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Development of Standard Reference Materials for Vitamins B6 and B12 in Human Serum


Vitamins are essential to normal functioning of the human body because they serve as co-factors for important biological processes. Deficiencies in vitamins B6 and B12 can lead to problems such as anemia and loss of cognitive function. It has been estimated that 10 % to 15 % of adults over the age of sixty are affected by vitamin B12 deficiency. Accurate determinations of serum levels of vitamins B6 and B12 are therefore important in identifying deficiencies as well as diagnosing other medical conditions. To date, a lack of accepted methodology and appropriate control materials has hindered broader testing for B6 and B12 deficiency. NIST is developing two Standard Reference Materials (SRMs) for these vitamins in serum to assist in accuracy of clinical tests for these vitamins.


Vitamin B6 is converted to pyridoxal 5'-phosphate (PLP), the analyte of clinical interest when assessing vitamin B6 dietary status. Vitamin B6 deficiency is associated with impairment of a variety of important functions in the body, and low levels of PLP are believed be associated with an increased risk of cardiovascular and other diseases. Vitamin B6 levels can be determined in biological samples through a variety of methods, including microbiological, enzymatic, and chromatographic approaches. Previous interlaboratory comparisons of PLP measurements in serum have revealed large differences in laboratory proficiency. Vitamin B12 (cobalamin compounds) is necessary for the formation of healthy red blood cells and in maintenance of the central nervous system. Vitamin B12 deficiency can lead to anemia and neurological damage, including dementia. Analytical techniques used to measure vitamin B12 in serum include microbiological, radioisotopic, and chemiluminescence assays. A workshop sponsored by the National Institutes of Health Office of Dietary Supplements (NIH-ODS) highlighted the fact that the different assay techniques often do not provide comparable results. Reference materials are needed to improve the agreement among routine tests for vitamins B6 and B12. NIST is addressing this need through the development of SRM 3950 Vitamin B6 in Human Serum and SRM Vitamin B12 in Human Serum.

Additional Technical Details:

The two reference materials currently in development at NIST, SRM 3950 Vitamin B6 in Human Serum and SRM 3951 Vitamin B12 in Human Serum, consist of pools of frozen human serum. SRM 3950 has two levels of vitamin B6 (as PLP), and SRM 3951 has three levels of vitamin B12. These SRMs were designed in consultation with a panel of experts, and levels of the analytes of interest in these two SRMs are intended to span the range typically found in patient samples. As part of the certification of these SRMs, NIST has been developing isotope dilution LC-MS/MS methodology for PLP in serum. An isotopically labeled form of PLP has been obtained, and preliminary screening of the two serum pools, as well as method validation studies, indicate that this approach will be suitable for certification of PLP in SRM 3950. Method development is also in progress for SRM 3951. Quantification of vitamin B12 in serum is particularly challenging because of the low levels present (pmol/L). Both LC-MS/MS and neutron activation approaches are being evaluated. In addition, NIST will investigate measurement of methylmalonic acid in SRM 3951. Methylmalonic acid has been identified as another potential marker of vitamin B12 deficiency.

Major Accomplishments:

  • Development of higher order methodology for vitamin B6 (as PLP) and vitamin B12 is currently underway.  An isotopically labeled form of PLP has been obtained for use as an internal standard.  
  • The candidate materials for SRM 3950 and SRM 3951 have been obtained.
Photograph of SRM 3950 Vitamin B6 in Serum

Start Date:

October 1, 2005

End Date:


Lead Organizational Unit:



Johanna Camara
Stephen E. Long
Karen W. Phinney
Bryan E. Tomlin

Associated Products:

SRM 3950 Vitamin B6 in Human Serum (in progress)
SRM 3951 Vitamin B12 in Human Serum (in progress)


Karen W. Phinney
Phone 301-975-4457