Edward Goucher (Presenter)
Thermo Fisher Scientific
Bio: Global Business Development Manager for Applied Markets at Thermo Fisher Scientific.
Authorship: Joe Di Bussolo (1), Marta Kozak (1), Edward Goucher (1), Ian White (2), Emily Herman (2), Raidiri Castillo (3), Amit Shah (3) and Hashim Othman (3)
(1) Thermo Fisher Scientific (2) West Chester University of PA (3) Bio-Reference Laboratories
| Short Abstract Pregnenolone is a biosynthetic precursor to other steroids such as corticosteroids, androgens, and estrogens. We developed a sensitive, robust, high-throughput quantitation assay for pregnenolone which can measure 10 to 500 ng/dL (0.3 to 1.5 nmol/L) in blood serum. The LC-MS/MS method can be multi-channeled with other HESI-MS/MS methods with a total runtime of 4.5 minutes. Derivatization with hydroxyl amine was necessary to reliably achieve the desired analytical range. Selective-reaction monitoring (SRM) within a 1-minute data window produced quantitation and conformation chromatographic peaks. |
Long Abstract
Background: Pregnenolone is a biosynthetic precursor to other steroids such as corticosteroids, androgens, and estrogens. It is converted to progesterone by 3-beta-hydroxysteroid dehydrogenase or to 17-OH-pregnenolone by 17-alpha-hydroxylase. Researchers investigating how these enzymes function need to quantify pregnenolone within an analytical range of 10 to 500 ng/dL (0.3 to 1.5 nmol/L) in blood serum. Since pregnenolone does not ionize well by either atmospheric-pressure chemical-ionization (APCI) or electrospray ionization (ESI), derivatization with hydroxyl amine was necessary to reliably achieve the desired analytical range.
Methods: Pregnenolone was measured in blood serum using a multi-channel ultra high-performance liquid chromatography (UHPLC) coupled to a triple-quadrupole mass spectrometer (MS) with heated electro-spray ionization (HESI). Sample preparation involved extracting specimens with methyl-t-butyl ether after spiking them with pregnenolone-D4 internal standard (IS). The extracts were evaporated and the residues were reacted with hydroxyl amine to form positive-ion oxime derivatives. The preparations were dried and reconstituted with water and methanol (1:1). Injections were made into a 4-channel UHPLC system. A 4.5-minute water-to-methanol gradient eluted the analyte and IS through a heated column, packed with solid-core silica with phenyl groups bonded to its surfaces, into the HESI source. Selective-reaction monitoring (SRM) within a 1-minute data window produced quantitation and conformation chromatographic peaks.
Results: Confirmation/quantitation ion ratios among calibrators, QCs, and specimens were within 20% of averages calculated from the calibrators. Method precision, assessed as percent coefficient of variation (%CV) of peak areas from 20 replicate injections of two pools of test specimens, were better than 5% and 8% for intra- and inter-batches, respectively. Carryover, measured in blanks immediately following injections of the highest calibrator, never exceeded 0.2%. Specimen IS peak areas averaged 65% relative to the averaged IS peak areas in calibrators and QCs, indicating moderate ion-suppression by matrix. However, the IS in each sample successfully compensated for matrix effects as proved by method comparison results. The desired analytical range from 10 to 500 ng/dL (0.3 to 1.5 nmol/L) was achieved and was consistently linear (r2 ≥ 0.999 with 1/X weighting). For method comparison, 40 donor samples were analyzed and results were compared with those from a reference lab. Pregnenolone values among these samples ranged from 13 to 130 ng/dL and the percent difference between two analytical methods did not exceed 20% for 93% of the samples. Sample throughputs were 13, 26, 39 or 52 injections per hour when multi-channeled across 1, 2, 3 or 4 channels, respectively.
Conclusion: We developed a sensitive, robust, high-throughput quantitation assay for pregnenolone which can measure 10 to 500 ng/dL (0.3 to 1.5 nmol/L) in blood serum. The LC-MS/MS method can be multi-channeled with other HESI-MS/MS methods.
References & Acknowledgements:
| Description | Y/N | Source |
| Grants | no | |
| Salary | yes | Thermo Fisher Scientific |
| Board Member | no | |
| Stock | no | |
| Expenses | no |
IP Royalty: no
| Planning to mention or discuss specific products or technology of the company(ies) listed above: | yes |