Development and Validation of Liquid Chromatography-Tandem Mass Spectrometry Method for Detection and Quantification of Flunisolide in Tissue Culture Medium

Abstract

A robust, sensitive, and selective liquid chromatography-tandem mass spectrometry (LC-MS/MS) method capable of quantifying flunisolide in the tissue culture matrix was developed and validated. Both flunisolide and dexamethasone (internal standard) were extracted from tissue culture medium, with 5% fetal bovine serum, 1% nonessential amino acids, and 1% penicillin/streptomycin by simple liquid–liquid extraction. The analytes were chromatographically separated (10 ng/mL for flunisolide) using a C8 column (4.6 mm × 150 mm; 5 µm particle size). The mobile phase was comprised of methanol:water (80:20 v/v). The analytes were separated at baseline within 2.5 min. using a flow rate of 1 mL/min. Mass spectrometry detection was carried out in negative atmospheric pressure chemical ionization mode. The calibration curves for the analyte were linear over the range of 10–200 ng/mL (R2 ≥ 0.9968, n = 6). The inter and intra-batch mean percent accuracy ranged within 97.7–110.6% and the precision range was 3.5–10.4% (% coefficient of variation ≤15%). Stability studies revealed that the analyte was stable in the matrix for at least six hours at room temperature and at the end of three successive freeze and thaw cycles. The method reported here is specific for flunisolide quantification was also used in monolayer efflux assay using CaCo2 cell lines, which will eventually aid P- glycoprotein transport studies for flunisolide.