Daniel Zhou (Presenter)
Stanford Health Care
Authorship: Daniel Zhou (1), El Taher M. El Gierari (1) Run Zhang Shi (2)
(1) Stanford Health Care, (2) Stanford University School of Medicine
| Short Abstract STAT 17-hydroxyprogesterone [17-OHP] is commonly ordered for confirmation of congenital adrenal hyperplasia [CAH] mostly in neonates and pediatric patients. Rapid test turnaround time [TAT] is critical, especially in life-threatening salt-wasting cases. Since conventional LC-MS/MS requires a time consuming manual sample preparation step, we investigated commercially available dispersive pipette extraction tips [DPX tips] to shorten the sample preparation time with the aim of building a STAT test to meet a clinical diagnostic need. Results indicate that DPX tips are effective for 17-OHP analysis, and may reduce sample preparation time by 75%. This rapid method is able to meet the STAT test requirement for CAH confirmation in children. |
Long Abstract
Background:
STAT 17-hydroxyprogesterone [17-OHP] is commonly ordered for confirmation of congenital adrenal hyperplasia [CAH] mostly in neonates and pediatric patients. Rapid test turnaround time [TAT] is critical, especially in life-threatening salt-wasting cases. Conventional LC-M/MS is the method of choice; however, a time consuming manual sample preparation step is required mostly by liquid-liquid extraction which involves centrifugation and drying steps. We investigated commercially available dispersive pipette extraction tips [DPX tips] to shorten the sample preparation time with the aim of building a STAT test to meet a clinical diagnostic need.
Methods:
Calibrator/QC/serum sample [100 µL] were first aspirated into a WAX tip [DPX Labs, LLC, Columbia, SC] followed by continued aspiration of 150 µL of IS [17OHP-d8] in acetonitrile. A small air gap is then pulled into the tip to completely mix the two solutions with the sorbent material within the WAX tip. The cleaned extract was dispensed into a new set of tubes and a 30 µL aliquot was diluted with 100 µL of H2O before injection [100 µL] to a LC-MS/MS system. LC separation was achieved by a Kinetex 2.6 µm C18 column [Phenomenex] using buffer A [0.1% formic acid in H2O] and buffer B [0.1% formic acid in methanol]. Quantification was performed on a 5500 tandem mass spectrometry [SCIEX] in the multiple reaction monitoring [MRM] positive mode after electrospray ionization [ESI]. All results were normalized by internal standard peak area by using Analyst 1.6.1 [SCIEX].
Results: Preliminary validation results showed good correlation compared with a conventional LC-MS/MS method currently in clinical use. The new sample preparation method was much more rapid [~30 minutes] compared with a conventional approach [~2 hours].
Conclusion: These results indicate that DPX tips with weak anion exchange sorbent is effective for 17-OHP analysis, and DPX tips may reduce sample preparation time by 75%. The rapid method is able to meet the STAT test requirement in a clinical lab for confirmation of CAH in children.
References & Acknowledgements:
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