Automated, fast and sensitive quantification of 17 alpha-hydroxy-progesterone, androstenedione and testosterone by tandem mass spectrometry with on-line extraction.
Plasma 17 alpha-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 18 e column allow a rapid and easy quantification. The lowest limit of detection was 0.03-0.06 microg/L. Our method has proved linear up to 250 microg/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=1.942 x+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=0.963 x+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.
Klinik für Kinder und Jugendliche, Universitätsklinikum Erlangen, Loschgestr. 15, 91054 Erlangen, Germany. manfred.rauh@kinder.imed.uni-erlangen.de
Manfred
M
Rauh
Michael
M
Gröschl
Wolfgang
W
Rascher
Helmuth G
HG
Dörr
D019326:17-alpha-Hydroxyprogesterone / Q000097:blood*; D000293:Adolescent; D000735:Androstenedione / Q000097:blood*; D002138:Calibration; D002648:Child; D002675:Child, Preschool; D002853:Chromatography, Liquid; D005260:Female; D006801:Humans; D007223:Infant; D007231:Infant, Newborn; D008297:Male; D013058:Mass Spectrometry; D012015:Reference Standards; D015203:Reproducibility of Results; D013739:Testosterone / Q000097:blood*; D013997:Time Factors
D013739:Testosterone; D000735:Androstenedione; D019326:17-alpha-Hydroxyprogesterone