Method For Determination Of Cortisol Research Articles

Highly selective and automated online SPE LC–MS3 method for determination of cortisol and cortisone in human hair as biomarker for stress related diseases

Hair analysis has been increasingly used to establish long-term biomarkers of exposure to both endogenous and exogenous substances, with a special emphasis on steroidal hormones. Hair cortisol and cortisone have been associated to physiological and psychological strains, anxiety and depression. Hair is a very complex matrix, which might jeopardize analyte detection at low concentrations. A new, highly selective and sensitive method based on fragments of second order, MS3 (MS/MS/MS), was developed and validated for the analysis of hair cortisol and cortisone. An online solid phase extraction was performed on a C8 restricted access material (RAM) phase following by separation on a reversed-phase C18 column using methanol and 0.02% ammonium hydroxide as mobile phase. The developed method required minimal sample preparation and the injection of only 50µL of sample leading to a LOQ of 2pgmg−1. Good linear responses were observed in the range 2–200pgmg−1 (R2>0.99) and extraction recoveries ranged between 77–125% and 70–123% for cortisol and cortisone, respectively. Intra- and inter-assay coefficients of variation were between 1.4 and 14%. In order to evaluate the applicability of the method, preliminary tests (N=33) were conducted in 3cm hair samples (close to scalp) of healthy volunteers with an age range of 4–63. Average concentrations in hair were 12.7±14pgmg−1 and 41.6±42pgmg−1 for cortisol and cortisone, respectively. Further investigations on cortisol and cortisone as biomarkers for chronic psychological strain will be assessed as a next step.

Determination of cortisol in human saliva using liquid chromatography–electrospray tandem mass spectrometry

The aim of this work was to develop a method for determination of cortisol in saliva by liquid chromatography–tandem mass spectrometry (LC–MS–MS). Saliva was sampled on Salivette tubes. These were centrifuged, deuterium-labeled cortisol was added as internal standard and the proteins precipitated by acetonitrile. The supernatant was evaporated, dissolved in methanol acidified with acetic acid and analyzed by LC–MS–MS. The with-in run precision, tested by pooling saliva samples from volunteers and then analyzing these in a single run, was found to be 7% at 0.7 μg l −1. The between-run precision was tested by analysis of the same samples at different days and found to be 11% at 2.5 μg l −1. The limit of quantification was 0.5 μg l −1. The method was applied for analysis of saliva samples from three volunteers during their last week before vacation and the first and second week on vacation. In addition, the method was compared to analysis by an immunological method. The values from the immunological method were 2.7 times higher than the LC–MS–MS results.

Automated bioanalysis of drugs using coupled column liquid chromatography for sample pretreatment aspects of quality assurance and collaborative automated handling of data

In this paper two new examples on the use of coupled column liquid chromatography (CCLC) for automated analysis of drugs in biosamples are given. A fully automated method for determination of cortisol in human plasma was realized by combining automated sample workup of plasma samples on an automated instrument for solid phase extraction on-line with a CCLC system which was used for further sample pretreatment. Coupled columns can also be used as an injection device in liquid chromatography allowing for automated sample pretreatment and interfacing to miniaturized columns. The potential of this approach for enhancement of sensitivity and selectivity is exemplified with the determination of a drug candidate at picomolar concentration. Examples of the performance characteristics of the two applications demonstrate the capability of these approaches for routine analysis. The paper also stresses the need for a quality assurance programme in order to assure the quality of the data generated by a laboratory. A laboratory information management system in which the analytical method forms an integral part is also demonstrated. This system is forming the basis for collaborative automated handling of data generated by e. g. a fully automated method based on CCLC.

Radioimmunoassay and chemical ionization/mass spectrometry compared for plasma cortisol determination.

We describe a method for determination of cortisol in plasma and urine, based on chemical ionization/mass spectrometry with deuterium-labeled cortisol as the internal standard. The within-run precision (CV) was 2.5-5.7%, the between-run precision 4.6%. Results by this method were compared with those by a radioimmunological method (RIANEN Cortisol, New England Nuclear) for 395 plasma samples. The latter method gave significantly higher (approx. 25%) cortisol values.