Journal of Chromatography. B, Biomedical Applications

Direct detection of several steroid glucuronide and sulfate conjugates was achieved with electrospray reversed-phase HPLC-mass spectrometry. Separation of steroid 17-OH or 5-H epimers conjugated with glucuronide or sulfate could be achieved using gradient elution. Testosterone glucuronide, testosterone sulfate, epitestosterone sulfate and epitestosterone glucuronide were chromatographically resolved, although significant variation in solvent strength was observed between methanol and acetonitrile. Positive ionization mode MS and MS-MS spectra were employed to obtain both quantitative and structural information...

In connection with a national anti-doping control program, including analysis of 8946 urine samples, 28 athletes were found to have delivered samples free from xenobiotic anabolic steroids but with an increased testosterone/epitestosterone (T/E) ratio (> 6). Unannounced testing of the above athletes produced 2-4 additional urine samples during the next 2-3 months. A low degree of variation of the T/E ratio, with a C.V. below 30% was found in 17 of the subjects whereas 10 had a C.V. varying from 31% to 43%...

Two chromatographic methods, reversed-phase liquid chromatography (LC) and immunoaffinity chromatography (IAC), were compared in the preparation of purified testosterone extracts suitable for gas chromatography-combustion/isotope ratio mass spectrometry (GC-C-IRMS) analysis. We have shown previously that GC-C-IRMS is a promising means of detection of testosterone misuse in sport. The two clean-up procedures afford sufficient recovery and adequate purity of testosterone. LC presents several advantages over IAC: access to other urinary steroids, longer column life, no need for special equipment and no antibody preparation...

Recent reports on the misuse of beta 2-agonists, both as stimulants and as "anabolic agents" in sports, highlight the importance of screening and confirmation methods for these compounds in anti-doping control procedures. Although only a few analytical methods have been developed for this purpose, the large experience gained, both in pharmacokinetic studies and above all in the control of the residues of beta 2-agonists in animal fluids and tissues, can be of great help in the anti-doping field. This paper reviews single-residue (SR) and multi-residue (MR) methods developed for the analysis of beta 2-agonists in urine, plasma and hair samples, based on hyphenated chromatographic and mass spectrometric techniques, published in the last ten-year period...

This paper describes a comprehensive method for the detection of natural and synthetic anabolic agents, including some veterinary preparations such as trenbolone, zeranol (a non-steroidal agent) and clenbuterol (a beta 2-agonist). For the natural steroids such as testosterone, the precise determination of urinary androgens during routine procedures allowed the description of statistical distribution of relevant parameters of the endogenous steroid profile amongst male athletes. The validity of the results is discussed, taking into account some factors that may cause the degradation of the specimen...

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A novel and rapid method for the separation and determination of R-(-)- and S-(+)-enantiomers of apomorphine in serum by high-performance liquid chromatography with UV detection is reported. The method involved a solid-phase extraction of the R-(-)- and S-(+)-enantiomers of apomorphine and the internal standard R-(-)-propylnorapomorphine from serum using a C8 Bond-Elut column. The HPLC system consisted of a reversed-phase cellulose-based chiral column (Chiralcel OD-R, 250 x 4.6 mm I.D.) with a mobile phase of 35:65 (v/v) acetonitrile-0...

A sensitive and reproducible method is described for the determination of the cytochrome P450 enzyme 2E1 substrate chlorzoxazone and its primary metabolite 6-hydroxychlorzoxazone in human plasma and urine. Plasma or diluted urine were acidified, incubated with beta-glucuronidase and then were extracted with diethyl ether. Separation of the analytes was achieved on a C18 column with UV detection set at 283 nm. Excellent linearity was observed over the concentration ranges of 100-3000 ng/ml and 4-400 micrograms/ml in plasma and urine, respectively...

A simple procedure for the determination of amphetamine in urine with minimal sample preparation is described. This method involves direct addition of human urine to an acetone-dansyl chloride solution for simultaneous deproteinization and fluorescence derivatization. The derivatized amphetamine is then measured by HPLC with fluorescence detection. It eliminates the extraction procedures often required by other HPLC or GC methods. The effects of pH, temperature and reaction time on the derivatization reaction were investigated...

A high-performance liquid chromatography (HPLC) method for the determination of suramin, its precursors and analogues in aqueous solutions and in plasma samples with advantages compared to earlier methods is described. Due to the method's high sensitivity (detection limit of suramin in plasma samples: 7 ng/ml; in aqueous solutions: 5 ng/ml) and selectivity (suramin tR: 7.05 min, precursor amine 2 tR: 4.68 min), it is possible to analyze degradation products, impurities and possible metabolites of suramin besides suramin...

A HPLC method was developed for the determination of the metabolites of coumarin and 7-hydroxycoumarin in plasma and serum. Separation was based on gradient elution of 7-hydroxycoumarin glucuronide, 7-hydroxycoumarin, coumarin and finally 4-hydroxycoumarin (which is used as an internal standard). Standards, prepared in plasma or serum, and samples were treated with trichloroacetic acid, mixed and centrifuged. The supernatant was removed and analyzed by reversed-phase high-performance liquid chromatography on a C18 column...

Camptothecins are indole alkaloids isolated from a Chinese tree, Camptotheca acuminata, and have a wide spectrum of anticancer activity in vitro and in vivo. A novel camptothecin congener 10-hydroxycamptothecin (HCPT) has been shown to be more active and less toxic than camptothecin, and the lactone HCPT is believed to be responsible for its anticancer activity. In the present study, a reversed-phase high-performance liquid chromatography, (HPLC) with fluorescence detection was developed and validated for the simultaneous analysis of HCPT for lactone from (I) and carboxylate form (II) in plasma, urine and feces and tissues...

Ifosforamide mustard is the active metabolite of ifosfamide, a cytostatic drug. In this study a sensitive and selective method for the analysis of ifosforamide mustard in plasma is described. The method consists of direct derivatisation of ifosforamide mustard in plasma with diethyldithiocarbamate and subsequent solid-phase extraction of the resulting derivative. The analysis of the derivatisation product was performed by high-performance liquid chromatography with UV detection. The calibration graph was linear in the concentration range 0...

An isocratic reversed-phase LC-MS method for measuring concentrations of 5-chloro-2',3'-dideoxy-3'-fluorouridine (935U83; I) directly and its 5'-glucuronide metabolite (5-chloro-2',3'-dideoxy-5'-O-beta-D-glucopyranuronosyl-3'-fluorour idine) indirectly in human plasma was developed, validated, and applied to a Phase I clinical study. The pyrimidine nucleoside, I, was extracted from human plasma by using anionic solid-phase extraction. The concentration of the glucuronide conjugate was determined from the difference between the molar concentration of I in a sample hydrolyzed with beta-glucuronidase and the nonhydrolyzed sample...

A simple HPLC method has been developed that allows the sensitive determination of rifabutin (RBT) in human serum using on-line concentration and column switching. After pretreatment of the serum with acetonitrile and centrifugation, the samples were applied to a concentration column (CC) (Zorbax CN). Washing with phosphate buffer-methanol removed most of the contaminating substances. Via a six-port valve the CC was switched to the analytical mode. RBT was separated on a Chromspher RP 8 column (acetonitrile-phosphate buffer pH 7...

It has been suggested that GTS-21 can improve the learning deficits and inhibit the neuro-degeneration in patients with Alzheimer's disease. This paper describes a reversed-phase high-performance liquid chromatographic assay with visible detection at 405 nm for determination of GTS-21 and its metabolite, 4-hydroxy-GTS-21 in rat plasma. The method uses solid-phase extraction with a Bond Elut C18 column. A quantitation limit of 1.0 ng/ml was achieved using 0.5 ml of rat plasma. In the validation study, the coefficients of variation and the relative errors of each compound were less than 10%...

A high-performance liquid chromatographic assay for the quantitative determination of the opioid analgesic tramadol and its metabolites is described. A homologue of tramadol [1-(m-hydroxyphenyl)-2-(N-ethyl-N-methylaminomethyl)cycloheptane-1 -ol hydrochloride] is used as internal standard. The assay allows the determination of tramadol O- and N-demethylation activity in vitro in microsomal fractions of human liver. Tramadol and its in vitro generated Phase I metabolites are extracted by a one-step extraction procedure from microsomal incubation mixtures using methylene chloride...

Nifedipine, a calcium-channel blocking drug was analysed in dog plasma after oral dosing with two different formulations. Sample preparation was automated with a laboratory robot. Quantitative determination of the drug was performed on a reversed-phase HPLC system with electrochemical detection (ED) using an internal standard. Validation of the analytical method showed that the system is well suited for pharmacokinetic studies on dogs. The assay was linear in the range 1-50 ng/ml. Inter-day and intra-day variability were between 6...

A selective and reproducible high-performance capillary electrophoretic (HPCE) method for the quantification of amikacin (AMK), an aminocyclitol antibiotic, in human plasma, has been developed for use in clinical laboratory tests. The method involves ultrafiltration (UF) of plasma before derivatization with the fluorescence derivatization reagent 1-methoxy-carbonylindolizine-3, 5-dicarbaldehyde at room temperature for 15 min in the dark. An aliquot of the derivatives is directly introduced into the fused-silica capillary [75 cm (effective length) x 50 microns I...

A gas chromatographic-negative ion chemical ionization mass spectrometric (GC-NCI-MS) method for the quantitative analysis of clonazepam in human plasma is described. Clonazepam (M(r) = 315) was derivatized by N,O-bis-(trimethylsilyl)trifluoroacetamide with 1% trimethylchlorosilane. A pre-conditioning procedure involving injection of a silyl-8 and ethyl acetate extraction solution from plasma reduces the interaction between clonazepam-TMS and the analytical system. The routine limit of quantification was set to be 0...