The Application Notebook
Pickering Laboratories, Inc.
Niacin (Nicotinic acid) is an essential nutrient important to human health. Free Nicotinic Acid and Nicotinamide have similar biological activity and both are used as dietary supplements to prevent Vitamin B3 deficiency. In supplements, the use of Nicotinamide is often preferred due to absence of common side effects of Nicotinic Acid, such as skin flushing.
Table I: HPLC gradient
The European Committee for Standardization has approved the HPLC method with post-column photochemical derivatization to measure Vitamin B3 in foodstuff. UV irradiation converts Nicotinic Acid and Nicotinamide into highly fluorescent derivatives. The addition of Pickering Laboratories UVETM Photochemical reactor to any HPLC system allows for highly sensitive and selective analysis of both Nicotinic Acid and Nicotinamide in a variety of different matrices.
Figure 1: Chromatogram of 1 µg/mL calibration standard of Nicotinic Acid and Nicotinamide.
Calibration Range: Nicotinic Acid: 0.1–50 µg/mL, R2 = 0.999;
Nicotinamide: 0.1–50 µg/mL, R2 = 0.998
Figure 2: Chromatogram of fresh peas sample.
Figure 3: Chromatogram of fresh tomatoes sample.
The sample preparation described below is designed to measure bioavailable (or free) Nicotinic Acid and Nicotinamide in foods. Different procedures can be employed to prepare samples for the analysis of total Niacin.
Figure 4: Chromatogram of raw pork sample.
Table II: Niacin analysis in foods
Analytical Conditions
Analytical Column: ThermoHypersil, Aquasil C18 (150 × 4.6 mm)
Temperature: 40 °C
Flow rate: 1 mL/min
Mobile Phase: Methanol/Phosphate buffer (0.035 mol/L of Potassium Phosphate Monobasic adjusted to pH 4.45). See Table I for gradient conditions.
Injection volume: 20 µL
Figure 5: Chromatogram of oat cereal sample.
Post-column Conditions
UVETM Photochemical Reactor
Detection: FLD, Ex 322 nm, Em 370 nm
Figure 6: Chromatogram of Vitamin B Complex dietary supplement.
Pickering Laboratories, Inc.
1280 Space Park Way, Mountain View, CA 94043
Tel. (800) 654-3330, (650) 694-6700
Website: www.pickeringlabs.com