Abstract
Accurate and precise cortisol measurements are requisite for ensuring appropriate diagnosis and management of diseases related with adrenal or pituitary gland disorders. Prompted by the needs in characterization of certified reference materials and quality assurance for serum cortisol measurements, we developed and evaluated a highly reliable measurement procedure based on isotope dilution liquid chromatography-tandem mass spectrometry (ID LC-MS/MS) combined with dextran sulfate-Mg2+ precipitation as the sample pretreatment. An appropriate amount of serum was accurately weighed and spiked with the isotope labelled internal standard. After precipitation, massive lipids and lipoproteins were removed from serum matrix. The clear supernatant was transferred and extracted with ethyl acetate-hexane solution. The cortisol was analyzed with LC-MS/MS in positive electrospray ionization mode. The within-run and total coefficient of variations (CVs) ranged from 0.3 to 0.6% and 0.7 to 1.2%, respectively, for a concentration range of 76.30 to 768.04 nmol/L. A regression comparison of the results obtained by the present method and the certified values of ERM-DA451 showed agreement with no statistical difference (Y = 1.0092 X-0.7455; 95% CI for the slope, 0.9940 to 1.0212; 95% CI for the intercept, − 3.6575 to 2.6390, r2 = 0.999). All structural analogs of cortisol tested were well resolved from cortisol in 12 min on a phenyl ligand column under an isocratic elution. The limit of quantification was estimated to 5 pg cortisol in absolute amount. This method is accurate and simple and can be served as a candidate reference measurement procedure in establishment of serum cortisol reference system.
Similar content being viewed by others
References
Knutsson U, Dahlgren J, Marcus C, Rosberg S, Brönnegård M, Stierna P, et al. Circadian cortisol rhythms in healthy boys and girls: relationship with age, growth, body composition, and pubertal development. J Clin Endocrinol Metab. 1997;82(2):536–40.
Reimondo G, Pia A, Bovio S, Allasino B, Daffara F, Paccotti P, et al. Laboratory differentiation of Cushing’s syndrome. Clin Chim Acta. 2008;388(1–2):5–14.
Hawley JM, Owen LJ, Lockhart SJ, Monaghan PJ, Armston A, Chadwick CA, et al. Serum cortisol: an up-to-date assessment of routine assay performance. Clin Chem. 2016;62(9):1220–9.
Siekmann L, Breuer H. Determination of cortisol in human plasma by isotope dilution-mass spectrometry. Definitive methods in clinical chemistry, I. J Clin Chem Clin Biochem. 1982;20(12):883–92.
Tai SS, Welch MJ. Development and evaluation of a candidate reference method for the determination of total cortisol in human serum using isotope dilution liquid chromatography/mass spectrometry and liquid chromatography/tandem mass spectrometry. Anal Chem. 2004;76(4):1008–14.
Thienpont LM, De Brabandere VI, Stöckl D, De Leenheer AP. Candidate reference method for determining serum cortisol based on isotope dilution-gas chromatography/mass spectrometry using heptafluorobutyrilation as derivatization method. Anal Biochem. 1996;234(2):204–9.
De Brabandere VI, Thienpont LM, Stöckl D, De Leenheer AP. Three routine methods for serum cortisol evaluated by comparison with an isotope dilution gas chromatography-mass spectrometry method. Clin Chem. 1995;41(12 Pt 1):1781–3.
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(10):853–60.
Botelho JC, Shacklady C, Cooper HC, Tai SS, Van Uytfanghe K, Thienpont LM, et al. Isotope-dilution liquid chromatography-tandem mass spectrometry candidate reference method for total testosterone in human serum. Clin Chem. 2013;59(2):372–80.
Botelho JC, Ribera A, Cooper HC, 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. 2016;88(22):11123–9.
Vamathevan V, Murby EJ. Accurate analysis of testosterone in human serum using a heart-cutting 2D-UPLC-MS/MS procedure. J Chromatogr B Anal Technol Biomed Life Sci. 2016;1038:49–56.
Little JL, Wempe MF, Buchanan CM. Liquid chromatography-mass spectrometry-mass spectrometry method development for drug metabolism studies: Examining lipid matrix ionization effects in plasma. J Chromatogr B Anal Technol Biomed Life Sci. 2006;833(2):219–30.
Dong J, Yu S, Yang R, Li H, Guo H, Zhao H, et al. A simple and precise method for direct measurement of fractional esterification rate of high density lipoprotein cholesterol by high performance liquid chromatography. Clin Chem Lab Med. 2014;52(4):557–64.
Warnick GR, Albers JJ. Heparin-Mn2+ quantitation of high-density-lipoprotein cholesterol: an ultrafiltration procedure for lipemic samples. Clin Chem. 1978;24(6):900–4.
Zhang T, Zhang C, Zhao H, Zeng J, Zhang J, Zhou W, et al. Determination of serum glucose by isotope dilution liquid chromatography-tandem mass spectrometry: a candidate reference measurement procedure. Anal Bioanal Chem. 2016;408(26):7403–11.
Warnick GR, Benderson J, Albers JJ. Dextran sulfate-Mg2+ precipitation procedure for quantitation of high-density-lipoprotein cholesterol. Clin Chem. 1982;28(6):1379–88.
Annesley TM. Ion suppression in mass spectrometry. Clin Chem. 2003;49(7):1041–4.
Matuszewski K, 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(13):3019–30.
Thienpont LM, Verhaeghe PG, Van Brussel KA, De Leenheer AP. Estradiol-17 beta quantified in serum by isotope dilution-gas chromatography-mass spectrometry: reversed-phase C18 high-performance liquid chromatography compared with immuno-affinity chromatography for sample pretreatment. Clin Chem. 1988;34(10):2066–9.
Andrade-Eiroa A, Canle M, Leroy-Cancellieri V, Cerdà V. Solid-phase extraction of organic compounds: A critical review. Part ii. Trends Anal Chem. 2016;80:655–67.
Bachorik PS, Albers JJ. Precipitation methods for quantification of lipoproteins. Methods Enzymol. 1986;129:78–100.
Kimberly MM, Leary ET, Cole TG, Waymack PP. Selection, validation, standardization, and performance of a designated comparison method for HDL-cholesterol for use in the cholesterol reference method laboratory network. Clin Chem. 1999;45(10):1803–12.
Owen LJ, Adaway JE, Davies S, Neale S, El-Farhan N, Ducroq D, et al. Development of a rapid assay for the analysis of serum cortisol and its implementation into a routine service laboratory. Ann Clin Biochem. 2013;50(4):345–52.
CLSI. C-62A: Liquid chromatography-mass spectrometry methods; approved guideline. Wayne, PA: Clinical and Laboratory Standards Institute; 2014.
ISO/IEC Guide 98–3:2008. Uncertainty of measurement - part 3: guide to the expression of uncertainty in measurement (GUM:1995). Geneva: ISO; 2008.
Ellison SLR. RossleinM, Williams A, editors. EURACHEM/CITAC Guide CG 4: quantifying uncertainty in analytical measurement. 2nd ed. EURACHEM: Czech Republic; 2000.
Funding
This study was supported by grants from the National Natural Science Foundation of China (Grant Number: 81301488).
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Anonymized left-over sera were collected from patients receiving physical examinations or hospitalized at Beijing Hospital (BJH, Beijing, China). The study was reviewed and approved by the BJH Ethics Committee.
Conflict of interest
The authors declare that they have no competing interests.
Additional information
Publisher’s note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Electronic supplementary material
ESM 1
(PDF 199 kb)
Rights and permissions
About this article
Cite this article
Zhang, T., Zhao, H., Li, M. et al. Development and validation of a candidate reference method for serum cortisol by isotope dilution liquid chromatography-tandem mass spectrometry combined with dextran sulfate-Mg2+ precipitation. Anal Bioanal Chem 412, 1325–1333 (2020). https://doi.org/10.1007/s00216-019-02356-2
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00216-019-02356-2