“World class” methods, comprising the best aspects of various methods purported to meet fitness-for-purpose for vitamin analysis in dietary supplements, are being developed by AOAC under an NIH/FDA/AOAC contract. AOAC Expert Review Panels (ERPs) recently reviewed over 50 methods for Vitamins B6 and B12 for their advantages and disadvantages and for completeness of validation. Methods were evaluated using the AOAC Acceptance Criteria for Single-Laboratory Validation (SLV). Additional criteria included appropriateness for the intended use, clarity of the method description, ruggedness, reproducibility, recovery, analytical range, and limit of quantitation.

A dozen of the most promising methods and possible best candidate methods for Vitamins B6 and B12 were further evaluated by the respective ERP on April 1-2 at AOAC headquarters in Gaithersburg, Maryland, USA, as part of the NIH/FDA/AOAC initiative on dietary supplements. Still not satisfied with choosing one method as a whole for further validation for either B6 or B12, the panels decided to develop their own methods, comprising the best aspects of different methods.

How is a world class method made? For B6, the ERP selected a method from Pharmavite for sample preparation, an LC-UV/MS method from Pei Chen of the U.S. Department of Agriculture with a recommendation of the addition of a fluorescence detector for added sensitivity, and a method from DSM (B6 in tablets) for chromatographic separation. The ERP agreed that the ideal candidate method would include UV, fluorescence, and MS detection in the SLV to generate as much information as possible. Panel members believed that this approach would not result in a significant amount of extra work for the SLV and would ultimately result in the best method moving forward.

Instead of addressing all the possible forms of B6, the ERP focused its efforts where significant progress could be made in terms of identifying a method for SLV. Thus, the panel selected pyridoxine HCl as the analyte of interest.

Similarly, for Vitamin B12, the ERP chose an ultra-performance liquid chromatography coupled with triple quadrupole MS method developed by Baiyi Lu et al. of China’s Zhejiang University, but modified with the sample preparation selected for Vitamin B6, as the best candidate method. The ERP decided that the candidate method should be useful for the quantitative analysis of the cyanocobalamin form of Vitamin B12 in the presence of other forms of Vitamin B12, degradation products of Vitamin B12, other vitamins, and constituents of dietary supplements.

These methods, as well as the ERP process by which they are chosen, serve as a means to providing analytical solutions to identified needs or concerns. An ERP for Vitamin D is tentatively scheduled for June 8, 2009.

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