Anal. Chem. 2007, 79, 6020-6026
Technical Notes
Rapid Screening of Doping Agents in Human Urine by Vacuum MALDI-Linear Ion Trap Mass Spectrometry Hari Kosanam,† P. K. Sai Prakash,‡ C. R. Yates,§ D. D. Miller,§ and Suma Ramagiri*,§
Department of Chemistry, University of Memphis, Memphis, Tennessee 38152, Department of Chemistry, Osmania University, Hyderabad, India 500007, and Department of Pharmaceutical Sciences, University of Tennessee Health Science Center, Memphis, Tennessee 38163
Detection of doping agents in urine frequently requires extensive separation prior to chemical analyses. Gas or liquid chromatography coupled to mass spectrometry has produced accurate and sensitive assays, but chromatographic separations require time and, sometimes, chemical derivatization. To avoid such tedious and lengthy procedures, vacuum matrix-assisted laser desorption ionization (vMALDI) coupled with the linear ion trap mass spectrometry (LIT/MS) technique is tested for its applicability as a rapid screening technique. Commonly used doping agents like nandrolone, boldenone, trenbolone, testosterone, and betamethasone were chosen as study compounds. Different MALDI matrixes like r-cyano-4-hydroxycinnamic acid (CHCA), dihyroxy benzoic acid (DHB) with and without cetyl trimethyl ammonium bromide (CTAB), a surfactant, and meso-tetrakis(pentafluorophenyl) porphyrin (F20TPP) were tested. Among them, F20TPP (MW 974.57 Da) was selected as the preferred matrix owing to the lack of interfering matrix peaks at the lower mass range (m/z 100-700). Urine samples spiked with study compounds were processed by solid-phase extraction (SPE) and consistently detected through a linear range of 0.1-100 ng/mL. The limit of detection and lower limit of quantification for all five analytes have been determined to be 0.03 and 0.1 ng/mL, respectively, in urine samples. Testosterone-d3 was used as an internal standard, and the quantitative measurements were achieved by the selective reaction monitoring (SRM) mode. The method was validated and showed consistency in the results. Hence, vMALDI-LIT/MS can be used as a rapid screening method to complement the traditional GC/MS and LC/MS techniques for simultaneous identification, confirmation, and quantification of doping agents in urine.
Doping is defined as “the use of an artifice, whether substance or method, potentially dangerous to an athlete’s health or capable of enhancing their performances or the presence in the athlete’s body of a substance or the ascertainment of the use of a method on the list annexed to the Olympic Movement Anti-Doping Code”. The practice of enhancing athletic performance through foreign substances was known from the earliest Olympic Games.1-4 In the past 50 years, steroids have been used as growth promoters in husbandry practice with beneficial effects on animal growth promotion and feed efficiency.5 Steroids are also prohibited in equesterian sports by most of the horse racing authorities including the United States national equestrian federation.6,7 Nowadays, steroids are even available as “nutritional” supplements, and it has been shown that some nutritional supplement cocktails contained banned substances not indicated on the label.8,9 World antidoping code has classified prohibited substances as10 (1) anabolic agents (exogenous anabolic androgenic steroids, e.g., boldenone, nandrolone, etc.; endogenous anabolic androgenic steroids, e.g., testosterone, androstenediol, etc., (2) β-2 agonists, e.g., bambuterol, trenbolone, etc., (3) agents with antiestrogenic activity (aromatase inhibitors, e.g., anastrozole, letrozole, etc.; selective estrogen receptor modulators (SERMs), e.g., raloxifene, tamoxifene, toremifene, etc.; other antiestrogenic substances, e.g., clomiphene, cyclofenil, fulvestrant, (4) diuretics and other masking agents, e.g., indapamide, metolazone, (5) hormones and related (1) (2) (3) (4) (5) (6) (7) (8)
* Corresponding author. E-mail:
[email protected]. Phone: 901-448-5259. Fax: 901-448-7842. † University of Memphis. ‡ Osmania University. § University of Tennessee Health Science Center.
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Bowers, L. Ther. Drug Monit. 2002, 24, 178-181. Vogel, G. Science 2004, 305, 632-635. Kicman, A. T.; Gower, D. B. Ann. Clin. Biochem. 2003, 40, 321-356. Van Helder, W. P.; Kofman, E.; Tremblay, M. S. Can. J. Sport Sci. 1991, 16, 248-257. Berthiaume, R.; Mandell, I.; Faucitano, L.; Lafreniere, C. J. Anim. Sci. 2006, 84, 2168-2177. Short, C. R.; Sams, R. A.; Soma, L. R.; Tobin, T. J. Vet. Pharmacol. Ther. 1998, 21, 145-153. Gowen, R. R.; Lengel, J. G. Vet. Clin. North Am.: Equine Pract. 1993, 9, 449-460. Geyer, H.; Parr, M. K.; Mareck, U.; Reinhart, Y.; Schrader, W. Scha¨nzer, V. Int. J. Sports Med. 2004, 25, 124-129. De Cock, K. J. S.; Delbeke, F. T.; Van Eenoo, P.; Desmet, N.; Roels, K.; De Backer, P. J. Pharm. Biomed. Anal. 2001, 25, 843-852. World Anti-Doping Agency, The World Anti-Doping Code The 2007 Prohibited List, International Standard. http://www.wada-ama.org/en/ prohibitedlist.ch2. 10.1021/ac070118z CCC: $37.00
© 2007 American Chemical Society Published on Web 06/30/2007
substances, e.g., erythropoietin (EPO), human growth hormone (hGH), insulinlike growth factor (IGF-1), gonadotrophins, insulin, and corticotrophins, (6) stimulants, e.g., amphetamine, cocaine, etc., (7) narcotics, e.g., morphine, oxycodone, etc., (8) cannabinoids, e.g., marijuana, hashish, etc., (9) glucocorticosteroids, and (10) alcohol. The illegal use of these compounds has raised concerns about human health after consumption, resulting in the introduction of monitoring programs to control their use. The standard technique for steroid analysis is gas chromatography-mass spectrometry (GC/MS)11-18 which requires chemical derivatization, depending on the individual properties of the steroid. The lack of a universal derivatization reagent hinders the availability of a method to statutory testing laboratories. GC/MS methods are time-consuming, and there are problems associated with the method, e.g., stability of the derivatives and their thermal decomposition during analysis. Steroids have also been investigated by liquid chromatographic-mass spectrometric (LC/MS) techniques.19-26 LC/MS provides good sensitivity but requires longer separation times, about 5-10 min per sample, and sometimes encumbered by complicated solvent handling and plumbing, and we all know that autosamplers are slow relative to plate readers and are notorious for carry over problems. Therefore, the development of more straightforward mass spectrometry methods is of great interest. Matrix-assisted laser desorption/ionization (MALDI) has recently seen an emergence in small molecule analysis and appears extremely promising for high-throughput analysis and imaging studies.27-33 MALDI has several advantages, and most importantly, MALDI can achieve a high sample throughput making it attractive (11) Van Thuyne, W.; Delbeke, F. T. Biomed. Chromatogr. 2004, 18, 155-159. (12) Catlin, D. H.; Ahrens, B. D.; Kucherova, Y. Rapid Commun. Mass Spectrom. 2002, 16, 1273-1286. (13) Kintz, P.; Cirimele, V.; Dumestre, T. V.; Villain, M.; Ludes, B. J. Chromatogr., B: Anal. Technol. Biomed. Life Sci. 2002, 766, 161-167. (14) Gaillard, Y.; Vayssette, F.; Balland, A.; Pepin, G. J. Chromatogr., B: Biomed. Sci. Appl. 1999, 735, 189-205. (15) Choi, M. H.; Chung, B. C.; Lee, W.; Lee, U. C.; Kim, Y. Rapid Commun. Mass Spectrom. 1999, 13, 376-380. (16) Casademont, G.; Perez, B.; Garcia Regueiro, J. A. J. Chromatogr., B: Biomed. Appl. 1996, 686, 189-198. (17) Choi, M. H.; Chung, B. C.; Kim, M.; Choi, J.; Kim, Y. Rapid Commun. Mass Spectrom. 1998, 12, 1749-1755. (18) Hooijerink, D.; Schilt, R.; Van Bennekom, E.; Brouwer, B. Analyst 1994, 119, 2617-2622. (19) Deventer, K.; Eenoo, P. V.; Delbeke, F. T. Biomed. Chromatogr. 2006, 20, 429-433. (20) Ho, E. N.; Leung, D. K.; Wan, T. S.; Yu, N. H. J. Chromatogr., A 2006, 1120, 38-53. (21) Yu, N. H.; Ho, E. N.; Leung, D. K.; Wan, T. S. J. Pharm. Biomed. Anal. 2005, 37, 1031-1038. (22) Reilly, C.A.; Crouch D. J. J. Anal. Toxicol. 2004, 28, 1-10. (23) Buiarelli, F.; Cartoni, G. P.; Coccioli, F.; De Rossi, A.; Neri, B. J. Chromatogr., B: Anal. Technol. Biomed. Life Sci. 2003, 784, 1-15. (24) Starcevic, B.; DiStefano, E.; Wang, C.; Catlin, D. H. J. Chromatogr., B: Anal. Technol. Biomed. Life Sci. 2003, 792, 197-204. (25) Deventer, K.; Delbeke, F. T.; Roels, K.; Van Eenoo, P. Biomed. Chromatogr. 2002, 16, 529-535. (26) Draisci, R.; Palleschi, L.; Ferretti, E.; Lucentini, L.; Cammarata, P. J. Chromatogr., A 2000, 870, 511-522. (27) Hatsis, P.; Brombacher, S.; Corr, J.; Kovarik, P.; Volmer, D. A. Rapid Commun. Mass Spectrom. 2003, 17 (20), 2303-2309. (28) Muddiman, D. C.; Gusev, A. I.; Langner, K. S.; Proctor, A.; Hercules, D. M.; Tata, P.; Venkataramanan, R.; Diven, W. J. Mass Spectrom. 1995, 30 (10), 1469-1479. (29) Sleno, L.; Volmer, D. A. Rapid Commun. Mass Spectrom. 2005, 19 (14), 1928-1936. (30) Hatsis, P.; Brombacher, S.; Corr, J.; Kovarik, P.; Volmer, D. A. Rapid Commun. Mass Spectrom. 2003, 17 (20), 2303-2309.
Figure 1. Chemical structures and nominal molecular weights of investigated anabolic steroids and the proposed product ion structure for SRM analysis.
to analytical laboratories for increasing productivity and efficiency. Furthermore, in comparison to ESI, MALDI is not as susceptible to ion suppression or enhancement. Therefore, we investigated the use of vacuum matrix-assisted laser desorption ionization coupled with linear ion trap mass spectrometry (vMALDI-LIT/ MS) as a high-throughput screening method for doping agents in spiked urine samples. The primary aim of this study was to develop a fast, sensitive, and validated method for the identification of doping agents spiked in urine by vMALDI-LIT/MS. For this study we have chosen five commercially available doping agents nandrolone, boldenone, testosterone, trenbolone, and betamethasone (Figure 1) with molecular weights ranging from 250 to 500 Da. The emphasis of this study was first to evaluate different MALDI matrixes for clean mass spectra without any interfering peaks at the low mass range (m/z 100-500) and to test its applicability for quantification. The method is validated in terms of linearity, precision, and accuracy (both inter- and intraday), and the linearity curves were compared between conventional LC-ESI/MS and the novel vMALDI-LIT/ MS to prove it as a complementary technique. EXPERIMENTAL SECTION Materials. Testosterone, betamethasone, testosterone-d3, nandrolone, boldenone, and trenbolone were purchased from Sigma (31) Hsieh, Y.; Casale, R.; Fukuda, E.; Chen, J.; Knemeyer, I.; Wingate, J.; Morrison, R. A.; Korfmacher, W. A. Rapid Commun. Mass Spectrom. 2006, 20 (6), 965-972. (32) Sleno, L.; Volmer, D. A. Rapid Commun. Mass Spectrom. 2006, 20 (10), 1517-1524. (33) Jespersen, S.; Niessen, W. M. A.; Tjaden, U. R.; Greef, V. J. J. Mass Spectrom. 1995, 30 (2), 357-364.
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(St. Louis, MO). The three MALDI matrixes used in this work, R-cyano-4-hydroxycinnamic acid (CHCA), dihyroxy benzoic acid (DHB), and meso-tetrakis(pentafluorophenyl) porphyrin (F20TPP), and cetyl trimethyl ammonium bromide (CTAB) were obtained from Sigma (St. Louis, MO). Acetonitrile, methanol, and other solvents and chemicals used in this study were also obtained from Sigma. Light Microscope Images. Light microscope images of the matrixes were acquired using an Olympus SZ-PT dissection microscope under 2× and the Nikon Eclipse E800 (Nikon, Tokyo, Japan) under 40× equipped with a Nikon Coolpix 4500 digital camera (Nikon, Tokyo, Japan) using MetaVue software. Urine Sample Preparation. The extraction of steroids from spiked human urine specimens was accomplished using solidphase extraction (SPE). A Bond Elute Certify cartridge (Varian, Inc. Palo Alto, CA) was preconditioned with 2 mL of methanol and deionized water. Then, 1 mL of urine spiked with testosteroned3 (30 ng/mL) as the internal standard (IS) was added followed by a washing step with 0.5 mL of deionized water and an elution step with 0.5 mL of methanol. The methanolic layer was collected and evaporated to dryness; the residue was reconstituted in 10 µL of methanol. Approximately 1 µL of the reconstituted sample was mixed with an equal amount of matrix solution (2.5 mg/mL in 50% aq acetonitrile for CHCA and DHB, 10 mg/mL of F20TPP in chloroform) was spotted onto a stainless steel target plate. Standard Calibration Solution Preparation. A stock solution of each steroid was prepared by dissolving 1 mg in 1 mL of methanol and was further diluted to 100 µg/mL. From these solutions, standard solutions with varying amounts of five steroids (0.1-100 ng/mL) containing 30 ng/mL of IS were prepared in water and in urine samples. Selection of MALDI Matrix. Three types of MALDI matrixes, CHCA, DHB, and F20TPP, were examined with and without the addition of CTAB. The dried droplet method was used for the spotting. Liquid Chromatography-Mass Spectrometry. An Agilent 1100 series LC system (Agilent Technologies, Palo Alto, CA) was coupled to an Agilent MSD single-quadrupole mass spectrometer equipped with an electrospray-ionization source. A reverse-phase, C18, 4 µm, 4.6 mm × 250 mm column (Novapak, Waters Corp, Milford, MA) was used for separations. The mobile phase consisted of eluent A (10 mM ammonium acetate pH 6.7) and eluent B (50:50 acetonitrile-methanol), and separations were achieved using a gradient program of 0 min, 80% A; 7 min, 50% A; 8 min, 20% A; 9 min. The flow rate was 1.0 mL/min with the column at ambient temperature. The electrospray interface was operated in positive ion mode with mass spectra acquired over a mass range of 100500 m/z. The internal capillary and fragmentor voltage were set at 3 kV and 70 V, respectively. Nitrogen drying gas was set at a flow rate of 12 L min-1 at 350 °C. Nebulization was aided with a nitrogen sheath gas provided at a pressure of 35 kPa. The selective ion monitoring (SIM) mode was used for analyte quantification. vMALDI-Linear Ion Trap Mass Spectrometer. A Finnigan LTQ linear ion trap coupled to the Finnigan vMALDI (Thermo Finnigan, San Jose, CA) ion source was used for MALDI ion trap analysis. The data were acquired on the vMALDI LTQ instrument using the Xcalibur 1.4 software in the data dependent mode. A survey scan (MS) was followed by MS/MS scans on the five most 6022
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abundant ions for selection of a SRM transition. This sequence of six scan events was repeated six times for each sample spot. Assay Validation for Steroids. A quantitative method for steroids spiked in human urine by vMALDI-LIT/MS was validated in terms of specificity, analytical recovery, accuracy, precision (both inter- and intraday), and linearity. Blank urine specimens necessary for the assay validation were obtained from six different healthy volunteers (three females, three males). Specificity was evaluated by analyzing urine samples from three male and three female volunteers. The absence of interferences at the m/z of the analytes was verified, and these samples were pooled and used in the validation studies. The effect of biological matrix ion suppression was investigated by analyzing a blank urine sample and water mixed separately with the porphyrin matrix. The limit of detection was defined as the lowest level at which a compound could be identified in urine samples, with diagnostic ions present with a signal-to-noise (S/N) ratio greater than 3. The lower limit of quantification (LLOQ) is the lowest standard on the calibration curve that meets the following conditions: (1) the analyte response is at least 5 times the blank response, (2) precision is e20%, (3) accuracy expressed as bias is e20%. The extraction recovery was determined by analyzing samples spiked before and after SPE extraction at three different concentrations (0.1, 50, and 100 ng/mL). Spiking after SPE extraction corresponded to 100% recovery. The results were attained by comparing mean values of the relative peak heights of the analytes. For evaluation of intraday precision and accuracy, three duplicate plasma samples containing IS (30 ng/mL) and all five analytes at the low (0.1 ng/mL), medium (50 ng/mL), and high (100 ng/ mL) concentrations were prepared, extracted, and analyzed on the same day. As for interday precision and accuracy, the same samples were analyzed each day for 2 days. Precision of the method was expressed as the relative standard deviations (RSD %) of the peak height ratios of the analytes to the IS at different concentrations. Calibration curves were obtained from analyte to internal standard peak-height ratios using linear regression with a 1/x weighting factor. For the intraassay precision and accuracy, one set of calibration samples and three sets of QC samples were analyzed. For interassay precision, three separate sets consisting of calibrations samples and QC samples were analyzed. RESULTS AND DISCUSSION Crystal Quality and Sample Cleanup. It has been suggested that the MALDI ionization process is less susceptible to ion suppression than ESI allowing greater latitude for direct sampling without prior cleanup. Urine samples processed by SPE and unprocessed urine samples were compared to avoid tedious sample preparation during the analysis, but the signal intensity and spectrum quality were not good for the unprocessed urine samples. Different ratios of sample to matrix solution were evaluated to overcome these problems, but this was found to be unsuccessful. Fortunately, simple established sample cleanup procedures are adequate for restoring analytical success. We also checked the MALDI matrix as an elution solvent, and the nonretentive behavior of the MALDI matrix ensured a homogeneous sample/matrix mixture. We also tried microelution plates to provide a small degree of sample concentration and avoid any further dry down/reconstitution steps. This SPE-type sample cleanup also showed good results. All these sample preparation
Figure 3. Full mass spectra of nandrolone spiked in human urine at a concentration of 100 ng/mL in (A) porphyrin matrix and (B) CHCA + DHB matrix.
Figure 2. Light microscope images of crystal spots after air-drying of 1 µL of (A) CHCA + DHB, (B) DHB, (C) porphyrin matrix solutions, and (D) porphyrin matrix crystals at high resolution.
Figure 4. Complex mixture analysis of five anabolic steroids spiked in human urine at 500 ng/mL in the porphyrin matrix.
methods proved to be advantageous and doing this allows preparation of 96 samples simultaneously. Furthermore, this method was amenable to automated sample spotting using common liquid handlers. meso-Tetrakis(pentafluorophenyl) Porphyrin (F20TPP) as MALDI Matrix. The use of MALDI mass spectrometry for
the quantitative analysis of drugs is normally hampered by interfering matrix peaks in the low-mass range with poor precision of signal abundances. F20TPP has a high molecular weight (MW 974.6 Da) compared to other more commonly used matrixes such as DHB (MW 154.1 Da) and CHCA (MW 189.2 Da). The crystal quality of CHCA, DHB, and F20TPP matrix were compared in Analytical Chemistry, Vol. 79, No. 15, August 1, 2007
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Figure 5. Product ion spectra of five anabolic steroids and the internal standard. The chosen product ions for SRM transition are indicated by an asterisk.
Figure 2. The data obtained from the F20TPP matrix were comparable to those obtained from the CHCA + DHB matrix, with the advantage that the F20TPP matrix spectra had no interfering signals in the low-mass range (m/z 100-700). Representative spectra of nandrolone spiked in urine at 100 ng/mL using F20TPP and CHCA + DHB as matrixes were shown in Figure 3. The use of CTAB, a surfactant, in CHCA and DHB not only suppressed the matrix related background ions but also the test compound signals. Therefore, the F20TPP matrix was 6024
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chosen for subsequent quantitation studies by vMALDI-LIT/ MS. Figure 4 shows the mass spectrum of all five steroids as a complex mixture spiked in human urine (each at 50 ng/mL) in the F20TPP matrix. Selection of Internal Standard. MALDI has become an established method for qualitative analysis with increasing applications in quantification. Quantitative analysis using MALDI is still difficult due to the poor point-to-point repeatability, sampleto-sample reproducibility, and shot-to-shot signal degradation. We
Table 1. Extraction Efficiency, Accuracy, and Precision for Quantification of Five Steroids Spiked in Human Urine added (ng/mL)
testosterone
testosterone-d3
nandrolone
Extraction Efficiency 99 98 94
boldenone
betamethasone
trenbolone
86 98 95
99 93 95
95 88 97
(%)a
0.1 50 100
98 91 90
91 96 99
0.1 50 100
95 94 101
98 99 98
Intraday Accuracy (%)b 89 92 90
93 98 91
96 94 92
100 98 99
0.1 50 100
97 99 100
93 99 97
Interday Accuracy (%)c 97 93 95
87 96 92
100 91 96
96 98 97
0.1 50 100
3.6 4.5 2.8
2.6 3.8 4.6
Intraday CV (%)b 3.1 4.3 5.6
2.8 3.3 4.5
2.3 4.5 5.7
3.6 5.2 3.6
0.1 50 100
5.1 3.5 2.8
2.9 4.9 3.5
Interday CV (%)c 4.1 4.9 3.8
3.9 4.9 2.4
4.8 3.9 3.3
3.2 2.7 4.4
a Extraction efficiency ) peak height of an analyte/peak height of the internal standard added to the blank urine before extraction. The value was the result averaged from n ) 6 on the same day. b Intraday accuracy and precision results were obtained for each concentration (n ) 6) of the analyte analyzed on a single day. c Interday accuracy and precision results were obtained for each concentration (n ) 6) of the analyte on two separate days.
tried to overcome these difficulties by using an internal standard. An ideal internal standard should be chemically similar to the analyte and stable during the analysis, completely resolved from the analytes, and close to the analytes in mass and concentration to avoid instrumental errors. An isotopically labeled analogue of the analyte is an ideal internal standard. We therefore used a structurally analogous compound, testosterone-d3, as an internal standard. The SRM transition 292 f 109 m/z was chosen for the internal standard based intensity of the product ion spectra. Similarly, SRM transitions for nandrolone (274 f 109 m/z), trenbolone (271 f 199 m/z), betamethasone (393 f 239 m/z), testosterone (289 f 109 m/z), and boldenone (287 f 121 m/z) were selected for quantitative analysis (Figure 5). Crystal Position System and Auto Spectrum Filter. The crystal positioning system (CPS) and automatic spectrum filtering (ASF) are two unique features of vMALDI-LIT/MS. Simultaneous usage of CPS and ASF dramatically reduces the acquisition time and increases the sensitivity, thereby making MALDI a rapid screening technique. CPS is an intelligent optical filter process in which the video image of a sample spot is used to determine the location of sample/matrix crystals. During data acquisition, CPS moves the sample plate, so that only these crystals will be irradiated by the laser beam. CPS determines “crystals” based on the intensities, densities, and distances of the sample image. CPS addresses all of the situations related to robotic sample placement, multichannel pipettes, and experimental variability on the MALDI plate as well as nonhomogeneous crystal formation, especially at low concentration and with particular matrixes. Auto spectrum filter determines the quality of a spectrum using user-defined MS or MSn signal thresholds, peak areas, or signalto-noise ratios. During acquisition, if a raw spectrum is above the threshold, the spectrum will be sent to the data processing system and the sample plate remains at this sweet spot to continue
collecting good spectra until the acquisition is complete or the filter criteria are no longer met. In this particular experiment, sample acquisition time and quantification were tested with and without the CPS and ASF modes. Data obtained with CPS and ASF showed more advantages for acquisition time and sensitivity. Method Validation and Optimization for Steroids. The results for assay validation by vMALDI-LIT/MS are summarized in Table 1. In order to prove specificity of the screening procedure, six different lots of blank urine specimens were analyzed for the test compound signals and only testosterone ions were observed. Therefore, endogenous testosterone from the blank urine was eliminated by solid-phase extraction before it was used for preparation of standard solutions. In the MALDI experiment, the variation in matrix ion signal was monitored as a general indicator of crystallization and ionization suppression events. Since the porphyrin ion was the main vehicle for ionization of the analyte during the MALDI process, the availability of this ion therefore represents a more general indication of suppression than a targeted detection of an ion of interest. Comparable peak height and area of the porphyrin in blank urine and water represents no ion suppression. Alternatively, QCs prepared in different lots of blank urine also proved there was no ion suppression by the biological matrix ions or by cross talk of the analyte ions. Extraction recovery for five doping agents varied between 86 and 99%. The accuracy for all analytes was between 87 and 101%, and the precision was within 5.7%. The LOD and LLOQ for all five of the analytes have been determined to be 0.03 and 0.1 ng/ mL, respectively, in urine samples. The method showed good linearity from 0.1 to 100 ng/mL with correlation coefficients higher than 0.9947 for all five steroids. Analytical Chemistry, Vol. 79, No. 15, August 1, 2007
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Comparison of vMALDI-LIT/MS vs LC-ESI/MS. Consideration and study of vMALDI-LIT/MS for small-molecule analysis inevitably leads to its comparison to LC-ESI/MS. The specific qualities for routine quantification important to an ionization technique are potential for speed and overall sensitivity. Potential for Speed. Small-molecule quantification by MALDI has its greatest utility when applied to higher throughput analysis of large numbers of samples generated from assays involving several hundred chemical entities. Current ESI practices in such an environment has several practical boundary conditions, such as the use of standardized mobile phases and chromatography conditions, the use of only a few template MS/MS condition settings, and the general inability to specifically optimize analytical conditions for any single compound. LC-ESI/MS analysis speed is mainly limited by the autosampler response and chromatographic elution times. Though it provides universality and high sensitivity, it is not considered as high throughput because of complicated solvent handling and plumbing, and vMALDI-LIT/ MS has features addressing these issues. Sensitivity. Sensitivity in a quantitative measurement is directly proportional to sample concentration and sample consumption. LC-ESI/MS can consume a larger proportion of the overall sample and deliver it to the ionization source in a concentrated band whereas MALDI sample consumption and delivery concentration is small and relatively fixed. In MALDI, the sample consumed in a single laser ablation spot is equivalent
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to just over 2 nL of the original sample. A typical desorption envelope measurement consumes about 25 nL of the original sample. This MALDI measurement provided an integrated area count of 1583. In comparison, 25 µL of the original sample injected for an LC-ESI/MS measurement provided an integrated area count of 132 450. These data suggest that MALDI provides a 10fold increase in efficiency over ESI for the mass spectrometer used in these experiments. CONCLUSION In summary, a high throughput method for screening doping agents spiked in urine by vMALDI-LIT/MS was developed and validated. Among the three matrixes that were used in the analysis, F20TPP was found to be advantageous owing to less interference in the mass range of 100-700 Da. This method yielded low LOD/LLOQ values and good dynamic ranges for all analytes and is therefore a very promising tool for rugged and rapid quantitative analyses. Potential for speed and overall sensitivity of vMALDI-LIT/MS offers a viable higher-throughput alternative to the LC-ESI/MS technique and other traditional methods in dope tests analysis. Extension of vMALDI-LIT/MS to authentic sample analysis in under progress. Received for review January 20, 2007. Accepted May 24, 2007. AC070118Z
