Isotopically Labeled Steroid Standards

Introduction

The use of anabolic steroids, such as testosterone, to enhance physical and athletic performance continues to plague competitive sports.  Testing is routinely performed on Olympic athletes and Tour de France participants.  Reports of baseball stars admitting to using performance-enhancing substances are also becoming more common. Authorities such as the WADA (World Anti-Doping Agency) monitor and test athletes to ensure that they are not using banned substances.¹ A variety of methods exist for testing for anabolic steroids or anabolic-androgenic steroids (AAS). However, they have limitations due to cross-reactivity with structurally similar compounds which interfere with a proper diagnosis. Specificity, sensitivity and precision concerns on several assays have driven the authorities involved to establish an effective clinical standardization process.² Several GC-MS and LC-MS/MS assays have been tested and developed for the determination of bioactive compounds with analytical sensitivity and specificity and these methods frequently use stable isotope labeled standards.

Section Overview

• Labeled Internal Standards
• Anabolic - Androgenic Steroids

Labeled Internal Standards

Analyses of biological samples such as urine or serum are often performed due to the ease of obtaining and storing a sample. Since the consequences for a positive result are so severe, laboratories have rigorous protocols and procedures to ensure the accuracy of the test methods and to limit the potential for false positive results. Many methods use an internal standard for quantitative analysis. Internal standards provide several advantages including minimizing or masking the variations resulting from sample preparation and extractions.³ Using a stable isotope labeled internal standard, because of its similar physico-chemical characteristics to the analyte, has the additional advantage of minimizing mass detector fluctuations. Other advantages include identical optimized GC/MS, or LC-MS conditions, similar elution patterns and improved selectivity due to the mass difference. Use of stable isotope dilution methods also offers high sensitivity and rapid quantitation.^4,5 The isotopic dilution method requires the use of labeled internal standards. They are used not only for quantitation, but also as an internal calibrant for accurate mass measurements. Selection criteria for such internal standards depends on the analysis method and the desired mass shift. The internal standard should have a mass close to the metabolite of interest. However, it also should not interfere with the peaks of interest. ISOTEC^® Stable Isotopes offers a range of labeled steroids and hormones to use as internal standards which offer an appropriate mass shift with various labeling patterns and different incorporated stable isotopes.

Anabolic - Androgenic Steroids

The detection of synthetic steroids is a continuing challenge for authorities and analytical laboratories. Some assays use an indirect approach targeting a derivative or metabolite of the steroids. For example, there is a method of analyzing the oxime derivatives of the steroids and using deuterated testosterone and androstanone as internal standards.⁷ As described in another article, use of indirect detection of hydrolyzed derivatives and the lack of a proper reference material led researchers to develop a direct approach to analyze AAS glucuronide conjugates⁸offering reliable and precise analytical data. Researchers used deuterated and non-deuterated analogs in urine samples to establish references for simultaneous and direct detection of anabolic-androgenic steroid (AAS) metabolites. In this case, all the metabolites were extracted and analyzed simultaneously by a LC–MS/MS method. Researchers are also keen to analyze steroid patterns for the diagnosis of hormone-related disorders such as hyperaldosteronism, congenital adrenal hyperplasia or cancer. LC-MS/MS is often used for developing effective clinical diagnostic methods and rapid simultaneous quantitation of steroid patterns in human biofluids using labeled internal standards.⁹

Researchers often prefer to use deuterated internal standards. However at times, it becomes necessary to consider alternate labeling patterns and stable isotopes, such as ¹³C. A recent article observed an unusual false positive for a steroid while conducting a post-race equine urinalysis.⁶ They observed a consistent false positive in a biological sample that had specific enzymatic and microbial content. Further research demonstrated that the internal standards (testosterone-16,16,17-d₃ and 5a-androstane-3a,17b-diol –d₃) were undergoing oxidation followed by H/D exchange to form the analytes resulting in a false positive result. The authors emphasize the need for caution when using internal standards and recommend not including internal standards when performing a qualitative confirmatory test. However, an alternative to consider would be to use an internal standard where the isotope cannot be exchanged, for example testosterone-2,3,4-¹³C₃. Using this material would preserve the M+3 mass shift and hopefully make it easier to prevent false positives from occurring due to this reaction pathway.

Stable isotope labeled steroids are reliable internal standards for researchers developing assays or laboratories conducting routine testing. ISOTEC^® Stable Isotopes offers a wide variety of labeled bioactive compounds including steroids, vitamins, and their metabolites. If you are interested in a specific labeling pattern which is not listed in our current product offering, please Contact Us. We would be happy to discuss your requirements and work with you to provide a cost effective solution.

References

1. 2006. World Anti-Doping Program Guideline-reporting and management of elevated T/E ratios.. WADA Standards and harmonization/sciences research –guideline for elevated T/E..

2. Vesper HW, Botelho JC, Shacklady C, Smith A, Myers GL. 2008. CDC project on standardizing steroid hormone measurements. Steroids. 73(13):1286-1292. https://doi.org/10.1016/j.steroids.2008.09.008

3. Wieling J. 2002. LC-MS-MS Experiences with Internal Standards. Chromatographia. Vol. 55 SUP 1): S-107-113.

4. Chen Y, Yazdanpanah M, Hoffman BR, Diamandis EP, Wong P. 2009. Rapid determination of serum testosterone by liquid chromatography-isotope dilution tandem mass spectrometry and a split sample comparison with three automated immunoassays. Clinical Biochemistry. 42(6):484-490. https://doi.org/10.1016/j.clinbiochem.2008.11.009

5. Thienpont LM, Van Uytfanghe K, Blincko S, Ramsay CS, Xie H, Doss RC, Keevil BG, Owen LJ, Rockwood AL, Kushnir MM, et al. 2008. State-of-the-Art of Serum Testosterone Measurement by Isotope Dilution?Liquid Chromatography? Tandem Mass Spectrometry. 54(8):1290-1297. https://doi.org/10.1373/clinchem.2008.105841

6. Kwok WH, Leung DKK, Leung GNW, Tang FPW, Wan TSM, Wong CHF, Wong JKY. 2008. Unusual observations during steroid analysis. Rapid Commun. Mass Spectrom.. 22(5):682-686. https://doi.org/10.1002/rcm.3420

7. Kalhorn TF, Page ST, Howald WN, Mostaghel EA, Nelson PS. 2007. Analysis of testosterone and dihydrotestosterone from biological fluids as the oxime derivatives using high-performance liquid chromatography/tandem mass spectrometry. Rapid Commun. Mass Spectrom.. 21(19):3200-3206. https://doi.org/10.1002/rcm.3205

8. Hintikka L, Kuuranne T, Leinonen A, Thevis M, Schänzer W, Halket J, Cowan D, Grosse J, Hemmersbach P, Nielen MWF, et al. 2008. Liquid chromatographic?mass spectrometric analysis of glucuronide?conjugated anabolic steroid metabolites: method validation and interlaboratory comparison. J. Mass Spectrom.. 43(7):965-973. https://doi.org/10.1002/jms.1434

9. Ceglarek U, Kortz L, Leichtle A, Fiedler GM, Kratzsch J, Thiery J. 2009. Rapid quantification of steroid patterns in human serum by on-line solid phase extraction combined with liquid chromatography?triple quadrupole linear ion trap mass spectrometry. Clinica Chimica Acta. 401(1-2):114-118. https://doi.org/10.1016/j.cca.2008.11.022