International Journal of Current Research and Review
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IJCRR - 13(19), October, 2021

Pages: 39-48

Date of Publication: 11-Oct-2021


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Validated Approach of High-performance Liquid Chromatography Method for Degradation Study of Saxagliptin and Metformin HCl

Author: Abhilasha Mittal, Manoj Charde, Imran A Sheikh

Category: Healthcare

Abstract:Introduction: The study focus on its degradation pattern well know antidiabetic drugs are Saxagliptin and Metformin HCl Aim: The research study aims to validate the high-performance liquid chromatography method for the degradation study of Saxagliptin and Metformin HCl. Method: In support of the determination and to study its degradation pattern in various pharmaceutical dosage forms a novel and simple reverse phase liquid chromatographic method has been established. Saxagliptin and Metformin HCl are well known to use in Type 2 Diabetes. The planned work was performed on Younglin (S.K) isocratic System UV Detector C18 column (150 mm × 4.6 mm). A mixture of Potassium Acetonitrile: Pot. Phosphate Buffer (ph 3.2) in the ratio of 25:75 was used as mobile phase in this method and the method was validated as per ICH guidelines along with flow rate 1.0 ml/min (UV detection at 212 nm) Result: By exposing the drug the Saxagliptin and Metformin HCl to acidic, alkaline, H2 O2 and Neutral degradations forced degradation studies were performed. This method (RP-HPLC) is suitable for kinetic studies as well as the assay of pharmaceutical dosage forms and was found to be robust and specific. Conclusion: In any pharmacopoeia, Saxagliptin is not an official drug. While MET was determined by United States Pharmacopeia (USP), and British Pharmacopoeia (BP) both suggest a non-aqueous titration method using anhydrous formic acid as a solvent and 0.1 M perchloric acid as titrant for the assay of MET. An endpoint is determined potentiometrically.

Keywords: Saxagliptin, Metformin HCl, RP-HPLC, validation, stability-indicating, pharmacopoeia., USP The study focus on its degradation pattern well know antidiabetic drugs are Saxagliptin and Metformin HCl

Full Text:

Introduction

The Metformin hydrochloride- biguanide in patients with type 2 diabetes improves glucose tolerance and is a highly recommended drug, especially when compared to other antihyperglycemic drugs. Its chemical name is N, N -Dimethyl imidodicarbonimi dicyandiamide. The recommended dose is 500 mg orally twice daily or 500 to 2000 mg once daily. Lactic acidosis is a rare, but serious, problem that can occur due to the accumulation of metformin. Hemodialysis is recommended to correct the acidosis and to remove accumulated Metformin.1

Saxagliptin competes with a DPP4 inhibitor that reduces the activity of incretin hormones, thereby increasing its concentration in the blood and reducing glucose uptake and glucose-based preparation in patients with type 2 diabetes. The recommended dose is 2.5mg or 5mg orally once daily.2

      

2. Material and Methods

Materials:

2.1 Chemicals and reagents Saxagliptin and Metformin HCl were provided from Merck Laboratories ltd. HPLC grade Potassium Acetonitrile, Pot. Phosphate Buffer was procured from Merck Ltd. Millipore Mili Q plus purification system was used to prepare highly pure water.

Commercial Formulation: Saxagliptin and Metformin HCl tablets were available in the market. The tablets were checked and stored properly.

2.2 HPLC instrumentation and conditions

Chromatographic conditions

Subsequent chromatographic conditions were established by trial and error and maintained consistently throughout the study

Chromatographic conditions for proposed method

HPLC: Younglin (S.K) isocratic System UV Detector

Software:Autochro -3000

Column:4.6 x 150 mm

Stationary phase:C18 (Primesil)

Particle size packinV: 5 mm

Mobile Phase: Acetonitrile: Pot.Phosphate Buffer(ph 3.2 with opa):25: 75

Detection Wavelenth: 212 nm

Flow rate:1.0 ml/min

Temperature: Ambient

Sample size: 20 ml

2.2.1 Standard Sample Preparation

Preparation of the Metformin & Saxagliptin Standard & Sample Solution:

Standard Solution Preparation: The drug’s standard stock solution was prepared by weighing accurately and transferred 10 mg Metformin and 10mg Saxagliptin working standard into a 10ml & 100ml clean dry volumetric flask respectively. To dissolve it completely about 7ml & 70ml of diluent were added and sonicated and with the same solvent, the volume was made up to the mark. Further pipette out 2ml of Metformin and Saxagliptin into a 10ml volumetric flask and diluted up to the mark with diluents from the above prepared Stock Solution.

Sample Solution Preparation: The Sample Stock Solution was prepared by weighing accurately and transferred 936.9 mg of Metformin and Saxagliptin Tablet powder into a 100ml clean dry volumetric flask. 70ml of the diluents was added and sonicated to dissolve it completely and the volume was made up to the mark with the same solvent.

Further from the above-prepared Stock                                   

  1. Std. Metformin Hcl 1000  mg and 10 mg Saxagliptin  in 100  ml NAOH= 10,000 µgm/ml Metformin Hcl and  100µgm/ml   Saxagliptin stock-001

  2. Take   0.1 ml stock  and make 10 ml with mobile phase   =

    1 µg/m/ml Saxagliptin & 100 µgm/ml Metformin Hcl                                                                                                 

  1.  Take   0.2 ml  stock  and make 10 ml with mobile phase   =

   2 µg/ml Saxagliptin  & Metformin Hcl   200 µgm/ml                                                                                                 

  1.  Take   0.3 ml  stock  and make 10 ml with mobile phase   =

     3µm/ml Saxagliptin & Metformin Hcl   300 µgm/ml                                                                                                                                                                                      

  1. Take   0.4 ml  stock  and make 40 ml with mobile phase   =

    4 µg/ml Saxagliptin & Metformin Hcl   400 µgm/ml                                                                                                 

  1.  Take   0.5 ml  stock  and make 50 ml with mobile phase   =

                     5 µgm/ml Saxagliptin & Metformin Hcl   500 µg/ml              

Tab solution Preparation

20 tablets were taken and were triturated to powder weighing Powder wt. =12.00 gms, having average Powder Weight = 1.200   gms. / Tablet  Eq.Wt for 1000 mg= 1000 x1200 / 500 = 2400 mg. 2400 mgs was taken in 100 ml NAOH i.e =   10000 µgm/ml Metformin Hcl and 100 µgm/ml Saxagliptin STOCK –I   (Sonicate 30 min then fitter with 0.45 µm) was taken as Tablet Stock solution    

Tablet Assay

Take 0.3 ml from tab stock and makeup 10 ml with MP     3+ 300    µgm/ml    for assay

 2.3 Forced degradation studies 5, 6

To prove the stability-indicating property of the method the forced degradation studies were performed on Metformin HCl and Saxagliptin. The stress conditions employed for degradation study of Metformin Hcl and Saxagliptin include light exposure, water hydrolysis and oxidation (3 % H2O2), acid hydrolysis (1 N HCl), base hydrolysis (1N NaOH), heat (105°C), Peak purity of the principal peak in the chromatogram of stressed samples of Saxagliptin and Metformin Hcl tablets was checked using PDA detector.  

2.4 Method Ruggedness 7

Ruggedness was tested by analyzing six samples from the corresponding combination of one group by a different analyst using a different column, format and date. % RSD for% assay of ruggedness found was 0.10% for Saxagliptin and 0.32% for Metformin Hcl and Overall% RSD found was 0.14% for Saxagliptin and 0.29% for Metformin Hcl as based in Table no. 3

Linearity 8, 11

With the emergence of the analysis method, the mixture of the standard solution of Saxagliptin and Metformin Hcl at concentration concentrations of 0.5123 μg / ml to 11.2299 μg / ml of Saxagliptin and 159.6713 μg / ml to 3500.0690 μg / ml according to the analysis method according to Metformin in the HPLC system. The coefficient (r) of Saxagliptin and Metformin Hcl using the regression equation and 1 / (concentration2) of the equilibrium weight were calculated. The correlation coefficient (r) found was 0.99942 for Saxagliptin and 0.99990 for Metformin HCl.

Method Precision 13, 15

From a homogenous mixture of a single batch six samples were prepared as per the test procedure of methodology and analyzed on HPLC system to evaluate the method of precision for the analytical method. The % RSD of six samples found was 0.16% and 0.15% for % assay of Saxagliptin and Metformin Hcl as tabulated in Table no.2

Accuracy 9, 12

For the determination of the accuracy of the method, a recovery study was carried out by analyzing the samples at three different concentrations at 50, 100 and 150%. The percentage of recoveries at three concentrations was calculated.

Robustness 10, 14

A robustness study of the method was carried because of the insignificant variations of the technique, such as changes in the composition of the mobile column temperature, altering flow rate, and mobile phase.

Results:

Tablet assay: The assay results for Saxagliptin and Metformin HCl were found within the specifications. The developed method was validated as per ICH guidelines. Results are shown in Table 1.

Forced Degradation Study: In these results of the study the report on the stability-indicating assay of combination Saxagliptin and Metformin HCl by HPLC in presence of degradation products. For analysis of combination, in this method, the isocratic elution method was selected. The reason behind it is, it gave better peak width and baseline separation, which is suitable for routine analysis of combination. In the study as per ICH guidelines, the developed method was validated.

The important part of the process of drug product development is the stability testing forms. How the drug quality substance varies with time under influence of various environmental factors such as light, humidity, temperature, and enables recommendations of retest periods, shelf life to be established, storage conditions is stability testing intends to provide evidence. Results are shown in Figures 3-6.

Linearity: The calibration curve showed good linearity in the range of 100-500 mg/ml for Metformin HCl and 1-5 mg/ml For Saxagliptin. In the results, a typical calibration curve has the regression equation of y = 59.68x – 3931 and R² = 0.999 for Metformin HCl and y = 190.7x + 22.55 and R² = 0.999 for Saxagliptin. Results are shown in table no. 2 and figure 7 and 8.

Precision: The results of system precision for Metformin HCl and Saxagliptin method precision are found within the prescribed limit of ICH guidelines. Results are shown in table 3.

Accuracy: For the accuracy study from all the samples, i.e. n=3 for 80 %, n=3 for 100 % and n=3 for 120 %. The results indicated that the mean of recovery for Saxagliptin was 100.08 %, and % RSD was 0.86, similarly, the mean of recovery for Metformin Hcl was 99.99 %, and % RSD was 1.17. As both % RSD values are less than 2 % hence, the reported % recovery by the developed method of the known added amount of analyte in the sample was within the confidence intervals. The results are shown in Tables No 4 and 5.

Robustness: Chromatographic small changes influence the conditions such as organic content in pH of buffer in the mobile phase (0.2%), the wavelength of detection (5%), mobile phase (2%), and change in flow rate (10%), various previous research supported to determine the robustness of the method are also in favour of the developed RP-HPLC. The results are shown in table number 6.

Repeatability: Here, the average area, standard deviation and % RSD were calculated for two drugs and obtained as 0.5% and 0.5% respectively for Dapagliflozin and Saxagliptin (Table 16, 17).

The combination Saxagliptin and Metformin HCl in presence of degradation products by HPLC is the report on the stability-indicating assay. For analysis of combination in this method, the isocratic elution method was selected. The reason, it gave better peak width, baseline separation, which is suitable for routine analysis of combination. As per ICH guidelines, the developed method was validated. The important part of the process of drug product development is stability testing.

The important part of the process of drug product development is the stability testing forms. How the drug quality substance varies with time under influence of various environmental factors such as light, humidity, temperature, and enables recommendations of retest periods, shelf life to be established, storage conditions is stability testing intends to provide evidence. The results are shown in table number  7.

Conclusion: The procured pure drug of Saxagliptin and Metformin HCl were found to melt in the range of 149-169°C respectively. The drug was found to be freely soluble in Acetonitrile, potassium buffer, Sparingly soluble in water, alcohol. UV absorption of 10µg/ml of Saxagliptin and Metformin HCl methanol was generated and absorbance was taken in the range of 200-400 nm. λmax of saxagliptin and Metformin HCl in acetonitrile was 214nm and 234 nm respectively. It has been observed that using the mobile phase of acetonitrile + pH buffer gave adequate retention time with good peak shape. Assay for % Label claim for %RSD Calculated. The analysis process was repeated five times with tablet formulations. Tablet Assay for % Label claim for % RSD Calculated. To validate the accuracy of the established method the recovery studies were done. To analysed tablet solution, a definite concentration of standard drug (80%,100%, and 120%), to ascertain the resolution and reproducibility of the proposed chromatographic system for estimation of Saxagliptin and Metformin HCl system suitability parameters were studied. Hereby analyzing various replicates standards of Saxagliptin and Metformin HCl, the method was established. To record any intra-day & inter-day variation in the result that concluded respectively all the solution was analyzed thrice. The Robustness of a method is its capability to remain unpretentious by small thoughtful changes in parameters. Small but careful variations in the optimized method parameters were done to assess the robustness of the proposed method. The effect of changes in mobile phase composition and tailing factor of drug peak, wavelength, flow rate, and retention time was studied. The mobile phase composition was changed in proportion to flow rate was varied and wavelength change of optimized chromatographic condition. %RSD for peak area for Metformin HCl was calculated which should be less than 2%. the result was revealed in the analytical method that was determined. %RSD for peak area for Saxagliptin was calculated which should be less than 2%. the result was revealed in the analytical method that was determined. Liquid chromatography is a technique of physical separation in which the components of a liquid mixture are distributed between two immiscible phases; this has provided us with information regarding the degradation part of the drug under study.

Acknowledgement: Authors are thankful to Principal NIMS Institute of Pharmacy Jaipur and RSITC Jalgaon for giving me the opportunity to carry out research work successfully.

Conflict of interest: None

Source of Funding: Research work is self-financed

 Author’s contribution:  Complete

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A Study by Ghaffar UB et al. entitled "Correlation between Height and Foot Length in Saudi Population in Majmaah, Saudi Arabia" is awarded Best Article for Vol 12 issue 21
A Study by Siti Sarah Binti Maidin entitled "Sleep Well: Mobile Application to Address Sleeping Problems" is awarded Best Article for Vol 12 issue 20
A Study by Avijit Singh"Comparison of Post Operative Clinical Outcomes Between “Made in India” TTK Chitra Mechanical Heart Valve Versus St Jude Mechanical Heart Valve in Valve Replacement Surgery" is awarded Best Article for Vol 12 issue 19
A Study by Sonali Banerjee and Mary Mathews N. entitled "Exploring Quality of Life and Perceived Experiences Among Couples Undergoing Fertility Treatment in Western India: A Mixed Methodology" is awarded Best Article for Vol 12 issue 18
A Study by Jabbar Desai et al. entitled "Prevalence of Obstructive Airway Disease in Patients with Ischemic Heart Disease and Hypertension" is awarded Best Article for Vol 12 issue 17
A Study by Juna Byun et al. entitled "Study on Difference in Coronavirus-19 Related Anxiety between Face-to-face and Non-face-to-face Classes among University Students in South Korea" is awarded Best Article for Vol 12 issue 16
A Study by Sudha Ramachandra & Vinay Chavan entitled "Enhanced-Hybrid-Age Layered Population Structure (E-Hybrid-ALPS): A Genetic Algorithm with Adaptive Crossover for Molecular Docking Studies of Drug Discovery Process" is awarded Best article for Vol 12 issue 15
A Study by Varsha M. Shindhe et al. entitled "A Study on Effect of Smokeless Tobacco on Pulmonary Function Tests in Class IV Workers of USM-KLE (Universiti Sains Malaysia-Karnataka Lingayat Education Society) International Medical Programme, Belagavi" is awarded Best article of Vol 12 issue 14, July 2020
A study by Amruta Choudhary et al. entitled "Family Planning Knowledge, Attitude and Practice Among Women of Reproductive Age from Rural Area of Central India" is awarded Best Article for special issue "Modern Therapeutics Applications"
A study by Raunak Das entitled "Study of Cardiovascular Dysfunctions in Interstitial Lung Diseas epatients by Correlating the Levels of Serum NT PRO BNP and Microalbuminuria (Biomarkers of Cardiovascular Dysfunction) with Echocardiographic, Bronchoscopic and HighResolution Computed Tomography Findings of These ILD Patients" is awarded Best Article of Vol 12 issue 13 
A Study by Kannamani Ramasamy et al. entitled "COVID-19 Situation at Chennai City – Forecasting for the Better Pandemic Management" is awarded best article for  Vol 12 issue 12
A Study by Muhammet Lutfi SELCUK and Fatma entitled "Distinction of Gray and White Matter for Some Histological Staining Methods in New Zealand Rabbit's Brain" is awarded best article for  Vol 12 issue 11
A Study by Anamul Haq et al. entitled "Etiology of Abnormal Uterine Bleeding in Adolescents – Emphasis Upon Polycystic Ovarian Syndrome" is awarded best article for  Vol 12 issue 10
A Study by entitled "Estimation of Reference Interval of Serum Progesterone During Three Trimesters of Normal Pregnancy in a Tertiary Care Hospital of Kolkata" is awarded best article for  Vol 12 issue 09
A Study by Ilona Gracie De Souza & Pavan Kumar G. entitled "Effect of Releasing Myofascial Chain in Patients with Patellofemoral Pain Syndrome - A Randomized Clinical Trial" is awarded best article for  Vol 12 issue 08
A Study by Virendra Atam et. al. entitled "Clinical Profile and Short - Term Mortality Predictors in Acute Stroke with Emphasis on Stress Hyperglycemia and THRIVE Score : An Observational Study" is awarded best article for  Vol 12 issue 07
A Study by K. Krupashree et. al. entitled "Protective Effects of Picrorhizakurroa Against Fumonisin B1 Induced Hepatotoxicity in Mice" is awarded best article for issue Vol 10 issue 20
A study by Mithun K.P. et al "Larvicidal Activity of Crude Solanum Nigrum Leaf and Berries Extract Against Dengue Vector-Aedesaegypti" is awarded Best Article for Vol 10 issue 14 of IJCRR
A study by Asha Menon "Women in Child Care and Early Education: Truly Nontraditional Work" is awarded Best Article for Vol 10 issue 13
A study by Deep J. M. "Prevalence of Molar-Incisor Hypomineralization in 7-13 Years Old Children of Biratnagar, Nepal: A Cross Sectional Study" is awarded Best Article for Vol 10 issue 11 of IJCRR
A review by Chitra et al to analyse relation between Obesity and Type 2 diabetes is awarded 'Best Article' for Vol 10 issue 10 by IJCRR. 
A study by Karanpreet et al "Pregnancy Induced Hypertension: A Study on Its Multisystem Involvement" is given Best Paper Award for Vol 10 issue 09

List of Awardees

A Study by Ese Anibor et al. "Evaluation of Temporomandibular Joint Disorders Among Delta State University Students in Abraka, Nigeria" from Vol 13 issue 16 received Emerging Researcher Award


A Study by Alkhansa Mahmoud et al. entitled "mRNA Expression of Somatostatin Receptors (1-5) in MCF7 and MDA-MB231 Breast Cancer Cells" from Vol 13 issue 06 received Emerging Researcher Award


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International Journal of Current Research and Review (IJCRR) provides platform for researchers to publish and discuss their original research and review work. IJCRR can not be held responsible for views, opinions and written statements of researchers published in this journal

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