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.
Rauh
Manfred
M
Klinik für Kinder und Jugendliche, Universitätsklinikum Erlangen, Loschgestr. 15, 91054 Erlangen, Germany. manfred.rauh@kinder.imed.uni-erlangen.de
Gröschl
Michael
M
Rascher
Wolfgang
W
Dörr
Helmuth G
HG
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