Monitoring of HAART regime antiretrovirals in serum of acquired immunodeficiency syndrome patients by micellar liquid chromatography

A methodology based on micellar liquid chromatography to monitor five antiretroviral drugs (lamivudine, stavudine, tenofovir, zidovudine and efavirenz) was proposed. Antiretrovirals were studied in sets of three, corresponding to each highly active antiretroviral therapy (HAART) regime, prescribed to acquired immunodeficiency syndrome (AIDS)-infected patients. Four aqueous micellar mobile phases buffered at pH 7 were optimized to separate these compounds, using sodium dodecyl sulfate as the tensioactive, and 1-propanol or 1-pentanol as the organic modifier. The composition of each mobile phase was optimized for each antiretroviral. The common separation conditions were: C18 apolar column (125 × 4.6 mm, 5 μm particle size), UV detection set at 214 nm, and mobile phase running at 1 mL min(-1) without controlling the temperature. The finally suggested method was validated for five analysed antiretroviral drugs following the US Food and Drug Administration guidelines in terms of: linearity between 0.5 and 50 ppm (r(2) > 0.9995), sensitivity (LOD lower than 0.25 ppm), intra- and inter-day precision (<7.1 and <5.2%, respectively) and accuracy (recovery 88.5-105.3% and 93.5-101.3%, respectively), as well as robustness (<6.5%). The proposed method was used to monitor the level of antiretrovirals in the serum of AIDS patients. The suggested methodology was found to be useful in the routine analysis of antiretrovirals in serum samples.     

Development and validation of a liquid chromatographic/tandem mass spectrometric assay for the quantitation of nucleoside HIV reverse transcriptase inhibitors in biological matrices

Besides liquid chromatographic (LC)/UV methods adapted to therapeutic drug monitoring, there is still a need for more powerful techniques that can be used for pharmacological research and clinical purposes. We developed an LC method coupled with tandem mass spectrometry (MS/MS) to separate, detect and quantify with high sensitivity the nucleoside analogues used in multitherapies (zidovudine, stavudine, zalcitabine, didanosine, lamivudine and abacavir) in plasma and in the intracellular medium. We worked on two essential issues: (i) the need to use two ionization modes in order to achieve the best sensitivity, which leads to the optimization of the chromatographic separation of drugs detected in the positive ionization mode and drugs detected in the negative ionization mode, and (ii) the need to optimize the extraction step in order to enhance sample recovery. The peripheral blood mononuclear cells were lysed in Tris buffer-MeOH. A clean-up procedure was performed by solid-phase extraction only for plasma samples. The LC separation was carried out on a Zorbax Stable Bond C(18) column followed by MS/MS analysis after electrospray ionization in either the negative or positive mode. The positive ionization mode was applied at the beginning of the run to detect zalcitabine and lamivudine, then the ionization mode was changed to negative for the detection of didanosine, stavudine, internal standard and zidovudine. The calibration range for all the analytes was 0.5-200 ng ml(-1). The recoveries were between 64 and 90%, with coefficients of variation (CVs) lower than 15%. The inaccuracy (bias) was +/-15% with CVs always lower than 12%. The analytes were stable at room temperature and in the extraction solvent for at least 24 h, after storage at -80 degrees C for 3 months, after three freeze-thaw cycles and in the injection solvent after 48 h at 4 degrees C. Together with the measurement of intracellular triphosphorylated metabolites thanks to the powerful plasma and intracellular assay method for intact drugs, it is possible to describe the behaviour of nucleoside analogues against HIV through plasma pharmacokinetics, cell membrane diffusion including drug transport involvement, and also the intracellular metabolism.     

Exploring dried blood spot sampling technique for simultaneous quantification of antiretrovirals: lamivudine, stavudine and nevirapine in a rodent pharmacokinetic study

A high‐performance liquid chromatography/positive ion electrospray tandem mass spectrometry method for the simultaneous quantification of lamivudine, stavudine and nevirapine was developed and validated in dried blood spot (DBS) cards. The analytes were separated using an isocratic mobile phase on a reverse phase column and analyzed by MS/MS in the MRM mode using the respective [M + H]⁺ ions, m/z 230–112 for lamivudine, m/z 225–127 for stavudine, m/z 267–226 for nevirapine, m/z 383–337 for zidovudine (IS). The lower limit of quantification was 1 ng/mL for both lamivudine and stavudine and 10 ng/mL for nevirapine. Acceptable precision and accuracy were obtained for concentrations over the standard curve range. The method was successfully applied to quantify them in a rat pharmacokinetic study in whole blood, plasma and DBS cards after a single oral co‐administration at the dose of 10, 2 and 13 mg/kg for lamivudine, stavudine and nevirapine, respectively, to male Wistar rats. Following oral administration the pharmacokinetic results in all the matrices are in close agreement. Thus accomplishment of this method would facilitate the ease of collection of clinical samples on DBS cards for lamivudine, stavudine and nevirapine during human clinical trials and therapeutic drug monitoring. Copyright © 2012 John Wiley & Sons, Ltd.     

Screening and monitoring antiretrovirals and antivirals in the serum of acquired immunodeficiency syndrome patients by micellar liquid chromatography

Thirteen different antiretrovirals are commonly used in hospital protocols for suppressing the activity of the human immunodeficiency virus (HIV) and associated opportunistic diseases in patients with acquired immunodeficiency syndrome (AIDS). In this work, three micellar mobile phases are recommended for screening these substances, using UV detection, and the process can be performed in less than 18 min. The first mobile phase (sodium dodecyl sulphate or SDS 50 mM) is used for the group consisting of acyclovir, didanosine, ganciclovir, stavudine and zidovudine. The second mobile phase (SDS 120 mM/4.5% propanol) is used for the group containing abacavir, lamivudine, nevirapine, valaciclovir and zalcitabine, whereas the third mobile phase (SDS 150 mM/5% pentanol) is used for efavirenz, indinavir and ritonavir. The use of micellar liquid chromatography (MLC) as an analytical tool allows serum samples to be injected directly. The method was validated over the range of 0–10 μg mL⁻¹. The limits of detection (signal-to-noise ratio of 3), which ranged from 6 to 30 ng mL⁻¹, were adequate for monitoring these substances. Intra- and inter-day relative standard deviations of the assay were below 3% for all compounds. The recoveries in spiked serum samples were in the 89.5–104.4% range. The method can be applied to the screening, monitoring and control of patients’ treatment with antiretrovirals and antivirals.