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Therapeutic drug monitoring (TDM) of antiretroviral drugs requires accurate and precise analysis of their trace amounts in plasma samples. Solid-phase extraction (SPE) coupled with dispersive liquid-liquid microextraction based on solidification of floating organic drop (DLLME-SFO) was introduced as a simple and sensitive method for extraction, pre-concentration and simultaneous determination of efavirenz (EFV), nelfinavir (NFV) and nevirapine (NVP) in human plasma and pharmaceutical formulations by using high performance liquid chromatography (HPLC) with UV detection. A response surface methodology (RSM) based on central composite design (CCD) was used for optimizing the main variables in the extraction procedure. Under the optimum experimental conditions, the calibration curves were linear in the range of 0.1-400 ng mL -1 with correlation coefficients from 0.9982 to 0.9997 and limits of detection (at S/N = 3) of 0.03 to 0.07 ng mL -1 . Moreover the intra-day and inter-day precision (RSD%) were in the range of 2.2-4.2% and 3.1-5.2%, respectively. The enrichment factors and relative recoveries of the anti-HIV drugs were 459-1507 and 93.7-105.4%. The method was successfully applied to the simultaneous determination of the trace amounts of the antiretroviral drugs in blood plasma and pharmaceutical formulations., (Copyright © 2019 Elsevier B.V. All rights reserved.)

A simple, rapid, reliable and highly sensitive on-line two-dimensional reversed-phase liquid chromatography-tandem mass spectrometric (2D-LC/MS/MS) method to determine antiretroviral drugs viz., abacavir (ABC), nevirapine (NVP) and indinavir (IDV) in rat serum and urine was developed and validated. The analytes were extracted on-line from rat serum and urine by a restricted access material (RAM) column and back-flushed into the reversed-phase C18 column for separation by LC. Detection was carried out by ESI-MS/MS. The developed method showed good selectivity, accuracy and precision for quantification of the antiretroviral drugs in rat serum and urine. Quantification limits for abacavir and nevirapine were 4.0 ng ml(-1), whereas for indinavir 4.7 ng ml(-1). The calibration graphs were linear in the range of 4-50 ng ml(-1)for abacavir, nevirapine and indinavir. The method was successfully applied to study the pharmacokinetics of antiretroviral in rats.

An accurate, sensitive and simple reverse-phase (RP) high-performance liquid chromatography (HPLC) assay has been developed and validated for the simultaneous quantitative determination of tipranavir with nine other antiretroviral drugs in plasma. A liquid-liquid extraction of the drugs in tert-butylmethylether (TBME) from 200 microL of plasma is followed by a reversed phase gradient HPLC assay with UV detection at 210 nm. The standard curve for the drug was linear in the range of 80-80,000 ng/mL for tipranavir; 10-10,000 ng/mL for nevirapine, indinavir, efavirenz, and saquinavir; and 25-10,000 ng/mL for amprenavir, atazanavir, ritonavir, lopinavir, and nelfinavir. The regression coefficient (r(2)) was greater than 0.998 for all analytes. This method has been fully validated and shown to be specific, accurate and precise. Due to an excellent extraction procedure giving good recovery and a clean baseline, this method is simple, rapid, accurate and provides excellent resolution and peak shape for all analytes. Thus this method is very suitable for therapeutic drug monitoring.

A sensitive and rapid liquid chromatography tandem mass spectrometry (LC-MS-MS) method has been developed to measure the levels of the HIV-1 non-nucleoside reverse transcriptase inhibitor nevirapine (NVP) in human plasma. The analyte and internal standard (IS) are isolated from plasma by a simple perchloric acid precipitation of plasma proteins followed by centrifugation. LC-MS-MS in positive mode used pairs of ions at m/z of 267/226 for NVP and 628/421 for the IS, respectively. Two linear calibration curves were established for quantitation of NVP with the low curve ranging from 25 to 1000 ng/ml and the high curve ranging from 1000 to 10,000 ng/ml. Mean inter- and intra-assay coefficients of variation (CVs) over the ranges of the two standard curves were less than 10%. The overall recovery of NVP was 92.4%., (Copyright 2003 Elsevier Science B.V.)

Micellar electrokinetic chromatography methods were developed and validated to separate and quantitate anti-HIV drug mixtures containing zidovudine(AZT)/didanosine(ddI)/nevirapine (mixture A) and AZT/ddI/ritonavir (mixture B) in human serum. Serum samples were prepared using a solid-phase extraction procedure. The effects of various factors such as buffer type, buffer and surfactant concentrations, and pH on the separations were investigated. The optimized resolution was achieved with a run buffer containing 18 mM sodium dodecylsulfate in 15 mM phosphate and borate buffer (pH 9.0). An uncoated 52 cm (effective length 30 cm)x50 micrometer ID fused-silica capillary operated at 30 degrees C was used in the analysis with UV detection at 210 nm. Aprobarbital was chosen as the internal standard. All analytes were separated within 14 min with a voltage of +15 kV and a current around 30 microA. The methods were validated over the range of 0.5-25.0 microgram/ml for AZT, 0.8-18.5 microgram/ml for ddI, 0.5-22.8 microgram/ml for nevirapine in mixture A and the range of 0.5-25.0 microgram/ml for AZT, 0.8-18.5 microgram/ml for ddI, 1.2-28.8 microgram/ml for ritonavir in mixture B. Intra-day and inter-day accuracy was less than 12.4% and intra-day and inter-day precision was less than 13.9% for both mixtures. Extraction recoveries of all analytes from serum were higher than 75.9%. The assay should be applicable to pharmacokinetic studies and routine monitoring of these drugs in serum.

A simple method for the simultaneous determination of zidovudine and nevirapine in human plasma by reversed-phase liquid chromatography with UV detection at 265 nm was developed. A solid-liquid extraction procedure with internal standard was applied to the samples prior to analysis. The system requires a Zorbax SB-C18 column, 250 x 4.6 mm I.D. and a mobile phase composed of potassium dihydrogen phosphate (10 mM; pH 6.5)-acetonitrile (83:17, v/v). Peak-areas are linear; correlation coefficients are better than 0.999; both inter- and intra-day accuracy and precision are lower than 15%. Extraction recoveries are higher than 90% for both zidovudine and nevirapine. The method proposed was employed to determine the levels of the two retroviral drugs in plasma from HIV infected human subjects.

A new high-performance liquid chromatography (HPLC) assay was developed for the simultaneous determination of zidovudine (AZT)/lamivudine (3TC)/nevirapine in human plasma. Plasma samples were treated using a solid-phase extraction procedure. The compounds were separated using a mobile phase of 20 mM sodium phosphate buffer (containing 8 mM 1-octanesulfonic acid sodium salt)-acetonitrile (86:14, v/v) with pH adjusted to 3.2 with phosphoric acid on an octylsilane column (150x3.9 mm i.d.) with UV detection at 265 nm. Aprobarbital was chosen as the internal standard (IS). The method was validated over the range of 57.6-2880 ng/ml for AZT, 59.0-17650 ng/ml for 3TC and 53.2-13300 ng/ml for nevirapine. The method was shown to be accurate, with intra-day and inter-day accuracy from 0.1 to 11% and precise, with intra-day and inter-day precision from 0.4 to 14%. Extraction recoveries of the analytes and IS from plasma were higher than 92%. The assay should be suitable for use in pharmacokinetic studies and routine plasma monitoring of this triple-drug therapy in AIDS patients.