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Simultaneous determination of testosterone, cortisol, and dehydroepiandrosterone in saliva by stable isotope dilution on-line in-tube solid-phase microextraction coupled with liquid chromatography–tandem mass spectrometry

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Abstract

We have developed a simple and sensitive method for the simultaneous determination of testosterone (TES), cortisol (CRT), and dehydroepiandrosterone (DHEA) in saliva by automated online in-tube solid-phase microextraction (SPME) coupled with liquid chromatography–tandem mass spectrometry (LC–MS/MS) using a Discovery HS F5 column. The optimum in-tube SPME conditions were 25 draw/eject cycles of 40 μL of sample at a flow rate of 200 μL/min using a Supel-Q PLOT capillary column as an extraction device. The extracted compounds were easily desorbed from the capillary by passage of the mobile phase, and no carryover was observed. The in-tube SPME LC–MS/MS method showed good linearity with correlation coefficients r ≥ 0.9998 for TES, CRT, and DHEA using their respective stable isotope-labeled internal standards. The intra-day and inter-day precisions (relative standard deviations) were below 4.9 and 8.5 % (n = 5), respectively. This method was successfully utilized to analyze TES, CRT, and DHEA in saliva samples without any other pretreatment or interference peaks, and the quantification limits (S/N = 10) of TES, CRT and DHEA were about 0.01, 0.03 and 0.29 ng/mL saliva, respectively. The recoveries of these compounds spiked into saliva samples were each above 94 %. This method was applied to analyze changes in salivary TES, CRT, and DHEA levels resulting from stress and fatigue load.

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Acknowledgments

This work was supported by The Promotion and Mutual Aid Corporation for Private Schools of Japan, The Science Research Promotion Fund, a Grant-in-Aid for Basic Scientific Research (C, No. 22590048), and the Cosmetology Research Foundation.

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Authors and Affiliations

  1. School of Pharmacy, Shujitsu University, Nishigawara, Okayama, 703-8516, Japan

    Hiroyuki Kataoka, Kentaro Ehara, Rie Yasuhara & Keita Saito

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  1. Hiroyuki Kataoka
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  2. Kentaro Ehara
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Correspondence to Hiroyuki Kataoka.

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Kataoka, H., Ehara, K., Yasuhara, R. et al. Simultaneous determination of testosterone, cortisol, and dehydroepiandrosterone in saliva by stable isotope dilution on-line in-tube solid-phase microextraction coupled with liquid chromatography–tandem mass spectrometry. Anal Bioanal Chem 405, 331–340 (2013). https://doi.org/10.1007/s00216-012-6479-4

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