Evaluation of allergens in propolis by ultra-performance liquid chromatography/tandem mass spectrometry

The purified extract of propolis is used as a traditional remedy for the treatment of several diseases. Its beneficial activities are mainly attributed to the polyphenolic fraction. Nevertheless, propolis can cause allergic dermatitis and the sensitization rate in humans is increasing significantly mainly in younger subjects. The aim of this study was to develop and validate a selective and sensitive ultra‐performance liquid chromatography tandem mass spectrometry analysis (UPLC/MS/MS) for the evaluation of the amount of caffeic acid and its esters with allergenic action in raw propolis samples and commercial formulations. The separation was carried out on a 1.7 μm C₁₈ BEH Shield column and the detection performed by means of electrospray ionization in negative ion mode with multiple reaction monitoring. The confirmation of formulae of the precursor and product ions was accomplished by injection into a high‐resolution system (FTICR‐MS) using accurate mass measurements. The error was below 1.4 ppm.The range of the standard curves was 0.5–10 μg/mL and dihydrocaffeic acid was used as internal standard (IS). The lower limit of detection (LLOD) for 3‐methyl‐2‐butenyl‐ (3M2B), 3‐methyl‐3‐butenyl‐ (3M3B), 2‐methyl‐2‐butenyl‐ (2M2B), benzyl‐ (CABE), phenylethylcaffeic acid (CAPE) and for caffeic acid (CA) and the IS was 0.1 and 0.3 μg/mL, respectively. The recoveries were in the range 96–104% and the intra‐ and inter‐day precisions were within 6.2%. In the European (n = 8) and Asiatic (n = 3) propolis the most abundant allergens were CABE>3M2B>CAPE>3M3B>CA>2M2B. These compounds were not found in the red (n = 1) and green (n = 1) Brazilian propolis. Hydroalcoholic extracts (n = 6) and tablets (n = 6) were analyzed by the proposed UPLC/MS/MS method. The results showed that in the commercial products CABE, 3M2B, CAPE and 3M3B were also the most abundant. Copyright © 2011 John Wiley & Sons, Ltd.     

Fast determination of herbicides in waters by ultra-performance liquid chromatography/tandem mass spectrometry

A rapid multiresidue method for the analysis of more than 40 herbicides (such as simazine, terbuthylazine and diuron) in waters has been developed and validated by ultra-performance liquid chromatography coupled to tandem mass spectrometry (UPLC/MS/MS). Prior to chromatographic determination, the samples were extracted using a solid-phase extraction procedure. The analysis was performed on an Acquity UPLC BEH C(18) column using a gradient elution profile and a mobile phase consisting of methanol and an aqueous solution of formic acid (0.01%). Other chromatographic and MS/MS parameters were optimised in order to improve selectivity and sensitivity of the analytes. The analytes were detected using electrospray ionisation (ESI)-MS/MS in positive ion mode with multiple reaction monitoring (MRM), optimising parameters such as voltage cone, capillary voltage, source and desolvation temperature, and desolvation and cone gas flow. The optimised method provides a rapid separation (less than 10 min) of the selected herbicides in the assayed matrices, and it was validated by the analysis of spiked blank matrix samples. Good linearity was obtained and the repeatability of the method was less than 20% for the lowest calibration point. The limits of detection ranged from 0.002 to 0.02 microg/L, and the limits of quantification from 0.005 to 0.05 microg/L, which were below the values specified by the European Union. Finally, the method was successfully applied to real environmental samples from Andalusia (southern Spain). Terbuthylazine, simazine, atrazine desisopropyl and desethyl terbuthylazine were the herbicides most frequently found in water samples.     

Multiresidue pesticide analysis of fruits by ultra-performance liquid chromatography tandem mass spectrometry

Ultra-performance liquid chromatography coupled to tandem mass spectrometry (UPLC–MS–MS) has been used for screening and quantification of 32 pesticides and metabolites in two fruit matrices. The compounds investigated belonged to different chemical families of insecticides, acaricides, fungicides, and herbicides; several metabolites were also included. Quantification was conducted using matrix-matched standards calibration; response was a linear function of concentration in the range tested (10–500 ng mL⁻¹). The method was validated with blank samples of lemon and raisin spiked at 0.01 and 0.1 mg kg⁻¹, and recoveries were satisfactory, between 70 and 110%, for most of the pesticides tested and relative standard deviations were below 15% (n = 5 at each spiking level). Excellent sensitivity resulted in limits of detection for all compounds well below 0.01 mg kg⁻¹, with the limit of quantification being validated at 0.01 mg kg⁻¹. The UPLC system generates narrow peaks (approx. 5 s), thus increasing peak height and improving sensitivity. This improved separation efficiency facilitates adequate resolution not only of the analytes but also of matrix interferences compared with conventional HPLC. The method developed could also resolve some geometric isomers. The main advantage of this approach is the high sample throughput achieved because of the short analysis time, which enables satisfactory separation of all the compounds in less than 5 min per sample.     

Analysis of thyreostatic drugs in thyroid samples by ultra-performance liquid chromatography tandem mass spectrometry detection

A method based on ultra-performance liquid chromatography-electrospray ionisation-tandem mass spectrometry for the determination of six thyreostatic drugs in thyroid tissue has been optimised and validated in accordance with the Decision 2002/657/EC. Samples are extracted with methanol and the extracts cleaned-up on silica cartridges. The recoveries range from 40% for 6-phenyl-2-thiouracil to 79% for 2-thiouracil. Quantification is carried out with blank tissue samples spiked with the analytes in the range 25-500 microg kg(-1). 5,6-Dimethyl-2-thiouracil is used as internal standard. CCalpha and CCbeta are in the ranges 4.3-16.1 microg kg(-1) and 8.7-20.7 microg kg(-1), respectively. Accuracy, expressed as percentage of error, is lower than 6% and relative standard deviation in reproducibility conditions falls between 5.6 and 10.3%. Nowadays, the proposed method is routinely implemented in the laboratory of the Agència de Salut Pública de Barcelona and allows processing of up to 20 samples per day.     

Rapid analysis of organic farming insecticides in soil and produce using ultra-performance liquid chromatography/tandem mass spectrometry

A new method for the analysis of three ecological insecticides, namely azadyrachtin, spinosad (sum of spinosyn A and spinosyn D) and rotenone, in produce and soil samples is presented. Investigated compounds are one of the most significant insecticides authorized for organic farming crop protection in many countries. Extraction of the pesticides from plant and soil matrices was performed by using a modified quick, easy, cheap, effective, rugged, and safe (QuEChERS) method. The method entailed a single extraction of the investigated compounds with acidified acetonitrile followed by a dispersive solid-phase extraction cleanup step prior to the final determination by reverse-phase ultra-performance liquid chromatography/tandem quadrupole mass spectrometry (UPLC-MS/MS). Validation studies were carried out on cabbage, tomato and soil samples. Recoveries of the spiked samples were in the range between 67% and 108%, depending on the matrix and the spiking level. Relative standard deviations for all matrix–compound combinations did not exceed 12%. The limits of quantification were ≤0.01 mg kg⁻¹ in all cases, except for azadirachtin. The developed method was applied to the analysis of real samples originating from organic farming production.     

Determination of lovastatin in human plasma by ultra-performance liquid chromatography/electrospray ionization tandem mass spectrometry

A fast and sensitive ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method was developed for the determination of lovastatin in human plasma. With simvastatin as internal standard, sample pretreatment involved one-step extraction with n-hexane-methylene dichloride-isopropanol (20:10:1, v/v/v) of 0.5 mL plasma. Chromatographic separation was carried out on an Acquity UPLC BEH C(18) column with mobile phase consisting of acetonitrile-water (containing 5 mmol/L ammonium acetate; 85:15, v/v) at a flow-rate of 0.35 mL/min. The detection was performed on a triple-quadrupole tandem mass spectrometer by multiple reaction monitoring (MRM) via electrospray ionization source with positive mode. The analysis time was shorter than 1.7 min per sample. The standard curve was linear (r2>or=0.99) over the concentration range 0.025-50.0 ng/mL with a lower limit of quantification of 0.025 ng/mL. The intra- and inter-day precision values were below 11% and the accuracy (relative error) was within 6.0% at three quality control levels. This is the first method of MS with MRM coupled to UPLC for the determination of lovastatin, which showed great advantages of high sensitivity, selectivity and high sample throughput. It was fully validated and successfully applied to the pharmacokinetic study of lovastatin tablets in healthy Chinese male volunteers after oral administration.     

Structural characterization of iridoid glucosides by ultra-performance liquid chromatography/electrospray ionization quadrupole time-of-flight tandem mass spectrometry

The mass spectral fragmentation behavior of ten iridoid glucosides (IGs) has been studied using electrospray ionization (ESI), collision-induced dissociation (CID), and quadrupole time-of-flight tandem mass spectrometry (Q-TOF MS/MS). In the negative ESI mass spectra, the deprotonated [M-H](-) ion was observed for all of the ten IGs except gardoside methyl ester, while the formate adduct [M+HCOO](-) ion appeared to be favored by the presence of a methyl ester or a lactone group in the C-4 position when formic acid was added to the mobile phase. The CID MS/MS spectra of the [M-H](-) ions have been used for structural elucidation. Ring cleavages of the aglycone moiety have been observed in the MS/MS spectra, corresponding to (1,4)F(-), (2,6)F(-), (2,7)F(-), and (2,7)F(0) (-) ions, based on accurate mass measurements and the elemental compositions of the product ions. These characteristic ions gave valuable information on the basic structural skeletons. Furthermore, on the basis of the relative abundances of the fragment ions (1,4)F(-) and (2,7)F(-), different sub-classes, such as cyclopentane-type and 7,8-cyclopentene-type IGs, can be differentiated. Ring cleavage of the sugar moieties was also observed, yielding useful information for their characterization. In addition, the neutral losses, such as H(2)O, CO(2), CH(3)OH, CH(3)COOH, and glucosidic units, have proved useful for confirming the presence of functional substituents in the structures of the IGs. Based on the fragmentation patterns of these standard IGs, twelve IGs have been characterized in an extract of Hedyotis diffusa Willd. by means of ultra-performance liquid chromatography/Q-TOF MS/MS, of which six have been unambiguously identified and the other six have been tentatively identified.     

Simultaneous analysis of thirteen diuretics residues in bovine milk by ultra-performance liquid chromatography/tandem mass spectrometry

A simple, rapid, sensitive and specific method used to screen and confirm multi-class diuretics residues in whole bovine milk is described. Thirteen drugs of four different classes including carbonic anhydrase inhibitors, loop, thiazide and potassium-sparing diuretics were extracted from whole milk by acetonitrile followed by further purification with hexane. The analytes were separated using an ACQUITY UPLC BEH C18 column and detected by electrospray ionization tandem mass spectrometry (ESI-MS/MS). MS data acquisition was performed by a time-scheduled multiple reaction monitoring program, selecting two ion transitions for each target compound. The overall average recoveries based on matrix-fortified curves fortified with diuretics at three levels ranged from 80.6 to 108.8% with the coefficients of variation ranging from 2.6 to 19.7% (n = 6). The limits of quantitation (LOQs) of diuretics in bovine milk were 5.0 microg/kg for spironolactone and 0.5 microg/kg for other analytes, respectively.     

Simultaneous determination of 24 sulfonamide residues in meat by ultra-performance liquid chromatography tandem mass spectrometry

The present study used the liquid extraction pretreatment method and developed an ultra-performance liquid chromatography triple quadrupole tandem mass spectrometry (UPLC-MS/MS) for the simultaneous determination of 24 kinds of sulfonamide residues in meat. The meat samples were homogenized, extracted and deproteinized by acetonitrile, defatted by n-hexane, and further liquid-liquid extracted by ethyl acetate. All of 24 sulfonamide residues were simultaneously separated and determined by UPLC-MS/MS within 15 min. The sulfonamide residues were monitored via the ESI(+) ionization method and quantified by six-channel multiple reaction monitoring (MRM). The calibrations were performed in sample matrixes by the isotope dilution method and the interference effect of sample matrixes on the ionization was effectively eliminated. Good linear relationship (R(2)=0.991-0.999) was observed within the concentration range of 0.2-50 microg/kg. Satisfied recoveries (67.8-113.9%) of all the sulfonamides were demonstrated in different standard-spiked levels except sulfanitran (SNT). The analytical category, separation speed, selectivity, sensitivity and repeatability of sulfonamides using UPLC-MS/MS were significantly improved compared to other analytical methods. Quantitative results of 240 meat samples demonstrated that the present method has a convenient operation and good practicability, which can be applied to the quantitative analysis of a large number of samples.     

Analysis of salazinic, norstictic, and usnic acids in Xanthoparmelia chlorochroa by ultra-performance liquid chromatography/tandem mass spectrometry

The lichen species Xanthoparmelia chlorochroa is toxic when consumed by domestic sheep, cattle, and Rocky Mountain elk. Clinical signs exhibited by poisoned animals include red urine, ataxia, and muscular weakness that rapidly progresses to recumbency. Elk are unable to recover once becoming recumbent; however, most affected cattle can recover if offered suitable feed shortly following the onset of signs. At present, the pathogenesis and specific toxin(s) are unknown. As part of an effort to elucidate the proximate toxin, a method using ultra-performance LC coupled to MS/MS with negative-ion electrospray ionization has been developed to compare salazinic, norstictic, and usnic acid concentrations in X. chlorochroa collected from locales associated with lichen poisonings. Compounds were extracted from lichen samples with acetone and sonication. The stationary phase was a Waters Acquity UPLCTM BEH Ca18 (50 x 2.1 mm; 1.7 microm particle size) column. The mobile phase consisted of an acetonitrile-water gradient. The precision of the method was confirmed by an SD below 0.4% (n=9) for triplicate samples. LOD values were 200, 100, and 50 ng/mL for salazinic, norstictic, and usnic acids, respectively.     

Metabolomic investigation of cholestasis in a rat model using ultra-performance liquid chromatography/tandem mass spectrometry

Metabolomics follows the changes in concentrations of endogenous metabolites, which may reflect various disease states as well as systemic responses to environmental, therapeutic, or genetic interventions. In this study, we applied metabolomic approaches to monitor dynamic changes in plasma and urine metabolites, and compared these metabolite profiles in Eisai hyperbilirubinemic rats (EHBR, an animal model of cholestasis) with those in the parent strain of EHBR - Sprague-Dawley (SD) rats - in order to characterize cholestasis pathophysiologically. Ultra-performance liquid chromatography/tandem mass spectrometry-based analytical methods were used to assay metabolite levels. More than 250 metabolites were detected in both plasma and urine, and metabolite profiles of EHBR differed from those of SD rats. The levels of antioxidative and cytoprotective metabolites, taurine and hypotaurine, were markedly increased in urine of EHBR. The levels of many bile acids were also elevated in plasma and urine of EHBR, but the extent of elevation depended on the particular bile acid. The levels of cytoprotective ursodeoxycholic acid and its conjugates were markedly elevated, while that of cytotoxic chenodeoxycholic acid remained unchanged, suggesting the balance of bile acids had shifted resulting in decreased toxicity. In EHBR, reduced biliary excretion leads to increased systemic exposure to harmful compounds including some endogenous metabolites. Our metabolomic data suggest that mechanisms exist in EHBR that compensate for cholestasis-related damage.     

Pharmacokinetic study of intraperitoneally administered troglitazone in mice using ultra-performance liquid chromatography/tandem mass spectrometry

A rapid and sensitive ultra-performance liquid chromatography/tandem mass spectrometry (UPLC/MS/MS) method was developed and validated for the determination of troglitazone in mouse plasma. Troglitazone and its internal standard (IS), rosiglitazone, were separated on an ACQUITY UPLC BEH C(18) column (1.7 microm particle size, 50 x 2.1 mm i.d.) by gradient elution with water and methanol at a flow rate of 0.5 mL/min. The cycle time of each analysis was 2.5 min. Rosiglitazone and troglitazone eluted at 1.13 and 1.57 min, respectively, and were chromatographically resolved from the ion suppression and enhancement zones due to the biological matrix effect. Quantitation of the analytes was performed in electrospray negative ionization mode (ESI -ve) using multiple reaction monitoring (MRM) experiments. The weighted (1/x) calibration curve was quadratic over the plasma concentration range 1-2500 ng/mL with a correlation coefficient (r(2)) of 0.9966. The limit of quantitation (LOQ) of troglitazone in mouse plasma was lower than 1 ng/mL. The inter- and intra-day variations of the assay were lower than 12.1%; the overall accuracy ranged from 86.4-110.2% and recovery from spiked plasma was more than 60%. The developed method was successfully applied to determine troglitazone in mouse plasma after intraperitoneal administration.     

High-throughput chiral liquid chromatography/tandem mass spectrometry

Chiral liquid chromatography is a well-established area of bioanalytical chemistry and is often used during the processes of drug discovery and development. The development and use of a chiral drug require the understanding of the pharmacokinetic characteristics of each of the enantiomers, including potential differences in their absorption, distribution, metabolism, and excretion. Chromatographic techniques coupled to atmospheric pressure ionization-tandem mass spectrometry have shown potential as sensitive and robust tools in the quantitative and qualitative determination of enantiomers in biologic fluids and tissue extracts. However, development of a chiral liquid chromatography method requires time-consuming procedures that are devised empirically. Clearly, there is an incentive to design chromatographic approaches that are easy to use, compatible with mass spectrometry ionization interface conditions, exhibit relatively short run times without compromising sensitivity, and offer a broad analyte specificity. For these reasons, the present paper explores the feasibility of the bonded macrocyclic glycopeptide phases (teicoplanin and vancomycin) for analysis by chiral liquid chromatography/tandem mass spectrometry. Ritalinic acid, pindolol, fluoxetine, oxazepam, propranolol, terbutaline, metoprolol, and nicardipine were tested in this study. Furthermore, an example of a simultaneous chiral LC/MS/MS detection (chromatographic run time approximately 10 min) of four pharmaceutical products resulting in baseline resolutions of all four pairs of enantiomers is presented. Methanol, an MS-compatible mobile phase, was utilized in all the experiments.     

Determination of iodoacetic acid using liquid chromatography/electrospray tandem mass spectrometry

A rapid analytical method based on liquid chromatography/tandem mass spectrometry (LC/MS/MS) using electrospray ionization in negative ion detection mode was developed for the analysis of underivatized iodoacetic acid in water. The method was applied to model reaction mixtures in the study of the formation of iodoacetic acid after chlorinated tap water was boiled in the presence of potassium iodide or iodized table salt. Samples can be directly analyzed by the LC/MS/MS system without extraction or chemical derivatization. Limit of detection was determined to be 0.3 microg/L (or 0.3 ng/mL) and limit of quantitation was about 1 microg/L (1 ng/mL).     

Determination of airborne isocyanates as di-n-butylamine derivatives using liquid chromatography and tandem mass spectrometry

A method for the determination of isocyanates as di-n-butyl amine (DBA) derivatives using tandem mass spectrometry (MS/MS) and electrospray ionisation (ESI) is presented. Multiple-reaction monitoring (MRM) of the protonated molecular ions and corresponding deuterium-labelled d₉-DBA derivatives resulted in selective quantifications with correlation coefficients >0.998 for the DBA derivatives of isocyanic acid (ICA), methyl isocyanate (MIC), ethyl isocyanate (EIC), propyl isocyanate (PIC), phenyl isocyanate (PhI), 1,6-hexamethylene diisocyanate (HDI), 2,4-, 2,6-toluene diisocyanate (TDI), isophorone diisocyanate (IPDI), 4,4′-methylenediphenyl diisocyanate (MDI), 3-ring MDI, 4-ring MDI, HDI-isocyanurate, HDI-diisocyanurate, HDI-biuret and HDI-dibiuret. The instrumental precision for 10 repeated injections of a solution containing 0.1 μg ml⁻¹ of the studied derivatives was <2%. Performing MRM of the product ion [DBA + H]⁺ (m/z = 130) from the protonated molecular ion resulted in the lowest detection limits, down to 10 amol (for TDI). Quantification of concentrations below 10⁻⁶ of the occupational exposure limit (OEL) for TDI during 10 min of air sampling was made possible. In an effort to control the formation of alkali adducts, addition of lithium acetate to the mobile phase and monitoring of lithium adducts was evaluated. Having lithium present in the mobile phase resulted in complete domination of [M + Li]⁺ adducts, but detection limits for the studied compounds were not improved. Different deuterium-labelled derivatives as internal standards were evaluated. (1) DBA derivatives of deuterium-labelled isocyanates (d₄-HDI, d₃-2,4-TDI, d₃-2,6-TDI and d₂-MDI), (2) d₉-DBA derivatives of the corresponding isocyanates and (3) d₁₈-DBA derivatives of the corresponding isocyanates. An increase in number of deuterium in the molecule of the internal standard resulted in an increase in instrumental precision and a decrease in correlation within calibration series.     

Analysis of tetracyclines in honey by high-performance liquid chromatography/tandem mass spectrometry

A confirmatory method coupling liquid chromatography to tandem mass spectrometry (LC/MS/MS) is described for the determination of tetracycline, oxytetracycline, doxycycline and chlortetracycline in honey. Demeclocycline, another tetracycline molecule not reported for its usage in honey, was used as internal standard to quantify the four analytes. The sample preparation entails a clean-up on an Oasis HLB solid-phase extraction cartridge and analyses were realised by LC/MS/MS in selected reaction monitoring mode. The stability of tetracyclines was checked under various storage conditions at -20, +4 and +20 degrees C (both under dark and light exposures). Indeed, tetracyclines are not stable molecules and the epimerisation phenomenon was evaluated in this work. Appropriate correction factors of the MS/MS responses of each epimer were studied for each of the four tetracyclines to accurately quantify them. Moreover, the matrix effects encountered during the LC/MS/MS analyses were also studied in spiked experiments from blank honey samples of various geographical origins and different flower types.     

Simultaneous determination of seventeen glucocorticoids residues in milk and eggs by ultra-performance liquid chromatography/electrospray tandem mass spectrometry

A comprehensive analytical method has been developed and validated for the simultaneous determination of seventeen glucocorticoid residues in eggs and milk. The mass spectrometer parameters, the composition of the mobile phase and the sample preparation method were firstly optimized to obtain maximum sensitivity. The samples were deconjugated with beta-glucuronidase/arylsulfatase enzyme and concentrated using an Oasis HLB solid-phase extraction cartridge, followed by cleanup with a dual Sep-pak silica and aminopropyl cartridge. The analytes were quantified by ultra-performance liquid chromatography (using a C18 column)/electrospray ionization tandem mass spectrometry (UPLC/ESI-MS/MS) operating in the negative ion mode. The assay for the 17 glucocorticoids was linear over the range of 1-200 microg/L for milk and egg samples with a high correlation coefficient (>0.99). The limits of quantification (LOQs) for the target analytes were 0.04-1.27 microg/kg for the egg samples and 0.03-0.73 microg/kg for the milk samples. The average extraction recoveries of the glucocorticoids from eggs and milk at two concentration levels (spiked at 0.40 and 2.00 microg/kg) were 65.6-118.7% and 61.5-119.6%, respectively, with relative standard deviations between 1.8-17.0% and 2.4-18.4%, respectively. Because of its high sensitivity, good precision and specificity, the method was found to be suitable for trace analysis of synthetic and natural glucocorticoids in complex biosamples such as eggs and milk.     

Determination of ephedra alkaloids by liquid chromatography/tandem mass spectrometry

In conjunction with an AOAC Task Group on dietary supplements, a liquid chromatography/tandem mass spectrometry (LC-MS/MS) method was validated for measurement of 6 major alkaloids in raw ephedra sinica herb, ephedra extracts, ephedra tablets, complex dietary supplements containing ephedra, and a high-protein drink mix containing ephedra. The amount of ephedrine-type alkaloids present was determined by LC with mass selective detection. Six replicates of each matrix were analyzed on 3 separate occasions. The presence of 6 ephedrine-type alkaloids was detected at a level > 0.5 microg/g based on a 0.5 g sample. The standard curve range for this assay is from 0.02 to 1.0 microg/mL. Appropriate dilutions covered a wide range of specific alkaloid concentrations. The calibration curves for all 6 analytes had correlation coefficients > 0.995.     

Analysis of urinary aromatic acids by liquid chromatography tandem mass spectrometry

The separation and detection of 11 urinary aromatic acids was developed using HPLC-MS/MS. The method features a simple sample preparation involving a single-step dilution with internal standard and a rapid 8 min chromatographic separation. The accuracy was evaluated by the recovery of known spikes between 87 and 110%. Inter- and intra-assay precision (CV) was below 11% in all cases and the analytes were observed to be stable for up to 8 weeks when stored at -20 degrees C. The method was validated based upon linearity, accuracy, precision and stability and was used to establish reference intervals for children and adults.     

High-throughput and sensitive screening by ultra-performance liquid chromatography tandem mass spectrometry of diuretics and other doping agents

The reliability of ultra-performance liquid chromatography coupled to tandem mass spectrometry (UPLC- MS/MS) for high throughput screening in anti-doping control has been tested. A method to screen for the presence of diuretics and other doping agents in urine has been optimised and validated. The extraction procedure consisted of an alkaline extraction (pH 9.5) with ethyl acetate and salting-out effect (sodium chloride). The extracts were analysed by UPLC-MS/MS. Analysis of 34 forbidden drugs and metabolites was achieved in a total run time of 5 min, using a C18 column (100 mm x 2.1 mm i.d., 1.7 microm particle size) and a mobile phase containing deionised water and acetonitrile with formic acid, with gradient elution at a flow-rate of 0.6 mL min(-1). Identification of the compounds was performed by multiple reaction monitoring, using electrospray ionisation in positive- or negative-ion mode. Precursor and product ions were studied for each compound and cone voltage and collision energy were optimised. Due to the different chemical structure of the compounds under study, extraction recoveries varied from less than 10% to 100% depending on the analyte. The limits of detection ranged from 50 ng mL(-1) to 200 ng mL(-1), and all the compounds comply with the requirements of quality established by the World Anti-doping Agency. Intra-assay precision was evaluated at two concentrations for each compound and, in most cases, a relative standard deviation of the signal ratio lower than 20% was obtained. The method has demonstrated to be reliable when analysing routine samples and the short analysis time resulting from a simple sample preparation and a rapid instrumental analysis allow a fast turn-around time and makes it of great interest for routine anti-doping control purposes.