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Journal of Analytical Toxicology, Vol. 29, January/February 2005 Review I Applications of Liquid Chromatography- Mass Sp
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Journal of Analytical Toxicology, Vol. 29, January/February 2005
Review I
Applicationsof Liquid ChromatographyMass Spectrometryin Doping Control* Lucia Politi, Angelo Groppi, and Aldo Polettini t Department of Legal Medicine and Public Health, University of Pavia, Via Forlanini 12, 27100 Pavia, Italy
Abstract paper reviews liquid chromatographic-massspectrometric (LC-MS) proceduresfor the screening, identification and quantification of doping agents in urine and other biological samples and devoted to drug testing in sports. Reviewed methods publishedapproximately within the last five years and cited in the PubMed database have been divided into groups usingthe same classificationof the 2004 W o r l d Anti-Doping Agency (WADA) Prohibited List. Together with proceduresspecifically developed for anti-doping analysis, I.C-MS applications used in other fields (e.g., therapeutic drug monitoring, clinical and forensic toxicology, and detection of drugs illicitly used in livestock production) have been includedwhen considered as potentially extensible to doping control. Information on the reasonsfor potential abuse by athletes, on the requirementsestablishedby W A D A for analysis, and on the W A D A rulesfor the interpretation of analytical findingsare provided for the different classesof drugs. This
Introduction Doping control is a particularly demanding task for both analyticaland interpretive reasons: 1. the extremelywide spectrum of doping substances, in terms of molecular weight, polarity, PKa,and chemical/thermalstability; 2. the high sensitivityof detection required for many compounds that, being administered long before competition, are expected to be present in urine at the low micrograms-per-liter level at the time of competition; 3. the short term often required to give results, particularly during high-level sport events; and 4. the discrimination of doping from other possible reasons for positive, such as the use of drugs for recreational purposes or physiological/pathological alterations of endogenous steroids levels. Athough gas chromatography-mass spectrometry (GC-MS) is currently still the standard technique in anti-doping analysis, ' Partof thecontentof this paperwas presentedat the 8th IATDMCTMeetingin Basel, Switzerland,September2003. + Author to whomcorrespondenceshould be addressed:Prof.Aldo Polettini,Ph.D., Dept. of LegalMedicineand Public Health,Universityof Pavia,Via Forlanini12, 271O0 Pavia,Italy.E-mail:[email protected].
because of its robustness and high level of standardization, liquid chromatography-mass spectrometry (LC-MS) has a number of features that can be effectivelyexploited in sports drug testing or that offer good perspectivesof application in the near future (1,2). First, LC-MS allows minimal sample preparation, thus increasing the sample throughput by means of 1. direct analysis of conjugated metabolites, 2. chromatographic separation of polar compounds with no need for derivatization, and 3. on-line sample preparation, because of the compatibility between aqueous sample and analytical system, at least in the reversed-phase (RP) mode. Furthermore, LC-MS makes it possible to detect the whole metabolic profile of drugs, from the parent compound to very polar conjugated metabolites, thus possibly providing an aid in the interpretation of resuits, as in the case of high testosterone-to-epitestosterone ratios (3) or in the discrimination between doping and recreational use of drugs such as stimulants or narcotics. LC-MS can be helpful also in the analysis of chemically unstable and/or volatile drugs, particularly when the evaporation/reconstitution step can be avoided (4). LC-MS allows the extension of the upper mass limit to dozens of thousands of Daltons by means of electrospray ionization (ESI),which is able to easily produce multiply charged ions, or the most recently developedtime-offlight (TOF) mass analyzers, thus permitting the detection of peptide hormones. The present article reviewsLC-MS methods published in the literature approximatelywithin the last 5 years and cited in the PubMed database (5). Most of the LC-MS applications specific to the anti-doping field available in the literature refer to drugs not amenable to GC separation (e.g., conjugated steroids, peptide hormones), or to substances suitable for GC after derivatization (e.g., diuretics, ~2-agonists, B-blockers, and corticosteroids), or to specific target compounds. Therefore, in the present review also LC-MS methods developed for other purposes (e.g., therapeutic drug monitoring, clinical and forensic toxicology, and detection of drugs illicitly used in livestock production) have been included when considered as potentially extensible to doping control. Particular attention was dedicated to the identification criteria proposed by the different authors (when available) for a rapid comparison with the
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recently established requirements of the World Anti-Doping Agency (WADA)(6). The classification of doping agents in the following paragraphs keeps that of the 2004 WADAProhibited List (7) in order to facilitate the finding of methods for specific classes.
W A D A Identification Criteria
WADA has recently established criteria for the identification of a compound by combined chromatographic-mass spectrometric analysis (6). Regarding LC separation, the retention time of the analyte is required not to "differ by more than 2% or • 0.4 rain (whichever is smaller) from that of the same substance" in the reference material. As for MS detection, when a single-mass full or partial scan is acquired, all diagnostic ions with a relative abundance greater than 10% in the reference spectrum must be present in the unknown. In addition, the relative abundance of three "diagnostic ions" (i.e., "molecular ion or fragment ions whose presence and abundance are characteristic of a substance and thereby may assist in its identification") shall not differ by more than a certain percentage-depending on the abundance of the ion relative to the base ion in the reference spectrum (see Table I)--from the relative intensities of the same ions in the reference material. Background subtraction can be applied if necessary, but it must be used consistently throughout the batch of samples. Computer-based library searching is also allowed but criteria for identification have to be established in advance by the laboratory and all the matches have to be reviewed by a qualified scientist as "the match factor for a reverse search does not guarantee identification" (6). When acquisition is performed in selected ion monitoring (SIM), at least three diagnostic ions must be acquired. The signal-to-noise ratio of the least intense diagnostic ion must be greater than 3:1. The relative intensities of any of the ions (preferably determined from the peak area or height of the integrated mass chromatogram) shall not differ by more than the amount reported in Table I from the relative intensities of the same ions acquired in the reference material. The concentration Table I. Maximum Tolerance Windows for Relative Ion Intensities to Ensure Appropriate Uncertainty in Identification when using LC-MS and LC-MS n* Relative Abundance (% of base peak)
tC-MS and LC-MS n
> 50% 25% to 50% < 25%
-+ 15% (absolute) t __.25% (relative)* +_ 10% (absolute) *
* Modified from 6.
Absolute difference is obtained by subtracting/adding the stated percentage from
the relative abundance of the ion in the referencematerial. * Relative difference is obtained by subtracting/adding from the relative abundance of the ion in the reference material the amount calculated by multiplying the stated percentage by the relative abundance of the ion in the reference material.
of the analyte should be "comparable" in the sample and in the reference material. Tandem MS can also be used for identification either in full scan or selected reaction monitoring (SRM) mode. Collision conditions have to be selected in order to ensure that the precursor ion is present in the product ion scan or in the SRM acquisition. In some cases, a single reaction (one precursor ion -~ one product ion) may be sufficiently unique to be definitive, although in such case the mass resolution of the first mass analyzer should be set to unity. When monitoring more than one product ion, the relative intensities of any of the ions shall not differ by more than the amount in Table I from those of the same ions in the reference material "analyzed contemporaneously". The signal-to-noise ratio of the least intense diagnostic ion must be greater than 3:1. Both in SIM and in tandem MS detection, when a diagnostic ion shows a relative abundance of less than 5% in the reference, the condition that has to be satisfied for positive identification is the mere presence of the ion in the unknown. Finally, if a sufficient number of diagnostic ions is not available, a derivative of the compound, or a second ionization or fragmentation technique (able to provide different diagnostic ions) shall be used.
Stimulants
According to the 2004 WADAProhibited List (7) this group of substances, prohibited in-competition only, includes amphetamine and methylenedioxyamphetamine derivatives, cocaine, strychnine, and drugs generally exerting a direct and indirect action on adrenergic receptors and therefore have a pronounced stimulating effect on the central nervous system. Both optical isomers (where relevant) and other substances with similar chemical structure or similar pharmacological effects are included in this group. A minimum required performance limit (MRPL) of 0.5 mg/L has been set by WADAfor stimulants (and of 0.2 mg/L for pipradol and strychnine), where MRPL is "a minimum concentration at which all laboratories must be able to operate", whereas it must not be considered as a threshold, a limit of detection (LOD) or quantification (LOQ) (8). Quantitative thresholds in urine have been established for the following stimulants: caffeine (12 rag/L), cathine (5 mg/L), ephedrine, methylephedrine (10 mg/L), phenylpropanolamine, and pseudoephedrine (25 mg/L). Some stimulants, such as caffeine, phenylephrine, phenylpropanolamine, pipradrol, pseudoephedrine, and synephrine will be the object of a WADA2004 Monitoring Program (9) in order to detect patterns of misuse in sport and have therefore been removed from the 2004 WADAProhibited List. No methods for the screening of stimulants in urine by LC-MS for anti-doping purposes have been published in the literature. Nevertheless, many methods devoted to the analysis of one or more stimulants, and to amphetamines and derivatives or to cocaine and metabolites in particular, for clinical and forensic toxicology purposes are available and have been recently reviewed (2,10). The following methods have been considered in this article because of their innovative features.
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Kataoka et al. (11) described the LC-MS determination of amphetamines and methylenedioxyamphetamines in urine after automated in-tube solid-phase microextraction (SPME). This extraction technique is based on the use of an open tubular fused-silica capillary as the SPME device instead of the standard SPME fiber. The capillary (GC capillary, 60 cm x 0.25-ram i.d., coated with Omegawax250, 0.25-~m film thickness) is placed between the injection loop and the injection needle of the high-performance liquid chromatograph (HPLC) autosampler and, by repeated draws and ejections of the sample from/into the vial, the analytes partition into the stationary phase of the capillary until a concentration equilibrium is reached. Analytes are then desorbed by mobile phase flow and transported to the LC column. Urine was diluted 1:10 with water, filtered (0.45 lJm), and buffered with 0.5M Tris-HCl buffer (pH 8.5) before SPME. Separation was achieved in RP mode under isocratic conditions. Analytes were detected in ESI-M8 positive selected ion monitoring (one ion per analyte) mode. The authors concluded that in-tube SPME provides a fast, easy,low-cost, and fully automated (and, therefore, more accurate and precise relative to manual techniques) tool for the isolation of amphetamines and other drugs from aqueous matrices. Nordgren and Beck (12) developed an RP-LC-MS-MS procedure based on the detection of positive ions after atmospheric pressure chemical ionization (APCI) for the direct measurement of methylenedioxyamphetamines (MDA and MDMA) in SRM mode (one transition per analyte) in 10-fold water-diluted urine with 8-rain analysis time. APCIwas found to provide better sensitivity than ESI and likely improved the robustness of the method because of the less pronounced ion suppression from the matrix. Nevertheless, ion suppression was claimedby the authors to explain a false-negativeresult obtained for a sample containing 0.103 mg/L MDMAas measured by GC-MS (LODfor MDMAby LC-MS-MS, 0.020 mg/L). The potential of direct analysis of cocaine and metabolites, as well as of opiates, in urine by LC-MS-MS using an ion trap detector was investigated by Dams et al. (13). The authors successfully achieved the simultaneous determination of 25 analytes with LOQs in the range 0.010-0.100 mg/L by monitoring 1-3 reactions per compound after RP gradient elution and APCI of centrifuged (5 min/510 g) urine fortified with deuterated internal standards. Validation criteria for specificity,precision, accuracy, dilution integrity, and stability were fulfilled. With continuous post-column infusion of morphine no matrix effect was observed by the authors for k' > 1 during the analysis of blank urine despite the lack of sample preparation. Direct injection of 0.22-1Jm filtered urine combined with fast RP chromatography (2.1 rain run time) has been proposed by Jeanville et al. (14,15) for the confirmation and quantification of cocaine/benzoylecgoninein immunoassay-positivesamples. Detection (with LODs in the low microgram-per-liter range) was carried out in MS-MS SRM (three reactions per analyte) positive mode. The method was first applied to blanks, matrix prepared standards, and quality controls (14) and later extended to positive urine samples (15). When sample preparation is required before LC-MS, the organic extract usually needs to be dried and reconstituted in a
solvent miscible and with less elution strength than the mobile phase in order to avoid band broadening of the analyte. However, some analytes are not suitable to evaporation/reconstitution because of volatilization, degradation, or adsorption phenomena. In order to avoid loss of pseudoephedrine during evaporation, Naidong et al. (4) proposed the extraction of the analyte from urine by automated 96-well solid-phase extraction (SPE) with 1% formic acid in acetonitrile followedby direct injection of the eluate in a normal-phase HPLC system consisting of a silica column and a low aqueous/high organic eluent. Identification of pseudoephedrine was performed monitoring one transition after ESI in positive mode. As a result, a batch of 96 samples could be processed in I h. The method fulfilled validation criteria recommended by FDA (16). The ability of the procedure to discriminate between close isomers of pseudoephedrine (e.g., ephedrine) was not investigated by the authors. Mortier et al. (17) evaluated the performance of a new type of ion source, sonic spray (SSI), in the determination of paramethoxyamphetamine and other amphetamine-related designer drugs. SSI is an atmospheric pressure ionization technique based on the use of a very high ("sonic") gas flow coaxial to the capillary. In such conditions ions are formed without applying heat or an electrical field, making this technique suitable for the analysis of unstable compounds. SSI proved to be sensitive enough and appropriate for the analysis of amphetamine-related drugs in biosamples, with ion suppression being negligible in urine (less than 3%), likely because of the applied sample preparation (liquid-liquid extraction), and chromatographic separation ("wash out" of endogenous interfering compounds during the first 12 min and elution of analytes after 14 min). One or two transitions per compound were monitored for identification and quantification. Complete validation was achieved for sensitivity, precision, and accuracy. Narcotics
Prohibited narcotics (in-competition only) include the opiates buprenorphine, heroin, hydromorphone, morphine, oxycodone, oxymorphone and the opioids methadone, pentazocine, pethidine, and dextromoramide. According to the 2004 WADAProhibited List (7), differentlyfrom the previous version (where the statement "...and related substances" was added at the bottom of the list) (18), the prohibited narcotics are restricted to the 10 mentioned compounds (7). The required MRPL for narcotics is 0.2 mg/L (8). As for stimulants, several methods dealing with the determination of opiates have been published in the literature (2,10), although none of them specifically dedicated to sports drug testing. Therefore, the methods described here have been limited to those allowing the determination of intact glucuronides (when applicable) together with the free metabolites in urine. In fact, the possibility to detect the whole metabolic profile of narcotics (i.e., conjugated vs. nonconjugated fraction, detection of 6-acetylmorphine as a marker of heroin intake) may provide an effectivemeans to discriminate use for doping purposes at competition from other forms of opiates intake (e.g., use of codeine, consumption of foods containing poppy seeds) or even from use for recreational purposes. This
Journal of Analytical Toxicology, Vo[, 29, January/February 2005
approach appears to be more informative than the adoption of a quantitative threshold for morphine in urine (> 1 mg/L, based on the sum of the glucuronide conjugate and the free drug concentrations) above which "a doping violation has occurred", as established by the 2004 WADAProhibited List (7,8). Bogusz et al. (19) reported an LC-APCI-MS method for the detection of morphine, morphine-3-glucuronide (M3G), morphine-6-glucuronide (M6G), codeine, codeine-6-glucuronide, and 6-acetylmorphine (6-AM) in biological fluids, including urine. After SPE with C18cartridges and RP isocratic LC separation, the determination was performed in SIM mode (one ion per analyte, two ions for the glucuronides, corresponding to the protonated conjugate and the protonated aglycone) with a total analysis time of 17 min. Sch~inzle et at. (20) reported an LC-APCI-MS assay for the detection of morphine, M3G, M6G, and normorphine in body fluids for pharmacokinetic purposes. SIM for the protonated molecular ions was adopted in order to enhance sensitivity, whereas the triple-quadrupole MS was operated in SRM only for the detection of M3G and M3G-d3, owing to an interfering peak. Normorphine coeluted with morphine but could be separated mass spectrometrically. LOQs were approximately in the range 0.003-0.020 mg/L with a 0.1-mL volume of urine. Some authors (19,21) have observed post-column deconjugation of M3G and M6G to morphine that may lead to overestimation of the parent compound. Even if the amount of conjugate undergoing in-source hydrolysis is lowered to a few percent, this may cause a huge error in the quantification of morphine as the relative concentration of the latter is usually much lower. Therefore, baseline separation of the analytes is mandatory in order to establish the performance of the LC-MS method in quantitation. Kronstrand and colleagues (22) determined buprenorphine (BUP) and its free and conjugated metabolites in the urine of 16 patients receiving Subutex| using LC-MS-MS. Direct analysis of filtered (0.22 pm) urine was adopted, although sample preparation (enzymatic hydrolysis followed by SPE) was required in order to detect buprenorphine and norbuprenorphine (NBUP) below the 0.020 mg/L cut-off (4 cases out of 16). It is rather difficult to achieve good fragmentation conditions [both using in source collision-induced dissociation (CID) and MS-MS] for BUP (23,24) owing likely to its stable moiety, and this is confirmed by Kronstrand et al. (22), who used the surviving parent ion (i.e., monitoring of the [M+H]+ ion both as precursor and product with a relatively high collision energy in order to fragment interfering ions having the same mass) for the quantitation of BUP, NBUP, their deuterated analogues, and one or two transitions as qualifiers. It is worth mentioning, however, that Moody et al. (25) were able to determine BUP at the subnanogram level using SRM (mlz 468 + mlz 396). Other opiates included among narcotics, such as oxymorphone, hydromorphone, oxycodone, and hydrocodone, can be detected in urine by LC-MS using the same separation conditions setup for morphine (26). On the other hand, we found only one paper describing the isolation and identification of hydromorphone-3-glucuronide and other free and conjugated metabolites of hydromorphone (27). Also, methadone and metabolites are amenable to LC-MS
using conditions similar to those adopted for morphine (13), whereas the search in PubMed did not reveal LC-MS methods for dextromoramide, pethidine (meperidine), and pentazocine in urine [the latter was determined in blood and human tissues using LC-fast atom bombardment MS by Imamura et al. (28)]. Cannabinoids Cannabinoids are included among the compounds prohibited by WADAin-competition only (7), although their diffusion as recreational drugs makes it difficult to prove doping based on their presence in urine. Owing likely to this reason, in the case of cannabinoids (as for alcohol and other substances generally available as medicinal drugs), the WADA Code allows for a reduced sanction if the "...Athlete can establish that the use of such a specified substance was not intended to enhance sport performance" (7). A quantitative threshold of 15 pg/L for the main tetrahydrocannabinol (THC) metabolite, carboxyTHC, in urine has been established by WADA(8). Two LC-MS methods for the determination of THC metabolites in urine have been published in the literature: one directed to carboxy-THC after basic hydrolysis (29), the other enabling also the direct detection of carboxy-THCglucuronide (although the latter could be quantified only indirectly, i.e., after hydrolysis, owing to the unavailability of pure standard) (30). A thorough discussion on the pros and cons of the application of LC-MS to the bioanalysis of cannabinoids can be found in the review articles of Van Bocxlaer et al. (10) and Marquet (2).
Anabolic agents Anabolic agents, prohibited in- and out-of-competition, include exogenous anabolic androgenic steroids (AAS) such as boldenone, clostebol, fluoxymesterone, nandrolone, stanozolol and analogues, endogenous AAS (androstenediol, androstenedione, dehydroepiandrosterone, dihydrotestosterone, testosterone and analogues), the !5~-agonist clenbuterol and zeranol, a non-steroidal estrogen analogue. The 2004 WADA Prohibited List (7) keeps the testosterone (T)-to-epitestosterone (E) ratio of 6 as a threshold to activate further investigations in order to discriminate doping with T from possible physiological and pathological conditions that may alter the T/E ratio. WADAalso keeps the quantitative threshold established for 19-norandrosterone (2 pg/L), the nandrolone metabolite used for detecting doping with this exogenous steroid (8). The MRPL is 10 pg/L for anabolic agents in general (as the main metabolite), 2 pg/L for clenbuterol, methandienone (as its metabolite 17[3-methyl-5[8-androst-l-ene3o~,17c~-diol), methyltestosterone (as 17c~-methyl-513-androstane-3c~,1718-diol),stanozolol (3'-hydroxystanozolol), and epitestosterone, and 1 pg/L for norandrosterone. Exogenous AAS. Among exogenous AAS, nandrolone (19nortestosterone, 19-NT) is probably one of the most famous because of the relevant number of professional athletes that recently tested positive for this AASand to the following debate on the possible explanations (31,32). The diagnosis of doping with 19-NT is an analytical challenge as 1. possible sources of involuntary intake of 19-NT have been discovered (pork and boar meat, some mislabelled nutritional supplements) and 2. the two target metabolites that are considered as proof of
Journal of Analytical Toxicology, VoL 29, January/February2005
19-NT intake, 19-norandrosterone (19-NA) and 19-noretiocholanolone (19-NE), have been demonstrated to be endogenous products in human urine (33,34). LC-MS-MS (ESI, positive mode,two transitions per compound) has been among the analytical techniques (together with low-resolution and highresolution GC-MS) used to prove the presence of 19-NA and 19-NE in human urine after consumption of boar meat (33). However,the laborious and time-consuming sample preparation procedure adopted, including enzymatic hydrolysis, did not allowthe authors to fully exploit the advantages of LC-MS. To this respect, the method proposed by Kuuranne and colleagues (35) appears as a substantial step forward as it enables the direct detection of 19-NT conjugated metabolites 3a-hydroxy-5~c-estran-17-one glucuronide and 3a-hydroxy-5[~-estran-17-one glucuronide. The method, based on liquid-phase microextraction (LPME) followed by RP gradient separation and ESI positive mode MS-MS, is actually multi-residue as the glucuronides of other exogenous AAS can be determined (bolasterone, boldenone, calusterone, drostanolone, mestanolone, mesterolone, methandriol, methenolone, and stanozolol). Ammonium adducts or [M+H]§ ions were selected as precursors and two product ions (corresponding to [MH-GIu]§ [MH-GIuH20] +, or [MH-GIu-2H20]+) were monitored. LPMEwas found to give cleaner samples compared to SPE and liquid-liquid extraction and to shorten analysis time. Moreover, the LPME device is inexpensiveand disposable. LODswere typicallyin the range 2-20 IJg/L. However, the coelution of the glucuronide metabolite of boldenone with epitestosterone glucuronide, both having the same SRM ion pairs, hampered the detection of boldenone intake and also the determination of the ratio of testosterone and epitestosterone glucuronides. 19-NT has been detected by APCI LC-MS-MS in the urine of treated bovines (36). Kim et al. (37) developed an LC-MS-MS method to determine 19-NTand its esters in equine plasma. This approach, although applied to non-human specimens, is interesting as it theoretically enables an unambiguous diagnosis of doping owing to the certain exogenous origin of the esters. Stanozolol (Stan) is one of the most difficult exogenous AAS to analyze by GC-MS as it adsorbs at parts of the injector and it derivatizes with difficulty. Moreover,it is almost completely metabolized to give 16-hydroxystanozolol(16-OHStan), 3'-hydroxystanozolol (3'-OHStan) and several other hydroxy-derivatives (38--40).In 1998 a multi-laboratory study for the analysis of Stan and metabolites in urine, feces, and blood of calveswas published (41): veal calves were treated with stanozolol and the presence of the parent drug and its metabolites was determined by five different laboratories using different cleanup procedures and analytical techniques (three of them by LC-MS-MS, one by LC-MS, one by GC-MS NCI mode). As expected LC-MS-MS provided better results than LC-MS in terms of selectivity (although extensive fragmentation, with more than 20 diagnostic ions, negatively affected sensitivity) and it was found to be the method of choice for 16-OHStan. 3'-OHStan was only detected in trace amounts by some laboratories and could not be detected by GC-MS owing to a coeluting interference. Van de Wiele et al. (40) described the detection of 16-OHStan by LC-MS-MS in bovine urine with two different approaches: a shorter and simpler method
without derivatization and a more sensitive and specific method with a phenyl boronic derivatization step (LOD, 0.03 IJg/L instead of 0.3 IJg/L).The 16-OHStan derivative exhibited better characteristics of the daughter ion spectrum (less fragmentation, more stable ion ratios). A method for the LC-MS-MS analysis of 16-OHStan in bovine urine has been developed by Van Poucke and Van Peteghem (42). These authors proposed one of the very few recent multi-analyte approaches to AASby LC-MS, their method being able to detect also 17r trenbolone, 4-chloroandrost-4-ene-3,17dione, and r and [3-boldenone all with an LOD of I IJg/L.After C18 SPE and enzymatic hydrolysis followed by further SPE purification by coupled C18 and NH2 cartridges, the extract was separated in gradient RP mode. Detection was carried out in ESI positive SRM mode (three collisions per analyte). Another screening approach for exogenous AAS, also including stanozolol (3'OHStan glucuronide) is the already discussed method proposed by Kuuranne et al. (35). In a previous paper this research group (43) compared ESI, APCI, atmospheric pressure photoionization (APPI) for the detection of free steroids noticing that ESI provides better sensitivity. Sporadic applications of LC-MS to exogenousAAS in animal urine [methyltestosterone, mibolerone, and boldenone (44); fluoxymesterone (45); and trenbolone (46)] are available in the literature. Kim et al. (47) applied LC-MS and LC-MS-MS to the structure elucidation of gestrinone metabolites in human urine. EndogenousAAS. Another challenge for sports drug testing is the ascertainment of testosterone intake, the standard GC-MS procedure having important technical and interpretive limitations. T and E are excreted mainly in conjugated forms [T mainly as glucuronide (TG) and E as glucuronide (EG) and sulfate (ES)] that require to be hydrolyzed and derivatized before GC-MS analysis (48). This process has a number of drawbacks (49): 1. possible conversion of androstenediol to T catalized by an isomerase contained in 13-glucuronidase/ arylsulfatase from Helix pomatia ([3-glucuronidase from E. coil eliminates the conversion problem, but it does not have arylsulfatase activity); 2. inability of arylsulfatase to cleave sulfate conjugates (when the sulfate is in 17-position); 3. acidcatalyzed rearrangement of epitestosterone sulfate under acidic hydrolysis conditions (50); and 4. relative instability of the derivative. Direct analysis of steroid conjugates by LC-MS overcomes these problems, can be more accurate, and reduces the risk of a false-positive result due to the underestimation of ES. Bowers and colleagues (3,51) applied positive ESI SRM (with aglycone as product ions) to the analysis of intact TG, TS, EG, and ES in urine after C18 SPE. These authors observed a strong signal suppression varying greatly between samples (range 20-99%) and also between the four conjugates of interest in individual urine samples and decided to use a deuterated internal standard for each analyte to achieve accurate quantitation (although the identification criterion of abundance ratios for the monitored product ions within + 20% of those observed for the reference compounds was not always met in urine samples). Ion suppression, still significant despite the adopted purification step, was explained by the authors with the relatively polar nature of steroid conjugates (and to the consequent difficulty to separate them from other matrix
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components in RP mode because of their poor retention), and their extremely low concentration as well. The challenging task of detecting intact testosterone esters in plasma was tackled by Shackleton et al. (52). In order to improve LC separation and ionization efficiency, steroids were converted to polar, water soluble Girard hydrazones, allowing the authors to detect T enanthate in plasma collected 96 h after intramuscular injection of a 110-rag dose and T undecanoate 1 h after oral administration of 80 mg of the ester. Dehydroepiandrosterone (DHEA), included in the list of prohibited endogenous AAS, is a 17-ketosteroid that has been studied as an indicator of adrenal function and biological aging (53). Jia et al. (54) developed an LC-MS method able to detect urinary 17-ketosteroid sulfates and glucuronides after a simple SPE. The method proved to be particularly sensitive thanks to the use of SSI. SSI had LODs more than 20 times lower than ES! when a high concentration buffer was used. On the other hand, very little fragmentation was produced. LC-MS methods for the determination of DHEA and its conjugated metabolites in body fluids have been recently reviewed by Marwah et al. (53). Clenbuterol and zeranol. Clenbuterol is a [32-agonistbronchodilator with stimulant activity on the central nervous system whose oral use at high doses is known to promote muscle gain and decrease fat deposition. According to the 2004 WADAProhibited List (7), clenbuterol is prohibited both inand out-of-competition. GC-MS analysis of clenbuterol, and of [32-agonists in general, requires derivatization not only to improve the chromatographic performance of the compound, but also to modify the poorly selective fragmentation under electron impact (55). Therefore, LC-MS is undoubtedly the technique of choice for clenbuterol and the other [32-agonists. A very sensitive and selective LC-MS-MS method for the detection of clenbuterol in equine urine was developed by Guan et al. (56). Clenbuterol was extracted from urine samples by liquid-liquid extraction after [3-glucuronidase hydrolysis (not necessary for human urine) and chromatographically separated using a CN column. The quadrupole-TOF hybrid mass analyzer was operated in positive ion with an ESI source. The adopted source parameters favored the formation of predominant [M+H]§ and CID in the collision cell was operated so that appropriate MS-MS conditions could be established for clenbuterol quantification (one pseudo-SRM).The use of high mass resolution of TOF resulted in low baseline noise in pseudoSRM, thus allowing an LOD of 50 pg/0.1 mL of urine. Direct injection of bovine and human urine was experimented by Hogendoorn et al. (57) using a column switching technique coupled with tandem MS with a thermospray (TSP) interface. Because of the widespread illicit use of clenbuterol as a growth promoter in zootechnics, a number of papers dealing with the LC-MS analysis of clenbuterol in serum, muscle, liver, and other animal matrices are available (58-60). Zeranol is a non-steroidal estrogen analogue reported to be used as a growth stimulant in cattle fattening. Its detection in trace amounts in pigs urine by LC-MS-MS was carried out by Kleinova et al. (61) after enzymatic hydrolysis (glucuronidase/arylsulfatase, from Helix pomatia) and C18
SPE. The APCI interface was operated in the negative ion mode, and two product ions originating from the deprotohated molecular species were selected for SRM. The procedure was applied to a 5-mL urine sample and resulted in an LOD of 0.1 I~g/L.
Peptide hormones This group of substances prohibited in-and out-of competition includes erythropoietin (EPO), growth hormone (hGH) and insulin-like growth factor 1 (IGF-1), insulin, corticotropins, chorionic gonadotropin (hCG), and gonadotropins (both pituitary and synthetic, LH), the latter two prohibited in males only. Electrospray LC-MS has a great potential in the analysis of peptides because of the possibility of generating ions directly in solution and easily producing multiply charged ions from peptides and proteins, thus extending the effective mass range of a quadrupole/ion trap MS. Recently developed TOF analyzers also provide an interesting tool for protein detection because of their greatly extended mass range (> 500.000 Da). Nevertheless, LC-MS is far from being applied on a routine basis to the detection of peptides in doping control. Analytical problems (microheterogeneity, extremely low levels in urine) on one side and interpretive problems (physiological levels not yet established; very short half-lives; effective biological markers not yet defined) on the other, make peptide analysis for anti-doping purposes still out of reach. Approaches to the LC-MS analysis of peptides are available in the literature for EPO, hGH, IGF-1, hCG, and insulin, whereas no LC-MS application was found for corticotropins and pituitary and synthetic gonadotropins. Erythropoietin. Recombinant human erythropoietin (rhEPO) is used as a doping agent in endurance sports because it enhances aerobic capacity by increasing red cell volume and it helps reduce physiologicalstrain during exercise and accelerate recovery after training (62).A preliminary study on the detection of exogenous EPO was carried out by Stanley and Poljak (63). Enzymatic deglycosylationwas used to overcome loss of sensitivity due to microheterogeneity (EPO undergoes post-translational carbohydrate chains attachment making this peptide a family of molecules instead of a single compound; consequently, the detected signal is split over numerous and therefore less intense peaks). The method was applied to the detection of two epoetins in the pharmaceutical preparations Eprex| rhEPO, and Aranesp| a glycoprotein produced by recombinant DNA technology that differs from human EPO at five positions of the amino acidic chain. After removing the carbohydrate chains by a single-step procedure (using N-glycosidase, O-glycosidase, and neuraminidase), matrix-assisted laser desorption ionization (MALDI)-TOF-MSanalysis enabled the detection of both intact glycoproteins and deglycosylated proteins in the picomole range. In order to lower the LOD obtained by MALDI-TOF-MS,the protein was digested with endoproteinase Glu-C and the El2 Glu-C peptide obtained by enzymatic cleavage, which is common to both epoetins, could be detected at the low femtomole level using gradient nano-HPLC-ESI-MS-MS. Although the sensitivity makes the method suitable for the detection of exogenous EPO intake with good mass spectral selectivity, the time taken to
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elute E12 from the column (over 100 rain) was found to be too long for routine application. Growth hormone and insulin-like growth factor 1. Human growth hormone is a peptide with a molecular weight of 22124 Da. LC-ESI-MSwas used to discriminate between recombinant human growth hormone (rhGH) and methionyl rhGH (metGH, 22255 Da), the addition of a single methionine to the carboxy-terminal end of the latter allowing a clear differentiation of the masses of the two proteins (64,65). rhGH was used by Wu et al. (66) as a model system for proteomic studies of low-level proteins in plasma. A plasma sample spiked at 10-fold (16 lJg/L) above the natural level of the hormone was analyzed by bi-dimensional chromatography and micro ESI-ion trap MS after trypsin-digestion, rhGH was identified by five different peptides. The method was able to detect growth hormone in the low femtomole level but, even though sample preparation was minimal, the adopted chromatographic separation was too time consuming for routine application. Insulin-like growth factor 1 (IGF-1) is reported to be a promising marker for the detection of GH abuse. IGF-1 is a 70 amino acid, 7.5 KDa protein reported to significantly increase in plasma following GH administration. De Kock et al. (67) developed a preliminary procedural study for the identification and quantitation of IGF-1 after enzymatic digestion. The ESI [M+H] 1§or [M+2H]2§ ions of the obtained peptides were analyzed in MS-MS mode and their product ion spectra showed good correlation with the theoretical fragmentation as generated by software computing. Quantitative analysis of the intact protein was also carried out using Arg3-IGF-1 as internal standard and full scan ESI MS of the [M+7H]7§ ions. The method was able to reach an LOQ of 30 ng on column. However, no data on the application of the method to biosamples were presented. hCG. hCG is a glycoprotein produced by the placenta that is known to stimulate general steroid production in males. The hormone is used to recover from anabolic steroid cycles or to directly increase testosterone secretion without affecting the T/E ratio (68). The hCG molecule is composed of two subunits: an ~-subunit of 92 amino acids and a [3-subunit of 145 amino acids. The r is essentially identical with those of the human pituitary glycoprotein hormones (thyroid stimulating hormone, follicle stimulating hormone, and lutropin). The [3-subunitdistinguishes hCG from the other glycoprotein hormones (69). Liu and Bowers (70) developed an immunoaffinity trapping method, specific for the intact hCG and the free [3-subunit, to extract hCG from urine. After elution from the immunoaffinity phase, the disulfide bonds of the peptides were reduced, alkylated, and extracted on a C18cartridge. After tryptic digestion, three tryptic fragments originating from the [3-subunit were selected for identification and quantitation in order to avoid interference from other glycoprotein hormones. The LOD of the method was 25 IU/L. The LC-MS method permitted also to confirm both the primary sequence of the ~-subunit and the attachment of five oligosaccharide groups to the molecule (after reductive alkylation with 4-vinylpyridine in order to add charge sites to the peptide), although such conditions resulted in poor sensitivity because of the microheterogeneity of the molecule (69). Gain et al.
(68) recently published an LC-MS-MS procedure for the analysis of hCG tryptic peptides in urine. After immunoaffinity extraction, the glycoprotein was digested with trypsin. The tryptic peptide 13T5was chosen as a marker as it was found to give the most abundant peak in the TIC and because of the uniqueness of its sequence (no interference from other human pituitary glycoprotein hormones). Moreover, the surface loop of peptide [3T5in the hCG molecule interacts with the hCG receptor in the testis, thus any change in the amino acid sequence in this region may affect the biological activity of the hormone. The product ion spectrum of ~T5 proved to be very useful in identifying and confirming the presence of the hormone in urine. The method enabled the quantification and confirmation of urinary hCG at 5 IU/L,that is the MRPL set by WADAfor hCG (8). Insulin. Because anabolic agents have been designated illegal, athletes have been looking for alternative drugs to help them put on muscle mass and burn off fat. Insulin might be used as a performance-enhancing agent because it facilitates glucose entry into cells, thus increasing muscle glycogen concentration; it inhibits muscle protein breakdown; and its use during training and post-event is reported to improve recovery (71). Darby et al. (72) demonstrated that LC-MS can discriminate between human insulin from bovine and porcine insulin, both used clinically for the treatment of diabetes. The human body activates insulin from a proinsulin precursor. Stimulation of insulin excretion causes proinsulin to break down to two constituents: insulin and C-peptide. According to the authors, the determination of the C-peptide could provide important information as, if present at low concentration compared to insulin, could suggest an exogenous administration. SPE with RP packing allowed the extraction/concentration of insulin from plasma without the use of antibodies. Instrumental analysis was carried out by RP-LC separation and ESI-MS of the [M+3H]3§ and [M+4H]4§ ions. The LOQ was 1.0 IJg/L for insulin, corresponding to a high physiological level, and 0.8 IJg/L for the C-peptide, which is above the normal range in plasma. Visser et al. (73) applied on-line SPE (anion-exchange followed by C8)-LC combined with ESI-MS (SIM of [M+4H]4§ and [M+5H]5§ to the separation and detection of bovine, porcine, human, and Arg-human insulin in plasma. However, the LOD of 100 nmol/L, that is at least 100 times higher than physiological levels, make this procedure currently unsuitable for the analysis of real samples.
~-Agonists According to the 2004 WADAProhibited List (7) all [32-agonists, including their d- and/-isomers, are prohibited in competition, with the exception of formoterol, salbutamol, salmeterol, and terbutaline that "are permitted by inhalation only to prevent and/or treat asthma and exercise-induced/asthma and broncho constriction" (a medical notification is required in such case). Similarly to clenbuterol, a 132-agonistwith "anabolic" properties at high doses, salbutamol is prohibited also out-of competition when its concentration in urine (flee drug plus glucuronide) is greater than 1000 lJg/L, "unless the athlete proves that the abnormal result was the consequence of the therapeutic use of inhaled salbutamol".
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As reported for clenbuterol earlier in this paper, LC-MS is the technique of choice in the bioanalysis of [~2-agonists (55). LC-MS screening procedures for 132-agonistsin urine/plasma have been published by a number of authors in the past (74-79). Within the last five years only one LC-MS screening procedure for anti-doping purposes (80) and a few methods devoted to single compounds, namely salmeterol (81), ractopamine (82), and salbutamol (83), have been published in the literature. Nineteen 132-agonistswere screened in human and equine urine using LC-MS-MS by Thevis et al. (80). After acidic hydrolysis followed by a solvent washing step (human urine only), analytes were extracted from the alkalized sample (saturated with NaCl) by liquid-liquid extraction. The extract was reconstituted in diluted HCl and the analytes were separated by gradient RP-LC. ESI-MS-MS detection was carried out in SRM mode monitoring at least four reactions per compound. LODs were in the range 2-50 lJg/L. In the case of equine urine the sample preparation was slightly modified and included enzymatic hydrolysis: LODs were in the range 2-100 I~g/L. Salmeterol was detected in equine urine until 3-12 h after inhalation (25 IJg x 20 puffs) with an LOQ of 0.25 IJg/L (81). The analyte was no longer detectable after 24 h. The sample, added with clenbuterol as internal standard, was submitted to enzymatic hydrolysis and liquid-liquid extraction. After gradient RP-LC separation, APCI and ESI either in positive and negative ion modes were evaluated and positive ESI was found to provide the best sensitivity. The MSn identification criteria established by the International Olympic Committee (84) were adopted by the authors, therefore the precursor ion and 2 product ions, corresponding to the loss of 1 and 2 H20 molecules, were monitored. Full method validation was carried out, and five different [32-agonistswere found not to interfere with separation and detection of the analyte. Ractopamine was extracted from bovine urine using a rather laborious procedure involving enzymatic hydrolysis and double SPE (using a polymeric and a mixed mode stationary phase) (82). Instrumental analysis was carried out by gradient RP-LC followed by ESI-MS-MS in SRM mode (six product ions monitored). The procedure enabled the detection of the analyte in urine and biological tissues at nanogramper-liter levels. The enantiomers of salbutamol and of its metabolite salbutamol-O-sulfate were determined in human plasma and urine using a high-throughput procedure by means of an automated 96-well SPE and of a teicoplanin-based LC column by Joyce et al. (83). The m/z 240 ~ m/z 148 transition was monitored for both salbutamol and metabolite (owing to partial thermal degradation of the latter in the source), and the m/z 342 (Na adduct) ~ m/z 244 was used as a confirmatory transition for the sulfate. LOQs for the enantiomers of salbutamol and its sulfate in plasma were 0.1 ~g/L and 5 ~g/L, respectively. However, by restricting the method to salbutamol only it was possible to lower the LOQ to 0.025 IJg/L.
Agents with anti-estrogenic activity Agents with anti-estrogenic activity include drugs used
for the stimulation of ovarian functions and for the treatment of postmenopausal osteoporosis, like cyclofenil and clomiphene, and drugs used in the therapy of breast cancer, like tamoxifen and aromatase inhibitors (e.g., the nonsteroidal anastrozole and the steroidal exemestane). Despite their genotoxic action (tamoxifen has been found to cause hepatic carcinoma in rats) these compounds may be abused in order to prevent side effects of anabolic steroids use in males (gynecomastia and reduced endogenous production of testosterone) and have been prohibited in- and out-of competition in males by WADA(7). Zweigenbaum and Henion (85) showed that highthroughput LC-MS-MS can be used for the determination of selected estrogen receptor modulators (SERMs) in human plasma with more than 2000 samples analyzed in a 24-h time. The method was aimed at the detection of two SERMs (tamoxifen and its major metabolite 4-hydroxytamoxifen, and raloxifen) and two structurally related compounds (nafoxidine and idoxifen). After automated 96-welt liquid-liquid extraction, the analytes were separated in less than 30 s and detected by ESI-MS-MS SRM mode. Only one transition per compound was monitored, but with Q1 operating at mass resolution of 0.7 Da peak width at half-height. The LODs achieved were in the range 5-50 IJg/L on spiked plasma sampies. Idoxifen and its pyrrolidinone metabolite were analyzed in real plasma samples by the same group using a similar procedure (86). The adapted instrumental conditions allowed the analysis of over 3700 samples per day.The obtained LOQs were 10 IJg/L for idoxifen and 30 IJg/L for the metabolite. The same authors were able to decrease the LOQ for idoxifene to 0.5 t~g/L by improving the chromatographic separation (87) [4-rain run instead of 0.4 min as in the previous paper (86)]. Also in this case only one transition was monitored for both detection and quantitation, but Q1 was operated at unit mass resolution. The authors evaluated also the performance of a TOF-MS detector as compared to the triple quadrupole instrument operated in SRM mode and found that TOF-MS provided an improved full-scan sensitivity and accurate mass measurement capability at the expenses of sensitivity (10 times lower) and of a reduced dynamic range (5-2000 IJg/L compared to 0.5-5000 ]Jg/L for the triple-quadrupole instrument). In a later paper (88) by the same research group, the use of APPI as compared to APCI was applied to the determination of idoxifene and metabolites in human plasma. APPI was found to give better sensitivity, particularly in the case of the neutral metabolite SB245420 where an LOD of 100 IJg/L was achieved when neither APCI nor ESI could produce any ion current from the same compound. Little difference was found, on the other hand, in matrix suppression effect between APCI and APPI. LC-MS-MS was used by Myung et al. (89) to characterize cyclofenil metabolites in human urine. Two glucuronide metabolites corresponding to the loss of both acetyl groups (M1) and to hydroxylation of M1 (M2) were identified, whereas the parent compound was not detected in urine. Both glucuronides exhibited a molecular adduct ion [M+NH4]§ that fragmented to an intense aglycone product ion under MS-MS analysis.
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Ninety-six-well Cz SPE combined with LC-MS-MS was used by Cenacchi et al. (90) for the quantitation of exemestane in human plasma. The method, based on APCI MS-MS (monitoring the m/z 297 ~ m/z 121 transition), was fully validated in the concentration range 0.05-25 pg/L.
Diuretics and probenedd Diuretics (e.g., acetazolamide, furosemide, benzthiazide, hydrochlorothiazide, spironolactone, ethacrynic acid, and triamterene) and probenecid, together with epitestosterone and plasma expanders, are grouped in the 2004 WADAProhibited List (7) under the definition "Masking Agents", that is substances that "have the potential to impair the excretion of Prohibited Substances, to conceal their presence in urine or other samples used in doping control, or to change haematological parameters". Masking agents are prohibited in- and out-ofcompetition. Diuretics have also a potential of abuse in sports where athletes must make a particular weight classification, such as wrestling, weightlifting, body building, and horseracing, or where weight loss may enhance performance. Moreover, diuretics are used to prevent the retention of water, a typical side effect of steroids. Diuretics are generally analyzed by GC-MS for anti-doping purposes. However,derivatization is required and the resulting derivatives may be unstable. Some diuretics (like amiloride and benzthiazide) may also decompose under the derivatization conditions or during GC analysis (91). LC-MS provides an advantageous alternative as it enables direct analysis of these compounds. This is probably the reason why a number of LC-MS screenings for diuretics specifically developed for sports drug testing purposes is available in the literature (91-94). Thieme et al. (91) reported an LC-MS-MS method able to determine 32 diuretics and masking agents in urine. Extraction was carried out using XAD columns, in contrast to the other published procedures (92-94) where liquid-liquid extraction was used, and after RP-LC gradient separation, detection was carried out by ESI negative and positive ionization modes (in subsequent runs). Negative ionization was generally preferred, with exception for basic compounds (amiloride, triamterene, and etozoline) and for canrenone (metabolite of potassium canrenoate and spironolactone). One reaction per analyte was monitored for confirmation purposes. The method proved to be specific and robust enough to be applied under routine conditions. The authors did not report LOQs because quantitative analysis of diuretics is not part of the routine doping tests, any administration of these drugs to athletes being prohibited (an LOD of I IJg/Lwas reported only for polythiazide). In this respect, it is worth mentioning that WADAhas established an MRPL for diuretics at 250 1Jg/L(8). Deventer et al. (92) performed the screening of 18 diuretics and probenecid in urine on an LC-MS-MS ion trap instrument equipped with ESI interface using scan by scan polarity change, thus allowing the detection of all the analytes within a single run. Diuretics were extracted by consecutive acidic and alkaline liquid-liquid extraction, and the two extracts were combined and, after gradient RP-LC separation, screened by full-scan MS or, for compounds pro-
viding a low S/N ratio at 100 IJg/L in urine, by MS-MS. For confirmation purposes analysis was performed in MS-MS mode in order to obtain sufficient structural information. The method appears to be suitable as a screening for doping control purposes because reported LODs are lower than 250 iJg/L. Sanz-Nebot et al. (93) optimized the separation of eight diuretics using a mathematical model able to predict retention as a function of acetonitrile concentration and pH of the mobile phase. The final mobile phase provided baseline separation for benzthiazide, bendroflumethiazide, trichlormethiazide, spironolactone, ethacrynic acid, bumetanide, potassium canrenoate, and furosemide. Detection was performed in positive ion full scan mode. A thorough discussion of the ESI fragmentation of the different diuretics is provided in the paper. However, the authors did not report LODs, and the quantitative performance of the method was not evaluated. An LC-MS method for the detection of three thiazidebased diuretics (chlorothiazide, CT, hydro chlorothiazide, HCT, and trichlormethiazide, TCM) in equine urine was reported by Gabris et al. (94). In-source CID with both ESI and APCI in negative ion mode was tested for their suitability to give sufficient fragmentation for confirmation purposes. Silicon hexafluoride was used as carrier gas instead of nitrogen in ESI experiments in order to eliminate electrical discharge problems, but it was found also to reduce overall signal intensity. APCI provided better sensitivity and fragmentation: at least three diagnostic ions for each compound could be selected. The combination of LC and negative ion CID-APCIMS with a conventional quadrupole mass analyzer yielded a reliable and relatively rapid method for the analysis of thiazides in urine. On the other hand, the reported LODs (270 pg/L for CT, 130 pg/L for HCT, and 380 IJg/L for TCM) are greater than the WADAMRPL (8), with the only exception of HCT. The determination of tripamide and its metabolites in human urine for sports drug testing purposes was investigated by Kim et al. (95) using LC-ESI-MS-MS. Tripamide is a sulfonamide derived anti-hypertensive agent with diuretic properties not yet included in the 2004 WADAProhibited List (7). The parent compound was never detected in the urine collected for 48 h after the administration of one Tripamol| table (15-rag dose). Two slightly different conjugated metabolites were detected after acidic hydrolysis (but not after hydrolysis with ~-glucuronidase/arilsulfatase), both with a [M+H]§ ion at m/z 385, likely resulting from hydroxylation, loss of-NH2 and dehydrogenation. Glucocorficosteroids Although local administration or intra-articular injection are permitted when therapeutically justified (medical notification required), the systemic use in-competition of corticosteroids (e.g., corticosterone, hydrocortisone, dexamethasone, betamethasone, flumethasone, triamcinolone acetonide, and prednisolone) is prohibited by WADA(7). Some of the typical effects of corticosteroids on nervous system (euphoria), on carbohydrate metabolism (stimulation of gluconeogenesis), on cardiovascular apparatus, and on blood (increase of the
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hemoglobin and red cell content) make these drugs attractive for doping purposes. Corticosteroids analysis presents a number of difficulties as, on one side, the low polarity and the neutral character of these molecules limit the possibility to obtain an efficient purification from the biological matrix (96), on the other, their thermal lability and low volatility make them amenable to GC repartition only after derivatization. Therefore, corticosteroids represent another class of doping agents where the potential of LC-MS can be fully exploited as demonstrated by the number of screening methods published in the literature for the detection of illicit uses of these drugs either in sports (97-99) or in livestock production (100,101). Deventer and Delbeke (97) published a validated method for the screening of 10 corticosteroids by LC-MS-MS. According to the authors, phase II metabolism of several synthetic corticosteroids is hampered by the presence of a double bond between C1 and C2 and a fluorine atom at C9 of the steroid moiety. Hence, these corticosteroids can be detected in human urine without hydrolysis and sample preparation can be limited to liquid-liquid extraction under mild alkaline conditions. ESI-MS-MS detection was carried out in SRM (two product ions per analyte) negative ion mode with the only exception of 16o~-hydroxyprednisolone for which polarity was switched to positive. The RP gradient chromatography did not allow baseline separation of the isomers betamethasone and dexamethasone. The discrimination between them could be achieved by MS3 analysis of the [M-HCH20]- ion or by isocratic elution. LODs in the range 0.5-4 lag/L were reported by the authors. An LC-ESI-MS method for the confirmation analysis of 11 corticosteroids in urine was developed by Fluri et al. (98) involving Extrelut-based liquid-liquid extraction and gradient RP-LC. Negative ionization was found to provide a better S/N ratio and a simpler fragmentation with a [M-31]- ion, likely resuiting from loss of CH2OH, as the base peak. Detection was carried out in SIM using three ions per analyte as required by WADA identification criteria (8). Limits of confirmation, defined as the LOD of the less intense confirmation ion, were in the range 1-20 1Jg/L. LC-MS-MS was applied to the screening of corticosteroids (100) and to the detection of selected phase II metabolites in bovine urine (101) by Antignac and colleagues, although the laborious and time consuming sample preparation adopted, justified by the complexity of the matrix and by the extremely low LODs required (40-70 pg/g), limits the application of this procedure in anti-doping analysis.
l~-Blockers [3-Blockers (e.g., alprenolol, atenolol, betaxolol, labetalol, metoprolol, pindolol, propranolol, sotalol, and timolol) reduce the stimulatory effects of the sympathetic nervous system by preventing the binding of noradrenaline to its receptors and are used by athletes to reduce heart rate and tremor, which can be valuable in sporting activities where steadiness is important (102). The 2004 WADAProhibited List includes a number of sports where 0-blockers are prohibited in-competition only (e.g., gymnastics, ski jumping, and diving). In the case of 10
archery and shooting the use of these drugs is forbidden also out of competition (7). Thevis et al. (103) described a method for the determination of 32 [3-blockingagents by LC-APCI-MS-MS,suitable for high throughput analysis of urine specimen in doping control. The product ion mass spectra generated with CID of the [M+H]§ ions exhibited common fragmentation schemes: m/z 116 is a typical fragment ion of I3-blockers bearing an oxypropanolamine side chain ending with an isopropyl group (e.g., acebutolol and propranolol). Another typical fragment is [MH-77]§ likely due to the combined loss of water and isopropanolamine. In the case of IS-blockers bearing an oxypropanolamine side chain ending with a t-butyl group (e.g., nadolol, penbutolol, and timolol), the main fragment results from the neutral loss of isobutene [MH-56]§ often followed by the loss of a water molecule. [3-blockers with a phenylethanolamine structure show typical loss-of-water ions [MH-18]§ and ions resulting from the subsequent elimination of propene [M-42]§ and those generated by the loss of the substituent present at the phenolic ring structure. After an enzymatic hydrolysis/liquid-liquid extraction based sample preparation and gradient RP-LC, screening was carried out by monitoring a single reaction per analyte, although for confirmation at least three reactions could be used. Chromatography was fast (less than 7 rain including re-equilibration) and sufficient for the separation of compounds detected by the same parent ion/product ion reaction (e.g., levobunolol and penbutolol). LODs in the range 10-100 lag/Lwere obtained, which are significantly lower than the WADAMRPL for this class (500 lag/L). Gergov and colleagues (104) presented an LC-MS method for the screening and confirmation of 16 [3-blockingagents in urine samples for clinical and forensic purposes. LC-MS with selected ion monitoring was chosen for pre-screening samples owing to its inherent simplicity. In case of tentative identification of a [3-blocker (presence of the [M+H]+ ion at the expected retention time) the sample was automatedly re-injected and the product ion mass spectrum was acquired and searched against a library containing 400 MS-MS spectra of drugs. During method development, the authors compared insource CID (single MS, summed spectra at low and high orifice voltage) and MS-MS at low, medium and high collision energy (20, 35, and 50 eV, respectively) and found that 35 eV MS-MS experiments provided the best performance in terms of selectivity of identification. Also, MS-MS based search produced fewer false negatives than library searching with mass spectra derived from single-stage quadrupole MS. The limit of identification was below 200 lag/Lfor the majority of the compounds although for atenolol and pindolol the reported limit of identification was higher than the WADAMRPL. Kataoka et al. (105) developed an automated in-tube SPMELC-MS method for the determination of nine [3-blockers in urine and serum samples similar to the one already described for amphetamines and methylenedioxyamphetamines (11). This sample preparation technique proved to be fast, simple and inexpensive. The reported LODswere sensibly lower than the WADAMRPL, although detection did not meet the WADA identification criteria (6) as it was carried out in SIM mode monitoring only the protonated molecular ion.
Journalof AnalyticalToxicology,Vol. 29, January/February2005
Conclusions The reviewed literature shows that LC-MS applications are available for almost all the classes of doping agents, although not always specifically developed for doping control purposes. For manyof the prohibited or controlled drugs, LC-MSappears not only to be able to overcome the typical drawbacks of GC-MS, but also to provide a further aid in the interpretation of analytical findings. Peptide hormones constitute the most important exception as, despite the strong potential of LC-MS for high molecular weight substances, both analytical and interpretive problems still strongly limit the use of this technique for the detection of many compounds belonging to this class. In general, however, it is foreseeable that the use of LC-MS in anti-doping labs will progressively increase with time at the decrease of purchase and operative costs of instrumentation, as it is already happening in other analytical fields. The low limits/high selectivity of detection that have to be achieved still force towards the adoption of separated screening procedures for the main classes of doping agents, not differently fromwhat is being done with GC-MS. The setup of general unknownscreening procedures able to detect a wide range of compounds within a single analysis, as applied in other fields of analytical toxicology (e.g., forensic and clinical toxicology) (106-108), is presently out of reach, not only because of the sensitivity required but also because of the low reproducibility of mass spectra (at least in terms of relative abundances) obtained through different ionization techniques and, within the same ionization technique, through instrumentation from different manufacturers (109). To this respect, good perspectives are offered by the increasing availability of high mass resolution capabilities in bench-top LC-MS instruments, providing the possibility to achieve the identification of a compound on the basis of its "exact" mass and on the ratio of isotopic peaks,leaving the structural informationcontained in the mass spectrum for confirmation purposes (110,111).
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