Sensitive and simultaneous determination of HIV protease inhibitors in rat biological samples by liquid chromatography–mass spectrometry

Abstract

A sensitive and simultaneous liquid chromatographic-mass spectrometric (LC/MS) method for the determination of current four HIV protease inhibitors (PIs), indinavir (IDV), saquinavir (SQV), nelfinavir (NFV) and amprenavir (APV) in rat plasma and liver dialysate by a microdialysis method was described. An isocratic LC/MS method in combination with atmospheric pressure chemical ionization was developed for the determination of these four PIs in biological samples in the same run. The analytes including an internal standard were extracted from 100 microL of plasma or 150 microL of liver dialysate samples by salting-out with 100 microL of ice-cold 2 M K(3)PO(4) followed by ether extraction. The separation of analytes was carried out on a reversed-phase semi-micro column using 50% of acetonitrile containing 1% acetic acid as mobile phase at a flow rate of 0.2mL/min(-1). The separation was completed within 5 min. Precision, recovery and limits of detection indicated that the method was suitable for the quantitative determination of these PIs in rat plasma or liver dialysate. This simple, sensitive and highly specific LC/MS method is suitable for pharmacokinetic studies and therapeutic drug monitoring in AIDS patients who receive double protease therapy.

Pharmacokinetics of Human Immunodeficiency Virus Protease Inhibitor, Nelfinavir, in Poloxamer 407-Induced Hyperlipidemic Model Rats

“…21) Atazanavir was used as an internal standard for LC-MS method as described below. Propylene glycol and acetonitrile (HPLC grade) were purchased from Wako Pure Chemical Industries (Osaka, Japan).…”

“…Assay Procedure NFV in plasma and the other samples in this study was assayed by the LC-MS method, developed by Gao et al 21) Briefly, 10 ml of atazanavir (ATV, internal standard, 100 mg/ml in methanol) and 100 ml of 2% ZnSO 4 in 50% methanol solution was added to aliquots of 100 ml plasma sample in a 1.5 ml microcentrifuge tube and vortexed vigorously for 15 s. Diethyl ether (1 ml) was then added to the tube, vortexed for 30 s, and centrifuged at 12000 g for 5 min. The aqueous phase in test tubes was frozen in a cold bath at Ϫ10°C and the ether phase was transferred to HPLC sample vials.…”

In Vivo Effects of Cyclosporin A and Ketoconazole on the Pharmacokinetics of Representative Substrates for P-Glycoprotein and Cytochrome P450 (CYP) 3A in Rats

“…NFV and IDV (an internal standard) in the bile and plasma samples were deproteinized with 100 ml of 2 M K 3 PO 4 and extracted with diethylether, and then measured using a previously described liquid chromatography-mass spectrometry (LC/MS) system with a negative APCI mode. 8) EM and IDV (an internal standard) in whole blood samples were deproteinized with 2% ZnSO 4 in 50% methanol and extracted with diethyl ether, and then measured using a liquid chromatography tandem mass spectrometry (LC/MS/MS) system. Briefly, the LC/MS/MS system consisted of a PE-Sciex API 365 triple quadrupole mass spectrometer equipped with turbo ion spray sample inlet as an interface for electrospray ionization (ESI) and Analyst TM workstation (Perkin-Elmer-Sciex, Ontario, Canada), an LC-10AD micropump (Shimadzu, Kyoto Japan), and an AS8020 automatic sample injector (Toso Co., Ltd., Tokyo, Japan).…”

Decrease in Brain Distribution of Fluvoxamine in Experimental Hyperlipidemic Rats

“…Pluronic F-127 ® (also known as Poloxamer 407, P-407) was obtained from Sigma (St. Louis, MO, USA). Nelfinavir (NFV, internal standard) was extracted from a commercial product, Viracept ® , and the crude extract was purified by a preparative HPLC method (26). Potassium bromide (KBr), potassium chloride (KCl) and glucose (Glu) were purchased from Wako Pure Chemical Industries Limited (Osaka, Japan).…”