A novel ultrapressure liquid chromatography tandem mass spectrometry method for the simultaneous determination of androstenedione, testosterone, and dihydrotestosterone in pediatric blood samples: age- and sex-specific reference data.
BACKGROUND
Current immunoassays for analysis of plasma androgens in children have several limitations due to antibody-specific variations of data and normal ranges. Mass spectrometry-based methods are available for individual steroids but need complex sample preparation and report only fragmentary reference data for the pediatric population.


OBJECTIVE
Our objective was to develop a state of the art sensitive and specific tandem mass spectrometry method for high-throughput simultaneous determination of plasma concentrations of androstenedione (A), testosterone (T), and dihydrotestosterone (DHT) and to report age-, sex-, and pubertal stage-specific reference levels for these steroids in children aged 0-18 yr.


METHODS
Plasma (100 microl) was mixed with internal standard and extracted by solid-phase extraction. Androgens were measured by ultrapressure liquid chromatography tandem mass spectrometry. Samples of 138 boys and 131 girls with neither signs of endocrine nor systemic disease were considered for the generation of reference data. The following age groups were used: less than 1 wk, 2 wk to 2 months, 3-5 months, 6-11 months, 1-3 yr, 4-6 yr, 7-9 yr, 10-12 yr, 13-15 yr, and over 16 yr.


RESULTS
Lower quantification limit was 2.9 ng/dl (0.1 nmol/liter) for A, T, and DHT. No relevant interference with other steroids was detected. Reference data for A, T, and DHT are reported as functions of age, sex, pubertal maturation, and testicular volume.


CONCLUSIONS
Simplicity, velocity, sensitivity, specificity, and the availability of pediatric reference data allow application of our new method in clinical routine as well as in research settings.
Kulle
A E
AE
Department of Pediatrics, Christian-Albrechts University of Kiel, 24105 Kiel, Germany.
Riepe
F G
FG
Melchior
D
D
Hiort
O
O
Holterhus
P M
PM
D000293:Adolescent; D000367:Age Factors; D000728:Androgens / Q000097:blood; D000735:Androstenedione / Q000097:blood*; D002648:Child; D002675:Child, Preschool; D002853:Chromatography, Liquid / Q000379:methods; D013196:Dihydrotestosterone / Q000097:blood*; D005260:Female; D006801:Humans; D007223:Infant; D008297:Male; D013058:Mass Spectrometry / Q000379:methods; D015203:Reproducibility of Results; D012727:Sex Characteristics; D013737:Testis / Q000033:anatomy & histology; D013739:Testosterone / Q000097:blood*
D000728:Androgens; D013196:Dihydrotestosterone; D013739:Testosterone; D000735:Androstenedione