Development of a Candidate Reference Measurement Procedure for the Determination of Vitamin B6 in Human Serum
Mon 3:30 PM - Track 3: Regulations and Standards
Johanna E. Camara
Johanna E. Camara, Eric L. Kilpatrick, Karen W. Phinney.

Analytical Chemistry Division, National Institute of Standards and Technology, Gaithersburg, MD
Vitamin B6 levels are of clinical interest due to their reflection of dietary status and association with disease states such as cardiovascular disease and stroke. The major circulating B6 vitamer is pyridoxal 5'-phosphate (PLP), which is the most common direct measure of vitamin B6 in plasma or serum. Currently, no certified reference materials or reference measurement procedures exist for vitamin B6 in serum, which would allow for objective comparison of multiple analytical methods and clinical laboratories.

To address this deficiency, the National Institute of Standards and Technology (NIST) is developing Standard Reference Materials (SRMs) and a candidate liquid chromatography-tandem mass spectrometry (LC/MS/MS) reference method for the analysis of vitamin B6 as PLP in human serum. In this method, the isotope-labeled internal standard pyridoxal-[2H3] 5'-phosphate was spiked into serum, which was then subjected to protein precipitation by the addition of trichloroacetic acid solution. After centrifugation, the supernatant was collected and injected without further processing. All samples were subjected to separation by reversed-phase chromatography with gradient elution followed by tandem mass spectrometry using multiple reaction monitoring (MRM).

Method parameters were developed using human serum pools containing 2 different levels of PLP. This method displayed linearity throughout the entire calibrant range (final PLP levels (5-10 ng/g) with R2=0.99. This method was applied to 2 separate frozen human serum pools with differing PLP concentrations that measured 4.78 ng/g and 9.40 ng/g, with within-set CVs ranging from 8.1-9.9 % and between-set CVs ranging from 8.8-9.2 %. Accuracy was evaluated with a recovery study in which PLP was added to serum at 4 levels (4-40 ng/g) with mean recovery at all levels approximately 98 %. An interference study was performed for the similar B6 vitamers pyridoxamine 5'-phosphate (PMP) and pyridoxine 5'-phosphate (PNP). Addition of biologically-relevant levels of these vitamers (5 nM) to human serum pools did not result in significant changes to PLP values measured by this method. A matrix effect study revealed that the usage of the stable-isotope labeled internal standard corrected for the absolute suppression of the MS/MS signal.

This method possesses suitable accuracy and precision for measuring PLP in human serum and has been appropriately validated for consideration as a candidate reference measurement procedure. This method has also been evaluated on an alternate LC/MS/MS system by an independent operator and will be applied to the value assignment of PLP in NIST SRM 3950 Vitamin B6 in Serum.