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.
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.
School of Pharmacy, Shujitsu University, Nishigawara, Okayama 703-8516, Japan. hkataoka@shujitsu.ac.jp
Hiroyuki
H
Kataoka
Kentaro
K
Ehara
Rie
R
Yasuhara
Keita
K
Saito
D002623:Chemistry Techniques, Analytical; D002853:Chromatography, Liquid / Q000379:methods*; D003687:Dehydroepiandrosterone / Q000032:analysis*; D006801:Humans; D006854:Hydrocortisone / Q000032:analysis*; D007554:Isotopes / Q000032:analysis; D008956:Models, Chemical; D015203:Reproducibility of Results; D012463:Saliva / Q000378:metabolism*; D052617:Solid Phase Microextraction / Q000379:methods*; D013314:Stress, Mechanical; D053719:Tandem Mass Spectrometry / Q000379:methods*; D013739:Testosterone / Q000032:analysis*; D013997:Time Factors
D007554:Isotopes; D013739:Testosterone; D003687:Dehydroepiandrosterone; D006854:Hydrocortisone