1a. Objectives (from AD-416):
Objective 1: Evaluate existing measurement systems and determine the need for improved measurements for water and lipid soluble vitamins (e.g., vitamins D and E and selected B vitamins), for which significant public health concern and inadequate composition data exists in foods and dietary supplements. Sub-Objective 1.A: Systematically evaluate public health concerns and adequacy of food composition data for vitamins and matrices to prioritize improved measurement needs, including methods and available Reference Materials. Sub-Objective 1.B. Develop detailed evaluations of measurement systems for priority analytes and matrices. Sub-Objective 1.C. Develop specific purpose statements for development and/or updating of methods for measurements of single or multiple vitamins. Objective 2: Develop and validate new or updated analytical methods using current technology to determine the levels of water-soluble vitamins (WSV), lipid soluble vitamins (LSV) and/or other components for foods and dietary supplements. Sub-Objective 2.A: Develop/update and optimize measurement procedures to establish validated capability for simultaneous measurements of multiple WSV (SimWSV). Sub-Objective 2.B: Develop and validate analytical methods for the determination and quantification of LSV (A, D, E, and K) and lipids in food matrices and dietary supplements. Sub-Objective 2.C Develop/update and optimize measurement procedures to establish validated capability for measurements of vitamin B12 in dietary supplements and foods. Sub-Objective 2.D Develop multivariate calibration methods for simultaneous determination of multiple vitamins in extracts with no prior chromatographic separations. Objective 3: Develop and validate sample preparation procedures to optimize extraction, remove interferences, and/or to concentrate difficult to analyze vitamins in foods and dietary supplements. Objective 4: Catalyze cooperative activities to identify and provide improved measurement systems and essential Reference Materials for vitamins in foods and dietary supplements. Provide analytical data to characterize the vitamin content of selected Reference Materials. Sub-Objective 4.A: Generate information with developed and validated methods to assign value-added information on vitamin content to available Reference Materials. Sub-Objective 4.B: Catalyze development of overall measurement systems for vitamins in foods and dietary supplements.
1b. Approach (from AD-416):
Objective 1: Evidence-based reviews will provide priorities for specific vitamins, define adequacy of existing data, and define quality of data required for future needs. The present state of the performance of individual labs and the adequacy of analysis of specific vitamin measurement systems will be evaluated from extensive data available from the USDA contract analyses conducted as part of NFNAP and DSID. Clearly defined purpose statements will be developed for specific applications. Objective 2: Improved procedures will be developed to simultaneously measure water soluble vitamins (SIMWSV) [thiamin, riboflavin, niacin, pyridoxine, folic acid, pantothenic acid, biotin, choline, and ascorbic acid] in foods and dietary supplements (DS). SIMWSV and LC-IDMS methods for DS will be extended to fortified foods. The additional challenges of natural levels of vitamins in unfortified foods require different approaches or compromise conditions to obtain acceptable analytical results. We will examine newer chromatographic separation modes such as hydrophilic interaction liquid chromatography (HILIC) and aqueous normal phase (ANP) chromatography to improve these separations. A defined protocol of intra-laboratory validation will be carried out following AOAC guidelines. The FCMDL Vitamin D method is implemented for analysis of food samples, and additional foods, dietary supplements and reference materials will be analyzed. IDMS methods will be initiated for other lipid soluble vitamins A, E, and K. The FCMDL method to determine low levels of B12 in vitamin supplements using dual column LC/UV will be extended to fortified foods using sensitive LC/MS techniques and collaboratively cross-validated to the microbiological method. The possibility to calibrate spectral fingerprints (information with no chromatographic separation) of extracts of various types of food and dietary supplement materials to obtain quantitative information about vitamin content will be explored. Calibration models will be developed and validated. Objective 3: Multiple extractions for WSV with different buffers, pHs, and multiple extraction approaches (including classical and modern methods such as pressurized liquid extraction [PLE], microwave-assisted extraction [MAE], ultrasonic irradiation, stirring, shaking, and Soxhlet) will be systematically explored to ensure complete extraction and compare extraction efficiencies of different procedures. The lipid soluble vitamins (LSV) (A, D, E, K) would be similarily extracted with a organic solvents of different polarities. Objective 4: FCMDL capability for high quality vitamin determinations will be applied to provide reference measurements to value to NIST SRMs such as the Adult/Infant Formula SRM and Fortified Cereal SRM. Through initiating and providing guidance for a number of nutrition metrology-related activities, FCMDL will catalyze improvement of the overall measurement system for vitamins. FCMDL will participate as collaborators in method validation studies as appropriate. FCMDL will continue to organize and advise the development and conduct of symposia, and other appropriate workshops.
3. Progress Report:
A method was validated for the determination of vitamin B6 in most vitamin and mineral supplements. A general extraction method was used that was modified slightly depending on whether the supplement was a tablet, soft gel, liquid, sustained release material, or a “gummy-bear” type of material. Chromatographic separation was based on both high performance liquid chromatography (HPLC) and ultra-HPLC (U-HPLC). The U-HPLC separation was accomplished in less than 20 minutes. The vitamin was detected using ultraviolet absorption, fluorescence, and mass spectrometry. Accuracy and precision of ±5% were achieved for all supplement materials.
1. A ‘triple parallel mass spectrometry’ experimental arrangement in which three mass spectrometers are used in parallel to provide both qualitative and quantitative information using complementary ionization techniques for mass spectrometry was applied to the analysis of Vitamin D. The data from these experiments allow a much higher degree of confidence in quantitative results, because multiple instruments acquire qualitative information on the same samples at the same time. We are now able to obtain quantitative results on one instrument plus qualitative analysis on three other instruments simultaneously, using different and complementary ionization sources, each producing different types of data. This unique instrument offers unprecedented accuracy and specificity.