scholarly journals Validation of Simultaneous Quantitative Method of HIV Protease Inhibitors Atazanavir, Darunavir and Ritonavir in Human Plasma by UPLC-MS/MS

2014 ◽  
Vol 2014 ◽  
pp. 1-12 ◽  
Author(s):  
Tulsidas Mishra ◽  
Pranav S. Shrivastav

Objectives. HIV protease inhibitors are used in the treatment of patients suffering from AIDS and they act at the final stage of viral replication by interfering with the HIV protease enzyme. The paper describes a selective, sensitive, and robust method for simultaneous determination of three protease inhibitors atazanavir, darunavir and ritonavir in human plasma by ultra performance liquid chromatography-tandem mass spectrometry.Materials and Methods. The sample pretreatment consisted of solid phase extraction of analytes and their deuterated analogs as internal standards from 50 μL human plasma. Chromatographic separation of analytes was performed on Waters Acquity UPLC C18 (50 × 2.1 mm, 1.7 μm) column under gradient conditions using 10 mM ammonium formate, pH 4.0, and acetonitrile as the mobile phase.Results. The method was established over a concentration range of 5.0–6000 ng/mL for atazanavir, 5.0–5000 ng/mL for darunavir and 1.0–500 ng/mL for ritonavir. Accuracy, precision, matrix effect, recovery, and stability of the analytes were evaluated as per US FDA guidelines.Conclusions. The efficiency of sample preparation, short analysis time, and high selectivity permit simultaneous estimation of these inhibitors. The validated method can be useful in determining plasma concentration of these protease inhibitors for therapeutic drug monitoring and in high throughput clinical studies.

2009 ◽  
Vol 55 (1) ◽  
pp. 170-174 ◽  
Author(s):  
Laure Elens ◽  
Sophie Veriter ◽  
Vincent Di Fazio ◽  
Roger Vanbinst ◽  
Daniel Boesmans ◽  
...  

Abstract Background: Most HPLC-UV methods for therapeutic drug monitoring of anti-HIV drugs have long run times, which reduce their applicability for high-throughput analysis. We developed an ultra-performance liquid chromatography (UPLC)–diode array detection method for the simultaneous quantification of the HIV-protease inhibitors (PIs) amprenavir, atazanavir, indinavir, lopinavir, nelfinavir, ritonavir, saquinavir, and tipranavir (TPV), and the nonnucleoside reverse transcriptase inhibitors (NNRTIs) efavirenz and nevirapine. Methods: Solid-phase extraction of 1 mL plasma was performed with Waters HLB cartridges. After 3 wash steps, we eluted the drugs with methanol, evaporated the alcohol, and reconstituted the residue with 50 μL methanol. We injected a 4-μL volume into the UPLC system (Waters ACQUITY UPLC BEH C8 column maintained at 60 °C) and used a linear gradient of 50 mmol/L ammonium acetate and 50 mmol/L formic acid in water versus acetonitrile to achieve chromatographic separation of the drugs and internal standard (A-86093). Three wavelengths (215, 240, and 260 nm) were monitored. Results: All drugs were eluted within 15 min. Calibration curves with concentrations of 0.025–10 mg/L (1.875–75 mg/L for TPV) showed coefficients of determination (r2) between 0.993 and 0.999. The lower limits of quantification were well below the trough concentrations reported in the literature. Inter- and intraassay CVs and the deviations between the nominal and measured concentrations were <15%. The method was validated by successful participation in an international interlaboratory QC program. Conclusions: This method allows fast and simultaneous quantification of all commercially available PIs and NNRTIs for therapeutic drug monitoring.


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