Hi,

(for mobile phase and column details)

(raw material chromatogram)

(premix production 10th batch)

(premix production 20th batch)

Our HPLC model is: Sh...zu Prominence lc-20A

Detector : SPD-20A, UV/VIS detector

Column Type: ODS-3

Mobile Phase: Dissolve 0,941 gram IPPC-06 (Sodium 1-Hexane Sulphanate) in double distilled water to make 1L solution.Add 2,35 ml of Phosphoric Acid(Purity :85%)into the volume flask.Sonicate(5mins) and filter under reduced pressure to remove insoluble substance (pore size: 0,45 mcm).

(ghost peak)

(b1 standard curve)

(b2 standard curve)

(b3 standard curve)

(b6 standard curve)

(b9 standard curve)

Calibration curve was made by using mix standards in mobile phase with four point calibrations, analyzed independently by HPLC and a standard curve was plotted between concentration and peak area.

Water was distilled and deionized by using Millipore.

Standards were individually weighed 0.1 g for each vitamins that desired to be analyzed by HPLC. But we had to use less than 0.1 g of standards. Then we have decided to use 0.025 g of standards (for 1000 ppm of vitamin standards).

Standard stock solution for vitamin B1 (thiamine HCl) was prepared by dissolving 25.2 mg of thiamine hydrochloride in 25 ml of double distilled water.

Standard stock solution for vitamin B2 (riboflavin) was prepared by dissolving 25.2 mg of riboflavin in 25 ml double distilled water.

Standard stock solution for vitamin B3 (nicotinamide) was prepared by dissolving 25.1 mg of nicotinamide in 25 ml of double distilled water.

Standard stock solution for vitamin B6 (pyridoxine) was prepared by dissolving 25.3 mg of pyridoxine in 25 ml of double distilled water.

Standard stock solution for vitamin B9 (folic acid) was prepared by dissolving 25.0 mg of folic acid in 25 ml of double distilled water. Additionally, for vitamins B2 and B9 , to improve solubility, 20 drops of NH3 were added to the solutions of standards.

At this stage, 1000 ppm standard solutions were obtained for each vitamin B groups. Then, ppm values of each vitamin B groups were determined by considering the mixes that would have been analyzed.

They will be diluted from 1000 ppm of standard solutions. We have to determine the maximum and minimum anticipated concentrations of the vitamin standards in the samples we intend to test on the HPLC.

Determined concentrations are shown in table :

Mix 1 Mix 2 Mix 3 Mix 4

B1 10 ppm 20 ppm 40 ppm 80 ppm

B2 10 ppm 20 ppm 40 ppm 80 ppm

B3 10 ppm 20 ppm 40 ppm 80 ppm

B6 10 ppm 20 ppm 40 ppm 80 ppm

B9 10 ppm 20 ppm 40 ppm 80 ppm

For the preparation of these mixes, they were diluted from standard stock solutions of vitamins 1000 ppm. You will see in the table :

Mix 1 Mix 2 Mix 3 Mix 4

B1 1 ml 2 ml 4 ml 8 ml

B2 1 ml 2 ml 4 ml 8 ml

B3 1 ml 2 ml 4 ml 8 ml

B6 1 ml 2 ml 4 ml 8 ml

B9 1 ml 2 ml 4 ml 8 ml

The working standards were prepared as follows:

Mix 1 contains 10 ppm of B1 were diluted from 1 ml of B1 vitamin which is 1000 ppm, 10 ppm of B2 were diluted from 1 ml of B2 vitamin which is 1000 ppm, 10 ppm of B3 were diluted from 1 ml of B3 vitamin which is 1000 ppm, 10 ppm of B6 were diluted from 1 ml of B6 vitamin which is 1000 ppm, 10 ppm of B9 were diluted from 1 ml of B9 vitamin which is 1000 ppm. The standard mixture 1 was diluted to mark with water (100ml) and mixture was sonicated (5 minutes) and filtered through a 0.45 µm filter.

Mix 2 contains 20 ppm of B1 were diluted from 2 ml of B1 vitamin which is 1000 ppm, 20 ppm of B2 were diluted from 2 ml of B2 vitamin which is 1000 ppm, 20 ppm of B3 were diluted from 2 ml of B3 vitamin which is 1000 ppm, 20 ppm of B6 were diluted from 2 ml of B6 vitamin which is 1000 ppm, 20 ppm of B9 were diluted from 2 ml of B9 vitamin which is 1000 ppm. The standard mixture 2 was diluted to mark with water (100ml) and mixture was sonicated (5 minutes) and filtered through a 0.45 µm filter.

Mix 3 contains 40 ppm of B1 were diluted from 4 ml of B1 vitamin which is 1000 ppm, 40 ppm of B2 were diluted from 4 ml of B2 vitamin which is 1000 ppm, 40 ppm of B3 were diluted from 4 ml of B3 vitamin which is 1000 ppm, 40 ppm of B6 were diluted from 4 ml of B6 vitamin which is 1000 ppm, 40 ppm of B9 were diluted from 4 ml of B9 vitamin which is 1000 ppm. The standard mixture 3 was diluted to mark with water (100ml) and mixture was sonicated (5 minutes) and filtered through a 0.45 µm filter.

Mix 4 contains 80 ppm of B1 were diluted from 8 ml of B1 vitamin which is 1000 ppm, 80 ppm of B2 were diluted from 8 ml of B2 vitamin which is 1000 ppm, 80 ppm of B3 were diluted from 8 ml of B3 vitamin which is 1000 ppm, 80 ppm of B6 were diluted from 8 ml of B6 vitamin which is 1000 ppm, 80 ppm of B9 were diluted from 8 ml of B9 vitamin which is 1000 ppm. The standard mixture 4 was diluted to mark with water (100ml) and mixture was sonicated (5 minutes) and filtered through a 0.45 µm filter.

And now we have a series of standard solutions with which to calibrate the HPLC.

Finally, I would like to share with you all chromatograms of working standard mixtures as you can see below :

Mix 1 (mixture that contains 10 ppm of each vitamins standard) :

Mix 2 (mixture that contains 20 ppm of each vitamins standard) :

Mix 3 (mixture that contains 40 ppm of each vitamins standard) :

Mix 4 (mixture that contains 80 ppm of each vitamins standard) :

Preparation of Sample (MIX) Solution

0,2 g of sample (fortification mix) was weighed and transferred into a 100 ml volumetric flask and then 20 drops of NH 3 were added to flask. The mixture was diluted to mark with water and mixture was sonicated (5 minutes) and filtered through a 0.45 µm filter.

If you can help me i will be appreciated.

Thanks.