Rapid RP-HPLC Quantification and Stress Degradation Studies on Alogliptin, Saxagliptin, Metformin in Pharmaceutical Dosage Formulation
Vipin Prakash G1*, A Jerad Suresh2, Sarif Niroush Konari3
1Department of Pharmaceutical Analysis, The Dale View College of Pharmacy and Research Centre,
Punalal, Thiruvananthapuram - 695575, Kerala.
2 Principal, Faculty of Pharmacy, SRMC Porur, Chennai - 600116, Tamilnadu, India.
3Department of Pharmaceutical Chemistry, JDT Islam college of Pharmacy,
Jamiath Hill, Vellimadukunnu, Calicut - 673012, Kerala, India.
*Corresponding Author E-mail: 1vipin1@gmail.com, niroush@gmail.com
ABSTRACT:
A Fast and simple isocratic method of stability indicating RP-HPLC technique was established for determination of Alogliptin, Saxagliptin and Metformin in pharmaceutical dosage forms. The proposed method is suitable for multicomponent analysis of commercially existing combinations in market. An LC separation of these drugs were achieved with Inertsil ODS C18 (4.6 x 250mm, 5mm) using potassium dihydrogen phosphate adjusted to pH 3: acetonitrile (85:15%V/V) in isocratic mode at 1mL min-1, at ambient temperature and detection at 280 nm. These drugs were exposed to forced degradation studies in altered stress conditions. The proposed method was found to be precise, stability indicating as no interfering peaks were noticed. Robustness studies and assay of the dosage forms were established within the limit of ICH guidelines.
KEYWORDS: Liquid chromatography; ICH guideline; Robustness studies.
INTRODUCTION:
Alogliptin and Saxagliptin are new set of Dipeptidyl peptidase-4 (DPP-4) class inhibitor used for the improvement of glycemic control in patients with type II diabetes mellitus. metformin1 belongs to the class of biguanide. Theses combinations were used to decrease both fasting and postprandial plasma glucose levels, ensure in lowering HbA1c with low risk for hypoglycemia and weight gain. Since high demand of DPP-4 class inhibitor drug in global pharmaceutical market, it is required to develop a new cost-effective, fast and precise analytical LC method for the estimation of drug in bulk as well as pharmaceutical dosage forms.
Literature survey point out that few simultaneous LC techniques have been reported for quantitative estimation of DPP-4 inhibitor class drugs such as aloglpitin with metformin3-4 and for metformin with saxagliptin5 without stability studies but multicomponent techniques were not developed or reported for the analytical estimation on titled drugs in bulk or pharmaceutical dosage form. Moreover, existing simultaneous techniques were less economical, more time consuming and not suitable for multicomponent estimation of ALG, SAXG and MET. Hence the main objective of the project is to develop single sensitive, fast, cost-effective LC method for the quantification of alogliptin, saxagliptin and metformin and as well as to establish stress degradation nature of the drug combination.
MATERIALS AND METHODS:
Alogliptin, Saxagliptin and Metformin standards were obtained from Pharmatrain research lab, Hyderabad, India. Methanol and water (HPLC-grade) were obtained from Lichrosolv Merck and Acetonitrile (LC-grade) from Molychem. Sodium hydroxide (NaOH), hydrogen peroxide (H2O2), hydrochloric acid (HCl), potassium hydrogen phosphate (KH2PO4) and ortho phosphoric acid were from Finar chemicals and Merck respectively. Two different formulations having ALG with MET and SAXG with MET were bought from the pharmaceutical market. Instrument used in the study was waters LC equipped with 2695 separation module, UV detector and Empower software.
OPTIMIZATION OF ANALYTICAL TECHNIQUE:
LC separation of the three drugs were achieved with Inertsil ODS C18 (4.6 x 250 mm, 5 m) using mobile phase 0.025M Phosphate buffer (adjusted to pH 4): acetonitrile (85:15 % V/V) in isocratic mode at a flow rate of 1 mL min-1, column at ambient temperature and detection of all the drugs were monitored at 280 nm. All the solutions were filtered through 0.45 m filter, and degassed in an ultrasonic bath prior to use.
STANDARD AND SAMPLE SOLUTIONS:
Accurately weighed and transferred 500mg of MET, 6.25mg of ALG and 2.5mg of SAXG pure drug into a 10 mL clean dry volumetric flask. Added about 7mL of Diluent (mobile phase) and sonicated to dissolve totally and made volume up to the mark with the same solvent. Finally pipetted out 0.3mL of the above stock solutions into a 10mL standard flask and diluted up to the mark with the diluent. For formulation, 10 tablets were taken and transferred an amount equivalent to 500mg of MET, 6.25mg of ALG and 2.5mg of SAXG into 10mL standard flasks. Same procedure was followed as that of working standard.
Validation of Chromatographic Technique:
Recovery and Specificity study:
Recovery was conducted at 50, 100 and 150% (with respect to targeted assay concentration). Specificity was carried out with working placebo solution (blank), standard and formulations were introduced into the RP-HPLC system.
Precision, ruggedness, Linearity and Robustness:
Precision and ruggedness of the analytical technique were established for both system and method by using concentration of 1500, 18.75, 7.5”g mL-1 of MET, ALG and SAXG by six replicate injections. Method precisions were attained by unique sample preparations each of the three drugs from the similar batch. Linearity was validated using standard preparation of different concentrations 500, 1000, 1500, 2000, 2500 ”g mL-1 for MET, 6.25, 12.5, 18.75, 25 and 31.25”g mL-1 for ALG and 2.5, 5, 7.5, 10 and 12.5”g mL-1 for SAXG. In place of Robustness, deliberate change in the flow rate, mobile phase composition and temperature were made to evaluate the impact on the method.
Limit of detection (LOD) and Limit of quantification (LOQ):
LOD and LOQ were calculated on signal to noise ratio by equating measured analyte signals of the samples with blank and establishing the minimum concentration that can be detected as well as quantified.
DEGRADATION STUDIES:
The International Conference on Harmonization (ICH) guideline titled stability testing of new drug substances and products need the stress testing to explain the intrinsic stability features of the active substance. The aim of this work was to carry out the stress degradation studies on the titled drug using the developed method.6-23
Preparation of Standard solution :
Standard solution of MET, ALG, and SAXG were prepared to get concentration of 1500, 18.75, 7.5”g mL-1 respectively.
Hydrolytic degradation in acidic and alkaline condition:
0.3mL of above solution, 3mL of 0.1 N HCl and 0.1 N NaOH were added into a 10mL volumetric flask for acidic and alkaline conditions respectively. The volumetric flasks were kept at 60șC for 6h and then subsequently neutralized with alkaline and acidic solutions. Finally made up to 10mL with the diluent. Filtered the solution with 0.45 microns syringe filters and placed in vials of LC system
Thermal induced degradation:
MET, ALG and SAXG samples were taken in petridish and kept in Hot air oven at 1100C for 24h. Then the sample was taken and diluted with diluents and injected into LC system.
Oxidative degradation and photo degradation:
Pipetted out 0.3mL of stock solution and transferred into a 10mL volumetric flask, 1mL of 3% w/v of hydrogen peroxide was added and the volume was made up to the mark with diluent. The volumetric flask was kept at room temperature for 15mins and photo degradation was carried after exposure to sunlight for continuous 6h. Filtered the solution with 0.45 microns syringe filters and placed in vials of LC system.
RESULTS:
Optimizations of chromatographic techniques were carried out through well planned experimental design based on nature of drugs and literature review. Significant reduction in the wastage of solvent system and ecofriendly mobile phase system were achieved through experimental design especially in robustness studies (Table 1). Separation of the three drugs were achieved with Inertsil ODS C18 column (4.6 x 250 mm, 5 μm) using mobile phase 0.025 M Phosphate buffer (adjusted to pH 4) : acetonitrile (85:15 % V/V) in isocratic mode at a flow rate of 1 mL min-1, column at ambient temperature and detection of all the drugs were monitored at 280 nm.
Developed method applied for the quantification of two different commercial preparations namely metformin and alogliptin, metformin and saxagliptin. LOD, LOQ and recovery studies indicated that the method was highly fast and accurate. Results were found within ICH limit and summarized in (Table 2). The method was validated24,25 as per ICH guidelines and results are summarized in (Table 3, 4 and 5). Degradation studies expose the specificity of the developed technique in presence of degradation products, that was carried out in bulk and pharmaceutical dosage forms. It was performed for three drugs and purity of drug peaks were proven by purity angles. Pharmaceutical dosage forms were exposed to different stress conditions. (Table 6, Fig. 1-5)
Table 1: Robustness study
|
Parameter |
Modification |
USP plate count |
USP Tailing factor |
||||
|
MET |
ALG |
SAXG |
MET |
ALG |
SAXG |
||
|
Change in flow rate
|
0.9mL min-1 1 mL min-1 1.1mL min-1 |
2263.65 2112 2151.29 |
3331.30 3186.09 2971.64 |
3035.38 3353.63 3465.98 |
1.40 1.45 1.44 |
1.29 1.33 1.41 |
1.40 1.27 1.41 |
|
Change in Organic Composition of the Mobile Phase |
10% less Actual * 10% more |
2445.83 2112 2104.64 |
3594.68 3186.09 2935.13 |
5094.60 3353.63 3252.62 |
1.40 1.45 1.39 |
1.29 1.33 1.48 |
1.32 1.27 1.37 |
|
Temperature Variation |
10C less Ambient 10C more |
2445.83 2112 2151.29 |
3594.45 3186.09 2935.13 |
5094.60 3353.63 3465.98 |
1.40 1.45 1.44 |
1.29 1.33 1.48 |
1.32 1.27 1.41 |
Actual-optimized mobile phase composition
Table 2: Analysis of formulation, LOD and LOQ
|
Formulation* |
Drugs |
Label claim mg |
% label claim |
LOD ”g mL-1 |
LOQ ”g mL-1 |
|
A(MET+ALG) B(MET+SAXG) |
MET |
1000 |
99.61 |
4.98l |
16.65 |
|
ALG |
12.5 |
100.31 |
0.51 |
1.70 |
|
|
SAXG |
5 |
99.5 |
0.09 |
0.29 |
MET-Metformin, ALG-Alogliptin, SAXG-Saxaglitpin, Avg Baseline noise obtained from Blank:66 ”V for LOD(Limit of detection) and LOQ (Limit of quantification)
Table 3: Recovery study
|
Drug |
Specification level |
The amount added (mg) |
The amount found (mg) |
% recovery |
Mean % recovery |
|
MET* |
50 |
250 |
248.46 |
99.38 |
99.98 |
|
100 |
500 |
499.23 |
99.85 |
||
|
150 |
750 |
755.36 |
100.72 |
||
|
ALG* |
50 |
3.12 |
3.13 |
100.25 |
99.78 |
|
100 |
6.25 |
6.22 |
99.57 |
||
|
150 |
9.37 |
9.33 |
99.52 |
||
|
SAXG* |
50 |
1.25 |
1.26 |
100.63 |
100.10 |
|
100 |
2.5 |
2.50 |
99.67 |
||
|
150 |
3.75 |
3.74 |
99.66 |
Mean of three replicates on each level, MET-Metformin, ALG-Alogliptin, SAXG-Saxaglitpin
Table 4: Precision
|
Injection |
Metformin |
Alogliptin |
Saxagliptin |
|
Injection-1 |
957498.0 |
158363.0 |
89485.0 |
|
Injection-2 |
958373.0 |
158376.0 |
89474.0 |
|
Injection-3 |
958377.0 |
158237.0 |
89648.0 |
|
Injection-4 |
958374.0 |
158373.0 |
89467.0 |
|
Injection-5 |
959484.0 |
158932.0 |
89364.0 |
|
Injection-6 |
954484.0 |
158383.0 |
89464.0 |
|
Average |
957765.0 |
158444.0 |
89483.7 |
|
Standard Deviation |
1726.6 |
245.3 |
91.7 |
|
% RSD |
0.2 |
0.2 |
0.1 |
Table V: Intermediate Precision/Ruggedness
|
Injection |
Metformin |
Alogliptin |
Saxagliptin |
|
Injection-1 |
959473.0 |
158387.0 |
87983.0 |
|
Injection-2 |
958474.0 |
158327.0 |
87838.0 |
|
Injection-3 |
958373.0 |
158363.0 |
87537.0 |
|
Injection-4 |
958363.0 |
158736.0 |
87538.0 |
|
Injection-5 |
959373.0 |
158373.0 |
87373.0 |
|
Injection-6 |
958363.0 |
157368.0 |
87293.0 |
|
Average |
958736.5 |
158259.0 |
87593.7 |
|
Standard Deviation |
534.3 |
461.8 |
267.1 |
|
% RSD |
0.1 |
0.3 |
0.3 |
Fig. 1 Acid Degradation
Fig. 2 Base Degradation
Table 6: Forced Degradation Stress Study
|
Parameter |
Metformin |
Alogliptin |
Saxagliptin |
||||||
|
PA * |
Degd * |
Deg* |
PA * |
Degd * |
Deg * |
PA * |
Degd* |
Deg * |
|
|
Standard |
956581 |
3.81 |
Nil |
154741 |
8.87 |
Nil |
89833 |
4.53 |
Nil |
|
Acid |
920116 |
3.02 |
Peak 2 |
141008 |
7.88 |
Peak 2 |
85764 |
3.92 |
Peak 2 |
|
Base |
927681 |
3.61 |
Peak 1,3 |
142549 |
6.58 |
Peak 1,3 |
86313 |
3.03 |
Peak1,3 |
|
Peroxide |
922023 |
3.64 |
Peak 2 |
144556 |
5.92 |
Peak 2 |
87109 |
3.47 |
Peak 2 |
|
Thermal |
921805 |
3.63 |
Peak 2 |
145584 |
5.85 |
Peak 2 |
86720 |
3.55 |
Peak 2 |
|
Photo |
921845 |
3.81 |
Peak 1 |
145686 |
8.87 |
Peak 1 |
86646 |
4.53 |
Peak 1 |
PA-Peak Area, % Degd-Degradation, Deg-degradent
Fig. 3 Peroxide Degradation
Fig. 4 Photolytic Degradation
Fig. 5 Thermal Degradation
DISCUSSION:
Rising demand of DPP-IV product in pharmaceutical market, it was required to develop a sensitive and rapid RP-HPLC quantification on alogliptin, saxagliptin and metformin in pharmaceutical dosage formulation. Furthermore, consumption of the mobile phase was very less since total eluting time was within 8 mins. Hence the developed method was economical, reliable, fast and sensitive. Moreover, overlay chromatogram and forced degradation study direct that the method was very selective and novel technique without the interferences of degradants produced from the formulation or from excipients (Fig. 2). Validation study for developed method as per ICH guidelines indicate that the method was highly precise, robust and accurate for multicomponent analysis of the titled drugs in combined dosage form. The developed technique point out the monitoring of the drug stability and quick routine analysis of pharmaceutical formulation in bio analytical Laboratory, therapeutic drug monitoring (TDM) for clinical trials, quality control division of pharmaceutical industries, bio-equivalence studies of combos and 54clinical pharmacokinetics.
ACKNOWLEDGEMENTS:
Authors are grateful to Pharma Train, Hyderabad for providing research facilities and drug samples. We express our gratitude to Mr. Madheesh senior researcher for his continuous inspiration to complete the project.
REFERENCES:
1. Ministry of Health and Family Welfare, Government of India., Indian Pharmacopoeia., The Controller of Publication., New Delhi., 2010, 1657-1660.
2. Hani N., Ramzi M. and Moammal Q. Development and Validation of an HPLC Method for Determination of Antidiabetic Drug Alogliptin Benzoate in Bulk and Tablets. J. Anal. Methods Chem. 2018; 1(1): 1-7.
3. Chinnalalaiah R., Ravi K. and Srinivasa R. Stability indicating RP-HPLC Method for Simultaneous Estimation of Alogliptin Benzoate and Metformin Hydrochloride in Tablet Dosage Form. Int. J. Pharm Sci. 2016; 8(1): 116-120.
4. Hanan A., Merey K., Sherine S., Diab A. and Moustafa. Chromatographic Methods for the Simultaneous Determination of Binary Mixture of Saxagliptin HCl and Metformin HCl. Bulletin of Faculty of Pharmacy Cairo University. 2017; 55(2): 311-317.
5. Blessy M., Ruchi D., Prajesh N., Prajapati. and Agrawal Y. Development of Forced Degradation and Stability Indicating Studies of Drugs A review. J. Pharm. Anal. 2014; 4(1): 159-165.
6. Bhavana Habib, Jyoti Mittha. Quality Evaluation of Generic Products of Metformin and Vildagliptin Tablets. Asian Journal of Pharmaceutical Analysis. 2021; 11(4): 255-8. doi: 10.52711/2231-5675.2021.00043
7. Vaishali P. Shelke. Development and Validation of RP-HPLC Method for the Estimation of Gemigliptin and Metformin Hydrochloride. Asian Journal of Pharmaceutical Analysis. 2021; 11(4): 259-2. doi: 10.52711/2231-5675.2021.00044
8. Ceema Mathew, Sunayana Varma. Green Analytical Methods based on Chemometrics and UV spectroscopy for the simultaneous estimation of Empagliflozin and Linagliptin. Asian Journal of Pharmaceutical Analysis. 2022; 12(1): 43-8. doi: 10.52711/2231-5675.2022.00008
9. M. M. Eswarudu, G. Ouchitya, N. Sudhakar Reddy, M. Deekshitha, P. Srinivasa Babu. Review on Analytical Methods for Estimation of Antidiabetic Drugs: Empagliflozin, Linagliptin and Metformin Hydrochloride. Asian Journal of Pharmaceutical Analysis. 2023; 13(1): 42-6. doi: 10.52711/2231-5675.2023.00007
10. Vashi Dhara, Chaudhari Hetvi. Development and Validation of UV Spectroscopic Method for Simultaneous Estimation of Remogliflozin Etabonate and Vildagliptin in bulk and Pharmaceutical Dosage Form. Asian Journal of Pharmaceutical Analysis. 2023; 13(2): 69-3. doi: 10.52711/2231-5675.2023.00012
11. Sushil D. Patil, Sunil V. Amurutkar, C.D. Upasani. Development and Validation of Stability Indicating RP-HPLC Method for Empagliflozin. Asian J. Pharm. Ana. 2016; 6(4): 201-206. doi: 10.5958/2231-5675.2016.00030.2
12. Konari S., and Jacob J. Stability Indicating Isocratic RP-HPLC DAD Method for the Simultaneous Estimation of Flucloxacillin and Ampicillin in Pharmaceutical Dosage Form: Application for Routine Drug. Inventi impact: Pharm Analysis and Quality Assurance. 2015; 3: 158-164
13. Priya Barbude, Mukund Tawar, Prashant Burange. Method Development using a UV Visible Spectrophotometer for the Simultaneous Estimation of Metformin (MET), Saxagliptin (SXG), and Dapagliflozin (DGF) in Marketed Formulation. Asian Journal of Pharmaceutical Analysis. 2022; 12(4): 243-7. doi: 10.52711/2231-5675.2022.00039
14. Amitkumar J. Vyas, Chirag D. Jadav, Ajay I. Patel, Ashok B. Patel, Sunny R. Shah, Devang Sheth, Sandip Dholakia. Review on Stability Indicating Assay Method or Forced Degradation Study: Strategy and Regulatory Consideration. Asian Journal of Pharmaceutical Analysis. 2023; 13(2): 131-9. doi: 10.52711/2231-5675.2023.00022
15. Sarif Niroush Konari, Jane T Jacob, Vipin Prakash. Stability Indicating UV Spectrophotometric Method for Linagliptin and Metformin in Pharmaceutical Dosage Form. Pharm Methods. 2017; 8(2): 121-126
16. Akash D. Rajmane, Komal P. Shinde. A Review of HPLC Method Development and Validation as per ICH Guidelines. Asian Journal of Pharmaceutical Analysis. 2023; 13(2): 143-1. doi: 10.52711/2231-5675.2023.00024
17. Darshan A. Salade, Kishor S. Arote, P. H. Patil, Pankaj S. Patil, Amol R. Pawar. A Review on Pharmaceutical Cleaning Validation. Asian Journal of Pharmaceutical Analysis. 2022; 12(3): 197-2. doi: 10.52711/2231-5675.2022.00033
18. Akhilesh Gupta, Vimal Yadav, Jaydeep S. Yadav, Swati Rawat. An Analytical Approach of Doxofylline: A Review. Asian J. Pharm. Ana. 2011; 1(4): 67-70
19. Sarif Niroush Konari, Jane T Jacob. Stability Indicating LC Analytical Method Development and Validation for the Simultaneous Estimation of Flucloxacillin and Amoxicillin in Pharmaceutical Dosage Form. J.Taibah Uni. Sci. 2015; 9(2): 167-176.
20. Konari S. and Jacob J. Stability indicating Validated UPLC Technique for the Simultaneous Analysis of Raltegravir and Lamivudine in Pharmaceutical Dosage Forms, HIV and AIDS Review. 2016; 15(4): 161-169.
21. Sarif Niroush Konari, Jane T Jacob. Stability indicating Validated RP-HPLC Technique for the Analysis of Multicomponent Anti-Diabetic Drug Combos in Pharmaceutical Dosage Forms. Karbala Int. J. Mod. Sci. 2015; 1(1): 39-48,
22. Lakshmana A., Prasanthi T. and Anusha L. RP-HPLC Method Development and Validation for Simultaneous Estimation of Linagliptin and Empagliflozin. Indian drugs. 2019; 56(5): 68-71.
23. Jacob J. and Konari S. Application of Analytical Validated High-Performance Thin-Layer Chromatographic Technique for the Multicomponent Analysis of Cardiovascular Drug Combos in Pharmaceutical Dosage Form. J. Planar Chromat. Modern TLC. 2015; 28(5): 354-361.
24. International Conference on Harmonization. ICH.Q2 (R1)., Validation of Analytical Procedures. Text and Methodology. Geneva. November 2005.
25. International Conference on Harmonization. ICH.Q1A (R2). Stability Testing of New Drug Substances and Products. Geneva. November 2005.
Received on 14.06.2023 Modified on 06.10.2023
Accepted on 20.12.2023 ©Asian Pharma Press All Right Reserved
Asian J. Pharm. Ana. 2024; 14(1):11-16.
DOI: 10.52711/2231-5675.2024.00003