Original research article| Volume 99, ISSUE 4, P244-250, April 2019

A sensitive and robust UPLC–MS/MS method for quantitation of estrogens and progestogens in human serum



      With the widespread use of sex-steroid hormones in contraceptives and hormone replacement therapy, there is an increasing need for reliable analytical methods. We report the development of a sensitive and robust UPLC–MS/MS method for quantitation of both endogenous and synthetic sex-steroid hormones in human serum.

      Study design

      We developed and validated a UPLC–MS/MS method to quantify progestogens (etonogestrel, levonorgestrel, medroxyprogesterone acetate, norethindrone, progesterone) and estrogens (estradiol and ethinyl estradiol) with good accuracy, high sensitivity, and excellent robustness. We then applied the method to the analysis of sex-steroid hormones in serum from 451 clinical research participants.


      Each UPLC–MS/MS analysis was 6.5 min. The lower limits of quantitation (LLOQs) were 25 pg/ml for the progestogens, and 2.5 and 5.0 pg/ml for estradiol and ethinyl estradiol, respectively. When estradiol was analyzed without assessment of progestogens, the LLOQ was reduced to 1 pg/ml. The calibration curves were linear from 25–50,000, 2.5–2000 (1–2000 for estrogens-only analysis) and 5–2000 pg/ml, respectively. Both the accuracy and precision were below±15% not only for routine validation (intraday and interday), but for long-term (>2 years) assay robustness with external controls, thereby, demonstrating the utility of this method for multi-year clinical trial assessments of progestogens and estrogens. We applied the method to quantify sex-steroid levels in 1804 clinical samples.


      We successfully developed a UPLC-MS/MS method, and overcame the matrix suppression to allow sensitive quantitation of both synthetic and endogenous sex-steroid hormones in human serum.


      We developed a sensitive and robust UPLC-MS/MS method to accurately measure the levels of sex-steroid hormones in serum. The method overcame matrix interference barriers and achieved excellent long-term stability and reproducibility (≥96.9% accuracy; ≤13.0% relative variability measured with external controls over 2 years), demonstrating its utility in clinical sample analysis.


      To read this article in full you will need to make a payment

      Purchase one-time access:

      Academic & Personal: 24 hour online accessCorporate R&D Professionals: 24 hour online access
      One-time access price info
      • For academic or personal research use, select 'Academic and Personal'
      • For corporate R&D use, select 'Corporate R&D Professionals'


      Subscribe to Contraception
      Already a print subscriber? Claim online access
      Already an online subscriber? Sign in
      Institutional Access: Sign in to ScienceDirect


        • Chao J.H.
        • Page S.T.
        The current state of male hormonal contraception.
        Pharmacol Ther. 2016; 163: 109-117
        • De Leo V.
        • Musacchio M.C.
        • Cappelli V.
        • Piomboni P.
        • Morgante G.
        Hormonal contraceptives: pharmacology tailored to women's health.
        Hum Reprod Update. 2016; 22: 634-646
        • Lobo R.A.
        Hormone-replacement therapy: current thinking.
        Nat Rev Endocrinol. 2017; 13: 220-231
        • Abraham G.E.
        Solid-phase radioimmunoassay of estradiol-17β.
        J Clin Endocrinol Metabol. 1969; 29: 866-870
        • Hanquez C.
        • Urios P.
        • Desfosses B.
        • Samake H.
        • Lince E.
        • Rajkowski K.M.
        • et al.
        Enzyme-linked immunosorbent assay (ELISA) for steroid hormones with polyclonal and monoclonal antibodies: an assay for urinary aldosterone.
        Clin Chim Acta. 1987; 164: 71-82
        • Soldin S.J.
        • Soldin O.P.
        Steroid hormone analysis by tandem mass spectrometry.
        Clin Chem. 2009; 55: 1061-1066
        • Stanczyk F.Z.
        • Clarke N.J.
        Advantages and challenges of mass spectrometry assays for steroid hormones.
        J Steroid Biochem Mol Biol. 2010; 121: 491-495
        • Stanczyk F.Z.
        • Jurow J.
        • Hsing A.W.
        Limitations of direct immunoassays for measuring circulating estradiol levels in postmenopausal women and men in epidemiologic studies.
        Cancer Epidemiol Biomarkers Prev. 2010; 19: 903-906
        • Sweeley C.C.
        • Horning E.C.
        Microanalytical separation of steroids by gas chromatography.
        Nature. 1960; 187: 144-145
        • Stanczyk F.Z.
        • Lee J.S.
        • Santen R.J.
        Standardization of steroid hormone assays: why, how, and when?.
        Cancer Epidemiol Biomarkers Prev. 2007; 16: 1713-1719
        • Ke Y.
        • Bertin J.
        • Gonthier R.
        • Simard J.N.
        • Labrie F.
        A sensitive, simple and robust LC-MS/MS method for the simultaneous quantification of seven androgen- and estrogen-related steroids in postmenopausal serum.
        J Steroid Biochem Mol Biol. 2014; 144: 523-534
        • Keevil B.G.
        LC-MS/MS analysis of steroids in the clinical laboratory.
        Clin Biochem. 2016; 49: 989-997
        • Anari M.R.
        • Bakhtiar R.
        • Zhu B.
        • Huskey S.
        • Franklin R.B.
        • Evans D.C.
        Derivatization of ethinylestradiol with dansyl chloride to enhance electrospray ionization: application in trace analysis of ethinylestradiol in rhesus monkey plasma.
        Anal Chem. 2002; 74: 4136-4144
        • Nelson R.E.
        • Grebe S.K.
        • OK D.J.
        • Singh R.J.
        Liquid chromatography-tandem mass spectrometry assay for simultaneous measurement of estradiol and estrone in human plasma.
        Clin Chem. 2004; 50: 373-384
        • Li W.
        • Li Y.H.
        • Li A.C.
        • Zhou S.
        • Naidong W.
        Simultaneous determination of norethindrone and ethinyl estradiol in human plasma by high performance liquid chromatography with tandem mass spectrometry--experiences on developing a highly selective method using derivatization reagent for enhancing sensitivity.
        J Chromatogr B Analyt Technol Biomed Life Sci. 2005; 825: 223-232
        • Achilles S.L.
        • Mhlanga F.G.
        • Musara P.
        • Poloyac S.M.
        • Chirenje Z.M.
        • Hillier S.L.
        Misreporting of contraceptive hormone use in clinical research participants.
        Contraception. 2018; 97: 346-353
      1. Food and Drug Adminstration guidance for industry-bioanalytical method validation. 2013
        • Koal T.
        • Schmiederer D.
        • Pham-Tuan H.
        • Rohring C.
        • Rauh M.
        Standardized LC-MS/MS based steroid hormone profile-analysis.
        J Steroid Biochem Mol Biol. 2012; 129: 129-138
        • Methlie P.
        • Hustad S.S.
        • Kellmann R.
        • Almas B.
        • Erichsen M.M.
        • Husebye E.
        • et al.
        Multisteroid LC-MS/MS assay for glucocorticoids and androgens, and its application in Addison's disease.
        Endocr Connect. 2013; 2: 125-136
        • Gandhi A.
        • Guttikar S.
        • Trivedi P.
        High-sensitivity simultaneous liquid chromatography–tandem mass spectrometry assay of ethinyl estradiol and levonorgestrel in human plasma.
        J Pharm Anal. 2015; 5: 316-326
        • Kushnir M.M.
        • Blamires T.
        • Rockwood A.L.
        • Roberts W.L.
        • 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: 1138-1147
        • Guedes-Alonso R.
        • Ciofi L.
        • Sosa-Ferrera Z.
        • Santana-Rodriguez J.J.
        • Bubba M.D.
        • Kabir A.
        • et al.
        Determination of androgens and progestogens in environmental and biological samples using fabric phase sorptive extraction coupled to ultra-high performance liquid chromatography tandem mass spectrometry.
        J Chromatogr A. 2016; 1437: 116-126
        • Kushnir M.M.
        • Rockwood A.L.
        • Bergquist J.
        • Varshavsky M.
        • Roberts W.L.
        • Yue B.
        • et al.
        High-sensitivity tandem mass spectrometry assay for serum estrone and estradiol.
        Am J Clin Pathol. 2008; 129: 530-539
        • Blue S.W.
        • Winchell A.J.
        • Kaucher A.V.
        • Lieberman R.A.
        • Gilles C.T.
        • Pyra M.N.
        • et al.
        Simultaneous quantitation of multiple contraceptive hormones in human serum by LC-MS/MS.
        Contraception. 2018; 97: 363-369
        • Zhao M.
        • Baker S.D.
        • Yan X.
        • Zhao Y.
        • Wright W.W.
        • Zirkin B.R.
        • et al.
        Simultaneous determination of steroid composition of human testicular fluid using liquid chromatography tandem mass spectrometry.
        Steroids. 2004; 69: 721-726
        • Guo T.
        • Taylor R.L.
        • Singh R.J.
        • Soldin S.J.
        Simultaneous determination of 12 steroids by isotope dilution liquid chromatography-photospray ionization tandem mass spectrometry.
        Clin Chim Acta. 2006; 372: 76-82
        • Regal P.
        • Vazquez B.I.
        • Franco C.M.
        • Cepeda A.
        • Fente C.
        Quantitative LC-MS/MS method for the sensitive and simultaneous determination of natural hormones in bovine serum.
        J Chromatogr B Analyt Technol Biomed Life Sci. 2009; 877: 2457-2464
        • Rothman M.S.
        • Carlson N.E.
        • 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: 177-182
        • Koren L.
        • Ng E.S.
        • Soma K.K.
        • Wynne-Edwards K.E.
        Sample preparation and liquid chromatography-tandem mass spectrometry for multiple steroids in mammalian and avian circulation.
        PLoS One. 2012; 7e32496
        • Moeller B.C.
        • Stanley S.D.
        The development and validation of a turbulent flow chromatography-tandem mass spectrometry method for the endogenous steroid profiling of equine serum.
        J Chromatogr B Analyt Technol Biomed Life Sci. 2012; 905: 1-9
        • Keefe C.C.
        • Goldman M.M.
        • Zhang K.
        • Clarke N.
        • Reitz R.E.
        • Welt C.K.
        Simultaneous measurement of thirteen steroid hormones in women with polycystic ovary syndrome and control women using liquid chromatography-tandem mass spectrometry.
        PLoS One. 2014; 9e93805
        • Weisser J.J.
        • Hansen C.H.
        • Poulsen R.
        • Larsen L.W.
        • Cornett C.
        • Styrishave B.
        Two simple cleanup methods combined with LC-MS/MS for quantification of steroid hormones in in vivo and in vitro assays.
        Anal Bioanal Chem. 2016; 408: 4883-4895
        • Eisenhofer G.
        • Peitzsch M.
        • Kaden D.
        • Langton K.
        • Pamporaki C.
        • Masjkur J.
        • et al.
        Reference intervals for plasma concentrations of adrenal steroids measured by LC-MS/MS: impact of gender, age, oral contraceptives, body mass index and blood pressure status.
        Clin Chim Acta. 2017; 470: 115-124
        • Hines J.M.
        • Bancos I.
        • Bancos C.
        • Singh R.D.
        • Avula A.V.
        • Young W.F.
        • et al.
        High-resolution, accurate-mass (HRAM) mass spectrometry urine steroid profiling in the diagnosis of adrenal disorders.
        Clin Chem. 2017; 63: 1824-1835
        • McCulloch R.D.
        • Robb D.B.
        Field-free atmospheric pressure photoionization-liquid chromatography-mass spectrometry for the analysis of steroids within complex biological matrices.
        Anal Chem. 2017; 89: 4169-4176
        • Crago E.A.
        • Sherwood P.R.
        • Bender C.
        • Balzer J.
        • Ren D.
        • Poloyac S.M.
        Plasma estrogen levels are associated with severity of injury and outcomes after aneurysmal subarachnoid hemorrhage.
        Biol Res Nurs. 2015; 17: 558-566
        • Thurston R.C.
        • Chang Y.
        • Barinas-Mitchell E.
        • Jennings J.R.
        • Landsittel D.P.
        • Santoro N.
        • et al.
        Menopausal hot flashes and carotid intima media thickness among midlife women.
        Stroke. 2016; 47: 2910-2915
        • Thurston R.C.
        • Chang Y.
        • Barinas-Mitchell E.
        • Jennings J.R.
        • von Kanel R.
        • Landsittel D.P.
        • et al.
        Physiologically assessed hot flashes and endothelial function among midlife women.
        Menopause. 2017; 24: 886-893
        • Chappell C.A.
        • Lamorde M.
        • Nakalema S.
        • Chen B.A.
        • Mackline H.
        • Riddler S.A.
        • et al.
        Efavirenz decreases etonogestrel exposure: a pharmacokinetic evaluation of implantable contraception with antiretroviral therapy.
        AIDS. 2017; 31: 1965-1972
        • Li J.
        • Oberly P.J.
        • Poloyac S.M.
        • Gibbs R.B.
        A microsomal based method to detect aromatase activity in different brain regions of the rat using ultra performance liquid chromatography-mass spectrometry.
        J Steroid Biochem Mol Biol. 2016; 163: 113-120
        • Polis C.B.
        • Curtis K.M.
        • Hannaford P.C.
        • Phillips S.J.
        • Chipato T.
        • Kiarie J.N.
        • et al.
        An updated systematic review of epidemiological evidence on hormonal contraceptive methods and HIV acquisition in women.
        AIDS. 2016; 30: 2665-2683