PROPOSED DEFINITIVE METHODS FOR MEASUREMENT OF PLASMA TESTOSTERONE AND 17-ALPHA-HYDROXYPROGESTERONE

Objectives: This report provides the results of the development and ev aluation of definitive isotope dilution/mass spectrometry (ID/MS) meth ods for the determination of testosterone and 17 alpha-hydroxyprogeste rone in human plasma at concentrations ranging from 1.4 to 37.9 nmol/L and 1.5 to 45.4 nmol/L, respectively. The internal standards were 16, 16,17-H-2-testosterone and 21,21,21,-H-2-17 alpha-hydroxyprogesterone. The development of optimum extraction and derivatization procedures, and studies of storage time, temperature effects, accuracy, and precis ion are presented. Results: The results indicate that the methods empl oying the TBDMS derivative of testosterone and MO-TMS derivative of 17 alpha-hydroxyprogesterone are capable of generating accurate and prec ise data at the inherently low concentrations given, with recovery gre ater than 95%. Accuracy of testosterone in the fortified steroid-free and pooled plasma by ID/MS measurement was good because the relative e rror ranged from +3.1% to -0.7, with a mean of 0.9% over the concentra tion levels of 1.4 to 37.9 nmol/L testosterone, and the imprecision ra nged from 4.2% to 0.7% CV, with a mean of 1.9%. Accuracy of 17 alpha-h ydroxyprogesterone in the fortified steroid-free and pooled plasma was also good, considering the inherently low concentrations. The relativ e error ranged from -2.1% to +1.5%, with a mean of 1.1%, and the impre cision ranged from 3.8% to 0.8% CV with a mean of 1.3% over concentrat ion levels of 1.5 to 15.1 nmol/L. The high precision and accuracy and absence of statistically significant bias qualifies these methods as c andidate definitive methods for plasma testosterone and 17 alpha-hydro xyprogesterone.