title: Quantitative analysis of cortisol and 6β-hydroxycortisol in urine by fully automated SPE and ultra-performance LC coupled with electrospray and atmospheric pressure chemical ionization (ESCi)-TOF-MS.
abstract: An ultra-performance LC TOF MS method for quantitative analysis of cortisol and 6β-hydroxycortisol in urine was developed. The method was used for determination of the ratio between 6β-hydroxycortisol and cortisol in urine received from autopsy cases and living persons as a measure of cytochrome P450 3A enzyme activity. Urine samples (0.25 mL) were extracted with an in-house developed fully automated 96-well SPE system. The compounds were quantified using a Waters ACQUITY UPLC system coupled to a Waters SYNAPT G2. The MS sensitivity was optimized by using negative ionization in sensitivity mode (resolution >10 000 full-width at half-maximum), and further optimized by using the enhanced duty cycle around the 410 m/z. ESCi (simultaneous electrospray and atmospheric pressure chemical ionization) mode was used to compensate for the matrix effects of postmortem urine. Finally, the SYNAPT G2 was tested as a quantitative instrument. The developed method has a measurement range from 2.5-300 ng/mL for cortisol to 10-1200 ng/mL for 6β-hydroxycortisol. Mean overall process efficiencies were 29.4 and 23.0% for cortisol and 6β-hydroxycortisol, respectively. In 20 forensic reference cases, the range of the 6β-hydroxycortisol/cortisol ratio was 0.29-14.2 with a median of 3.04.
authors.affiliation: Section of Forensic Chemistry, Department of Forensic Medicine, Faculty of Health Sciences, University of Copenhagen, Copenhagen, Denmark. lotte.lang@forensic.ku.dk
authors.forename: Lotte M
authors.initials: LM
authors.lastname: Lang
authors.forename: Petur W
authors.initials: PW
authors.lastname: Dalsgaard
authors.forename: Kristian
authors.initials: K
authors.lastname: Linnet
mesh_terms: D001331:Automation; D002851:Chromatography, High Pressure Liquid / Q000295:instrumentation / Q000379:methods*; D006801:Humans; D006854:Hydrocortisone / Q000031:analogs & derivatives* / Q000652:urine*; D052616:Solid Phase Extraction / Q000295:instrumentation / Q000379:methods*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000295:instrumentation / Q000379:methods; D053719:Tandem Mass Spectrometry / Q000295:instrumentation / Q000379:methods*
chemical_list: C001380:6 beta-hydroxycortisol; D006854:Hydrocortisone