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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 ( ESC i)‐ TOF ‐ MS
Author(s) -
Lang Lotte M.,
Dalsgaard Petur W.,
Linnet Kristian
Publication year - 2013
Publication title -
journal of separation science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.72
H-Index - 102
eISSN - 1615-9314
pISSN - 1615-9306
DOI - 10.1002/jssc.201200736
Subject(s) - chromatography , chemistry , urine , electrospray ionization , mass spectrometry , high performance liquid chromatography , atmospheric pressure chemical ionization , ionization , analytical chemistry (journal) , chemical ionization , biochemistry , ion , organic chemistry
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 P 450 3 A 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 W aters ACQUITY UPLC system coupled to a W aters SYNAPT G 2. 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 . ESC i (simultaneous electrospray and atmospheric pressure chemical ionization) mode was used to compensate for the matrix effects of postmortem urine. Finally, the SYNAPT G 2 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.