A High-Performance Liquid Chromatography–Electrospray–Tandem Mass Spectrometry Analysis of Cortisol and Metabolites in Placental Perfusate

A reversed-phase high–performance liquid chromatography–electrospray–tandem mass spectrometry assay (HPLC–ESI–MS/MS) was developed to quantitate cortisol, cortisone, 20α- and β-dihydrocortisol, 20α- and β-dihydrocortisone, tetrahydrocortisol, and tetrahydrocortisone. The technique was used to analyz...

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Bibliographic Details
Published in:Analytical biochemistry 1997-05, Vol.247 (2), p.342-347
Main Authors: Dodds, H.M., Taylor, P.J., Cannell, G.R., Pond, S.M.
Format: Article
Language:English
Subjects:
11-beta-Hydroxysteroid Dehydrogenases
Chromatography, High Pressure Liquid - methods
Cortisone - analogs & derivatives
Cortisone - analysis
Cortisone - metabolism
Evaluation Studies as Topic
Female
Humans
Hydrocortisone - analogs & derivatives
Hydrocortisone - analysis
Hydrocortisone - metabolism
Hydroxysteroid Dehydrogenases - antagonists & inhibitors
In Vitro Techniques
Mass Spectrometry - methods
Perfusion
Placenta - chemistry
Placenta - metabolism
Pregnancy
Tetrahydrocortisol - analysis
Tetrahydrocortisol - metabolism
Tetrahydrocortisone - analysis
Tetrahydrocortisone - metabolism
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Summary:A reversed-phase high–performance liquid chromatography–electrospray–tandem mass spectrometry assay (HPLC–ESI–MS/MS) was developed to quantitate cortisol, cortisone, 20α- and β-dihydrocortisol, 20α- and β-dihydrocortisone, tetrahydrocortisol, and tetrahydrocortisone. The technique was used to analyze perfusate from the isolated human placental lobule for cortisol and its metabolites. Analytes were prepared from the perfusion medium using C18 solid-phase extraction cartridges. The internal standard for the analyses was 6α-methylprednisolone. Chromatography was performed on a Novapak C18 column at ambient temperature using 53% methanol and 47% 10 mm ammonium formate buffer (pH 4.0) as mobile phase, at a flow rate of 80 μL/min. A PE-SCIEX API III triple quadrupole instrument was used for mass spectrometric detection. An ionspray (pneumatically assisted electrospray) interface was used in negative and positive ionization mode. The assay was linear over the range 100–2000 μg/L for each analyte. The instrumental limit of detection was 50 pg. Assay imprecision at 400 and 800 μg/L was ≤ 10% (total coefficient of variation). Accuracy ranged between 83.2% for 20β-dihydrocortisone to 102.6% for cortisone. Recovery of 1000 μg/L analyte ranged from 91.3% for cortisone to 109.7% for tetrahydrocortisol.
ISSN:0003-2697
1096-0309
DOI:10.1006/abio.1997.2074