Determination of mixtures of benzo[a]pyrene, 2,6-dimethylnaphthalene and their metabolites by high-performance liquid chromatography with fluorescence detection

Nonradiometric techniques were used to identify and quantitate two aromatic hydrocarbons and several of their metabolites in biological samples. High-performance liquid chromatographic separation combined with ultraviolet fluorescence detection provided a sensitive and selective method of trace analysis. Metabolites of benzo[a]pyrene and 2,6-dimethylnaphthalene were prepared by in vitro incubation of these substrates, singly and together, with liver microsomes of coho salmon (Oncorhynchus kisutch). Individual metabolites were separated by high-performance liquid chromatography and quantitated at fluorescence excitation and emission wavelengths characteristic of the corresponding parent hydrocarbon. Future refinement of these techniques may allow the determination of more complex mixtures of xenobiotics and their metabolites in marine organisms.     

Determination of heroin and its metabolites by high-performance liquid chromatography

A method is described for the simultaneous determination of heroin (3, 6-diacetylmorphine, DAM) and its two active metabolites 6-acetylmorphine and morphine in blood by high-performance liquid chromatography using a normal-phase column and a UV detector at 218 nm. The compounds are stabilized in blood by rapid freezing and recovered by a multistep liquid—liquid extraction. The mobile phase is acetonitrile—methanol (75:25, v/v) buffered to apparent pH 7 with ammonium hydroxide and acetic acid. Usingl--acetylmethadol as an internal standard, UV detection and a 1-ml biofluid sample, the lower limit of sensitivity is 12.5 ng/ml. Commonly used narcotic analgesics including codeine, propoxyphene, meperidine, methadone and levorphanol do not interfere with the analysis. The method has been applied to blood samples from humans and rats. Extracts of blood from a patient who had received an intravenous dose of 14 mg of DAM contained DAM and both of its active metabolites.     

Determination of 3,4-dihydroxyphenyl glycol in plasma by gas chromatography-mass spectrometry and high-performance liquid chromatography methods

Several modifications of GC-MS and HPLC methods for plasma level DHPG have been described. The effects of storage temperature and stabilizing agents on DHPG stability have been studied. The stabilizing agent has been found to play a more important role than low-temperature storage in preventing DHPG from decomposition during sample storage. A specific and sensitive GC-MS method (electron impact) has been established using stable isotope-labeled DHPG as an internal standard. HPLC has been improved by modifying the conditions, resulting in a good separation of DHPG and internal standard from solvent front and other early eluting compounds. Comparison of the GC-MS and HPLC procedures demonstrates a strong correlation between these two methods.     

Determination of acetylmethadol and metabolites by use of high-performance liquid chromatography

A method is described for the simultaneous determination of l,α-acetylmethadol (LAAM) and five active metabolites — noracetylmethadol, dinoracetylmethadol, methadol, normethadol, and dinormethadol — in biofluids by high-performance liquid chromatography using a normal-phase column and a UV detector at 218 nm. The compounds are recovered from biofluids by a multistep liquid—liquid extraction. The mobile phase is methanol—acetonitrile (70:30, v/v) containing 0.015% ammonium hydroxide as the modifier. Retention times can be varied by adjusting the composition of the mobile phase to maximize peak height for quantitation using l-propranolol as the internal standard or peak separation for the collection of fractions. Using a UV detector the lower limit of sensitivity is 10 ng/ml of biofluid. Using fraction collection of radiolabeled drug and metabolites followed by liquid scintillation counting the lower limit of sensitivity is 1.0 ng/ml. Commonly used or abused narcotics including morphine, heroin, meperidine, methadone and propoxyphene do not interfere with the analysis. The method has been applied to plasma and urine samples from humans, sheep and rats. Extracts of urine from patients receiving maintenance treatment with LAAM contain LAAM and each of the five active metabolites.     

Determination of Quinidine and its major metabolites by high-performance liquid chromatography

A specific and precise assay, capable of quantitating in human plasma simultaneously but separately quinidine, dihydroquinidine and the quinidine metabolites 2′-quinidinone, 3-OH-quinidine and a third metabolite found — tentatively identified as the product formed by rearrangement of quinidine-N-oxide — is reported. The assay uses a normal phase high-performance liquid chromatographic (HPLC) system with a variable-wavelength UV detector at 235 nm and has a limit of sensitivity at approximately 20 ng/ml. The mobile phase consists of hexanes—ethanol—ethanolamine (91.5:8.47:0.03). A 2-ml plasma sample is worked up by adding primaquine base as an internal standard and extracting with ether—dichloromethane—isopropanol (6:4:1). The organic extract is evaporated and the residue reconstituted in 100—600 μl of mobile phase and an aliquot injected onto the column.Comparison of this procedure with the Edgar and Sokolow (dichloroethane) extraction—fluorescence procedure and with the Cramer and Isaksson (benzene) double extraction—fluorescence assay indicates that both fluorescence procedures give quinidine concentrations up to 2.3 times those determined by HPLC. These discrepancies were shown to be due to carry-over of metabolites and some extraneous background fluorescence.     

Determination of [d-Ala2, d-Leu5]enkephalin and the metabolites containing C-terminal d-leucine by high-performance liquid chromatography

A high-performance liquid chromatographic procedure has been developed for the determination of [d-Ala², d-Leu⁵]enkephalin (DADLE) and the fragments containing d-leucine in rat blood. The procedure was applied to the determination of blood levels of [³H-d-Leu⁵]DADLE and the C-terminal fragments after intravenous administration of [³H-d-Leu⁵]DADLE to a rat. Unlabelled DADLE and the C-terminal fragments were spiked as carriers to rat blood samples and the blood samples were extracted with 1% trifluoroacetic acid in methanol. The recoveries from rat blood were quantitative for all compounds. DADLE and the C-terminal four fragments were well separated on a reversed-phase column with gradient elution using a mobile phase composed of 0.14% HClO₄ and acetonitrile.     

Determination of disulfiram and metabolites from biological fluids by high-performance liquid chromatography

A high-performance liquid chromatographic method is described for the determination of disulfiram, diethyldithiocarbamate, diethyldithiocarbamate methyl ester, carbon disulfide, and diethylamine from a single sample of plasma or urine. The analytical procedure is based on a quantitative stepwise extraction of disulfiram and diethyldithiocarbamate methyl ester, or the conversion of diethyldithiocarbamic acid, carbon disulfide and diethylamine to diethyldithiocarbamate methyl ester for chromatographical determination. The procedure is specific, precise and simple. The application of the analytical methods developed for the determination of disulfiram and the various metabolites in plasma from mice given disulfiram intraperitoneally or humans given Antabuse orally is illustrated.     

Determination of lansoprazole and five metabolites in plasma by high-performance liquid chromatography

A high-performance liquid chromatographic method for the determination of lansoprazole, a new proton-pump inhibitor, and five of its metabolites in human plasma is described. Lansoprazole, its metabolites, and internal standard (omeprazole) were extracted into diethyl ether-methylene chloride and separation was obtained using a reversed-phase column under isocratic conditions. The method features monochromatic ultraviolet detection at 285 nm, and single extraction, single evaporation sample handling. The lower limit of quantitation, based on standards with acceptable coefficients of variation, was 10 ng/ml for all compounds. No endogenous compounds were found to interfere. This method has been demonstrated to be suitable for pharmacokinetic studies in humans.     

Determination of aromatic metabolites in ruminant urine by high-performance liquid chromatography

A method based on reversed-phase HPLC is reported for the separation and quantification of various urinary aromatic metabolites: hippuric, phenylaceturic, salicyluric, benzoic, phenylacetic, salicylic. 3-phenylpropionic and cinnamic acids and several phenols in ruminant urine. In this method, a Nova-Pak C₁₈ (4 μm) 150×3.9 mm I.D. column, two solvents [A: 15°b methanol in 20 mM acetic acid (pH 3.3); B: methanol] in a gradient mode at a flow-rate of 0.8 ml/min, and UV detection at 210 nm were used. Quantification of the total (free and conjugated) benzoic, phenylacetic and salicylic acids present in urine was achieved by hydrolysis of the samples in 3 M HCl at 100°C for 24 h prior to HPLC analysis. The lowest detection concentration was 50 μmol/I. This method is useful for scanning the profile of aromatic metabolites in urine of ruminants, which provides information on the diets the animals receive.     

Determination of bufuralol and its major metabolites in plasma by high-performance liquid chromatography

A high-performance liquid chromatographic method for the determination of bufaralol, a benzofuran analogue, in plasma is described.The unchanged drug, the major metabolites and an internal standard are extracted from plasma, purified by back-extraction steps and thereafter separated using a reversed-phase liquid chromatographic system. The detection is carried out by means of a fluorescence detector and an UV detector connected in series. The sensitivity of the assay for the unchanged drug and the major metabolite is about 1 ng/ml plasma using a 0.5 ml specimen per analysis and the relative standard deviation of the whole assay lies in the range ± 4–5%.The procedure was successfully used to determine plasma levels in volunteers following a single oral dose of 40 mg of bufaralol. The results obtained using the new high-performance liquid chromatographic method were compared with those determined by another method which combines gas chromatography with mass fragmentography, and it was found that these two sets of results coincided quite well.     

Determination of piribedil and its basic metabolites in plasma by high-performance liquid chromatography

A high-performance liquid chromatographic method for the determination of piribedil and its p-hydroxylated, catechol and N-oxide metabolites in plasma is described. After addition of an internal standard (buspirone), the plasma samples were subjected to a three-step extraction procedure. The final extracts were evaporated to dryness under nitrogen, and the residues were reconstituted in 100 μl of mobile phase (0.01 M phosphate buffer—acetonitrile, 50:50, v/v) and chromatographed by acetonitrile gradient elution on a C₁₈ reversed-phase column coupled to an ultraviolet detector set at 240 nm. The method was selective for piribedil and its metabolites, and sufficiently sensitive and precise for studies aimed at elucidating the role of the metabolites in the parent drug's pharmacological effects.     

Determination of pyrimidine dimers in DNA by high-performance liquid chromatography/gas chromatography and electron capture detection

Exposure of DNA to uv radiation results in the formation of a number of photoproducts including the cyclobutyl pyrimidine dimers. At low uv fluences the concentrations of these dimeric compounds are only a small fraction of the corresponding DNA pyrimidine concentration (e.g., as low as 0.02% or less of the total thymine content). Sensitive methods of analysis are therefore required for accurate determinations. Analytical methodology based upon HPLC fractionation and electrophore labeling followed by GC/electron capture detection (ECD) has been developed to quantitate these species. Separation of thymine-thymine, thymine-uracil, and uracil-uracil from the monomeric bases and from other constituents present in acid-hydrolyzed DNA is achieved by reversed-phase HPLC. Isolation of the dimeric fractions is followed by off-line derivatization to form pentafluorobenzyl products for analysis by GC/ECD. All active hydrogens are alkylated, yielding products with high response factors and detection limits in the low femtomole range. The overall analytical scheme for the determination of pyrimidine dimers in DNA is presented.     

Determination of nicotine and its main metabolites in urine by high-performance liquid chromatography

Nicotine and its main metabolites (cotinine, trans-3'-hydroxycotinine, trans-3'-hydroxycotinine glucuronide, nicotine-1'-N-oxide and 3-pyridylcarbinol) were analysed in urine after liquid—liquid extraction by high-performance liquid chromatography using norephedrine as internal standard, ultraviolet detection at 260 nm and scanning ultraviolet spectra with a photodiode-array detector. The conjugated trans-3'-hydroxycotinine was determined after enzymatic hydrolysis. Specific determination of 3-pyridylcarbinol was also carried out. Owing to its good selectivity, sensitivity and reproducibility, the method was applied to the analysis of urine samples from smokers and non-smokers. The results obtained suggest that the urinary markers used to assess active smoking or exposure to environmental tobacco smoke must be not only nicotine and cotinine, but also their main free and conjugated metabolites.     

Detection and determination of the major metabolites of [3H]cytosine arabinoside by high-performance liquid chromatography

An ion-pair high-performance liquid chromatographic assay involving solid-phase scintillation detection was established for the rapid identification and determination of all major metabolites of tritium-labelled cytosine arabinoside (Ara-C) in an in vitro system. In a single run of 50 min, Ara-C, Ara-CMP, Ara-CDP-choline, Ara-CDP, Ara-U, Ara-UMP, Ara-CTP, Ara-UDP can be measured. The method is fast, sensitive, with limits of detection ranging from 40 to 200 pg (absolute), and highly reproducible.     

Assessment of [18F]fluoroethylflumazenil metabolites using high-performance liquid chromatography and tandem mass spectrometry

A simple procedure using HPLC and tandem mass spectrometry has been developed for the determination of fluoroethylflumazenil metabolites. Samples were precipitated with acetonitrile, evaporated to dryness followed by reconstitution with methanol. As mobile phase, 50 mM ammonium formate–methanol (58:42, v/v) was used. The method is valid both for cold and radiolabelled metabolites. Various cold metabolites (hydroxylated and/or dealkylated) were identified in rat and human microsome preparations. Radiolabelled metabolites arise from two or more transformations including hydroxylation. The methodology developed can be applied for further characterisation of metabolites, and for the determination of non metabolised [¹⁸F]fluoroethylflumazenil in routine clinical analysis.     

Determination of serotonin,its precursors,metabolites and [3H]serotonin in lung by high-performance liquid chromatography with fluorescence detection

A rapid, simple, sensitive method for the determination of serotonin, its precursors, metabolites and [³H]serotonin in lung is described. Tissue preparation requires only homogenization in dilute perchloric acid and centrifugation prior to separation by high-performance liquid chromatography using a reversed-phase column. Detection is based on the native fluorescence of indole compounds and detection limits ranged between 30–90 pg injected. The method has been used to determine these compounds in mouse lung and plasma.     

Determination of pyrazinamide and its main metabolites in rat urine by high-performance liquid chromatography

A new high-performance liquid chromatograhic procedure for simultaneous determination of pyrazinamide (PZA) and its three metabolites 5-hydroxypyrazinamide (5-OH-PZA), pyrazinoic acid (PA), and 5-hydroxypyrazinoic acid (5-OH-PA), in rat urine was developed. 5-OH-PZA and 5-OH-PA standards were obtained by enzymatic synthesis (xanthine oxidase) and checked by HPLC and GC–MS. Chromatographic separation was achieved in 0.01 M KH₂PO₄ (pH 5.2), circulating at 0.9 ml/min, on a C₁₈ silica column, at 22°C. The limits of detection were 300 μg/l for PZA, 125 μg/l for PA, 90 μg/l for 5-OH-PZA and 70 μg/l for 5-OH-PA. Good linearity (r²>0.99) was observed within the calibration ranges studied: 0.375–7.50 mg/l for PZA, 0.416–3.33 mg/l for PA, 0.830–6.64 mg/l for 5-OH-PZA and 2.83–22.6 mg/l for 5-OHPA. Accuracy was always lower than ±10.8%. Precision was in the range 0.33–5.7%. The method will constitute a useful tool for studies on the influence of drug interactions in tuberculosis treatment.     

Determination of cystamine by high-performance liquid chromatography

A highly sensitive and specific assay method for cystamine using high-performance liquid chromatography has been developed. The method is based on postcolumn derivatization of cystamine with o-phthaladehyde in the presence of 2-mercaptoethanol and sodium hypochlorite. The separation of cystamine was achieved using a cation exchange column (ISC-05/S0504). The assay was linear over the concentration range of 2 to 200 pmol. For the application of this assay method to biological materials, the pretreatment with a cation exchange column (Dowex 50W X 8) was necessary to remove interfering o-phthaladehyde-reactive substances. Since cysteamine in biological materials was quantitatively converted to cystamine during these sampling procedures, this method was found to be suitable for assaying the cysteamine plus cystamine content in various organs and tissues. The cysteamine-cystamine content in various tissues of rat determined by the present assay method has been presented.