Quantitative determination of tolbutamide and its metabolites in human plasma and urine by high-performance liquid chromatography and UV detection

An isocratic, high-performance liquid chromatography method has been developed for simultaneous determination of the oral antidiabetic tolbutamide and two of its metabolites, 4-hydroxytolbutamide and carboxytolbutamide, in human plasma and urine. The method was based on simple one-step liquid-liquid extraction with tertiary-butyl methyl ether as extraction solvent. The chromatographic eluent was 23:77 (v/v) methanol: 0.01 M aqueous sodium acetate buffer pH 3.0, and the UV detection was performed at a wavelength of 230 nm. The limit of detection was 0.1 microM for tolbutamide in plasma and 1.5 microM, 0.5 microM, and 0.75 microM for carboxytolbutamide, 4-hydroxytolbutamide, and tolbutamide, respectively, in urine. The limit of quantitation was 0.5 micro for tolbutamide in plasma and 2 microM, 0.75 microM, and 1.25 microM for carboxytolbutamide, 4-hydroxytolbutamide, and tolbutamide, respectively, in urine. The overall mean recoveries ranged from 91% to 109% for tolbutamide in plasma and from 80% to 98% in urine for all three compounds.     

High-performance liquid chromatographic determination of aspirin and its metabolites in plasma and urine

A simple quantitative method for the rapid determination of aspirin and its metabolites, salicylic acid, salicyluric acid, and gentisic acid, in plasma and urine using o-toluic and o-anisic acids, respectively, as internal standards was developed. Plasma proteins were precipitated by the addition of acetonitrile and, after centrifugation, the supernatant fluid was injected directly onto a reverse-phase column. The mobile phase consisted of an isocratic mixture of water, methanol, and glacial acetic acid (64:25:1, v/v/v) and the separated components were detected at 238 nm using a UV detector. Concentrations greater than or equal to 0.5 microgram/ml could be quantitated for aspirin or its metabolites in plasma. The peak heights and peak height ratios to the internal standard, o-toluic acid, were linear for the concentration range of 0.5-200 micrograms/ml. The aspirin metabolites in urine were isolated by extracting the acidified urine with either and then reextracting the material into an aqueous buffer solution at pH 7.0. Twenty microliters of the buffer extract was directly injected onto the column. The separated components were detected and quantitated at 305 nm. Concentrations greater than or equal to 5 micrograms/ml of salicyluric acid, salicylic acid, and gentisic acid could be determined accurately. The peak heights and peak height ratios to the internal standard, o-anisic acid, were found to be linear for the concentration range of 5-200 micrograms/ml in urine.     

Quantitative liquid chromatographic determination of bromadoline and its N-demethylated metabolites in blood, plasma, serum, and urine samples

Bromadoline and its two N-demethylated metabolites were extracted into ether:butyl chloride after the addition of internal standard and basification of the various biological fluids (blood, plasma, serum, and urine). These compounds were then extracted into dilute phosphoric acid from the organic phase and separated on a reversed-phase chromatographic system using a mobile phase containing acetonitrile and a buffer of 1,4-dimethylpiperazine and perchloric acid. The overall absolute extraction recoveries of these compounds were approximately 50-80%. The background interferences from the biological fluids were negligible and allowed quantitative determination of bromadoline and the metabolites at levels as low as 2-5 ng/mL. At mobile phase flow rate of 1 mL/min, the sample components and the internal standard were eluted at the retention times within approximately 7-12 min. The drug- and metabolite-to-internal standard peak height ratios showed excellent linear relationships with their corresponding concentrations. The analytical method showed satisfactory within- and between-run assay precision and accuracy, and has been utilized in the simultaneous determination of bromadoline and its two N-demethylated metabolites in biological fluids collected from humans and from dogs after administration of bromadoline maleate.     

High-performance liquid chromatographic-ultraviolet determination of primaquine and its metabolites in human plasma and urine

A high-performance liquid chromatographic method was developed for the simultaneous determination of primaquine and its metabolites from plasma and urine samples obtained after oral administration of primaquine diphosphate. Following partial deproteinization with acetonitrile, samples were chromatographed by direct injection onto a cyano column with UV detection at 254 nm. Levels as low as 100 ng/mL per 20-microL injection were quantitated. Preliminary pharmacokinetic analysis is reported for two human volunteers after oral doses of 60 mg and 90 mg. Two apparent plasma metabolites and two possible urinary metabolites of primaquine are also reported.     

Quantitative determination of antipyrine and its metabolites in urine by the method of microcolumn HPLC

Translated from Khimiko-farmatsevticheskii Zhurnal, Vol. 23, No. 3, pp. 351–354, March, 1989.     

Analytical methods for the determination of naftifine and its metabolites in human plasma and urine

Analytical procedures have been worked out for the determination both of naftifine, the antifungal constituent of Exoderil, and its demethyl derivative in plasma, and of the metabolites p-hydroxyphenyl-, 3,4-dihydrodiol- and 7,8-dihydrodiol-naftifine and naphthoic acid in urine. For plasma a HPLC-method with UV-detection after extraction of the samples with hexane is used. In urine samples the metabolites are deconjugated, extracted with chloroform, silylated and measured by GC with flame ionization detection. The standard calibration curves for the parent compound and metabolites are linear. The detection limit for naftifine and its demethyl derivative is ca. 5 ng/ml, for naphthoic acid 1 microgram/ml and for the other metabolites 2 micrograms/ml.     

高效液相色谱法测定尿中咖啡因主要代谢物浓度

本文建立了测定尿中咖啡因5种主要代谢物（AFMU、1X、1U、17X、17U）浓度的高效波相色谱法。尿样用硫酸铵饱和后加氯仿及异丙醇混合液（85：15）提取2次，空气流吹干，蒸馏水溶解进样。以ShimPackC18为固定相，甲醇-动腈-0．05％醋酸（10：1：89）为流动相，扑热息痛为内标，流速为1．2ml／min，在280um处定量检测。本法精确稳定可靠。用此方法测定了120名健康志愿者口服咖啡后的尿样，对人体内N-乙酰化转移酶和CYP1A2酶活性作了初步分析。     

Simultaneous high-performance liquid chromatographic determination of carbamazepine and its principal metabolites in human plasma and urine

A rapid high-performance liquid chromatographic method is described for the simultaneous determination of carbamazepine and the 10,11-epoxide, 10,11-dihydroxy, and 2-hydroxy metabolites of carbamazepine. The chromatographic system involves the use of a 18C-microsorb, reversed-phase column with acetonitrile/water (28:72) as the mobile phase. Detection and quantitation are monitored by ultraviolet absorption at 212 nm. The compounds are extracted from 250 microliters of plasma or from 100 microliters urine with methyl-t-butyl ether and 0.1 M sodium hydroxide; 2-methylcarbamazepine is added as internal standard. If phenytoin and/or phenobarbital are present in plasma or urine samples, it is necessary to use 1.0 M sodium hydroxide. The limits of quantitation for carbamazepine and its metabolites are 10 ng/ml.     

Identification of trimetazidine metabolites in human urine and plasma

1. The objective was to use modern mass spectrometric techniques to update current information on the metabolism of trimetazidine in human subjects found by previous studies.

2. Urine and plasma samples were taken from four healthy human volunteers taking part in a larger kinetic study. Each subject received an oral dose of 80-mg trimetazidine daily for 4 days.

3. Identification and quantitation of trimetazidine and its metabolites in urine and plasma were achieved using modern liquid chromatography-mass spectrometric methods.

4. The major drug-related component observed in urine and plasma was unchanged trimetazidine. In addition to the parent drug, 10 metabolites were detected in urine in concentrations ranging from 0.008 (0.01% dose) to 1.094 μg.ml^-1 (1.4% dose). Metabolic profiles following acute and chronic doses of trimetazidine were qualitatively similar.     

Quantitative levels of aripiprazole parent drug and metabolites in urine

Objective

The aim of this study is to assess urine levels of aripiprazole and metabolites among patients receiving steady-state dosing of aripiprazole.

Methods

One hundred fifty adults, judged compliant with a stable aripiprazole regimen, had observed dosing for 5 consecutive days. Urine specimens, obtained on days 1, 4, and 5, were analyzed for pH, creatinine, specific gravity, and for aripiprazole, OPC3373, and dehydroaripiprazole. Linear regression was used to assess the association between unadjusted urine levels of each drug/metabolite and dose taken, and linear stepwise multiple regression was performed to identify variables that added to the explanation of the variance.

Results

OPC3373 was found in 97 % of urine samples, whereas unchanged aripiprazole and dehydroaripiprazole were found in only 58 and 39 % of samples, respectively. Variance in urine metabolite levels accounted for by medication dose was relatively low for each individual drug/metabolite, r ² only 0.13 to 0.23. However, when OPC3373 was adjusted for age, weight, sex, and urine creatinine values, the r ² improved to 0.63, and further improved to 0.70, when height, urine specific gravity, and the presence of dehydroaripiprazole were added in a stepwise multiple regression model.

Conclusions

Unadjusted urine levels of aripiprazole and metabolites are not strongly related to aripiprazole dosing, however, accounting for key variables yields a strong relationship between measurable urine parameters and dose taken. By defining the expected range of adjusted urine levels for each dose, the potential exists for a clinical test to identify partially nonadherent individuals who would not have been identified by conventional “present vs. absent” urine drug testing.     

高效液相色谱-串联质谱法测定人血浆中色氨酸及其代谢物浓度

目的建立一种基于高效液相色谱-串联质谱(LC-MS/MS)技术同时快速测定人血浆中色氨酸及其代谢产物(犬尿氨酸、犬尿喹啉酸和吲哚丙酸)含量的方法。方法采用甲醇/乙腈(v/v,50/50)沉淀蛋白,以色氨酸-d_5为内标,色谱柱:Ultimate XB-C_(18) HPLC柱(2.1 mm×100 mm,3μm),流动相:水-甲醇(45/55,水相含5 mmol/L醋酸铵和0.2%甲酸),流速:0.3 ml/min,柱温:40℃,分析时间共3 min。质谱采用API4000 QTrap三重四极杆质谱仪的正离子多反应监测模式监测。考察该方法的专属性、线性及定量范围、精密度与准确度、回收率与基质效应以及稳定性。结果色氨酸、犬尿氨酸、犬尿喹啉酸及吲哚丙酸的线性范围分别在100～2×10~4、25～5 000、5～1 000、10～2 000 ng/ml;平均回收率均>90%;日内和日间精密度(RSD)和准确度(RE)均小于15%;稳定性良好。该方法成功测定了90例冠心病患者血浆中的色氨酸、犬尿氨酸、犬尿喹啉酸及吲哚丙酸的浓度。结论该方法简便、快捷、灵敏,可用于人体血浆中色氨酸及其代谢产物浓度的测定。     

Simultaneous determination of levodopa, carbidopa and their metabolites in human plasma and urine samples using LC-EC

In this study levodopa (L-DOPA), carbidopa (C-DOPA) and their metabolites were resolved from other endogenous components present in human plasma and urine and determined quantitatively. The developed technique involved the use of a second pump, a switching valve, and a pre-column in the LC system in order to perform on-line sample clean-up and enrichment. This procedure is dependent on an effective removal of the many interfering matrix components that vitiate HPLC analysis. Several unknown endogenous electroactive compounds, present in plasma, were eliminated by the purification step, or suppressed by the pre-treatment or detection conditions. The analyses were separated on an Octyl-bonded reversed-phase column followed by amperometric detection using a carbon fibre microelectrode flow cell operated at +0.8 V versus silver/silver phosphate reference electrode. The cell was compatible with the mobile and the stationary phase used in the flow system without any complex surface reaction. The peak currents obtained for the different analytes were directly proportional to the analyse over the concentration range 0.02-4.0 microg ml(-1). Using this method, the minimum detectable concentration was estimated to be 5 and 8 ng ml(-1) for L-DOPA and C-DOPA, respectively. Recovery studies performed on human plasma samples ranged from 93.83 to 89.76%, with a relative standard deviation of < 6%. The intra- and inter-assay coefficients of variation were < 7%. The accuracy of the assay, which was defined as the percentage difference between the mean concentration found and the theoretical (true) concentration, was 12% or better. The electrochemical pre-treatment regime described in this work permitted a longer application of the same microelectrode. The method showed a good agreement with other available methods described in the introduction and offers the advantages of being simple, less time and labour consuming, does not require additional solvents for extraction, inexpensive and suitable for routine analysis and kinetic purposes.     

Analytical methods for the determination of terbinafine and its metabolites in human plasma, milk and urine

Analytical procedures have been developed for the determination of the allylamine antimycotic terbinafine (1) and its demethylderivate (2) in plasma, milk and urine, and the metabolite carboxy-terbinafine (3) in plasma and urine, as well as the further metabolites demethyl-carboxy-terbinafine (4) and naphthoic acid (5) in urine. HPLC-methods for plasma analysis employed either electrochemical detection (for 1 and 2) or UV-detection (for 3) following a protein precipitation step with methanol or sample extraction with hexane as appropriate. For quantitative urine analysis of substances 1-4 native urine samples were deconjugated, mixed with internal standard and injected by an autosampler into a microprocessor controlled HPLC-system. The substances were monitored by UV-absorption. The metabolite 5 was determined in urine after deconjugation, sample preparation with commercially available cartridges and silylation by automatized GC with fused silica capillary column and FID-detection. The standard calibration curves for the parent compound (1) and metabolites (2-5) are linear within the required analytical ranges. The detection limit for 1 and 2 is 50 ng/ml in plasma and 150 ng/ml in milk and for 3 in plasma 100 ng/ml. The detection limit in urine is 300 ng/ml for all substances (1-4) analyzed by HPLC and 50 ng/ml for 5 analyzed by GC.     

GLC determination of ticrynafen and its metabolites in urine, serum, and plasma of humans and animals

A sensitive GLC assay for ticrynafen, a diuretic agent with uricosuric properties, and its two metabolites in urine, serum, and plasma is described. The method employs methylation of carboxylic acid groups and trimethylsilyation of the hydroxyl group on one metabolite that cannot otherwise be separated readily from ticrynafen as a simple methyl ester. Urinary output and serum or plasma levels of ticrynafen and its two metabolites were measured in specimens from human volunteers receiving one 250-mg tablet.     

Quantitative determination of captopril in blood and captopril and its disulfide metabolites in plasma by gas chromatography

A sensitive, quantitative gas chromatographic-electron capture (GC-EC) method for the determination of captopril in blood and captopril and its disulfide metabolites (collectively) in plasma was developed. After addition of an internal standard and N-ethylmaleimide to the biological samples, excess N-ethylmaleimide and naturally occurring interfering substances were removed by extraction with benzene followed by acidification and extraction with hexane. The N-ethylmaleimide adducts of captopril and of the internal standard were then extracted with benzene and converted to their hexafluoroisopropyl esters. For the assay of captopril and its disulfide metabolites, tributylphosphine was used to reduce the disulfide metabolites to captopril prior to derivatization. The hexafluoroisopropyl esters of the N-ethylmaleimide adducts of captopril and of the internal standard, the 4-ethoxyproline analogue of captopril, were separated by GC on a column packed with 3% OV-101 on Chromosorb W-HP. The lower limits of sensitivity were 20 ng/mL for captopril in blood and 50 ng/mL for captopril and its disulfide metabolites in plasma. Linearity, precision, and accuracy were excellent. The method was validated by comparison of results obtained for total captopril in dog plasma by the GC-EC assay with results obtained by a published GC-MS method. The assay was applied to dog and human samples to explore its general utility.     

Identification and determination of four metabolites of mangiferin in rat urine

Four metabolites of mangiferin were firstly isolated and identified from rat urine. The structures of the four metabolites were determined to be 1,3,7-trihydroxyxanthone (M-1), 1,3,6,7-tetrahydroxyxanthone (M-2), 1,3,6-trihydroxy-7-methoxyxanthone (M-3) and 1,7-dihydroxyxanthone (M-4), respectively. A simple and specific analytical method for determination of the four metabolites in rat urine was developed by high performance liquid chromatography (HPLC). Quercetin was employed as an internal standard. The correlation coefficients of the calibration curves were higher than 0.997, both intra- and inter-day precision of four metabolites were determined and their R.S.D. did not exceed 10%. The accuracy and linear range had been investigated in detail. The cumulative urinary excretions of the four metabolites were measured and the possible metabolic pathway of the metabolites was discussed.     

GLC determination of heroin and its metabolites in human urine.

Heroin and its metabolites, 6-monoacetylmorphine, morphine, and normorphine, were determined in human urine with a GLC procedure. Heroin was extracted with chloroform at pH 4.5 and chromatographed at a temperature programmed from 200-250 degrees by 8 degrees/min. 6-Monoacetylmorphine and morphine were extracted with ethylene dichloride containing 30% isopropanol at pH 8.5, and normorphine was extracted at pH 10.4 wtih the same solvent. The extract was derivatized with trimethylsilylimidazole and chromatographed at 230 degrees for the determination of 6-monoacetylmorphine and morphine and at 220 degrees for normorphine and morphine.