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)

As part of our on-going study of the analysis of anti-human immunodeficiency virus (HIV) nucleosides, a capillary electrochromatography (CEC) method has been developed for the concurrent analysis of nucleoside HIV reverse transcriptase inhibitors (NRTIs) in a pool of endogenous nucleosides. Up to now, beta-cyclodextrin-bonded silica stationary phases have mainly been dedicated to the separation of enantiomers; however, these polysaccharides can be also be used in achiral way. This work aims at showing how CEC performed on a beta-cyclodextrin-bonded silica stationary phase can be used to concurrently resolve zidovudine (AZT), lamivudine (3TC), didanosine (ddA) and its administrated form (ddI), stavudine (d4T) and hivid (ddC) in a mixture of adenosine (A), cytidine (C), guanosine (G), thymidine (T) and uridine (U). The influence of several parameters (pH buffer, ionic strength, acetonitrile content, temperature and voltage) on both the retention times and the retention factors has been investigated using the short-end injection technique to achieve baseline separation in a short-time analysis before quantitation. Moreover, the retention factors of the charged solutes in short-end injection-CEC were calculated using theoretically derived equations, allowing for the actual voltage drop in the packed section of the semipacked CEC capillary.
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PMID:Concurrent analysis of nucleoside reverse transcriptase inhibitors in a pool of endogenous nucleosides by short-end injection-capillary electrochromatography on a beta-cyclodextrin-bonded stationary phase. 1200 25

A selective and sensitive high-performance liquid chromatographic (HPLC) method has been developed for the determination of the six human immunodeficiency virus (HIV)-protease inhibitors (amprenavir, indinavir, lopinavir, nelfinavir, ritonavir, and saquinavir) and the non-nucleoside reverse transcriptase inhibitors (efavirenz and nevirapine) in a single run. After a liquid-liquid extraction with diethyl ether, the six protease inhibitors and the two non-nucleoside reverse transcriptase inhibitors are separated on a Stability RP18 column eluted with a gradient of acetonitrile and phosphate buffer 50 mmol/L pH 5.65. A sequential ultraviolet detection (5-minute sequence set at 240 nm for nevirapine acquisition, 22-minute sequence set at 215 nm for other antiretroviral drugs acquisition followed by a sequence set at 260 nm for internal standard acquisition) allowed for simultaneous quantitation of the six protease inhibitors, nevirapine, and efavirenz. Calibration curves were linear in the range 100 ng/mL to 10,000 ng/mL. The limit of quantitation was 50 ng/mL for all drugs except nevirapine (100 ng/mL). Average accuracy at four concentrations ranged from 88.2% to 110.9%. Both interday and intraday coefficients of variation were less than 11% for all drugs. The extraction recoveries were greater than 62%. This method is simple and shows a good specificity with respect to commonly co-prescribed drugs. This method allows accurate therapeutic monitoring of amprenavir, indinavir, lopinavir, nelfinavir, ritonavir, saquinavir, efavirenz, and nevirapine.
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PMID:High-performance liquid chromatographic method for the simultaneous determination of the six HIV-protease inhibitors and two non-nucleoside reverse transcriptase inhibitors in human plasma. 1202 35

A rapid (less than 30 min), sensitive, and specific liquid chromatography method for simultaneous assay of nine antiretroviral drugs in human plasma is described. This technique allows therapeutic drug monitoring of six approved protease inhibitors (amprenavir, indinavir, lopinavir, nelfinavir, ritonavir, and saquinavir) and two approved non-nucleoside reverse transcriptase inhibitors (efavirenz and nevirapine). Assays were performed after diethyl ether liquid-liquid extraction from 250-microL plasma samples. Chromatographic separation was achieved on an X-TERRA (Waters; Saint Quentin, France) column using a 58% water (with 3 mmol/L pyrrolidine) and 42% acetonitrile mobile phase. Three ultraviolet wavelengths were used for detection with a diode array detector. This method allowed quantitative assay of all nine antiretroviral drugs within a concentration range of 25 ng/mL to 9000 ng/mL. The method has been validated extensively and has been in routine use in our laboratory for several months for drug monitoring in plasma samples from patients treated with antiretroviral drugs.
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PMID:Simultaneous quantitative assay of six HIV protease inhibitors, one metabolite, and two non-nucleoside reverse transcriptase inhibitors in human plasma by isocratic reversed-phase liquid chromatography. 1214 42

Syntheses of [carbonyl-11C]2-(2-benzoylphenoxy)-N-phenylacetamide, a radiolabeled inhibitor of human immunodeficiency virus type 1 (HIV 1) reverse transcriptase, were achieved by applying palladium-mediated cross-coupling reactions with insertion of [11C]carbon monoxide. Our interest was focused for the present on a comparison of the Stille and Suzuki methods, using trimethylphenylstannane or phenylboronic acid as alternative coupling reagents, respectively. The Suzuki variant gave a much higher amount of [11C]CO radioactivity trapped in the reaction mixture, but a significant loss of product occurred due to adsorption phenomena on the potassium carbonate present in the heterogeneous reaction mixture. The labeled product was isolated in only 20% yield (based on trapped [11C]CO, not corrected for decay). According to Stille, the reaction provided a product that could be isolated more easily but it did not increase the final yield of the target compound due to a low trapping efficiency for [11C]CO. Both methods were performed in an overall synthesis time of 30min, starting from [11C]CO2, and gave a product with a specific radioactivity of at least 30GBq/micromol. The Stille method as well as the Suzuki reaction allowed the synthesis of a radiochemically pure product in aqueous acetonitrile.
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PMID:Syntheses of [carbonyl-11C]2-(2-benzoylphenoxy)-N-phenylacetamide from [11C]carbon monoxide by the Suzuki and the Stille reactions. 1243 42

A sensitive high-performance liquid chromatography method was developed with spectrofluorimetric detection for the determination of tenofovir, a new HIV reverse transcriptase inhibitor, in human plasma. After precipitation of 200 microl of plasma samples by methanol and evaporation of the supernatant, fluorescent derivatized compounds were obtained by a 40-min incubation at 80 degrees C with chloroacetaldehyde 0.34% at pH 4.5. The assay was performed isocratically using 5 mM Na(2)HPO(4) (pH 6), containing tetrabutylammonium (TBA) chloride 5 mM, and acetonitrile (85:15, v/v) as mobile phase, and a Cluzeau C(8) plus satisfaction column maintained at 35 degrees C. Detection was performed at excitation and emission wavelengths set at 236 and 420 nm, respectively. In these conditions, tenofovir can be separated from adefovir, the internal standard, and endogenous substances. The method was found to be linear and has been validated over a concentration range of 5-1000 microg/l. The average coefficient of the limit of quantification (5 microg/l) was 5.38% and at this concentration, a signal-to-noise ratio of 500 was measured
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PMID:Determination of tenofovir in human plasma by high-performance liquid chromatography with spectrofluorimetric detection. 1255 52

In the present investigation, a novel approach towards a complete separation of all 15 protease and reverse transcriptase inhibitors which are currently approved for use in highly active antiretroviral therapy in a single analytical run is presented. The developed method employs an acidic background electrolyte with sodium polyanethol sulfonate (SPAS) as polyanionic electroosmotic flow (EOF) modifier to establish a strong cathodic EOF, sodium dodecyl sulfate (SDS) as pseudostationary selector, and acetonitrile and ethanol as organic modifiers. Separation of the analytes is based on two different mechanisms. The more basic analytes are protonated at the prevailing pH conditions and thus migrate in front of the cathodic EOF, whereas the less basic and neutral analytes interact with the SDS and are retained after the EOF. By optimizing electrolyte pH, the amount of solvents and SDS concentrations in the background electrolyte it is possible to completely separate all compounds of interest.
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PMID:Simultaneous separation of fifteen approved protease and reverse transcriptase inhibitors for human immunodeficiency virus therapy by capillary electrophoresis. 1260 35

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 microl 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.
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PMID:Simple and rapid method for the simultaneous determination of the non-nucleoside reverse transcriptase inhibitors efavirenz and nevirapine in human plasma using liquid chromatography. 1286 43

An accurate, sensitive, and specific reverse-phase high-performance liquid chromatography (HPLC) assay for the simultaneous quantitative determination of HIV-protease inhibitors (PIs) (indinavir, IDV; amprenavir, APV; saquinavir, SQV; nelfinavir, NFV; ritonavir, RTV; and lopinavir, LPV) and non-nucleoside reverse transcriptase inhibitors (NNRTIs) (nevirapine, NVP; delavirdine, DLV; and efavirenz, EFV) in human blood plasma is described. The method provides excellent resolution and peak shape for nine analytes through a linear gradient (36-86%) of 25% phosphate buffer (pH 4.5), 60% acetonitrile, 15% methanol, and 0.75 ml TFA, with a gradient mobile phase flow rate (0.9-1.1 ml) over 30 min run time. The optimized solid phase extraction (SPE) extraction method using (1.0 ml, 100mg BOND ELUT-C18 Varian) column provides a clean base line and high extraction efficiency using a 550 microl plasma sample. The method was validated over the range of 10-10,000 ng/ml for NVP, IDV, and SQV; 10-5000 ng/ml for EFV; 25-10000 ng/ml for APV; and 25-5000 ng/ml for DLV, NFV, RTV, and LPV. This method is accurate (average accuracies of three different concentrations ranged from 91 to 112%), and precise (within- and between-day precision measures ranged from 0.2 to 5.7% and 0.1 to 5.4%, respectively). This method is suitable for use in clinical pharmacokinetic studies as well as in therapeutic drug monitoring (TDM).
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PMID:High-performance liquid chromatography assay for the quantification of HIV protease inhibitors and non-nucleoside reverse transcriptase inhibitors in human plasma. 1513 96

A rapid and simple high-performance liquid chromatography method has been developed for the determination of the HIV-1 reverse transcriptase inhibitor abacavir in human plasma. It included a single liquid-liquid extraction procedure with a mixture of ethyl acetate-diethyl ether prior to reversed-phase chromatography on a C18 column and C18 precolumn insert. Ultraviolet detection was set at 285 nm. The mobile phase consisted of water-acetonitrile (83:17, v/v) and the flow rate was kept at 1 mL/min. The total run time for a single analysis was 10 min. The method has been validated over the range 50-2500 ng/mL. The assay was linear over the entire concentration range (r2 = 0.9993). Intra- and inter-day precision and accuracy were less than 8.1 and -5.2%, respectively. The extraction recovery was greater than 94.3%. Abacavir was stable under the relevant storage conditions tested. After the validation, the analytical error function was established as standard deviation (SD; ng/mL) = -1.072 + 0.037C (C = theoretical concentration value). The method developed and its associated analytical error function will be suitable for pharmacokinetic studies and monitoring of HIV-1 patients.
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PMID:Determination of abacavir in human plasma by high-performance liquid chromatography with ultraviolet detection and the analytical error function. 1538 66

A sensitive and accurate liquid chromatography-tandem mass spectrometric (LC-MS/MS) method for the intracellular determination of nine antiretroviral drugs in human peripheral blood mononuclear cells (PBMCs) is proposed. PBMCs are isolated by density gradient centrifugation using Vacutainer CPT tubes and cell count is performed with a Coulter instrument. Single-step extraction of drugs from PBMCs pellets is performed with MeOH 50% (with clozapine added as internal standard, I.S.) and the supernatant is injected onto a 2.1 mm x 30 mm SymmetryShield 3.5 microm-RP18 column equipped with a 2.1 x 10 mm guard column. Chromatographic separations are performed using a gradient program with a mixture of 2 mM ammonium acetate containing 0.1% formic acid and acetonitrile with 0.1% formic acid. Analytes quantification is performed by electro-spray ionisation-triple quadrupole mass spectrometry using the selected reaction monitoring (SRM) detection mode. The positive mode is used for the HIV protease inhibitors (PIs) indinavir, amprenavir, saquinavir, ritonavir, nelfinavir, lopinavir, atazanavir and the non-nucleoside reverse transcriptase inhibitors (NNRTIs) nevirapine, and the negative mode is applied for efavirenz. The calibration curves are prepared using blank PBMCs spiked with antiretroviral drugs at concentrations ranging from 0.5 to 100 ng/ml of cell extracts and fitted to a quadratic regression model weighted by 1/(concentration)(2). The lower limit of quantification is less than 0.5 ng/ml. The mean extraction recovery for all PIs/NNRTIs is always above 88%. The method is precise, with mean inter-day CV% within 0.6-10.2%, and accurate (range of inter-day deviation from nominal values -7.2 to +8.3%). This analytical method can be conveniently used in clinical research for the assessment of intracellular levels of all PIs/NNRTIs commercially available at present using a simple one-step cell extraction of PBMCs followed by liquid chromatography coupled with tandem triple quadripole mass detection.
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PMID:Intracellular measurements of anti-HIV drugs indinavir, amprenavir, saquinavir, ritonavir, nelfinavir, lopinavir, atazanavir, efavirenz and nevirapine in peripheral blood mononuclear cells by liquid chromatography coupled to tandem mass spectrometry. 1583 90


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