Measurement of serum testosterone using high-performance liquid chromatography/tandem mass spectrometry
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Faye B. Vicente
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.
References
1. Wheeler MJ. The determination of bio-available testosterone. Ann Clin Biochem 1995; 32:345–57.10.1177/000456329503200401Suche 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; 49:1381–95.10.1373/49.8.1381Suche 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)00261-7Suche in Google Scholar
4. Herold DA, Fitzgerald RL. Immunoassays for testosterone in women: better than a guess? Clin Chem 2003; 49:1250–1.10.1373/49.8.1250Suche 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)90260-7Suche 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-XSuche 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; 49:322–5.10.1373/49.2.322Suche 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-JSuche 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-XSuche 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.328Suche 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)00232-0Suche 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)00403-3Suche 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.2003-031287Suche 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)01293-5Suche 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.2004.044503Suche 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/48.9.1511Suche 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.Suche in Google Scholar
18. Tietz NW. Clinical guide to laboratory tests, 3rd ed. Philadelphia, PA: W.B. Saunders, 1995.Suche in Google Scholar
19. Annesley TM. Ion suppression in mass spectrometry. Clin Chem 2003; 49:1041–4.10.1373/49.7.1041Suche 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.876Suche in Google Scholar PubMed
©2006 by Walter de Gruyter Berlin New York
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