Pascale Macours (Presenter)
Leiden University Medical Center
Bio: Pascale Macours studied chemistry at the Free University of Brussels (ULB) in Belgium where she performed her PhD on the absolute configuration determination and asymmetric synthesis of Tetraponerines, defense alkaloids of the ant Tetraponera sp. in 1995. After a post-doc on the study of thyroid metabolism, she was responsible of the chromatography laboratory at the Erasme Hospital in Brussels. In 2013, she moved to the Leiden University Medical Center where she is responsible for the development of new chromatographic and mass spectrometry methods for the patient care. Her main expertise is chromatography and small molecules mass spectrometry, including method development and validation.
Authorship: Pascale Macours, Bart Ballieux, Pascal Schippers, Christa Cobbaert
Leiden University Medical Center (LUMC), Department of Clinical Chemistry and Laboratory Medicine, Albinusdreef 2, 2333 ZA Leiden, The Netherlands
| Short Abstract We developed an in house test for simultaneous quantification of six clinically relevant steroids [11-deoxycortisol, 21-deoxycortisol,delta-4-androstenedione, free dehydroepiandrosterone (DHEA), 17-alpha-hydroxyprogesterone and testosterone] in human serum. After extraction of steroids using ยต-elution Oasis MCX SPE sorbent, an UPLC-MSMS test was developed using an Agilent UPLC and 6495 triple quadrupole. Matrix-matched calibrators were used which showed excellent linearity and reproducibility. Total imprecision, measured on pool sera, ranged between 3.6 and 18 % for the six steroids. Accuracy of testosterone was of 103%, checked using NIST human serum reference material. Lowest limits of quantification were <0.5 nmol/L for all steroids, except for DHEA which was 3.0 nmol/L. The calculated total error met the desirable analytical performance criteria required for clinical care. |
Long Abstract
Background: Steroid determinations by immunoassays are prone to cross reactivity of antibodies with chemically similar compounds. In medical laboratories, immunoassays are performed in uniplex, requiring separate samples and runs for each compound, whereas LC-MS/MS offers improved selectivity and the ability to multiplex methods. Just like any medical test, in house tests have to be fit-for-clinical purpose (1). Therefore, all steps of a typical MS workflow, including sample preparation, and the analytical and post-analytical phases, have to be considered. We describe here a thorough analytical validation of an UHPLC-MS/MS method for quantifying six serum steroids. Predefined analytical performance criteria are based on desirable analytical performance requirements derived from biological variation.
Method: Sample preparation consists of a serum protein precipitation step with zinc sulfate and methanol followed by SPE extraction of steroids using an Oasis cation exchange MCX micro-elution SPE. The UPLC separation was achieved on a Zorbax Eclipse Plus column whereas MS-measurement was done by multiple reaction monitoring (MRM) on a triple quadrupole mass spectrometer. MS- parameters have been optimized to get the highest sensitivity whereas the choice of MRM transitions guarantees the highest selectivity. Isotopically labelled steroid analogues are used as internal standards. Calibration was done using standards that were spiked into a steroid free serum matrix. NIST human serum material was used to determine testosterone accuracy. Bias and imprecision were validated according to CLSI EP-15. Furthermore patient sera were used for method comparison between LC-MS/MS and the routinely used immunoassays.
Results: Protein precipitation followed by MCX SPE resulted in consistent and highly reproducible recoveries of all six steroids with minimal ion suppression and without matrix effect. The calibration with matrix-matched standards showed excellent linearity (> 0.995) and reproducibility (1.5-12.3%) for all six steroids. After spiking, serum recoveries ranged between 86 - 115 % at concentrations varying between 0.3 - 30 nmol/L. Total imprecision for all six steroids in female and male pool sera was between 3.6 % (androstenedione in female serum pool) and 18 % (DHEA in male serum pool). Recovery of testosterone, assessed on value-assigned NIST male and female human serum reference material, was respectively 103 and 104 %. The average total error for quantification of testosterone was below 11%. Lowest limits of quantification (LOQ) were < 0.5 nmol/L for all steroids, except for DHEA which has an LOQ of 2.0 nmol/L.
Conclusion: We conclude that the lab-developed-LC-MS/MS test for simultaneous quantification of six endogenous serum steroids is selective and has analytical performance characteristics that make it fit-for-clinical-purpose. Before implementing this MS-based steroid profile for patient care, new reference values and decision limits should be determined.
References & Acknowledgements:
1. From Biomarkers to Medical Tests: The Changing Landscape of Test Evaluation. Andrea R. Horvath, Sarah J. Lord, Andrew StJohn, Sverre Sandberg, Christa M. Cobbaert, Stefan Lorenz, Phillip J. Monaghan, Wilma D.J. Verhagen-Kamerbeek, Christoph Ebert and Patrick M. M. Bossuyt for the Test Evaluation Working Group of the European Federation of Clinical Chemistry and Laboratory Medicine. CCA 2014; 427: 49-57.
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