Athletic drug testing: an analyst's view of science and the law.

Detection of performance-enhancing drugs in sports has received increasing visibility. Athletic drug testing uses sophisticated technology and both interindividual (population) and intraindividual reference ranges to interpret data. An effective program must incorporate educational and adjudication components in addition to testing. The difficult interface between science and the law is evident in many recent sports arbitration decisions.

Direct measurement of urinary testosterone and epitestosterone conjugates using high-performance liquid chromatography/tandem mass spectrometry.

Measurement of the ratio of testosterone (T) and epitestosterone (E) in urine has been used as an indication of 'natural' steroid supplementation for a decade. The direct measurement of the glucuronide and sulfate conjugates of testosterone and epitestosterone by high-performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS) should resolve a number of issues regarding unusual metabolism due to either genetic disposition or attempts to avoid detection of abuse. Determination of nanomoles per liter (0.1 ppb) concentrations of analytes in a complex biological matrix by HPLC/MS/MS is complicated by sample matrix-specific ion suppression during ESI. Deuterated internal standards of all compounds were used to overcome the effects of suppression. Comparison of the HPLC/MS/MS method with a two-part gas chromatographic/mass spectrometric method showed statistical equivalence in urine samples. Analysis of urine samples with elevated T-glucuronide to E-glucuronide ratios did not show that a significant number could be explained by an elevated excretion of epitestosterone sulfate. The HPLC/MS/MS method was also used further to characterize genetic and metabolic factors that give rise to unusual T/E ratios.

Athletic drug testing.

A drug-control program requires testing to ensure compliance and to deter use. In the athletic drug testing area, measurement of performance-enhancing substances is complex partly because of the large number of prohibited substances. A number of sophisticated analytical techniques, such as high-resolution mass spectrometry, are increasingly used to provide the maximum detection time window. Endogenous steroids pose an increasing challenge because of their availability in "nutritional supplements". Continued vigilance is required to prevent the pharmacologic enhancement of performance.

Analytical goals in therapeutic drug monitoring.

The practice of therapeutic drug monitoring is based on several disciplines, including pharmacokinetics, pharmacodynamics, and chemical analysis. The impact of the analysis on the determination of pharmacokinetic parameters is not well appreciated. Analytical goals in therapeutic drug monitoring should be established by determining the nature of the problem to be solved, selecting the appropriate matrix and methodology to solve the problem, and developing valid analytical schemes that are performed competently with appropriate quality and interpreted within the framework of the problem.

Mass spectrometric characterization of nicked fragments of the beta-subunit of human chorionic gonadotropin.

We describe the use of an HPLC/MS technique for the characterization of nicked fragments of hCG beta-subunit. After reductive alkylation of the nicked hCG beta-subunit with vinylpyridine, endoproteinase Glu-C or trypsin was used to digest the protein to produce peptides that could be analyzed by HPLC/electrospray ionization MS. Human leukocyte elastase digestion was used to produce an experimentally nicked hCG. Two nicking sites were observed, between amino acids 42Thr and 43Arg and between 44Val and 45Leu. The former site has not been previously reported for elastase digestion. The structures of the fragments were confirmed by HPLC/MS after removal of the oligosaccharide by direct mass measurement and by mass determination of their proteolytic digests. Without the glycopeptidase treatment, the microheterogeneity of the two N-linked oligosaccharides could be deduced from the spectra of the proteolytic fragments. Nicking with elastase was found to alter the oligosaccharide structures. Nicked beta-subunit samples isolated from the urine of choriocarcinoma patients were also analyzed and the location of the nicking site(s) agreed with that determined by classical techniques. Important differences in the oligosaccharide structures were also observed in these samples, including the presence of triantennary oligosaccharides not found in hCG from healthy subjects. These findings demonstrate the potential of HPLC/MS for characterization of glycoprotein standard preparations.

Analytical advances in detection of performance-enhancing compounds.

The use and abuse of performance-enhancing substances has been an issue in sports since the ancient Greeks. The availability of numerous synthetic steroids and recombinant peptide hormones has made testing an analytical challenge. Recent advances in mass spectrometry have provided an opportunity to decrease detection limits. The Atlanta Olympic Games in 1996 marked the first time every specimen was screened by gas chromatography (GC) coupled to high-resolution mass spectrometry (MS). A further improvement may be seen with GC/MS/MS and quadrupole ion traps. Electrospray HPLC/MS has also been applied to the detection and confirmation of peptide hormones in urine. The ability to detect subtle differences in oligosaccharide structure may provide a way to detect abuse of recombinant glycoproteins. Simply decreasing detection limits is not enough; new technology also allows development of a foundation on which to base interpretation. Application of HPLC/MS/MS has allowed direct measurement of steroid conjugates in urine. The relative importance of sulfate, glucuronide, and other conjugates and metabolites of testosterone and epitestosterone can now be assessed. In the international sports arena, the impact of genetic metabolic disposition must also be considered if we are to provide an equitable system. Further research will establish more-refined criteria for the detection threshold of abused substances.

Evaluation of selected-ion storage ion-trap mass spectrometry for detecting urinary anabolic agents.

Limits of detection are important issues for GC/MS screening for anabolic agents and for confirmation of various drugs of abuse. We compared a quadrupole ion trap (QIT) operated in two different selected-ion storage modes and a quadrupole mass filter (QMF) operated in the selected-ion monitoring mode. Results with the model compound tetrachlorobenzene indicate that, for simultaneous monitoring of more than four ions, the QIT operated in a frequency-modulated selected-ion storage mode has better limits of detection than the QMF. Use of a single-ion storage technique gave results similar to those of the QMF. We also evaluated both QIT selected-ion storage approaches for the limits of detection of the trimethylsilyl derivatives of four anabolic steroid metabolites and the beta-agonist clenbuterol. We found no improvement in detection limits over that of a similar method with selected-ion monitoring and a QMF when four anabolic steroid metabolites and clenbuterol were extracted from a urine matrix. The lack of improvement in the limit of detection resulted from matrix background signals at masses similar to those of the steroids.

Mass spectrometric characterization of the beta-subunit of human chorionic gonadotropin.

A high-performance liquid chromatographic/electrospray mass spectrometric (HPLC/MS) technique is described for the characterization of the beta-subunit of the glycopeptide human chorionic gonadotropin (hCG). The beta-subunit of hCG was dissociated from the alpha-subunit using 0.1% trifluoroacetic acid (TFA) and separated by reversed-phase HPLC using a 0.1% TFA-acetonitrile gradient. Although reductive alkylation with 4-vinylpyridine allowed direct observation of the intact beta-subunit of hCG by HPLC/MS due to the increase in charge, the heterogeneity of the carbohydrate fractions resulted in poor detection limits and extremely complex spectra. After reductive alkylation with either iodoacetate or 4-vinylpyridine, tryptic fragments of either the alpha- or beta-subunit can be observed using reversed-phase HPLC/MS. HPLC/MS data were consistent with the reported primary sequence, although oligosaccharide attachment sites at both 127Ser and 132Ser could not be documented. Microheterogeneity of the carbohydrate moiety on both N-glycosylation sites on the beta-subunit could be readily observed. A larger degree of heterogeneity was observed on 13Asn. Differences were also observed in the oligosaccharide distribution in three commercial preparations of hCG. Detection of the C-terminal portion of the beta-subunit required enzymatic deglycosylation prior to HPLC/MS analysis.

Direct measurement of steroid sulfate and glucuronide conjugates with high-performance liquid chromatography-mass spectrometry.

Direct detection of several steroid glucuronide and sulfate conjugates was achieved with electrospray reversed-phase HPLC-mass spectrometry. Separation of steroid 17-OH or 5-H epimers conjugated with glucuronide or sulfate could be achieved using gradient elution. Testosterone glucuronide, testosterone sulfate, epitestosterone sulfate and epitestosterone glucuronide were chromatographically resolved, although significant variation in solvent strength was observed between methanol and acetonitrile. Positive ionization mode MS and MS-MS spectra were employed to obtain both quantitative and structural information. Some differences were noted with respect to steroid structure and adduct formation, including significant differences in the stability of epimers in the declustering region of the interface. Negative ionization mode, although having lower limits of detection, did not provide useful structural information in either the MS or MS-MS mode. Using a packed capillary column (300 microns I.D.), a detection limit of 25 pg was achieved for epitestosterone glucuronide.

Separation and confirmation of anabolic steroids with quadrupole ion trap tandem mass spectrometry.

Gas chromatography-mass spectrometry (GC-MS) is the method of choice for separation and detection of anabolic steroids in urine. Recently, there have been advances in the areas of gas chromatography columns, tandem mass spectrometry using ion traps, and large volume sample injection that have promise for lowering detection limits and extending the utility of GC-MS for steroid analysis. In this work, a Varian Saturn III GC-MS system has been used in its tandem mass spectrometry mode to detect low picogram levels of model steroids in standard solution and the urine matrix. Application of MS-MS-MS provided structurally informative spectra for 3'-hydroxystanozolol at concentrations of 1 ng/ml. In addition, four polysilphenylene-polydimethylsiloxane capillary columns were examined for background and selectivity. The columns had bleed several-fold lower than conventional polysiloxane columns. The columns also exhibited significant differences in selectivity for structurally similar steroids. Finally, a new temperature-programmed split-splitless injector was used to inject as much as 25 microliters on column. The resulting limits of detection were 5 pg/ml for norandrosterone.

Immunoaffinity trapping of urinary human chorionic gonadotropin and its high-performance liquid chromatographic-mass spectrometric confirmation.

A method has been developed for confirmation of the glycopeptide human chorionic gonadotropin (hCG) in urine. A solid-phase immunoaffinity trapping technique utilizing a monoclonal antibody recognizing both intact hCG and free hCG beta-subunits was developed for the extraction of hCG from urine. Recovery of hCG from a urine matrix was essentially quantitative. The hCG was quantitatively eluted with 6 M guanidine hydrochloride, reductively alkylated with vinylpyridine, and subjected to tryptic digestion. The tryptic digest was analyzed by HPLC-MS. Ions from three tryptic fragments were monitored with selected ion monitoring to provide specific detection of hCG. The signal observed for a concentration of 25 mIU/ml of hCG could be clearly distinguished from background with a signal-to-noise ratio of 12:1.

Facile synthesis of [16,16,17-2H3]-testosterone, -epitestosterone and their glucuronides and sulfates.

The analysis of trace components in complex biological matrices requires the use of reliable internal standards. For the gas chromatography/mass spectrometry (GC/MS) and high performance liquid chromatography/mass spectrometry (HPLC/MS) analyses, the stable isotope-labelled analogues of the analyte molecules are the most appropriate internal standards. In this work high-yield synthetic procedures for stably labelled and isotopically pure [16,16,17-2H3]-testosterone and- epitestosterone are reported. Synthetic methodologies for the glucuronidation and sulfation were established with the commercially available epitestosterone. Structure characterization of 4-androsten-17 alpha-ol-3-one methyl-2',3',4'-tri-O-acetyl-beta-D-glucuronate was made by two-dimensional nuclear magnetic resonance (COSY). Subsequently glucuronidation of [16,16,17-2H3]-testosterone and sulfation of [16,16,17-2H3]-epitestosterone were carried out at greater than 60% yield. However, the yield from the glucuronidation of epitestosterone was not as high. Electrospray mass spectrometry of four conjugates: testosterone sulfate, epitestosterone sulfate, testosterone glucuronide and epitestosterone glucuronide was carried out in the positive ion mode at a number of orifice voltages (50-95 V). Studies of the collisionally induced dissociation at both the interface and in the collision cell (MSMS) confirmed that the glycosidic bond of epitestosterone glucuronide was more labile than that of testosterone glucuronide. Use of the deuterated internal standards is reported to demonstrate the direct analysis of the steroid conjugates by HPLC/MS.

The dose-dependent inhibition of rat renal translation elongation seen after in vivo cyclosporin A is not caused by cyclosporin metabolites.

Cyclosporin A (CsA) given to Sprague-Dawley rats in vivo produced a tissue-specific, dose-dependent inhibition of translation elongation in renal microsomes. CsA at an oral dose of 50 mg/kg/day for 6 days reduced renal microsomal translation by 70.5%. Renal cytoplasm from rats treated in vivo with CsA inhibited translation by 55% when added to renal microsomes isolated from tissues of control animals. In contrast, CsA added to renal microsomes in vitro did not inhibit translation. Renal cytoplasm from CsA-treated rats containing translation inhibitory factor was found by HPLC to contain CsA and CsA metabolites M1 and M17. CsA metabolites M1, M17, M18 and M21 were isolated from human bile and tested in vitro for translation elongation inhibitory activity in renal microsomes. CsA, M18 and M21 did not inhibit translation elongation at concentrations of up to 2500 ng/ml. M17 inhibited translation elongation, but only by 8.4% at the highest concentration tested (2500 ng/ml), a level 20-fold higher than that measured in renal cytoplasm (125 ng/ml). M1 produced a concentration-dependent inhibition of translation elongation, beginning at 500 ng/ml, or approximately 2-fold higher than that found in renal cytoplasm (260 ng/ml). M1 at 2500 ng/ml or approximately 10-fold higher than the concentration measured in renal cytoplasm, inhibited translation elongation by 23.8%, only 1/3 that observed upon addition of renal cytoplasm containing translation inhibitory factor. We conclude from these findings that the dose-dependent inhibition of renal translation elongation following in vivo CsA cannot be explained by the renal formation or uptake of known CsA metabolites.

Hepatic levels of cyclosporine and metabolites in patients after liver transplantation.

Despite the critical role of the liver in the metabolism of cyclosporine, only a few studies have measured hepatic levels (CSAH) in patients receiving the drug, and none has directly assayed hepatic levels of the metabolites. In this study we measured CSAH and its principal metabolites (mono-OH and di-OH CSA) by HPLC/mass spectroscopy in 19 liver biopsy specimens collected from 14 patients who had undergone liver transplantation, in order to determine how they correlated with blood levels (CSAB). The hepatic concentrations were also compared with biochemical and histological parameters of cholestasis. A positive correlation was observed between CSAH and CSAB (r = 0.47), irrespective of the length of time the patients had received the drug (7 to 1662 days) as defined by the relationship: CSAH(ng/g wet weight) = 6.7 x CSAB(ng/ml)+338. Hepatic levels of metabolites exceeded those of the parent compound in 11 biopsy specimens. No correlation was found for CSAH and the metabolites and serum bilirubin or the degree of cholestasis in the liver biopsy specimens. These findings indicate that: (1) CSA is concentrated in liver tissue several-fold over blood; (2) The hepatic concentration can be estimated from the blood concentration even in the presence of cholestasis; (3) Significant levels of CSA metabolites are found in liver tissue, frequently exceeding the concentration of the parent compound.

Recent advances in high-performance liquid chromatography/mass spectrometry and high-performance liquid chromatography/tandem mass spectrometry: detection of cyclosporine and metabolites in kidney and liver tissue.

Recent advances in both ionization methods and mass spectrometers have resulted in powerful new techniques for the study of drug metabolism and disposition. The interest in high-performance liquid chromatography/mass spectrometry (HPLC/MS) is the result of the lack of a sensitive universal detector for HPLC. Although it is not the ideal detector, HPLC/MS has become a reliable technique for xenobiotic analysis. The application of HPLC/MS to studies of the pharmacology and toxicology of molecules of mass < 1,500 daltons is most advantageous in three areas: development of specific methods for trace analysis, detection and characterization of metabolites, and studies of interactions between drug molecules and peptides/proteins. We have used HPLC/MS to study the deposition of cyclosporine and its metabolites in needle biopsy samples from kidney and liver in which sample size is severely limited. The limit of detection in the single-ion monitoring mode was 500 fg (450 amol), which is about a thousandfold lower than UV limits of detection.