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Title: Detection and measurement of stress hormones with mass spectrometry
Author: Tanner, Amy Vivien
ISNI:       0000 0004 2737 9751
Awarding Body: University of Greenwich
Current Institution: University of Greenwich
Date of Award: 2012
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The purpose of this thesis was to develop and validate a liquid chromatography mass spectrometry (LC-MS) method to measure salivary cortisol and testosterone. Furthermore, to use this method to analyse saliva samples collected during an exercise study measuring the hormonal response to acute exercise. The ongoing contribution to knowledge provided by this research is the application of a developed and validated LC-MS method to analysis of cortisol from pre and post exercise saliva samples, and evaluation of the cortisol and testosterone response to three different training sessions; including an interval session (INT), tempo run (TEMP) and aerobic circuit training (CIR). An LC-MS method was developed to measure cortisol and testosterone. The mass spectrometer used was a triple quadrupole BioQ (Waters/Micromass, UK) coupled with a 1200 series HPLC machine (Agilent Technologies, UK). Optimisation of various parameters was undertaken including: cone voltage and capillary voltage, followed by optimisation of liquid chromatography (LC) parameters such as; mobile phase gradient and flow rate. Saliva samples were collected at rest, and hormones measured with the new LC-MS method and ELISA to validate the developed method. Finally, heart rate and salivary and plasma cortisol and testosterone response to acute exercise were tested, to compare the response to different training protocols in runners. Following this, the MS method underwent further validation against ELISA. Results revealed optimum MS parameters were: cone voltage 25 V; capillary voltage 3.5 kV and LM and HM resolution 8 Da. For the LC method, mobile phase flow rate was optimised at 0.1 mL/min, with a gradient profile ranging from 50 to 95% methanol (MeOH), and a run time of 15 minutes. Sample preparation was also considered (solid phase extraction); and wash phase of 10% MeOH, 10 times sample concentration and reconstitution with 80% MeOH were optimised to improve analyte recovery and detection. Comparison of cortisol measured at rest with LC-MS and ELISA (n=22) revealed a correlation between methods (r=0.83, P<0.001). The exercise study showed the INT elicited a higher peak heart rate (172±11) than CIR (148±10) or TEMP trials (163±10). INT also produced a higher RPE (15 ±2) than CIR (13±1) and TEMP (14±2) trials. Salivary cortisol increased at all time points post exercise in INT and remained higher than rest (1.29 ± 0.51 ng/mL) at 60 minutes recovery (3.13 ±1.59 ng/mL). Salivary testosterone increased pre to post exercise in CIR (119.7±39.5 pg/mL to 150.1 ±51.5 pg/mL), TEMP (142.4±76.5 pg/mL to 185.0±49.1 pg/mL) and INT (117.5±23.5 pg/mL to 176.3±46.0 pg/mL) trials. There was also a correlation between cortisol measured in plasma and saliva (r=0.813, p=0.0001). Additionally, comparison of salivary cortisol measurements obtained with mass spectrometry and ELISA (n=85) showed a correlation (r=0.934, p=00001). In conclusion, validation revealed cortisol could be quantfied accurately and precisely with LC-MS. Additionally, INT elicited a higher mean RPE and cortisol response than TEMP or CIR.
Supervisor: Nielsen, Birthe ; Allgrove, Judith Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: QC Physics ; QP Physiology