“ESTIMATION OF ANAGLIPTIN AND METFORMIN HCl IN BULK ...

“ESTIMATION OF ANAGLIPTIN AND METFORMIN HCl IN BULK &

PHARMACEUTICAL DOSAGE FORM USING STABILITY

INDICATING HIGH PERFORMANCE LIQUID

CHROMATOGRAPHY”

Ruchi H. Majithia*1 & Dr. Akruti Khodadiya2

*Assistant Professor, SAL Institute of Pharmacy, Ahmedabad, Gujarat- 380060

1Research Scholar, C. U. Shah College of Pharmacy and Research, Gujarat- 363030

1Mail ID: [email protected]

1Mobile No: +91-8155921801

2Associate Professor, C.U. Shah University, Wadhwan city, Gujarat- 363030

2Mail ID: [email protected]

*Corresponding author

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http://aegaeum.com/ Page No: 46

AEGAEUM JOURNAL

Volume 8, Issue 7, 2020

ABSTRACT:

A simple, accurate and precise stability indicating high performance liquid chromatography

was developed and validated for estimation of Anagliptin and Metformin HCl in bulk and

pharmaceutical dosage form. The mobile phase Potassium Dihydrogen Phosphate Buffer (pH

2.7): Acetonitrile solution in the ratio of (15:85% V/V) at detection wavelength 243 nm for

Anagliptin and Metformin and flow rate 2 mL/min and the retention time for Anagliptin and

Metformin HCl was found to be 11.23 and 6.02 respectively. The method was linear over the

concentration ranges 10-60 μg/mL for Anagliptin and 25-150 μg/mL for Metformin. The

LOD was 1.158 μg/mL for Anagliptin and 2.344 μg/mL for Metformin. The LOQ was 3.508

μg/mL for Anagliptin and 7.102 μg/mL for Metformin. During stress conditions, ANA

degraded significantly under acidic, alkaline, oxidative and thermal stress conditions;

degraded moderately under photolytic stress conditions; and showed negligible degradation

under elevated temperature and humidity conditions. MET degraded significantly under

acidic, alkaline and oxidative stress conditions; degraded moderately under thermal and

humidity stress conditions and showed negligible degradation under photolytic stress

condition.

KEYWORDS: Anagliptin (ANA), Metformin HCl (MET), High Performance/ Pressure

Liquid Chromatography(HPLC), Simultaneous estimation.

INTRODUCTION

Anagliptin is used for type 2 diabetus mellitus and longer duration of action for treatment of

type 2 non-insulin dependent diabetus mellitus disease as a Dipeptidyl Peptidase 4 inhibitor

Anagliptin, chemically N-[2-[[2-[(2S)-2-Cyanopyrrolodin-1-yl]-2-oxoethyl] amino]-2-

methylpropyl]-2-methylpyrazolo [1, 5-a] pyrimidine-6-carboxamide (Figure 1) is a

Dipeptidyl Peptidase 4 (DPP 4) inhibitor which is used in treatment of type 2 NIDDM [01].

Dipeptidyl Peptidase 4 enzyme breaks down the incretins GLP-1 gastrointestinal hormones

released in response to a meal. By preventing GLP-1 inactivation, they are able to increase

the secretion of insulin and suppress the release of glucagon by the alpha cells of the

pancreas. This drives blood glucose levels towards normal level [02-03]. This drug is not

official in any of the pharmacopeia. Anagliptin is a very effective pill with minimum risk

profile for type 2 diabetus mellitus and longer duration of action for treatment of type 2 non-

insulin dependent diabetus mellitus disease [04]. A literature survey revealed that few methods

are reported for determination of ANA, either alone or in combination, by spectrophotometric

[05-07], HPLC [08-09], LC/MS [10].

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Figure 1: Chemical Structure of Anagliptin

Figure 2: Chemical structure of Metformin HCl

Metformin is chemically a 1-Caramimidamido-N, N-Dimethylmethanimidamide [11] (Figure

2) and has pharmacological action based on Biguanides category [12-13]. It suppresses hepatic

gluconeogenesis and glucose output from liver. This is the major action responsible for

lowering blood glucose in diabetics. It is official in IP [14], BP [15], and USP [16]. Literature

review revels that many spectrophotometric [17-18], HPLC [19-20], HPTLC [21] methods are

reported for determination of Metformin hydrochloride (HCl), either alone or in

combination[08].

The aim of the present work was to develop a stability indicating HPLC method for

simultaneous estimation of ANA and MET in bulk and pharmaceutical dosage form. It is

pertinent to note that, some of the published methods enabled estimation of drugs in

combination products containing two drugs via spectrophotometric. However, so far, not any

HPLC method was reported in pharmaceutical dosage form. Hence, HPLC chromatographic

conditions were developed with forced degradation studies and the method was validated to

establish selectivity with respect to excipients and degradation products.

MATERIALS AND METHOD:

Apparatus

Chromatographic separation of drugs was performed on a Shimadzu HPLC instrument

(LC_2010 CHT) [software LC Solution, equipped with photo diode array detector (SPD-

.HCl

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M20A), Auto-sampler]. The system contains a quaternary gradient pump, auto sampler,

column oven and a PDA detector. Software referred to above was used to record and

integrates the chromatograms. Among other instruments, analytical balance (Acculab ALC-

210.4, Huntingdon Valley, PA), Photostability chamber (TH-90S, Thermolab, Mumbai,

India) , Hot air oven (TO-90S, Thermolab), pH meter (Thermo Electron Crop., Pune, India),

Sonicator (EN 30 US, Enertech Fast Clean, Mumbai, India) were also used at different stages

of development and validation of stability indicating assay method.

Chemicals and Reagents

The API of Anagliptin and Metfromin procured from Intas Pharmaceuticals LTD,

Ahmedabad. Combination test product METOANA Tablets 100/250 mg (manufactured by

Sanwa Kagaku Kenkyusho Co. LTD) was procured from the market. All the solvents like

Methanol, Acetonitrile, Phosphate Buffer and Potassium dihydrogen phosphate were of

HPLC grade from Finar Chemicals Ltd, Ahmedabad, India. All the chemical reagents were of

analytical grade.

Preparation of Standard Stock Solution

The standard solution was prepared by weighing accurately about 10 mg of ANA and 25 mg

of MET transferred into clean and dry 10 mL volumetric flask. Initially about 5 mL methanol

was added to the flask respectively and sonicated. The volume was made up to the mark with

the methanol to achieve 1000 µg/mL of ANA and 2500 µg/mL of MET standard stock

solutions.

Preparation of working standard solution

From the above prepared Stock solutions, pipette out 1 mL of ANA and 1 mL MET solution

and transferred into a clean and dry 10 mL volumetric flask, methanol was added upto the

mark to get final concentration 100 µg/mL & 250 µg/mL respectively, in mixture.

Preparation of test solution

Twenty METOANA tablets were accurately weighed, their average weight was calculated.

Amount of finely powdered tablet equivalent to 100 mg ANA and 250 mg MET was weighed

and transferred into a 100 mL volumetric flask and the volume was adjusted to mark with

methanol. The contents of the flask were sonicated for 30 min to dissolve the active

ingredients completely. The solution was filtered through a Whatman filter paper no. 41.

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From this 1 mL aliquot was transferred into a 100 mL volumetric flask and the volume was

made with methanol. This corresponds to 10 µg/ mL of ANA & 25 µg/ mL MET, in mixture

was analyzed for assay determination.

Standardized Chromatographic conditions

The analyte drugs and degradation products were well separated with ZORBAX RX C8 (250

mm × 4.6 mm, 5 µm) as a stationary phase and gradient system of Potassium Dihydrogen

Phosphate Buffer (pH 2.7): Acetonitrile solution in the ratio of (15:85% V/V). The flow rate

was adjusted to 2 mL/min. Determination was done at wavelength 243 nm. Column

temperature was set to 300 C. Total run time was 20 min. Injection volume was 10 µL.

Methanol was used as a diluent.

METHOD VALIDATION

The proposed method was validated as per ICH guidelines Q2 R1 [22].

System suitability test parameters

System suitability tests are used to verify that the resolution and repeatability of the system

were adequate for the analysis intended. The parameters used in this test were the

chromatographic peak resolution (>2), theoretical plate number (>2000) and tailing factor

(<1.8). The repeatability of these parameters was checked by injecting six solutions of ANA

and MET.

Linearity

From the standard stock solution containing 100 µg/mL ANA and 250 µg/mL MET, aliquots

of 1 mL, 2 mL, 3 mL, 4 mL, 5 mL and 6 mL were transferred in clean and dry 10 mL

volumetric flasks respectively. The volume was made up to the mark with diluent. This

yielded solution of 10, 20, 30, 40, 50 and 60 µg/mL of ANA and; 25, 50, 75, 100, 125 and

150 µg/mL of MET respectively. An injection volume of 10 μL of each solution was injected

under operating chromatographic condition. Six replicates of each concentration were

performed and then calibration plots were determined by linear least – squares regression.

The plate was developed on previously described mobile phase. The peak areas were plotted

against the corresponding concentrations to obtain the calibration graphs.

Precision

Repeatability was determined by applying six replicates of test solution (10 µg/mL ANA and

25 µg/mL MET). The intraday and interday precisions were determined by responses of six

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replicates on same and different days for the test concentration. The results were reported in

terms of % RSD.

Accuracy

Recovery study was carried out by standard addition method where known amount of

standard concentration at 50%, 100% and 150% of the test solution were spiked in the test

solution in triplicate. The amount of drugs was estimated by substituting values in regression

equation. The % RSD of the recovery was calculated.

LOD and LOQ

The LOD and LOQ of the developed method were calculated from the calibration curve using

equations, LOD = 3.3 ×ϭ/S and LOQ = 10 × ϭ/S where ϭ is the standard deviation of y-

intercept and S is the slope of the curve.

Robustness

By introducing small changes in the detection wavelength (±2 nm) and saturation time for the

chamber before plate development (±2 min), the effects on the results were determined. One

factor at a time was changed and the effect on peak area of the drug was studied. Robustness

of the method was done on single level for six replicates and %RSD was calculated.

FORCED DEGRADATION STUDIES:

Force degradation study was intended to ensure the effective separation of ANA and MET

and their potential degradation products which are generated under different stress conditions

like acid, alkaline and neutral hydrolysis, oxidative degradation, thermal and photolytic

degradation.

Acid Hydrolysis

Accurately weighed 10 mg ANA and 25 mg MET were transferred in 10 mL volumetric flask

individually and in combination. To this were added 5 mL methanol and 5 mL of 0.1 N HCl

and it was kept at room temperature for 5 h. From that solution 1 mL was transferred into 10

mL volumetric flask, neutralized with 0.1 N NaOH and diluted to mark with methanol. This

corresponds to 100 µg/mL ANA and 250 µg/mL MET were injected under operating

condition.

Alkaline Hydrolysis

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individually and in combination. To this were added 5 mL methanol and 5 mL of 0.1 N

NaOH and it was kept at room temperature for 1 h. From that solution 1 mL was transferred

into 10 mL volumetric flask, neutralized with 0.1 N HCl and diluted to mark with methanol.

This corresponds to 100 µg/mL ANA and 250 µg/mL MET were injected under operating

condition.

Oxidative Hydrolysis


individually and in combination. To this were added 5 mL methanol and 5 mL of 3% H2O2

and it was kept at RT for 3 h. From that solution 1 mL was transferred into 10 mL volumetric

flask, neutralized with 0.1 N HCl and diluted to mark with methanol. This corresponds to 100

µg/mL ANA and 250 µg/mL MET were injected under operating condition.

Thermal Degradation

Accurately weighed 10 mg ANA and 25 mg MET were transferred in 10 mL volumetric

flask individually and in combination in petridish. Those were kept at 700 C, 4 h for 4 h.

After that those were dissolved in 10 mL methanol. From that solution 1 mL was transferred

into 10 mL volumetric flask and diluted to mark with mobile phase. This corresponds to 100

µg/mL ANA and 250 µg/mL MET were injected under operating condition.

Photolytic degradation


individually and in combination in petridish. It was exposed in photo stability chamber (TH-

90S, Thermo lab, Mumbai, India) in UV light at 254 nm for 36 h to get 200 watt hours / m2

intensity. After that those were dissolved in 10 mL methanol. From that solution 1 mL was

transferred into 10 mL volumetric flask and diluted to mark with mobile phase. This

corresponds to 100 µg/mL ANA and 250 µg/mL MET were injected under operating

condition.

RESULT AND DISCUSSION

RESULT AND DISCUSSION

OPTIMIZED CHROMATOGRAPHIC CONDITION

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For separation of ANA and MET, and their degradation peaks, different mobile phases with

different solvents in different ratio were tried like (1) Water: ACN (50:50 %V/V) (2) Buffer:

ACN (pH 3) (50:50 % V/V) (3) Acetonitrile: Buffer (pH 2.7) (70:30 %V/V). The mobile

phase Potassium Dihydrogen Phosphate Buffer (pH 2.7): Acetonitrile solution in the ratio of

(15:85% V/V) showed well resolved peak with better peak shape. The drugs were resolved

with tR of 6.02 ± 0.41 min and 11.23 ± 0.37 min of MET and ANA, respectively.

Determination of all three drugs was done at wavelength 243 nm with column temperature

300 C and adjusted flow rate 2.0 mL/min. Injection Volume was set 20 µL for a good peak

shape. (Figure 3)

Figure 3: Chromatogram of STD ANA (100 µg/mL) and MET (250 µg/ mL)

Linearity

The method was found linear over the concentration range of 10-60 µg/ mL 25-150 µg/ mL

for ANA and MET respectively. The calibration curve obtained by the least square regression

analysis between average peak area and concentration showed linear relationship with a

correlation coefficient of 0.9991 and 0.999 for ANA and MET respectively. The linear

regression equation were y = 35.448x - 71.612, and y = 178.98x + 793.84 for ANA and MET

respectively. (Table 1, Figure 4, Figure 5)

Table 1: Linearity range of ANA and MET

Sr

No.

ANA MET

Conc

(µg/ mL) Peak Area* ± SD %RSD

Conc

(µg/ mL) Peak Area * ± SD %RSD

1 10 309.33 ± 3.20 1.03 25 5357 ± 66.15 1.23

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2 20 623.16 ± 7.54 1.21 50 9873 ± 132.89 1.34

3 30 971.66 ± 9.72 1.00 75 14161 ± 193.53 1.36

4 40 1345.33 ± 15.16 1.12 100 18349 ± 206.07 1.12

5 50 1687.5 ± 18.11 1.07 125 22751 ± 245.14 1.07

6 60 2077.33 ± 24.17 1.16 150 28153 ± 360.00 1.27

*Average of six determinations

Figure 4: Calibration Curve of Linearity of ANA at 243 nm

Figure 5: Calibration Curve of Linearity of MET at 243 nm

Precision

The % RSD for repeatability was found to be 1.23 for ANA and 1.14 for MET. The % RSD

of Intraday Precision was found to be 1.37 for ANA and 1.21 for MET. The % RSD of

y = 35.448x - 71.612 R² = 0.9991

0

500

1000

1500

2000

2500

0 10 20 30 40 50 60 70

Calibration curve of ANA at 243 nm

y = 178.98x + 793.84 R² = 0.999

0

5000

10000

15000

20000

25000

30000

0 20 40 60 80 100 120 140 160

Calibration curve of MET at 243 nm

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Interday precision was found to be 1.41 for ANA and 1.39 for MET. Hence, confirming

precision of the developed method. (Table 2)

Table 2: Repeatability, Intraday and Interday Precision of ANA and MET

Parameter

ANA MET

Conc

(µg/ mL) Peak Area* ± SD %RSD

Conc

(µg/ mL) Peak Area * ± SD %RSD

Repeatability 10 372 ± 4.50 1.23 25 4603.19 ± 52.73 1.14

Intraday

Precision 10 376.66 ± 5.16 1.37 25 4632.80 ± 56.40 1.21

Interday

Precision 10 371 ± 5.25 1.41 25 4626.13 ± 64.59 1.39


Accuracy

The accuracy of the developed method was established by standard addition method by

adding known standard concentration solutions to the pre-analysed samples. Recoveries were

in between 99.69-101.69 % for ANA and 99.68-101.45 % for MET which is in accordance

with ICH guidelines which proves method to be accurate. (Table 3)

Table 3: Recovery study data of ANA and MET

Drug

Amount of

Test

Solution

(µg/ mL)

Amount

of Stand.

added

(µg/ mL)

Peak Area* ± SD

Total

Amount

Found

(µg/ mL)

Recovered

amount

(µg/ mL)

%

Recovery

%

RSD

ANA

10 0 425 ± 5.29 9.96 -- 99.69 1.49

10 5 612.36 ± 7.07 15.25 10.25 101.69 1.30

10 10 790.66 ± 10.40 20.28 10.28 101.42 1.44

10 15 959 ± 15.71 25.11 10.11 100.47 1.76

MET

25 0 5252.46 ± 50.43 24.92 -- 99.68 1.13

25 12.5 7588.7 ± 88.09 37.98 25.48 101.28 1.29

25 25 9849 ± 94.69 50.61 25.61 101.23 1.04

25 37.5 12137 ± 120.37 63.40 25.9 101.45 1.06

*Average of three determinations

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LOD and LOQ

The LOD calculated by formulae was found to be 1.158 μg/mL for ANA and 2.344 μg/mL

for MET. The LOQ calculated by formulae was found to be 3.508 μg/mL for ANA and 7.102

μg/mL for MET.

Robustness

Slight change in the chromatographic condition of the developed method like small changes

in the Flow rate (±0.1 mL), column temperature (± 50 C), wavelength (± 2 nm) and pH of

buffer solution in mobile phase (± 0.2) did not affect the result significantly. The % RSD

values were found below 2 indicated the method to be robust. (Table 4)

Table 4: Robustness study of ANA and MET

Condition Variation % Assay* ± SD % RSD#

ANA MET ANA MET

Normal N/A 100.53 ± 1.07 100.21 ± 1.20

1.26 1.31

Flow rate

(± 0.1 mL/min)

2.1

mL/min 101.10 ± 1.23 99.98 ± 1.24

1.9

mL/min 101.04 ± 0.87 100.96 ± 1.35

Detection

Wavelength (243±2

nm)

241 nm 100.32 ± 1.11 101.2 ± 1.74

245 nm 100.02 ± 0.83 101.67 ± 1.03

Column

temperature

(± 50 C)

250 C 101.23 ± 1.23 99.45 ± 1.34

350 C 101.86 ± 1.45 99.75 ± 1.26

pH (±0.2) 2.5 100.85 ± 1.18 101.15 ± 1.21

2.9 101.15 ± 1.25 101.69 ± 1.24

*RSD of original and modified conditions

Analysis of marketed formulation

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The developed method was applied to marketed tablet preparation. The assay results of ANA

and MET were 100.53 ± 3.89 % and 100.21 ± 47.99 %, respectively of the labelled amount.

(Table 5)

Table 5: Analysis of marketed tablet formulation

Drug Amount of drug

% Label claimed

(% Assay* ± SD) % RSD

Labeled Estimated

ANA 100 100.53 100.53 ± 3.89 1.09

MET 250 250.43 100.21 ± 47.99 1.07


Forced degradation studies

The result of forced degradation studies are summarized in (Table 6). Under the optimized

chromatographic conditions degradation products of analytes were well resolved and the

percent degradation was calculated by comparing peak area with standard preparation.

During stress degradation experiments, it was observed that ANA degraded significantly

under acidic, alkaline, oxidative and thermal stress conditions; and showed negligible

degradation under photolytic stress conditions and neutral hydrolysis condition. MET

degraded significantly under alkaline and oxidative stress conditions; degraded moderately

under acidic, neutral and thermal stress condition and showed negligible degradation under

photolytic stress condition.

The purity of the drug (analyte peak) was ascertained by analysing spectrum at peak start,

peak end and peak apex position which showed no interference of any excipients or process

impurities in analyte peak. The method is also deemed to be specific from potential

degradation products as most of the degradation products are adequately resolved from both

analyte peaks. The sample tablet was exposed to thermal and photolytic degradation

conditions as per ICH Q1A R2 guidelines [23]. The method was therefore being considered to

be stability indicating for tablet solid dosage form. (Figure 6, Figure 7, Figure 8, Figure 9,

Figure 10 and Figure 11)

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Figure 6: Chromatogram of Acid hydrolysis of mixture of ANA and MET at 243 nm

Figure 7: Chromatogram of Alkali hydrolysis of mixture of ANA and MET at 243 nm

Figure 8: Chromatogram of Neutral hydrolysis of mixture of ANA and MET at 243 nm

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Figure 9: Chromatogram of Oxidative hydrolysis of mixture of ANA and MET at 243

nm

Figure 10: Chromatogram of Thermal degradation of mixture of ANA and MET at 243

nm

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Figure 11: Chromatogram of Photolytic (UV Light) degradation of mixture of ANA and

MET at 243 nm

Table 6: Summary of Forced Degradation study of individual API in mixture of ANA

and MET

Degradation

condition

tR of Analyte

No. of

degradati

on peaks

tR of Degradation

peak %Degradation

ANA MET ANA ANA MET

0.1 N HCl at

RT, 5 h 11.453 6.011 4

1.903, 3.019, 7.511,

8.801 4.79 16.97

0.1 N NaOH

at RT, 1 h 11.45 6.011 5

5.225, 6.617,

14.138, 15.057,

15.561

26.63 23.75

Water, RT, 3

h 11.23 6.02 -- -- 1.02 1.17

3% H2O2, RT,

3 h 11.453 6.011 5

1.9, 3.1, 8.8, 14,

15.1 13.69 15.64

Thermal at

700 C, 4 h 11.453 6.011 3 3.01, 7.5, 10.0 1.83 9.09

UV light, 254

nm, 36 h 11.23 6.02 2 1.9, 3.0 1.15 3.77

Table 7: Summary of Validation Parameters

Sr.

No. Parameter ANA MET

1 Specificity Specific Specific

2 tR value 11.23 ± 0.37 6.02 ± 0.41

3 Linearity Range 10-60 μg/mL 25-150 μg/mL

4 Regression Line equation y = 35.448x - 71.612 y = 178.98x + 793.84

5 Correlation Coefficient 0.9991 0.999

6 Precision

Repeatability 1.23 1.14

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Intraday Precision 1.37 1.21

Interday Precision 1.41 1.39

7 Accuracy (% Recovery) 99.69-101.69 % 99.68-101.45 %

8 LOD (μg/mL) 1.15 2.34

9 LOQ (μg/mL) 3.50 7.10

10 Robustness Robust Robust

CONCLUSION

The proposed HPLC method is precise, specific, linear and accurate for the estimation of

ANA and MET in pharmaceutical dosage form without interference from the excipients and

potential degradation products in various stress conditions like acid hydrolysis, alkaline

hydrolysis, neutral hydrolysis, oxidative, thermal and photolytic degradation conditions. The

developed method is validated as per ICH guidelines. The results showed the suitability of

developed method for stability studies of the pharmaceutical dosage form.

AKNOWLEDGEMENT

The authors are graceful to INTAS Pharmaceuticals, Ahmedabad for providing a gift sample

of Anagliptin and Metformin. The authors are grateful to Global Analytical Laboratory,

Ahmedabad for providing all the facilities to carry out research work.

CONFLICT OF INTEREST: Nil

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