Brief Communication

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

Published online January 1, 2021

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

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

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 ( 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–

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

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