Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.7.49 (reverse transcriptase)
31,746 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We report the development of a simple, economical and reliable chromatographic method for the simultaneous determination of six HIV protease inhibitors (PIs; amprenavir, indinavir, lopinavir, nelfinavir, ritonavir and saquinavir), the active metabolite of nelfinavir (M8) and the non-nucleoside reverse transcriptase inhibitor (NNRTI; efavirenz) in human plasma. After extraction from plasma with an ethyl acetate-acetonitrile mixture, the analytes were separated on a phenyl column with a gradient of acetonitrile and phosphate solutions, and detected at three ultraviolet wavelengths. Calibration curves were linear over the range 0.025-15 microg/mL for saquinavir and 0.05-15 microg/mL for the other analytes. The accuracies ranged from -6.9% to +7.6%, and the intra-assay and inter-assay precisions were <9.2 and <11.8%, respectively. Our method, covering most of the PIs and NNRTIs currently used, facilitates ready therapeutic drug monitoring in hospital laboratories.
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PMID:Simultaneous determination of six HIV protease inhibitors (amprenavir, indinavir, lopinavir, nelfinavir, ritonavir and saquinavir), the active metabolite of nelfinavir (M8) and non-nucleoside reverse transcriptase inhibitor (efavirenz) in human plasma by high-performance liquid chromatography. 1597 Dec 89

In this work, a simple and rapid electrokinetic chromatography method for the simultaneous separation of different protease inhibitors (indinavir, ritonavir, saquinavir, nelfinavir), nucleoside reverse transcriptase inhibitors (stavudine, zidovudine, didanosine) and non-nucleoside reverse transcriptase inhibitors (nevirapine, efavirenz) was developed. The analyses were performed in a 75 microm i.d. uncoated fused-silica capillary with 48.5 cm length (effective length of 40 cm) using a running buffer consisting of 20 mmol L(-1) sodium dodecyl sulfate, 10 mmol L(-1) sodium tetraborate, 30% acetonitrile and 5% ethanol. Samples were injected hydrodynamically by applying 50 mbar pressure during 6 s. All analytes were separated within 10 min with a voltage of 20 kV. The proposed method was validated for zidovudine, didanosine and efavirenz in human serum. Serum samples were prepared using a solid-phase extraction procedure (Waters Oasis HLB cartridges). For quantitative purposes, stavudine was chosen as the internal standard (IS). Method validation parameters were determined revealing good migration time repeatability (<0.7% RSD) and peak area repeatability (<1.2% RSD). Intra- and inter-day precisions were less than 1.7% and 4.4% RSD, respectively. Matrix matching analytical curves for each drug were linear in the 1.0-20.0 microg mL(-1) interval (r > 0.998). Limits of detection (LOD) were in range of 0.3-0.5 microg mL(-1). The extraction recoveries were higher than 90% with exception of efavirenz, which was 77.4%. Based on the performance characteristics, the proposed method was found suitable for the determination of zidovudine, didanosine and efavirenz in serum samples.
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PMID:Determination of antiretroviral agents in human serum by capillary electrophoresis. 1639 7

Therapeutic drug monitoring (TDM) is pivotal to improve the management of HIV infection. Here, a HPLC-UV method has been developed to quantify simultaneously seven HIV protease inhibitors (amprenavir, atazanavir, indinavir, lopinavir, nelfinavir, ritonavir, and saquinavir; PIs), seven nucleoside reverse transcriptase inhibitors (abacavir, didanosine, emtricitabine, lamivudine, stavudine, zalcitabine, and zidovudine; NRTIs), and two non-nucleoside reverse transcriptase inhibitors (efavirenz and nevirapine; NNRTIs) in human plasma. The volume of the plasma sample was 600 microL. This method involved automated solid-phase extraction with Oasis HLB Cartridge 1 cc (divinylbenzene and N-vinylpyrrolidone) and evaporation in a water bath under nitrogen stream. The extracted samples were reconstituted with 100 microL methanol. Twenty microliters of these samples were injected into a HPLC-UV system, the analytes were eluted on an analytical C(18) Symmetry column (250 mm x 4.6mm I.D.) with a particle size of 5 microm. The mobile phase (0.01 M KH(2)PO(4) and acetonitrile) was delivered at 1.0 mL/min with linear gradient elution. The total run time for a single analysis was 35 min, the anti-HIV drugs were detected by UV at 240 and 260 nm. The calibration curves were linear up to 10 microg/mL. The absolute recovery ranged between 88 and 120%. The in vitro stability of anti-HIV drugs (0.005-10 microg/mL) in plasma has been studied at 24.0 degrees C. On these bases, a two to four analyte method has been tailored to the individual needs of the HIV-infected patient. The HPLC-UV method here reported has been validated and is currently applied to monitor PIs, NRTIs, and NNRTIs in plasma of HIV-infected patients. It allows to monitor the largest number of anti-HIV drugs simultaneously, appearing useful in a routine laboratory, and represents an essential step to elucidate the utility of a formal therapeutic drug monitoring for the optimal follow-up of HIV-infected patients.
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PMID:Simultaneous determination of 16 anti-HIV drugs in human plasma by high-performance liquid chromatography. 1640 32

For the quantification of all currently approved non-nucleoside reverse transcriptase inhibitors and protease inhibitors, including the new protease inhibitor darunavir and the active nelfinavir metabolite M8, an assay was developed, using liquid chromatography coupled with tandem mass spectrometry. The sample pretreatment consisted of a protein precipitation with a mixture of methanol and acetonitrile using only 100 microL plasma. Chromatographic separation was performed on a reversed-phase C18 column (150 x 2.0 mm, particle size 5 microm) with a quick stepwise gradient using an acetate buffer (pH 5) and methanol, at a flow rate of 0.25 mL/min. The analytical run time was only 10 min. 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 a range of 0.1 to 20 microg/mL for amprenavir, atazanavir, efavirenz, indinavir, lopinavir, nelfinavir, the active nelfinavir metabolite M8, nevirapine and ritonavir, a range of 0.05 to 10 microg/mL for saquinavir and darunavir and a range of 0.5 to 100 microg/mL for tipranavir, based on observed concentration ranges in patients treated with these drugs. D5-squinavir, D6-indinavir, 13C6-efavirenz and dibenzepine were used as internal standards. The method was proven to be specific, accurate, precise and robust. Accuracies ranged from 88.5% to 102.2% and all precisions were less than 9.5%. Furthermore, the assay demonstrates a high sensitivity for all analytes and the stepwise gradient allows addition of new analytes into the same method. The method is now successfully applied for routine therapeutic drug monitoring and pharmacokinetic studies in HIV-infected patients.
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PMID:Fast and simultaneous determination of darunavir and eleven other antiretroviral drugs for therapeutic drug monitoring: method development and validation for the determination of all currently approved HIV protease inhibitors and non-nucleoside reverse transcriptase inhibitors in human plasma by liquid chromatography coupled with electrospray ionization tandem mass spectrometry. 1761 Feb 14

The determination of antiretroviral drug concentrations in patients treated with highly active antiretroviral therapy (HAART) is an essential part of optimum patient management because of the multitude of pharmacokinetic drug interactions between these drugs and the risk of treatment failure or viral resistance if therapeutic concentrations are not reached. Currently, 21 different antiretrovirals are used in various combinations rendering therapeutic drug monitoring a laborious task. We therefore aimed to simultaneously determine as many antiretrovirals as possible using triple quadrupole mass spectroscopy with electrospray ionisation. For this purpose, spectra and fragmentation patterns of the protease inhibitors amprenavir, atazanavir, indinavir, lopinavir, nelfinavir, ritonavir, and saquinavir, the non-nucleoside reverse transcriptase inhibitors delavirdine, efavirenz, and nevirapine, the nucleoside reverse transcription inhibitors abacavir, didanosine, emtricitabine, lamivudine, stavudine, zalcitabine, and zidovudine, and the nucleotide reverse transcriptase inhibitor tenofovir were evaluated. A bioanalytical method to determine all protease and non-nucleoside reverse transcriptase inhibitors, and zalcitabine and zidovudine concentrations in biological matrices was developed. Samples were prepared by protein precipitation with methanol after addition of three different internal standards. Antiretrovirals were separated by high-performance liquid chromatography on a Nucleosil C18-100 Nautilus column using a gradient of 20 mM ammonium acetate including 0.1% aqueous acetic acid and acetonitrile and detected by electrospray ionisation/tandem mass spectrometry in the negative (efavirenz, stavudine, zidovudine) or positive ionisation mode (all other compounds). The bioanalytical method was successfully validated according to FDA guidelines and applied to plasma and cerebrospinal fluid samples of patients treated for acquired immunodeficiency syndrome (AIDS).
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PMID:Electrospray tandem mass spectroscopic characterisation of 18 antiretroviral drugs and simultaneous quantification of 12 antiretrovirals in plasma. 1763 76

A simple analytical method was developed in 100 microL of plasma for the simultaneous assay of the 7 nucleoside/nucleotide reverse transcriptase inhibitors (abacavir, didanosine, emtricitabine, lamivudine, stavudine, tenofovir, and zidovudine) currently used for the treatment of HIV-infected patients. After adding the internal standard, 6-beta-hydroxy-theophyline, plasma samples were precipitated with 500 microL acetonitrile and the supernatants were evaporated to dryness. The residues were reconstituted with 500 microL of water and 10 microL of the extracts were injected in the chromatographic system. The chromatographic separation was performed with a C-18 column and a gradient mobile phase consisting of a mixture of water and acetonitrile, both containing 0.05% formic acid. Analytes quantification was performed by electrospray ionisation triple quadrupole mass-spectrometry in the positive mode using selected reaction monitoring (SRM). Intra- and inter-assay precision and accuracy were lower than 20% for the limit of quantification, and 15% for higher concentrations. The method has been implemented to assess plasma concentrations of patients infected by HIV and was found suitable for therapeutic drug monitoring.
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PMID:Quantification of seven nucleoside/nucleotide reverse transcriptase inhibitors in human plasma by high-performance liquid chromatography with tandem mass-spectrometry. 1832 57

A bioanalytical method for the determination of most commonly prescribed protease inhibitors (atazanavir, darunavir, lopinavir and ritonavir) and non-nucleoside reverse transcriptase inhibitors (efavirenz and nevirapine) was developed and validated according to FDA guidelines. In brief, dried blood spots were punched out of a collection paper with a 0.25 in. diameter punch. The analytes were extracted from the punched-out disc using a mixture of acetonitrile, methanol and 0.2M zinc sulphate in water (1:1:2, v/v/v) containing the internal standards dibenzepine, 13C6-efavirenz and D5-saquinavir. 20 microL of the extract was injected onto the reversed-phase C18 column (150 mm x 2.0 mm) for separation from endogenous compounds and the analytes were quantified using a triple quadrupole mass spectrometer. The analytical run time was only 10 min. Validated concentration ranges covered the ranges encountered in routine clinical practice. The assay was linear over the concentration ranges tested (0.1-20 mg/L for atazanavir, lopinavir, nevirapine and efavirenz and 0.05-10 mg/L for darunavir and ritonavir). Accuracies and inter- and intra-run precisions at all levels ranged from 96.2 to 113.9% and 3.1 to 13.3%, respectively. Analytes in dried blood spots were stable for at least 7 days at 30 degrees C. The method enabled patient-friendly sample collection, easy and cheap sample shipment and non-hospital based sampling for therapeutic drug monitoring and pharmacokinetic studies.
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PMID:Quantification of protease inhibitors and non-nucleoside reverse transcriptase inhibitors in dried blood spots by liquid chromatography-triple quadrupole mass spectrometry. 1845 82

In the present paper, a new chromatographic method for the determination of acquired immune deficiency syndrome (AIDS) drugs in plasma samples is proposed. The method consists of solid-phase extraction for sample pretreatment and further chromatographic analysis. Drugs have been separated on a C(18) column using an elution gradient based on an increase in the acetonitrile percentage. Analytes have been detected spectrophotometrically at 240, 250, 260 and 280nm. Chromatographic conditions have been thoroughly optimized using experimental design and multicriteria functions. Analytical parameters of the method have been established for both synthetic and plasma samples. Limits of detection are around 5ngmL(-1) for reverse transcriptase inhibitors (nucleoside and non-nucleoside) and 20ngmL(-1) for protease inhibitors. The method has been validated through a spiking/recovery procedure at three concentration levels. Results obtained are highly satisfactory, with recovery values around 100% for all drugs. The method has been applied to the determination of various drug mixtures of medical interest in plasma samples.
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PMID:Reversed-phase liquid chromatographic method with spectrophotometric detection for the determination of antiretroviral drugs. 1847 88

A new method using high-performance liquid chromatography coupled with photo diode array detection was developed and validated for the quantification of plasma concentrations of the human immunodeficiency syndrome integrase inhibitor raltegravir (RGV), the new nonnucleoside reverse transcriptase inhibitor etravirine (ETV), and 11 other antiretroviral agents: ritonavir, atazanavir, lopinavir, nevirapine, efavirenz, saquinavir, indinavir, nelfinavir, and its metabolite M-8, amprenavir, and darunavir. A simple solid phase extraction procedure was applied to 500 microL aliquots of plasma, and chromatographic separation of the drugs and internal standard (quinoxaline) was achieved with a gradient (acetonitrile and phosphate buffer) on an C-18 reverse-phase analytical column with a 28-minute analytical run time. Calibration curves were optimized according to expected ranges of drug concentrations in patients, and the coefficient of determination (r) was higher than 0.998 for all analytes. Mean intraday and interday precisions (percent relative SD) for all compounds were 3.67% and 6.39%, respectively, and mean accuracy (percent deviation from nominal concentration) was -1.17%. Extraction recovery ranged within 75% and 83% for all drugs analyzed. The solid phase extraction and high-performance liquid chromatography coupled with photo diode array method described allow a specific, sensitive, and reliable simultaneous determination of RGV, ETV, and 11 antiretroviral agents in plasma by a single assay. Good extraction efficiency and low limit of quantification make this a suitable method for use in clinical trials and for therapeutic drug monitoring of RGV, protease inhibitors, and nonnucleoside reverse transcriptase inhibitors, including ETV.
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PMID:An HPLC-PDA method for the simultaneous quantification of the HIV integrase inhibitor raltegravir, the new nonnucleoside reverse transcriptase inhibitor etravirine, and 11 other antiretroviral agents in the plasma of HIV-infected patients. 1882 56

For the quantification of the novel non-nucleoside reverse transcriptase inhibitor etravirine in human plasma, dried blood spots and peripheral blood mononuclear cell (PBMC) lysate, an assay was developed and validated, using liquid chromatography coupled with tandem mass spectrometry. Etravirine was extracted from plasma by means of protein precipitation with a mixture of methanol and acetonitrile using only 50 microL plasma. Extraction from dried blood spots was performed with a one-step extraction with a mixture of methanol, acetonitrile and 0.2M zinc sulphate in water (1:1:2, v/v/v) and extraction from cell lysate was performed in 50% methanol in water. Chromatographic separation was performed on a reversed phase C18 column (150mmx2.0mm, particle size 5microm) with a quick stepwise gradient using an acetate buffer (pH 5) and methanol, at a flow rate of 0.25mL/min. (13)C(6)-efavirenz was used as an internal standard. The analytical run time was only 10min. 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 a range of 25-5000ng/mL in plasma, 50-10,000ng/mL in dried blood spots and a range of 5-2500ng/mL in PBMC lysate. Accuracies ranged from 89% to 106% in plasma, from 94% to 109% in dried blood spots and from 91% to 105% in PBMC lysate. Precisions at the all concentration levels ranged from 1.9% to 14% in plasma, 4.7% to 20% in dried blood spots and from 3.1% to 11% in PBMC lysate. The bioanalytical assay was successfully incorporated with previously developed assays for the determination of all currently approved PIs and NNRTIs in plasma and dried blood spots and it is now applied for therapeutic drug monitoring and pharmacological research in HIV-infected patients treated with etravirine.
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PMID:Quantification of etravirine (TMC125) in plasma, dried blood spots and peripheral blood mononuclear cell lysate by liquid chromatography tandem mass spectrometry. 1905 44


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