Optimized Simultaneous Estimation of Metformin, Teneligliptin, and Pioglitazone in Tablet and Bulk Drug: A Box-Behnken Design Approach for RP-UFLC Method Development and Validation.

The combination of metformin (MET), teneligliptin (TEN), and pioglitazone (PIO) is newest fixed-dose combination for the treatment type 2 diabetes launched in India. An accurate, rapid, and cost-effective reversed phase ultra-fast liquid chromatography method was developed, validated, and applied for the quantification of MET, TEN, and PIO in bulk and tablet with a very short runtime. Box-Behnken design was implemented to optimize the ultra-fast liquid chromatography conditions. A C18 column (150 × 4.6 mm, 5μ) was utilized with a mobile phase buffer (0.01 M phosphate buffer, pH = 6.2) and acetonitrile in a 51:49 ratio with flow rate of 1.5mL/min at column oven temperature 40°C. Detection was carried out at 255nm by 20 μL injection volume. Retention times were found to be 0.94, 1.36, and 2.07 min, whereas the limit of quantification were 0.209, 0.712, and 57.030 μg/mL for MET, TEN, and PIO, respectively. The linearity of anticipated development was studied in the range of 250-1250 μg/mL (MET, r2 = 0.99974), 10-50 μg/mL (TEN, r2 = 0.99997), and 7.5-37.5μg/mL (PIO, r2 = 0.99999). The relative standard deviation values for method and intermediate precision were <2%. The method was validated as per International Conference on Harmonization guideline for accuracy, precision, linearity, limit of detection, limit of quantification, specificity, robustness, and forced degradation. This is the only study that presents reversed phase ultra-fast liquid chromatography method for the new fixed-dose combination of MET, TEN, and PIO, with the shortest run time of 3.0 min. The proposed method is innovative, efficient, precise, quickest, and economical with high accuracy which makes the study novel.