Simultaneous HPLC Assay for Quantification of Indinavir, Nelfinavir, Ritonavir, and Saquinavir in Human Plasma

Abstract

Background: HIV protease inhibitors are recommended as part of combination antiretroviral therapy. Dual protease inhibitor therapy is also being used clinically. Consequently, a simultaneous assay for indinavir, nelfinavir, ritonavir, and saquinavir was developed. Methods: Indinavir, nelfinavir, ritonavir, and saquinavir were extracted from plasma (250 μL) with methyl-t-butyl ether at basic pH after addition of an internal standard (A-86093). The compounds were separated on a Keystone BetaBasic C4 column (250 × 3 mm i.d.) at 40 °C with a mobile phase of acetonitrile-50 mmol/L ammonium formate buffer, pH 4.1 (52:48, by volume) at a flow rate of 0.5 mL/min. Indinavir, nelfinavir, ritonavir, and the internal standard (A-86093) were detected at 218 nm, and saquinavir was detected at 235 nm. The method was validated by analysis of five triplicate analyses of calibrators along with quality-control samples at three different concentrations prepared in human plasma. Results: The extraction recovery was 87–92%. Within-run accuracy for quality-control samples was 6–8%, with CVs of 2–8%. Limits of quantification were 40–50 μg/L for indinavir, nelfinavir, and ritonavir, and 20 μg/L for saquinavir. Cross-validation with a liquid chromatography-mass spectroscopy method for saquinavir and nelfinavir was conducted with patient samples. Regression analysis revealed a good correlation (r2 >0.94) between methods. Larger variations at concentrations >4000 μg/L were observed with nelfinavir. Interference with drugs commonly used in AIDS patients was not observed. Pharmacokinetic profiles for two patients on dual protease therapy were determined. Conclusions: A reliable and rugged simultaneous HPLC assay for four HIV protease inhibitors was developed. The assay method is convenient for clinical laboratories involved in therapeutic drug monitoring for HIV protease inhibitors. The assay has enough sensitivity to conduct pharmacokinetic studies in patients taking more than one HIV protease inhibitor along with other antiretroviral medications.

Intracellular accumulation of Praziquantel in T lymphoblastoid cell lines, CEM (parental) and CEMvbl(P-gp-overexpressing)

“…We chose to use SQV, a PI as PZQ may be co-administered with PIs, since there is geographic overlap between the regions afflicted by both Schistosomiasis and HIV/AIDS [ 55 ]. The method was validated over a concentration range of 1.6 to 19.2 μM for both drugs (50 to 600 ng/ml for PZQ and 107 to 1288 ng/ml for SQV); which is within the detectable range for both drugs in human plasma [ 56 , 57 ].…”

Quantification of antiretroviral drugs in dried blood spot samples by means of liquid chromatography/tandem mass spectrometry

“…Relative high steady‐state trough levels between ca. 100 and 6000 ng/mL of PI and NNRTI allow the use of an LC‐UV analysis method resulting in sufficient limits of detection (LOD) 16–23. However, UV responses are often not selective enough to distinguish between drug and coeluted matrix component responses, which can result in too high drug levels due to false positive peak signals.…”

Bio-analytical Assay Methods used in Therapeutic Drug Monitoring of Antiretroviral Drugs-A Review

“…As the bioavailability of SQV is very low, two HPLC-UV assays were developed with a detection limit close to 1 ng/mL plasma. Few studies have shown simultaneous quantification of SQV with other PIs with higher limits of quantification [264,265]. In addition, SQV was also simultaneously assayed with the NNRTI's like delavirdine and EFV [149,151].…”

“…This method involved extraction of RTV and SQV from serum on solid-phase extraction using C 18 cartridges followed by HPLC separation on C8 column with a UV detector set at 240 nm [260]. Several other sensitive simultaneous assay meth-ods were reported with UV and MS detectors for RTV [202,205,209,211,264,265,278].…”