Fast accurate quantification of salivary cortisol and cortisone in a large-scale clinical stress study by micro-UHPLC-ESI-MS/MS using a surrogate calibrant approach.

Cortisol and cortisone are common markers for stress and thus preferentially analyzed in matrices that allow non-invasive sampling such as saliva. Though the major drawback of immunoassays is lack of specificity due to cross reactivities, they are still most commonly used for quantification of steroid hormones. To overcome such problems, sensitive methods based on liquid chromatography-mass spectrometry are becoming more and more accepted as the golden standard for steroid bioanalysis as they achieve accurate quantification at trace levels for multiple analytes in the same run. Along this line, the aim of this study was the development of a new microflow UHPLC-ESI-MS/MS method for the measurement of salivary cortisol and cortisone, which due to its microflow regime provides enhanced sensitivity and is more ecofriendly. The developed method implemented sample preparation by Solid-Phase Extraction (SPE) in a 96-well plate format. Data acquisitions were carried out in MRM (multiple reaction monitoring) mode. The quantitative determination of endogenous compounds in saliva remains a challenge since analyte-free matrix is lacking. Hence, a surrogate calibrant approach with cortisol-d ₄ andcortisone- ¹³ C ₃ was applied for the target compounds in the presented method. A number of factors were optimized and the method validated. The lower limit of quantitation (LLOQ) was 72 and 62 pg mL ^-1 for cortisol and cortisone, respectively. Linear calibration was achieved in the range from 0.062 to 75.5 ng mL ^-1 for cortisol-d ₄ and 0.072 to 44 ng mL ^-1 forcortisone- ¹³ C ₃ . The performance of the method was also evaluated via proficiency test for salivary cortisol. Finally, it was applied successfully to evaluate cortisol and cortisone concentrations in multiple batches in routine clinical stress study samples (4056 total injections with 1983 study samples). Moreover, the instrument performance (in particular retention time variability) within each batch, between different batches and lot-to-lot of 5 investigated capillary columns over time is described. The work documents that micro-UHPLC-ESI-MS/MS is suitable and robust enough to carry out a full clinical study with greater than 1000s of samples over an extended period if adequate internal standards can be used.
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