A sensitive and accurate LC-MS/MS assay with the derivatization of 1-Amino-4-methylpiperazine applied to serum allopregnanolone, pregnenolone and androsterone in pre- and postmenopausal women

- An accurate and simultaneous LC-MS/MS assay of Allopregnanolone, Pregnenolone and Androsterone.
- A novel keto derivatization applied to Allopregnanolone, Pregnenolone and Androsterone.
- Low limit of quantitation for Allopregnanolone, Pregnenolone and Androsterone at 5 pg/mL.

The concentrations of allopregnanolone (Allopreg), pregnenolone (Preg) and androsterone (ADT) are very low in the circulation, especially in postmenopausal women, resulting in a considerable challenge for their accurate measurements in serum or plasma. In this report, a sensitive and reliable LC-MS/MS assay method has been developed using a simple sample preparation and the 1-Amino-4-methylpiperazine (AMP) derivatization procedure. A 5 pg/ml (0.1 pg on column) of low limit of quantitation has been achieved for Allopreg, Preg and ADT, with a sensitivity comparable to data obtained with the commercial reagent. The major benefit of this reagent is to limit the matrix effect since the excess amount of reagent can be removed during the reaction. Multiple reaction monitoring (MRM) from the derivatization of AMP not only increases the detection of these compounds but also provides a good resolution for Allopreg, Preg and ADT from interferences, especially for Allopreg from its isomers. Within the calibration range of 5 pg/ml to 2000 pg/ml, a good linearity was obtained with R > 0.99 where the weighing factor is 1/X. Bias and coefficients of variance are within 15% for all QC levels. The matrix effect has been evaluated, well meeting the acceptance criteria according to the FDA guidelines. With this method, the concentrations of Allopreg, Preg and ADT in postmenopausal serum are in the range of 6.4-53.6 pg/ml, 16.2-68.0 pg/ml and 23.9-114.0 pg/ml, respectively, while the ranges in premenopausal serum are 8.2-701.5 pg/ml, 31.2-135.2 pg/ml and 47.8-310.0 pg/ml, respectively.