Method Development and Validation of Metformin Hydrochloride and Canagliflozin in Bulk and Tablet Dosage Forms by RP-HPLC

On March 29, 2013, the FDA authorized canagliflozin, making it the country's first sodium glucose co-transportor 2 (SGLT2) inhibitor. For individuals with type 2 diabetes mellitus, canagliflozin is recommended as a supplement to diet and exercise to improve glycemic control. Canagliflozin should not be used to treat diabetic ketoacidosis or in people with type 1 diabetes mellitus. In 1950, French physician Jean Sterne started studying metformin in humans after it was discovered in 1922. Metformin is mostly used to treat type-2 diabetes, however its usage in polycystic ovarian syndrome is growing. Diarrhea, nausea, and abdominal pain are common adverse effects of metformin, which is normally well tolerated. The first-line treatment for type-2 diabetes is metformin, which is sold under the trade name Glucophage. By specifically blocking renal sodium-glucose transporter 2 (SGLT2), canagliflozin increases the excretion of glucose in the urine. This mode of action is independent of insulin and may be used in conjunction with other oral antidiabetic medications.

Fig 1: Structure of Canagliflozin.

SGLT2, which is responsible for over 90% of renal glucose reabsorption, is inhibited by canagliflozin. As a result, the amount of glucose that is filtered through the glomeruli and enters the tubular lumen determines how effective this medication is. As a result, it works best in individuals with uncontrolled type 2 diabetes. In addition to lowering blood glucose, it has numerous additional positive effects, such as lowering glycosylated hemoglobin levels as a result of improved blood glucose regulation. Additionally, it increased the liver's sensitivity to insulin by lowering blood glucose levels, which in turn decreased the liver's synthesis of glucose. In T2DM patients, this lowers the body's overall gluco-toxic condition and aids in lowering serum insulin levels. Patients using this medication have a negative energy balance and weight loss as a result of the calories being expelled from their bodies in the form of glucose in their urine, which is advantageous for T2DM patients once more. Because of its diuretic impact and slight weight loss, this medication also lowers blood pressure. Because it causes minor weight reduction, it also has a favorable effect on blood lipids.

A collection of metabolic disorders characterized by elevated blood glucose levels is referred to as diabetes mellitus.  The bigunaide class of oral antihyperglycemic medications includes the anti-diabetic medication metformin hydrochloride.  6.2-hour half-life. The activity lasts for eight to twelve hours.  Metformin lowers blood glucose levels via reducing intestinal glucose absorption and hepatic glucose synthesis. Different concentrations of polymers of different grades of HPMC, carboxymethyl cellulose, calcium phosphate dibasic anhydrous, micro crystalline cellulose, PVP, and lactose monohydrate were used to make metformin hydrochloride and gliclazide. Different pellet forms, such as disintegrating pellets, coated pellets, and metformin pellets, were found to have separate effects on the formulation. An HPMC/PVA-based TDS-patch was created to assess the impact of pH on transdermal medication delivery. Floating Metformin HCl tablets are intended to extend the stomach residence time following oral delivery and have demonstrated sustained release through appropriate duration of action at a specific region. Metformin HCl's chemical formula is -1-carbamimidamido-N, N-dimethymethanimidamide. C4H11N5 is the molecular formula.HCL has a molecular weight of 165.63 and a melting point of 165.63. The solubility is nearly insoluble in acetone, ether, and chloroform but freely soluble in water and methanol.

Fig 2: Structure of Metformin HCl

Compared to other classes of oral antihyperglycemic medications, metformin has a different mode of action. Metformin lowers blood glucose levels by reducing intestinal glucose absorption and hepatic glucose synthesis. It also improves insulin sensitivity by enhancing peripheral glucose uptake and utilization. Metformin's early activation of AMP-activated protein kinase (AMPK), a liver enzyme crucial to insulin signaling, the body's overall energy balance, and the metabolism of lipids and carbohydrates, mediates these effects. Improved insulin binding to insulin receptors may be the cause of increased peripheral glucose consumption.
In skeletal muscle, metformin treatment also raises AMPK activation. Insulin-independent glucose absorption is known to occur when GLUT4 is deployed to the plasma membrane by AMPK.

MATERIAL AND METHOD:

An isocratic RP-HPLC method was performed on a Waters Alliance e2695 HPLC system with 515 HPLC pump, equipped with 2998 Photo Diode Array (PDA) detector and Empower 2 software for processing and data collecting. Kromasil particle size) is used as a stationary phase. An ultrasonic bath sonicator (Frontline FS 4, Mumbai, India), semi-micro analytical balance (India) and Whatman filter paper No. 41 is used in the study.

Preparation of mobile phase

An accurately weighed quantity of 0.77 g of Ammonium acetate was taken into a 1000 mL beaker and diluted to 1000 mL with HPLC grade water and degassed in ultrasonic water bath and filtered through 0.45 μm nylon membrane filter using vacuum filtration gives required buffer concentration of 0.01 M Ammonium acetate buffer and the pH was adjusted to 3.5 with orthophosphoric acid. 0.01 M Ammonium acetate buffer with pH adjusted to 3.5 with orthophosphoric acid were mixed with HPLC grade Acetonitrile in the proportion of 65:35, v/v and it was filtered through 0.45 μm nylon membrane filter and degassed by ultrasonication.

Preparation of MET and CAN mixed standard drug stock solutions

The mixed standard drug stock solutions of Metformin Hydrochloride and Canagliflozin were prepared by dissolving 500 mg of Metformin Hydrochloride and 50 mg of Canagliflozin in 100 mL of mobile phase into a 100 mL of volumetric flask and then sonicated to dissolve it completely to get the concentration of 5000 μg/mL of Metformin Hydrochloride and 500 μg/mL of Canagliflozin.

Chromatographic Parameters:

Equipment: Waters Alliance e2695 HPLC system with 2998 PDA detector

Mobile Phase : 0.01 M Ammonium acetate buffer (pH adjusted to 3.5 with orthophosphoric acid) and Acetonitrile (65:35, v/v)

Flow rate : 1 mL/min

Detection Wavelength: 254 nm

Column temperature: Ambient

Run time : 8 minutes

Method validation for bio-analytical studies consist of procedures that shows a suitable method for quantitative analysis of drug analytes present in the biological fluids such as blood, plasma, serum and urine was reproducible and reliable for the future purpose. The essential factors for bio-analytical method validation consist of: (1) Accuracy (2) Precision (3) Selectivity (4) Sensitivity (5) Reproducibility and (6) Stability.

RESULTS AND DISCUSSION:

Method optimization

For the optimisation of RP-HPLC method several parameters and mobile phase compositions were tried. A satisfactory separation and good peak symmetry for Metformin Hydrochloride and Canagliflozin were obtained with Kromasil C18 column (250 mm×4.6 mm, 5 m particle size) and mobile phase containing a mixture of 0.01 M Ammonium acetate buffer (pH adjusted to 3.5 with orthophosphoric acid) and Acetonitrile (65:35, v/v) was delivered at a flow rate of 1 mL/min to get better reproducibility and repeatability. Both Metformin Hydrochloride and Canagliflozin were scanned in the wavelength region of 200-400 nm by using photo diode array (PDA) detector. Quantitation was attained with a PDA detector at 254 nm depends on peak area. Therefore 254 nm was selected as detection wavelength in the present study. The retention time of Metformin Hydrochloride and Canagliflozin was found to be 2.440 min and 3.713 min respectively. A typical chromatogram of blank, standard and sample solution of Metformin Hydrochloride and Canagliflozin is shown in Figure 3.

Figure 3 Chromatogram of blank, standard and sample solution of Metformin Hydrochloride and Canagliflozin

Figure 4 Standard calibration curves of MET and CAN

CONCLUSION:

A simple, rapid, accurate, precise, sensitive, and robust RP-HPLC method was successfully developed and validated for the simultaneous estimation of Metformin Hydrochloride (MET) and Canagliflozin (CAN) in bulk and pharmaceutical dosage forms in accordance with ICH guidelines. The method demonstrated excellent linearity, accuracy, precision, specificity, and robustness, with satisfactory recovery and low detection and quantification limits. Stability and forced degradation studies confirmed that the method is stability-indicating and capable of effectively separating degradation products from the analytes. Therefore, the developed RP-HPLC method is reliable and suitable for routine quality control, stability testing, and pharmaceutical analysis of MET and CAN in combined dosage formulations.

REFERENCES

1. Niessen WMA. Liquid chromatography-mass spectrometry, chromatographic science series, 2006, 97, 3rd edition, Taylor and Francis, London.
2. Marvin CM. LC/MS: A Practical User's Guide, Wiley, Hoboken, NJ, 2005.
3.  Niessen WMA, Voyksner RD. Current Practice in Liquid Chromatography-Mass Spectrometry, 1st Edition, Elsevier, Amsterdam, 1998.
4. Snyder LR, Kirkland JJ, Dolan JW. Introduction to Modern Liquid Chromatography, 2010, 3rd Edition, 185.
5. The European Medicines Agency, Pre-Authorisation Evaluation of Medicines for Human Use, ICH Q2 (R1), Validation of Analytical Procedures: Text and Methodology, 2009, EMEA/410412/2007, London.
6. Validation of Analytical Procedures: Methodology, ICH Harmonised Tripartite Guidelines, 1996, 1-8.
7. United States Pharmacopoeia and National Formulary, Asian Edition 24, the United States Pharmacopoeia Convention Inc., U.S.A., 2149- 2152.
8. Quality Assurance of Pharmaceuticals, (A compendium of guidelines and related materials), 1997, 1, WHO, Geneva, 119-124.
9. ICH Guidelines Q2 (R1) Validation of Analytical Procedures: Text and Methodology, Current Step 4 version Parent Guideline, 1994, 1-13.
10. Takahashi Y, Amano Y, Yuki T, Ose T, Miyake T, Kushiyama Y, Sato S, Ishihara S and Kinoshita Y. Influence of acid suppressants on gastric emptying: cross-over analysis in healthy volunteers. J Gastroenterol Hepatol. 2006; 21(11):1664-8.
11. Porro PB. Famotidine in the treatment of gastric and duodenal ulceration: overview of clinical experience. Digestion 1985; 32 (1): 62- 69.
12. Langtry HD, Grant SM, Goa KL. Famotidine, an Updated Review of its Pharmacodynamic and Pharmacokinetic Properties, and Therapeutic Use in Peptic Ulcer Disease and Other Allied Diseases. Drugs 1989; 38 (4): 551–590.
13. Schunack W. Pharmacology of H2-receptor antagonists: an overview. The Journal of International Medical Research 1989; 17(1): 9A-16A.
14. Savio CR, Irfan S and Richard WM. Domperidone: Review of Pharmacology and Clinical Applications in Gastroenterology. The American Journal of Gastroenterology 2007; 102: 2036–2045.
15. Deshpande P, Gandhi S, Vandana B, Raviraj B, Abhijeet D, Vrushali Diwale. High Performance Thin Layer Chromatographic Determination of Famotidine and Domperidone in Combined Tablet Dosage Form. Research Journal of Pharmaceutical, Biological and Chemical Sciences 2010; 1(4): 354-359.
16. Helali N, Darghouth F, Monser L. RP-HPLC Determination of Famotidine and its Potential Impurities in Pharmaceuticals. Chromatographia 2004; 60(7): 455–460.
17. Ahsanul Haque Md, Shahriar Md, Parvin MN and Ashraful Islam S M. Validated RP-HPLC Method for Estimation of Ranitidine Hydrochloride, Domperidone and Naproxen in Solid Dosage Form. Asian J. Pharm. Ana. 2011; 1(3): 59-63.
18.  Sahu R, Preeti Nagar, Bhattacharya S, Deepti Jain. Simultaneous spectrophotometric estimation of famotidine and domperidone in combined tablet dosage form. Indian Journal of Pharmaceutical Sciences 2006; 68(4): 503-506.
19. Dipali D. Tajane, Sacchidanand R. Gite, Aditi R. Shah, Arun B. Kale, Ranjit V. Gadhave and Vishnu P. Choudhari. Spectrophotometric Simultaneous Determination of Famotidine and Domperidone in Combined Tablet Dosage Form by Ratio Derivative and Area under Curve Method. Der Pharmacia Sinica 2011; 2(3): 60-66.
20. Rajani Sekhar V, Reddy YP, Ramalingam P, Thej DH. RP-HPLC and UV-derivative spectrophotometry technique for the simultaneous estimation of ibuprofen and famotidine in pharmaceutical dosage form. Der Pharmacia Sinica 2013; 4(2):160-170.
21. Patel AH, Patel JK, Patel KN, Rajput GC, Rajgor NB. Development and Validation of Derivative Spectrophotometric Method for Simultaneous Estimation of Domperidone and Rabeprazole Sodium in Bulk andDosage Forms. International Journal on Pharmaceutical and Biological Research 2010; 1(1): 1-5.