Simultaneous Analysis of Vitamins B1, B2, B3, and B6 in Protein Powders and Supplements

B vitamins are a group of water soluble vitamins that play an important role in cell metabolism. This group consists of a number of compounds including thiamine (vitamin B1), riboflavin (vitamin B2), niacin and nicotinamide (vitamin B3), and pyridoxine and pyridoxal (vitamin B6). B vitamins are found in plant and animal food sources, such as legumes, nuts, green leafy vegetables, red meat, and poultry. Many commercial food products are fortified with vitamin B complex, and people could take multivitamins supplements to help fight vitamin B deficiencies.

Figure 1: Chromatogram of mixed B vitamins standard solution. Niacin, riboflavin, pyridoxine, pyridoxal, and thiamine – 1 ug/mL; nicotinamide – 10 ug/mL.

Pickering Laboratories offers a method for simultaneous determination of vitamins B1, B2, B3, and B6 in supplements and protein powders. The method uses chemical and photochemical post-column derivatization with fluorescence detection that increases sensitivity and selectivity of analysis. Photochemical derivatization required for niacin and nicotinamide and chemical derivatization is needed for thiamine. Vitamins B2 and B6 have natural fluorescence.

Figure 2: Chromatogram of NIST multivitamin tablets sample.

Method

Calibration

• Calibration ranges: thiamine: 0.03–10 μg/mL; niacin: 0.125–10 μg/mL; nicotinamide: 0.3–100 μg/mL; riboflavin: 0.03–10 μg/mL; pyridoxal: 0.125–10 μg/mL; pyridoxine: 0.125–10 μg/mL.

• To make riboflavin stock solution, dissolve 20 mg of riboflavin in 5 mL of 0.1 M NaOH and immediately add 50 mL of 0.1 M HCl and make up the solution with DI water to 500 mL. Make working standards by diluting the stock solution with 0.01 M HCl. Store protected from light.

• Stock and working standards for thiamine, niacin, nicotinamide, pyridoxal, and pyridoxine are made in 0.01 M HCl and stored protected from light.

Figure 3: Chromatogram of soy protein shake powder sample.

Instrument Set Up

Connect the instruments in the following order: HPLC pump – HPLC autosampler – analytical column – Pinnacle PCX post-column derivatization instrument – UVE™ photochemical reactor – fluorescence detector.

Figure 4: Chromatogram of NIST chocolate protein drink mix sample.

Sample Preparation

For protein powders: To 0.5 g of samples add 50 mL of extraction buffer (0.1N NaOH adjusted to pH 2 with phosphoric acid). Homogenize using hand held homogenizer for 30 s and heat on a water bath at 100 ºC for 30 min. Cool the solution down, filter through 0.45 μm nylon filter, and inject. Protect from light.

Table I: HPLC gradient

Table II: Pinnacle PCX pump program

For multivitamins supplements tablets: Blend at least 10 tablets to a fine powder and mix the entire sample thoroughly. Weigh 250 mg of sample and add 90 mL of DI water acidified to pH 2.6 with 0.1 N HCl. Stir using magnetic stirring plate for 2 h, protecting from light. Make the volume up to 100 mL with acidified water. Filter the sample through 0.45 μm nylon filter and inject. Protect from light.

Table III: FLD program

Table IV: Analysis of NIST samples

Analytical Conditions

Post-Column Conditions

Pickering Laboratories, Inc.
1280 Space Park Way, Mountain View, CA 94043
tel. (800) 654-3330, (650) 694-6700
Website: www.pickeringlabs.com