Saxagliptin (API) was obtained as a gift sample from Mylan laboratories Ltd. (Hyderabad, India). Onglyza (Saxagliptin-5 mg) tablets were purchased from Bristol-Myers Squibb (USA). Solvents like Chloroform and Methanol were purchased from SD fine chemicals Ltd. (Mumbai, India).

It consists of an applicator of model LINOMAT-5 and a scanner (CAMAG, Switzerland) with UV detector. The data was monitored and processed using WINCAT software. The sample was loaded on to the TLC plates by Hamilton Rheodyne injector syringe of 100 µL (CAMAG) and the plate was developed in twin trough chamber (20 × 20).

Standard stock solution was prepared by dissolving 10 mg of Saxagliptin in 100 mL of methanol to obtain a solution with final concentration, 100 µg/mL.

Ten tablets of ONGLYZA were weighed and finely powdered. An accurately weighed powder equivalent to 10 mg was taken to 100 mL volumetric flask and dissolved in 50 mL Methanol and finally volume was made up to the mark with the Methanol to obtain a concentration of 100 µg/mL. This solution was sonicated for 10 min and filtered. Filtration is done to remove the excipients and to avoid clogging of sample applicator syringe.

Different concentration solutions of Saxagliptin like 400, 600, 800, 1000 and 1200 ng/spot were applied on the TLC plate from the standard stock solution. After the development the TLC plate was scanned under UV at 222 nm and the peaks were recorded using WINCATS software version 1.4.3. The calibration curve was obtained by plotting peak area vs. concentration, using linear regression model. According to ICH guidelines, an acceptance criterion of linearity for an analytical method is correlation coefficient should be not less than 0.998.

The limit of detection (LOD) and limit of quantification (LOQ) of Saxagliptin by the developed HPTLC method were calculated using the following formulae, respectively.

where σ is the standard deviation of the response and S is the slope of the calibration curve.

where σ is the standard deviation of the response and S is the slope of the calibration curve.

Accuracy is calculated as the percentage of recovery by the assay of known added amount of analyte in the sample, or as the difference between the mean and the accepted true value, together with the confidence intervals. As per ICH guidelines accuracy should be assessed using a minimum of nine determinations i.e., three replicates at three concentrations across the specified range of the procedure. Three different levels (50%, 100% and 150%) of standard solutions were added to a pre-analyzed sample and were analyzed for % recovery of Saxagliptin. Acceptance criterion is the mean % recovery should not be less than 98% and not more than 102%.

ICH guidelines recommend that repeatability should be assessed using a minimum of nine determinations covering the specified range of the procedure (i.e., three replicates of three concentrations) or using a minimum of six determinations at 100% of the test concentration. It can be determined by measuring repeatability, intermediate precision and reproducibility. Three different concentration solutions of standard Saxagliptin were applied to the TLC plate and the chromatograms were recorded. The precision of an analytical procedure is usually expressed as the variance, standard deviation or coefficient of variation of a series of measurements and the acceptance criterion is % RSD of the peak areas should not be more than 2.0%.

The robustness of an analytical method is a measure of its capacity to remain unaffected by small but deliberate variations in method parameters and provides an indication of its reliability during normal usage. It was done by changing the mobile phase volume and development time while constantly maintaining the remaining parameters. The acceptance criterion is % RSD of the peak areas should not be more than 2.0%.

After multiple trials an optimized mobile phase was developed for the HPTLC analysis of Saxagliptin. The standard and test samples were spotted in the form of bands of width 8 mm using a 100 µL syringe on precoated silica gel aluminum plate 60 F₂₅₄ (10 × 10 cm) using a CAMAG Linomat-5 sample applicator. The development was carried out in 20 × 20 cm twin trough glass chamber using Methanol:Chloroform (6:4 v/v) as mobile phase. The saturation time for mobile phase was 20 min. The development time was 20 to 30 minutes. After the development, the TLC plates were air dried and were densitometrically scanned using CAMAG thin layer chromatographic scanner at 222 nm. The chromatograms were recorded using WINCATS software version 1.4.3. The chromatogram of Saxagliptin is shown in Figure 1. This method has shown good resolution of Saxagliptin in both standard and test samples and the R_f value was found to be 0.50 ± 0.02.

The chromatograms of standard as well as test Saxagliptin solutions were recorded and the percentage of drug content was calculated. The percentage assay for Saxagliptin was found to be 98.90%, which lies within the limits mentioned by ICH (98% - 120%). Hence the developed method can be routinely used for the estimation of Saxagliptin in the tablet formulations. The chromatogram of Saxagliptin test solution (sample solution) was shown in Figure 2.