Congenital Adrenal Hyperplasia (CAH) is caused by inherited defects in steroid biosynthesis, in particular 21-hydroxylase deficiency, the classical form of CAH. Hormonal imbalances are reflected in decreased levels of aldosterone and cortisol and excessive secretion of 17?-hydroxyprogesterone (17-OHP) and androstenedione. Diagnosis of CAH is based on the quantification of 17-OHP, usually by immunoassay. Compared with other neonatal screening tests, the specificity of screening for 21-CAH by immunoassays is low, characterized by high false-positive results. This is due to cross-reactivity with steroids other than 17-OHP, especially in pre-term neonates and in critically ill newborns.

Methods

We have developed a simple method to determine the levels of 17-OHP, cortisol, 11-deoxycortisol, 21-deoxycortisol, and androstenedione in serum using ultra performance liquid chromatography-tandem mass spectrometry (UPLC/MS/MS). Calibrators were prepared in stripped serum over the concentration range 5 to 500ng/mL for cortisol, 1 to 100ng/ML for 11-deoxycortisol, 21-deoxycortisol, and androstenedione and 1 to 300ng/mL for 17-OHP; QC samples were prepared independently. A simple liquid-liquid extraction procedure was developed using 50uL of serum/plasma and methyl-tert-butyl ether as the extraction solvent. The analytes were separated using a gradient of 45-98% methanol with 0.1% formic acid and detected by multiple reaction monitoring mass spectrometry.

Results

The assay was linear over each analyte concentration range with all correlation co-efficients (R2) > 0.997. Inter- and intra-day precision of the assay was better than 10%CV across the analytical range.

Conclusions

A second-tier test for CAH to reduce the incidence of false positive test results has been developed. The method allows for detection and quantification of cortisol, 21-deoxycortisol, 11-deoxycortisol, androstenedione and 17-OHP over the range of the clinical assay with good linearity, sensitivity and precision.