Simultaneous determination of trimipramine and its major metabolites by high-performance liquid chromatography

Trimipramine is a tricyclic antidepressant drug often assayed by gas chromatographic or gas chromatographic-mass spectrometry techniques. A high-performance liquid chromatographic method with electrochemical detection is described for the assay of trimipramine and its major metabolites, monodesmethyltrimipramine and 2-hydroxytrimipramine, in plasma. The method is sensitive, accurate and robust and thus suitable for routinely assaying samples following single doses of trimipramine to man. The assay was applied to plasma samples obtained following a single 50-mg dose of trimipramine to healthy volunteers.     

Measurement of N-methyl-2-pyridone-5-carboxamide in urine by high performance liquid chromatography

We have previously described a simple and reproducible method for the measurement of nicotinamide and its major metabolite N-methyl-2-pyridone-5-carboxamide (2-pyr) in human plasma. We now describe a low-cost high-throughput method for measurement of urinary 2-pyr, and demonstrate that Isolute C18 bulk can replace use of the column to clean up the samples prior to injection into the HPLC apparatus. Using a standard curve together with an internal standard for each sample, with mean recovery of 2-pyr greater than 95%, the assay has proved reproducible, with considerable savings in cost and time. The principal advantages of this method are the rapid column clean up of samples prior to injection and the simple but effective methodology.     

Simultaneous determination of nefiracetam and its metabolites by high-performance liquid chromatography.

A reversed-phase high-performance liquid chromatographic assay was developed to simultaneously quantitate nefiracetam (NEF), a novel nootropic agent, and its three known oxidized metabolites (N-[(2,6-dimethylphenylcarbamoyl)methyl]succinamic acid (5-COOH-NEF), 4-hydroxy-NEF and 5-hydroxy-NEF) in human serum and urine. The quantitative procedure was based on solid-phase extraction with Sep-Pak C18 and ultraviolet detection at 210 nm. The calibration curves of NEF and the metabolites were linear over a wide range of concentrations (0.5-21.5 nmol/ml for NEF and 0.4-9.5 nmol/ml for metabolites in serum and 4-86 nmol/ml for NEF and 8-190 nmol/ml for metabolites in urine). Intra- and inter-day assay coefficients of variation for the compounds were less than 10%. The limit of detection was 0.1 nmol/ml for NEF, 5-COOH-NEF and 4-hydroxy-NEF, and 0.2 nmol/ml for 5-hydroxy-NEF in both serum and urine. This method is applicable for the determination of NEF and its metabolites in human serum and urine with satisfactory accuracy and precision.     

Simultaneous determination of cytosine arabinoside, its nucleotides and metabolites by ion-pair high-performance liquid chromatography

Currently available high-performance liquid chromatographic assays for cytosine arabinoside (ara-C) and its metabolites suffer from two major shortcomings: inability to resolve both ara-C and its nucleotides in a single chromatographic step and/or inadequate sensitivity to allow quantitation of intracellular cytosine arabinofuranoside-5'-triphosphate (ara-CTP) without the use of radiolabelled drug. In this paper, we describe a new ion-pairing high-performance liquid chromatographic assay for ara-C in biological samples that can separate ara-C from its nucleotides, metabolites, and naturally occurring ribonucleotides in a single chromatographic step with a lower limit of quantitation of 5 pmol for ara-C and 10 pmol for ara-CTP. Examples of the utility of this assay are shown in studies of intracellular pharmacokinetics of ara-C in cultured human breast cancer cells and in analysis of plasma nucleoside levels in patients receiving high-dose thymidine chemotherapy. We conclude that this assay provides a rapid and versatile system that can be applied to the study of both cellular and plasma nucleoside pharmacokinetics.     

Simultaneous analysis of prednisone, prednisolone and their major hydroxylated metabolites in urine by high-performance liquid chromatography

A high-performance liquid chromatographic technique for the simultaneous determination of prednisone, prednisolone and their major hydroxylated metabolites, viz., 20 beta-hydroxyprednisone, 6 beta-, 20 alpha- and 20 beta-hydroxyprednisolone, in human urine is presented. The retention times were 6.5, 11.4, 18.1, 24.2, 31.6 and 35.3 min, respectively. The technique employs betamethasone as the internal standard. Samples are extracted with ethyl acetate using a diatomaceous earth extraction column, and the extract was dried and injected onto a silica gel column with ultraviolet detection at 254 nm. The calibration curve is linear within the studied range 50-1500 ng/ml for prednisolone and 50-750 ng ml for the other steroids. The intra-day and inter-day coefficients of variation are less than 10% for prednisone and prednisolone but higher for the metabolites. The assay was used to study the excretion rate profile of each of these steroids in the urine of a normal male subject receiving a 49.3-mg intravenous dose of prednisolone. The results indicate that prednisone, 6 beta-, 20 alpha- and 20 beta-hydroxyprednisolone may be the major unconjugated metabolites of prednisolone while 20 beta-hydroxyprednisone may be a minor metabolite.     

Simultaneous determination of isbufylline and its major metabolites in rabbit blood and urine by reversed-phase high-performance liquid chromatography.

A sensitive high-performance liquid chromatographic assay for isbufylline and its major metabolites in rabbit blood and urine is described. After extraction, samples were eluted by a linear reversed-phase gradient. Specimens obtained after intravenous administration of isbufylline to rabbits were analysed to identify and subsequently quantify the potential metabolites. Using the ultraviolet absorption trace on the recorder as a reference, elution fractions were collected and analysed by mass spectrometry with the direct inlet system and gas chromatography-mass spectrometry after derivatization. Seven metabolites were identified and another five quantified. The method is specific, accurate, reproducible and recommended for pharmacokinetic studies.     

Determination of ibuprofen and its major metabolites in human urine by high-performance liquid chromatography

A sensitive and selective high-performance liquid chromatographic assay for free and total ibuprofen and its major metabolites in human urine is described. Urine is acidified, drug and metabolites are extracted into hexane-propanol, back-extracted into sodium bicarbonate, neutralized and chromatographed. Ibufenac (4-isobutylphenylacetic acid) and 2-phenylpropionic acid were employed as internal standards. The extraction efficiencies were 94-100% for all compounds. The two metabolites and their internal standard were separated using an isocratic chromatographic system, followed by an abrupt step gradient to a second eluent for separation of ibuprofen and its internal standard with a total run time of 18 min. Detection was by a fixed-wavelength detector (214 nm). Sample-to-sample and day-to-day reproducibility studies yielded coefficients of variability of less than 9% for all compounds. The sensitivity was sufficient to determine 2.5 micrograms/ml free ibuprofen in 100 microliters urine.     

Measurement of dothiepin and its major metabolites in plasma by high-performance liquid chromatography.

This paper describes a reversed-phase high-performance liquid chromatographic method which will simultaneously measure dothiepin and its three major metabolites (northiaden, northiaden-S-oxide and dothiepin-S-oxide) in plasma using trimipramine as internal standard. Sample preparation involved a basic extraction using diethyl ether followed by an acid back-extraction. The method we report is linear over the range 50-1000 ng/ml (r = 0.999), for all analytes. Total imprecision is less than 11% (coefficient of variation) and accuracy is greater than 94% (n = 20). Recovery of analytes varied considerably from 51.7% for northiaden-S-oxide to 90.2% for dothiepin-S-oxide.     

3-正丙基-4-氨基-5-酰胺基-1-甲基吡唑的合成

以2－戊酮为起始原料，经Claisen缩合、环化、甲基化、皂化、硝化、酰胺化和还原反应，制得目标化合物。总收率比文献报道值提高一倍，达46．8％。     

Simultaneous determination of aceclofenac and three of its metabolites in human plasma by high-performance liquid chromatography

Aceclofenac [[2-(2',6'-dichlorophenyl)amino]phenylacetoxyacetic acid] is a phenylacetic acid derivative with potent analgesic and anti-inflammatory properties and an improved gastro-intestinal tolerance. In the present study, a liquid-liquid extraction-based reversed-phase HPLC method with UV detection was validated and applied for the analysis of aceclofenac and three of its metabolites (4'-hydroxy-aceclofenac, diclofenac, 4'-hydroxy-diclofenac) in human plasma. The analytes were separated using an acetonitrile-phosphate buffer gradient at a flow rate of 1 mL/min, and UV detection at 282 nm. The retention times for aceclofenac, diclofenac, 4'-hydroxy-aceclofenac, 4'-hydroxy-diclofenac and ketoprofen (internal standard) were 69.1, 60.9, 46.9, 28.4 and 21.2 min, respectively. The validated quantitation range of the method was 10-10000 ng/mL for aceclofenac, 4'-hydroxy-aceclofenac and diclofenac, and 25-10000 ng/mL for 4'-hydroxy-diclofenac. The developed procedure was applied to assess the pharmacokinetics of aceclofenac and its metabolites following administration of a single 100 mg oral dose of aceclofenac to three healthy male volunteers.     

Simultaneous determination of fluzinamide and three of its active metabolites in plasma by high-performance liquid chromatography

A sensitive and selective high-performance liquid chromatographic method has been developed for a new anticonvulsant, fluzinamide, and three of its active metabolites. This method requires only 0.5 ml of plasma, and it involves a single extraction with a mixture of hexane--dichloromethane--butanol (55:40:5). The plasma extract is chromatographed on a 10-micron, C18 reversed-phase column and quantitated by ultraviolet absorbance at 220 nm. The concentration--response curves for all four compounds are linear from 0.05 micrograms/ml to at least 10 micrograms/ml. The extraction efficiency of this method is greater than 90%. The accuracy and precision of the method were tested by analyzing spiked unknown samples that had been randomly distributed across the concentration range. The mean concentrations found were within +/- 9% of the various amounts added with a standard deviation of +/- 3.5%. This method has been successfully applied to the analysis of samples obtained from fluzinamide-dosed dogs, healthy unmedicated volunteers, and patients who were at steady state with phenytoin, carbamazepine, and fluzinamide.