Revisiting hyper- and hypo-androgenism by tandem mass spectrometry

  • Flaminia Fanelli
  • Alessandra Gambineri
  • Marco Mezzullo
  • Valentina Vicennati
  • Carla Pelusi
  • Renato Pasquali
  • Uberto PagottoEmail author


Modern endocrinology is living a critical age of transition as far as laboratory testing and biochemical diagnosis are concerned. Novel liquid chromatography—tandem mass spectrometry (LC-MS/MS) assays for steroid measurement in biological fluids have abundantly demonstrated their analytical superiority over immunometric platforms that until now have dominated the world of steroid hormones determination in clinical laboratories. One of the most useful applications of LC-MS/MS is in the hypogonadism and hyperandrogenism field: LC-MS/MS has proved particularly suitable for the detection of low levels of testosterone typical of women and children, and in general more reliable in accurately determining hypogonadal male levels. This technique also offers increased informative power by allowing multi-analytical profiles that give a more comprehensive picture of the overall hormonal asset. Several LC-MS/MS methods for testosterone have been published in the last decade, some of them included other androgen or more comprehensive steroid profiles. LC-MS/MS offers the concrete possibility of achieving a definitive standardization of testosterone measurements and the generation of widely accepted reference intervals, that will set the basis for a consensus on the diagnostic value of biochemical testing. The present review is aimed at summarizing technological advancements in androgen measurements in serum and saliva. We also provide a picture of the state of advancement of standardization of testosterone assays, of the redefinition of androgen reference intervals by novel assays and of studies using LC-MS/MS for the characterization and diagnosis of female hyperandrogenism and male hypogonadism.


LC-MS/MS Testosterone Androgens Hyperandrogenism Hypogonadism 



This work was supported by a Grant from “Programma di ricerca Giovani Ricercatori Regione Emilia Romagna-Università 2010–2012”, Bologna, Italy (to F.F.). We thank Ms. Susan West for language editing of the manuscript.


  1. 1.
    Rosner W, Vesper H, Endocrine Society, American Association for Clinical Chemistry, American Association of Clinical Endocrinologists, Androgen Excess/PCOS Society, et al. Toward excellence in testosterone testing: a consensus statement. J Clin Endocrinol Metab. 2010;95:4542–8.PubMedCrossRefGoogle Scholar
  2. 2.
    Hankinson SE, Manson JE, London SJ, Willett WC, Speizer FE. Laboratory reproducibility of endogenous hormone levels in postmenopausal women. Cancer Epidemiol Biomarkers Prev. 1994;3:51–6.PubMedGoogle Scholar
  3. 3.
    Potischman N, Falk RT, Laiming VA, Siiteri PK, Hoover RN. Reproducibility of laboratory assays for steroid hormones and sex hormone-binding globulin. Cancer Res. 1994;54:5363–7.PubMedGoogle Scholar
  4. 4.
    Steinberger E, Ayala C, Hsi B, Smith KD, Rodriguez-Rigau LJ, Weidman ER, et al. Utilization of commercial laboratory results in management of hyperandrogenism in women. Endocr Pract. 1998;4:1–10.PubMedCrossRefGoogle Scholar
  5. 5.
    Boots LR, Potter S, Potter D, Azziz R. Measurement of total serum testosterone levels using commercially available kits: high degree of between-kit variability. Fertil Steril. 1998;69:286–92.PubMedCrossRefGoogle Scholar
  6. 6.
    Herold DA, Fitzgerald RL. Immunoassays for testosterone in women: better than a guess? Clin Chem. 2003;49(8):1250–1.PubMedCrossRefGoogle Scholar
  7. 7.
    Matsumoto AM, Bremner WJ. Serum testosterone assays—accuracy matters. J Clin Endocrinol Metab. 2004;89:520–4.PubMedCrossRefGoogle Scholar
  8. 8.
    Wu AH, French D. Implementation of liquid chromatography/mass spectrometry into the clinical laboratory. Clin Chim Acta. 2012. doi: 10.1016/j.cca.2012.10.026.
  9. 9.
    Miller WL, Auchus RJ. The molecular biology, biochemistry, and physiology of human steroidogenesis and its disorders. Endocr Rev. 2011;32:81–151.PubMedCrossRefGoogle Scholar
  10. 10.
    Burger HG. Androgen production in women. Fertil Steril. 2002;77 Suppl 4:S3–5.PubMedCrossRefGoogle Scholar
  11. 11.
    Stanczyk FZ. Diagnosis of hyperandrogenism: biochemical criteria. Best Pract Res Clin Endocrinol Metab. 2006;20:177–91.PubMedCrossRefGoogle Scholar
  12. 12.
    Manni A, Pardridge WM, Cefalu W, Nisula BC, Bardin CW, Santner SJ, et al. Bioavailability of albumin-bound testosterone. J Clin Endocrinol Metab. 1985;61:705–10.PubMedCrossRefGoogle Scholar
  13. 13.
    Muller M, Grobbee DE, Thijssen JH, van den Beld AW, van der Schouw YT. Sex hormones and male health: effects on components of the frailty syndrome. Trends Endocrinol Metab. 2003;14:289–96.PubMedCrossRefGoogle Scholar
  14. 14.
    Resko JA, Eik-nes KB. Diurnal testosterone levels in peripheral plasma of human male subjects. J Clin Endocrinol Metab. 1966;26:573–6.PubMedCrossRefGoogle Scholar
  15. 15.
    Davison SL, Bell R. Androgen physiology. Semin Reprod Med. 2006;24:71–7.PubMedCrossRefGoogle Scholar
  16. 16.
    Goodarzi MO, Dumesic DA, Chazenbalk G, Azziz R. Polycystic ovary syndrome: etiology, pathogenesis and diagnosis. Nat Rev Endocrinol. 2011;7:219–31.PubMedCrossRefGoogle Scholar
  17. 17.
    Pasquali R, Gambineri A. Polycystic ovary syndrome: a multifaceted disease from adolescence to adult age. Ann N Y Acad Sci. 2006;1092:158–74.PubMedCrossRefGoogle Scholar
  18. 18.
    The Rotterdam ESHRE/ASRM-Sponsored PCOS Consensus Workshop Group. Revised 2003 consensus on diagnostic criteria and long-term health risks related to polycystic ovary syndrome (PCOS). Hum Reprod. 2004;19:41–7.CrossRefGoogle Scholar
  19. 19.
    Azziz R, Carmina E, Dewailly D, Diamanti-Kandarakis E, Escobar-Morreale HF, Futterweit W, et al. Positions statement: criteria for defining polycystic ovary syndrome as a predominantly hyperandrogenic syndrome: an Androgen Excess Society guideline. J Clin Endocrinol Metab. 2006;91:4237–45.PubMedCrossRefGoogle Scholar
  20. 20.
    Taieb J, Mathian B, Millot F, Patricot MC, Mathieu E, Queyrel N, et al. Testosterone measured by 10 immunoassays and by isotope-dilution gas chromatography-mass spectrometry in sera from 116 men, women, and children. Clin Chem. 2003;49:1381–95.PubMedCrossRefGoogle Scholar
  21. 21.
    Fauser BC, Tarlatzis BC, Rebar RW, Legro RS, Balen AH, Lobo R, et al. Consensus on women’s health aspects of polycystic ovary syndrome (PCOS): the Amsterdam ESHRE/ASRM-Sponsored 3rd PCOS Consensus Workshop Group. Fertil Steril. 2012;97:28–38.PubMedCrossRefGoogle Scholar
  22. 22.
    Vermeulen A, Verdonck L, Kaufman JM. A critical evaluation of simple methods for the estimation of free testosterone in serum. J Clin Endocrinol Metab. 1999;84:3666–72.PubMedCrossRefGoogle Scholar
  23. 23.
    Chang WY, Knochenhauer ES, Bartolucci AA, Azziz R. Phenotypic spectrum of polycystic ovary syndrome: clinical and biochemical characterization of the three major clinical subgroups. Fertil Steril. 2005;83:1717–23.PubMedCrossRefGoogle Scholar
  24. 24.
    Kumar A, Woods KS, Bartolucci AA, Azziz R. Prevalence of adrenal androgen excess in patients with the polycystic ovary syndrome (PCOS). Clin Endocrinol (Oxf). 2005;62:644–9.CrossRefGoogle Scholar
  25. 25.
    Yildiz BO, Azziz R. The adrenal and polycystic ovary syndrome. Rev Endocr Metab Disord. 2007;8:331–42.PubMedCrossRefGoogle Scholar
  26. 26.
    Carmina E, Stanczyk FZ, Matteri RK, Lobo RA. Serum androsterone conjugates differentiate between acne and hirsutism in hyperandrogenic women. Fertil Steril. 1991;55:872–6.PubMedGoogle Scholar
  27. 27.
    Matteri RK, Stanczyk FZ, Gentzschein EE, Delgado C, Lobo RA. Androgen sulfate and glucuronide conjugates in nonhirsute and hirsute women with polycystic ovarian syndrome. Am J Obstet Gynecol. 1989;161:1704–9.PubMedCrossRefGoogle Scholar
  28. 28.
    Carmina E, Lobo RA. Evidence for increased androsterone metabolism in some normoandrogenic women with acne. J Clin Endocrinol Metab. 1993;76:1111–4.PubMedCrossRefGoogle Scholar
  29. 29.
    Legro RS, Carmina E, Stanczyk FZ, Gentzschein E, Lobo RA. Alterations in androgen conjugate levels in women and men with alopecia. Fertil Steril. 1994;62:744–50.PubMedGoogle Scholar
  30. 30.
    Seftel AD. Male hypogonadism. Part I: epidemiology of hypogonadism. Int J Impot Res. 2006;18:115–20.PubMedCrossRefGoogle Scholar
  31. 31.
    Araujo AB, Travison TG, Bhasin S, Esche GR, Williams RE, Clark RV, et al. Association between testosterone and estradiol and age-related decline in physical function in a diverse sample of men. J Am Geriatr Soc. 2008;56:2000–8.PubMedCrossRefGoogle Scholar
  32. 32.
    Traish AM, Miner MM, Morgentaler A, Zitzmann M. Testosterone deficiency. Am J Med. 2011;124:578–87.PubMedCrossRefGoogle Scholar
  33. 33.
    Bhasin S, Zhang A, Coviello A, Jasuja R, Ulloor J, Singh R, et al. The impact of assay quality and reference ranges on clinical decision making in the diagnosis of androgen disorders. Steroids. 2008;73:1311–7.PubMedCrossRefGoogle Scholar
  34. 34.
    Kelleher S, Conway AJ, Handelsman DJ. Blood testosterone threshold for androgen deficiency symptoms. J Clin Endocrinol Metab. 2004;89:3813–7.PubMedCrossRefGoogle Scholar
  35. 35.
    Dobs AS. The role of accurate testosterone testing in the treatment and management of male hypogonadism. Steroids. 2008;73:1305–10.PubMedCrossRefGoogle Scholar
  36. 36.
    Rosner W, Auchus RJ, Azziz R, Sluss PM, Raff H. Position statement: utility, limitations, and pitfalls in measuring testosterone: an endocrine society position statement. J Clin Endocrinol Metab. 2007;92:405–13.PubMedCrossRefGoogle Scholar
  37. 37.
    Feldman HA, Longcope C, Derby CA, Johannes CB, Araujo AB, Coviello AD, et al. Age trends in the level of serum testosterone and other hormones in middle-aged men: longitudinal results from the Massachusetts male aging study. J Clin Endocrinol Metab. 2002;87:589–98.PubMedCrossRefGoogle Scholar
  38. 38.
    Mohr BA, Guay AT, O’Donnell AB, McKinlay JB. Normal, bound and nonbound testosterone levels in normally ageing men: results from the Massachusetts Male Ageing Study. Clin Endocrinol. 2005;62:64–73.CrossRefGoogle Scholar
  39. 39.
    Wu FC, Tajar A, Beynon JM, Pye SR, Silman AJ, Finn JD, et al. Identification of late-onset hypogonadism in middle-aged and elderly men. N Engl J Med. 2010;363:123–35.PubMedCrossRefGoogle Scholar
  40. 40.
    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.PubMedCrossRefGoogle Scholar
  41. 41.
    Sikaris K, McLachlan RI, Kazlauskas R, de Kretser D, Holden CA, Handelsman DJ. Reproductive hormone reference intervals for healthy fertile young men: evaluation of automated platform assays. J Clin Endocrinol Metab. 2005;90:5928–36.PubMedCrossRefGoogle Scholar
  42. 42.
    Furuyama S, Mayes DM, Nugent CA. A radioimmunoassay for plasma testosterone. Steroids. 1970;16:415–28.PubMedCrossRefGoogle Scholar
  43. 43.
    Jeffcoate SL. A radioimmunoassay for testosterone, androstenedione and other 3-oxo-4-unsaturated steroids. J Endocrinol. 1971;49:2–3.PubMedGoogle Scholar
  44. 44.
    Krone N, Hughes BA, Lavery GG, Stewart PM, Arlt W, Shackleton CH. Gas chromatography/mass spectrometry (GC/MS) remains a pre-eminent discovery tool in clinical steroid investigations even in the era of fast liquid chromatography tandem mass spectrometry (LC/MS/MS). J Steroid Biochem Mol Biol. 2010;121:496–504.PubMedCrossRefGoogle Scholar
  45. 45.
    Shackleton C. Clinical steroid mass spectrometry: a 45-year history culminating in HPLC-MS/MS becoming an essential tool for patient diagnosis. J Steroid Biochem Mol Biol. 2010;121:481–90.PubMedCrossRefGoogle Scholar
  46. 46.
    Whitehouse CM, Dreyer RN, Yamashita M, Fenn JB. Electrospray interface for liquid chromatographs and mass spectrometers. Anal Chem. 1985;57:675–9.PubMedCrossRefGoogle Scholar
  47. 47.
    Ceglarek U, Kortz L, Leichtle A, Fiedler GM, Kratzsch J, Thiery J. 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. Clin Chim Acta. 2009;401:114–8.PubMedCrossRefGoogle Scholar
  48. 48.
    Guo T, Taylor RL, Singh RJ, Soldin SJ. Simultaneous determination of 12 steroids by isotope dilution liquid chromatography-photospray ionization tandem mass spectrometry. Clin Chim Acta. 2006;372:76–82.PubMedCrossRefGoogle Scholar
  49. 49.
    Kushnir MM, Rockwood AL, Roberts WL, Pattison EG, Bunker AM, Fitzgerald RL, et al. Performance characteristics of a novel tandem mass spectrometry assay for serum testosterone. Clin Chem. 2006;52:120–8.PubMedCrossRefGoogle Scholar
  50. 50.
    Annesley TM. Ion suppression in mass spectrometry. Clin Chem. 2003;49:1041–4.PubMedCrossRefGoogle Scholar
  51. 51.
    Matuszewski BK, Constanzer ML, Chavez-Eng CM. Strategies for the assessment of matrix effect in quantitative bioanalytical methods based on HPLC-MS/MS. Anal Chem. 2003;75:3019–30.PubMedCrossRefGoogle Scholar
  52. 52.
    Kushnir MM, Rockwood AL, Roberts WL, Yue B, Bergquist J, Meikle AW. Liquid chromatography tandem mass spectrometry for analysis of steroids in clinical laboratories. Clin Biochem. 2011;44:77–88.PubMedCrossRefGoogle Scholar
  53. 53.
    Moal V, Mathieu E, Reynier P, Malthièry Y, Gallois Y. Low serum testosterone assayed by liquid chromatography-tandem mass spectrometry. Comparison with five immunoassay techniques. Clin Chim Acta. 2007;386:12–9.PubMedCrossRefGoogle Scholar
  54. 54.
    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.PubMedCrossRefGoogle Scholar
  55. 55.
    Yamashita K, Miyashiro Y, Maekubo H, Okuyama M, Honma S, Takahashi M, et al. Development of highly sensitive quantification method for testosterone and dihydrotestosterone in human serum and prostate tissue by liquid chromatography-electrospray ionization tandem mass spectrometry. Steroids. 2009;74:920–6.PubMedCrossRefGoogle Scholar
  56. 56.
    Harwood DT, Handelsman DJ. Development and validation of a sensitive liquid chromatography-tandem mass spectrometry assay to simultaneously measure androgens and estrogens in serum without derivatization. Clin Chim Acta. 2009;409:78–84.PubMedCrossRefGoogle Scholar
  57. 57.
    Fitzgerald RL, Griffin TL, Herold DA. Analysis of testosterone in serum using mass spectrometry. Methods Mol Biol. 2010;603:489–500.PubMedCrossRefGoogle Scholar
  58. 58.
    Kushnir MM, Blamires T, Rockwood AL, Roberts WL, Yue B, Erdogan E, et al. Liquid chromatography-tandem mass spectrometry assay for androstenedione, dehydroepiandrosterone, and testosterone with pediatric and adult reference intervals. Clin Chem. 2010;56:1138–47.PubMedCrossRefGoogle Scholar
  59. 59.
    Rauh M, Gröschl M, Rascher W, Dörr HG. Automated, fast and sensitive quantification of 17 alpha-hydroxy-progesterone, androstenedione and testosterone by tandem mass spectrometry with on-line extraction. Steroids. 2006;71:450–8.PubMedCrossRefGoogle Scholar
  60. 60.
    Fanelli F, Belluomo I, Di Lallo VD, Cuomo G, De Iasio R, Baccini M, et al. Serum steroid profiling by isotopic dilution-liquid chromatography-mass spectrometry: comparison with current immunoassays and reference intervals in healthy adults. Steroids. 2011;76:244–53.PubMedCrossRefGoogle Scholar
  61. 61.
    Owen LJ, Keevil BG. Testosterone measurement by liquid chromatography tandem mass spectrometry: the importance of internal standard choice. Ann Clin Biochem. 2012;49:600–2.PubMedCrossRefGoogle Scholar
  62. 62.
    Vogeser M, Seger C. Pitfalls associated with the use of liquid chromatography-tandem mass spectrometry in the clinical laboratory. Clin Chem. 2010;56:1234–44.PubMedCrossRefGoogle Scholar
  63. 63.
    Clinical and Laboratory Standard Institute (CLSI). Mass spectrometry in the clinical laboratory: General principles and guidance. CLSI document C50-A. (ISBN 1-56238-648-4).Google Scholar
  64. 64.
    Rosner W, Vesper H. Preface. CDC workshop report improving steroid hormone measurements in patient care and research translation. Steroids. 2008;73:1285.PubMedCrossRefGoogle Scholar
  65. 65.
    Thienpont LM, Van Uytfanghe K, Blincko S, Ramsay CS, Xie H, Doss RC, et al. State-of-the-art of serum testosterone measurement by isotope dilution-liquid chromatography-tandem mass spectrometry. Clin Chem. 2008;54:1290–7.PubMedCrossRefGoogle Scholar
  66. 66.
    Borrey D, Moerman E, Cockx A, Engelrelst V, Langlois MR. Column-switching LC-MS/MS analysis for quantitative determination of testosterone in human serum. Clin Chim Acta. 2007;382:134–7.PubMedCrossRefGoogle Scholar
  67. 67.
    Tai SS, Xu B, Welch MJ, Phinney KW. Development and evaluation of a candidate reference measurement procedure for the determination of testosterone in human serum using isotope dilution liquid chromatography/tandem mass spectrometry. Anal Bioanal Chem. 2007;388:1087–94.PubMedCrossRefGoogle Scholar
  68. 68.
    Singh RJ. Validation of a high throughput method for serum/plasma testosterone using liquid chromatography tandem mass spectrometry (LC-MS/MS). Steroids. 2008;73:1339–44.PubMedCrossRefGoogle Scholar
  69. 69.
    Turpeinen U, Linko S, Itkonen O, Hämäläinen E. Determination of testosterone in serum by liquid chromatography-tandem mass spectrometry. Scand J Clin Lab Invest. 2008;68:50–7.PubMedCrossRefGoogle Scholar
  70. 70.
    Chen Y, Yazdanpanah M, Hoffman BR, Diamandis EP, Wong PY. Rapid determination of serum testosterone by liquid chromatography-isotope dilution tandem mass spectrometry and a split sample comparison with three automated immunoassays. Clin Biochem. 2009;42:484–90.PubMedCrossRefGoogle Scholar
  71. 71.
    Salameh WA, Redor-Goldman MM, Clarke NJ, Reitz RE, Caulfield MP. Validation of a total testosterone assay using high-turbulence liquid chromatography tandem mass spectrometry: total and free testosterone reference ranges. Steroids. 2010;75:169–75.PubMedCrossRefGoogle Scholar
  72. 72.
    Bui HN, Struys EA, Martens F, de Ronde W, Thienpont LM, Kenemans P, et al. Serum testosterone levels measured by isotope dilution-liquid chromatography-tandem mass spectrometry in postmenopausal women versus those in women who underwent bilateral oophorectomy. Ann Clin Biochem. 2010;47:248–52.PubMedCrossRefGoogle Scholar
  73. 73.
    Savolainen K, Kiimamaa R, Halonen T. High-throughput analysis of testosterone in serum samples by on-line solid phase extraction liquid chromatography-tandem mass spectrometry. Clin Chem Lab Med. 2011;49:1845–8.PubMedCrossRefGoogle Scholar
  74. 74.
    Shi RZ, van Rossum HH, Bowen RA. Serum testosterone quantitation by liquid chromatography-tandem mass spectrometry: interference from blood collection tubes. Clin Biochem. 2012;45(18):1706–9. doi: 10.1016/j.clinbiochem.2012.08.008.PubMedCrossRefGoogle Scholar
  75. 75.
    Botelho JC, Shacklady C, Cooper HC, Tai SS, Uytfanghe KV, Thienpont LM, et al. Isotope-dilution liquid chromatography-tandem mass spectrometry candidate reference method for total testosterone in human serum. Clin Chem. 2013;59:372–80.PubMedCrossRefGoogle Scholar
  76. 76.
    French D. Development and validation of a serum total testosterone liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay calibrated to NIST SRM 971. Clin Chim Acta. 2013;415:109–17.PubMedCrossRefGoogle Scholar
  77. 77.
    Rhea JM, French D, Molinaro RJ. Direct total and free testosterone measurement by liquid chromatography tandem mass spectrometry across two different platforms. Clin Biochem. 2013. doi: 10.1016/j.clinbiochem2013.PubMedGoogle Scholar
  78. 78.
    Kulle AE, Riepe FG, Melchior D, Hiort O, Holterhus PM. A novel ultrapressure liquid chromatography tandem mass spectrometry method for the simultaneous determination of androstenedione, testosterone, and dihydrotestosterone in pediatric blood samples: age- and sex-specific reference data. J Clin Endocrinol Metab. 2010;95:2399–409.PubMedCrossRefGoogle Scholar
  79. 79.
    Guo T, Chan M, Soldin SJ. Steroid profiles using liquid chromatography-tandem mass spectrometry with atmospheric pressure photoionization source. Arch Pathol Lab Med. 2004;128:469–75.PubMedGoogle Scholar
  80. 80.
    Janzen N, Sander S, Terhardt M, Peter M, Sander J. Fast and direct quantification of adrenal steroids by tandem mass spectrometry in serum and dried blood spots. J Chromatogr B Anal Technol Biomed Life Sci. 2008;861:117–22.CrossRefGoogle Scholar
  81. 81.
    Keski-Rahkonen P, Huhtinen K, Poutanen M, Auriola S. Fast and sensitive liquid chromatography-mass spectrometry assay for seven androgenic and progestagenic steroids in human serum. J Steroid Biochem Mol Biol. 2011;127:396–404.PubMedCrossRefGoogle Scholar
  82. 82.
    Kalhorn TF, Page ST, Howald WN, Mostaghel EA, Nelson PS. Analysis of testosterone and dihydrotestosterone from biological fluids as the oxime derivatives using high-performance liquid chromatography/tandem mass spectrometry. Rapid Commun Mass Spectrom. 2007;21:3200–6.PubMedCrossRefGoogle Scholar
  83. 83.
    Licea-Perez H, Wang S, Szapacs ME, Yang E. Development of a highly sensitive and selective UPLC/MS/MS method for the simultaneous determination of testosterone and 5alpha-dihydrotestosterone in human serum to support testosterone replacement therapy for hypogonadism. Steroids. 2008;73:601–10.PubMedCrossRefGoogle Scholar
  84. 84.
    Shiraishi S, Lee PW, Leung A, Goh VH, Swerdloff RS, Wang C. Simultaneous measurement of serum testosterone and dihydrotestosterone by liquid chromatography-tandem mass spectrometry. Clin Chem. 2008;54:1855–63.PubMedCrossRefGoogle Scholar
  85. 85.
    Labrie F, Bélanger A, Bélanger P, Bérubé R, Martel C, Cusan L, et al. Androgen glucuronides, instead of testosterone, as the new markers of androgenic activity in women. J Steroid Biochem Mol Biol. 2006;99:182–8.PubMedCrossRefGoogle Scholar
  86. 86.
    Rosner W. Errors in the measurement of plasma free testosterone. J Clin Endocrinol Metab. 1997;82:2014–5.PubMedGoogle Scholar
  87. 87.
    Van Uytfanghe K, Stöckl D, Kaufman JM, Fiers T, Ross HA, De Leenheer AP, et al. Evaluation of a candidate reference measurement procedure for serum free testosterone based on ultrafiltration and isotope dilution-gas chromatography-mass spectrometry. Clin Chem. 2004;50:2101–10.PubMedCrossRefGoogle Scholar
  88. 88.
    Chen Y, Yazdanpanah M, Wang XY, Hoffman BR, Diamandis EP, Wong PY. Direct measurement of serum free testosterone by ultrafiltration followed by liquid chromatography tandem mass spectrometry. Clin Biochem. 2010;43:490–6.PubMedCrossRefGoogle Scholar
  89. 89.
    Dabbs Jr JM, Campbell BC, Gladue BA, Midgley AR, Navarro MA, Read GF, et al. Reliability of salivary testosterone measurements: a multicenter evaluation. Clin Chem. 1995;41:1581–4.PubMedGoogle Scholar
  90. 90.
    Sakaguchi K, Hasegawa T. Analysis of salivary testosterone by liquid chromatography-tandem mass spectrometry: correlation with serum bioavailable testosterone and aging. Rinsho Byori. 2005;53:388–94.PubMedGoogle Scholar
  91. 91.
    Lee S, Kwon S, Shin HJ, Park J, Lim HS, Lee KR, et al. Quantitative measurement of salivary testosterone in Korean adults by stable isotope-dilution liquid chromatography electrospray-tandem mass spectrometry. BMB Rep. 2010;43:761–5.PubMedCrossRefGoogle Scholar
  92. 92.
    Macdonald PR, Owen LJ, Wu FC, Macdowall W, Keevil BG, NATSAL team. A liquid chromatography-tandem mass spectrometry method for salivary testosterone with adult male reference interval determination. Clin Chem. 2011;57:774–5.PubMedCrossRefGoogle Scholar
  93. 93.
    Bui HN, Schagen SE, Klink DT, Delemarre-van De Waal HA, Blankenstein MA, Heijboer AC. Salivary testosterone in female-to-male transgender adolescents during treatment with intra-muscular injectable testosterone esters. Steroids. 2013;78:91–5.PubMedCrossRefGoogle Scholar
  94. 94.
    Matsui F, Koh E, Yamamoto K, Sugimoto K, Sin HS, Maeda Y, et al. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for simultaneous measurement of salivary testosterone and cortisol in healthy men for utilization in the diagnosis of late-onset hypogonadism in males. Endocr J. 2009;56:1083–93.PubMedCrossRefGoogle Scholar
  95. 95.
    Shibayama Y, Higashi T, Shimada K, Odani A, Mizokami A, Konaka H, et al. Simultaneous determination of salivary testosterone and dehydroepiandrosterone using LC-MS/MS: Method development and evaluation of applicability for diagnosis and medication for late-onset hypogonadism. J Chromatogr B Anal Technol Biomed Life Sci. 2009;877:2615–23.CrossRefGoogle Scholar
  96. 96.
    Jensen MA, Hansen AM, Abrahamsson P, Nørgaard AW. Development and evaluation of a liquid chromatography tandem mass spectrometry method for simultaneous determination of salivary melatonin, cortisol and testosterone. J Chromatogr B Anal Technol Biomed Life Sci. 2011;879:2527–32.CrossRefGoogle Scholar
  97. 97.
    Turpeinen U, Hämäläinen E, Haanpää M, Dunkel L. Determination of salivary testosterone and androstendione by liquid chromatography-tandem mass spectrometry. Clin Chim Acta. 2012;413:594–9.PubMedCrossRefGoogle Scholar
  98. 98.
    Kataoka H, Ehara K, Yasuhara R, Saito K. Simultaneous determination of testosterone, cortisol, and dehydroepiandrosterone in saliva by stable isotope dilution on-line in-tube solid-phase microextraction coupled with liquid chromatography-tandem mass spectrometry. Anal Bioanal Chem. 2013;405:331–40.PubMedCrossRefGoogle Scholar
  99. 99.
    van den Ouweland JM, Kema IP. The role of liquid chromatography-tandem mass spectrometry in the clinical laboratory. J Chromatogr B Anal Technol Biomed Life Sci. 2012;883–884:18–32.CrossRefGoogle Scholar
  100. 100.
    Westgard QC. Desirable biological variation database specifications. Accessed 18 Jan 2013.
  101. 101.
    Vesper HW, Botelho JC. Standardization of testosterone measurements in humans. J Steroid Biochem Mol Biol. 2010;121:513–9.PubMedCrossRefGoogle Scholar
  102. 102.
    Yun YM, Botelho JC, Chandler DW, Katayev A, Roberts WL, Stanczyk FZ, et al. Performance criteria for testosterone measurements based on biological variation in adult males: recommendations from the Partnership for the Accurate Testing of Hormones. Clin Chem. 2012;58:1703–10.PubMedCrossRefGoogle Scholar
  103. 103.
    Sartorius G, Spasevska S, Idan A, Turner L, Forbes E, Zamojska A, et al. Serum testosterone, dihydrotestosterone and estradiol concentrations in older men self-reporting very good health: the healthy man study. Clin Endocrinol (Oxf). 2012;77:755–63.CrossRefGoogle Scholar
  104. 104.
    Siekmann L. Determination of steroid hormones by the use of isotope dilution—mass spectrometry: a definitive method in clinical chemistry. J Steroid Biochem. 1979;11:117–23.PubMedCrossRefGoogle Scholar
  105. 105.
    Thienpont LM, Van Nieuwenhove B, Stöckl D, Reinauer H, De Leenheer AP. Determination of reference method values by isotope dilution-gas chromatography/mass spectrometry: a five years’ experience of two European Reference Laboratories. Eur J Clin Chem Clin Biochem. 1996;34:853–60.PubMedGoogle Scholar
  106. 106.
    Vesper HW, Bhasin S, Wang C, Tai SS, Dodge LA, Singh RJ, et al. Interlaboratory comparison study of serum total testosterone [corrected] measurements performed by mass spectrometry methods. Steroids. 2009;74:498–503.PubMedCrossRefGoogle Scholar
  107. 107.
    Solberg HE. The IFCC recommendation on estimation of reference intervals. The RefVal program. Clin Chem Lab Med. 2004;42:710–4.PubMedCrossRefGoogle Scholar
  108. 108.
    Sunderman FW Jr. Current concepts of “normal values,” “reference values,” and “discrimination values,” in clinical chemistry. Clin Chem. 1975;21:1873–7.Google Scholar
  109. 109.
    Haring R, Hannemann A, John U, Radke D, Nauck M, Wallaschofski H, et al. Age-specific reference ranges for serum testosterone and androstenedione concentrations in women measured by liquid chromatography-tandem mass spectrometry. J Clin Endocrinol Metab. 2012;97:408–15.PubMedCrossRefGoogle Scholar
  110. 110.
    Barth JH, Field HP, Yasmin E, Balen AH. Defining hyperandrogenism in polycystic ovary syndrome: measurement of testosterone and androstenedione by liquid chromatography-tandem mass spectrometry and analysis by receiver operator characteristic plots. Eur J Endocrinol. 2010;162:611–5.PubMedCrossRefGoogle Scholar
  111. 111.
    Rothman MS, Carlson NE, Xu M, Wang C, Swerdloff R, Lee P, et al. Reexamination of testosterone, dihydrotestosterone, estradiol and estrone levels across the menstrual cycle and in postmenopausal women measured by liquid chromatography-tandem mass spectrometry. Steroids. 2011;76:177–82.PubMedCrossRefGoogle Scholar
  112. 112.
    Bui HN, Sluss PM, Blincko S, Knol DL, Blankenstein MA, Heijboer AC. Dynamics of serum testosterone during the menstrual cycle evaluated by daily measurements with an ID-LC-MS/MS method and a 2nd generation automated immunoassay. Steroids. 2013;78:96–101.PubMedCrossRefGoogle Scholar
  113. 113.
    Fanelli F, Belluomo I, Di Lallo V, Prontera O, Repaci A, Di Dalmazi G, et al. Androgen profiling in adolescent females by liquid chromatography-tandem mass spectrometry. In 15th International & 14th European Congress of Endocrinology, 5–9 May 2012, Florence, Italy.Google Scholar
  114. 114.
    Pasquali R, Stener-Victorin E, Yildiz BO, Duleba AJ, Hoeger K, Mason H, et al. PCOS Forum: research in polycystic ovary syndrome today and tomorrow. Clin Endocrinol (Oxf). 2011;74:424–33.CrossRefGoogle Scholar
  115. 115.
    Escobar-Morreale HF, Asunción M, Calvo RM, Sancho J, San Millán JL. Receiver operating characteristic analysis of the performance of basal serum hormone profiles for the diagnosis of polycystic ovary syndrome in epidemiological studies. Eur J Endocrinol. 2001;145:619–24.PubMedCrossRefGoogle Scholar
  116. 116.
    Hahn S, Kuehnel W, Tan S, Kramer K, Schmidt M, Roesler S, et al. Diagnostic value of calculated testosterone indices in the assessment of polycystic ovary syndrome. Clin Chem Lab Med. 2007;45:202–7.PubMedCrossRefGoogle Scholar
  117. 117.
    Villarroel C, Trejo L, Muñoz A, Kohen P, Fuentes A, Devoto L. Assessment of diagnostic competence of plasmatic androgens on polycystic ovary syndrome based on receiver operator characteristic curves. Gynecol Endocrinol. 2010;26:600–6.PubMedCrossRefGoogle Scholar
  118. 118.
    Cho LW, Kilpatrick ES, Jayagopal V, Diver MJ, Atkin SL. Biological variation of total testosterone, free androgen index and bioavailable testosterone in polycystic ovarian syndrome: implications for identifying hyperandrogenaemia. Clin Endocrinol. 2008;68:390–4.Google Scholar
  119. 119.
    Golbahar J, Al-Ayadhi M, Das NM, Gumaa K. Sensitive and specific markers for insulin resistance, hyperandrogenemia, and inappropriate gonadotrophin secretion in women with polycystic ovary syndrome: a case-control study from Bahrain. Int J Womens Health. 2012;4:201–6.PubMedCrossRefGoogle Scholar
  120. 120.
    Stener-Victorin E, Holm G, Labrie F, Nilsson L, Janson PO, Ohlsson C. Are there any sensitive and specific sex steroid markers for polycystic ovary syndrome? J Clin Endocrinol Metab. 2010;95:810–9.PubMedCrossRefGoogle Scholar
  121. 121.
    Yasmin E, Balen AH, Barth JH. The association of body mass index and biochemical hyperandrogenaemia in women with and without polycystic ovary syndrome. Eur J Obstet Gynecol Reprod Biol. 2013;166:173–7.PubMedCrossRefGoogle Scholar
  122. 122.
    Bell A, Meek CL, Viljoen A. Evidence of biochemical hyperandrogenism in women: the limitations of serum testosterone quantitation. J Obstet Gynaecol. 2012;32:367–71.PubMedCrossRefGoogle Scholar
  123. 123.
    Janse F, Eijkemans MJ, Goverde AJ, Lentjes EG, Hoek A, Lambalk CB, et al. Assessment of androgen concentration in women: liquid chromatography-tandem mass spectrometry and extraction RIA show comparable results. Eur J Endocrinol. 2011;165:925–33.PubMedCrossRefGoogle Scholar
  124. 124.
    Legro RS, Schlaff WD, Diamond MP, Coutifaris C, Casson PR, Brzyski RG, et al. Total testosterone assays in women with polycystic ovary syndrome: precision and correlation with hirsutism. J Clin Endocrinol Metab. 2010;95:5305–13.PubMedCrossRefGoogle Scholar
  125. 125.
    Gambineri A, Fanelli F, Prontera O, Repaci A, Di Dalmazi G, Zanotti L, et al. Prevalence of hyperandrogenic states in late adolescent and young women: epidemiological survey on high-school students in Northern Italy. J Clin Endocrinol Metab. 2013;98:1641–50.Google Scholar
  126. 126.
    Naessen T, Kushnir MM, Chaika A, Nosenko J, Mogilevkina I, Rockwood AL, et al. Steroid profiles in ovarian follicular fluid in women with and without polycystic ovary syndrome, analyzed by liquid chromatography-tandem mass spectrometry. Fertil Steril. 2010;94:2228–33.PubMedCrossRefGoogle Scholar
  127. 127.
    Harman SM, Metter EJ, Tobin JD, Pearson J, Blackman MR, Baltimore Longitudinal Study of Aging. Longitudinal effects of aging on serum total and free testosterone levels in healthy men. Baltimore Longitudinal Study of Aging. J Clin Endocrinol Metab. 2001;86:724–31.PubMedCrossRefGoogle Scholar
  128. 128.
    Bhasin S, Pencina M, Jasuja GK, Travison TG, Coviello A, Orwoll E, et al. Reference ranges for testosterone in men generated using liquid chromatography tandem mass spectrometry in a community-based sample of healthy nonobese young men in the Framingham Heart Study and applied to three geographically distinct cohorts. J Clin Endocrinol Metab. 2011;96:2430–9.PubMedCrossRefGoogle Scholar
  129. 129.
    Yeap BB, Alfonso H, Chubb SA, Handelsman DJ, Hankey GJ, Norman PE, et al. Reference ranges and determinants of testosterone, dihydrotestosterone, and estradiol levels measured using liquid chromatography-tandem mass spectrometry in a population-based cohort of older men. J Clin Endocrinol Metab. 2012;97:4030–9.PubMedCrossRefGoogle Scholar
  130. 130.
    Huhtaniemi IT, Tajar A, Lee DM, O’Neill TW, Finn JD, Bartfai G, et al. Comparison of serum testosterone and estradiol measurements in 3174 European men using platform immunoassay and mass spectrometry; relevance for the diagnostics in aging men. Eur J Endocrinol. 2012;166:983–91.PubMedCrossRefGoogle Scholar
  131. 131.
    Brandhorst G, Streit F, Kratzsch J, Schiettecatte J, Roth HJ, Luppa PB, et al. Multicenter evaluation of a new automated electrochemiluminescence immunoassay for the quantification of testosterone compared to liquid chromatography tandem mass spectrometry. Clin Biochem. 2011;44:264–7.PubMedCrossRefGoogle Scholar
  132. 132.
    Travison TG, Nguyen AH, Naganathan V, Stanaway FF, Blyth FM, Cumming RG, et al. Changes in reproductive hormone concentrations predict the prevalence and progression of the frailty syndrome in older men: the concord health and ageing in men project. J Clin Endocrinol Metab. 2011;96:2464–74.PubMedCrossRefGoogle Scholar
  133. 133.
    Bhasin S, Jasjua GK, Pencina M, D’Agostino Sr R, Coviello AD, Vasan RS, et al. Sex hormone-binding globulin, but not testosterone, is associated prospectively and independently with incident metabolic syndrome in men: the Framingham heart study. Diabetes Care. 2011;34:2464–70.PubMedCrossRefGoogle Scholar
  134. 134.
    Haring R, Teng Z, Xanthakis V, Coviello A, Sullivan L, Bhasin S, et al. Association of sex steroids, gonadotrophins, and their trajectories with clinical cardiovascular disease and all-cause mortality in elderly men from the Framingham Heart Study. Clin Endocrinol (Oxf). 2012. doi: 10.1111/cen.12013.Google Scholar
  135. 135.
    Lakshman KM, Kaplan B, Travison TG, Basaria S, Knapp PE, Singh AB, et al. The effects of injected testosterone dose and age on the conversion of testosterone to estradiol and dihydrotestosterone in young and older men. J Clin Endocrinol Metab. 2010;95:3955–64.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  • Flaminia Fanelli
    • 1
  • Alessandra Gambineri
    • 1
  • Marco Mezzullo
    • 1
  • Valentina Vicennati
    • 1
  • Carla Pelusi
    • 1
  • Renato Pasquali
    • 1
  • Uberto Pagotto
    • 1
    Email author
  1. 1.Endocrinology Unit, Department of Medical and Surgical Sciences, and Center for Applied Biomedical Sciences, S.Orsola – Malpighi HospitalUniversity Alma Mater StudiorumBolognaItaly

Personalised recommendations