Automated isotope dilution liquid chromatography–tandem mass spectrometry with on-line dilution and solid phase extraction for the measurement of cortisol in human serum sample

•Automated analysis.•We use empty column for the online dilution.•Simply and high sensitivity.•High accuracy.

A candidate reference measurement procedure involving automated isotope dilution coupled with liquid chromatography–tandem mass spectrometry (ID-LC–MS/MS) with on-line dilution and solid phase extraction (SPE) has been developed and critically evaluated. We constructed the LC–MS/MS with on-line dilution and SPE system. An isotopically labelled internal standard, cortisol-d4, was added to serum sample. After equilibration, the methanol was added to the sample, and deproteination was performed. Then, the sample was applied to the LC–MS/MS system. The limit of detection (LOD) and limit of quantification (LOQ) were 0.2 and 1 ng g−1, respectively. Excellent precision was obtained with within-day variation (RSD) of 1.9% for ID-LC–MS/MS analysis (n = 6). This method, which demonstrates simple, easy, good accuracy, high precision, and is free from interferences from structural analogues, qualifies as a reference measurement procedure.

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