Quantitative analysis of cortisol and 6beta-hydroxycortisol in urine by fully automated SPE and ultra-performance LC coupled with electrospray and atmospheric pressure Chemical ionization (ESCi)-TOF-MS

TY - JOUR

T1 - Quantitative analysis of cortisol and 6beta-hydroxycortisol in urine by fully automated SPE and ultra-performance LC coupled with electrospray and atmospheric pressure Chemical ionization (ESCi)-TOF-MS

AU - Lang, Lotte Markussen

AU - Dalsgaard, Petur Weihe

AU - Linnet, Kristian

PY - 2013/1

Y1 - 2013/1

N2 - 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.

AB - 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.

M3 - Journal article

SN - 1615-9306

VL - 36

SP - 246

EP - 251

JO - Journal of Separation Science

JF - Journal of Separation Science

ER -