An accurate, precise and derivatized HPTLC method has been developed for the simultaneous estimation of Metformin and Sitagliptin in tablet formulation. In this method standard and sample solutions of Metformin and Sitagliptin were applied on pre-coated silica gel 60F254 TLC plate, and developed using Ammonium sulphate (0.5%)-2-Propanol-Methanol (8:1.6 :1.6v/v ), as mobile phase[1] and derivatized using CAMAG-REPROSTAR-3. A camag HPTLC system comprising of Camag Linomat-5-applicator, camag twin trough chamber, camag reprostar-3, camag TLC -3- scanner was used for the analysis. The drugs on the plate were scanned at 254 nm. The dynamic linearity range was 700-1500 ng/spot for Sitagliptin and 7-15μg/spot for Metformin. The method was validated for precision, accuracy and reproducibility
Keywords: Simultaneous estimation; HPTLC; Metformin and Sitagliptin.
Sitagliptin [SGT] (figure 1) is an oral dipeptidyl peptidase-4 (DPP-4) inhibitor, which improves glycaemic control by inhibiting DPP-4 inactivation of the incretin hormones glucagon-like peptide1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP). This increases active incretin and insulin levels and decreases glucagon levels and postglucose-load glucose excursion [2].
Metformin [MET] (Metformin HCl, N,N- dimethylimidodicarbonimidic diamide hydrochloride), is an oral antidiabetic drug [3] (figure 2). Literature survey © revealed that various analytical methods like spectrophotometric [4], HPLC [5,6] , LC-MS [7,8] and UPLC [9] methods, have been reported for the determination of Metformin and Sitagliptin , individually and combination with some other drugs. A very few HPTLC methods for Sitagliptin in combined dosage forms has so far been reported. The review of literature prompted us to develop an simultaneous method for the estimation of Sitagliptin and Metformin in combined
JANUMET Tablet used for the formulation analysis contains Metformin (500 mg) and Sitagliptin (50 mg) and it is manufactured and marketed by MSD Pharmaceuticals Pvt. Ltd, Maharashtra. Pure samples were procured from, Metformin– Orchid Pharmaceuticals, Chennai and Sitagliptin – Micro Labs, Bangalore. All the chemicals and reagents used were of analytical grade. A Camag HPTLC system comprising of Camag Linomat-5-applicator, Hamilton syringe, Camag twin trough chamber, Camag TLC scanner, and stationary phase pre coated with Silica gel 60F254 were used.
The given standard Sitagliptin 1 mg was dissolved in 1 ml Methanol, this solution used as working standard solution (1μg/1μl) for the analysis. The given standard Metformin 1 mg was dissolved in 1 ml Methanol, this solution used as working standard solution (1μg/1μl) for the analysis. Standard solutions having concentration ranging from 700-1500 ng/spot for Sitagliptin and 7-15μg/spot for Metformin were applied on TLC plates.
The given Janumet 14 tablets were powdered using Pestle & Mortar to fine powder. From this, powder 2 mg was weighed in an Electronic balance (Afcoset) and dissolved in 1 ml of Methanol, centrifuged and the supernatant liquid was taken for the HPTLC studies. This solution contains 1.41 μg of Sitagliptin and 14.1 μg of Metformin drug sample as test solution per μl. From this 1μl of Sample solution was applied on the pre-coated silica gel 60F254 plate and from the peak area obtained, the amount of Sitagliptin and Metformin in formulation was simultaneously calculated using the respective calibration graph.
The TLC plates were pre washed with ° methanol and activated by keeping at 115 for about 30 min. The samples were spotted in the form of bands of width 4 mm with 100 μl Hamilton syringe on the pre-coated silica gel 60F plate (12×10cm) and the 254 slit dimension was kept at 15 min respectively. The mobile phase Ammonium sulphate (0.5%)-2-Propanol-Methanol (8 : 1.6 : 1.6 v/v) used was in chamber and the plate saturation time was 15 min, migration distance was allowed up to 90 mm, linear ascending development was carried out in (20×10cm) twin trough glass chamber. Subsequent to the development, TLC plates were dried in current of air and kept in photo documentation chamber. The images of developed plate were captured at white light, UV 254 nm and UV 366 nm using Camag - Reprostar - 3 instrument. The developed plate was derivatized with iodine 70 An average value ± relative standard deviation of 5 observations. vapor and the images were done in white light using Camag - Reprostar -3 instrument. The derivatized plate was scanned at 254 nm using Camag-TLC-scanner-3 instrument.
The method was validated in accordance 1 with WHO guidelines ⁰ for linearity, accuracy, limit of detection, limit of quantification, inter-day and intra-day assay precision, repeatability of measurement and repeatability of sample application. Samples applied on the plate were developed with the mobile phase and the peak areas were noted. The mobile phase, Ammonium sulphate (0.5%)-2-Propanol-Methanol (8: 1.6:1.6v/v) gave Rf value of 0.35 ± 0.02 for Sitagliptin and 0.63 ± 0.04 for Metformin.
A good linear relationship was obtained over the concentration range700-1500 ng/spot for Sitagliptin and 7-15μg/spot for Metformin respectively. The linear regression data showed a regression coefficient of 0.999 for Sitagliptin and 0.999 for Metformin.
The LOD with signal/ noise ratio were found to be 25 ng and 500 ng/spot for Sitagliptin and Metformin respectively. The LOQ with signal/ noise ratio was found to be 900 ng and 1500 ng /spot for Sitagliptin and Metformin respectively.
Intra-day assay precision was found by analysis of standard drug at three times on
Formulation Label Claim Amount MET STG MET STG Janumet 500 50 495.8 48.6 * the same day. Inter-day assay precision was carried out using at three different days, and percentage relative standard deviation (%RSD) was calculated. The RSD was found to be less than 2 for both intra-day and inter-day precision. Repeatability of sample application was assessed by spotting 1 μl of drug solution, six times. From the peak areas, the percentage RSD was determined. The complete validation parameters are shown in Table 1.
Rf - resolution factor, RSD- relative standard deviation, LOD – limit of detection, LOQ – limit of quantification
The recovery study was carried out at two levels, 50%, 100 %. To the powdered formulation, the standard drugs of Sitagliptin and Metformin were added at 50 % and 100 % levels, dilutions were made and analyzed by the method. The % recovery and % RSD were calculated and found to be within the limit, as listed in
% Assay Standard % RSD MET STG MET STG MET STG 99.16 97.8 0.4743 0.8456 0.095 1.73 71
An average value ± relative standard deviation of 5 observations.
During the stage of method development different mobile phases were tried and the mobile phase comprising of Ammonium sulphate (0.5%)-2-Propanol-Methanol (8:1.
6:1.6v/v) was confirmed. The Rf value was found to be 0.35 & 0.63 for Sitagliptin and Metformin respectively. Linearity of the drug was determined by the calibration curves and the linearity based on the peak area was in the range of 700-1500 ng & 7- 15μg respectively (table 1). The regression coefficient value for Sitagliptin and Metformin are 0.999 (figure 3) and 0.999 Amount Amount MET SIT MET SIT 50% 250 25 252.42 25.5 100% 500 50 505 51.6 *
S3 1 0.35 879.6 S4 1 0.35 1087.8 S5 1 0.35 1300.4 F 1 0.63 4074.9 F 2 0.35 1170.5 M1 1 0.63 2351.9 M2 1 0.63 2889.7 M3 1 0.63 3445.9 M4 1 0.63 3505.9 M5 1 0.63 4435.3 (figure 4) respectively. The limit of quantification was determined by injecting minimum concentration of the drugs. The limit of quantification was found as 1000 ng/spot, & 50 ng/spot. The recovery was 99.5%, and 97.7% for sitagliptin and metformin (table 1) and the repeatability showed excellent % RSD less than 1.4 (table 2, table 3). The method passes all the validation parameter limits and proves to be selective, sensitive and precise. Hence the proposed method can be used for the routine assay of Metformin and Sitagliptin using HPTLC.
% Recovery % RSD MET SIT MET SIT 100.96 102 0.94 1.03 101 103.2 1.02 1.33 Assigned substance Sitagliptin standard Sitagliptin standard Sitagliptin standard Sitagliptin standard Sitagliptin standard Metformin Sitagliptin Metformin standard Metformin standard Metformin standard Metformin standard Metformin standard 72
Hence, the developed HPTLC method is precise, specific and accurate; the statistical analysis proved that the method is repeatable and selective for the simultaneous analysis of Metformin and Sitagliptin in bulk drugs and in pharmaceutical dosage forms without any interference from the excipients.
The authors are grateful to the Management, RVS College of Pharmaceutical Sciences, Sulur, Coimbatore, and Dalmiah Research Centre, Coimbatore, for providing the required facilities