Documentation

Method Abstract 239

MA 239 / 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 multi-vitamins supplements to help fight vitamin B deficiencies.

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.

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.

Analytical Conditions

Analytical Column: Thermo Hypersil, Aquasil C18 (4.6×150 mm)
Column Temperature: 40 ºC
Flow Rate: 1 mL/min
Mobile Phase:
     Solvent A: Dissolve 4.77 g of Potassium Phosphate Monobasic in 900 mL of DI water,
     adjust pH to 5.9 with KOH. Bring volume to 1 L with DI water.
     Solvent B: Acetonitrile. See Table 1 for gradient conditions.
Injection Volume: 20 uL

Post-column Conditions

Post-column Derivatization System: Pinnacle PCX
Reactor Volume: 0.5 mL
Reactor Temperature: 30 ºC
Reagent: Dissolve 10 g of Sodium Hydroxide in 500 mL of DI water, add 2 g of Sodium Sulfite, mix till fully dissolved
Reagent Flow Rate: Initial flow rate 0 mL/min. See Table 2 for pump program
Detection: FLD, see Table 3 for detector program UVE™ Photochemical Reactor

TABLE 1. HPLC GRADIENT

TIME (Min)

TIME (Min)

ACETONITRILE, %

0

100

0

8

100 0

15

90 10

22

90 10

30

40 60

35

40 60
 
35.1 100 0
45 100 0
 

         

TABLE 3. FLD Program

TIME (Min)

EXCITATION, nm

EMISSION, nm

0

322

400

17.9

322 400

18

370 440

25

370 440

25.1

350 470

35

350 470
 

 

TABLE 2. Pinnacle PCX Pump Program

TIME (Min)

FLOW RATE, ML/MIN

0

0

16.9

0

17

0.5

23

0.5

23.1

0

35

0
 

TABLE 4. Analysis of NIST Samples

  FOUND IN
PROTEIN
POWDER
RSD, %;
N=3
NIST VALUE
FOR PROTEIN
POWDER
FOUND IN
VITAMINS
TABLETS
RSD;
N=3
NIST VALUE
FOR VITAMINS
TABLETS
NICOTINAMIDE 282 ug/g 4.5% 258 ug/g 15288 ug/g  0.8% 13907 ug/g
THIAMINE 12.6 ug/g 9.0% 15.8 ug/g 1145 ug/g 0.6% 1045 ug/g
RIBOFLAVIN 25.9 ug/g 11.2% 26.9 ug/g 1204 ug/g 1.8%  1302 ug/g
 

 

MA239