A New HPLC-Fluorescence Method for the Simultaneous Determination of Fluticasone Propionate and Salmeterol Xinafoate in Their Solutions Involving Schiff-Base Derivatization with Hydrazine

Abstract

A partially validated high-performance liquid chromatographic method with UV and fluorescence detection was developed to determine fluticasone propionate (FP) and salmeterol xinafoate (SX) simultaneously in their solutions. RP-HPLC (Hypersil C18 column, 250 mm × 4.6 mm, 5 µm) was used for chromatographic separation employing an isocratic mobile phase consisting of methanol, acetonitrile and water (50/20/30, v/v/v) at a flow rate of 0.8 mL.min–1. With the aid of the Schiff-base reaction, the hydrazine derivative of FP was prepared to enhance the fluorescence signal of FP. The method was employed at an excitation maximum of λ = 246, 262 and 298 nm for FP, SX and FP derivatives, respectively. The method is accurate, precise and linear (R2 ≥ 0.999) in the range of 0.246–86.957 µg×mL–1 for FP in its derivatives mixture and 0.156–50.00 µg×mL–1 for SX. The calculated LOD values are 0.081 and 0.052 µg×mL–1, respectively. It was noted that the fluorescence detection sensitivity of FP and SX is better than that of UV (LOD = 0.396 and 0.254 µg×mL–1 for FP and SX, respectively). The advantage of the validated HPLC-Fluorescence method compared with expensive and sophisticated LC-MS methods is its simplicity. When compared with other HPLC-UV/Fluorescence methods, the proposed method includes simple chromatographic conditions (no column oven nor high flow rate) and uses non-buffered mobile phase solvents; it is the first HPLC-Fluorescence method for the simultaneous determination of SX and FP. Moreover, the Fluorescence signal of FP was enhanced by derivatization with hydrazine. This was confirmed by computational and Mass spectrometric analysis.