Dean Carlow (Presenter)
Memorial Sloan Kettering Cancer Center
Bio: I am a clinical pathologist who serves as Director of the Clinical Mass Spectrometry Laboratory and Section Chief of Clinical Consultation within the department of Laboratory Medicine at the Memorial Sloan Kettering Cancer Center. In these roles, I act as a liaison between the laboratory’s members and the clinical faculty and oversee the operations of a busy mass spectrometry laboratory. My research interests include therapeutic drug monitoring and new test development using a variety of analytical techniques, including liquid chromatography tandem mass spectrometry. I received my PhD and MD degrees from the University of North Carolina School of Medicine at Chapel Hill and completed my residency at the Hospital of the University of Pennsylvania.
Authorship: Ryan C. Schofield, Lakshmi V. Ramanathan, Melissa S. Pessin, Dean C. Carlow
Department of Laboratory Medicine, Memorial Sloan Kettering Cancer Center
| Short Abstract Our objective was to develop a very sensitive LC-MS/MS assay for both testosterone and estradiol in serum in a single analysis without the need for chemical derivatization. Serum samples were prepared by the addition of deuterated internal standards followed by a liquid-liquid extraction and LC-MS/MS in both positive and negative ion modes. The assay was very sensitive, had wide analytical measurement ranges and was precise. The accuracy was assessed by comparison with established LC-MS/MS assays, recovery studies, and comparison with several different reference materials. We have successfully developed an accurate and highly sensitive assay to simultaneously measure testosterone and estradiol levels in serum. |
Long Abstract
Background: Very sensitive measurements of serum estrogens and testosterone are important in adult and pediatric endocrinology and oncology. Sensitive measurements of estradiol (E2) are needed for determination of menopausal status, estrogen deficiency, estrogen measurements in men, during antiestrogen treatment and in the diagnosis of other sex-hormone-related disorders. E2 levels in postmenopausal women, men and children are typically <20 pg/mL. Ultrasensitive testosterone (Te) measurements are needed for adult women, whose values are routinely <50 ng/dL, in children, and in men undergoing antiandrogen therapy whose values are usually less than 10 ng/dL. Many automated immunoassays lack the required sensitivity and specificity for the measurement of these very low concentrations of E2 and Te. Our objective was to develop a very sensitive LC-MS/MS assay for both Te and E2 in serum in a single analysis without the need for chemical derivatization and extended extraction protocols.
Methods: Serum samples (500 μL) were prepared by the addition of deuterated internal standards of both compounds followed by a liquid-liquid extraction using hexane:ethyl acetate (9:1, v/v). The supernatant was evaporated at 35°C under a stream of nitrogen and reconstituted in methanol:water (1:1, v/v). 40 μL was injected into the LC-MS/MS system. Chromatographic separation was performed on a Thermo Scientific TLX-2 HPLC system interfaced to an ABSciex 6500 mass spectrometer operated in both positive and negative ion ESI mode. Chromatographic separation was achieved using an Accucore C18 (50 X 3 mm i.d.) analytical column. Mobile phase comprised of A: 0.02 mM ammonium fluoride in water and B: acetonitrile. The HPLC gradient elution was 15-50% of mobile phase B over 2 minutes that was then held for 1.5 minutes. MRM transitions were as follows: Te 289.2>97.1 and 289.2>109.1 m/z; E2 271.0>145.1 and 271.0>143.1 m/z. Calibrators and controls were purchased from Chromsystems using their multilevel calibrators and tri-level controls.
Results: The LOQs of Te and E2 were 1 ng/dL and 5 pg/mL, respectively. The analytical measurement range (AMR) for Te was 1-1,170 ng/dL and 5-600 pg/mL for E2. The calibration curves were linear over the AMR with correlation coefficients R2≥0.998. Within-day (N=10) and between-day (N=20) CVs at concentrations spanning the AMR were less than 5% for both analytes. Assay accuracy was determined 3 different ways: (1) by comparison with a LC-MS/MS method performed at a national laboratory; (2) by recovery studies; (3) by comparison with NIST standard reference material (SRM) 971M and 971F for testosterone, and European Commission Institute Reference Materials and Measurements (IRMM) certified reference material (CRM) BCR-576, 577 and 578 for estradiol; (4) by comparison with 40 samples for both testosterone and estradiol obtained from the Center for Disease Control (CDC) Hormone Assay Standardization (HOST) program. Comparison with samples analysed by LC-MS/MS at the reference laboratory showed the following: Te had a regression slope of 1.05, R2=0.986; E2 had a regression slope of 0.93, R2=0.996. Te recoveries at three concentrations spanning the AMR were between 101.4 and 105.4% and E2 recoveries were between 100.3 and 105.2%. For testosterone, analysis of NIST SRM 971F and 971M (certified values of 27.7 and 642.9 ng/dL, respectively) yielded measured values of 27.6 ng/dL (95% CI: 27.1-28.1) and 624.7 ng/dL (95% CI: 624.8-656.4); a bias of less than 0.4%. For estradiol, analysis of IRMM CRM BCR 576, 577 and 578 (certified values of 31.1, 188.0 and 365.0 pg/mL, respectively) yielded measured values of 32.2 pg/mL (95% CI: 29.3-35.3), 181.2 pg/mL (95% CI: 176.7-185.6), and 352.6 pg/mL (95% CI: 326.1-379.1); a bias of less than 4%. In addition, both testosterone and estradiol showed excellent correlation with specimens obtained from the CDC HOST program. A solution containing 13 naturally occurring steroids with the potential for isobaric interferences or with similar retention times were analyzed and showed no interferences.
Conclusion: We have developed an accurate and highly sensitive assay to simultaneously measure Te and E2 levels in serum by LC-MS/MS without the need for chemical derivatization. Unlike many immunoassays, this method is free of cross reactivity from structurally similar analogs.
References & Acknowledgements:
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