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  • Development And Validation of a Simple, LCMS Compatible New Assay Method for The Simultaneous Estimation of Anti-Diabetic Drugs Metformin, Sitagliptin and Glimepiride

  • Post Graduate and Research Laboratory, Department of Chemistry C. S.’S Patkar – Varde College, Goregaon (W), Mumbai – 400062.

Abstract

A simple, rapid, precise, accurate and reproducible RP- HPLC method was developed for the simultaneous estimation of Metformin, Glimepiride and Sitagliptin in tablet dosage forms. The developed method was validated with different parameters such as linearity, precision, accuracy and solution stability as per ICH guidelines. It was further applied to simultaneous quantitative estimation of Metformin, Glimepiride and Sitagliptin in tablet dosage forms. The method was developed using Hemochrom C8 column (250mm x 4.6mm, 5µm) using Acetonitrile and Trifluoroacetic acid combination as a mobile phase in gradient mode with PDA detector. The detection wavelength was set at 210 nm for Metformin and Sitagliptin and 228 nm Glimepiride quantification respectively. The retention times of Sitagliptin, Metformin and Glimepiride were found to be around 14 min, 4.8 min and 18.5 min. The assay of the developed method was found to be within 98-102% for all the three APIs in the formulations. The % RSD for reproducibility was found to be ? 2.0% Conclusion: The proposed method was statistically evaluated by validation and can be applied for routine quality control analysis of Sitagliptin, Metformin and Glimepiride in bulk and in the tablet dosage form.

Keywords

Sitagliptin, Metformin Hydrochloride, Glimepiride, anti-diabetic, Validation, Assay.

Introduction

Metformin hydrochloride (MET) is chemically 1, 1-dimethylbiguanide hydrochloride (Fig. 1 a.) is the main first-line medication for the treatment of type 2 diabetes, particularly in people who are obese. It is also used in the treatment of polycystic ovary syndrome. Similarly, Glimepiride (GLM) is chemically 1- [ [p- [2- (3-ethyl-4-methyl-2-oxo-3-pyrroline-1 carboxamido) ethyl] phenyl] sulfonyl]-3- (trans-4-methylcyclohexyl) urea (Fig.1 b.) is an anti-diabetic medication used to treat type 2 diabetes. Glimepiride was patented in 1979 and approved for medical use in 1995. It is available as a generic medication and sitagliptin phosphate monohydrate (SIT) is (3R)-3-amino-1-[3-(trifluoromethyl)-5,6-dihydro-triazolo[4,3-a]pyrazin-7(8H)-yl]-4-(2,4,5-trifluorophenyl)butan-1-one phosphate onohydrate (Fig 1 c.) Sitagliptin is an anti-diabetic medication used to treat type 2 diabetes.  It is available in oral dosage form. It is also available in a fixed-dose combination as sitagliptin/metformin. Recently, Combination therapy in Type 2 diabetes is more efficacious and is better tolerated than high dose of Individual drug products. It offers convenient dosing and improves patient compliance in Diabetes treatment. The literature review reveals that the HPLC methods for the simultaneous estimation of Metformin, Glimepiride and Sitagliptin in tablet dosage are quite tedious in sample preparation, using complex buffers and longer run times. Hence the present study was aimed at developing simple, short, reliable and accurate RP- HPLC method for simultaneous estimation of Metformin, Glimepiride and Sitagliptin in tablet dosage form which can also be extended to the LCMS analysis for Metabolite identification.

Fig 1: a) Structure of Metformin Hydrochloride, b) Glimepiride and c) Sitagliptin phosphate

MATERIALS AND METHODS:                                 

Materials and Methods:

The API Ref Standards of Metformin, Glimepiride and Sitagliptin phoosphate were provided as a gift sample FDC Ltd.    Mumbai, India. The solvents used were of following make: Acetonitrile, (HPLC grade Thermofisher), HPLC Water (MilliQ), Trifluoro acetic acid (AR grade Spectrochem), Methanol (HPLC grade Thermofisher), 0.45µm filter (Millipore, Bangalore). Glimepiride 2mg and Metformin HCl 1000mg combination Tablet & Sitagliptin 50mg and Metformin 1000mg tablet were procured from a local pharmacy. (Alkem Lab – Glimepiride and Metformin tablet 2mg / 1000mg; Sun pharma – Sitagliptin and Metformin tablet 50 mg / 1000 mg) Various C18 and C8 columns with different dimensions and carbon load were tried during development with different combination of Acetonitrile, Methanol and various volatile buffers and the best results were obtained on Hemochrom C8 column (4.6mm x 250mm; 5µm).

Chromatographic Conditions: 

The method development, validation and analysis of the drug was carried out Waters Alliance HPLC 2695 Separation module equipped with 2996   PDA Detector. The column used was Reverse Phase Hemochrom C8 column (4.6mm x 250mm; 5µ), ambient temperature at the flow rate of 1 ml/min using Empower software. Mobile phase used was Acetonitrile and 0.05% Aq. TFA in gradient mode.  The PDA detector was set at detection wavelength of 228 nm for Glimepiride and 210 nm for Sitagliptin and Metformin respectively.  The Standard and sample solutions for Metformin and Sitagliptin estimation were prepared in 0.05% TFA whereas Methanol was used as a diluent for Glimepiride.

Preparation of Standard Stock Solution: Preparation of Std. Metformin Solution (Stock A): An accurately weighed quantity, 50 mg of Metformin was taken in 50 ml Volumetric flask, about 25ml of Diluent was added and sonicated for 15 mins and diluted up to 50 ml to produce a solution of 1000 ppm. From the freshly prepared standard stock solution (1000 ppm), 100 ppm working standard solution was prepared by diluting 1 ml to 10 ml in a volumetric flask using the diluent

Preparation of Std. Sitagliptin Solution (Stock B): An accurately weighed quantity, 25 mg of Sitagliptin was taken in a 50 ml volumetric flask, about 25ml of Diluent was added and sonicated for 15 mins and diluted up to 50 ml to produce a solution of 500 ppm. From the freshly prepared standard stock solution (500 ppm), 1ml stock solution was pipetted out in 10 ml of a volumetric flask, the volume was made up to 10 ml with mobile phase to get a final concentration of 50 ppm.

Preparation of Std. Glimepiride Solution (Stock C): An accurately weighed quantity, 50 mg of Glimepiride was taken in a 50 ml volumetric flask. About 25ml of the Diluent was added, sonicated for 15 mins and diluted upto 50 ml to produce a solution of 1000 ppm. From the freshly prepared standard stock solution (1000 ppm), 1ml stock solution was pipetted out in 10 ml of a volumetric flask, and volume was made up to 10 ml with mobile phase to get a final concentration of 100 ppm.

Method Validation: The working standard of various concentrations was prepared by taking aliquots of respective Standard stock solutions and diluting to get the required concentration for injection to obtain a calibration plot  11

Assay Preparation for Commercial Formulation:

1) For estimation of Metformin (500 mg) and Sitagliptin (50 mg) from tablets:

Twenty tablets were finely powdered in a mortar and pestle. Sample powder equivalent to weight of one tablet was accurately weighed and transferred into a 500 ml volumetric flask. About 250 ml of the diluent was added and sonicated for 15 mins. And diluted up to 500 ml with the diluent (500 ppm). Further 2 ml of this solution was diluted to 10 ml with the diluent. (100ppm). Few ml of the 100 ppm solution was filtered through a PTFE 0.45 micron membrane filter paper and used for assay of Metformin. For Sitagliptin, 1 ml of the tablet solution was diluted to 10 ml (50 ppm) The representative chromatograms of test Metformin and Sitagliptin is shown in Fig. 2. The amounts of Metformin and Sitagliptin per tablet were calculated by from the calibration curve. The analysis procedure was repeated three times with tablet formulation. Tablet Assay for %Label claim for % RSD was calculated and the result are as shown in Table 1.

1) For estimation of Metformin (1000 mg) and Glimepiride (2 mg) from tablets:

Twenty tablets were finely powdered in a mortar and pestle. Sample powder equivalent to weight of one tablet was accurately weighed and transferred into a 1000 ml volumetric flask. About 500 ml of the diluent was added and sonicated for 15 mins. and diluted up to 1000 ml with the diluent (1000 ppm). Further 1 ml of this solution was diluted to 10 ml with the diluent. (100ppm). Few ml of the 100 ppm solution was filtered through a PTFE 0.45 micron membrane filter paper and used for assay of Metformin. For Glimepiride, weight equivalent to 1 tablet was taken in a 100 ml flask, about 25 ml Diluent Methanol was added, sonicated for 15 mins, and then diluted to 100 ml with Methanol (200 ppm). 5 ml of the above  tablet solution was further diluted to 10 ml (100 ppm) The representative chromatogram of test Metformin and Glimepiride is shown in Fig. 3. The amounts of Metformin and Glimepiride per tablet were calculated by from the calibration curve. The analysis procedure was repeated three times with tablet formulation. Tablet Assay for %Label claim result was shown in Table 1.

Fig 2: Representative Chromatogram of Metformin Sitagliptin Formulation

Fig 3: Representative Chromatogram of Glimepiride Metformin Formulation

Table 1: Analysis Of Marketed Sample

Drug

RT (min)

Area (mV*s)

Area%

Glimepiride

17.5

3116050

98.26

Metformin

4.7

4245317

98.62

Sitagliptin

14.0

1381070

100.76

RESULTS:

Linearity and Range: The data obtained in the calibration experiments when subjected to linear regression analysis showed a linear relationship between peak areas and concentrations in the range 50 -150 µg/ml for Metformin & Glimepiride and 25-75µg/ml for Sitagliptin. The respective linear equation for Metformin was y = 43392x + 142874, Glimepiride equation y = 29092x + 26589 and Sitagliptin equation y = 8478.2x + 13608, where x is the concentration and y is area of peak. The correlation coefficient was 0.999. The calibration curve of Metformin, Glimepiride and Sitagliptin is depicted in Fig. 4, 5 and 6 respectively.

Fig 4: Calibration Curve of Glimepiride

Fig 5: Calibration Curve of Metformin

Fig 6: Calibration Curve of Sitagliptin

Accuracy: It is defined as the closeness of agreement between the actual (true) value and analytical value and obtained by applying the test method a number of times. The accuracy of the methods was determined at three different concentration levels, i.e., 50%, 100%, and 150% in triplicate for each drug as per ICH guidelines. From the total amount of drug found, the percentage recovery was found in a range of     98 - 102% Table 2.

Precision: The precision of method was established by carrying out (n=6) analysis of analyte using the proposed method. The low volume of standard deviation showed that the method is precise. The result obtained are shown in Table 1.

Table 2: Statistical Validation of Recovery Studies of Glimepiride, Metformin and Sitagliptin

Drug concentration level

Drug

% Mean Recovery

 

Glimepiride

101.26%

50%

Metformin

98.62%

 

Sitagliptin

98.86%

100.0%

Glimepiride

98.26%

 

Metformin

98.62%

 

Sitagliptin

99.12%

150%

Glimepiride

100.70%

 

Metformin

101.30%

 

Sitagliptin

100.53%

DISCUSSION: The proposed method for simultaneous estimation of Metformin, Glimepiride and Sitagliptin in tablet dosage forms is simple, accurate, economical, rapid and LCMS compatible. The method was validated as per the ICH guidelines. Standard calibration curves for Metformin, Glimepiride and Sitagliptin were linear with correlation coefficients (r2) values in the range. The values of % RSD are within the prescribed limit of 2%, showing high precision of method, and recovery was within 98-102% for all the three drugs. The analysis of pharmaceutical formulations reveals that the proposed methods is suitable for their simultaneous determination with virtually no interference of usual additive present in pharmaceutical formulations. Hence, the above method can be applied successfully to estimate Metformin, Glimepiride and Sitagliptin in formulations simultaneously.

CONCLUSION: A simple, rapid, accurate and precise RP-HPLC methods has been developed and validated for the routine analysis of Metformin, Glimepiride and Sitagliptin in API and tablet dosage forms. The developed method can be applied for routine and quality control analysis of the investigated drugs in combination formulations. The amount found from the proposed methods was in good agreement with the label claim of the formulation. Also, the value of standard deviation and coefficient of variation calculated were satisfactorily low, indicating the suitability of the proposed methods for the routine estimation of tablet dosage forms.

ACKNOWLEDGEMENT: The authors are thankful to the Director of Ultrapure Analytics Mumbai, Maharashtra for providing necessary facilities for research work. They are also grateful to FDC Lab for providing the gift samples of pure drugs and finally my guide Dr. Ramesh Shriranga Yamgar, Professor & Head - Department of chemistry C. S.’s Patkar - Varde college, Goregaon (w), Mumbai – 400062 for all the guidance provided to me during my research work.

REFERENCES

  1. Simultaneous Determination of anti-diabetic drugs by Nawab Sher, Nasreen Fatima1,  
  2. Shahnaz Perveen, Farhan Ahmed Siddiqui, Braz. J. Pharm. Sci. 2019;55:e17394
  3. Applied science Received: 31 May 2019; Accepted: 22 July 2019; Published: 29 July 2019
  4. Tablet Scoring: Current Practice, Fundamentals, and Knowledge Gaps by Emmanuel
  5. Epidemiology of type 2 diabetes in India, Indian Journal of Ophthalmology Vol.69 (11), Nov 2021
  6. Guidance for Industry: Tablet Scoring, Nomenclature, Labelling, and Data for Evaluation.
  7. IP 2022, Government of India, Ministry of health and family welfare, Ghaziyabad,
  8. British Pharmacopoeia 2023, Tablets general Notices.
  9. U.S. Pharmacopoeia/NF 2022 Issued 2 Published February 01 2022.
  10. London Medicines and Health Care Product Regulatory Agency. British Pharmacopoeia. Vol. I. London: Stationary Office, London Medicines and Health Care Product Regulatory Agency; 2010. p. 254-5.
  11. Council of Europe. European Pharmacopoeia 6.0. Vol. II. 6th ed. Starboary: Council of Europe; 2008. p. 1292.
  12. ICH Guideline Q2 (R2) Guideline Glimepiride, Metformin and Sitagliptin Information available from Public domain.

Reference

  1. Simultaneous Determination of anti-diabetic drugs by Nawab Sher, Nasreen Fatima1,  
  2. Shahnaz Perveen, Farhan Ahmed Siddiqui, Braz. J. Pharm. Sci. 2019;55:e17394
  3. Applied science Received: 31 May 2019; Accepted: 22 July 2019; Published: 29 July 2019
  4. Tablet Scoring: Current Practice, Fundamentals, and Knowledge Gaps by Emmanuel
  5. Epidemiology of type 2 diabetes in India, Indian Journal of Ophthalmology Vol.69 (11), Nov 2021
  6. Guidance for Industry: Tablet Scoring, Nomenclature, Labelling, and Data for Evaluation.
  7. IP 2022, Government of India, Ministry of health and family welfare, Ghaziyabad,
  8. British Pharmacopoeia 2023, Tablets general Notices.
  9. U.S. Pharmacopoeia/NF 2022 Issued 2 Published February 01 2022.
  10. London Medicines and Health Care Product Regulatory Agency. British Pharmacopoeia. Vol. I. London: Stationary Office, London Medicines and Health Care Product Regulatory Agency; 2010. p. 254-5.
  11. Council of Europe. European Pharmacopoeia 6.0. Vol. II. 6th ed. Starboary: Council of Europe; 2008. p. 1292.
  12. ICH Guideline Q2 (R2) Guideline Glimepiride, Metformin and Sitagliptin Information available from Public domain.

Photo
Nandini Joshi
Corresponding author

Post Graduate and Research Laboratory, Department of Chemistry C. S.’S Patkar – Varde College, Goregaon (W), Mumbai – 400062.

Photo
Dr. Ramesh Yamgar
Co-author

Post Graduate and Research Laboratory, Department of Chemistry C. S.’S Patkar – Varde College, Goregaon (W), Mumbai – 400062.

Photo
Dr. Dattatraya Desai
Co-author

Post Graduate and Research Laboratory, Department of Chemistry C. S.’S Patkar – Varde College, Goregaon (W), Mumbai – 400062.

Nandini Joshi, Dr. Ramesh Shrirang Yamgar, Dr. Dattatraya Desai, Development and Validation of a Simple, LCMS Compatible New Assay Method for The Simultaneous Estimation of Anti-Diabetic Drugs Metformin, Sitagliptin and Glimepiride, Int. J. of Pharm. Sci., 2025, Vol 3, Issue 5, 116-122 https://doi.org/10.5281/zenodo.15321767

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