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 4.6mm, 5 microm HPLC column, with 75:25 methanol:0.6% aqueous ammonium acetate as the mobile phase. Under these near neutral conditions, SB demonstrated a good peak shape (tailing 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 microgmL(-1)) and SB (LOD for SX=0.26 microgmL(-1)). The limit of detection for FP was 0.19 microgmL(-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 SB 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.