Article

Brief Communication

Ann Lab Med 2021; 41(1): 108-113

Published online January 1, 2021 https://doi.org/10.3343/alm.2021.41.1.108

Copyright © Korean Society for Laboratory Medicine.

A Sensitive and Specific Liquid Chromatography-Tandem Mass Spectrometry Assay for Simultaneous Quantification of Salivary Melatonin and Cortisol: Development and Comparison With Immunoassays

Sunghwan Shin, M.D.1 , Hyeonju Oh, M.S.1 , Hea Ree Park, M.D.2 , Eun Yeon Joo, M.D., Ph.D.3 , and Soo-Youn Lee, M.D., Ph.D.1

1Department of Laboratory Medicine and Genetics, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea; 2Department of Neurology, Ilsan Paik Hospital, Inje University College of Medicine, Goyang, Korea; 3Department of Neurology, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea

Correspondence to: Soo-Youn Lee, M.D., Ph.D.
Department of Laboratory Medicine and Genetics, Samsung Medical Center, Sungkyunkwan University School of Medicine, 81 Irwon-ro, Gangnam-gu, Seoul 06351, Korea
Tel: +82-2-3410-1834
Fax: +82-2-3410-2719
E-mail: suddenbz@skku.edu

Eun Yeon Joo, M.D., Ph.D.
Department of Neurology, Samsung Medical Center, Sungkyunkwan University School of Medicine, 81 Irwon-ro, Gangnam-gu, Seoul 06351, Korea
Tel: +82-2-3410-3597
Fax: +82-2-3410-0052
E-mails: ejoo@skku.edu; eunyeon.joo@gmail.com

Received: December 19, 2019; Revised: April 12, 2020; Accepted: July 20, 2020

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Melatonin and cortisol are clinically important for diagnosing sleep and mood disorders. We developed and validated a liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for simultaneous measurement of salivary melatonin and cortisol concentrations according to the Clinical and Laboratory Standards Institute guidelines. Additionally, we compared the LC-MS/MS assay with immunoassays, ELISA (Direct Salivary Melatonin Elisa EK-DSM, Bühlmann Laboratories AG, Schönenbuch, Switzerland) and electrochemiluminescence immunoassay (Cortisol II, Roche, Mannheim, Germany), using 121 saliva samples. The LC-MS/MS assay exhibited good performance in terms of linearity, precision, accuracy, limit of detection, lower limit of quantification, extraction recovery, carry-over, and matrix effect. The LC-MS/MS assay and immunoassays showed strong correlation (Pearson’s r=0.910 for melatonin, r=0.955 for cortisol), but demonstrated a significant mean bias of 23.2% (range 54.0–143.7%) for melatonin and 48.9% (range 59.7–184.7%) for cortisol. Our LC-MS/MS assay provided more sensitive and reliable salivary melatonin and cortisol quantification results compared with immunoassays.

Keywords: Melatonin, Cortisol, Liquid chromatography-tandem mass spectrometry (LC-MS/MS), Immunoassay, Saliva, Performance

Mobile phase parameters for LC-MS/MS

(A) 2 mmol/L ammonium acetate in deionized water;

(B) 0.1% (v/v) formic acid in acetonitrile.

Abbreviation: LC-MS/MS, liquid chromatography-tandem mass spectrometry.

Analyte and internal standard MS parameters

Abbreviations: MS, mass spectrometry; MRM, multiple reaction monitoring; RT, retention time.

Fig. 1.

Comparison plots between LC-MS/MS and immunoassays. Top: (A) Passing-Bablok regression and (B) Bland-Altman plot to compare salivary melatonin concentrations measured by LC-MS/MS assay and ELISA. Bottom: (C) Passing-Bablok regression and (D) Bland-Altman plot to compare salivary cortisol concentrations measured by LC-MS/MS assay and ECLIA.

Abbreviations: LC-MS/MS, liquid chromatography-tandem mass spectrometry; ECLIA, electrochemiluminescence immunoassay.


Fig. 2.

Melatonin and cortisol profiles of two participants measured by LC-MS/MS assay and immunoassays.

Abbreviations: ECLIA, electrochemiluminescence immunoassay; LC-MS/MS, liquid chromatography-tandem mass spectrometry.


The performance of LC-MS/MS assay to measure melatonin and cortisol concentrations

AnalyteAssay rangerLODLLOQ*Accuracy (%)Precision (CV, %)

Intra-assay (N=5)Inter-assay (N=30)


8.6 (0.55)86 (5.52)344 (22.07)8.6 (0.55)86 (5.52)344 (22.07)
Melatonin2.15–430 pmol/L0.9970.43 pmol/L2.15 pmol/L100.3–102.23.63.34.96.85.43.5

Cortisol0.14–27.59 nmol/L0.9990.03 nmol/L0.14 nmol/L96.9–107.82.63.13.14.74.73.7

  1. Claustrat B, Leston J. Melatonin: physiological effects in humans. Neurochirurgie 2015;61:77-84.
    Pubmed
  2. Debono M, Ghobadi C, Rostami-Hodjegan A, Huatan H, Campbell MJ, Newell-Price J, et al. Modified-release hydrocortisone to provide circadian cortisol profiles. J Clin Endocrinol Metab 2009;94:1548-54.
    Pubmed
  3. Blair J, Adaway J, Keevil B, Ross R. Salivary cortisol and cortisone in the clinical setting. Curr Opin Endocrinol Diabetes Obes 2017;24:161-8.
    Pubmed
  4. Voultsios A, Kennaway DJ, Dawson D. Salivary melatonin as a circadian phase marker: validation and comparison to plasma melatonin. J Biol Rhythms 1997;12:457-66.
    Pubmed
  5. Jung C, Greco S, Nguyen HH, Ho JT, Lewis JG, Torpy DJ, et al. Plasma, salivary and urinary cortisol levels following physiological and stress doses of hydrocortisone in normal volunteers. BMC Endocr Disord 2014;14:91.
    Pubmed
  6. Taylor AE, Keevil B, Huhtaniemi IT. Mass spectrometry and immunoassay: how to measure steroid hormones today and tomorrow. Eur J Endocrinol 2015;173:D1-12.
    Pubmed
  7. Kennaway DJ. A critical review of melatonin assays: past and present. J Pineal Res 2019;67:e12572.
    Pubmed
  8. Doi M, Sekizawa N, Tani Y, Tsuchiya K, Kouyama R, Tateno T, et al. Late-night salivary cortisol as a screening test for the diagnosis of Cushing’s syndrome in Japan. Endocr J 2008;55:121-6.
    Pubmed
  9. Turpeinen U, Hämäläinen E. Determination of cortisol in serum, saliva and urine. Best Pract Res Clin Endocrinol Metab 2013;27:795-801.
    Pubmed
  10. van Faassen M, Bischoff R, Kema IP. Relationship between plasma and salivary melatonin and cortisol investigated by LC-MS/MS. Clin Chem Lab Med 2017;55:1340-8.
    Pubmed
  11. Fustinoni S, Polledri E, Mercadante R. High-throughput determination of cortisol, cortisone, and melatonin in oral fluid by on-line turbulent flow liquid chromatography interfaced with liquid chromatography/tandem mass spectrometry. Rapid Commun Mass Spectrom 2013;27:1450-60.
    Pubmed
  12. Jensen MA, Hansen AM, Abrahamsson P, N⊘rgaard AW. Development and evaluation of a liquid chromatography tandem mass spectrometry method for simultaneous determination of salivary melatonin, cortisol and testosterone. J Chromatogr B Analyt Technol Biomed Life Sci 2011;879:2527-32.
  13. Jensen MA, Mortier L, Koh E, Keevil B, Hyttinen S, Hansen ÅM. An interlaboratory comparison between similar methods for determination of melatonin, cortisol and testosterone in saliva. Scand J Clin Lab Invest 2014;74:454-61.
    Pubmed
  14. CLSI, Liquid chromatography-mass spectrometry methods. 1st ed. Wayne, PA: Clinical and Laboratory Standards Institute. CLSI C62-A
  15. Vogeser M, Kratzsch J, Ju Bae Y, Bruegel M, Ceglarek U, Fiers T, et al. Multicenter performance evaluation of a second generation cortisol assay. Clin Chem Lab Med 2017;55:826-35.
    Pubmed
  16. Shirtcliff EA, Granger DA, Schwartz E, Curran MJ. Use of salivary biomarkers in biobehavioral research: cotton-based sample collection methods can interfere with salivary immunoassay results. Psychoneuroendocrinology 2001;26:165-73.
    Pubmed
  17. Nölte I, Lütkhoff AT, Stuck BA, Lemmer B, Schredl M, Findeisen P, et al. Pineal volume and circadian melatonin profile in healthy volunteers: an interdisciplinary approach. J Magn Reson Imaging 2009;30:499-505.
    Pubmed
  18. Riemann D, Klein T, Rodenbeck A, Feige B, Horny A, Hummel R, et al. Nocturnal cortisol and melatonin secretion in primary insomnia. Psychiatry Res 2002;113:17-27.
    Pubmed
  19. Corbalán-Tutau D, Madrid JA, Nicolás F, Garaulet M. Daily profile in two circadian markers “melatonin and cortisol” and associations with metabolic syndrome components. Physiol Behav 2014;123:231-5.
    Pubmed