Development and validation of a reversed-phase liquid chromatographic method with fluorescence detection for the study of Saquinavir pharmacokinetics in rat plasma

This research work aims to exploit the high selectivity and sensitivity of fluorescence detector to develop and validate a high performance liquid chromatography (HPLC) method having very small sampling volume, much better mass-sensitive detection limit and lower operating cost for the determination of Saquinavir (SQV), known to have low oral bioavailability, in rat plasma. Unlike the traditional methods that require at least 0.25 mL of plasma for each measurement, the present method requires only a 0.1 mL sample volume. This is very useful in reducing the blood collection from study rats, offering the possibility to make sufficient number of samples for pharmacokinetic study and minimizing the amount of blood-derived biological waste. After liquid-liquid extraction, the compounds were separated on a Vydac C₁₈ monomeric column (250 mm × 4.6 mm i.d. × 5 μm particle size) using a mobile phase composed of acetonitrile and potassium dihydrogen phosphate buffer (45:55, v/v). Fluorescence detection was performed at 237 nm (excitation) and 380 nm (emission). Validity of the method was studied and the method was found to be precise and accurate with a linearity range from 0.005 to 1.000 μg mL^-1 (r > 0.9980). The limit of detection (LOD) was found to be 0.001 μg mL^-1. The intra-day and inter-day precision studies showed good reproducibility with coefficients of variation (C.V.) less than 11.4%. The developed method was applied successfully to monitor the pharmacokinetic profile following oral administration of SQV to rats.