Applications of Mass Spectrometry in Analyses of Steroid Hormones

  • Zimeng YanEmail author
  • Chang Cheng
  • Shaorong Liu


Steroid hormones are endogenous chemicals controlling many endocrinology functions. Mass spectrometry technologies have been applied for analyses of steroid hormones as biomarkers in endocrinology and pathology diagnoses, doping drugs in athletes and racing horses, residuals in food safety concerns, and environmental pollutants in water and sediments. Both liquid chromatography mass spectrometry (LC-MS or LC-MS/MS) and gas chromatography mass spectrometry (GC-MS or GC-MS/MS) are broadly used in research, clinical, pharmaceutical industry, competition sports, food safety, and environmental testing laboratories. Sample preparation techniques, such as deconjugation and extraction, are critical procedures for isolating steroid hormone from sample matrices, including biological fluids, tissues, environmental water and sediments. Chemical derivatization modifies the physicochemical properties of steroid hormone molecules to improve their chromatographic performances and to enhance their sensitivities to mass detection. Chromatographic techniques such as HPLC, UPLC, and GC have direct impact on separation of analytes, MS interface, and analysis throughput. The method sensitivity and specificity of LC-MS and GC-MS depend largely on the analyte status, i.e., easiness of ionization, derivatization, sample matrix, and MS detection mode, e.g., ESI, APCI, APPI, MAILDI, or EI. LC-MS and GC-MS methodologies should be developed and validated following scientific and regulatory guidelines, and the steroid hormones analyses should be standardized.


Atmospheric Pressure Chemical Ionization Supercritical Fluid Chromatography Derivatization Reagent Helix Pomatia Sulfonyl Chloride 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


  1. 1.
    Shimada K, Mitamura K, Higashi T (2001) Gas chromatography and high-performance liquid chromatography of natural steroids. J Chromatogr A 935:141–172CrossRefGoogle Scholar
  2. 2.
    Kushnir MM, Rockwood AL, Bergquist J (2010) Liquid chromatography–tandem mass spectrometry applications in endocrinology. Mass Spectrom Rev 29:480–502CrossRefGoogle Scholar
  3. 3.
    Soldin SJ, Soldin OP (2009) Steroid hormone analysis by tandem mass spectrometry. Clin Chem 55:1061–1066CrossRefGoogle Scholar
  4. 4.
    Blair IA (2010) Analysis of estrogens in serum from postmenopausal women: past, present and future. Steroids 75:297–306CrossRefGoogle Scholar
  5. 5.
    Rathahao E, Page A, Jouanin I, Paris A, Debrauwer L (2004) Liquid chromatography coupled to negative electrospray/ion trap mass spectrometry for the identification of isomeric glutathione conjugates of catechol estrogens. Int J Mass spectrom 231:119–129CrossRefGoogle Scholar
  6. 6.
    Moon J, Jung H, Moon M, Chung B, Choi M (2009) Heat-map visualization of gas chromatography-mass spectrometry based quantitative signatures on second steroids metabolism. J Am Soc Mass Spectrom 20:1626–1637CrossRefGoogle Scholar
  7. 7.
    Gomes RL, Meredith W, Snape CE, Sephton MA (2009) Analysis of conjugated steroid androgens: deconjugation, derivatization and associated issues. J Pharm Biomed Anal 49:1133–1140CrossRefGoogle Scholar
  8. 8.
    Xu X, Roman JM, Issaq HJ, Keefer LK, Veenstra TD, Ziegler RG (2007) Quantitative measurement of endogenous estrogens and estrogen metabolites in human serum by liquid chromatography-tandem mass spectrometry. Anal Chem 79:7813–7821CrossRefGoogle Scholar
  9. 9.
    Rauh M (2010) Steroid measurement with LC–MS/MS. Application examples in pediatrics. J Steroid Biochem Mol Biol 121:520–527CrossRefGoogle Scholar
  10. 10.
    Deventer K, Delbeke FT (2003) Validation of a quantitative screening method for corticosteroids by liquid chromatography tandem mass spectrometry. In: Shanzer W, Geyer H, Gotzmann A, Mareck U (eds) Recent advances in doping analysis, vol 11, Spert and Buch Strauβ, Köln, pp 23–31Google Scholar
  11. 11.
    Leung GNW, Chung EW, Ho ENM, Kwok WH, Leung DKK, Tang FPW, Wan TSM, Yu NH (2005) High throughput screening of corticosteroids and basic drugs in horse urine by liquid chromatography-tandem mass spectrometry. J Chromatogr B 825:47–56CrossRefGoogle Scholar
  12. 12.
    Noppe H, Le Bizec B, Verheyden K, De Brabander HF (2008) Novel analytical methods for the determination of steroid hormones in edible matrices. Anal Chim Acta 611:1–16CrossRefGoogle Scholar
  13. 13.
    Pacakova V, Loukotkova L, Bosakova Z, Stulik K (2009) Analysis for estrogens as environmental pollutants—a review. J Sep Sci 32:867–882Google Scholar
  14. 14.
    Stanczyk FZ, Clarke NJ (2010) Advantages and challenges of mass spectrometry assays for steroid hormones. J Steroid Biochem Mol Biol 121:491–495CrossRefGoogle Scholar
  15. 15.
    Giese RW (2003) Measurement of endogenous estrogens: analytical challenges and recent advances. J Chromatogr A 1000:401–412CrossRefGoogle Scholar
  16. 16.
    Higashi T (2006) Trace determination of steroids causing age-related diseases using LC/MS combined with detection-oriented derivatization. Chem Pharm Bull 54:1479–1485CrossRefGoogle Scholar
  17. 17.
    Higashi T, Shimada K (2004) Derivatization of neutral steroids to enhance their detection characteristics in liquid chromatography–mass spectrometry. Anal Bioanal Chem 378:875–882CrossRefGoogle Scholar
  18. 18.
    Stanczyk FZ, Lee JS, Santen RJ (2007) Standardization of steroid hormone assays: why, how, and when? Cancer Epidemiol Biomarkers Prev 16:1713–1719CrossRefGoogle Scholar
  19. 19.
    Ziegler RG, Faupel-Badger JM, Sue L, Fuhrman BJ, Falk RT, Boyd-Morin J, Henderson MK, Hoover RN, Veenstra TD, Keefer LK, Xu X (2010) A new approach to measure estrogen exposure and metabolism in epidemiologic studies. J Steroid Biochem Mol Biol 121:538–545CrossRefGoogle Scholar
  20. 20.
    Hauser B, Deschner T, Boesch C (2008) Development of a liquid chromatography–tandem mass spectrometry method for the determination of 23 endogenous steroids in small quantities of primate urine. J Chromatogr B 862:100–112CrossRefGoogle Scholar
  21. 21.
    Yang W, Regnierb FE, Slivac D, Adame J (2008) Stable isotope-coded quaternization for comparative quantification of estrogen metabolites by high-performance liquid chromatography–electrospray ionization mass spectrometry. J Chromatogr B 870:233–240CrossRefGoogle Scholar
  22. 22.
    Harwood DT, Handelsman DJ (2009) 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 409:78–84CrossRefGoogle Scholar
  23. 23.
    Santen RJ, Demers L, Ohorodnik S, Settlage J, Langecker P, Blanchettd D, Gosse PE, Wang S (2007) Superiority of gas chromatography/tandem mass spectrometry assay (GC/MS/MS) for estradiol for monitoring of aromatase inhibitor therapy. Steroids 72:666–671CrossRefGoogle Scholar
  24. 24.
    Zhao M, Bakera SD, Yan X, Zhao Y, Wright WW, Zirkinb BR, Jarow JP (2004) Simultaneous determination of steroid composition of human testicular fluid using liquid chromatography tandem mass spectrometry. Steroids 69:721–726CrossRefGoogle Scholar
  25. 25.
    Regal P, Vázquez BI, Franco CM, Cepeda A, Fente C (2009) Quantitative LC–MS/MS method for the sensitive and simultaneous determination of natural hormones in bovine serum. J Chromatogr B 877:2457–2464CrossRefGoogle Scholar
  26. 26.
    Taioli E, Im A, Xu X, Veenstra TD, Ahrendt G, Garte S (2010) Comparison of estrogens and estrogen metabolites in human breast tissue and urine. Reprod Biol Endocrinol 8:93–99CrossRefGoogle Scholar
  27. 27.
    Xia Y, Chang SW, Patel S, Bakhtiar R, Karanam B, Evans DC (2004) Trace level quantitation of deuterated 17β-estradiol and estrone in ovarietomized mouse plasma and brain using liquid chromatography/tandem mass spectrometry following dansylation reaction. Rapid Commun Mass Spectrom 18:1621–1628CrossRefGoogle Scholar
  28. 28.
    Xu X, Veenstra TD, Fox SD, Roman JM, Issaq HJ, Falk R, Saavedra JE, Keefer LK, Ziegler RG (2005) Measuring fifteen endogenous estrogens simultaneously in human urine by high-performance liquid chromatography-mass spectrometry. Anal Chem 77:6646–6654CrossRefGoogle Scholar
  29. 29.
    Hsu J, Chang Y, Chen T, Lin L, Liao P (2007) Evaluation of electrospray ionization and atmospheric pressure chemical ionization for simultaneous detection of estrone and its metabolites using high-performance liquid chromatography/tandem mass spectrometry. J Chromatogr B 860:49–56CrossRefGoogle Scholar
  30. 30.
    Li Q, Lam MHW, Wu RSS, Jiang B (2010) Rapid magnetic-mediated solid-phase extraction and pre-concentration of selected endocrine disrupting chemicals in natural waters by poly(divinylbenzene-co-methacrylic acid) coated Fe3O4 core-shell magnetite microspheres for their liquid chromatography–tandem mass spectrometry determination. J Chromatogr A 1217:1219–1226CrossRefGoogle Scholar
  31. 31.
    O’Brien Z, Post N, Brown M, Madan A, Coon T, Luo R, Kohout TA (2009) Validation and application of a liquid chromatography–tandem mass spectrometric method for the simultaneous determination of testosterone and dihydrotestosterone in rat prostatic tissue using a 96-well format. J Chromatogr B 877:3515–3521CrossRefGoogle Scholar
  32. 32.
    Yokokawa A, Yamamoto K, Omori Y, Shibasaki H, Shinohara Y, Kasuya Y, Furuta T (2009) Simultaneous determination of androstenedione, 11β-hydroxyandrostenedione, and testosterone in human plasma by stable isotope dilution mass spectrometry. J Chromatogr B 877:621–626CrossRefGoogle Scholar
  33. 33.
    Gomes RL, Avcioglu E, Scrimshaw MD, Lester JN (2004) Steroid estrogen determination in sediment and sewage sludge: a critique of sample preparation and chromatographic/mass spectrometry considerations, incorporating a case study in method development. Trends Anal Chem 23:737–744CrossRefGoogle Scholar
  34. 34.
    Zuo Y, Zhang K, Lin Y (2007) Microwave-accelerated derivatization for the simultaneous gas chromatographic–mass spectrometric analysis of natural and synthetic estrogenic steroids. J Chromatogr A 1148:211–218CrossRefGoogle Scholar
  35. 35.
    Xu X, Ziegler RG, Waterhouseb DJ, Saavedrac JE, Keefer LK (2002) Stable isotope dilution high-performance liquid chromatography–electrospray ionization mass spectrometry method for endogenous 2- and 4-hydroxyestrones in human urine. J Chromatogr B 780:315–330CrossRefGoogle Scholar
  36. 36.
    Xu X, Keefer LK, Waterhouse DJ, Saavedra JE, Veenstra TD, Ziegler RG (2004) Measuring seven endogenous ketolic estrogens simultaneously in human urine by high-performance liquid chromatography-mass spectrometry. Anal Chem 76:5829–5836CrossRefGoogle Scholar
  37. 37.
    Knust U, Strowitzki T, Spiegelhalder B, Bartsch H, Owen RW (2007) Optimization of an isotope dilution gas chromatography/mass spectrometry method for the detection of endogenous estrogen metabolites in urine samples. Rapid Commun Mass Spectrom 21:2245–2254CrossRefGoogle Scholar
  38. 38.
    Moon J, Jung H, Moon M, Chung B, Choi M (2008) Inclusion complex-based solid-phase extraction of steroidal compounds with entrapped β-cycodextrin polymer. Steroids 73:1090–1097CrossRefGoogle Scholar
  39. 39.
    Moon J, Kim K, Moon M, Chung B, Choi M (2011) A novel GC-MS method in urine estrogen analysis from postmenopausal women with osteoporosis. J Lipid Res 52:1595–1603CrossRefGoogle Scholar
  40. 40.
    Kumar V, Nakada N, Yasojima M, Yamashita N, Johnson AC, Tanaka H (2009) Rapid determination of free and conjugated estrogen in different water matrices by liquid chromatography–tandem mass spectrometry. Chemosphere 77:1440–1446CrossRefGoogle Scholar
  41. 41.
    Nguyen HP, Yang SH, Wigginton JG, Simpkins JW, Schug KA (2010) Retention behavior of estrogen metabolites on hydrophilic interaction chromatography stationary phases. J Sep Sci 33:793–802CrossRefGoogle Scholar
  42. 42.
    Ramanathan R, Cao K, Cavalieri E, Gross ML (1998) Mass spectrometric methods for distinguishing structural isomers of glutathione conjugates of estrone and estradiol. J Am Soc Mass Spectrom 9:612–619CrossRefGoogle Scholar
  43. 43.
    Reddy S, Iden CR, Brownawell BJ (2005) Analysis of steroid conjugates in sewage influent and effluent by liquid chromatography-tandem mass spectrometry. Anal Chem 77:7032–7038CrossRefGoogle Scholar
  44. 44.
    Schlusener MP, Bester K (2005) Determination of steroid hormones, hormone conjugates and macrolide antibiotics in influents and effluents of sewage treatment plants utilizing high-performance liquid chromatography/tandem mass spectrometry with electrospray and atmospheric pressure chemical ionization. Rapid Commun Mass Spectrom 19:3269–3278CrossRefGoogle Scholar
  45. 45.
    Liu Z, Kanjo Y, Mizutani S (2011) Removal of natural free estrogens and their conjugates in a municipal wastewater treatment plant. Clean – Soil, Air, Water 39:128–135CrossRefGoogle Scholar
  46. 46.
    Hauser B, Mugisha L, Preis A, Deschner T (2011) LC–MS analysis of androgen metabolites in serum and urine from east African chimpanzees (Pan troglodytes schweinfurthii). Gen Comp Endocrinol 170:92–98CrossRefGoogle Scholar
  47. 47.
    Tang PW, Law WC, Wan TSM (2001) Analysis of corticosteroids in equine urine by liquid chromatography-mass spectrometry. J Chromatogr B 754:229–244CrossRefGoogle Scholar
  48. 48.
    Moon J, Ha Y, Moon M, Chung B, Choi M (2010) Systematic error in gas chromatography–mass spectrometry based quantitation of hydrolyzed urinary steroids. Cancer Epidemiol Biomarkers Prev 19:388–397CrossRefGoogle Scholar
  49. 49.
    Zhang H, Henion J (1999) Quantitative and qualitative determination of estrogen sulfates in human urine by liquid chromatography/tandem mass spectrometry using 96-well technology. Anal Chem 71:3955–3964CrossRefGoogle Scholar
  50. 50.
    Qin F, Zhao Y, Sawyer MB, Li X (2008) Hydrophilic interaction liquid chromatography-tandem mass spectrometry determination of estrogens conjugates in human urine. Anal Chem 80:3404–3411CrossRefGoogle Scholar
  51. 51.
    Qin F, Zhao Y, Sawyer MB, Li X (2008) Column-switching reversed phase–hydrophilic interaction liquid chromatography/tandem mass spectrometry method for determination of free estrogens and their conjugates in river water. Anal Chim Acta 627:91–98CrossRefGoogle Scholar
  52. 52.
    Tso J, Aga DS (2010) A systematic investigation to optimize simultaneous extraction and liquid chromatography tandem mass spectrometry analysis of estrogens and their conjugated metabolites in milk. J Chromatogr A 1217:4784–4795CrossRefGoogle Scholar
  53. 53.
    Yan W, Zhao L, Feng Q, Wei Y, Lin J (2009) Simultaneous determination of ten estrogens and their metabolites in waters by improved two-step SPE followed by LC–MS. Chromatographia 69:621–628CrossRefGoogle Scholar
  54. 54.
    Nishio T, Higashi T, Funaishi A, Tanaka J, Shimada K (2007) Develpment and application of electrospray-active derivatization reagents for hydroxysteroids. J Pharm Biomed Anal 44:786–795CrossRefGoogle Scholar
  55. 55.
    Penning TM, Lee S, Jin Y, Gutierrez A, Blair IA (2010) Liquid chromatography-mass spectrometry (LC-MS) of teroid hormone metabolites and its applications. J Steroid Biochem Mol Biol 121:546–555CrossRefGoogle Scholar
  56. 56.
    Johnson DW (2005) Ketosteroid profiling using Girard T derivatives and electrospray ionization tandem mass spectrometry: direct plasma analysis of androstenedione, 17-hydroxyprogesterone and cortisol. Rapid Commun Mass Spectrom 19:193–200CrossRefGoogle Scholar
  57. 57.
    Hala D, Overturf MD, Petersen LH, Huggett DB (2011) Quantification of 2-hydrazinopyridine derivatized steroid hormones in fathead minnow (pimephales promelas) blood plasma using LC-ESI+/MS/MS. J Chromatogr B 879:591–598CrossRefGoogle Scholar
  58. 58.
    Griffiths WJ, Liu S, Alvelius G, Sjovall J (2003) Derivatization for the characterisation of neutral oxosteroids by electrospray and matrix-assisted laser desporption/ionization tandem mass spectrometry: the Girard P derivatives. Rapid Commun Mass Spectrom 17:924–935CrossRefGoogle Scholar
  59. 59.
    Arai S, Miyashiro Y, Shibata Y, Kashiwagi B, Tomaru Y, Kobayashi M, Watanabe Y, Honma S, Suzuki K (2010) New quantification method for estradioal in the prostatic tissues of nenign prostatic hyperplasia using liquid chromatogrophy-dandem mass spectromentry. Steroids 75:13–19CrossRefGoogle Scholar
  60. 60.
    Higashi T, Takayama N, Kyutoku M, Shimada K, Kohb E, Namiki M (2006) Liquid chromatography–mass spectrometric assay of androstenediol in prostatic tissue: influence of androgen deprivation therapy on its level. Steroids 71:1007–1013CrossRefGoogle Scholar
  61. 61.
    Lin Y, Chen C, Wang G (2007) Analysis of steroid estrogens in water using liquid chromatography/tandem mass spectrometry with chemical derivatizations. Rapid Commun Mass Spectrom 21:1973–1983CrossRefGoogle Scholar
  62. 62.
    Xu L, Spink DC (2008) Analysis of steroidal estrogens as pyridine-3-sulfonyl derivatives by liquid chromatography electrospray tandem mass spectrometry. Anal Biochem 375:105–114CrossRefGoogle Scholar
  63. 63.
    You J, Zhao H, Sun Z, Suo Y, Chen G (2009) 10-Ethyl-acridine-2-sulfonyl chloride: a new derivatization agent for enhancement of atmospheric pressure chemical ionization of estrogens in urine. Chromatographia 70:45–55CrossRefGoogle Scholar
  64. 64.
    Yamashita K, Okuyama M, Watanabe Y, Honma S, Kobayashi S, Numazawa M (2007) Highly sensitive determination of estrone and estradiol in human serum by liquid chromatography-electrospray ionization tandem mass spectrometry. Steroids 72:819–827CrossRefGoogle Scholar
  65. 65.
    Giton F, Caron P, Bérubé R, Bélanger A, Barbier O, Fiet J (2010) Plasma estrone sulfate assay in men: comparison of radioimmunoassay, mass spectrometry coupled to gas chromatography (GC–MS), and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Clin Chim Acta 411:1208–1213CrossRefGoogle Scholar
  66. 66.
    Mitamura K, Yatera M, Shimada K (2000) Studies on neurosteroids Part XIII. Characterization of catechol estrogens in rat brains using liquid chromatography-mass spectrometry-mass spectrometry. Analyst 125:811–814CrossRefGoogle Scholar
  67. 67.
    Yamashita K, Masuda A, Hoshino Y, Komatsu S, Numazawa M (2010) Assay of labile estrogen o-quinones, potent carcinogenic molecular species, by high performance liquid chromatography–electrospray ionization tandem mass spectrometry with phenazine derivatization. J Steroid Biochem Mol Biol 119:141–148CrossRefGoogle Scholar
  68. 68.
    Díaz-Cruz MS, López de Alda MJ, López R, Barceló D (2003) Determination of estrogens and progestogens by mass spectrometry techniques (GC/MS, LC/MS and LC/MS/MS). J Mass Spectrom 38:917–923CrossRefGoogle Scholar
  69. 69.
    Cheng C, Hou J, Wang S, Xu B, Liu S, Yan Z (2011) Development and validation of an LC-MS/MS method for determination of fifteen estrogens and metabolites in human serum. In: Pittsburgh conference, Atlanta, GA, 18 March 2011Google Scholar
  70. 70.
    Nordstrom A, O’Maille G, Qin C, Siuzdak G (2006) Nonlinear data alignment for UPLC-MS and HPLC-MS based metabolomics: quantitative analysis of endogenous and exogenous metabolites in human serum. Anal Chem 78:3289–3295CrossRefGoogle Scholar
  71. 71.
    Novakoca L, Matysova L, Solich P (2006) Advantages of application of UPLC in pharmaceutical analysis. Talanta 68:908–918CrossRefGoogle Scholar
  72. 72.
    Zelena E, Dunn WB, Broadhurst D, Francis-McIntyre S, Carroll KM, Begley P, O’Hagan S, Knowles JD, Halsall A, Consortium H, Wilson ID, Kell DB (2009) Development of a robust and repeatable UPLC-MS method for the long-term metabolomic study of human serum. Anal Chem 81:1357–1364CrossRefGoogle Scholar
  73. 73.
    Matějíček D (2011) On-line two-dimensional liquid chromatography–tandem mass spectrometric determination of estrogens in sediments. J Chromatogr A 1218:2292–2300CrossRefGoogle Scholar
  74. 74.
    Lien G, Chen C, Wang G (2009) Comparison of electrospray ionization, atmospheric pressure chemical ionization and atmospheric pressure photoionization for determining estrogenic chemicals in water by liquid chromatography tandem mass spectrometry with chemical derivatizations. J Chromatogr A 1216:956–966CrossRefGoogle Scholar
  75. 75.
    Xu X, Roman JM, Veenstra TD, Van Anda J, Ziegler RG, Issaq HJ (2006) Analysis of fifteen estrogen metabolites using packed column supercritical fluid chromatography-mass spectrometry. Anal Chem 78:1553–1558CrossRefGoogle Scholar
  76. 76.
    Labadie P, Hill EM (2007) Analysis of estrogens in river sediments by liquid chromatography–electrospray ionisation mass spectrometry—comparison of tandem mass spectrometry and time-of-flight mass spectrometry. J Chromatogr A 1141:174–181CrossRefGoogle Scholar
  77. 77.
    U.S. Environmental protection agency method 539: determination of hormones in drinking water by solid phase extraction (SPE) and liquid chromatography electrospray ionization tandem mass spectrometry (LC-MS/MS). EPA document No. 815-B-10-001, November 2010.
  78. 78.
    Mouatassim-Souali A, Tamisier-Karolak S, Perdiz D, Cargouet M, Levi Y (2003) Validation of a quantitative assay using GC/MS for trace determination of free and conjugated estrogens in environmental water samples. J Sep Sci 26:105–111CrossRefGoogle Scholar
  79. 79.
    Zhou Y, Zhou J, Xu Y, Zha J, Ma M, Wang Z (2009) An alternative method for the determination of estrogens in surface water and wastewater treatment plant effluent using pre-column trimethylsilyl derivatization and gas chromatography/mass spectrometry. Environ Monit Assess 158:35–49CrossRefGoogle Scholar
  80. 80.
    Hsing AW, Stanczyk FZ, Bélanger A, Schroeder P, Chang L, Falk RT, Fears TR (2007) Reproducibility of serum sex steroid assays in men by RIA and mass spectrometry. Cancer Epidemiol Biomarkers Prev 16:1004–1008CrossRefGoogle Scholar
  81. 81.
    Myers GL (2008) Introduction to standardization of laboratory results. Steroids 73:1293–1296CrossRefGoogle Scholar
  82. 82.
    Rosner W, Vesper H (2008) CDC workshop report improving steroid hormone measurements in patient care and translation research. Steroids 7:1285CrossRefGoogle Scholar
  83. 83.
    Vesper HW, Botelho JC, Shacklady C, Smith A, Myers GL (2008) CDC project on standardizing steroid hormone measurements. Steroids 73:1286–1292CrossRefGoogle Scholar
  84. 84.
    Tai SS-C, Welch MJ (2005) Development and evaluation of a reference measurement procedure for the determination of estradiol-17β in human serum using ID-LC/MS/MS. Anal Chem 77:6359–6363CrossRefGoogle Scholar
  85. 85.
    Tai SS-C, Xu B, Welch MJ, Phinney KW (2007) 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 388:1087–1094CrossRefGoogle Scholar
  86. 86.
    Tai SS-C, Xu B, Welch MJ (2006) Development and evaluation of a candidate reference measurement procedure for the determination of progesterone in human serum using ID-LC/MS/MS. Anal Chem 78:6628–6633CrossRefGoogle Scholar
  87. 87.
    Peters FT, Maurer HH (2002) Bioanalytical method validation and its implications for forensic and clinical toxicology—a review. Accred Qual Assur 7:441–449CrossRefGoogle Scholar
  88. 88.
    Nelson RE, Grebe SK, O’Kane DJ, Singh RJ (2004) Liquid chromatography mass spectrometry assay for simultaneous measurement of estradiol and estrone in human plasma. Clin Chem 50:373–384CrossRefGoogle Scholar
  89. 89.
    Lai C, Tsai C, Tsai F, Wu J, Lin W, Lee C (2002) Monitoring of congenital adrenal hyperplasia by microbore HPLC–electrospray ionization tandem mass spectrometry of dried blood spots. Clin Chem 48:354–356Google Scholar
  90. 90.
    Naessen T, Sjogren U, Bergquist J, Larsson M, Lind L, Kushnir MM (2010) Endogenous steroids measured by high-specificity liquid chromatography-tandem mass spectrometry and prevalent cardiovascular disease in 70-year-old men and women. J Clin Endocrinol Metab 95:1889–1897CrossRefGoogle Scholar
  91. 91.
    Santen RJ, Lee JS, Wang S, Demers LM, Mauras N, Wang H et al (2008) Potential role of ultra-sensitive estradiol assays in estimating the risk of breast cancer and fractures. Steroids 73:1318–1321CrossRefGoogle Scholar
  92. 92.
    Ionita IA, Akhlaghi F (2010) Quantification of unbound prednisolone, prednisone, cortisol and cortisone in human plasma by ultrafiltration and direct injection into liquid chromatography tandem mass spectrometry. Ann Clin Biochem 47:350–357CrossRefGoogle Scholar
  93. 93.
    Nguyen HP, Li L, Gatson JW, Maass D, Wigginton JG, Simpkins JW, Schug KA (2011) Simultaneous quantification of four native estrogen hormones at trace levels in human cerebrospinal fluid using liquid chromatography–tandem mass spectrometry. J Pharm Biomed Anal 54:830–837CrossRefGoogle Scholar
  94. 94.
    Xu X, Othman ER, Issaq HJ, Hornung D, Al-Hendy A, Veenstra TD (2008) Multiplexed quantitation of endogenous estrogens and estrogen metabolites in human peritoneal fluid. Electrophoresis 29:2706–2713CrossRefGoogle Scholar
  95. 95.
    Nielen MWF, van Bennekom EO, Heskamp HH, van Rhijn JA, Bovee TFH, Hoogenboom LAP (2004) Bioassay-directed identification of estrogen residues in urine by liquid chromatography electrospray quadrupole time-of-flight mass spectrometry. Anal Chem 76:6600–6608CrossRefGoogle Scholar
  96. 96.
    Xu X, Keefer LK, Ziegler RG, Veenstra TD (2007) A liquid chromatography–mass spectrometry method for the quantitative analysis of urinary endogenous estrogen metabolites. Nat Protoc 2:1350–1355CrossRefGoogle Scholar
  97. 97.
    Wilkinson AP, Wahala K, Williamson G (2002) Identification and quantitation of polyphenol phytoestrogens in foods and human biological fluids. J Chromatogr B 777:93–109CrossRefGoogle Scholar
  98. 98.
    Bowers LD (1997) Analytical advances in detection of performance-enhancing compounds. Clin Chem 43:1299–1304Google Scholar
  99. 99.
    Peng L, Farcase T, McGinley Identification of steroids in urine and plasma by LC/MS/MS using strata X and Gemini C18. Application note: TN-1026.
  100. 100.
    Ingrand V, Herry G, Beausse J, de Roubin M (2003) Analysis of steroid hormones in effluents of wastewater treatment plants by liquid chromatography-tandem mass spectrometry. J Chromatogr A 1020:99–104CrossRefGoogle Scholar
  101. 101.
    Habauzit D, Armentgaud J, Roig B, Chopineau J (2008) Determination of estrogen presence in water by SPR using estrogen receptor dimerization. Anal Bioanal Chem 390:873–883CrossRefGoogle Scholar
  102. 102.
    Miége C, Bados P, Brosse C, Coquery M (2009) Method validation for the analysis of estrogens (including conjugated compounds) in aqueous matrices. Trends Anal Chem 28:237–244CrossRefGoogle Scholar
  103. 103.
    Grover DP, Zhanga ZL, Readman JW, Zhou JL (2009) A comparison of three analytical techniques for the measurement of steroidal estrogens in environmental water samples. Talanta 78:1204–1210CrossRefGoogle Scholar


  1. International Conference on Harmonisation of Technical Requirements for Registrations of Pharmaceuticals for Human Use, ICH Harmonised Tripartite Guideline, Validation of Analytical Procedures: Text and Methodology, Q2(R1), Current Step 4 version, 2005, 13 pp.
  2. U.S. Food and Drug Administration CFR—Code of Federal Regulations Title 21, Part 58—Good laboratory practice for nonclinical laboratory studies.
  3. U.S. Food and Drug Administration, Center for Drug Evaluation and Research (CDER), Center for Veterinary Medicine (CVM), May 2001, Guidance for industry—bioanalytical method validation.
  4. The United States Pharmacopeia–National Formulary (USP–NF), USP34-NF29 S1. General Requirements/<11>USP Reference Standards, pp 38–40. Pharmacopeial Forum 35(6):1507Google Scholar
  5. The United States Pharmacopeia–National Formulary (USP–NF), USP34-NF29 S1. General Information/<1225>Validation of Compendial Procedures, pp 779–782. Pharmacopeial Forum 35(2):444Google Scholar
  6. U.S. Environmental protection agency CFR—Code of Federal Regulations Title 40—Protection of Environment PART 792—Good laboratory practice standards.
  7. U.S. Environmental protection agency, Chemical QC guidelines.
  8. The National Institute of Standards and Technology, Analytical chemistry division, Development of Reference Methods and Reference Materials for the Determination of Hormones in Human Serum.
  9. The National Institute of Standards and Technology, Taylor BN, Kuyatt CE (1994) Guidelines for evaluating and expressing the uncertainty of NIST measurement results. NIST Technical Note 1297, pp 1–20.
  10. Thomson M, Ellison SLR, Wood R (2002) International union of pure and applied chemistry, harmonized guidelines for single laboratory validation of method of analysis. Pure Appl Chem74:835–855.

International Organization of Standardization, Guide:

  1. ISO Guide 30:1992 Terms and definitions used in connection with reference materialsGoogle Scholar
  2. ISO Guide 30:1992/Amd 1:2008 Revision of definitions for reference material and certified reference materialGoogle Scholar
  3. ISO Guide 31:2000 Reference materials—contents of certificates and labelsGoogle Scholar
  4. ISO Guide 32:1997 Calibration in analytical chemistry and use of certified reference materialsGoogle Scholar
  5. ISO Guide 33:2000 Uses of certified reference materialsGoogle Scholar
  6. ISO Guide 34:2009 General requirements for the competence of reference material producersGoogle Scholar
  7. ISO Guide 35:2006 Reference materials—general and statistical principles for certification.

International Organization of Standardization, Published:

  1. ISO 5725-1:1994 Accuracy (trueness and precision) of measurement methods and results—Part 1: general principles and definitionsGoogle Scholar
  2. ISO 5725-2:1994 Accuracy (trueness and precision) of measurement methods and results—Part 2: basic method for the determination of repeatability and reproducibility of a standard measurement methodGoogle Scholar
  3. ISO 5725-3:1994 Accuracy (trueness and precision) of measurement methods and results—Part 3: intermediate measures of the precision of a standard measurement methodGoogle Scholar
  4. ISO 5725-4:1994 Accuracy (trueness and precision) of measurement methods and results—Part 4: basic methods for the determination of the trueness of a standard measurement methodGoogle Scholar
  5. ISO 5725-5:1998 Accuracy (trueness and precision) of measurement methods and results—Part 5: alternative methods for the determination of the precision of a standard measurement methodGoogle Scholar
  6. ISO 5725-6:1994 Accuracy (trueness and precision) of measurement methods and results—Part 6: use in practice of accuracy values.

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  1. 1.Research Pharmaceutical Services, Inc.Fort WashingtonUSA
  2. 2.Analytical DevelopmentAlbany Molecular Research, Inc.RensselaerUSA
  3. 3.Department of Chemistry and BiochemistryUniversity of OklahomaNormanUSA

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