The experience in the development and validation of method for testosterone measurement in blood serum of premenopausal women using HPLC-MS/MS

Testosterone assessment is essential for detecting biochemical hyperandrogenism, one of the important diagnostic criteria of polycystic ovary syndrome (PCOS) both in clinical practice and in epidemiological studies. Currently, tandem liquid chromatography-mass spectrometry (LC-MS/MS) is the most preferred technique to measure testosterone level in women. Its validation is important to reproducibility of androgen tests results for clinical practice and for epidemiological studies of the prevalence PCOS.
The aim of the study. To develop and validate a method for determining total testosterone in blood serum using highly efficient LC-MS/MS to assess androgenemia in the epidemiological study of the prevalence of PCOS and its phenotypes in Eastern Siberia (ESPEP STUDY).
Materials and methods. We determined a total testosterone level in serum blood using triple quadrupole mass spectrometer LCMS-8060 (Shimadzu, Japan). The protocol of technique was developed using self-prepared purified human testosteronefree serum with a known concentration of analyzed compound. We used the serum samples of women of reproductive age to test the developed method.
Results. Optimum chromatographic conditions were obtained with a Kromasil 100-2.5-C18 column (2.1 mm × 100 mm; AkzoNobel, Netherlands), and an isocratic elution mode using a mobile phase consisting of acetonitrile and 0.1 % aqueous solution of formic acid. The total flow rate was 0.35 ml/min. The lower limit of quantification was 5 ng/dl with an average accuracy of 100.2 %. During the approbation of the method in a test population sample of 1138 premenopausal women (mean age – 34.3 ± 6.3 years), the median testosterone concentration was 26.9 ng/dl.
Conclusion. It was found that the proposed method for determining testosterone in blood serum has acceptable linearity and reproducibility and meets the requirements for bioanalytical methods under the regulatory documentation. This method can be used for clinical practice and epidemiological study of the prevalence of PCOS.

1. Lizneva D, Suturina L, Walker W, Brakta S, Gavrilova-Jordan L, Azziz R. Criteria, prevalence, and phenotypes of polycystic ovary syndrome. Fertil Steril. 2016; 106(1): 6-15. doi: 10.1016/j.fertnstert.2016.05.003

2. Teede HJ, Misso ML, Costello MF, Dokras A, Laven J, Moran L, et al. Recommendations from the international evidence-based guideline for the assessment and management of polycystic ovary syndrome. Fertil Steril. 2018; 110(3): 364-379. doi: 10.1016/j.fertnstert.2018.05.004

3. 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(1): 28-38.e25. doi: 10.1016/j.fertnstert.2011.09.024

4. Lizneva D, Gavrilova-Jordan L, Walker W, Azziz R. Androgen excess: Investigations and management. Best Pract Res Clin Obstet Gynaecol. 2016; 37: 98-118. doi: 10.1016/j.bpobgyn.2016.05.003

5. Bozdag G, Mumusoglu S, Zengin D, Karabulut E, Yildiz BO. The prevalence and phenotypic features of polycystic ovary syndrome: A systematic review and meta-analysis. Hum Reprod. 2016; 31(12): 2841-2855. doi: 10.1093/humrep/dew218

6. Boyle JA, Cunningham J, O’Dea K, Dunbar T, Norman RJ. Prevalence of polycystic ovary syndrome in a sample of Indigenous women in Darwin, Australia. Med J Aust. 2012; 196(1): 62-66. doi: 10.5694/mja11.10553

7. Azziz R, Kintziger K, Li R, Laven J, Morin-Papunen L, Merkin SS, et al. Recommendations for epidemiologic and phenotypic research in polycystic ovary syndrome: An androgen excess and PCOS society resource. Hum Reprod. 2019; 34(11): 2254-2265. doi: 10.1093/humrep/dez185

8. 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(6): 484-490. doi: 10.1016/j.clinbiochem.2008.11.009

9. 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(2): 405-413. doi: 10.1210/jc.2006-1864

10. Salameh WA, Redor-Goldman MM, Clarke NJ, Mathur R, Azziz R, Reitz RE. Specificity and predictive value of circulating testosterone assessed by tandem mass spectrometry for the diagnosis of polycystic ovary syndrome by the National Institutes of Health 1990 criteria. Fertil Steril. 2014; 101(4): 1135-1141.e2. doi: 10.1016/j.fertnstert.2013.12.056

11. Stanczyk FZ, Clarke NJ. Advantages and challenges of mass spectrometry assays for steroid hormones. J Steroid Biochem Mol Bio. 2010; 121(3-5): 491-495. doi: 10.1016/j.jsbmb.2010.05.001

12. Fanelli F, Gambineri A, Mezzullo M, Vicennati V, Pelusi C, Pasquali R, et al. Revisiting hyper- and hypo-androgenism by tandem mass spectrometry. Rev Endocr Metab Disord. 2013; 14(2): 185-205. doi: 10.1007/s11154-013-9243-y

13. Wierman ME, Auchus RJ, Haisenleder DJ, Hall JE, Handelsman D, Hankinson S, et al. Editorial: The new instructions to authors for the reporting of steroid hormone measurements. Mol Endocrinol. 2014; 28(12): 1917. doi: 10.1210/me.2014-1285

14. Tosi F, Fiers T, Kaufman JM, Dall’Alda M, Moretta R, Giagulli VA, et al. Implications of androgen assay accuracy in the phenotyping of women with polycystic ovary syndrome. J Clin Endocrinol Metab. 2016; 101(2): 610-618. doi: 10.1210/jc.2015-2807

15. Trabert B, Xu X, Falk RT, Guillemette C, Stanczyk FZ, McGlynn KA. Assay reproducibility of serum androgen measurements using liquid chromatography-tandem mass spectrometry. J Steroid Biochem Mol Biol. 2016; 155(PtA): 56-62. doi: 10.1016/j.jsbmb.2015.09.032

16. Suturina L, Lizneva D, Danusevich I, Lazareva L, Belenkaya L, Nadeliaeva I, et al. The design, methodology, and recruitment rate for the Eastern Siberia PCOS epidemiology&phenotype (ES-PEP) Study. Abstracts of the 41st Annual Meeting of the Androgen Excess & PCOS Society. Lorne, Victoria, Australia; 2016: 76.

17. Deshpande M, Kasture S, Mohan M, Chavan M. Bioanalytical method development and validation: A review. In: Muharrem L, Olcay Kaplan I (еds) Recent Advances in Analytical Chemistry. London: Intech Open; 2019.

18. Boterman M, Doig M, Breda M, Lowes S, Jersey J, Shoup R, et al. Recommendations on the interpretation of the new European Medicines Agency Guideline on Bioanalytical Method Validation by Global CRO Council for Bioanalysis (GCC). Bioanalysis. 2012; 4(6): 651-660. doi: 10.4155/bio.12.18

19. Fiandalo MV, Wilton JH, Mantione KM, Wrzosek C, Attwood KM, Wu Y, et al. Serum-free complete medium, an alternative medium to mimic androgen deprivation in human prostate cancer cell line models. Prostate. 2018; 78(3): 213-221. doi: 10.1002/pros.23459

20. Zhang J, Wu H, Kim E, El-Shourbagy TA. Salting-out assisted liquid/liquid extraction with acetonitrile: A new high through put sample preparation technique for good laboratory practice bioanalysis using liquid chromatography-mass spectrometry. Biomed Chromatogr. 2019; 23(4): 419-425. doi: 10.1002/bmc.1135

21. Salameh WA, Redor-Goldman MM, Clarke NJ, Reitz RE, Caulfield MP. Validation of a total testosterone assay using highturbulence liquid chromatography tandem mass spectrometry: total and free testosterone reference ranges. Steroids. 2010; 75(2): 169-175. doi: 10.1016/j.steroids.2009.11.004

22. Alvi SN, Hammami MM. An improved method for measurement of testosterone in human plasma and saliva by ultraperformance liquid chromatography-tandem mass spectrometry. J Adv Pharm Technol Res. 2020; 11(2): 64-68. doi: 10.4103/japtr.JAPTR_162_19

23. van Nuland M, Venekamp N, Wouters WME, van Rossum HH, Rosing H, Beijnen JH. LC-MS/MS assay for the quantification of testosterone, dihydrotestosterone, androstenedione, cortisol and prednisone in plasma from castrated prostate cancer patients treated with abiraterone acetate or enzalutamide. J Pharm Biomed Anal. 2019; 170: 161-168. doi: 10.1016/j.jpba.2019.03.043