High-performance liquid chromatographic determination of proquazone and its m-hydroxy metabolite in spiked human plasma and urine

A simple and rapid high-performance liquid chromatographic method for the determination of proquazone (PQZ) and its major metabolite, m-hydroxyproquazone, in spiked human plasma and urine was developed. Plasma samples were purified using acetonitrile as a protein precipitant, while urine samples were diluted only with the mobile phase and filtered prior to injection. Samples containing the parent compounds and glafenine (internal standard) were eluted from a reversed-phase C8 column using acetonitrile-0.025 M sodium acetate (60 + 40) adjusted to pH 5 as the mobile phase and detected at 234 nm. Peak area ratios of the analytes versus internal standard were used for calibration. The mean recoveries from plasma and urine samples spiked with PQZ and its m-hydroxy metabolite ranged from 97.87 to 103.88%. The relative standard deviation for the within- and between-day analyses were < 4%. The proposed method was applied for the assay of PQZ in laboratory-made tablets.     

Determination of clozapine and its metabolites in serum and urine by reversed phase HPLC

A rapid, specific and sensitive method using reversed phase HPLC for the simultaneous determination of clozapine and its two metabolites in serum and urine has been developed. The mobile phase was a mixture of 67% (v/v) methanol in water containing 0.4% tetramethylethylenediamine and 0.32% acetic acid (pH 5.5). The influence of methanol content, the pH of the mobile phase and the effect of adding alkylammonium ions as peak tailing reducer in the mobile phase have been investigated. The solvent for extracting clozapine from serum and urine was ether. 50 microliters of 0.25 M H2SO4 solution was used to redissolve the dry residue to eliminate the endogenous compounds which could otherwise be eluted together with clozapine from the HPLC column. The analysis of a single sample was accomplished within half an hour. The identities of the chromatographic peaks of clozapine and its N-demethyl metabolite collected from the patient urine sample were confirmed by mass spectrometry. The method is sufficiently sensitive (5 ng/ml) and reproducible (CV 2.9%-6.7%) for clinical and pharmacokinetic studies, and preliminary results in these respects are presented.     

Quantitative determination of tolazoline in serum and urine

A high-performance liquid chromatographic procedure is described for the analysis of tolazoline in serum and urine. This assay procedure is suitable for the analysis of micro-samples (50 or 100 microliters serum and 100 microliters urine). Samples are extracted in a single step and injected into a reversed-phase high-performance liquid chromatography system for detection at 210 nm. The clinical applicability of this assay is demonstrated by the determination of tolazoline serum and urine concentrations in neonates. In addition, the presence of urine conjugates and the extent of serum protein binding were investigated. This assay procedure has the required sensitivity (0.1 microgram/ml), accuracy and precision for both routine monitoring and pharmacokinetic characterization of tolazoline in neonates and adults.     

High-performance liquid chromatographic method for simultaneous determination of enantiomers of 5-dimethylsulphamoyl-6,7-dichloro-2-dihydrobenzofuran-2, carboxylic acid and its N-monodemethyl metabolite in monkey plasma and urine after chiral derivatization

A quantitative method for the simultaneous high-performance liquid chromatographic (HPLC) resolution and determination of the enantiomers of 5-dimethylsulphamoyl-6,7-dichloro-2,3-dihydrobenzofuran-2-carboxyl ic acid, a new diuretic, and its N-monodemethylated metabolite in monkey plasma and urine is described. The method includes diethyl ether extraction of the samples and S-(-)-alpha-methylbenzylamide derivatization of the extract, followed by reversed-phase solid-phase extraction and injection of the resulting diastereoisomers onto a reversed-phase HPLC column. Baseline separation was obtained. The assay showed linearity over the range 0.1-50 micrograms/ml of plasma and 0.25-500 microliters of urine, with a lower limit of detection of ca. 0.01 micrograms/ml for each of the enantiomers. The method is adequate for pharmacokinetic and enantioselective disposition studies of both the diuretic and its metabolite.     

Simultaneous determination of cocaine and its metabolites with caffeine in rat serum microsamples by high-performance liquid chromatography

A single, isocratic high-performance liquid chromatographic method is described for the determination of cocaine and three of its metabolites along with caffeine in serum microsamples (50 microliters). The small sample size permits the tracking of pharmacokinetic data over time in individual, small animals. The method also was used to demonstrate that cocaine, benzoylecgonine and norcocaine in rat serum samples were stable for at least a month without the presence of sodium fluoride.     

Simultaneous determination of carbamazepine and its epoxide metabolite in plasma and urine by high-performance liquid chromatography

A simple procedure for the simultaneous determination of carbamazepine and its major metabolite, carbamazepine epoxide, in plasma and urine is described. The assay involves two extractions of the drugs and an internal marker, clonazepam, from the alkalinized sample. The extract is evaporated to dryness at 45 degrees C and the residue is redissolved in methanol (30 microliters). A 25-microliters aliquot is injected into the liquid chromatograph and eluted with acetonitrile-water (40:60, v/v) on a C18 pre-column linked to a 5-microns C8 reversed-phase column. The eluent is detected at 215 nm. The method has been used to investigate the steady-state concentrations of carbamazepine and carbamazepine epoxide in the plasma and urine of a manic-depressive patient.     

High-performance liquid chromatographic determination of alpha-keto acids in human serum and urine using 1,2-diamino-4,5-methylenedioxybenzene as a precolumn fluorescence derivatization reagent

A simple and highly sensitive high-performance liquid chromatographic (HPLC) method for the determination of alpha-keto acids in human serum and urine is described. In an acidic solution, twelve species of alpha-keto acids examined were converted by reaction with 1,2-diamino-4,5-methylenedioxy-benzene into highly fluorescent derivatives. The derivatives were separated isocratically by reversed-phase HPLC on a TSK gel ODS-80TM column and detected fluorimetrically. Eight alpha-keto acids in human serum and eleven alpha-keto acids in human urine can be determined simultaneously. The detection limits (signal-to-noise ratio = 5) are 6-44 fmol in an injection volume of 5 microliters. The intra-assay relative standard deviations for both serum and urine sample analyses are usually ca. 5%.     

High-performance liquid chromatographic determination of the H+/K+ ATPase inhibitor (BY 1023/SK&F 96,022) and its sulphone metabolite in serum or plasma by direct injection and fully automated pre-column sample clean-up

A fully automated high-performance liquid chromatographic method is described for the determination of the new H+/K+ ATPase inhibitor BY 1023/SK&F 96,022 and its major metabolite occurring in dog serum. The method uses direct sample injection of up to 200 microliters and a pre-column switching technique. In order to optimize the recovery, pre-column conditions were varied systematically with respect to the pH of the pre-column eluent, its buffering capacity and content of acetonitrile. Optimization resulted in near 100% recovery for both compounds, thus allowing the use of external standardization. The linearity range, precision and detection limits were determined and the method shown to be applicable to both serum and plasma. The method was applied to define the pharmacokinetics in dogs and humans.     

Automatic preparation of human serum samples for analysis of the drug enoximone and its sulphoxide metabolite using high-performance liquid chromatography

A method for the estimation of the cardiotonic drug enoximone and its major sulphoxide metabolite, in serum, is described. The method uses a new technique for the preparation of biological material prior to the separation of analytes using high-performance liquid chromatography. This technique has been described as the automated sequential trace enrichment of dialysates (ASTED). The sample preparation process operates concurrently with the chromatographic separation. Aliquots of 500 microliters of serum are required for the analysis of serum from patients who have received a single 75-mg dose of enoximone. Within- and between-run coefficients of variation were found to be 2.8 and 3.9% for enoximone and 2.5 and 3.7% for enoximone sulphoxide. These values were obtained from estimating serum supplemented with enoximone and its metabolite, at concentrations of 0.5 and 1.0 mg/l, respectively.     

Bio-analysis of vinorelbine by high-performance liquid chromatography with fluorescence detection.

A simple and selective procedure for the determination of vinorelbine, a new semi-synthetic vinca alkaloid, is presented. The method is based on ion-exchange high-performance liquid chromatography on normal-phase silica with fluorescence detection, combined with liquid-liquid extraction using diethyl ether for sample clean-up. The absence of endogenous interferences and the excellent chromatographic behaviour of vinca alkaloids provides accurate results even at low concentrations. The limit of determination in plasma is 1.5 micrograms/l (500-microliters sample). Reproducible recoveries in urine were obtained if 10-50 microliters of sample were processed supplemented with 500 microliters of blank plasma.     

Determination of pholcodine and its metabolites in urine by capillary gas chromatography

A sensitive and selective method for the determination of pholcodine and its metabolites in urine using capillary gas chromatography with nitrogen detection is described. The procedure includes enzymatic hydrolysis of urine by beta-glucuronidase and sample pretreatment on C2 solid-phase extraction columns. Validation of the method showed good sensitivity, precision and reproducibility. The method was useful for the study of pholcodine metabolism in man. Pholcodine was found to conjugate with glucuronic acid. Morphine was identified as a metabolite and another unidentified metabolite was also detected.     

Evaluation of a radioimmunoassay of alpha 1-microglobulin (alpha 1-m) with simplified procedures

A newly established double antibody radioimmunoassay (RIA) was fundamentally and clinically evaluated. Original procedures were partially modified as follows: Sample volume for serum and urine was changed to 25 microliters, and thus 200 mg/l of alpha 1-m standard was prepared using 50 microliters of original standard solution (100 mg/l). The results were satisfactory in sensitivity (0.3 mg/l obtained from -2SD method), intra-assay precision with its coefficient variation (CV) ranging from 3.0 to 7.4%, interassay precision with its CV ranging from 3.0 to 10.7%, and recovery with the mean value of 102.4% in serum and 108.2% in urine respectively. There were no changes about alpha 1-m value between diluted (2 times) and undiluted with high concentration samples. Normal levels of alpha 1-m were less than 25 mg/l in serum and less than 10 mg/l in urine. The present results indicate that the determination of alpha 1-m could be very simple and useful for the most sensitive screening test for the evaluation of renal function.     

Determination of picotamide in human plasma and urine by high-performance liquid chromatography.

A high-performance liquid chromatographic method for the determination of picotamide in human plasma and urine is described. After addition of an internal standard (bamifylline), the plasma and urine samples were subjected to liquid-liquid extraction and clean-up procedures. The final extracts were evaporated to dryness and the resulting residues were reconstituted in 100 microliters of methanol-water (50:50, v/v) and chromatographed on a LiChrosorb RP-SELECT B reversed-phase column coupled to an ultraviolet detector monitored at 230 nm. Chromatographic analysis takes about 10 min per sample. The assay was linear over a wide range and has a limit of detection of 0.005 and 0.1 micrograms/ml in plasma and urine, respectively. It was selective for picotamide, accurate and robust and thus suitable for routine assays after therapeutic doses of picotamide.     

Determination of the antimitotic agents N-desacetylcolchicine, demecolcine and colchicine in serum and urine

In an effort to characterize the pharmacokinetic behavior of the antimitotic agent N-desacetylcolchicine a selective, sensitive high-performance liquid chromatographic method was developed for the determination of N-desacetylcolchicine, demecolcine and colchicine in serum or urine. To 0.5 ml of serum or 0.1 ml of urine diluted to 0.5 ml were added 50 microliters demecolcine (2 micrograms/ml) which serves as the internal standard. The sample was extracted using a C2 reversed-phase solid extraction column. N-Desacetyl-colchicine, colchicine and the internal standard were eluted from the column with methanol. The combined eluates were evaporated to dryness and the residue was reconstituted with water. The reconstituted sample was injected into a C18 reversed-phase column and eluted using a mobile phase consisting of 0.1 M potassium dihydrogenphosphate, 5 mM 1-pentanesulfonic acid in methanol and acetonitrile with a final pH of 6.0, at a flow rate of 1.5 ml/min. N-Desacetylcolchicine, colchicine and the internal standard were detected using a variable-wavelength ultraviolet detector at 254 nm. The limit of detection was 0.4 ng/ml for desacetylcolchicine and 4.0 ng/ml for colchicine. The method is linear over a concentration range of 1.0-200 ng/ml. The method has been shown to be a rapid, reliable method to monitor N-desacetylcolchicine levels in clinical trials in cancer patients.     

Liquid Chromatographic Determination of 4-Amino-N-(2,6-dimethylphenyl)-benzamide in Rat Serum and Urine

Abstract

The compound 4-amino-N-(2,6-dimethylphenyl)-benzamide has shown potential as a new anticonvulsant. A method for the liquid chromatographic determination of serum and urine concentrations of the compound and its N-acetylated metabolite was developed for pharmacokinetic studies. Quantitation was achieved via UV detection at 275 nm following isocratic reversed phase (C₁₈) separation using a ternary solvent system of water:acetonitrile:acetic acid (60:39:1) at a flow rate of 1.5 mL/min. The compounds were isolated from a 50 μL sample of serum using solid phase extraction with prior protein precipitation. The compounds and internal standard were eluted from the extraction column with acetonitrile. Isolation from urine was achieved similarly with the exclusion of protein precipitation. The assay procedure is useful for the determination of concentrations of parent compound from 0.68 to 204.6 μg/mL.     

Enzyme immunoassay screening procedure for the synthetic anabolic estrogens and androgens diethylstilbestrol, nortestosterone, methyltestosterone and trenbolone in bovine urine

Immunoassays are often used for the screening of anabolic residues in edible tissues and excreta (urine, faeces) from inspected animals. Radioimmunoassays have been used for ten years for the determination in biological samples of the main natural and synthetic anabolic estrogens and androgens. In order to simplify the sample preparation and analysis and to reduce the cost, competitive enzyme immunoassays (EIA) were developed for the main synthetic anabolics used illegally in livestock fattening. EIA are based on a competition between the analyte (hormone or metabolite) and the enzyme-labelled hormone for binding to specific antibodies immobilized in wells of a microtitration plate. Two enzymes were evaluated: horseradish peroxidase (HRP) and Bacillus licheniformis beta-lactamase (BLL) using hydrogen peroxide-o-phenylenediamine or benzylpenicillin-starch-iodine as substrates, respectively. The same derivative was used for chemical coupling of the hormone to enzyme (tracer preparation) and to bovine serum albumin to produce specific antibodies in rabbits. Hormone doses that inhibited 50% of the tracer (HRP-hormone) binding to antibody (ID50) were 18, 8, 6 and 11 pg per well for diethylstilbestrol, nortestosterone, methyltestosterone and trenbolone, respectively. These values were lower than those observed in RIA. The reproducibility and accuracy of EIA in urine analysis were similar to those of RIA. Very small amounts of urine were needed (2.5 microliters). This simple method may require less than 2 h. With the BLL-hormone tracer, the enzymatic activity remaining in the wells and hence the hormone content of the sample could be estimated with the naked eye using benzylpenicillin-starch-iodine as substrate.     

Microanalytical high-performance liquid chromatographic assay for cefpirome in human milk and urine.

To permit the characterization of cefpirome disposition in lactating females, a previously published high-performance liquid chromatographic (HPLC) method for determining the drug in serum was adapted for use with milk and urine. This automated, microanalytical technique requires 50 microliters of biological matrix, which is subjected to an isopropanol extraction. Chromatography was accomplished using a microbore HPLC system, a reversed-phase C18 column and a mobile phase of 0.3% triethylamine in water (pH 5.1). Cefpirome and the internal standard (beta-hydroxypropyltheophylline) were monitored using UV detection at 240 nm and had retention times of 2.84 and 5.05 min, respectively. The method was linear up to 500 mg/l for both matrices and had a limit of detection of 0.6 mg/l. The interday variation (relative standard deviation) at concentrations of 5.0, 50.0 and 500.0 mg/l was consistently less than 5% in both urine and breast milk. The method was found to be free from interference by other commonly administered medications and readily adaptable for use in clinical investigations. The ease of sample preparation, small sample volume requirement, short chromatographic time, apparent lack of interferences, analytical sensitivity and high precision and accuracy make this method ideal for use in pharmacokinetic investigations involving the determination of cefpirome in human milk and urine.     

High-performance liquid chromatographic determination of 3 alpha,5 beta-tetrahydroaldosterone in human urine with chemiluminescence detection.

A sensitive method for the determination of 3 alpha,5 beta-tetrahydroaldosterone (THALD) in human urine is described. The method uses high-performance liquid chromatography with chemiluminescence detection. Urinary THALD, released by enzyme hydrolysis, is isolated and concentrated using a Sephadex G-25M column and Bond-Elut C1 cartridges, and then oxidized by copper(II) acetate to form the corresponding glyoxal derivative. The glyoxal derivative is converted into the chemiluminescent quinoxaline by reaction with 4,5-diaminophthalhydrazide. The chemiluminescent quinoxaline is separated within 50 min on a reversed-phase column (TSKgel ODS-120T) with isocratic elution, followed by chemiluminescence detection; the chemiluminescence is produced by the reaction of the quinoxaline with hydrogen peroxide in the presence of potassium hexacyanoferrate(III) in alkaline solution. The detection limit for THALD is 0.6 pmol (220 pg) ml-1 in urine [1.5 fmol (0.53 pg) per 20 microliters injection] at a signal-to-noise ratio of 3. This method permits the sensitive and precise determination of THALD in human urine (50 microliters) from normal subjects and a patient with primary aldosteronism.     

Quantitative determination of valproic acid and 14 metabolites in serum and urine by gas chromatography/mass spectrometry.

A method for the determination of the antiepileptic drug valproic acid and 14 of its metabolites in serum and urine by gas chromatography/mass spectrometry with selected ion monitoring of the trimethylsilylated derivatives has been developed. Sample preparation, including hydrolysis of VPA-conjugates and removal of urea in urine is carried out at pH 5.0 and is rapid and simple. The samples are extracted with ethyl acetate and the concentrated extracts are trimethylsilylated. Analysis with adequate separation of metabolites is achieved with a DB 1701 fused silica (Megabore) capillary column. The method exhibits high recovery and reproducibility and is sufficiently sensitive and selective for analysis of small sample volumes. Application of the method for screening patient serum and urine samples for unusual metabolite patterns, with possible predictive value for early detection of liver injury, is presented.     

Determination of ethyl biscoumacetate and its metabolite 7-hydroxyethyl biscoumacetate in human serum by high-performance liquid chromatography and mass spectrometry.

An efficient reversed-phase high-performance liquid chromatographic method has been developed for the determination of ethyl biscoumacetate (EBA) and its metabolite in human serum, using the mu Bondapak C18 column and methanol-water-phosphoric acid (56:46.8:0.2, v/v/v) as the mobile phase. This method permitted the determination of both EBA and a metabolite in human serum. The latter has been mentioned by other authors only in urine samples, where significant concentrations were found. Identification of the metabolite as 7-hydroxyethyl biscoumacetate was based on its chromatographic separation, followed by isolation from the eluate and direct mass spectrometric identification. It has been found that the higher EBA concentrations in human serum described by Brodie et al. [J. Pharmacol. Exp. Ther., 106 (1952) 453] were caused by the insufficient resolving power of the spectrophotometric method used, leading to overlapping of the UV spectra of the parent drug and its metabolite.