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.
| Original language | English |
|---|---|
| Journal | Journal of Separation Science |
| Volume | 36 |
| Pages (from-to) | 246-251 |
| Number of pages | 6 |
| ISSN | 1615-9306 |
| Publication status | Published - Jan 2013 |