Proposed definitive methods for measurement of plasma testosterone and 17α-hydroxyprogesterone

Objectives: This report provides the results of the development and evaluation of definitive isotope dilution/mass spectrometry (ID/MS) methods for the determination of testosterone and 17α-hydroxyprogesterone 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 16,16,17-2H-testosterone and 21,21,21,-2H-17α-hydroxyprogesterone. The development of optimum extraction and derivatization procedures, and studies of storage time, temperature effects, accuracy, and precision are presented. Results: The results indicate that the methods employing the TBDMS derivative of testosterone and MO-TMS derivative of 117α-hydroxyprogesterone are capable of generating accurate and precise data at the inherently low concentrations given, with recovery greater than 95%. Accuracy of testosterone in the fortified steroid-free and pooled plasma by ID/MS measurement was good because the relative error ranged from +3.1% to −0.7, with a mean of 0.9% over the concentration levels of 1.4 to 37.9 nmol/L testosterone, and the imprecision ranged from 4.2% to 0.7% CV, with a mean of 1.9%. Accuracy of 17α-hydroxyprogesterone in the fortified steroid-free and pooled plasma was also good, considering the inherently low concentrations. The relative error ranged from −2.1% to +1.5%, with a mean of 1.1%, and the imprecision ranged from 3.8% to 0.8% CV with a mean of 1.3% over concentration levels of 1.5 to 15.1 nmol/L. The high precision and accuracy and absence of statistically significant bias qualifies these methods as candidate definitive methods for plasma testosterone and 17α-hydroxyprogesterone.