Development of Liquid Chromatography? UV Method for Simultaneous Determination of Leflunomide and NSAIDs in API and Pharmaceutical Formulations: It?s Application to In vitro Interaction Studies

Abstract

An efficient analytical method for the simultaneous determination of leflunomide and non steroidal anti-inflammatory drugs in API and formulations by LC-UV has been developed. The analytes were separated on Purospher Star, C 18 (5 µm, 250×4.6 mm) column at ambient temperature with methanol: water (80:20, v/v, pH at 2.7) at flow rate of 1.5 mL min -1 . Experiment was conducted in two phases. Leflunomide was separated with flurbiprofen and ibuprofen (phase-I) and diclofenac sodium and mefenamic acid (phase II). Calibration curves were linear over the range 0.625–5 µg mL -1 in both phases for leflunomide while for flurbiprofen, ibuprofen, diclofenac sodium, mefenamic acid linearity were achieved in the range of 0.625-5, 11.25-90, 1.56-50 and 0.78-25 µg mL -1 , respectively with r 2 >0.9998. Intra-day variation was <1.2 and <1.4 %, while in inter-day ranged between 0.042-1.45% and 0.08-1.27% in phase-I and II, respectively. Mean recovery values for intra-day ranged from 99.04-100.4% and 98.48-100.2% and for inter-day were between 98.54-100.29% and 98.85-100.54% in phase-I and II, respectively. The LLOD of leflunomide was 13 ng mL -1 , while LLOQ was 39ng mL -1 , respectively. LLOD and LLOQ for flurbiprofen, ibuprofen, diclofenac sodium and mefenamic acid were 6.9, 296, 71 and 1.2 ng mL -1 and 21, 897, 214.3 and 3.676 ng mL -1 , respectively. Present study showed that nanogram quantities of all the compounds can be estimated accurately. The newly established method was successfully applied to study in vitro interactions between leflunomide and NSAIDs.