Quantification of clenbuterol at trace level in human urine by ultra-high pressure liquid chromatography-tandem mass spectrometry.

Clenbuterol is a β2 agonist agent with anabolic properties given by the increase in the muscular mass in parallel to the decrease of the body fat. For this reason, the use of clenbuterol is forbidden by the World Anti-Doping Agency (WADA) in the practice of sport. This compound is of particular interest for anti-doping authorities and WADA-accredited laboratories due to the recent reporting of risk of unintentional doping following the eating of meat contaminated with traces of clenbuterol in some countries. In this work, the development and the validation of an ultra-high pressure liquid chromatography coupled to electrospray ionization tandem mass spectrometry (UHPLC-ESI-MS/MS) method for the quantification of clenbuterol in human urine is described. The analyte was extracted from urine samples by liquid-liquid extraction (LLE) in basic conditions using tert butyl-methyl ether (TBME) and analyzed by UHPLC-MS/MS with a linear gradient of acetonitrile in 9min only. The simple and rapid method presented here was validated in compliance with authority guidelines and showed a limit of quantification at 5pg/mL and a linearity range from 5pg/mL to 300pg/mL. Good trueness (85.8-105%), repeatability (5.7-10.6% RSD) and intermediate precision (5.9-14.9% RSD) results were obtained. The method was then applied to real samples from eighteen volunteers collecting urines after single oral doses administration (1, 5 and 10μg) of clenbuterol-enriched yogurts.

Fast analysis of doping agents in urine by ultra-high-pressure liquid chromatography-quadrupole time-of-flight mass spectrometry I. Screening analysis.

The general strategy to perform anti-doping analyses of urine samples starts with the screening for a wide range of compounds. This step should be fast, generic and able to detect any sample that may contain a prohibited substance while avoiding false negatives and reducing false positive results. The experiments presented in this work were based on ultra-high-pressure liquid chromatography coupled to hybrid quadrupole time-of-flight mass spectrometry. Thanks to the high sensitivity of the method, urine samples could be diluted 2-fold prior to injection. One hundred and three forbidden substances from various classes (such as stimulants, diuretics, narcotics, anti-estrogens) were analysed on a C(18) reversed-phase column in two gradients of 9min (including two 3min equilibration periods) for positive and negative electrospray ionisation and detected in the MS full scan mode. The automatic identification of analytes was based on retention time and mass accuracy, with an automated tool for peak picking. The method was validated according to the International Standard for Laboratories described in the World Anti-Doping Code and was selective enough to comply with the World Anti-Doping Agency recommendations. In addition, the matrix effect on MS response was measured on all investigated analytes spiked in urine samples. The limits of detection ranged from 1 to 500ng/mL, allowing the identification of all tested compounds in urine. When a sample was reported positive during the screening, a fast additional pre-confirmatory step was performed to reduce the number of confirmatory analyses.

Profiling of steroid metabolites after transdermal and oral administration of testosterone by ultra-high pressure liquid chromatography coupled to quadrupole time-of-flight mass spectrometry.

The screening of testosterone (T) misuse for doping control is based on the urinary steroid profile, including T, its precursors and metabolites. Modifications of individual levels and ratio between those metabolites are indicators of T misuse. In the context of screening analysis, the most discriminant criterion known to date is based on the T glucuronide (TG) to epitestosterone glucuronide (EG) ratio (TG/EG). Following the World Anti-Doping Agency (WADA) recommendations, there is suspicion of T misuse when the ratio reaches 4 or beyond. While this marker remains very sensitive and specific, it suffers from large inter-individual variability, with important influence of enzyme polymorphisms. Moreover, use of low dose or topical administration forms makes the screening of endogenous steroids difficult while the detection window no longer suits the doping habit. As reference limits are estimated on the basis of population studies, which encompass inter-individual and inter-ethnic variability, new strategies including individual threshold monitoring and alternative biomarkers were proposed to detect T misuse. The purpose of this study was to evaluate the potential of ultra-high pressure liquid chromatography (UHPLC) coupled with a new generation high resolution quadrupole time-of-flight mass spectrometer (QTOF-MS) to investigate the steroid metabolism after transdermal and oral T administration. An approach was developed to quantify 12 targeted urinary steroids as direct glucuro- and sulfo-conjugated metabolites, allowing the conservation of the phase II metabolism information, reflecting genetic and environmental influences. The UHPLC-QTOF-MS(E) platform was applied to clinical study samples from 19 healthy male volunteers, having different genotypes for the UGT2B17 enzyme responsible for the glucuroconjugation of T. Based on reference population ranges, none of the traditional markers of T misuse could detect doping after topical administration of T, while the detection window was short after oral TU ingestion. The detection ability of the 12 targeted steroids was thus evaluated by using individual thresholds following both transdermal and oral administration. Other relevant biomarkers and minor metabolites were studied for complementary information to the steroid profile, including sulfoconjugated analytes and hydroxy forms of glucuroconjugated metabolites. While sulfoconjugated steroids may provide helpful screening information for individuals with homozygotous UGT2B17 deletion, hydroxy-glucuroconjugated analytes could enhance the detection window of oral T undecanoate (TU) doping.

Ultra-high pressure liquid chromatography-mass spectrometry for plant metabolomics: a systematic comparison of high-resolution quadrupole-time-of-flight and single stage Orbitrap mass spectrometers.

The response of Arabidopsis to stress caused by mechanical wounding was chosen as a model to compare the performances of high resolution quadrupole-time-of-flight (Q-TOF) and single stage Orbitrap (Exactive Plus) mass spectrometers in untargeted metabolomics. Both instruments were coupled to ultra-high pressure liquid chromatography (UHPLC) systems set under identical conditions. The experiment was divided in two steps: the first analyses involved sixteen unwounded plants, half of which were spiked with pure standards that are not present in Arabidopsis. The second analyses compared the metabolomes of mechanically wounded plants to unwounded plants. Data from both systems were extracted using the same feature detection software and submitted to unsupervised and supervised multivariate analysis methods. Both mass spectrometers were compared in terms of number and identity of detected features, capacity to discriminate between samples, repeatability and sensitivity. Although analytical variability was lower for the UHPLC-Q-TOF, generally the results for the two detectors were quite similar, both of them proving to be highly efficient at detecting even subtle differences between plant groups. Overall, sensitivity was found to be comparable, although the Exactive Plus Orbitrap provided slightly lower detection limits for specific compounds. Finally, to evaluate the potential of the two mass spectrometers for the identification of unknown markers, mass and spectral accuracies were calculated on selected identified compounds. While both instruments showed excellent mass accuracy (<2.5ppm for all measured compounds), better spectral accuracy was recorded on the Q-TOF. Taken together, our results demonstrate that comparable performances can be obtained at acquisition frequencies compatible with UHPLC on Q-TOF and Exactive Plus MS, which may thus be equivalently used for plant metabolomics.

Fast analysis of doping agents in urine by ultra-high-pressure liquid chromatography-quadrupole time-of-flight mass spectrometry. II: Confirmatory analysis.

For doping control, analyses of samples are generally achieved in two steps: a rapid screening and, in the case of a positive result, a confirmatory analysis. A two-step methodology based on ultra-high-pressure liquid chromatography coupled to a quadrupole time-of-flight mass spectrometry (UHPLC-QTOF-MS) was developed to screen and confirm 103 doping agents from various classes (e.g., beta-blockers, stimulants, diuretics, and narcotics). The screening method was presented in a previous article as part I (i.e., Fast analysis of doping agents in urine by ultra-high-pressure liquid chromatography-quadrupole time-of-flight mass spectrometry. Part I: screening analysis). For the confirmatory method, basic, neutral and acidic compounds were extracted by a dedicated solid-phase extraction (SPE) in a 96-well plate format and detected by MS in the tandem mode to obtain precursor and characteristic product ions. The mass accuracy and the elemental composition of precursor and product ions were used for compound identification. After validation including matrix effect determination, the method was considered reliable to confirm suspect results without ambiguity according to the positivity criteria established by the World Anti-Doping Agency (WADA). Moreover, an isocratic method was developed to separate ephedrine from its isomer pseudoephedrine and cathine from phenylpropanolamine in a single run, what allowed their direct quantification in urine.

Quantification of glucuronidated and sulfated steroids in human urine by ultra-high pressure liquid chromatography quadrupole time-of-flight mass spectrometry.

The urinary steroid profile is constituted by anabolic androgenic steroids, including testosterone and its relatives, that are extensively metabolized into phase II sulfated or glucuronidated steroids. The use of liquid chromatography coupled to mass spectrometry (LC-MS) is an issue for the direct analysis of conjugated steroids, which can be used as urinary markers of exogenous steroid administration in doping analysis, without hydrolysis of the conjugated moiety. In this study, a sensitive and selective ultra high-pressure liquid chromatography coupled to quadrupole time-of-flight mass spectrometer (UHPLC-QTOF-MS) method was developed to quantify major urinary metabolites simultaneously after testosterone intake. The sample preparation of the urine (1 mL) was performed by solid-phase extraction on Oasis HLB sorbent using a 96-well plate format. The conjugated steroids were analyzed by UHPLC-QTOF-MS(E) with a single-gradient elution of 36 min (including re-equilibration time) in the negative electrospray ionization mode. MS(E) analysis involved parallel alternating acquisitions of both low- and high-collision energy functions. The method was validated and applied to samples collected from a clinical study performed with a group of healthy human volunteers who had taken testosterone, which were compared with samples from a placebo group. Quantitative results were also compared to GC-MS and LC-MS/MS measurements, and the correlations between data were found appropriate. The acquisition of full mass spectra over the entire mass range with QTOF mass analyzers gives promise of the opportunity to extend the steroid profile to a higher number of conjugated steroids.

Analytical interference of 4-hydroxy-3-methoxymethamphetamine with the measurement of plasma free normetanephrine by ultra-high pressure liquid chromatography-tandem mass spectrometry.

OBJECTIVES: The diagnosis of pheochromocytoma relies on the measurement of plasma free metanephrines assay whose reliability has been considerably improved by ultra-high pressure liquid chromatography tandem mass spectrometry (UHPLC-MS/MS). Here we report an analytical interference occurring between 4-hydroxy-3-methoxymethamphetamine (HMMA), a metabolite of 3,4-methylenedioxymethamphetamine (MDMA, "Ecstasy"), and normetanephrine (NMN) since they share a common pharmacophore resulting in the same product ion after fragmentation. DESIGN AND METHODS: Synthetic HMMA was spiked into plasma samples containing various concentrations of NMN and the intensity of the interference was determined by UPLC-MS/MS before and after improvement of the analytical method. RESULTS: Using a careful adjustment of chromatographic conditions including the change of the UPLC analytical column, we were able to distinguish both compounds. HMMA interference for NMN determination should be seriously considered since MDMA activates the sympathetic nervous system and if confounded with NMN may lead to false-positive tests when performing a differential diagnostic of pheochromocytoma.

Detection of metabolite induction in fungal co-cultures on solid media by high-throughput differential ultra-high pressure liquid chromatography-time-of-flight mass spectrometry fingerprinting.

Access to new biological sources is a key element of natural product research. A particularly large number of biologically active molecules have been found to originate from microorganisms. Very recently, the use of fungal co-culture to activate the silent genes involved in metabolite biosynthesis was found to be a successful method for the induction of new compounds. However, the detection and identification of the induced metabolites in the confrontation zone where fungi interact remain very challenging. To tackle this issue, a high-throughput UHPLC-TOF-MS-based metabolomic approach has been developed for the screening of fungal co-cultures in solid media at the petri dish level. The metabolites that were overexpressed because of fungal interactions were highlighted by comparing the LC-MS data obtained from the co-cultures and their corresponding mono-cultures. This comparison was achieved by subjecting automatically generated peak lists to statistical treatments. This strategy has been applied to more than 600 co-culture experiments that mainly involved fungal strains from the Fusarium genera, although experiments were also completed with a selection of several other filamentous fungi. This strategy was found to provide satisfactory repeatability and was used to detect the biomarkers of fungal induction in a large panel of filamentous fungi. This study demonstrates that co-culture results in consistent induction of potentially new metabolites.

Rapid high-performance liquid chromatographic determination with fluorescence detection of furosemide in human body fluids and its confirmation by gas chromatography-mass spectrometry.

Furosemide (FD: Lasix) is a loop diuretic which strongly increases both urine flow and electrolyte urinary excretion. Healthy volunteers were administered 40 mg orally (dissolved in water) and concentrations of FD were determined in serum and urine for up to 6 h for eight subjects, who absorbed water at a rate of 400 ml/h. Quantification was performed by HPLC with fluorescence detection (excitation at 233 nm, emission at 389 nm) with a limit of detection of 5 ng/ml for a 300-microliters sample. The elution of FD was completed within 4 min using a gradient of acetonitrile concentration rising from 30 to 50% in 0.08 M phosphoric acid. The delay to the peak serum concentration ranged from 60 to 120 min. FD was still easily measurable in the sera from all subjects 6 h after administration. In urine, the excretion rates reached their maximum between 1 and 3 h. The total amount of FD excreted in the urine averaged 11.2 mg (range 7.6-14.0 mg), with a mean urine volume of 3024 ml (range 2620-3596 ml). Moreover, the urine density was lower than 1.010 (recommended as an upper limit in doping analysis to screen diuretics) only for 2 h. An additional volunteer was administered 40 mg of FD and his urine was collected over a longer period. FD was still detectable 48 h after intake. Gas chromatography-mass spectrometry with different types of ionization was used to confirm the occurrence of FD after permethylation of the extract. Negative-ion chemical ionization, with ammonia as reactant gas, was found to be the most sensitive method of detection.

Variability of voriconazole plasma levels measured by new high-performance liquid chromatography and bioassay methods.

Voriconazole (VRC) is a broad-spectrum antifungal triazole with nonlinear pharmacokinetics. The utility of measurement of voriconazole blood levels for optimizing therapy is a matter of debate. Available high-performance liquid chromatography (HPLC) and bioassay methods are technically complex, time-consuming, or have a narrow analytical range. Objectives of the present study were to develop new, simple analytical methods and to assess variability of voriconazole blood levels in patients with invasive mycoses. Acetonitrile precipitation, reverse-phase separation, and UV detection were used for HPLC. A voriconazole-hypersusceptible Candida albicans mutant lacking multidrug efflux transporters (cdr1Delta/cdr1Delta, cdr2Delta/cdr2Delta, flu1Delta/flu1Delta, and mdr1Delta/mdr1Delta) and calcineurin subunit A (cnaDelta/cnaDelta) was used for bioassay. Mean intra-/interrun accuracies over the VRC concentration range from 0.25 to 16 mg/liter were 93.7% +/- 5.0%/96.5% +/- 2.4% (HPLC) and 94.9% +/- 6.1%/94.7% +/- 3.3% (bioassay). Mean intra-/interrun coefficients of variation were 5.2% +/- 1.5%/5.4% +/- 0.9% and 6.5% +/- 2.5%/4.0% +/- 1.6% for HPLC and bioassay, respectively. The coefficient of concordance between HPLC and bioassay was 0.96. Sequential measurements in 10 patients with invasive mycoses showed important inter- and intraindividual variations of estimated voriconazole area under the concentration-time curve (AUC): median, 43.9 mg x h/liter (range, 12.9 to 71.1) on the first and 27.4 mg x h/liter (range, 2.9 to 93.1) on the last day of therapy. During therapy, AUC decreased in five patients, increased in three, and remained unchanged in two. A toxic encephalopathy probably related to the increase of the VRC AUC (from 71.1 to 93.1 mg x h/liter) was observed. The VRC AUC decreased (from 12.9 to 2.9 mg x h/liter) in a patient with persistent signs of invasive aspergillosis. These preliminary observations suggest that voriconazole over- or underexposure resulting from variability of blood levels might have clinical implications. Simple HPLC and bioassay methods offer new tools for monitoring voriconazole therapy.

Single-step extraction of fluconazole from plasma by ultra-filtration for the measurement of its free concentration by high performance liquid chromatography.

High performance liquid chromatography (HPLC) is the reference method for measuring concentrations of antimicrobials in blood. This technique requires careful sample preparation. Protocols using organic solvents and/or solid extraction phases are time consuming and entail several manipulations, which can lead to partial loss of the determined compound and increased analytical variability. Moreover, to obtain sufficient material for analysis, at least 1 ml of plasma is required. This constraint makes it difficult to determine drug levels when blood sample volumes are limited. However, drugs with low plasma-protein binding can be reliably extracted from plasma by ultra-filtration with a minimal loss due to the protein-bound fraction. This study validated a single-step ultra-filtration method for extracting fluconazole (FLC), a first-line antifungal agent with a weak plasma-protein binding, from plasma to determine its concentration by HPLC. Spiked FLC standards and unknowns were prepared in human and rat plasma. Samples (240 microl) were transferred into disposable microtube filtration units containing cellulose or polysulfone filters with a 5 kDa cut-off. After centrifugation for 60 min at 15000g, FLC concentrations were measured by direct injection of the filtrate into the HPLC. Using cellulose filters, low molecular weight proteins were eluted early in the chromatogram and well separated from FLC that eluted at 8.40 min as a sharp single peak. In contrast, with polysulfone filters several additional peaks interfering with the FLC peak were observed. Moreover, the FLC recovery using cellulose filters compared to polysulfone filters was higher and had a better reproducibility. Cellulose filters were therefore used for the subsequent validation procedure. The quantification limit was 0.195 mgl(-1). Standard curves with a quadratic regression coefficient &gt; or = 0.9999 were obtained in the concentration range of 0.195-100 mgl(-1). The inter and intra-run accuracies and precisions over the clinically relevant concentration range, 1.875-60 mgl(-1), fell well within the +/-15% variation recommended by the current guidelines for the validation of analytical methods. Furthermore, no analytical interference was observed with commonly used antibiotics, antifungals, antivirals and immunosuppressive agents. Ultra-filtration of plasma with cellulose filters permits the extraction of FLC from small volumes (240 microl). The determination of FLC concentrations by HPLC after this single-step procedure is selective, precise and accurate.

Sensitive determination of D-lactic acid and L-lactic acid in urine by high-performance liquid chromatography-tandem mass spectrometry.

D-lactic acid in urine originates mainly from bacterial production in the intestinal tract. Increased D-lactate excretion as observed in patients affected by short bowel syndrome or necrotizing enterocolitis reflects D-lactic overproduction. Therefore, there is a need for a reliable and sensitive method able to detect D-lactic acid even at subclinical elevation levels. A new and highly sensitive method for the simultaneous determination of L- and D-lactic acid by a two-step procedure has been developed. This method is based on the concentration of lactic acid enantiomers from urine by supported liquid extraction followed by high-performance liquid chromatography-tandem mass spectrometry. The separation was achieved by the use of an Astec Chirobiotic? R chiral column under isocratic conditions. The calibration curves were linear over the ranges of 2-400 and 0.5-100 µmol/L respectively for L- and D-lactic acid. The limit of detection of D-lactic acid was 0.125 µmol/L and its limit of quantification was 0.5 µmol/L. The overall accuracy and precision were well within 10% of the nominal values. The developed method is suitable for production of reference values in children and could be applied for accurate routine analysis.

High-performance liquid chromatography of the renal blood flow marker p-aminohippuric acid (PAH) and its metabolite N-acetyl PAH improves PAH clearance measurements.

PAH (N-(4-aminobenzoyl)glycin) clearance measurements have been used for 50 years in clinical research for the determination of renal plasma flow. The quantitation of PAH in plasma or urine is generally performed by colorimetric method after diazotation reaction but the measurements must be corrected for the unspecific residual response observed in blank plasma. We have developed a HPLC method to specifically determine PAH and its metabolite NAc-PAH using a gradient elution ion-pair reversed-phase chromatography with UV detection at 273 and 265 nm, respectively. The separations were performed at room temperature on a ChromCart (125 mmx4 mm I.D.) Nucleosil 100-5 microm C18AB cartridge column, using a gradient elution of MeOH-buffer pH 3.9 1:99-->15:85 over 15 min. The pH 3.9 buffered aqueous solution consisted in a mixture of 375 ml sodium citrate-citric acid solution (21.01 g citric acid and 8.0 g NaOH per liter), added up with 2.7 ml H3PO4 85%, 1.0 g of sodium heptanesulfonate and completed ad 1000 ml with ultrapure water. The N-acetyltransferase activity does not seem to notably affect PAH clearances, although NAc-PAH represents 10.2+/-2.7% of PAH excreted unchanged in 12 healthy subjects. The performance of the HPLC and the colorimetric method have been compared using urine and plasma samples collected from healthy volunteers. Good correlations (r=0.94 and 0.97, for plasma and urine, respectively) are found between the results obtained with both techniques. However, the colorimetric method gives higher concentrations of PAH in urine and lower concentrations in plasma than those determined by HPLC. Hence, both renal (ClR) and systemic (Cls) clearances are systematically higher (35.1 and 17.8%, respectively) with the colorimetric method. The fraction of PAH excreted by the kidney ClR/ClS calculated from HPLC data (n=143) is, as expected, always <1 (mean=0.73+/-0.11), whereas the colorimetric method gives a mean extraction ratio of 0.87+/-0.13 implying some unphysiological values (>1). In conclusion, HPLC not only enables the simultaneous quantitation of PAH and NAc-PAH, but may also provide more accurate and precise PAH clearance measurements.

Analysis of the enantiomers of citalopram and its demethylated metabolites using chiral liquid chromatography.

A procedure using a chirobiotic V column is presented which allows separation of the enantiomers of citalopram and its two N-demethylated metabolites, and of the internal standard, alprenolol, in human plasma. Citalopram, demethylcitalopram and didemethylcitalopram, as well as the internal standard, were recovered from plasma by liquid-liquid extraction. The limits of quantification were found to be 5 ng/ml for each enantiomer of citalopram and demethylcitalopram, and 7.5 ng/ml for each enantiomer of didemethylcitalopram. Inter- and intra-day coefficients of variation varied from 2.4% to 8.6% for S- and R-citalopram, from 2.9% to 7.4% for S- and R-demethylcitalopram, and from 5.6% to 12.4% for S- and R- didemethylcitalopram. No interference was observed from endogenous compounds following the extraction of plasma samples from 10 different patients treated with citalopram. This method allows accurate quantification for each enantiomer and is, therefore, well suited for pharmacokinetic and drug interaction investigations. The presented method replaces a previously described highly sensitive and selective high-performance liquid chromatography procedure using an acetylated 3-cyclobond column which, because of manufactural problems, is no longer usable for the separation of the enantiomers of citalopram and its demethylated metabolites.

Determination of trimipramine and its demethylated and hydroxylated metabolites in plasma by gas chromatography-mass spectrometry.

A gas chromatographic-mass spectrometric (GC-MS) method has been developed, for the determination of trimipramine (TRI), desmethyltrimipramine (DTRI), didesmethyltrimipramine (DDTRI), 2-hydroxytrimipramine (2-OH-TRI) and 2-hydroxydesmethyltrimipramine (2-OH-DTRI). The method includes two derivatization steps with trifluoroacetic acid anhydride and N-methyl-N-(tert.-butyldimethyl silyl)trifluoroacetamide and the use of an SE-54 capillary silica column. The limits of quantitation were found to be 2 ng/ml for DTRI and 4 ng/ml for all other substances. Besides, methods have been optimized for the hydrolysis of the glucuronic acid conjugated metabolites. This specific detection method is useful, as polymedication is a usual practice in clinical situations, and its sensitivity allows its use for single-dose pharmacokinetic studies.