Rapid quantification of HIV protease inhibitors in human plasma by high‐performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry

Abstract

HIV protease inhibitors are important antiretroviral drugs which have substantially reduced the morbidity and mortality associated with HIV-1 infection. Recent data have shown relationships between plasma concentrations of the protease inhibitors and clinical response, which makes therapeutic drug monitoring valuable. We have developed and validated an assay, using liquid chromatography coupled with electrospray tandem mass spectrometry (LC/MS/MS), for the routine quantification of the six licensed protease inhibitors (amprenavir, indinavir, lopinavir, nelfinavir, ritonavir and saquinavir) and the pharmacologically active nelfinavir metabolite M8 in plasma. The sample pretreatment consisted of protein precipitation with a mixture of methanol and acetronitrile using only 100 microl of plasma. Chromatographic separation was performed on an Inertsil ODS3 column (50 x 2.0 mm i.d., particle size 5 microm), with a quick stepwise gradient using an acetate buffer (pH 5) and methanol, at a flow rate of 0.5 ml min(-1). The analytical run time was 5.5 min. The use of a 96-well plate autosampler allowed batch sizes up to 150 patient samples. The triple-quadrupole mass spectrometer was operated in the positive ion mode and multiple reaction monitoring was used for drug quantification. The method was validated over the concentration ranges 0.01-10 microg ml(-1) for indinavir and saquinavir, 0.1-10 microg ml(-1) for amprenavir, 0.05-10 microg ml(-1) for nelfinavir and ritonavir, 0.1-20 microg ml(-1) for lopinavir and 0.01-5 microg ml(-1) for M8. Saquinavir-d(5) and indinavir-d(6) were used as internal standards. The coefficients of variation were always <10% for both intra-day and inter-day precisions for each compound. Mean accuracies were also between the designated limits (+/-15%). The validated concentration ranges proved to be adequate in daily practice. This robust and fast LC/MS/MS assay is now successfully applied for routine therapeutic drug monitoring and pharmacokinetic studies in our hospital.

Population pharmacokinetics of indinavir alone and in combination with ritonavir in HIV‐1‐infected patients

“…Recoveries of indinavir and ritonavir were 105.4% and 91.7%, respectively. Within-and between-day precisions were always less than 9.4% for all quality control samples [17].…”

Quantitative Determination Method for Trace Amount of Penicillin Contaminants in Commercially Available Drug Product by HPLC Coupled with Tandem Mass Spectrometry

“…[15][16][17] Parameters of the mass spectrometer were investigated to maximize the intensity of the product ion. The peaks of m/z 349.1 for Amoxicillin, m/z 106.2 for Ampicillin, and m/z 160.2 for Flucloxacillin were found to show the highest intensity among several product ions.…”

Applications of LC‐MS/MS in Clinical Laboratory Diagnostics

“…Apart from the analysis of immunosuppressants the above approach is also routinely used for the determination of anti-depressants, neuroleptics, hypnotics, oral anti-coagulants, HIV-protease-inhibitors, amiodaron, finasterid, and other drugs in plasma and serum. It is also possible to simultaneously quantify opioids, cocaine, and their metabolites in urine [84][85][86][87][88][89][90]. …”