Simultaneous determination of 17α-hydroxypregnenolone and 17α-hydroxyprogesterone in dried blood spots from low birth weight infants using LC-MS/MS

17α-Hydroxypregnenolone (17OHPreg) has heretofore been considered to be the major cause of the false elevated 17α-hydroxyprogesterone (17OHP) value in the immunoassay-based newborn screening for congenital adrenal hyperplasia (CAH). To verify this point, we developed a liquid chromatography-tandem mass spectrometric (LC-MS/MS) method that enables the simultaneous quantification of 17OHPreg and 17OHP in the dried blood filter papers and measured their blood levels in infants, especially in infants with low birth weights. Steroids were extracted from the filter papers with methanol, purified using a Strata-X cartridge, derivatized with 2-hydrazinopyridine and subjected to LC-MS/MS. Validation tests proved that this method was specific and reproducible; endogenous steroids did not interfere with the quantifications, and the intra- and inter-assay coefficients of variation were below 5.2%. The limits of quantitation were 1.0 and 0.5 ng/mL for 17OHPreg and 17OHP, respectively, when 3 disks (3 mm in diameter) of the filter papers (corresponding to 8 μL of whole blood) were used. The blood 17OHPreg level was elevated in the very low birth weight (1000-1500 g) infants and extremely low birth weight (<1000 g) infants, compared to those in the normal birth weight (>2500 g) infants (P < 0.05). However, the 17OHPreg concentration was not high enough to cause the false positive results in the enzyme immunoassay-based screening, and it was considered that the false positive results come from other endogenous components rather than 17OHPreg.