Quantification of Human Plasma 18-Hydroxycorticosterone by Isotope Dilution Ultra Performance Liquid Chromatography Tandem Mass Spectrometry

  Objective  To establish a method for quantification of 18-Hydroxycorticosterone (18-OHB) in plasma by isotope dilution ultra performance liquid chromatography tandem mass spectrometry (ID-UPLC-MS/MS).

  Methods  This study was a methodology-validation on the evaluation of plasma 18-OHB with LC-MS/MS. Two hundreds microliter(μL) of serum samples or standard solution were mixed with 18-OHB-d4 (internal standard) and treated with methanol solution to precipitate protein and anion-exchange solID-phase extraction(SPE). After SPE, the eluates were detected in the positive electro-spray ionization mode and multiple reaction-monitor mode. The precision, recovery, lower limits of quantification, linearity and the matrix-effect of LC-MS/MS have been evaluated. Since November 2019, healthy participants were recruited continuously to the study to validate the reference intervals of Mayo Clinical Laboratory.

  Results  The analyzing time was 3.0 min. The repeatability coefficient of variation and laboratory imprecision for detecting low, medium and high levels of 18-OHB were 2.2%-3.5% and 3.7%-5.0%, respectively. The average recovery of 18-OHB ranged between 98.1% and 101.7%.The lower limit of quantification was 0.1 μg/L. The matrix of plasma had no significant effect on the measurement of 18-OHB. The 2.5th to 97.5th percentiles of 18-OHB measured by ID-UPLC-MS/MS in apparently healthy population was 0.01 and 0.60 μg/L, The plasma level of 18-OHB is outside the reference range provided by the Mayo Medical Laboratory Center in 66%(48/73) of the population.

  Conclusions  A reliable and specific LC-MS/MS method for evaluating plasma 18-OHB was established in the clinical laboratory. The method was simple, rapid and suitable for the diagnosis and classification of primary aldosteronism.