MSACL 2019 EU Abstract

Confident Quantitation of 25-Hydroxyvitamin D2 and D3 in Human Plasma for Clinical Research by LC-MSMS

Mariana Barcenas (1), Claudio De Nardi (2), Paolo Brambilla (3), Maura Brambilla (3), Chiara Fania (4)
(1)Thermo Fisher Scientific, Les Ulis, France (2)Thermo Fisher Scientific GmbH, Dreieich, Germany (3)Ospedale di Desio, Desio, Italy (4)Università degli Studi Milano-Bicocca, Milano, Italy

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Mariana Barcenas (Presenter) Thermo Fisher Scientific >> POSTER (PDF)

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Introduction: Vitamin D is hydrolized to its prohormone 25-hydroxyvitamin D in the liver, and being a predominant metabolite, circulating 25-hydroxyvitamin D serves as the preferred analyte that is monitored to determine nutritional status of Vitamin D.
Owing to its increased selectivity and specificity while addressing sensitivity, robustness, and reliability requirements, liquid chromatography (LC) coupled to mass spectrometry (MS) has become the platform of choice for analysis and quantitation of Vitamin D in biological matrices. An analytical method for quantitation of 25 hydroxyvitamin D2 and D3 in human plasma with simple sample preparation is reported for clinical research.

Objectives: Implementation of a high throughput analytical method for quantification of 25-hydroxyvitamin D2 and D3 in human plasma using a Thermo Scientific™ Transcend™ LX-2 system and a Thermo Scientific™ TSQ Quantis™ triple-stage quadrupole mass spectrometer.

Methods: Samples were prepared by precipitating 50 µL of plasma with 150 µL of Acetonitrile containing the internal standards. Calibrators obtained from RECIPE® Chemicals + Instruments GmbH (Munich, Germany), ranged from 9.84 to 81 ng/mL, and 9.04 to 78.9 ng/mL for 25-hydroxyvitamin D2 and D3 respectively. Chromatography was performed on a Transcend LX-2 system, connected to a TSQ Quantis triple-stage quadrupole mass spectrometer. LC separation was achieved in a Thermo Scientific™ Hypersil GOLD™ (50 x 2.1 x 1.9 µm) column. Mobile phases consisted of Water and Methanol, both containing 10mM Ammonium Formate and 0.1% Formic Acid. Total runtime was 3.5 minutes. Analytes and Internal Standards were detected using SRM mode with Atmospheric Pressure Chemical Ionization in positive mode.

Results: Regression coefficients for both analytes were above 0.999. The percentage bias between nominal and back-calculated concentration was within ±10% for all the calibrators in all the runs.
The data demonstrated outstanding accuracy of the method with the percentage bias between nominal and average back-calculated concentration for the used control samples ranging between -2.5% and 2.6%. The %CV for intra-assay precision was less than 3.6% for all the analytes. The maximum %CV observed for inter-assay precision, including all the analytes, was 4.1%.

Conclusion: A robust, reproducible, and reliable assay exhibiting best-in-class sensitivity was developed using liquid chromatography-tandem mass spectrometry for clinical research for quantification of 25-hydroxyvitamin D2 and D3 in human plasma. The method was analytically validated with a multichannel LC system connected to a mass spectrometer. Sample preparation consisted of simple and robust protein precipitation of the calibrators and controls were obtained from RECIPE.The data obtained with the described method successfully met sensitivity, reliability, accuracy, and precision expectations typically demanded by clinical research laboratories.