Quantitative determination of human serum testosterone via isotope dilution ultra‑performance liquid chromatography tandem mass spectrometry

Sun,Guang; Xue,Jinmei; Li,Liuxu; Li,Xue; Cui,Yaqiong; Qiao,Bin; Wei,Dianjun; Li,Huiqiang

doi:10.3892/mmr.2020.11235

Quantitative determination of human serum testosterone via isotope dilution ultra‑performance liquid chromatography tandem mass spectrometry

Authors:
- Guang Sun
- Jinmei Xue
- Liuxu Li
- Xue Li
- Yaqiong Cui
- Bin Qiao
- Dianjun Wei
- Huiqiang Li
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Affiliations: Department of Clinical Immunology, School of Medical Laboratory, Tianjin Medical University, Hexi, Tianjin 300203, P.R. China, School of Chemical Engineering and Technology, Tianjin University, Jinnan, Tianjin 300350, P.R. China, Department of Clinical Laboratory, Hebei Yanda Hospital, Yanda International Health City, Sanhe, Hebei 065201, P.R. China
Published online on: June 15, 2020 https://doi.org/10.3892/mmr.2020.11235
Pages: 1576-1582
Copyright: © Sun et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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Abstract

Quantification of testosterone serves an important role in the differential diagnosis of androgen‑related endocrine diseases. Mass spectrometry exhibits higher accuracy and lower variability than immunoassays, especially at low testosterone concentrations. The present study developed and validated an isotope dilution ultra‑performance liquid chromatography tandem mass spectrometry method for determination of human serum testosterone. The serum was equilibrated with an isotopic internal standard and treated with acidic buffer to release hormones from their binding proteins. Testosterone was extracted via two serial liquid‑liquid extractions. In the first stage, the lipid fractions from an acidic buffer solution were isolated using ethyl acetate and n‑hexane. The organic phase was evaporated and reconstituted in a basic buffer solution. In the second stage, the polar impurities of n‑hexane extraction were removed. Total testosterone in serum was quantified via ultra‑performance liquid chromatography tandem mass spectrometry in multiple reaction monitoring mode with positive electrospray ionization. The coefficient of variation of the method for intra‑ and inter‑assay was 2.13% (1.40‑2.77%) and 3.44% (3.06‑3.66%), respectively. The recovery ranged from 94.32 to 108.60% for different samples. The limit of detection was 0.50 ng/dl and the linear range was from 1.00 to 1,000.00 ng/dl. In addition, the extraction efficiency in three different levels of quality control of the serum ranged from 85.02 to 93.29%. Moreover, structural analogues were investigated and were not indicated to affect the quantification of testosterone. The present method may enable quantification of testosterone in a clinical setting with high precision and accuracy.

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1	Bhasin S, Brito JP, Cunningham GR, Hayes FJ, Hodis HN, Matsumoto AM, Snyder PJ, Swerdloff RS, Wu FC and Yialamas MA: Testosterone therapy in men with hypogonadism: An endocrine society clinical practice guideline. J Clin Endocrinol Metab. 103:1715–1744. 2018. View Article : Google Scholar : PubMed/NCBI
2	Kushnir MM, Rockwood AL, Roberts WL, Pattison EG, Bunker AM, Fitzgerald RL and Meikle AW: Performance characteristics of a novel tandem mass spectrometry assay for serum testosterone. Clin Chem. 52:120–128. 2006. View Article : Google Scholar : PubMed/NCBI
3	Salonia A, Rastrelli G, Hackett G, Seminara SB, Huhtaniemi IT, Rey RA, Hellstrom WJG, Palmert MR, Corona G, Dohle GR, et al: Paediatric and adult-onset male hypogonadism. Nat Rev Dis Primers. 5:382019. View Article : Google Scholar : PubMed/NCBI
4	Martin KA, Anderson RR, Chang RJ, Ehrmann DA, Lobo RA, Murad MH, Pugeat MM and Rosenfield RL: Evaluation and treatment of hirsutism in premenopausal women: An endocrine society clinical practice guideline. J Clin Endocrinol Metab. 103:1233–1257. 2018. View Article : Google Scholar : PubMed/NCBI
5	La'ulu SL, Kalp KJ and Straseski JA: How low can you go? Analytical performance of five automated testosterone immunoassays. Clin Biochem. 58:64–71. 2018. View Article : Google Scholar : PubMed/NCBI
6	Cui Y, She T, Zhao H, Li J, Li L, Gao W and Li H: Competitive light-initiated chemiluminescent assay: Using 5-α-dihydrotestosterone-BSA as competitive antigen for quantitation of total testosterone in human sera. Anal Bioanal Chem. 411:745–754. 2019. View Article : Google Scholar : PubMed/NCBI
7	Taieb J, Mathian B, Millot F, Patricot MC, Mathieu E, Queyrel N, Lacroix I, Somma-Delpero C and Boudou P: Testosterone measured by 10 immunoassays and by isotope-dilution gas chromatography-mass spectrometry in sera from 116 men, women, and children. Clin Chem. 49:1381–1395. 2003. View Article : Google Scholar : PubMed/NCBI
8	Rosner W, Auchus RJ, Azziz R, Sluss PM and Raff H: Position statement: Utility, limitations, and pitfalls in measuring testosterone: An Endocrine Society position statement. J Clin Endocrinol Metab. 92:405–413. 2007. View Article : Google Scholar : PubMed/NCBI
9	Wang C, Catlin DH, Demers LM, Starcevic B and 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. 89:534–543. 2004. View Article : Google Scholar : PubMed/NCBI
10	Kushnir MM, Rockwood AL and Bergquist J: Liquid chromatography-tandem mass spectrometry applications in endocrinology. Mass Spectrom Rev. 29:480–502. 2010. View Article : Google Scholar : PubMed/NCBI
11	International Organization for Standardization, . In vitro diagnostic medical devices-measurement of quantities in samples of biological origin-metrological traceability of values assigned to calibrators and control Materials. ISO 17511 (Geneva). ISO. 2003.
12	Stanczyk FZ and Clarke NJ: Advantages and challenges of mass spectrometry assays for steroid hormones. J Steroid Biochem Mol Biol. 121:491–495. 2010. View Article : Google Scholar : PubMed/NCBI
13	Vesper HW, Bhasin S, Wang C, Tai SS, Dodge LA, Singh RJ, Nelson J, Ohorodnik S, Clarke NJ, Salameh WA, et al: Interlaboratory comparison study of serum total testosterone [corrected] measurements performed by mass spectrometry methods. Steroids. 74:498–503. 2009. View Article : Google Scholar : PubMed/NCBI
14	Star-Weinstock M, Williamson BL, Dey S, Pillai S and Purkayastha S: LC-ESI-MS/MS analysis of testosterone at sub-picogram levels using a novel derivatization reagent. Anal Chem. 84:9310–9317. 2012. View Article : Google Scholar : PubMed/NCBI
15	Keski-Rahkonen P, Huhtinen K, Poutanen M and Auriola S: Fast and sensitive liquid chromatography-mass spectrometry assay for seven androgenic and progestagenic steroids in human serum. J Steroid Biochem Mol Biol. 127:396–404. 2011. View Article : Google Scholar : PubMed/NCBI
16	Yuan TF, Le J, Cui Y, Peng R, Wang ST and Li Y: An LC-MS/MS analysis for seven sex hormones in serum. J Pharm Biomed Anal. 162:34–40. 2019. View Article : Google Scholar : PubMed/NCBI
17	Tai SS, Xu B, Welch MJ and 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. 388:1087–1094. 2007. View Article : Google Scholar : PubMed/NCBI
18	Lynch KL: CLSI C62-A: A New standard for clinical mass spectrometry. Clin Chem. 62:24–29. 2016. View Article : Google Scholar : PubMed/NCBI
19	Guidance on the verification of quantitative measurement procedures used in the clinical chemistry. GL037. CNAS. 2019.
20	Rodriguez M and Orescan DB: Confirmation and quantitation of selected sulfonylurea, imidazolinone, and sulfonamide herbicides in surface water using electrospray LC/MS. Anal Chem. 70:2710–2717. 1998. View Article : Google Scholar : PubMed/NCBI
21	Botelho JC, Ribera A, Cooper HC and Vesper HW: Evaluation of an isotope dilution HPLC tandem mass spectrometry candidate reference measurement procedure for Total 17-β estradiol in human serum. Anal Chem. 88:11123–11129. 2016. View Article : Google Scholar : PubMed/NCBI
22	Matuszewski BK, Constanzer ML and Chavez-Eng CM: Strategies for the assessment of matrix effect in quantitative bioanalytical methods based on HPLC-MS/MS. Anal Chem. 75:3019–3030. 2003. View Article : Google Scholar : PubMed/NCBI
23	Yun YM, Botelho JC, Chandler DW, Katayev A, Roberts WL, Stanczyk FZ, Vesper HW, Nakamoto JM, Garibaldi L, Clarke NJ and Fitzgerald RL: Performance criteria for testosterone measurements based on biological variation in adult males: Recommendations from the partnership for the accurate testing of hormones. Clin Chem. 58:1703–1710. 2012. View Article : Google Scholar : PubMed/NCBI
24	Legro RS, Schlaff WD, Diamond MP, Coutifaris C, Casson PR, Brzyski RG, Christman GM, Trussell JC, Krawetz SA, Snyder PJ, et al: Total testosterone assays in women with polycystic ovary syndrome: Precision and correlation with hirsutism. J Clin Endocrinol Metab. 95:5305–5313. 2010. View Article : Google Scholar : PubMed/NCBI
25	Neville D, Houghton R and Garrett S: Efficacy of plasma phospholipid removal during sample preparation and subsequent retention under typical UHPLC conditions. Bioanalysis. 4:795–807. 2012. View Article : Google Scholar : PubMed/NCBI