Validation of a reverse-phase high performance liquid chromatographic method for concurrent assay of a weak base (salmeterol xinafoate) and a pharmacologically active steroid (fluticasone propionate)

The analysis of weakly basic drugs Such as salmeterol xinafoate (SX) by reverse-phase liquid chromatography remains a problem, particularly when present in combination with other drugs such as steroids and weak acids. This study describes the validation of an assay for a weakly basic drug, salmeterol (SB), its weakly acidic counter-ion, 1-hydroxy-2-naphthoic acid (XA), and the neutral glucocorticoid, fluticasone propionate (FP) using a second-generation silica stationary phase (Inertsil ODS-2). The assay utilized an Inertsil ODS-2 base-deactivated 250 mm x 4.6 mm, 5 mu m HPLC column, with 75:25 methanol:0.6% aqueous ammonium acetate as the mobile phase. Under these near neutral conditions, SB demonstrated good peak shape (failing factor= 1.21 +/- 0.02, n = 85). The method provided a short analysis time: XA, t(R) = 2.96 min; SB, t(R) = 5.23 min and FP, t(R) = 7.01 min. The assay displayed good sensitivity for both XA (LOD for SX = 0.22 mu g mL(-1))and SB (LOD for SX = 0.26 mu g mL(-1)). The limit of detection for FP was 0.19 mu mL(-1). Neither of the drugs was found to interfere in the determination of the other and the assay accuracy (% recovery) was high (the recoveries were: 99.58 +/- 1.85% for XA, 99.49 +/- 1.88% for SIB and 100.24 +/- 1.28% for FP). The assay reproducibility was determined with a mean coefficient of variance for the five calibration concentrations of XA = 0.71 +/- 0.18%; SB = 1.11 +/- 0.64% and FP = 0.92 +/- 0.14%. Analysis of a pressurized metered dose inhaler formulation demonstrated recovery of the analytes that are within pharmacopoeial limits. It was shown that RP-HPLC was Suitable for the high throughput analysis of the combination of SX and FP. (c) 2005 Elsevier B.V. All rights. reserved