Suparna Mundodi (Presenter)
Thermo Fisher
Bio: I am currently working as a Global Marketing Manager for Clinical vertical market for Thermo Fisher Scientific. I like combining my scientific background with a business outlook to solve every day problems. I am passionate about working with my clinical customers to understand their needs and learn the deeper science they are all involved on a daily basis to make this world a safer and healthier place.
Authorship: Joe Di Bussolo1, Ian White2, Raidiri Castillo3, Amit Shah3, Hashim Othman3, and Suparna Mundodi1
1Thermo Fisher Scientific, 2West Chester University of PA, 3Bio-Reference Laboratories.
| Short Abstract Here we present a sensitive, robust, high-throughput quantitation workflow for analysis of 17-Hydroxyprogesterone in serum implemented on 4-channel UHPLC system coupled to a triple-quadrupole mass spectrometer with atmospheric-pressure chemical ionization. Sample preparation involved liquid-liquid extraction. Processed samples were analyzed with a 4-minute water-to-methanol gradient LC method. A two SRM transitions were collected for analyte and for internal standard to calculate ion ratio. The limit of quantitation was 10 ng/dL, method precision was better than 6%RSD, negligible ionization suppression was observed. For method comparison, 40 donor samples were analyzed and results were consistent with those from reference lab. |
Long Abstract
Background: 17-Hydroxyprogesterone (17-OHP) is a biosynthetic precursor to other steroids such as corticosteroids, androgens, and estrogens. It is converted to 11-deoxycortisol by 21-alpha-hydroxylase or to androstenedione by 17, 20 lyase. Researchers investigating how these enzymes function need to measure 17-OHP within an analytical range of 10 to 1,000 ng/dL (0.3 to 30 nmol/L) in blood serum.
Methods: 17-0HP was measured in blood serum using a multi-channel ultra high-performance liquid chromatography (UHPLC) coupled to a triple-quadrupole mass spectrometer (MS) with atmospheric-pressure chemical ionization (APCI). Sample preparation involved extracting specimens with methyl-t-butyl ether after spiking them with 17-OHP-D8 internal standard (IS). After evaporating and reconstituting the extracts to concentrate them two-fold, injections were made into a 4-channel UHPLC system. A 4-minute water-to-methanol gradient eluted the analyte and IS through a column, packed with solid-core silica with hydrocarbon groups bonded to its surfaces, into the APCI 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 6% for intra- and inter-batches, respectively. Carryover, measured in blanks immediately following injections of the highest calibrator, never exceeded 0.1%. Specimen IS peak areas averaged 87% relative to the averaged IS peak areas in calibrators and QCs, indicating low ion-suppression by matrix. The desired analytical range from 10 to 1,000 ng/dL (0.3 to 30 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. 17-OHP values among these samples ranged from 13 to 789 ng/dL and the percent difference between two analytical methods did not exceed 20% for 95% of the samples. Sample throughputs were 14, 28, 42 or 56 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 17-hydroxyprogesterone which can measure 10 to 1,000 ng/dL (0.3 to 30 nmol/L) in blood serum. The LC-MS/MS method can be multi-channeled with other APCI methods.
References & Acknowledgements:
| Description | Y/N | Source |
| Grants | no | |
| Salary | yes | Thermo Fisher |
| 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 |