Background:Congenital adrenal hyperplasia is a group of autosomal recessive disorders caused by a deficiency of 1 of 4 enzymes required for the synthesis of glucocorticoids, mineralocorticoids, and sex hormones. Analysis of 11-deoxycortisol (11DC), 17-hydroxyprogesterone (17OHP), 17-hydroxypregnenolone (17OHPr), and pregnenolone (Pr) in blood allows detection of these enzyme defects. Methods: The steroids were extracted from 200 L of serum or plasma by solid-phase extraction, derivatized to form oximes, and extracted again with methyl t-butyl ether. Instrumental analysis was performed on an API 4000 tandem mass spectrometer with electrospray ionization in positive mode and multiple reaction-monitoring acquisition. Results: The limits of detection were 0.025 g/L for 11DC, 17OHP, and Pr and 0.10 g/L for 17OHPr. The method was linear to 100 g/L for 11DC, 17OHP, and Pr, respectively, and to 40 g/L for 17OHPr. Within-and between-run (total) imprecision (CVs) were <7.1% and 11%, respectively. Reference intervals for children in Tanner stages 1 through 5 and adult males and females for 17OHP, 11DC, Pr, and 17OHPr were established. Prepared samples were stable for >72 h. Conclusions: The detection limit and selectivity of this method and its small sample volume requirement allow analysis of endogenous concentrations of adrenal ste-

Background: Choline is critical for a variety of biological functions and has been investigated as a biomarker for various pathological conditions including acute coronary syndrome. Methods: A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was used to quantify choline in whole blood and plasma in freshly collected samples prepared with ultrafiltration or protein precipitation. We investigated the effects of preanalytical variables including types of anticoagulants and storage temperature and time. Results: We observed no significant differences in whole-blood choline concentration in EDTA-anticoagulated vs heparin-anticoagulated samples: mean (SD) difference 0.9% (3.2%), P = 0.80. For plasma, choline concentrations with heparin in 5 of 12 volunteers were &gt;10% higher than with EDTA, P = 0.01. One freeze-thaw cycle led to significant mean (SD) increases in choline concentrations in heparin whole blood, 19.3% (11.4%), P &lt;0.01, and the effect was not significant for other sample types studied (P &gt;0.33). For freshly collected samples stored at ambient temperature, choline concentrations in all types of samples increased with storage time. For EDTA whole blood, EDTA plasma, and heparin plasma, the choline concentration increased for the first 60 min and then stabilized. For heparin whole blood, the choline concentration continued to increase linearly with storage time for &gt;4 h, at which time the choline concentrations were increased by approximately 50%. Conclusions: Sample collection, storage, and sample preparation procedures are critical for clinical measurements of choline in whole blood and plasma.

During puberty, serum steroid concentrations change dramatically. The objective of this study was to determine the adrenal steroid concentrations in children from 7 to 17 years of age. Tanner stage was determined in each child by physical examination. 11-Deoxycortisol, pregnenolone, 17-hydroxypregnenolone, 17-hydroxyprogesterone and testosterone were quantified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Androstenedione and dehydroepiandrosterone sulfate were measured by immunoassay. The median and central 95% of the steroid concentrations were determined for age, gender, and Tanner stage. Except for 11-deoxycortisol, all of the steroids exhibited an increase in concentration after age 7-9 years in both boys and girls. 11-Deoxycortisol, which is made exclusively in the adrenal cortex, declined with age and Tanner stage. This suggests that a rise in gonadal function and decreased efficiency of 11beta-hydroxylase with age may contribute to an increase in the remaining steroids. Testosterone concentrations increased more dramatically in boys, but increases were seen with each Tanner stage in girls.