Determination of Active Vitamin B 12 (Cobalamin) in Dietary Supplements and Ingredients by Reversed-phase Liquid Chromatography: Single-laboratory Validation

a Xiao Qiu, a Yuhao Yin, a HaoShu Zhang, b Hillel Brandes, b Teresa Marsala, b Scott Krepich, b Katherine Stenerson, b Hugh Cramer and a Hong You a Eurofins Supplement Analysis Center, Eurofins Scientific, Inc., b MilliporeSigma Corresponding author: Hong You, Ph.D. Email: [email protected] Introduction Results Results

• Minority population, such as vegan/vegetarian, pregnant women, infants, and eating Figure 1. Improved Peak Resolution and Response by using 228nm as Table 4. AOAC SMPR 2016.017 requirements and HPLC method results detection wavelength. disorder patients, can be vulnerable to vitamin B12 deficiency because of their special dietary habits and physiological needs [1]. Vitamin B12 dietary supplement was HPLC Method Results recognized as an alternative food nutrient resource for group at risk of Vitamin B12 deficiency [2]. Parameter Requirement Hydroxo- Cyano- Adenosyl- Methyl- cobalamin cobalamin cobalamin cobalamin • Standard practices identify and quantify bioactive forms of vitamin B12 from dietary supplements simultaneously by HPLC/UV-Vis. Additionally, Chromatographic Analytical 0.001 ppm to 0.0005-85% w/w performance at different wavelength are compared to determine optimal peak range 100% resolution for all four vitamin B12 . 0.00040% ≤0.0005 ppm 0.00041% w/w 0.00052% w/w 0.00033% w/w • The Association of Analytical Communities (AOAC) publishes Standard Method LOQ w/w Performance Requirements (SMPR) to develop globally accepted official methods. This study targeted to meet the AOAC SMPR 2016.017 requirements for main forms < 5ppm 75-115 77.7 N/A N/A N/A of active vitamin B12 determination[3]. 5-20ppm 80-110 N/A 102.1 98.4 103.4 Recovery (%) >0.1 to 97-102 100.7 98.3 97.4 96.6 1% Objective >1% 98-102 98.5 99.3 99.8 102.2 Table 3. Label Claim Recovery for Samples Screened in the Repeatability Study < 5ppm ≤12 ≤4 N/A N/A N/A • To develop and validate a method for identifying and quantifying four bioactive forms Repeatability Recove Claim ID code Matrices Sample form Estimated level of vitamin B12 (adenosylcobalamin, cyanocobalamin, hydroxocobalamin, test result ry met? 5-20ppm ≤8 N/A ≤3 ≤4 ≤1 methylcobalamin) from dietary ingredients and supplements, as a consensus quality Finished RSD 111 Tablet 0.018% w/w cyanocobalamine 0.026% w/w 144% Yes r (%) >0.1 to ≤4 ≤1 ≤1 ≤1 ≤1 control protocol for dietary supplement labelling and manufacturing. product 1% 0.72% w/w hydroxocobalamin 0.78% w/w 108% Yes Finished >1% ≤2 ≤1 ≤1 ≤2 ≤1 708 Capsule Method product 20- 0.72% w/w adenosylcobalamin 0.98% w/w 136% Yes ≤8 N/A ≤1 N/A N/A 1000ppm Table 1. Reference Material Table 2. Mobile Phase Gradient 0.81% w/w adenosylcobalamin 0.87% w/w 107% Yes >0.1 to Finished RSD R (%) ≤6 ≤5 N/A ≤5 ≤4 205 Tablet 1% Compound Concentration Time 20 mM product Acetonitrile (%) 0.81% w/w methylcobalamin 0.81% w/w 100% Yes (mg/mL) (min) KH2PO4(%) >1% ≤3 N/A N/A N/A ≤3 Finished 0 95 5 670 Powder 0.86% w/w methylcobalamin 0.80% w/w 93% No Hydroxocobalamin 0.04 product 8 50 50 * Table only summarizes the results of AOAC SMPR 2016.017 required analytes (hydroxocobalamin, 226 Powder Raw material 86% w/w methylcobalamin 89.60% w/w 104% Yes Cyanocobalamin 0.04 8.1 20 80 cyanocobalamin, adenosylcobalamin, methylcobalamin) in required matrices (tablets, capsules, and powders). 11.6 20 80 Adenosylcobalamin 0.04 Discussion 11.7 95 5 References Methylcobalamin 0.04 15 95 5 • Compared with other published methods, • All SMPR 2016.017 requirements were met This simpler and efficient method is s for matrices in question. The proposed [1] Pawlak, R., Parrott, S. J., Raj, S., Cullum-Dugan, D., & Lucus, D. (2013). How prevalent is Chromatographic Conditions able to simultaneously identify and method is selective, linear, accurate, precise vitamin B12 deficiency among vegetarians?. Nutrition reviews, 71(2), 110-117. quantify four bioactive forms of vitamin and robust (Table 3) ; [2] US Department of Health and Human Services. Dietary guidelines for Americans 2015-2020, Column: Ascentis Express C8 column (2.7µm, 3 x 100 mm) B (Figure 1) ; 12 • The results of a variety of dietary ingredients eighth edition. Column Temperature: 30 °C [3] Van Breemen, R., Betz, J. M., Evans, L., Hall, A., Jennens, M., Kreider, R., ... & Schaneberg, and supplements tested in the Repeatability • UV wavelength 228nm provided high B. T. (2017). AOAC SMPR 2016.017. Journal of AOAC International, 100(1), 294-296. Detection: 230 nm signal-to-noise ratios improving accuracy study were compared to their label claims. Flow Rate: 0.4 mL/minute and precision, especially for low levels Data suggested that dietary supplement Acknowledgement Diluent: 0.04 mg/mL phosphate buffer (pH=5.8 with 0.1N analytes (Figure 1) ; manufacturers added excessive overage of potassium hydroxide) vitamin B12 maybe due to poor performance The funding was provided through an inter-company research & development grant. of methods that they currently use (Table 4) .