Simultaneous Quantitative Assay of Six HIV Protease Inhibitors, One Metabolite, And Two Non-Nucleoside Reverse Transcriptase Inhibitors in Human Plasma by Isocratic Reversed-Phase Liquid Chromatography

Abstract

A rapid (less than 30 min), sensitive, and specific liquid chromatography method for simultaneous assay of nine antiretroviral drugs in human plasma is described. This technique allows therapeutic drug monitoring of six approved protease inhibitors (amprenavir, indinavir, lopinavir, nelfinavir, ritonavir, and saquinavir) and two approved non-nucleoside reverse transcriptase inhibitors (efavirenz and nevirapine). Assays were performed after diethyl ether liquid-liquid extraction from 250-microL plasma samples. Chromatographic separation was achieved on an X-TERRA (Waters; Saint Quentin, France) column using a 58% water (with 3 mmol/L pyrrolidine) and 42% acetonitrile mobile phase. Three ultraviolet wavelengths were used for detection with a diode array detector. This method allowed quantitative assay of all nine antiretroviral drugs within a concentration range of 25 ng/mL to 9000 ng/mL. The method has been validated extensively and has been in routine use in our laboratory for several months for drug monitoring in plasma samples from patients treated with antiretroviral drugs.

Conventional HPLC Method Used for Simultaneous Determination of the Seven HIV Protease Inhibitors and Nonnucleoside Reverse Transcription Inhibitor Efavirenz in Human Plasma

“…[1][2][3][4][5][6][7][8][9][10][11][12][13][14][15] However, these methods have several disadvantages in terms of cost performance, time consumption and necessary equipment; for example, the use of expensive disposable cartridges at the solid-phase drug extraction, gradient elution control by a gradient HPLC pump system, and the ultraviolet detection at multiple wavelengths.…”

“…14) In fact, some previously reported assays used the mobile phase buffer at a variety of pH values. We sought the optimum pH of the mobile phase buffer by changing pH every 0.5 from pH 2 to pH 11.…”

“…Both interday and intraday CVs for all drugs were less than 8.5%, which is similar to or much lower than previously reported values. [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15] Mean extraction recoveries varied from 81.2% (IDV) to 98.4% (APV). These results indicate that the method developed here achieves a high degree of reproducibility and accuracy.…”

Population Pharmacokinetic Modeling of the Association between 63396C→T Pregnane X Receptor Polymorphism and Unboosted Atazanavir Clearance

“…ATV concentrations were quantified using a validated high-pressure liquid chromatography (HPLC)-photodiode array detector (PDA) method for cohort A (12) and cohort B (43), whereas cohort C analyses were performed using validated liquid chromatography-tandem mass spectrometry (LC-MS-MS) methods (15). The methods were validated internally using standard bioanalytical assay guidelines, and participation in the external KKGT (Association for Quality Assessment in Therapeutic Drug Monitoring and Clinical Toxicology) quality control (QC) program helps ensure that the laboratory quality assurance is maintained.…”

Effects of Concentration and Temperature on the Stability of Nevirapine in Whole Blood and Serum

“…Two methods of DNA isolation were used in this study. One was the QIAamp DNA Blood Midi Kit (Qiagen, Inc.), which has been widely used for isolating circulating DNA from serum or plasma (1)(2)(3)(4)(5). The other is the modified Guanidine/Promega Resin (G/R) method that was developed to isolate DNA from urine (6 ).…”