A general screening method for doping agents in human urine by solid phase extraction and liquid chromatography/time-of-flight mass spectrometry

A general screening method based on solid phase extraction (SPE) and liquid chromatography/time-of-flight mass spectrometry (LC/TOFMS) was developed and investigated with 124 different doping agents, including stimulants, -blockers, narcotics, -adrenergic agonists, agents with anti-estrogenic activity, diuretics and cannabinoids. Mixed mode cation exchange/C8 cartridges were applied to SPE, and chromatography was based on gradient elution on a C18 column. Ionization of the analytes was achieved with electrospray ionization in the positive mode. Identification by LC/TOFMS was based on retention time, accurate mass and isotopic pattern. Validation of the method consisted of analysis of specificity, analytical recovery, limit of detection and repeatability. The minimum required performance limit (MRPL), established by World Anti-Doping Agency (WADA), was attained to 97 doping agents. The extraction recoveries varied between 33 and 98% and the median was 58%. Mass accuracy was always better than 5 ppm, corresponding to a maximum mass error of 0.7 mDa. The repeatability of the method for spiked urine samples, expressed as median of relative standard deviations (RSD%) at concentrations of MRPL and 10 times MRPL, were 14% and 9%, respectively. The suitability of the LC/TOFMS method for doping control was demonstrated with authentic urine samples.     

A gas-liquid chromatographic method for the analysis of diphenoxylic acid in urine

Summary A gas-liquid chromatographic method has been developed and evaluated for the measurement of diphenoxylic acid in urine. This method uses base hydrolysis to liberate diphenoxylic acid from compounds conjugated in urine, followed by removal of interfering substances in urine by column chromatography on alumina. Quantitation was carried out using p-chlorodiphenoxylic acid as an internal standard.     

Estimation of disaccharides in plasma and urine by gas-liquid chromatography

A technique for the estimation of disaccharides in plasma and urine using gas-liquid chromatography is described. The procedure involves the formation of trimethylsilyl derivatives followed by injection of the reaction mixture directly onto the column. The method is precise, linear over a wide range and gives recoveries of 93--99%. The limit of sensitivity is 80 micrograms per 100 ml, but with modification of the volumes used, levels of 40 micrograms per 100 ml may be quantitated.     

Determination of xylitol in human urine by gas-liquid chromatography.

A rapid and specific method for the quantitative determination of xylitol in human urine has been developed. The method consists of the gas-liquid chromatographic analysis of the acetate ester derivative of the alditol in deionized urine using dulcitol as an internal standard. As little as 20 ng xylitol can be detected. At concentrations ranging from 25 to 400 micrograms/ml urine, the accuracy is +/- 4.0%.     

New screening method for basic compounds in urine by on-line extraction-high-performance liquid chromatography with photodiode-array detection

A fully automated, qualitative screening HPLC method for the identification of basic compounds in urine has been developed. A 1-ml volume of urine was extracted by on-line extraction and separated on two coupled strong cation-exchange (SCX) columns (2 x LunaSCX, 150 mm x 4.6 mm, 5 microm) under isocratic conditions. The mobile phase consisted of a mixture of potassium dihydrogenphosphate buffer (pH 2.3) and acetonitrile. The use of photodiode-array detection (DAD, lambda = 190-800 nm) gave access to a library of approximately 2600 toxicologically relevant compounds. The validated method is reliable, simple and in addition successfully proven with the analysis of real biological specimen for the routine use.     

Lattice-fluid model for gas-liquid chromatography

Lattice-fluid models describe molecular ensembles in terms of the number of lattice sites occupied by molecular species (r-mers) and the interactions between neighboring molecules. The lattice-fluid model proposed by Sanchez and Lacombe (Macromolecules, 1978;11:1145-1156) was used to model specific retention volume data for a series of n-alkane solutes with n-alkane, polystyrene, and poly(dimethylsiloxane) stationary liquid phases. Theoretical equations were derived for the specific retention volume and also for the temperature dependence and limiting (high temperature) values for the specific retention volume. The model was used to predict retention volumes within 10% for the n-alkanes phases; 22% for polystyrene; and from 20 to 70% for PDMS using no adjustable parameters. The temperature derivative (enthalpy) could be calculated within 5% for all of the solutes in nine stationary liquid phases. The limiting value for the specific retention volume at high temperature (entropy controlled state) could be calculated within 10% for all of the systems. The limiting data also provided a new chromatographic method to measure the size parameter, r, for any chromatographic solute using characteristic and size parameters for the stationary phase only. The calculated size parameters of the solutes were consistent, i.e. independent of the stationary phase and agreed within experimental error with the size parameters previously reported from saturated vapor pressure, latent heat of vaporization or density data.     

Determination of indomethacin in serum and urine by electron-capture gas-liquid chromatography.

A specific and sensitive method for the quantitative determination of indomethacin in serum and urine is described. The drug is extracted at pH 5.0 with 1,2-dichloroethane and a portion of the organic extract is concentrated and made to react with diazoethane in diethyl ether. The ethyl ester derivative is analyzed by electron-capture gas-liquid chromatography, quantitation being achieved by comparison of peak areas for samples and standards, which are prepared in serum or urine and treated in the same manner as the samples. The limit of sensitivity is 50 ng/ml and the relative standard derivation for repeat determinations on the same sample is about 3%.     

Comprehensive screening procedure for diuretics in urine by high-performance liquid chromatography

A rapid and reliable screening procedure using high-performance liquid chromatography for the detection of 23 diuretics (belonging to five different pharmacological groups) in urine has been developed. Two aliquots of 2-ml urine samples were extracted separately under acidic and basic conditions. The acidic and basic extracts were pooled, evaporated to dryness and reconstituted in methanol. The methanolic extract was injected onto a Hewlett-Packard Hypersil ODS C18 (5 microns) column (column I) and a Hewlett-Packard LiChrosorb RP-18 (5 microns) column (column II; an alternative column). The same gradient mobile phase was used for both columns. A diode array ultraviolet detector was set to monitor the signal to the integrator (Chem Station) at 230 and 275 nm. Recovery studies of the 23 diuretics were performed under acidic and basic conditions. The overall lower limits for detection on column I using both extraction procedures ranged from 0.5 to 1.5 micrograms/ml of urine (average 1.0 micrograms/ml). Amiloride, ethacrynic acid and probenecid could not be detected below 5 micrograms/ml of urine. No interference from the biological matrix was apparent. Amiloride could be detected in urine 4 h after oral administration of 15 mg of amiloride to a healthy volunteer, when the sample was extracted under alkaline conditions. The suitability of the screening method for the analysis of urine samples was tested by studying the variation with time of chlorthalidone, furosemide, probenecid, acetazolamide, quinethazone, spironolactone, bendroflumethiazide, bumetanide, triameterene and hydrochlorothiazide concentrations in the urine of normal human volunteers after minimum single or multiple (probenecid) doses.(ABSTRACT TRUNCATED AT 250 WORDS)     

A broad-spectrum equine urine screening method for free and enzyme-hydrolysed conjugated drugs with ultra performance liquid chromatography/tandem mass spectrometry

The authors' laboratory at one time employed four liquid chromatography/mass spectrometric (LC/MS) methods for the detection of a large variety of drugs in equine urine. Drug classes covered by these methods included anti-diabetics, anti-ulcers, cyclooxygenase-2 (COX-2) inhibitors, sedatives, corticosteroids, anabolic steroids, sulfur diuretics, xanthines, etc. With the objective to reduce labour and instrumental workload, a new ultra performance liquid chromatography/tandem mass spectrometric (UPLC/MS/MS) method has been developed, which encompasses all target analytes detected by the original four LC/MS methods. The new method has better detection limits than the superseded methods. In addition, it covers new target analytes that could not be adequately detected by the four LC/MS methods. The new method involves solid-phase extraction (SPE) of two aliquots of equine urine using two Abs Elut Nexus cartridges. One aliquot of the urine sample is treated with β-glucuronidase before subjecting to SPE. A second aliquot of the same urine sample is processed directly using another SPE cartridge, so that drugs that are prone to decomposition during enzyme hydrolysis can be preserved. The combined eluate is analysed by UPLC/MS/MS using alternating positive and negative electrospray ionisation in the selected-reaction-monitoring mode. Exceptional chromatographic separation is achieved using an UPLC system equipped with a UPLC(?) BEH C18 column (10 cm L×2.1 mm ID with 1.7 μm particles). With this newly developed UPLC/MS/MS method, the simultaneous detection of 140 drugs at ppb to sub-ppb levels in equine urine can be achieved in less than 13 min inclusive of post-run equilibration. Matrix interference for the selected transitions at the expected retention times is minimised by the excellent UPLC chromatographic separation. The method has been validated for recovery and precision, and is being used regularly in the authors' laboratory as an important component of the array of screening methods for doping control analyses of equine urine samples.     

Improved gas-liquid chromatographic method for the determination of baclofen in plasma and urine

A simple, rapid and sensitive assay for baclofen analysis has been developed. Baclofen and the internal standard are analyzed by gas-liquid chromatography with electron-capture detection after esterification of the carboxyl group to the butyl ester and acylation of the amino group to the pentafluoropropionylamide. Recovery from biological matrixes is accomplished by ion-pair extraction. The limit of quantitation of the entire assay as stated is about 10 ng/ml baclofen in plasma.     

Determination of trimethylsilyl methylated nucleic acid bases in urine by gas-liquid chromatography.

A method for the determination of the urinary excretion level of methylated nucleic acid bases by gas-liquid chromatography (GLC) has been developed. A clean-up procedure prior to GLC analysis consisted of hydrolysis, filtration, charcoal adsorption, and anion exchange. Studies to determine optimum derivatization conditions for conversion of the methylated bases to their trimethylsilyl derivatives and to evaluate GLC parameters and columns to obtain the best separation were conducted. Application of the method was shown by determining the excretion levels of methylated bases in urine of normal controls and of patients with various types of malignancy.     

Rapid micro-method for the measurement of ethchlorvynol in blood plasma and in urine by gas-liquid chromatography

A rapid gas-liquid chromatographic method has been developed for use in the measurement of the hypnotic drug ethchlorvynol in small (50 microliter) volumes of either blood plasma or urine. Neither solvent transfer nor evaporation steps are used in the procedure and sources of interference have proved to be minimal. The method has been applied primarily to the analysis of specimens obtained from patients who had ingested an overdose of this drug. However with slight modification, the technique may be used in the measurement of the plasma concentrations of ethchlorvynol attained during therapy.