Nevirapine In Human Plasma Research Articles

Determination of lamivudine and nevirapine in human plasma by Lc-Ms/Ms method

A sensitive and selective LC-MS/MS method to quantify Lamivudine and Nevirapine in K3EDTA human plasma over the concentration range of 25.0240 to 3997.1740 ng/mL and 37.5080 to 5991.0460 ng/mL, respectively were developed and validated. Lamivudine and Nevirapine and its internal standard (Lamivudine 13C 15N2 and Nevirapine D5) were selectively extracted from 300 µL plasma by direct elution using solid phase extraction technique. Elution was achieved by reverse phase liquid chromatography on Hypurity C18 (100 mm × 4.6 mm, 5.0 µm) column was done. LLOQ of 25.0240 ng/mL and 37.5080 ng/mL were resolute for this technique for Lamivudine and Nevirapine respectively. Accuracy and precision values for Lamivudine were 99.55 & 1.50 and for Nevirapine the accuracy and precision values are 99.33 & 1.03%, respectively.

The relationship between zidovudine, lamivudine and nevirapine plasma drug levels and antiretroviral treatment outcomes in Nigeria children living with HIV

Plasma concentrations of antiretrovirals are significant and important determinants of treatment failure and toxicity. The relationship between antiretroviral pharmacokinetic exposures and immunovirological outcomes has not been extensively studied in our setting. The aim of this study was to investigate the relationship between antiretroviral plasma concentrations and virological and immunological treatment outcomes in children living with human immunodeficiency virus (HIV) A retrospective collection of demographic, clinical , laboratory data and a prospective determination of plasma drug concentrations in 120 children aged 2-14 years after two years of receiving fixed dose zidovudine, lamivudine and nevirapine tablets using a simple, rapid, sensitive and validated method of high performance liquid chromatography with UV detection for simultaneous quantification of zidovudine, lamivudine and nevirapine in human plasma. All analyses were performed using graph pad prism version 5.0. A perfect agreement (p 100 cells/mm3). At 2 years zidovudine, lamivudine and nevirapine therapeutic levels, zidovudine supra therapeutic levels ,and nevirapine subtherapeutic levels were respectively significantly associated with immunologic success (CD4%>15 %). Low nevirapine levels can be used to identify those that require adherence counseling. Despite good virological and immunological outcomes, plasma concentrations of zidovudine, lamivudine and nevirapine were not significantly associated with virological and immunological outcomes (Absolute CD4 counts) but was significantly associated with immunological outcomes (CD4%). Plasma drug levels may be good surrogates of adherence but not of treatment outcomes. Monitoring CD4% remains important to optimize paediatric HIV treatment.Keywords: Antiretroviral treatment, children, therapeutic drug monitoring, treatment outcomes, immune-virological outcomes, plasma concentrations, zidovudine, lamivudine and nevirapine

A LC–MS/MS method with column coupling technique for simultaneous estimation of lamivudine, zidovudine, and nevirapine in human plasma

A new LC–MS/MS method for the simultaneous determination of lamivudine, zidovudine, and nevirapine in human plasma is developed using column-coupling technique. Labeled compound of respective analyte was used as an internal standard. After extraction from 100 μL plasma by solid phase extraction method, analytes were separated on a C18 column coupled with a cation exchange column. Total run time was 4.5 min. A tandem mass spectrometric detection was conducted using multiple reaction monitoring under positive ionization mode with an electrospray ionization interface. The method was validated as per the FDA guidelines over the concentration range of 9.47–1466.67 ng/mL for lamivudine, 10.32–1600.00 ng/mL for zidovudine, and 15.05–2426.67 ng/mL for nevirapine. Precision was in the range 0.86–5.77 (intraday) and 1.92–8.19 (interday) while accuracy was 93.25–104.36 % (intraday) and 96.83–103.28 % (interday). Stabilities of stock in aqueous solutions and in plasma were also determined. The method can be applied to the pharmacokinetic study of a combination treatment.

Comparative chromatography–mass spectrometry studies on the antiretroviral drug nevirapine—Analytical performance characteristics in human plasma determination

A contrast between the analytical performance characteristics using gas chromatography-mass spectrometry (GC-MS) liquid chromatography-mass spectrometry (LC-MS) and liquid chromatography-ultraviolet (LC-UV) detection for the determination of the antiretroviral drug (ARV) nevirapine (NVP) in fortified human plasma after QuEChERS extraction has been made. Analytical performance characteristics, i.e. linearities, instrument detection limits (IDLs), limits of quantitation (LOQs), method detection limits (MDLs), % mean recoveries and the corresponding relative standard deviations (%RSDs) were estimated using techniques above. Using GC-MS, the correlation coefficients (r(2)) were ≥0.990, which were deemed acceptable linearities. The MDLs ranged between 11.1-29.8μg/L and 13.7-36.0μg/L using helium and hydrogen carrier gases respectively. The LOQs ranged between 16.5-66.7μg/L and 28.4-98.7μg/L using helium and hydrogen carrier gases respectively with a % mean recovery of 83% and %RSD of 4.6%. Using LC-MS and LC-UV, the correlation coefficients (r(2)) were ≥0.990. The MDLs were ranged between 3.14 and 47.1μg/L. The LOQs ranged between 2.85 and 90.0μg/L respectively. The MDLs using GC-MS, LC-MS and LC-UV were below the therapeutic range for NVP in human plasma is considered to be between 2300μg/L (Cmin) and 8000μg/L (Cmax). This study also demonstrated that helium can be substituted with hydrogen which is relatively cheaper and easily obtainable even by use of a generator.

High throughput LC–MS/MS method for simultaneous determination of zidovudine, lamivudine and nevirapine in human plasma

A selective and sensitive high performance liquid chromatography-tandem mass spectrometry method has been developed and validated for simultaneous determination of zidovudine (ZDV), lamivudine (3TC) and nevirapine (NVP) in human plasma. After Solid phase extraction (SPE), analytes and ISTDs were run on Peerless Basic C18 column with an injection volume of 3μL and run time of 3.0min. An isocratic mobile phase of 0.1% formic acid in water:methanol (15:85, v/v) was used with positive mass spectrometric detection. The method was validated over a concentration range of 5-1500ng/mL for ZDV and 3TC and over the concentration range of 10-3000ng/mL for NVP. The intraday and interday precision and accuracy across four validation runs were ranged from 1.6 to 10.1% and 93.8 to 110.8% respectively.

High throughput LC–MS/MS method for simultaneous determination of tenofovir, lamivudine and nevirapine in human plasma

A selective and high throughput liquid chromatography-mass spectrometry method has been developed and validated for the simultaneous quantification of tenofovir (TFV), lamivudine (3TC) and nevirapine (NVP) in human plasma using emtricitabine (FTC) as internal standard (ISTD). Following solid phase extraction (SPE), the analytes and ISTD were run on Prontosil C18AQ column (100mm×4.6mm, 3μm) using an isocratic mobile phase consisting of 1mM ammonium acetate in water (pH 6.5±0.3):acetonitrile (50:50, v/v). The precursor and product ions of the drugs were monitored on a triple quadrupole instrument operated in the negative ionization mode. The method was validated over a concentration range of 2-500ng/mL for TFV and over a concentration range of 10-4000ng/mL for 3TC and NVP with relative recoveries ranging from 61 to 85%. The intra and inter batch precision (%CV) across four validation runs was less than 12.2%. The accuracy determined at four QC levels (LLOQ, LQC, MQC and HQC) was within ±8.5%, in terms of relative error.

Simultaneous quantitation of lamivudine, zidovudine and nevirapine in human plasma by liquid chromatography–tandem mass spectrometry and application to a pharmacokinetic study

A rapid and sensitive LC–MS/MS method for the simultaneous quantitation of lamivudine, zidovudine and nevirapine in human plasma using abacavir as internal standard has been developed and validated. The analytes and IS were extracted from plasma by solid phase extraction using Oasis HLB cartridges and separated on a Hypurity Advance C18 column using a mixture of acetonitrile:0.1% formic acid (76:24, v/v) at a flow rate of 0.8mL/min. Detection involved an API-4000 LC–MS/MS with electrospray ionization in the positive ion mode and multiple-reaction monitoring for analysis. The method was validated according to FDA guidelines and shown to provide intra- and inter-day precision and accuracy within acceptable limits in a run time of only 3.5min. The method was successfully applied to a pharmacokinetic study involving a single oral administration of a combination tablet to human male volunteers.

Development and validation of an HPLC-UV method for simultaneous determination of zidovudine, lamivudine, and nevirapine in human plasma and its application to pharmacokinetic study in human volunteers

A simple, rapid, and sensitive high performance liquid chromatographic method with UV detection has been developed and validated according to the FDA guidelines for the quantitation of zidovudine (ZDV), lamivudine (LMV), and nevirapine (NVR) in human plasma. The sample was prepared by simple liquid-liquid extraction. Chromatographic separation was carried out in a Hypersil BDS, C(18) column (250 mm × 4.6 mm; 5 µm particle size) with simple mobile phase composition of 0.1 M ammonium acetate buffer in 0.5% acetic acid, v/v and methanol (40:60, v/v) at a flow rate of 0.85 ml min(-1) where detector was set at 270 nm with a total run time of 10 min which is very short for simultaneous estimation of three analytes in plasma. The method was linear over the concentration range of 50-3000, 50-2000 and 10-3000 ng ml(-1) with lower limit of quantifications (LLOQ) of 50, 50, and 10 ng ml(-1) for ZDV, LMV, and NVR, respectively. Accuracy and precision values of both within-run and between-run obtained from six different sets of three quality control (QC) samples along with the LLOQ analyzed in separate occasions for all the analytes ranged from 94.47-99.71% and 0.298-3.507%, respectively. Extraction recovery of analytes in plasma samples was above 90.16%. In stability tests, all the analytes in human plasma were stable during storage and assay procedure. The developed and validated method was successfully applied to quantitative determination of the three analytes in plasma for pharmacokinetic study in 12 healthy human volunteers.

Simultaneous determination of lamivudine, stavudine and nevirapine in human plasma by LC–MS/MS and its application to pharmacokinetic study in clinic

A new high-throughput LC-MS/MS method for the simultaneous determination of lamivudine (3TC), stavudine (d4T) and nevirapine (NVP) in human plasma is presented, with zidovudine as an internal standard. The analytes were extracted from plasma by protein precipitation and only 150 microL plasma was needed. Chromatographic separation was achieved on a Shiseido C(8) column (150 x 2.0 mm, 5 microm) with a total run time of 6 min. A tandem mass spectrometric detection was conducted using multiple reaction monitoring under positive ionization mode with an electrospray ionization interface. The method was developed and validated over the concentration range of 25-5000 ng/mL for 3TC and NVP and 20-4000 ng/mL for d4T. The method was validated in terms of intra- and inter-day precision (< or = 8.6%), accuracy (within +/- 8.4%), linearity and specificity. The method has been successfully applied to the pharmacokinetic study of a combination treatment of 300 mg lamivudine, 30 mg stavudine and 200 mg nevirapine in 22 healthy male volunteers under fasting conditions.

A New Validated RP‐ HPLC Method for the Determination of Nevirapine in Human Plasma

A rapid, selective and sensitive high performance liquid chromatographic method for the estimation of nevirapine in human plasma has been developed. Chromatography was carried out on a Hypersil BDS C18 column using a mixture of ammonium acetate buffer (pH 4.0 ± 0.05) and acetonitrile (85:15 v/v) as the mobile phase. The eluents were monitored for the drug by UV detection at 254 nm. Oxcarbazepine was used as an internal standard for this study. The retention times for nevirapine and oxcarbazepine were found to be 7.2 and 14.7 min respectively. The method was found to be linear in the concentration range of 50 ng/mL to 5003.7 ng/mL. The method was validated as per FDA guidelines and was found to be suitable for bioequivalence and pharmacokinetic studies.

Simultaneous quantitative determination of zidovudine and nevirapine in human plasma using isocratic, reverse phase high performance liquid chromatography

Purpose: To develop a sensitive and rapid reverse phase high performance liquid chromatography (HPLC) method for the measurement of the levels of zidovudine (ZVD) and nevirapine (NVP) in human plasma. Methods: Standard stock solutions for HPLC analysis were prepared by dissolving ZVD and NVP in methanol. In the HPLC measurement, sample detection was carried out at 246 nm using an ultraviolet (UV)-photo diode array (PDA) detector. Plasma sample pretreatment consisted of protein precipitation extraction with methanol. The compounds were separated using a mobile phase consisting of a pH 3.0 solution (obtained by adjusting the pH of water with orthophosphoric acid): acetonitrile (73:27 v/v) on a Phenomenex LUNA C18, column (250×4.6 mm i.d., 5&#956;m) at a flow rate of 0.9 mL min-1. The total run time for the assay was 10.2 min. The method was validated over the range of 300-9600 ng mL-1 and 200-6400 ng mL-1 for ZVD and NVP, respectively. Results: The lowest limits of quantification (LLOQ) and of detection (LOD) were 300 and 63 ng mL-1 for ZVD and 200 and 17 ng mL-1 for NVP, respectively. The method was found to be accurate, with accuracy ranging from -10.92 to +9.57 % and precise, with intra-day, inter-day as well as analyst to analyst precision of 0.68 to 9.38 %. Extraction recoveries of the drugs from plasma were 91.39, 95.01, 89.51 % for ZVD and 90.93, 93.26, 92.13 % for NVP, for LQC (low quality control), MQC (medium quality control) and HQC (high quality control) samples, respectively. Stability data revealed that the drugs were stable in plasma under various test conditions. Conclusion: This assay can be suitably used for the determination of zidovudine (ZVD) and nevirapine (NVP) in human plasma and should be useful in HIV clinical trials and clinical therapeutic drug monitoring (TDM) programs. It would also be potentially useful in the determination of pharmacokinetic profiles and in bioequivalence studies in HIV research.. Keywords: Assay, Zidovudine, Nevirapine, Human plasma, Reverse phase high- performance liquid chromatography.Tropical Journal of Pharmaceutical Research Vol. 8 (1) 2009: pp. 79-86

Development and Validation of a Rapid Liquid Chromatography Tandem Mass Spectrometry Method to Quantify Nevirapine in Human Plasma and its Application to Bioequivalence Study in Healthy Human Subjects

A simple, accurate and robust liquid chromatography tandem mass spectrometry (LC‐ESI‐MS/MS) method has been developed for estimation of nevirapine in human plasma. Abacavir sulphate was used as an internal standard (IS). The plasma filtrate obtained after solid phase extraction (SPE), using a hydrophilic‐lipophilic balance (HLB) cartridge, was submitted directly to short column liquid chromatography‐tandem mass spectrometric assay, with negligible matrix effect on the analysis. Turbo ion spray, operating in positive mode and multiple reaction monitoring (MRM) scan type was used to quantify nevirapine in human plasma. The extraction procedure yielded extremely clean extracts with a recovery of 91.85 and 97.71% for nevirapine and IS, respectively. The method is simple and reliable with a chromatographic run time of 2.5 min. The lower limit of quantification (LLOQ) found was 25 ng ml−1 with good accuracy and precision. The developed method was validated in the linear dynamic range of 25–5000 ng ml−1 with correlation coefficient (r)≥0.9996. The intra‐and inter‐batch precision for the samples at the LLOQ were 7.04 and 7.71%, respectively. The intra‐ and inter‐batch accuracy ranged from 91.17– 102.17%. The method was successfully applied for bioequivalence studies in 36 healthy Indian human subjects after oral administration of 10 mg ml−1 suspension formulations.

High throughput LC–MS/MS method for simultaneous quantification of lamivudine, stavudine and nevirapine in human plasma

A selective and high throughput liquid chromatography/tandem mass spectrometry (LC–MS/MS) method has been developed and validated to separate, detect and simultaneously quantify lamivudine (3TC), stavudine (d4T) and nevirapine (NVP) in human plasma using metaxalone as internal standard (IS). After solid phase extraction (SPE), the analytes and the IS were chromatographed on a Symmetry C18 (150mm×3.9mm i.d., 5μm particle size) column using 5μL injection volume with a run time of 4.5min. An isocratic mobile phase consisting of 0.5% glacial acetic acid in water:acetonitrile (20:80, v/v) was used to separate all these drugs. The precursor and product ions of these drugs were monitored on a triple quadrupole mass spectrometer, operating in the multiple reaction monitoring mode (MRM) without polarity switch. The method was validated over the range of 25–3000ng/mL for 3TC, 20–2000ng/mL for d4T and 50–5000ng/mL for NVP. The absolute recoveries for analytes (≥86%) and IS (98.12%) achieved from spiked plasma samples were consistent and reproducible. Inter-batch and intra-batch precision (%CV) across four validation runs (LLOQ, LQC, MQC and HQC) was less than 10. The accuracy determined at these levels was within ±8% in terms of relative error. The method was successfully applied to a pivotal bioequivalence study of [60 (3TC)+12 (d4T)+100 (NVP)]mg dispersible tablets in 60 healthy human subjects under fasting condition.

Development of a one-step immunochromatographic strip test for the rapid detection of nevirapine (NVP), a commonly used antiretroviral drug for the treatment of HIV/AIDS

Currently, high-performance liquid chromatographic (HPLC) methods are mainly used to measure antiretroviral plasma concentrations in HIV-infected patients. Although the utility of routine therapeutic drug monitoring (TDM) as an additional tool to optimize long-term antiretroviral therapy is unclear, if TDM is to be widely used, the availability of simple, cheap and reliable methods for the measurement of antiretroviral drug levels are needed, particularly in resource-limited settings. In this study, an immunochromatograhic (IC) strip test to detect the presence of nevirapine (NVP) in body fluids has been developed. Antiserum to NVP was first raised in rabbits by immunization against NVP chemically conjugated with bovine serum albumin, and subsequently validated by Western immunoblotting and competitive indirect ELISA. The partially purified anti-NVP antibodies were conjugated with colloidal gold particles. The conjugation of the colloidal gold and polyclonal antibodies was monitored by UV–vis spectroscopy, while transmission electron microscopy images were used to characterize the particle size and shape of the conjugates. The resulting colloidal gold conjugates were used for the production of an IC strip test to detect nevirapine in human plasma. Preliminary assessment suggests no-cross reactivity of the NVP polyclonal antibodies but assessment of plasma samples from HIV-infected patients receiving HAART needs to be conducted. This assay could potentially be used for drug monitoring as part of the clinical care of HIV infected patients.

Detection of nevirapine in plasma using thin-layer chromatography.

Nevirapine (NVP) is widely prescribed in resource-poor settings to pregnant women for treatment and prevention of HIV infection. High rates of misreported adherence, however, have compelled clinicians to find alternative methods to ensure systemic drug exposure. This report describes a fast, inexpensive thin-layer chromatography (TLC) method to detect the presence of NVP in human plasma. Human plasma was spiked with various concentrations of NVP. NVP was subsequently isolated using solid-phase extraction and visualized with TLC. Clinical samples with NVP concentrations predetermined by high-performance liquid chromatography were used to validate the TLC method. NVP was detected at concentrations as low as 60 ng/mL. The lower limit of detection was set at 100 ng/mL due to the clear spot definition at this concentration. The turnaround time for assay results averages several hours, and costs associated with the assay are considerably below standard drug quantitation techniques. TLC provides a rapid, sensitive, and economical tool to qualitatively measure NVP in plasma. This method offers clinicians in resource-poor settings an alternative approach for measuring adherence, particularly in developing-world regions where NVP use is common and there is an immediate need to prevent mother-to-child HIV transmission.

Simultaneous determination of the HIV drugs indinavir, amprenavir, saquinavir, ritonavir, lopinavir, nelfinavir, the nelfinavir hydroxymetabolite M8, and nevirapine in human plasma by reversed-phase high-performance liquid chromatography.

A reversed-phase high-performance liquid chromatography method for the simultaneous quantitative determination of the currently available HIV protease inhibitors amprenavir, indinavir, lopinavir, nelfinavir, ritonavir, saquinavir, the active nelfinavir metabolite M8, and the nonnucleoside reverse transcriptase inhibitor nevirapine in human plasma is described. The method involved liquid-liquid extraction from plasma, followed by high-performance liquid chromatography with an OmniSpher 5 C18 column and ultraviolet detection set at a wavelength of 215 nm for the protease inhibitors and 280 nm for nevirapine. The runtime was 25 minutes. The assay has been validated over the concentration range of 0.05 to 30 mg/L for indinavir, nelfinavir, ritonavir, and saquinavir, 0.07 to 30 mg/L for amprenavir and lopinavir, and 0.05 to 15 mg/L for M8 and nevirapine. This method proved to be simple, accurate, and precise and is useful for the therapeutic drug monitoring of protease inhibitors and the nonnucleoside reverse transcriptase inhibitor nevirapine on a routine basis.

Simple and rapid method for the simultaneous determination of the non-nucleoside reverse transcriptase inhibitors efavirenz and nevirapine in human plasma using liquid chromatography

Efavirenz and nevirapine are non-nucleoside reverse transcriptase inhibitors for the treatment of HIV-1-infected individuals. A simple and rapid high-performance liquid chromatographic method for the simultaneous quantification of efavirenz and nevirapine in human plasma suitable for therapeutic drug monitoring is described. Sample pre-treatment consisted of protein precipitation with acetonitrile and subsequently dilution with distilled water. The drugs were separated from endogenous compounds by isocratic reversed-phase high-performance liquid chromatography with ultraviolet detection at 275 nm. The method was validated over the therapeutically relevant concentration range of 0.05–15.0 mg l −1 and 0.25–15.0 mg l −1 for efavirenz and nevirapine, respectively, using a volume of 100 μl of plasma. The calibration curves were linear over this concentration range. Carbamazepine was used as internal standard. The assay proved to be accurate (accuracies varied between −12.7 and 8.5%) and precise (intra- and inter-assay precisions were less then 5.9%). The tested batches of control human plasma and frequently co-administered drugs did not interfere with the described methodology. Efavirenz and nevirapine were stable under various relevant storage conditions. This validated assay is suited for use in pharmacokinetic studies with efavirenz and nevirapine and can readily be implemented in the setting of a hospital laboratory for the monitoring of efavirenz and nevirapine concentrations.

Simultaneous determination of zidovudine and nevirapine in human plasma by RP-LC

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, 250×4.6 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.

Rapid determination of nevirapine in human plasma by gas chromatography

A sensitive and rapid gas chromatographic method has been developed to determine the levels of the HIV-1 non-nucleoside reverse transcriptase inhibitor nevirapine in human plasma. Quantitative recovery following liquid–liquid-extraction with diethylether from 500 μl of human plasma was achieved. Subsequently, the assay was performed with a CP-Sil 5CB capillary column, 15 m×0.32 mm×1.0 μm film thickness with a nitrogen–phosphorous-detector (NPD), Helium 5.0 was used as carrier gas with a constant inlet pressure of 7 p.s.i. Linear standard curves were obtained for concentrations ranging from 10 to 20 000 ng/ml. The calculated intra- and inter-day coefficients of variation were below 8%.

Determination of zidovudine/lamivudine/nevirapine in human plasma using ion-pair HPLC

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 (150×3.9 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, 59.0–17 650 ng/ml for 3TC and 53.2–13 300 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.