| Summary: | 碩士 === 國立成功大學 === 環境醫學研究所 === 107 === Cortisol measured in saliva, blood, and urine is used to examine the hypothalamic–pituitary–adrenal (HPA) axis and psychological conditions, considered as stress marker. However, daily cortisol concentrations variations are likely to be affected by acute stress, the diurnal rhythm and pulsatile secretion. Cortisol measurements in saliva, blood and urine are challenging to reflect long-term level variations. Hair incorporated with cortisol during growth might be a suitable long-term biomonitoring sample. The longitudinal distribution of cortisol in the hair could reflect an accumulation of cortisol secreted over the past months. Liquid chromatography-tandem mass spectrometry (LC-MS) has emerged as an analytical method for hair cortisol analysis. Moreover, the use of LC-MS with higher sensitivity is an advantage for analyzed limited hair and lower cortisol levels. This study aims to develop a nanoflow UHPLC-MS3 system for a single hair cortisol analysis. This system constructed by capillary columns and the use of tandem MS (MS/MS and MS3) to enhance specificity and sensitivity for cortisol detection in a single hair, and method validation of this analytical platform was performed. The MS3 method could detect molecule with the use of the specific fragment pattern in the MS. The signal-to-noise ratio (S/N) of extracted ion chromatogram (EIC) by MS3 analysis was enhanced approximately 15-fold comparing to MS/MS analysis. Thus, the MS3 was employed in method validation and hair cortisol analysis. The sensitivity improvement of this analytical platform was evaluated by the signal intensity, sensitivity, S/N and limit of detection (LOD), comparing to microbore UHPLC-MS3 system. Comparing to microbore UHPLC-MS3, the signal response and sensitivity of nanoflow UHPLC-MS3 were increased by about 3-order of magnitude at 1-ng cortisol standard and about 500-fold respectively, and the LOD of cortisol in neat solution and hair extract by nanoflow UHPLC-MS3 was improved about 100- and 30-fold respectively. This analytical platform was applied for hair cortisol analysis. Hair samples were provided from National Cheng Kung University Hospital, and cortisol levels were evaluated in a single hair and 20 hairs by pair samples t-test. The average of cortisol level in a single hair and 20 hairs was closed (27.45 vs. 31.82 pg/mg hair), and there was no significant difference between a single hair and 20 hairs (p=0.66). The trends of cortisol level in hair was closed to plasma during pregnancy, so one hair could reflect the variation of cortisol level over the past period. This study presented a highly sensitive analytical platform for cortisol in a single hair analysis. In the future, this approach using only a single hair is powerful for diagnosis, exposure markers retrospective studies, and novel but low abundant biomarker discovery.
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