Automated, fast and sensitive quantification of 17α-hydroxy-progesterone, androstenedione and testosterone by tandem mass spectrometry with on-line extraction

Plasma 17α-hydroxyprogesterone (17-OHP), androstenedione and testosterone measurements are important for the diagnosis and monitoring of hyperandrogenic disorders, most importantly for congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency. The reliability of immunoassays has proved questionable especially for newborns and children. In order to reduce the analytical interferences due to cross-reactivity or matrix effects, to improve accuracy and shorten the analysis time, we have developed a liquid chromatography–tandem mass spectrometry (LC–MS/MS) method with atmospheric pressure chemical ionization (APCI) for simultaneous measurement. An on-line extraction cartridge with column-switching technique and liquid chromatography over a Chromolith RP 18e column allow a rapid and easy quantification.The lowest limit of detection was 0.03–0.06 μg/L. Our method has proved linear up to 250 μg/L (r = 0.999). Recoveries (S.D.) of 17-OHP, androstenedione and testosterone in plasma were 100% (5), 102% (2) and 92% (4), respectively. The regression equation for the LC–MS/MS (x) and immunoassay (y) methods for 17-OHP (excluding neonate samples) was y = 1.942x + 0.255 nmol/L (r = 0.695; n = 97). In comparison to our values, the immunoassay generally overestimates steroid concentration. The regression equation for the LC–MS/MS (x) and immunoassay (y) methods for testosterone was y = 0.963x + 0.035 nmol/L (r = 0.955; n = 107). Preliminary reference intervals for children were determined as a function of age and sex.The sensitivity and specificity of the LC–MS/MS method offer advantages over routine immunoassays due to the elimination of interferences especially for newborns, high throughput and short chromatographic run time.