Theresa Swift (Presenter)
University of Michigan Health System
Bio: I am earned my bachelors degree in medical laboratory science from Oakland University (Rochester, MI). From there I completed a clinical internship in all areas of the laboratory at the Detroit Medical Center (Detroit, MI). After completing my internship I worked as a medical laboratory scientist at a local hospital for a few years working in all areas of the lab. I then decided to further my education by completing a professional science masters degree from the Pennsylvania State University in biotechnology. After graduation I returned to the hospital and began learning more about the method development process and developing tests using HPLC and LC/MS/MS in the clinical laboratory. I am currently employed in the drug analysis/toxicology lab at the University of Michigan Health System.
Authorship: Theresa Swift, Larry Clayton, Donald Giacherio and Hemamalini Ketha
Department of Pathology, University of Michigan Health System, Ann Arbor, MI
| Short Abstract Quantitation of steroid metabolites including 17-hydroxyprogesterone (17OHP), testosterone (TTST), androstenedione (ANDRO) and dehydroepiandrostenedione (DHEA) is useful in the diagnosis and clinical management of adrenal disorders like congenital adrenal hyperplasia. We have developed a UPLC-MS/MS method that can be used for simultaneous measurement of 17OHP, TTST and ANDRO. DHEA was also quantified using the same sample preparation and chromatographical method but with the requirement of relatively higher sample volume. Since our method does not need a derivatization step, complete automation of the sample extraction process using a liquid handling system is feasible in the future. |
Long Abstract
Quantitation of steroid metabolites including 17-hydroxyprogesterone (17OHP), testosterone (TTST), Androstenedione (ANDRO) and dehydroepiandrostenedione (DHEA) is useful in the diagnosis and clinical management of adrenal disorders like congenital adrenal hyperplasia. Challenges with existing immunoassay methods for these analytes including high assay imprecision at low concentrations and lack of standardization are well documented. The objective of our study was to develop a UPLC-MS/MS method for 17OHP, TTST, ANDRO and DHEA which has simple sample extraction steps and is amenable for future automation.
We developed a UPLC-MS/MS method for quantitation of serum 17OHP, TTST, ANDRO and DHEA involving protein precipitation and solid phase extraction (SPE) using a C18 cartridge followed by separation on an analytical column and analysis on the mass spectrometer. Briefly, to 100µL serum sample 400 µL water, 100 µL zinc sulfate solution (0.25M in water) and 500µL solution of internal standard in methanol (0.5ng/ml 17OHP 13C3; TTST 13C3; ANDRO 13C3 and 5ng/ml DHEA-d6) was added. The samples were mixed by vortexing vigorously for 1 minute at room temperature and centrifuged at 13500 rpm for four minutes at room temperature. The supernatant was applied to a SPE cartridge and passed through it using positive pressure. The cartridge was washed once each with water, methanol:water (60:40) mixture, eluted with methanol and eluants dried under a stream of N2 at 45ºC. The residue was reconstituted with 150µL of methanol:water (30:70) mixture. 10 uL of the extract was separated on the analytical column (Acquity UPLC BEH C18, 1.7um, 2.1x100 mm) using water/0.1% formic acid and acetonitrile/0.1% formic acid as the mobile phase and analyzed on Waters XEVO TQ-S mass spectrometer. The relatively lower signal to noise observed for DHEA necessitated a higher sample volume (500 µL) to be extracted to achieve acceptable imprecision at the limit of quantitation of the assay.
The ion pairs used for quantitation of 17OHP, TTST, ANDRO and DHEA were 331.5>109.1, 289.4>109.1, 287.4>109 and 253.5>197.2 respectively. The linear analytical measurement range was 0.1-15.1ng/ml for 17OHP, 0.05-11.8ng/ml for TTST, 0.18-14.0ng/ml for ANDRO and 0.4-55.9ng/ml for DHEA. The LOQ for 17OHP, TTST, ANDRO and DHEA were 0.1ng/ml, 0.05ng/ml, 0.5 ng/mL, and 0.5 ng/mL respectively. Intra assay imprecision ranged between 1.33-13.31% for all analytes across the respective AMRs. 17OHP and TTST values compared very well with the Mayo Clinic LC-MS/MS assay (Y=1.0783x+0.1071 R2=0.985; X=Mayo LC-MS/MS, Y=UPLC-MS/MS; N=20). The correlation between radioimmunoassy (RIA) and LC-MS/MS for DHEA was Y=0.9886x-0.6872 where R2=0.9108 (X=RIA; Y=UPLC-MS/MS; N=40) and a significant positive bias was observed between TTST immunoassay (Siemens, Centaur) and LC-MS/MS results (y=1.2348x-0.2406 R2=0.9987; X=LC-MS/MS; X=immunoassay; N=20). No interference/cross talk was observed in any of the four steroid ion pairs at physiological or supra-physiological concentrations of Dihydrotestosterone, Epitestosterone, ANDRO, DHEA-S at 1.5ng/ml, 12ng/ml, 3ng/ml and 5ug/ml respectively.
In conclusion, we have developed a UPLC-MS/MS method that can be used for simultaneous measurement of 17OHP, TTST and ANDRO. DHEA was also quantified using the same sample preparation and chromatographical method but with the requirement of relatively higher sample volume. Since our method does not need a derivatization step, complete automation of the sample extraction process using a liquid handling system is feasible in the future.
References & Acknowledgements:
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