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Clinical Vitamin B₆ Analysis: An Interlaboratory Comparison of Pyridoxal 5'-Phosphate Measurements in Serum

¹ Inorganic Toxicology and Nutrition Branch, Division of Laboratory Sciences, National Center for Environmental Health, Centers for Disease Control and Prevention, Atlanta, GA.

^aAddress correspondence to this author at: Centers for Disease Control and Prevention, 4770 Buford Hwy, Atlanta, GA 30341. Fax 770-488-4139; e-mail CPfeiffer{at}cdc.gov.

Background: Recent investigations into the role vitamin B₆ plays in reducing risk of stroke and cardiovascular disease have heightened interest in vitamin B₆ intake and its relationship to clinical status indicators. Because a true reference method and certified reference materials are lacking, little is known about the relative analytical performance of clinical vitamin B₆ assays.

Methods: Ten laboratories experienced in clinical vitamin B₆ analysis participated in a 3-day analysis of 69 serum and 3 aqueous specimens for pyridoxal 5'-phosphate (PLP). Laboratories used either HPLC-based or enzymatic assays. Results were analyzed for imprecision, recovery, and bias relative to consensus means.

Results: Among laboratories, mean within-day CVs (3 specimens x 3 measurements/day) were 0.6%–37% and between-day CVs (20 specimens x 1 measurement/day x 3 days) were 1.4%–26%. Mean recoveries of added PLP were 53%–144%, and mean sample pool mixing recoveries were 75%–119%. Consensus means calculated for 20 serum specimens gave mean relative biases between measurement of –10.0% to 24.3% among participating laboratories over a range of 15.8–319 nmol/L PLP. Measurement imprecision and biases were evaluated against empirically derived performance criteria based on biological variation. Three of 10 laboratories met optimum imprecision requirements and had 90% or more of measurements satisfy optimum criteria for biases among methods. All 10 laboratories met minimum imprecision requirements, but 25%–53% of the results reported by 4 of the 7 suboptimal laboratories failed to satisfy the minimum criteria for bias.

Conclusion: Agreement among vitamin B₆ methods is good, but large differences in laboratory proficiency exist, pointing to the need for vitamin B₆ reference materials and external quality assurance programs.

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