All sample preparation was conducted in dimmed light environment and with amber glassware.
Standard Preparation
Thiamine, thiamine pyrophosphate (TPP), riboflavin, flavin mononucleotide (FMN), nicotinic acid, nicotinamide, pantothenic acid, pyridoxine, pyridoxamine, pyridoxal, pyridoxal 5’-phosphate (PLP), biotin, folic acid, 5-methyltetrahydrofolate (5MTHF), cyanocobalamin (CN B12), methylcobalamin (Me B12), adenosylcobalamin (Aden B12) were purchased from Sigma-Aldrich (St. Louis, MO). Stable isotope labeled B vitamins: 13C5, 15N-folic acid, 13C4, 15N2-riboflavin, 13C4 -thiamine, 2H4 -biotin, 13C6, 15N-pantothenic acid, 2H2 -pyridoxine, and 2H4 -nicotinic acid were purchased from Isosciences (isosciences.com, Ambler, PA). Individual B vitamin stock solutions were prepared by dissolving individual vitamins with deionized water at concentrations of 0.5 mg/mL (recorded to 3 significant figures) except riboflavin and biotin, which were prepared as a mixed stock solution at 0.05 mg/mL (recorded to 3 significant figures) for each riboflavin and biotin standard. The folic acid stock solution may need a small amount (one drop or two) of ammonium hydroxide solution (28–30% NH3) to assist the dissolution. The PLP stock solution was heated to 50 °C to assist dissolution.
Three intermediate B vitamin mix solutions were prepared by diluting aliquots of the individual stock solutions with deionized water as follows, one intermediate standard mix solution of CN B12 (0.1 mg/mL) and Me B12 (0.1 mg/mL), one intermediate standard mix solution of thiamine (0.01 mg/mL), pantothenic acid (0.1 mg/mL), pyridoxine (0.01 mg/mL), nicotinamide (0.1 mg/mL), nicotinic acid (0.1 mg/mL), and pyridoxal (0.01 mg/mL), and one intermediate standard mix solution of folic acid (0.1 mg/mL) and 5MTHF (0.1 mg/mL). The working standard solutions were prepared from these intermediate B vitamin mix solutions and the remaining individual standard stock solutions by mixing and dilution with deionized water to various concentration levels.
The individual stable isotope labeled B vitamin standard stock solutions were prepared in a similar way as previously described for the individual B vitamin stock standards. A mixture of all stable isotope standard stock solution was prepared from these individual stock solutions by mixing appropriate volumes of the individual stock solutions with deionized water to obtain a concentration of 0.02 mg/mL for each standard except 13C5, 15N - folic acid which was prepared at 0.2 mg/mL. This stable isotope standard mixture stock solution was diluted with deionized water 10 times to make an intermediate stable isotope mixture solution, which was later used to fortify the working standard solutions and the sample solutions as internal reference standards at 0.1 μg/mL, except 13C5, 15N - folic acid which was added at 1 μg/mL, in the final solutions.
Sample Preparation
The energy drinks (ED) from major brands, such as Red Bull, Monster Energy, and Rockstar, were purchased from a local store. They were filtered through a 0.45 µm GMF membrane syringe filter. An aliquot of the filtrate was analyzed without dilution for low concentration B vitamin (CN B12). Another aliquot of the filtrate was diluted with deionized water 50 times (1:50 by volume) and analyzed for other B vitamins. A vitamin B complex dietary supplement (DS, liquid form) was also tested. 50 µL of DS was taken and diluted with 200 mL deionized water first, then filtered through a 0.45 µm GMF membrane syringe filter and further diluted with deionized water at different ratios (0.95:1, 1:10, 1:100 by volume) prior to the analysis.
