solution was analyzed using different

stationary phases like C18, C8, Cyano and mobile phases, containing

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buffers like phosphate, sulphate and acetate with different pH (2-7) and

using organic modifiers like acetonitrile and methanol in the mobile

phase.

3.3.1 Selection of wavelength:

The two drugs sitagliptin phosphate and metformin hydrochloride

spectrums were collected using Water PDA system. The two drugs were

having UV maxima at around 210 nm (Fig 3.3.F1). Hence detection at

210 nm was selected for method development purpose.

Fig. 3.3.F1: Typical UV spectrums of Sitagliptin phosphate andMetformin hydrochloride.

(a) UV Spectrum of Sitagliptin phosphate

7.075 Sitagliptin190.3

267.2

AU

0.00

0.05

0.10

0.15

nm200.00 250.00 300.00 350.00

(b) UV Spectrum of Metformin hydrochloride2.200 Metformin

195.8 232.9

AU

0.00

1.00

2.00

nm200.00 220.00 240.00 260.00 280.00 300.00 320.00 340.00 360.00 380.00

Wavelength (nm)

135

3.3.2 Column Selection:

Two different C18 (Aquity BEH C18, 100 x 2.1 mm, 1.7 microns and

Zorbax C-18, 50 x 4.6 mm, 1.8 microns) Columns, one C8 Column

(Aquity BEH C8, 100 x 2.1 mm; 1.7 microns) and Zorbax SB-CN, 50 x

4.6 mm, 1.8 microns columns were used for method development. The

stationary phase had a great influence on the retention time, resolution

and peak shape.

The main difficulty of the chromatographic method was to get the

separation of metformin impurities from metformin. When Aquity BEH

C18, 100 x 2.1 mm; 1.7 micron and Zorbax C18, 50 x 4.6 mm, 1.8

microns columns were used in initial experiment with a mobile phase

composition of potassium dihydrogen phosphate buffer adjusted to pH

5.5 and acetonitrile in the various ratios in gradient mode. The flow rate

was 0.3 ml/min. The separation was achieved between metformin and

sitagliptin. But metformin imputiy-1 and metformin impurity-2 were not

separated from metformin. The typical chromatograms on BEH C18 and

Zorbax C18 are shown in Fig. 3.3.F2 & F3.

Fig. 3.3.F2: Trial chromatogram on BEH C18 column

Metforminimp1-0.599

Metformin+Metforminimp2-0.780

Sitagliptin-13.377

Sitagliptinimp-14.810

AU

0.00

1.00

2.00

Minutes0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00

136

Fig. 3.3.F3: Trial chromatogram on Zorbax C18 column

Metformin+Metimps-3.140

Sitagliptin-5.630

SitagliptinImpurity-5.909

AU

0.00

0.02

0.04

0.06

Minutes0.00 2. 00 4.00 6.00 8.00 10 .00

X-axis: Retention time in min and Y-axis: Peak response in AUWhen Zorbax SB-CN, 100 x 4.6 mm, 1.8 microns column was used

with the mobile phase consists buffer (10mM potassium dihydrogen

phosphate whose pH was adjusted to 5.50 with diluted phosphoric acid)

and acetonitrile in a gradient program metformin impurities were not

separated properly from metformin. But sitagliptin impurity was

separated from sitagliptin (Fig. 3.3.F4).

Fig. 3.3.F4: Trial chromatogram on Zorbax SB-CN column

Metformin-1.359

MetImpuirty1-1.505

MetImpurity2-1.608

Sitagliptin-5.582

SitagliptinImpurity-6.061

AU

0.00

0.20

0.40

0.60

Minutes0.00 2.00 4.00 6.00 8.00 10.00

X-axis: Retention time in min and Y-axis: Peak response in AU

When Aquity UPLC BEH C8, 100 x 2.1 mm, 1.7 microns column

used as stationary phase with buffer (10mM potassium dihydrogen

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phosphate whose pH was adjusted to 5.50 with diluted phosphoric acid)

and acetonitrile in a gradient program, the resolution was not achieved

between metformin impurities from metformin.

The further trail was carried out by using the same column with

mobile phase consists buffer (10mM potassium dihydrogen phosphate

and 2mM hexane-1- sulfonic acid sodium salt, pH was adjusted to 5.50

with diluted phosphoric acid) and acetonitrile in a gradient program,

metformin impurities are well separated from metformin along with the

separation of sitagliptin impurity from sitagliptin. After adding hexane-1-

sulfonic acid sodium salt as ion pair reagent to buffer solution, the

complete separation was achieved between metformin impurities and

metformin. The blend chromatogram of sitagliptin and metformin with

impurities (metformin impurity1, metformin impurity2 and sitagliptin

inpurity) is shown in Fig.3.3.F5.

Fig. 3.3.F5: Trial chromatogram on UPLC BEH C-8 column

MetforminImp1-1.705

MetforminImp2-1.988

Metformin-2.185

Sitagliptin-6.969

SitagliptinImp-7.332

AU

-0.20

0.00

0.20

0.40

0.60

0.80

1.00

1.20

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

X-axis: Retention time in min and Y-axis: Peak response in AU3.3.3 Effect of Organic solvent:

When methanol was used as a solvent in the mobile phase, methanol:

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buffer in the ratio 85: 15 (v/v) the retention time of metformin was

around 5 min with broad peak and resolution was not achieved between

metformin and metformin impurities. The sitagliptin was not retained

properly. The acetonitrile was instead of methanol, improved the peak

shape for metformin and sitagliptin.

3.3.4 Effect of pH:

The method was optimized to get the gussion peaks for metformin

and sitagliptin under varies pH of buffer. The pH 5.5 was found more

appropriate for peak shape for metformin, sitagliptin and related

impurities.

Based on method development trails it indicated that the gradient

elution with ion pair mobile phase was successful in separating drugs,

related impurities and all chromatographic degradation products.

3.3.5 Optimized chromatographic conditions:

Column: Aquity UPLC BEH C8 (100 x 2.1 mm, 1.7 µm)

Mobile phase: The mobile phase composition used was the buffer 10mM

potassium dihydrogen phosphate buffer and 2mM hexane-1- sulfonic

acid sodium salt (pH adjusted to 5.5 with diluted phosphoric acid) and

acetonitrile with gradient program.

Flow rate : 0.2 ml min-1

Column temperature : 27 °C

Wavelength : 210 nm

Injection volume : 0.5 µL

139

Run time : 10 min

Diluent : Water

Gradient program (Time(min)/%acetonitrile): 0/8, 2/8, 4/45, 6/45, 8/8

and 10/8.

Retention time: sitagliptin is about 7 min and metformin is about 2 min .

The interference of inactive ingredients microcrystalline cellulose,

polyvinylpyrrolidone, sodium lauryl sulfate, and sodium stearyl fumarate

was also checked by injecting sample solutions of excipients. There was

no interference of excipients with sitagliptin and metformin peaks.

Fig. 3.3.F6: Typical tablet chromatogram

Metformin-2.199

Sitagliptin-7.033

AU

-0.20

0.00

0.20

0.40

0.60

0.80

1.00

1.20

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

X-axis: Retention time in min and Y-axis: Peak response in AU3.4 Degradation behavior:

Degradation studies on sitagliptin phosphate and metformin

hydrochloride tablets under different stress conditions suggested the

following degradation behavior.

3.4.1 Degradation in acidic solution:

Sitagliptin phosphate and metformin hydrochloride drugs were

exposed to 0.1 N HCl for 48 hours reflux at 70°C. Slight degradation was

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observed (Fig. 3.4.F1 (a) and (b)).

Fig. 3.4.F1(a): Typical Blank chromatogram of acid hydrolysis

1.822

AU

-0.10

0.00

0.10

0.20

0.30

0.40

0.50

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

X-axis: Retention time in min and Y-axis: Peak response in AU

Fig. 3.4.F1(b): Typical chromatogram of acid hydrolysis

Metformin-2.212

Peak2-2.668

Peak3-6.392

Sitagliptin-7.135

AU

-0.10

0.00

0.10

0.20

0.30

0.40

0.50

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

X-axis: Retention time in min and Y-axis: Peak response in AUPeak Purity Results

Name RetentionTime

PurityAngle

PurityThreshold

PurityFlag

PeakPurity

Metformin 2.21 0.46 1.00 No pass

Sitagliptin 7.13 0.55 1.02 No pass3.4.2 Degradation in Basic solution:


exposed to 0.1 N NaOH for 48 hours reflux at 70°C. Major degradation

products were observed (Fig 5.4.F2(a) and (b)).

141

Fig. 3.4.F2(a): Typical Blank chromatogram of base hydrolysis

1.480

AU

-0.10

0.00

0.10

0.20

0.30

0.40

0.50

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

Fig. 3.4.F2(b): Typical chromatogram of base hydrolysis

Metformin-2.212

Peak2-2.668

Peak3-6.392

Sitagliptin-7.135

AU

-0.10

0.00

0.10

0.20

0.30

0.40

0.50

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


Name RetentionTime

PurityAngle

PurityThreshold

PurityFlag

PeakPurity


Sitagliptin 7.15 0.80 1.07 No pass

3.4.3 Degradation in peroxide (Oxidative degradation):


exposed to 3 % peroxide, for 4 hours reflux at room temperature. Major

degradation products observed (Fig. 3.4.F3(a) and (b)).

142

Fig. 3.4.F3(a): Typical Blank chromatogram of oxidative degradation

1.566

1.732

AU

-0.10

0.00

0.10

0.20

0.30

0.40

0.50

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

Fig. 3.4.F3(b): Typical chromatogram of oxidative degradation

Peak1-1.979

Metformin-2.194

Peak3-2.617

Peak4-3.369

Peak5-4.839

Sitagliptin-7.111

AU

-0.10

0.00

0.10

0.20

0.30

0.40

0.50

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


Name RetentionTime

PurityAngle

PurityThreshold

PurityFlag

PeakPurity

Metformin 2.19 0.72 1.00 No passSitagliptin 7.11 0.53 1.02 No pass

3.4.4 Degradation in Neutral (Water) solution:

No major degradation products were observed when stressed

conditions were applied (in water 48 hours reflux at 70 ºC). Minor

degradation products observed (Fig. 3.4.F4(a) and (b)).

143

Fig. 3.4.F4(a): Blank chromatogram of Water degradation

AU

-0.10

0.00

0.10

0.20

0.30

0.40

0.50

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

Fig. 3.4.F4(b): Typical chromatogram of Water degradation

Peak1-1.753

Metformin-2.212

Peak3-2.627

Peak4-6.425

Sitagliptin-7.169

AU

-0.10

0.00

0.10

0.20

0.30

0.40

0.50

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


Name RetentionTime

PurityAngle

PurityThreshold

PurityFlag

PeakPurity



3.4.5 Photolytic conditions:

When the sitagliptin phosphate and metformin hydrochloride drugs

powder was exposed to UV and visible light for 10 days, no degradation

was observed (Fig. 3.4.F5). The drugs were stable to photolysis.

144

Fig. 3.4.F5 : Typical chromatogram of photolytic degradation

Metformin-2.194

Sitagliptin-7.056

AU

-0.10

0.00

0.10

0.20

0.30

0.40

0.50

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


Name RetentionTime

PurityAngle

PurityThreshold

PurityFlag

PeakPurity



3.4.6 Thermal Degradation:

No significant degradation was observed when the sitagliptin

phosphate and metformin hydrochloride drugs powder was exposed to

heat at 105°C for 10 days (Fig. 3.4.F6 ).

Fig. 3.4.F6: Typical chromatogram of thermal degradation

145

Metformin-2.216

Sitagliptin-7.057

AU

-0.10

0.00

0.10

0.20

0.30

0.40

0.50

Minutes0.00 2.00 4.00 6.00 8.00 10.00


Name RetentionTime

PurityAngle

PurityThreshold

PurityFlag

PeakPurity



Peak purity test performed for the sitagliptin and metformin peaks

using photodiode array (PDA) detector data confirmed the purity of the

peak for all the stressed samples. In all the samples the peaks of

sitagliptin phosphate and metformin hydrochloride were pure and

homogenous. Assay of all stressed samples were calculated using

qualified standards of sitagliptin phosphate and metformin

hydrochloride. Considering the purities from the respective

chromatograms of stressed samples, mass balance (%assay + %

degradation products + % impurities) was calculated for each stress

sample. The mass balance of stressed samples was close to 99.0%. This

clearly demonstrates that the developed HPLC method was found to be

146

specific for sitagliptin phosphate and metformin in presence of its

degradation products.

3.5 Method Validation

The developed and optimized HPLC method was taken up for

validation. The analytical method validation was carried out in

accordance with ICH guidelines [22-24].

3.5.1 System Suitability Test:

Standard containing sitagliptin phosphate and metformin

hydrochloride was injected into UPLC system for 5 times and the system

suitability results were tabulated (Table 3.5.T1)

Table 3.5.T1: System Suitability results

Compound(n=5)

USPTailing factor (T)

% RSD for 5replicate injections

Metforminhydrochloride 1.2 0.5%

Sitagliptinphosphate

1.1 0.2%

n = Number of determinations

3.5.2 Precision:

The precision of an analytical procedure expresses the closeness of

agreement between a series of measurements obtained from multiple

sampling of the same homogenous sample under the prescribed

conditions. Assay method precision study was evaluated by carrying out

six independent assays of sitagliptin phosphate and metformin

147

hydrochloride test sample against qualified reference standards and RSD

of six consecutive assays was 0.60% and 0.60% for sitagliptin phosphate

and metformin hydrochloride respectively (intra-day) (Table 3.5.T2) and

0.50% and 0.72% for sitagliptin phosphate and metformin hydrochloride

respectively (inter-day) (Table 3.5.T3 and Table 3.5.T4). Results showed

no significant variation in measured response, which demonstrated that

the method was repeatable with RSDs below 1.0 %.

Table 3.5.T2: Precision results of the assay method (intra-day)

Preparation

% Assay

Sitagliptin phosphate Metformin hydrochloride

1 98.56 98.38

2 99.12 99.17

3 99.44 98.65

4 100.10 97.59

5 99.80 98.14

Average 99.40 98.39

SD 0.60 0.59

% RSD 0.60 0.60

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Table 3.5.T3: Precision results of the assay method (inter-day)

Preparation% Assay

Sitagliptin phosphate Metformin hydrochloride

1 99.45 99.56

2 98.92 98.49

3 99.16 99.18

4 99.53 97.93

5 98.29 98.05

Average 99.07 98.64

SD 0.50 0.71

% RSD 0.50 0.72

Table 3.5.T4: Results of repeatability and intermediate precision

S.No Parameter Variation

% RSDfor Assay


Metforminhydrochloride

1

Intermediate

precision

(intra-day)

(a) Analyst-2

(b) Waters AcquityUPLC system withUV detector

(c) Day-2

0.6 0.6

2

Repeatability

(inter-day)

(a) Analyst-1

(b) Waters AcquityUPLC system withPDA detector

(c) Day-1

0.5 0.7

149

3.5.3 Linearity of the assay method:

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. The linearity of the

assay method was established by injecting test sample from 50% to

150% of two drugs sitagliptin phosphate and metformin hydrochloride

assay concentrations (i.e. 50 µg/ml of sitagliptin phosphate and 500

µg/ml of metformin hydrochloride). Each solution injected into UPLC and

from that calculated the correlation coefficient (Table 3.5.T5 and Table

3.5.T6). The slope and Y-Intercept of the metformin peak in the linearity

study was found to be 8545 and 41987 respectively and the slope and Y-

Intercept of the sitagliptin peak in the linearity study was found to be

3975 and -17863 respectively.

Calibration curve obtained by least square regression analysis

between average peak area and the concentration showed (Fig. 3.5.F1 to

Fig. 3.5.F2) linear relationship with a regression coefficient of 0.999. The

best fit linear equation obtained was Y =3975 Con - 17863 for sitagliptin

phosphate and Y =18545 Con + 41987 for metformin hydrochloride.

150

Table 3.5.T5: Linearity results of the assay method for Sitagliptinphosphate

Concentration (µg mL-1) Peak area

25 81392

37.5 131354

50 180767

62.5 230935

75 280056

Correlation Coefficient(r) 0.999

Slope 3975

Intercept -17863

Fig. 3.5.F1: Linearity plot for Assay Method for Sitagliptinphosphate

X-axis: Concentration in µg mL-1 and Y-axis: Peak area mAU

151

Table 3.5.T6: Linearity results of the assay method for Metforminhydrochloride

Concentration (µg mL-1) Peak area

250 2129272

375 3265303

500 4338078

625 5476560

750 6364618

Correlation Coefficient(r) 0.998

Slope 8545

Intercept 41987

Fig. 3.5.F2: Linearity plot for Assay Method for Metforminhydrochloride

X-axis: Concentration in µg mL-1 and Y-axis: Peak area mAU

152

3.5.4 Accuracy:

The accuracy of an analytical procedure expresses the closeness of

agreement between the value, which is accepted either as a conventional

true value or an accepted reference value and the value found.

Accuracy of the assay method was established by injecting three

preparations of test sample using tablets at 50%, 100% and 150% of

analyte concentration (i.e. 50 µg/ml of sitagliptin phosphate and 500

µg/ml of metformin hydrochloride). Each solution was injected thrice

(n=3) into UPLC and the mean peak area was calculated. % Assay of test

solution was determined against three injections (n=3) of qualified

sitagliptin phosphate and metformin hydrochloride standards (Table

3.5.T7). The method showed consistent and high absolute recoveries at

all three concentration (50, 100 and 150%) levels with mean absolute

recovery ranging from 99.1 to 100.5% for sitagliptin phosphate and from

99.0 to 100.8% for metformin hydrochloride.

Table 3.5.T7: Recovery of the assay method

S.No Concentration(%)Mean recovery (%) % RSD

SP MH SP MH

1 50 99.75 100.6 0.45 0.50

2 100 101.25 100.0 0.26 0.47

3 150 100.18 98.32 0.29 0.61

SP = Sitagliptin phosphate

MH= Metformin hydrochloride

153

3.5.5 Solution stability:

The solution state stability of sitagliptin phosphate and metformin

hydrochloride in diluent in the assay method was carried out by leaving

the test solutions of sample in tightly capped volumetric flasks at room

temperature for two days. The same sample solution were assayed for

every six hours interval up to the study period, each time freshly

prepared reference standard was used to estimate the assay of test

sample. The % RSD of assay of sitagliptin phosphate and metformin

hydrochloride during solution stability experiments was within 0.5% for

two drugs. Hence sample solutions are stable for at least 48 hours in the

developed method. Standard and test solution injected at each 0 h, 6 h,

12 h, 18 h, 24 h, 30 h, 36 h, 42 h and 48 h.

Table 3.5.T8: Solution stability results of the assay method

S.No. Interval% Assay


Metforminhydrochloride

1 0 h 99.62 99.312 6 h 99.63 98.893 12 h 99.11 99.213 18 h 99.55 98.764 24 h 99.44 99.135 30 h 98.99 98.496 36 h 99.14 99.127 42 h 99.01 98.658 48 h 99.26 99.17

Average 99.31 98.97SD 0.259 0.284

% RSD 0.261 0.287

154

3.5.6 Robustness:

To determine the robustness of the developed method experimental

conditions were purposely altered and the tailing factor for metformin

peak and sitagliptin peak and also % RSD for 5 replicate injections of

metformin and sitagliptin were evaluated. In each of the deliberately

altered chromatographic condition (flow rate 0.18 mL min-1 and 0.22 mL

min-1, column temperature 20 ºC and 30 ºC and pH of the buffer 5.30

and 5.50) the tailing factor, and % RSD for 5 replicate injections of

metformin and sitagliptin peaks were within the limits illustrating the

robustness of the method (Table 3.5.T9).

Table 3.5.T9: Results of robustness study

Parameter Temperature

(± 5 0C of settemperature)

Flow rate(± 0.02 mL min-1

of the set flow) pH of the buffer(± 0.2 of pH)

Variation 200C 300C 0.18mL min-1

0.22mL min-1 5.30 5.50

USP Tailingfactor (T) SP 1.1 1.0 1.0 1.1 1.1 1.0

MH 1.0 1.1 1.1 1.0 1.1 1.1% RSD for

5 replicateinjections

SP 0.2 0.3 0.3 0.2 0.4 0.3

MH 0.3 0.4 0.4 0.3 0.5 0.6

SP= Sitagliptin phosphate ,

MH= Metformin hydrochloride

155

3.5.7 Tablet application

Analysis was performed for commercially available innovator tablets.

The Mean assay (n = 6) for SP and MH was 100.2% and 99.6%,

respectively. The percentage RSD value for the six assay values was

0.5%, 0.6% for SP and MH, respectively.

3.6 Summary and Conclusion

Validated stability-indicating UPLC method was developed for

simultaneous determination of sitagliptin phosphate and metformin

hydrochloride in combined tablets formulation. The RP-UPLC method

developed is rapid, precise, accurate and selective. The method was

completely validated showing satisfactory results for all the method

validation tested parameters. The developed method was found “specific”

to the drugs and for dosage form in combined tablets, as the peaks of the

degradation products did not interfere with the two drugs. Thus the

proposed method can be employed for assessing the stability of

sitagliptin phosphate and metformin hydrochloride for its individual

dosage form and for its combined dosage forms.

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simultaneous determination of sitagliptin phosphate...

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