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Measurement of serum testosterone using high-performance liquid chromatography/tandem mass spectrometry

  • Faye B. Vicente , Frederick A. Smith , Rafael Sierra and Sihe Wang
Published/Copyright: September 21, 2011
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Clinical Chemistry and Laboratory Medicine (CCLM)
From the journal Clinical Chemistry and Laboratory Medicine (CCLM) Volume 44 Issue 1

Abstract

Low levels of serum testosterone typically found in women and children cannot be reliably measured by immunoassay. We developed a simple and sensitive method using high-performance liquid chromatography/tandem mass spectrometry (HPLC-MS/MS). Sample preparation involved protein precipitation of serum (1.0mL) with acetonitrile containing the internal standard (testosterone-d3) followed by liquid extraction with methylene chloride. The chromatographic cycle per specimen was 10min. The performance was evaluated according to the CLSI EP10-A2 protocol. Within- and between-run imprecision was 20.9%, 2.29% and 1.80%, and 1.81%, 3.58% and 2.97% at mean concentrations of 0.17, 14.1 and 28.8nM, respectively, with no apparent carryover. The method was linear from 0.21 to 53.1nM and the analytical recovery was 100.5–106.2% across the concentrations tested. There was no interference observed from other steroids that were tested. Correlation using de-identified patient specimens with a commercial HPLC-MS/MS method showed a slope of 0.991, an intercept of −0.017 and a correlation coefficient (R2) of 0.998 by linear regression over concentrations ranging from 0.21 to 16.7nM. In conclusion, we report here an HPLC-MS/MS method suitable for clinical measurement of serum testosterone.

Keywords: liquid chromatography; mass spectrometry; testosterone
Corresponding author: Sihe Wang, 2300 Children's Plaza, Box 53, Chicago, IL 60614, USA Phone: +1-773-880-3676, Fax: +1-773-880-4687,

References

1. Wheeler MJ. The determination of bio-available testosterone. Ann Clin Biochem 1995; 32:345–57.10.1177/000456329503200401Search in Google Scholar

2. Taieb J, Mathian B, Millot F, Patricot MC, Mathieu E, Queyrel N, et al. Testosterone measured by 10 immunoassays and by isotope-dilution gas chromatographymass spectrometry in sera from 116 men, women, and children. Clin Chem 2003; 49:1381–95.10.1373/49.8.1381Search in Google Scholar

3. Starcevic B, Distefano E, Wang C, Catlin DH. Liquid chromatography-tandem mass spectrometry assay for human serum testosterone and trideuterated testosterone. J Chromatogr B Analyt Technol Biomed Life Sci 2003; 792:197–204.10.1016/S1570-0232(03)00261-7Search in Google Scholar

4. Herold DA, Fitzgerald RL. Immunoassays for testosterone in women: better than a guess? Clin Chem 2003; 49:1250–1.10.1373/49.8.1250Search in Google Scholar

5. Boschi S, Deiasio R, Mesini P, Bolelli GF, Sciajno R, Pasquali R, et al. Measurement of steroid-hormones in plasma by isocratic high-performance liquid-chromatography coupled to radioimmunoassay. Clin Chim Acta 1994; 231:107–13.10.1016/0009-8981(94)90260-7Search in Google Scholar

6. Dooley KC. Tandem mass spectrometry in the clinical chemistry laboratory. Clin Biochem 2003; 36:471–81.10.1016/S0009-9120(03)00105-XSearch in Google Scholar

7. Choi MH, Kim JN, Chung BC. Rapid HPLC-electrospray tandem mass spectrometric assay for urinary testosterone and dihydrotestosterone glucuronides from patients with benign prostate hyperplasia. Clin Chem 2003; 49:322–5.10.1373/49.2.322Search in Google Scholar

8. Borts DJ, Bowers LD. Direct measurement of urinary testosterone and epitestosterone conjugates using high-performance liquid chromatography/tandem mass spectrometry. J Mass Spectrom 2000; 35:50–61.10.1002/(SICI)1096-9888(200001)35:1<50::AID-JMS912>3.0.CO;2-JSearch in Google Scholar

9. Navajas R, Imaz C, Carreras D, Garcia M, Perez M, Rodriguez C, et al. Determination of epitestosterone and testosterone in urine by high-performance liquid-chromatography. J Chromatogr B Biomed Appl 1995; 673:159–64.10.1016/0378-4347(95)00260-XSearch in Google Scholar

10. Leinonen A, Kuuranne T, Kostiainen R. Liquid chromatography/mass spectrometry in anabolic steroid analysis-optimization and comparison of three ionization techniques: electrospray ionization, atmospheric pressure chemical ionization and atmospheric pressure photoionization. J Mass Spectrom 2002; 37:693–8.10.1002/jms.328Search in Google Scholar

11. Bowers LD, Sanaullah. Direct measurement of steroid sulfate and glucuronide conjugates with high-performance liquid chromatography mass spectrometry. J Chromatogr B Biomed Appl 1996; 687:61–8.10.1016/S0378-4347(96)00232-0Search in Google Scholar

12. Bean KA, Henion JD. Direct determination of anabolic steroid conjugates in human urine by combined high-performance liquid chromatography and tandem mass spectrometry. J Chromatogr B Biomed Sci Appl 1997; 690:65–75.10.1016/S0378-4347(96)00403-3Search in Google Scholar

13. Wang C, Catlin DH, Demers LM, Starcevic B, Swerdloff RS. Measurement of total serum testosterone in adult men: comparison of current laboratory methods versus liquid chromatography-tandem mass spectrometry. J Clin Endocrinol Metab 2004; 89:534–43.10.1210/jc.2003-031287Search in Google Scholar

14. Draisci R, Palleschi L, Ferretti E, Lucentini L, Cammarata P. Quantitation of anabolic hormones and their metabolites in bovine serum and urine by liquid chromatography-tandem mass spectrometry. J Chromatogr A 2000; 870:511–22.10.1016/S0021-9673(99)01293-5Search in Google Scholar

15. Cawood ML, Field HP, Ford CG, Gillingwater S, Kicman A, Cowan D, et al. Testosterone measurement by isotope-dilution liquid chromatography-tandem mass spectrometry: validation of a method for routine clinical practice. Clin Chem 2005; 51:1472–9.10.1373/clinchem.2004.044503Search in Google Scholar PubMed

16. Taylor RL, Machacek D, Singh RJ. Validation of a high-throughput liquid chromatography-tandem mass spectrometry method for urinary cortisol and cortisone. Clin Chem 2002; 48:1511–9.10.1093/clinchem/48.9.1511Search in Google Scholar

17. Clinical and Laboratory Standards Institute (CLSI). Preliminary evaluation of quantitative clinical laboratory methods: approved guideline, 2nd ed, Vol 22. Document EP10-A2. Wayne, PA: CLSI, 2002.Search in Google Scholar

18. Tietz NW. Clinical guide to laboratory tests, 3rd ed. Philadelphia, PA: W.B. Saunders, 1995.Search in Google Scholar

19. Annesley TM. Ion suppression in mass spectrometry. Clin Chem 2003; 49:1041–4.10.1373/49.7.1041Search in Google Scholar PubMed

20. Mei H, Hsieh YS, Nardo C, Xu XY, Wang SY, Ng K, et al. Investigation of matrix effects in bioanalytical high-performance liquid chromatography/tandem mass spectrometric assays: application to drug discovery. Rapid Commun Mass Spectrom 2003; 17:97–103.10.1002/rcm.876Search in Google Scholar PubMed

Received: 2005-9-9
Accepted: 2005-10-18
Published Online: 2011-9-21
Published in Print: 2006-1-1

©2006 by Walter de Gruyter Berlin New York

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https://doi.org/10.1515/CCLM.2006.014
Keywords for this article
liquid chromatography; mass spectrometry; testosterone

Articles in the same Issue

  1. Where does the evidence come from?
  2. NASBA: a novel approach to assess hormonal receptors and ERBB2 status in breast cancer
  3. Plasma cell-free DNA as an indicator of severity of injury in burn patients
  4. Bivariate statistical approach to evaluate laboratory performance by analysis of standard curves in an External Quality Assurance program for quantitative assays based on real-time PCR with Taq-Man™ probes
  5. Erythrocyte membrane acetylcholinesterase activity in subjects with MTHFR 677C→T genotype
  6. Plasma levels of tumor M2-pyruvate kinase should not be used as a tumor marker for hematological malignancies and solid tumors
  7. APO A-V–1131T→C polymorphism frequency and its association with morbidity in a Brazilian elderly population
  8. Association study between fibronectin and coronary heart disease
  9. Serum calcium and phosphorus associate with the occurrence and severity of angiographically documented coronary heart disease, possibly through correlation with atherogenic (apo)lipoproteins
  10. Urinary calcium excretion in severe preeclampsia and eclampsia
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  12. Measurement of serum amyloid A1 (SAA1), a major isotype of acute phase SAA
  13. Disturbed lipoprotein composition in non-dialyzed, hemodialysis, continuous ambulatory peritoneal dialysis and post-transplant patients with chronic renal failure
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  16. N-Terminal pro-brain natriuretic peptide: normal ranges in the pediatric population including method comparison and interlaboratory variability
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  21. EC4 European Syllabus for Post-Graduate Training in Clinical Chemistry and Laboratory Medicine: version 3 – 2005
  22. POX-Act assay and d-ROMs test – what are the facts?
  23. External quality control of urinary methyl malonic acid quantification – announcement of a pilot study
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