MSACL 2019 EU Abstract

Simultaneous Determination of Cortisol and Cortisone in Saliva by LC-MS/MS: Method Validation

Marco Bagnati (1), Marta Lamonaca (1), Ilaria Crespi (1), Giorgio Bellomo (1), Thomas Matulli Cavedagna (2), Vincenza Flora Dibari (2), Umberto Dianzani (1)
(1) Clinical Chemistry Unit, Department of Health Sciences, University of Piemonte Orientale, Maggiore della Carità Hospital, Novara and (2) R&D Laboratory, B.S.N., Gerenzano (VA), Italy

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Marco Bagnati (Presenter) Maggiore della Carità Hospital >> POSTER (PDF)

Relevant Financial Disclosures (within past 24 months) Honorarium/Expenses B.S.N. Srl, Castelleone, Cremona, Italy Conflict mitigation NOT REQUIRED.

INTRODUCTION: Cortisol measurement is a key-test in the diagnosis of Cushing syndrome and in other clinically relevant conditions. It can be performed in different biological samples: serum, urine and, in more recent years, saliva. In salivary measurements only free hormone is detected, samples can be collected during normal daily routine and stress-induced cortisol release is less likely to occur than after venipuncture. Although immunometric assays for salivary cortisol are employed, but suffer from non-specificity due to cross reactivity with steroid metabolites, synthetic corticosteroids or drugs and lack of standardization between labs.
OBJECTIVE: We developed and validated an analytical method for routine measurement of cortisol and cortisone in salivary samples.
METHODS: Saliva can be obtained by standard procedures using available devices (Salivette). 200 µL were added to an equal volume of dilution solution (containing cortisol-D4 as internal standard) and 1 ml of extraction solution. After extensive vortexing and centrifugation, 800 µL of the upper phase are collected, dried under nitrogen and resuspended with 30 µL of the reconstitution solution and 30 µL of the dilution solution. After vortexing and resuspension, 60 µL were transferred in a new vial placed in the LC-MS/MS autosampler. Samples were then analyzed using an AB SCIEX Triple Quad™ 6500 LC-MS/MS system (column: Hypersil Gold, ID 2.1x50 mm, 1.9 µm; flow: gradient; T 30°C; inj vol: 10 µL; MRM: cortisol: 363.1>121.1, 363.1>105.1; cortisone 361.1>121.1, 361.1>105.1; cortisol-D4: 367.1>121.1, 367.1>97.1; ESI+).
Within-, between-run and within-lab repeatability were calculated by ANOVA.
RESULTS: The validated method displayed as LLOD 5,7 pg/mL (cortisol) and 2.7 pg/mL (cortisone), as LLOQ 19.1 pg/mL (cortisol) and 9.0 pg/mL (cortisone), linearity 19.1-50000 pg/mL (cortisol) and 9.0-160000 pg/mL (cortisone), recovery 96-103% (cortisol) and 89-110% (cortisone); within-run, between-run and total repeatability was respectively 3.7 %, 4.9 % and 6.1 % for cortisol (at a concentration of 15 ng/ml) and 2.5 %, 5.3 % and 5.9 % for cortisone (at a concentration of 38 ng/ml). Samples can be stored up to three months at 4°C or up to one year at -20°C.
CONCLUSION: A LC-MS/MS method was developed and validated for the routine measurement of cortisol and cortisone in salivary samples. This method will allow to reduce laboratory errors due to both sample collection and unspecific measures, may assist the clinicians in the diagnosis of endocrinological diseases and will avoid unnecessary hospitalization (for late night collection) and invasive procedures.