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Clinical Chemistry 47: 292-300, 2001;
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(Clinical Chemistry. 2001;47:292-300.)
© 2001 American Association for Clinical Chemistry, Inc.

Articles

Performance Characteristics of a Carbon Isotope Ratio Method for Detecting Doping with Testosterone Based on Urine Diols: Controls and Athletes with Elevated Testosterone/Epitestosterone Ratios

Rodrigo Aguilera1,a, Thomas E. Chapman1, Borislav Starcevic1, Caroline K. Hatton1 and Don H. Catlin1,2

1 UCLA Olympic Analytical Laboratory, Department of Molecular and Medical Pharmacology and
2 Department of Medicine, University of California at Los Angeles, Los Angeles, CA 90025.

3 The word "elevated" is used to refer to a result that is above the administrative cutoff.
a Address correspondence to this author at: UCLA Olympic Analytical Laboratory, Department of Molecular and Medical Pharmacology, University of California at Los Angeles, 2122 Granville Ave., Los Angeles, CA 90025-6106. Fax 310-206-9077; e-mail rodrigoa{at}ucla.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Background: Carbon isotope ratio methods are used in doping control to determine whether urinary steroids are endogenous or pharmaceutical.

Methods: Gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS) was used to determine the {delta}13C values for 5{beta}-androstane-3{alpha},17{beta}-diyl diacetate (5{beta}A), 5{alpha}-androstane-3{alpha},17{beta}-diyl diacetate (5{alpha}A), and 5{beta}-pregnane-3{alpha},20{alpha}-diyl diacetate (5{beta}P) in a control group of 73 healthy males and 6 athletes with testosterone/epitestosterone ratios (T/E) >6.

Results: The within-assay precision SDs for 5{beta}A, 5{alpha}A, and 5{beta}P were ± 0.27{per thousand}, ± 0.38{per thousand}, and ± 0.28{per thousand}, respectively. The between-assay precision SDs ranged from ± 0.40{per thousand} to ± 0.52{per thousand}. The system suitability and batch acceptance scheme is based on SDs. For the control group, the mean {delta}13C (SD) values were -25.69{per thousand} (± 0.92{per thousand}), -26.35{per thousand} (± 0.68{per thousand}), and -24.26{per thousand} (± 0.70{per thousand}), for 5{beta}A, 5{alpha}A, and 5{beta}P, respectively. 5{beta}P was greater than 5{beta}A and 5{alpha}A (P <0.01), and 5{beta}A was greater than 5{alpha}A (P <0.01). The means - 3 SD were -28.46{per thousand}, -28.39{per thousand}, and -26.37{per thousand} for 5{beta}A, 5{alpha}A, and 5{beta}P, respectively. The maximum difference between 5{beta}P and 5{beta}A was 3.2{per thousand}, and the maximum 5{beta}A/5{beta}P was 1.13. Three athletes with chronically elevated T/Es had {delta}13C values consistent with testosterone administration and three did not.

Conclusions: This GC-C-IRMS assay of urine diols has low within- and between-assay SDs; therefore, analysis of one urine sample suffices for doping control. The means, SDs, ±3 SDs, and ranges of {delta}13C values in a control group are established. In comparison, testosterone users have low 5{beta}A and 5{alpha}A, large differences between 5{beta}A or 5{alpha}A and 5{beta}P, and high 5{beta}A/5{beta}P and 5{alpha}A/5{beta}P ratios.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
A critical issue in doping control is establishing the origin of testosterone and other steroids typically found in human urine. If the origin can be shown to be pharmaceutical, as opposed to endogenous, a doping offense has occurred. Androgens for pharmaceutical use are not synthesized de novo; they are obtained by semisynthesis from starting materials such as diosgenin and stigmasterol, which are derived from plants (1). Furthermore, these compounds have less 13C than their endogenous homologs (2)(3); therefore, urinary steroids with a low 13C/12C ratio are likely to have originated from pharmaceutical sources. This thesis has led to the use of gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS)1 in doping control because the method determines the {delta}13C value of steroids extracted from urine, based on the equation:

where 13C/12C refers to the isotope ratio in the sample or an international standard (a reference gas calibrated relative to Pee Dee Belemnite). Accordingly, for several years, GC-C-IRMS has been applied to detect the use of exogenous steroids such as testosterone (3)(4), dihydrotestosterone (5)(6), and dehydroepiandrosterone (6)(7) by athletes.

Testosterone administration increases the ratio of urine testosterone to epitestosterone (T/E). This finding led to the use of T/E as a screening test for testosterone administration (8). If the ratio exceeds 6, laboratories accredited by the International Olympic Committee report the result to the sport authority (9). It is known, however, that factors other than testosterone administration may also increase the T/E ratios (10)(11)(12). Furthermore, testosterone administration does not always lead to an elevated4 T/E ratio (3)(13). These factors complicate the interpretation of elevated T/E ratios and limit the significance of T/E ratios <6. Determining the T/E time profile of an individual is useful, but it requires consideration of the results of past tests or the collection of additional samples. Our previous work demonstrated that the measurement of {delta}13C values of testosterone and its metabolites can detect testosterone use (4)(14)(15). Others have reported that {delta}13C values may be abnormally low even in samples with T/E ratios <6 (3).

With the introduction of new methods, it is essential to fully characterize the assay and to establish the values in the control group. This is particularly true with {delta}13C measurements when applied to drug control because the method is inherently difficult, the analytical results are likely to be litigated, and it has been suggested (3)(5) that diet or ethnicity may influence the 13C/12C ratio of urine steroids. Accordingly, herein we characterize the {delta}13C measurements of the diacetates of 5{beta}- and 5{alpha}-androstane-3{alpha},17{beta}-diol (5{beta}A and 5{alpha}A, respectively), and 5{beta}-pregnane-3{alpha},20{alpha}-diol (5{beta}P) with respect to precision, linearity, system suitability, and batch acceptance. In addition, we determined {delta}13C values for these steroids in a control group of 73 healthy male subjects from four different ethnic groups. Finally, data gathered from the control group were used to interpret {delta}13C values obtained on athletes with elevated T/E ratios and to discuss diagnostic criteria for doping.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
steroid calibration solution
A steroid calibration solution was prepared that contained 25 mg/L 5{beta}-androstane-3{alpha},17{beta}-diol, 5{alpha}-androstane-3{alpha},17{beta}-diol, and 5{beta}-pregnane-3{alpha},20{alpha}-diol, all as diacetates (Steraloids, Inc.).

control group
The control group consisted of 74 male medical students (age range, 19–29 years) from the University of California at Los Angeles. Body weights were 45–110 kg. The students had the option to give a medical and drug history and to describe their ethnicity. No student declared taking steroids or reported any chronic disease. The ethnicities were "Asian" (n = 16), "African American" (n = 9), "Caucasian" (n = 25), "Hispanic" (n = 13), "other" (n = 7), and "unknown" (n = 4). The students collected 24-h urine samples, and 50-mL aliquots were stored at 4 °C. The study was conducted under the guidance of the Human Subject Protection Committee of the University of California at Los Angeles.

athletes with elevated T/E RATIOS
Of the large number of anonymous urine samples that our laboratory analyzes for sport drug control programs in the US, some have a urine T/E ratio >6. In such cases, the sport organization typically collects additional samples to track the urine T/E over weeks or months. We selected some of these urine samples for GC-C-IRMS analysis based on the availability of at least two samples per person, sufficient volume to perform the analysis, and permission from the sport organization.

quality-control urine samples
Quality-control urines (QC-H and QC-L) were obtained from two subjects known not to be using testosterone or any substance likely to alter the {delta}13C values of 5{beta}-androstane-3{alpha},17{beta}-diol, 5{alpha}-androstane-3{alpha},17{beta}-diol, and 5{beta}-pregnane-3{alpha},20{alpha}-diol (measured as diacetates). Each subject donated one 8-h urine, which was aliquoted into 10-mL cryogenic tubes and stored at -20 °C until analysis.

urinary steroid concentrations and sample preparation for GC-C-IRMS
The T/E ratio was estimated by GC-MS as described previously (16). The concentrations of 5{beta}- and 5{alpha}-androstane-3{alpha},17{beta}-diol were estimated from a QC sample prepared by adding to steroid-free urine 5{beta}- and 5{alpha}-androstane-3{alpha},17{beta}-diol. [16,16,17-2H]-testosterone (CDN Isotopes) was used as an internal standard. The 5{beta}-pregnane-3{alpha},20{alpha}-diol was not quantified. The sample preparation methods for GC-C-IRMS analysis and the instrument conditions have been described (15). The volume of urine extracted for GC-C-IRMS was 10 mL.

gc-c-irms determination of 5{alpha}A, 5{beta}A, AND5{beta}P
The GC-C-IRMS analyses were conducted on a Finnigan Delta Plus Isotope Ratio Mass Spectrometer. The IRMS was connected to a Hewlett Packard Model 6890 gas chromatograph via a Finnigan Combustion III interface. The GC was equipped with an HP-50+ capillary column [30 m x 0.25 mm (i.d.); 0.15 µm film thickness] and a Finnigan A200S autosampler. The combustion oven was oxidized by back-flushing with oxygen for 1 h every 30 samples. The oxidation reactor in the combustion oven was replaced every 500 samples. The injection volume was 1 µL. The recoveries for 5{beta}- and 5{alpha}-androstane-3{alpha},17{beta}-diol were 85% and 86%, respectively.

GC-C-IRMS RESPONSE LINEARITY
The linearity of the GC-C-IRMS response was determined by preparing seven solutions containing all three analytes at 2.5, 5, 10, 25, 50, 100, and 150 mg/L, respectively. One microliter of each solution was injected three times on 1 day, and the three {delta}13C values for each compound were averaged.

precision studies
Instrument precision for 5{beta}P, 5{beta}A, and 5{alpha}A was determined by extracting one aliquot of each QC urine and injecting each extract four times in succession. The within-day precision was determined by extracting 20 aliquots of QC-H in the same batch and injecting each one once. The between-assay precision was determined by extracting one aliquot of QC-H and QC-L per day for 16 days, spanning 15 months, and injecting each aliquot once.

daily system suitability test
To establish a tolerance range, the steroid calibrator was injected five times on each of 5 different days over 2 weeks (n = 25), and the mean {delta}13C values and SDs were calculated for each steroid. Each day, before urine sample analysis, the system suitability was assessed by injecting the calibrator three times and calculating the mean {delta}13C values. The system was considered suitable for batch analyses if the mean {delta}13C values of at least two of the three steroids were within ± 2 SD of the means described above. If the system suitability test failed, maintenance was performed on the gas chromatograph injection port, the GC column and/or the oxidation reactor were replaced, and the calibrator was re-injected. Data acceptance criteria included absence of peak tailing, retention times of the steroids within ± 1% of established values, and a minimum 0.8 V response at m/z 44 for each steroid.

criteria for batch acceptance
To establish a tolerance range for the QC urines for batch data acceptance, two aliquots of each QC urine were extracted and each injected twice per day for 5 days spanning 2 weeks (n = 20). ANOVA was performed using the factors day (5), injection (2), duplicate (2), and QC (2). The mean {delta}13C values and SDs were calculated for each of the three steroids. For batch analysis, each unknown urine sample was extracted and injected once. One aliquot of the two QC urines and one aliquot of the steroid calibrator were analyzed with each batch of samples. These three controls were injected at the beginning, in the middle, and at the end of the batch. The batch was accepted if at least six of the nine means for the three steroids were within ± 2 SD of the previously established means. The acceptance criteria were as noted above except that an instrument response of 0.3 V at m/z 44 was accepted.

statistical analyses
The Smirnov-Grubbs method was used to test the control group for outliers; otherwise, all statistical tests utilized statistical software (SAS). The linearity of the GC-C-IRMS response was assessed by least-squares linear regression. The response was considered linear if the slope was zero at P = 0.01. The method of Koch and Peters (17) was used to determine the SDs of duplicates. The normality of the distributions of 5{alpha}A, 5{beta}A, and 5{beta}P, differences, and ratios was determined by the Anderson-Darling test. The values for 5{alpha}A, 5{beta}A, and 5{beta}P were correlated with Pearson’s correlation coefficient, r. Two-sided paired t-tests were used to compare the means of 5{alpha}A, 5{beta}A, and 5{beta}P in the control group. The general linear model procedure was used to assess differences between the mean {delta}13C values of the ethnic groups, and the power of the analysis was assessed by one-way ANOVA.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
linearity of GC-C-IRMS RESPONSE
The IRMS response was linear for 5{beta}- and 5{alpha}-androstane-3{alpha},17{beta}-diyl diacetate from 2.5 to 150 mg/L (Fig. 1 ). However, for 5{beta}-pregnane-3{alpha},20{alpha}-diyl diacetate, the response was linear only between 5 and 150 mg/L. The concentrations of 5{beta}- and 5{alpha}-androstane-3{alpha},17{beta}-diol in the control group were 23–430 and 25–204 µg/L, respectively. Therefore, assuming 85% recovery, the amounts of 5{beta}- and 5{alpha}-androstane-3{alpha},17{beta}-diol extracted from the 10-mL urine and reconstituted in 25 µL of cyclohexane were analyzed in the linear range of the IRMS.



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  		Figure 1. Relationships between concentration of 5{beta}-androstane-3{alpha},17{beta}-diyl diacetate (x), 5{alpha}-androstane-3{alpha},17{beta}-diyl diacetate ({circ}), 5{beta}-pregnane-3{alpha},20{alpha}-diyl diacetate ({square}; right axis) and {delta}13C values.

precision of the GC-C-IRMS INSTRUMENT AND ASSAY
Instrument precision (SD) for 5{beta}A in QC-H and QC-L was 0.32{per thousand} and 0.41{per thousand}, respectively. For 5{alpha}A, it was 0.08{per thousand} and 0.59{per thousand}, respectively, and for 5{beta}P, it was 0.27{per thousand} and 0.16{per thousand}, respectively. The descriptive statistics for the within-assay experiment on QC-H are shown in Table 1 . The SDs were 0.27{per thousand}, 0.38{per thousand}, and 0.28{per thousand} for 5{beta}A, 5{alpha}A, and 5{beta}P, respectively, and the CVs were <=1.4%. The range of values was 0.9{per thousand} for 5{beta}A, 1.2{per thousand} for 5{beta}P, and 1.8{per thousand} for 5{alpha}A. The between-assay SDs for QC-H were 0.40{per thousand}, 0.42{per thousand}, and 0.44{per thousand} for 5{beta}A, 5{alpha}A, and 5{beta}P, respectively, and the CVs were <=1.8% (Table 2 ). For QC-L, the values were slightly higher. The mean {delta}13C values for the three steroids in QC-L were significantly lower than the means for QC-H.


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  		Table 1. Within-assay precision for 5{beta}A, 5{alpha}A, and 5{beta}P.


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  		Table 2. Between-assay precision for 5{beta}A, 5{alpha}A, and 5{beta}P.

system suitability and batch acceptance
In the system suitability test, the SDs of 5{beta}A, 5{alpha}A, and 5{beta}P were 0.31{per thousand}, 0.59{per thousand}, and 0.54{per thousand}, respectively. The tolerance range for the batch acceptance data provided SDs for 5{beta}A, 5{alpha}A, and 5{beta}P of 0.68{per thousand}, 0.67{per thousand}, and 0.65{per thousand} for QC-H, and 0.66{per thousand}, 0.56{per thousand}, and 0.39{per thousand} for QC-L, respectively. Because these data consisted of duplicate analyses, we calculated the SDs of duplicates and performed an ANOVA. The SDs of duplicates were 0.24{per thousand}, 0.32{per thousand}, and 0.37{per thousand} for 5{beta}A, 5{alpha}A, and 5{beta}P, respectively. These values were approximately one-half the SDs obtained for the factor injection. ANOVA revealed that QC-H and QC-L were different, and there was no difference for the factors injection, duplicate, or day.

control subjects: descriptive statistics for the concentrations and {delta}13C OF URINARY STEROIDS
The geometric means for urine testosterone, epitestosterone, and 5{beta}- and 5{alpha}-androstane-3{alpha},17{beta}-diol concentrations were 20, 29, 98, and 58 µg/L, respectively. The geometric mean for urine T/E was 0.7 (arithmetic range, 0.2–5.3). The concentration of urine 5{beta}-pregnane-3{alpha},20{alpha}-diol was not determined. Table 3 and Fig. 2 show the descriptive statistics and the histograms for 5{beta}A, 5{alpha}A, and 5{beta}P. The outlier test was applied, and no data points were excluded. The distributions of 5{alpha}A and 5{beta}P were gaussian (A2 = 0.28, P >0.25; and A2 = 0.21, P >0.25, respectively). The distribution of 5{beta}A was gaussian at P = 0.01, but not gaussian at P = 0.05, A2 = 0.85.


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  		Table 3. Control group of 73 subjects: Descriptive statistics for {delta}13C, differences between the means, and ratios of the means.



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  		Figure 2. Histograms of {delta}13C values for 5{beta}A (top), 5{alpha}A (middle), and 5{beta}P (bottom) for a control group of 73 healthy males.

There were significant (P <0.0001) correlations between 5{beta}A and 5{alpha}A (r = 0.70), between 5{beta}A and 5{beta}P (r = 0.67), and between 5{beta}P and 5{alpha}A (r = 0.58). The CVs of the {delta}13C values for the diacetates were 2.6–3.6%. The means - 3 SD for 5{beta}A, 5{alpha}A, and 5{beta}P were -28.46{per thousand}, -28.39{per thousand}, and -26.37{per thousand}, respectively. The mean 5{alpha}A was lower (-26.35{per thousand}) than the mean 5{beta}A (-25.69{per thousand}; P <0.001). The Wilcoxon nonparametric two-sample test confirmed that 5{alpha}A was lower than 5{beta}A. The means for 5{beta}A and 5{alpha}A were lower than the mean for 5{beta}P (both P <0.001).

Table 3Up also summarizes the statistics of the differences between the means of 5{beta}P and 5{beta}A (5{beta}P - 5{beta}A) and 5{beta}P and 5{alpha}A (5{beta}P - 5{alpha}A), and the ratios 5{beta}A/5{beta}P and 5{alpha}A/5{beta}P. The distributions of 5{beta}P - 5{beta}A and 5{beta}P - 5{alpha}A were gaussian (A2 = 0.23 and 0.19, respectively). The 5{beta}P - 5{beta}A differences ranged from -0.08{per thousand} to 3.17{per thousand}, and the mean + 3 SD was 3.47{per thousand}. The 5{beta}P - 5{alpha}A differences ranged from 0.16{per thousand} to 3.72{per thousand}, and the mean + 3 SD was 3.99{per thousand}. The distributions of 5{beta}A/5{beta}P and 5{alpha}A/5{beta}P were gaussian (A2 = 0.19 and 0.21, respectively). The means of 5{beta}A/5{beta}P and 5{alpha}A/5{beta}P were 1.06 and 1.09, respectively, and their corresponding means + 3 SD were 1.14 and 1.17. There were no differences between ethnic groups for the means of 5{alpha}A, 5{beta}A, or 5{beta}P (Table 4 ).


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  		Table 4. Control group: Descriptive statistics for 5{beta}A, 5{alpha}A, and 5{beta}P by ethnicity.

{delta}13C VALUES IN ATHLETES WITH ELEVATEDT/E RATIOS
The urine concentrations of testosterone and epitestosterone, the T/E ratios, and the {delta}13C values for the six male athletes with urine T/E ratios >6 are summarized in Table 5 . Of the six, athletes 1 and 2 were known to be taking testosterone for medical reasons. According to the sport organizations ordering the tests, athletes 3–6 denied taking any substance known to influence the T/E ratio. The number of samples per athlete ranged from three to eight. The T/E ratio of all samples was >6 for four of the six athletes. Athlete 4 had one T/E ratio of 5.0, and athlete 5 had one T/E ratio of 5.1; otherwise, all T/E ratios were >6.


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  		Table 5. Concentrations of testosterone and epitestosterone; T/E ratios; {delta}13C values for 5{beta}A, 5{alpha}A, and 5{beta}P; difference scores between 5{beta}P and testosterone metabolites (5{beta}A and 5{alpha}A); and ratios of metabolites to 5{beta}P (5{beta}A/5{beta}P and 5{alpha}A/5{beta}P) for multiple urine samples obtained from two athletes permitted to take testosterone for medical purposes and four athletes whose drug-taking behavior was unknown.

Table 5Up reveals that for athletes 1–3, all 5{beta}A and 5{alpha}A values were lower than the mean - 3 SD values of the controls in Table 3Up , whereas the 5{beta}P values were all close to the mean 5{beta}P value of the controls (-24.26{per thousand}). The cells in Table 5Up that are outside the ± 3 SD values of Table 4Up are outlined or shaded to facilitate comparisons. The combination of 5{beta}P values that are near the mean and low 5{beta}A and 5{alpha}A values produce large differences in 5{beta}P - 5{beta}A and 5{beta}P - 5{alpha}A, and large 5{beta}A/5{beta}P and 5{alpha}A/5{beta}P ratios. For athletes 1–3, all differences and ratios were outside the + 3 SD range. The means of athletes 1–3 and 4–6 and their z-scores are presented at the bottom of Table 5Up . Another aspect of athletes 1–3 was that all 5{alpha}A values were lower than the 5{beta}A values for the same urine: the means were -29.84{per thousand} for 5{beta}A and -31.83{per thousand} for 5{alpha}A (P = 0.04). The means of 5{beta}P - 5{beta}A (5.9{per thousand}; z-score = 6.5) and of 5{beta}P - 5{alpha}A (7.9{per thousand}; z-score = 9.1) were substantially greater than the + 3 SD values determined for the control group. Similarly, the mean 5{beta}A/5{beta}P ratio was 1.25 (z-score = 6.6), and the mean 5{alpha}A/5{beta}P ratio was 1.33 (z-score = 8.9).

To compare the testosterone concentrations in Table 5Up to the general population of athletes that we routinely test, we determined the log mean (3.22 µg/L) and log SD (1.19) of the distribution of the latest 11 938 male urine samples tested in our laboratory. The urine testosterone concentrations in subjects 1 and 2, the two permitted testosterone users, ranged from a low of 2.3 µg/L on the first urine ever collected to 186 µg/L (z-score = 1.5, in log units). For subject 3, the urine testosterone concentrations (mean, 613 µg/L; z-score = 2.6) and T/E ratios (mean = 64) were very high. In addition, like athletes 1 and 2, athlete 3 was characterized by low 5{beta}A and 5{alpha}A values, and large differences and ratios outside the ± 3 SD range.

In contrast to athletes 1–3, for athletes 4–6, the values for 5{beta}A, 5{alpha}A, and 5{beta}P and their differences and ratios were similar to the averages for the control group, and none of the values were remarkable except for one 5{beta}P - 5{alpha}A value of -0.4{per thousand} and one 5{alpha}A/5{beta}P ratio of 0.99, which were slightly lower than the mean - 3 SD. For subjects 4–6, 5{beta}P - 5{beta}A and 5{beta}P - 5{alpha}A were less than 2.1{per thousand} and 3.7{per thousand}, respectively, i.e., within ± 3 SD of the control group values. Similarly, all but one of the ratios to 5{beta}P were inside the ± 3 SD range. In addition, the urine testosterone concentrations for subjects 5 and 6 were within + 1.5 SD of the log mean for 11 938 athletes. Thus, compared with the control group, athletes 5 and 6 were characterized by elevated T/E ratios; whereas the values for 5{beta}A, 5{alpha}A, and 5{beta}P, their differences, and their ratios for athletes 5 and 6 were, with two minor exceptions, within ± 3 SD of the means of the controls. Athlete 4 had a relatively high mean urine testosterone (234 µg/L; z-score = 1.9).

The use of testosterone or testosterone precursors is indicated by low 5{alpha}A and 5{beta}A values, large 5{beta}P - 5{alpha}A and 5{beta}P - 5{beta}A differences, and large 5{alpha}A/5{beta}P and 5{beta}A/5{beta}P ratios. Of these six variables (two {delta}13C values, two differences, and two ratios), for athletes 1–3, the ratio 5{beta}A/5{beta}P (z-score = 8.9) and the difference 5{beta}P - 5{beta}A (z-score = 9.1) were the most sensitive indicators of testosterone administration as judged by z-scores. The value for 5{alpha}A was also a good indicator (z-score = 8.0). For differences and ratios that utilized 5{beta}A (5{beta}P - 5{beta}A and 5{beta}A/5{beta}P), the z-scores were 6.5 and 4.5, respectively. The least discriminating variable was 5{beta}A (z-score = 4.5). In contrast, for athletes 4–6 in Table 5Up , the z-scores were all low (range, -0.02 to 0.9).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
characteristics of the GC-C-IRMS ASSAY
GC-C-IRMS measurement of urinary steroids has become a potent analytical method in doping control, although scrupulous attention to detail is necessary. Successful implementation of the method requires strict adherence to QC, system suitability, and batch acceptance criteria such as those described herein. In the present study, after the tolerance ranges for the system and batches were established, the system suitability test never failed and the batch acceptance criteria were always met over the 14 months of the study.

In the instrument precision study, the mean SDs were less than ± 0.33{per thousand}; thus, when the variability attributable to extraction and derivatization was eliminated, all of the SDs were low. In the within-assay study, the SDs were also low: ± 0.27{per thousand}, ± 0.38{per thousand}, and ± 0.28{per thousand} for 5{beta}A, 5{alpha}A, and 5{beta}P, respectively. The between-assay SDs ranged from ± 0.40{per thousand} to ± 0.52{per thousand}, which is slightly higher than the within-assay values, but they indicate the excellent repeatability and reproducibility of the entire assay.

We did not find comparable precision studies for urinary steroids in the literature; however, Shackleton et al. (3) provided evidence that {delta}13C values for diols are reproducible based on duplicate analysis of 13 samples from one subject. We used the data in Table 1Up from Shackleton et al. (5) to calculate the SD of duplicates and found values of ± 0.36{per thousand}, ± 0.15{per thousand}, and ± 0.28{per thousand} for 5{beta}A, 5{alpha}A, and 5{beta}P, respectively. These data show that the SD for duplicates in another laboratory was similar to the SD that we obtained for duplicates in our batch acceptance studies. The concentrations of 5{beta}-androstane-3{alpha},17{beta}-diol and 5{alpha}-androstane-3{alpha},17{beta}-diol in the control urines were 23–430 µg/L; thus, given the 85% recovery of steroids from the 10-mL sample volume used in the analysis, the concentrations of 5{beta}-androstane-3{alpha},17{beta}-diol and 5{alpha}-androstane-3{alpha},17{beta}-diol in the 25 µL of the injection solution were 8–146 mg/L, and the samples were analyzed in the linear range of the system (Fig. 1Up ).

healthy control male subjects
The {delta}13C values in Table 3Up represent the largest group of healthy males living in the US measured to date. The ranges for the T/E ratios (0.1–3.6) and urine testosterone concentrations (2–116 µg/L) are consistent with the fact that no student declared taking testosterone or a steroid supplement. In addition, none of the students declared any endocrine disorders. Of the 74 urines, we were able to measure 5{beta}A, 5{alpha}A, and 5{beta}P values for all but 1. Sixty-eight of the samples provided satisfactory data on the first extraction of 10 mL of urine. For the other six, three provided an adequate signal on repeat analysis. Two samples required a 20-mL sample volume to obtain an adequate signal. In one sample with low diol concentrations (<25 µg/L), the voltage criteria were not met even with a 20-mL sample. Thus, our signal criteria were met on the first analysis with 10 mL of urine in 94% of the 74 cases, and all but 1 of the remaining samples provided satisfactory data on repeat analysis with 10 or 20 mL of urine. One person with one instrument can perform approximately three assays per week with a batch size of 20 samples plus 5 controls. The software to process the samples is somewhat cumbersome; thus, ~2 days per week are needed for data processing.

The mean 5{alpha}A and 5{beta}A values for our control group were -26.35{per thousand} and -25.69{per thousand}, respectively. The mean {delta} values - 3 SD for 5{beta}A and 5{alpha}A were approximately -28.4{per thousand}, which is similar to the {delta} values reported for synthetic testosterone (6)(7); however, this comparison should not be made. This is because the diols measured herein were acetylated, which makes the {delta} value more negative by approximately two units, whereas the synthetic testosterone (6)(7) was underivatized.

There are no comparable control group values in the literature; however, the baseline values in Fig. 6 of Shackleton et al. (3), which was based on analysis of 20 samples from individuals with mixed nationalities, are very similar to ours. Their lowest value for a diol was -28.2{per thousand}, whereas our lowest diol value was -27.89{per thousand}. Ueki and Okano (7) also measured 5{alpha}A and 5{beta}A; however, their data are not directly comparable to ours because they used a correction factor to convert all measured {delta}13C values of the acetylated compounds to give values for underivatized steroids, which we could not estimate. The correction factor would be the same for 5{alpha}A and 5{beta}A; thus, their difference of 2.6{per thousand} between the means of 5{alpha}A (-16.4{per thousand}) and 5{beta}A (-19.0{per thousand}) for 10 Japanese male samples can be compared to our difference of 0.66{per thousand} (Table 3Up ). In addition, the value for 5{alpha}A was lower than 5{beta}A in the study by Ueki and Okano (7), which is the opposite of what we found. We can also compare the range of values in our control subjects to the range reported by Ueki and Okano (7) in their Table 2Up for 20 healthy Japanese males. Our ranges for 5{alpha}A and 5{beta}A were 2.9{per thousand} and 3.9 {per thousand} (Table 4Up ), whereas they found ranges of 10.8{per thousand} and 7.2{per thousand}, respectively. The striking difference between the ranges found in the two laboratories could reflect differences in method, calibration, ethnicity, or diet. The explanation that Japanese subjects have different steroid biochemistry and metabolism was deemed unlikely because equally large ranges of 11.0{per thousand} (5{beta}A) and 14.1{per thousand} (5{alpha}A) were reported for urine samples from >350 Olympic athletes, and Olympic athletes are multiracial. In addition, Shackleton et al. (3) found similar values for Chinese and other nationalities, and we found no difference among four ethnic groups in our control group. That dietary factors could explain the differences was also considered, but diet is an unlikely explanation for the large differences in Olympic athletes because it is likely to take several weeks on a local diet before a measurable change in urinary steroid {delta}13C develops. Therefore, it is likely that our analytical method differs from that of Ueki and Okano (7).

The diversity of the control group was an advantage in that four major ethnic groups were present; however, it was a disadvantage because the n for each ethnic group was relatively small and therefore the statistical power of the GLM procedure was relatively low. Thus, we could not attribute any differences to ethnicity. Similarly, Shackleton et al. (5), who compared {delta}13C values for 20 individuals from 12 nationalities, did not attribute any of the differences to ethnicity or diet.

In the present study, the mean {delta}13C values for 5{alpha}A (-26.35{per thousand}) and 5{beta}A (-25.69{per thousand}) in the control group were different (P <0.001), whereas in our previous study with only 10 subjects, we did not observe such a difference (15). In addition, it appears from the data in Table 5Up of the present study and from Table 1Up in our previous study (15) that testosterone administration may decrease 5{alpha}A more than it decreases 5{beta}A. This difference occurs despite the fact that the percentage of conversion of a tracer dose of [14C]testosterone to 5{beta}-androstane-3{alpha},17{beta}-diol (3.2%) is greater than the percentage of conversion to 5{alpha}-androstane-3{alpha},17{beta}-diol (1.2%) (18). Urinary 5{alpha}-androstane-3{alpha},17{beta}-diol is known to arise from both hepatic and peripheral metabolism, whereas urinary 5{beta}-androstane-3{alpha},17{beta}-diol is considered to arise only from hepatic metabolism (19). After pharmaceutical doses of testosterone, 5{alpha}A may decrease more than 5{beta}A because peripheral paths to 5{alpha}-androstane-3{alpha},17{beta}-diol are favored. However, there is also a path from dehydroepiandrosterone sulfate to 5{beta}-androstane-3{alpha},17{beta}-diol that does not pass through testosterone (20). Utilization of this path could explain why 5{beta}A is higher than 5{alpha}A. Finally, the observed differences may be an analytical artifact. Similarly, the higher mean {delta}13C value for 5{beta}P (-24.26{per thousand}) might be related to metabolism or artifacts.

athletes with elevated T/E RATIOS
To clarify the status of an athlete with an elevated T/E ratio, the International Olympic Committee recommends that additional samples be obtained (9). The relevant sport authority typically charts the course of the urine T/E values over time and interprets the data pattern to decide whether to penalize the athlete. Unless there is a record of three or more past samples, this involves collecting additional samples. There are no guidelines stating how many samples are needed, how often they should be collected, and for how long, and there are no published criteria for determining whether the elevated T/E ratio is natural or is attributable to taking an exogenous substance. The data in Tables 1–3Up Up Up and 5 support the argument that by measuring {delta}13C, the decision to penalize the athlete can be made on a single sample. The six athletes described in Table 5Up were undergoing such testing, although at the time of the analysis the laboratory was not aware that subjects 1 and 2 were permitted testosterone users. No additional information was available on the status of athletes 3–6; however, inspection of the data for subject 3 indicates that it is reasonable to classify him as a testosterone or testosterone precursor user.

The six subjects in Table 5Up with increased T/E ratios may be characterized by the concentration of testosterone in their urines and by their 5{beta}A and 5{alpha}A values and the associated differences and ratios to 5{beta}P. A classification based on {delta}13C values, differences, and ratios revealed that athletes 1–3 were outside the control range ± 3 SD except for 5{beta}P. For athletes 1 and 2, this fits with our understanding of the basis of the carbon isotope ratio method, specifically, that after testosterone administration only the {delta}13C values of testosterone and testosterone metabolites will decrease and that those of other urinary steroids will not. Because 5{beta}-pregnane-3{alpha},20{alpha}-diol is not a metabolite of testosterone, its {delta}13C values are not expected to change with testosterone administration (3)(4)(15).

Applying the means ± 3 SD criteria to the {delta}13C data for athlete 3 requires classifying him as a testosterone or testosterone precursor user. This possibility is further supported by the very high urine T/E ratios and testosterone concentrations. The z-scores for his urine testosterone concentrations range between 2.2 and 2.9, which is a further indication of testosterone or testosterone precursor administration. Once the GC-C-IRMS method is validated to the point of full acceptance by the sport and legal community, we expect that athlete 3 could be classified as a user on one urine showing the pattern of a high T/E ratio, high testosterone concentration, and {delta}13C values less than - 3 SD.

Athletes with naturally elevated urine T/E ratios would be expected to have persistently elevated T/E ratios, unremarkable urine testosterone concentrations, and values for 5{beta}A and 5{alpha}A that are within ± 3 SD of the mean of the control group. The data for athletes 5 and 6 fit this pattern. Athlete 4 also fits, but some of his urine testosterone concentrations were relatively high. The z-score for the values 383 and 394 µg/L was ~2.3, and all but one of the others were >1.3. Until additional data are available, athlete 4 has been classified as having a naturally elevated T/E ratio, although we cannot completely exclude the possibility that athlete 4 is a testosterone or testosterone precursor user with normal {delta}13C values.

The data on mean z-scores in Table 5Up reveal that both the differences and ratios are better indices of testosterone use than the absolute {delta}13C values, 5{beta}A and 5{alpha}A. This is not unexpected given that 5{beta}P correlates with both 5{beta}A and 5{alpha}A. Furthermore, the difference 5{beta}P - 5{beta}A and the ratio 5{beta}A/5{beta}P are more robust indicators of testosterone use than the corresponding differences 5{beta}P - 5{alpha}A and the ratio 5{alpha}A/5{beta}P. This follows from the finding (Tables 3Up and 5Up ) that the {delta}13C values for 5{alpha}A were lower than the values for 5{beta}A.

In conclusion, a fundamental issue in doping control is whether GC-C-IRMS techniques will resolve ambiguities in the interpretation of the T/E test. The methods presented herein provide compelling evidence that the test has excellent precision and that when it is coupled with strict system suitability and batch acceptance criteria, it can be used in a routine fashion to obtain valid {delta}13C data. By comparing the {delta}13C values of a control group to data obtained from athletes, it is possible to make informed decisions regarding the origin of urinary 5{beta}-androstane-3{alpha},17{beta}-diol, 5{alpha}-androstane-3{alpha},17{beta}-diol, and 5{beta}-pregnane-3{alpha},20{alpha}-diol.


   Acknowledgments
 
We are grateful for financial support from the National Collegiate Athletic Association, the National Football League, and the United States Olympic Committee. We thank K. Schramm for technical assistance.


   Footnotes
 
1 Nonstandard abbreviations: GC-C-IRMS, gas chromatography-combustion-isotope ratio mass spectrometry; T/E, testosterone/epitestosterone ratio; 5{beta}A, {delta}13C value for 5{beta}-androstane-3{alpha},17{beta}-diyl diacetate; 5{alpha}A, {delta}13C value for 5{alpha}-androstane-3{alpha},17{beta}-diyl diacetate; 5{beta}P, {delta}13C value for 5{beta}-pregnane-3{alpha},20{alpha}-diyl diacetate; and QC, quality control.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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