Self-Classified Topic Area(s): Small Molecules / Tox / TDM
A High-throughput LC-MS/MS Method for Vitamin A and Vitamin E
Helge Berland (1), Kristin Viste (1), Iren Hjellestad (1), Jennifer Gjerde (1), Jørn V. Sagen (1,2), Bjørg Almås (1) (1) Hormone Laboratory, Haukeland University Hospital, Bergen, Norway (2) Department of Clinical science, University of Bergen, Norway
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Helge Berland (Presenter) Hormone Laboratory, Haukeland University Hospital
Presenter Bio: Working as a Method Developer at Haukeland University Hospital, Hormone Laboratory, with a background in analytical chemistry, and currently also working towards a Ph.D in Chemistry.
Relevant Financial Disclosures
(within past 24 months)
No relevant financial relationship(s) to disclose.
Abstract
Background: Assessment of vitamin status is of clinical as well as nutritional importance. We wanted to develop a robust high-throughput LCMS-method for routine analysis and quantification of serum all-trans-retinol (Vitamin A) and α-tocopherol (Vitamin E). The method was based on comprehensive optimization of an assay currently in use at the Hormone Laboratory.
Method: Automated procedure for sample preparation was performed using liquid-liquid extraction on a Tecan Evo Freedom 150. In brief: serum was added to ethanol with C13-labelled analyte internal standards (IS), then hexan was added for extraction and further transferred to tubes for evaporation and reconstitution in methanol where 0.2 g/L Butylated Hydroxytoluene (BHT) was added for stabilization of the analytes. Chromatographic separation of Vitamin A and Vitamin E was achieved on a Nexera X2 UPLC-system equipped with an Agilent ZORBAX Eclipse Plus C18 column. Gradient: water:methanol containing 0.1 % FA from 71-91 % methanol over 9.5 min, sample injection volume 1 µl. Analysis by MS/MS was performed on a Sciex TQ API4500 in positive ESI mode. Standard reference material (SRM), and quality controls (QC) were used to validate accuracy and precision of the method.
Results: The method demands only 25 µl patient serum. Sample preparation with LLE, evaporation and reconstitution is completed within 2 hours. Measurement range for routine use is defined by the upper and lower calibrators; 0.16-12.4 µmol/L for vitamin A and 1.86-125 µmol/L for Vitamin E. Linearity was tested for Vitamin A also beyond the measurement range and found between 0,05-60 µmol/L. Using in-house made QC’s at three levels (pooled patient sera) and ClinChek1-3 (Chromsystems) total presicion was found to be below 10.2 % for Vitamin A and 9.6 % for Vitamin E at all levels. The method was robust towards matrix effects, and is tracable to the NIST SRM 968f. Reference ranges were adapted from the Mayo Clinic and verified with 20 healthy fasting volunteers (employees).
Conclusions: We have optimized and further developed a robust LCMS/MS-method for measurement of Vitamin A and Vitamin E. The method is compatible with the work-flow and capacity needed in a medical laboratory.
= Discovery stage.
= Translation stage.
= Clinically available.
