Influence of Anticoagulants and Storage Conditions During Blood Sample Collection on Determination of the 6β-hydroxycortisol/cortisol Ratio by LC-MS/MS

• Published in: BUNSEKI KAGAKU Vol. 71; no. 6; pp. 357 - 363
• Main Authors: SHIBASAKI, Hiromi, YOKOKAWA, Akitomo, FURIHATA, Tomomi
• Format: Journal Article
• Language: Japanese
• Published: Tokyo The Japan Society for Analytical Chemistry 2022; Japan Science and Technology Agency
• Subjects: 6β-hydroxycortisol; anticoagulant; Anticoagulants; Blood; cortisol; Ethylenediaminetetraacetic acids; Heparin; High performance liquid chromatography; Hormones; LC-MS/MS; Mass spectrometry; Separation; storage condition; Tubes
• ISSN: 0525-1931
• DOI: 10.2116/bunsekikagaku.71.357

Here, we evaluated the effects of anticoagulants and storage conditions on the measurement of plasma cortisol and 6β-hydroxycortisol (6β-OHF) by high-performance liquid chromatography-electrospray ionization tandem mass spectrometry (LC–MS/MS), using stable isotope-labeled compounds as internal standards for calculating plasma or serum 6β-OHF/cortisol concentration ratios. Blood samples were obtained from five healthy subjects and divided into tubes containing three anticoagulants: sodium heparin, serum separation gel, and EDTA-2 potassium. There was no statistically significant difference in cortisol and 6β-OHF concentrations and 6β-OHF/cortisol ratios in the samples obtained from the three anticoagulants measured on the day of blood sampling. Serum samples obtained from serum separation gel were stored at 4°C for 4 days then at –20°C for 3 days, or at 4°C for 7 days. Whole blood samples with EDTA were stored at 4°C for 7 days. The cortisol and 6β-OHF concentrations in serum samples after 7 days of storage under two storage conditions were slightly higher and lower, respectively, than the value on day 1. The concentration of 6β-OHF stored as whole blood was higher than that of the serum storage sample due to the high blood/plasma ratio of 6β-OHF. We compared 6β-OHF/cortisol ratios with different anticoagulants and storage conditions. The change in the ratio was within 15 % in the serum sample, but –4.9 % ∼ 17.7 % in the whole blood sample. Although the storage condition affected the 6β-OHF/cortisol ratio, it ded not affect the subject's ratio order. Therefore, blood samples with any anticoagulant and under any storage conditions can be used; however, it is more desirable to use serum samples stored at –20°C than whole blood samples.