MSACL 2019 EU Abstract

Simultaneous Determination of Vitamins B1, B2 and B6 in Whole Blood by LC-MSMS

Stefanie Wernisch (1), Katharina Kern (2), Jörg Thomer (2)
(1) Waters, Eschborn, Germany (2) Recipe, Chemicals + Instruments, Munich, Germany

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Stefanie Wernisch (Presenter) Waters GmbH >> POSTER (PDF)

Presenter Bio: Specialist for clinical LC-MS applications at Waters
Mass spec instrumentation enthusiast

Research experience:
Research Fellow, University of Michigan Medical School (Metabolomics- Nephrology)
Post-doctoral fellow, Indiana University (Glycomics)
PhD, University of Vienna (Enantioselective HPLC)

Relevant Financial Disclosures (within past 24 months) Grant/Research Support Waters Salary Waters Conflict mitigation NOT REQUIRED.

Introduction:
The clinical manifestations of vitamin B deficiencies can involve alterations of the skin, neurological diseases and various forms of anemia. Accurate diagnosis and treatment of the underlying disease requires an analytical method which facilitates the simultaneous quantification of the most critical B vitamins in a single assay.
Analytical challenges arise from the complexity of whole blood as a sample matrix, the chemical diversity of the B vitamins, and the requirement for a highly selective method that reliably quantifies the biologically active forms of each vitamin.

Objectives:
a) Develop a method for the simultaneous quantification of vitamin B1 (thiamine diphosphate TDP), vitamin B2 (flavin adenine dinucleotide FAD) and vitamin B6 (pyridoxal phosphate PLP and pyridoxal PL) in human whole blood using liquid chromatography and tandem mass spectrometry.
b) Determine analytical figures of merit on two analytical platforms.

Methods:
The analytical method for the quantification of Vitamin B1, B2 and B6 in human whole blood was based on protein precipitation followed by direct injection of the supernatant. Analyte separation was achieved on a reversed-phase-type column using a binary gradient (total run time: 6 minutes). Analytes were ionized in positive mode and detected by multiple reaction monitoring (MRM).
Quantification was based on stable isotope-labeled internal standards and 4 levels of whole blood matrix calibrators.
Method performance was evaluated using the ClinMass® LC-MS/MS Complete Kit for the Determination of Vitamin B1, B2 and B6 in Whole Blood (RECIPE Chemicals + Instruments GmbH, Munich, Germany).
The method was first implemented on a Waters Acquity LC system connected to a Xevo TQ-S triple quadrupole mass spectrometer (Waters, Eschborn, Germany) for research use only. It was then verified on a Xevo TQ-D triple quadrupole instrument (Waters, Germany).

Results:
Fast processing of the samples was required due to the limited stability of B vitamins in biological matrix. Protein precipitation followed by mixing and centrifugation yielded a supernatant suitable for direct injection into the LC-MS/MS system.
Conclusion: The method facilitates the simultaneous determination of members of the B vitamin complex from 50 microliters of whole blood. It meets research laboratory requirements in terms of precision, accuracy, linear range and detection limits.