METHOD

Calibration:

Calibration Ranges:
Thiamine: 0.03 – 10 ug/mL; Niacin: 0.125-10 ug/mL;
Nicotinamide: 0.3 – 100 ug/mL; Riboflavin: 0.03-10 ug/mL;
Pyridoxal: 0.125-10 ug/mL, Pyridoxine: 0.125-10 ug/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, Ryridoxal and Pyridoxine are made in 0.01 M HCl and stored protected from light.

Instrument set up

Connect the instruments in the following order:
Pinnacle PCX post-column derivatization instrument – UVE™ photochemical reactor – Fluorescence detector.

Sample Preparation

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

For multi-vitamins supplements tablets:
Blend at least 10 tablets to a fine powder and mix the entire sample thoroughly. Weight 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 hours, protecting from light. Make the volume up to 100 mL with acidified water. Filter the sample through 0.45 um nylon filter and inject. Protect from light.

TABLE 2. Pinnacle PCX Pump Program