troubles with B vitamins

Discussions about HPLC, CE, TLC, SFC, and other "liquid phase" separation techniques.

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Hi,

we are producing flour fortifications(vitamin and mineral premixes)in powder form. In our quality laboratory we have hplc and aas to check&confirm the content.

The premix contains vitamins B(b1,b2,b3,b6,b9,b12), vitamin A, iron and zinc.

we noticed that any premix contains Vitamin B1, causes ghost peak which is almost at the same time with B3 peak and thus which effects the are of B3 and then we receive lower result of B3 than expected.

the second problem is: the results of Vitamin B1 are always higher about 20-30% which is unacceptable.
the form of B1 is: thiamine mononitrate and the standard of B1 is also thiamine mononitrate.

Do you have any idea about these problems? can you suggest anyting?
By the way, what is the most convenient solvent for b2 and b9 and how much will be enough.(we are using 10 drop of ammonia.

we are preparing the standards as 10ppm,20ppm,40ppm and 80 ppm. this range is ok or should we enlarge the range?

Thanks.
Hi,

It's hard to accurately diagnose your issue because I think there's a lot of critical information missing. As always, posting chromatograms is really helpful for us, to help you!

Can you provide the mobile phases, the LC program, the column and how you prepare the standards and samples?

1) The ghost peak: based on your brief description, do you mean that B3 is interfered by something in the sample? Because a ghost peak in HPLC means an unknown peak eluting at random retention times, also in blanks. Or do you mean that B1 and B3 aren't separated? Is B3 nicotinamide or nicotinic acid in your samples?

2) B1 recovery: It could be a calculation or dilution error somewhere. Next, thiamine is rather hygroscopic. Did you take into account the purity and the moisture of the analytical standard? Is it perhaps an old standard, or is it not stored appropriately? Another possibility is the degradation of thiamine in your standard solutions, and not in your samples. Thiamine is not a stable molecule in solution - it is the worst, in this respect, of the B-vitamins. If, for example, the pH of your standards is in the alkaline region, and the pH of your sample extract is in the acid region, that would be the most likely source of error (it is much more stable in acid). Try to get a match in chemical environment of standards and samples - using a buffer is a good idea.

3) Solvent for B2: Getting riboflavin to dissolve in anything is annoying, other than alkaline solutions. But the catch is: it is NOT stable in alkaline solutions, so you should not use them. I suggest using water / mildly acidic buffer at a maximum of 100ppm (10mg/100mL). That said I also found protocols mentioning dissolution of B2 in alkaline solutions, do it at your own risk...

4) Solvent for B9: Folic acid dissolves well, and is rather stable, in alkaline solutions (I use 0.1M NaOH).

5) Range: this is entirely dependent on the concentration in your samples and the sample preparation procedure. If all the samples fall in this range, there's no reason to expand it. However, I find it hard to believe that all your B-vitamins are at the same concentration. Typically, vitamin fortifications are based on reference daily intake, meaning that for example B1 will be about 500x higher than B12 in concentration. This presents a challenge for multi-vitamin analytical methods, but it's still possible.
hi Rndirk,

thanks for the information. tomorrow when i reach to the lab. i will send you more details , chromatograms, procedure, etc.

Thanks again.

Rndirk wrote:
hi

It's hard to accurately diagnose your issue because I think there's a lot of critical information missing. As always, posting chromatograms is really helpful for us, to help you!

Can you provide the mobile phases, the LC program, the column and how you prepare the standards and samples?

1) The ghost peak: based on your brief description, do you mean that B3 is interfered by something in the sample? Because a ghost peak in HPLC means an unknown peak eluting at random retention times, also in blanks. Or do you mean that B1 and B3 aren't separated? Is B3 nicotinamide or nicotinic acid in your samples?

2) B1 recovery: It could be a calculation or dilution error somewhere. Next, thiamine is rather hygroscopic. Did you take into account the purity and the moisture of the analytical standard? Is it perhaps an old standard, or is it not stored appropriately? Another possibility is the degradation of thiamine in your standard solutions, and not in your samples. Thiamine is not a stable molecule in solution - it is the worst, in this respect, of the B-vitamins. If, for example, the pH of your standards is in the alkaline region, and the pH of your sample extract is in the acid region, that would be the most likely source of error (it is much more stable in acid). Try to get a match in chemical environment of standards and samples - using a buffer is a good idea.

3) Solvent for B2: Getting riboflavin to dissolve in anything is annoying, other than alkaline solutions. But the catch is: it is NOT stable in alkaline solutions, so you should not use them. I suggest using water / mildly acidic buffer at a maximum of 100ppm (10mg/100mL). That said I also found protocols mentioning dissolution of B2 in alkaline solutions, do it at your own risk...

4) Solvent for B9: Folic acid dissolves well, and is rather stable, in alkaline solutions (I use 0.1M NaOH).

5) Range: this is entirely dependent on the concentration in your samples and the sample preparation procedure. If all the samples fall in this range, there's no reason to expand it. However, I find it hard to believe that all your B-vitamins are at the same concentration. Typically, vitamin fortifications are based on reference daily intake, meaning that for example B1 will be about 500x higher than B12 in concentration. This presents a challenge for multi-vitamin analytical methods, but it's still possible.
Hi,

Image (for mobile phase and column details)

Image
(raw material chromatogram)

Image (premix production 10th batch)

Image (premix production 20th batch)

Image
Image
Image
Image
Image
Image
Image
Image

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).

Image (ghost peak)

Image (b1 standard curve)
Image (b2 standard curve)
Image (b3 standard curve)
Image (b6 standard curve)
Image (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) :
Image

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

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

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

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.
That's a lot of information. I will not be reviewing the whole SOP, let's focus on the problems you are having.

None of the images seem to show. See the sticky for guidelines to embed chromatograms. You don't have to re-upload every image from the post above, let's start with just 1: a chromatogram that shows your "ghost" peak.

Both samples and standards are prepared in plain water, with some drops of ammonia here and there. This means you are working at high pH - where several vitamins are unstable. I suggest to make use of mildly acidic buffer, which is the best middle ground you can get in stability for all B-vitamins. B9 stock can still be dissolved in an alkaline solution, and when you mix this with other stocks diluted with buffer, the final mixtures should be pretty close to the buffer pH.
thank you for the information.

sorry, i am new in this forum :) i was not familiar with image uploading.

Anyway, we will use acidic buffer but do you think that it helps to get correct vitamin B1 content? Because it is always higher around 20-30% than expected.


Rndirk wrote:
That's a lot of information. I will not be reviewing the whole SOP, let's focus on the problems you are having.

None of the images seem to show. See the sticky for guidelines to embed chromatograms. You don't have to re-upload every image from the post above, let's start with just 1: a chromatogram that shows your "ghost" peak.

Both samples and standards are prepared in plain water, with some drops of ammonia here and there. This means you are working at high pH - where several vitamins are unstable. I suggest to make use of mildly acidic buffer, which is the best middle ground you can get in stability for all B-vitamins. B9 stock can still be dissolved in an alkaline solution, and when you mix this with other stocks diluted with buffer, the final mixtures should be pretty close to the buffer pH.
Let's call it an educated guess, but it's not the only source of error I suspect (see my first post). Did you check the purity, age and moisture of your B1 analytical standard? This should be on the certificate of the standard.

Is there a reason to think that there is something eluting beneath the B1 peak in your samples? For example, is the peak shape different than in the standards?
If you can't work out the quantitation and separation problems with your ODS column, then consider using a neutral, high-surface area material for HILIC. HILIC works well for the B vitamins.
PolyLC Inc.
(410) 992-5400
aalpert@polylc.com
Few remarks.
pnrrdl wrote:
the form of B1 is: thiamine mononitrate and the standard of B1 is also thiamine mononitrate.
...
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.

At least part of the problem with B1 may be due to incorrect calculations (nitrate vs. hydrochloride).

pnrrdl wrote:
we noticed that any premix contains Vitamin B1, causes ghost peak which is almost at the same time with B3 peak and thus which effects the are of B3 and then we receive lower result of B3 than expected.

The "ghost" peaks (tR = 2.0-2.5 min) marked on one of the figures are far from the peak of B3 (tR = 4.0 min). B3 is well separated from the "ghosts". No problem here.
What about the peaks on the chromatograms of blanks (solvent for standards, solvent for samples)?
What about the peaks potentially interfering with B3 on the chromatograms of the solution containing all the components except for B3?
What about the peaks potentially interfering with B1 on the chromatograms of the solution containing all the components except for B1?
I worked up a method several years ago for the full list of B vitamins using HPLC/MS/MS and found that to get consistent results I had to do both an acid extraction and a base extraction, and use two different analytical runs with standards prepared in both acid and base to match the extraction solution. One of my test extractions was simply grinding up Cheerio's cereal and extracting and doing both post and pre extraction spiking to determine which pH worked best for each Vitamin.

I don't have all the list handy at the moment but can say that B1 and B6 work best in acidic extraction and B3 worked better in basic extraction.

I found some research by Tee E-Siong from Kuala Lumpur that was very helpful where he describes extracting with both sulfuric acid or sodium hydroxide solutions. It took some time but after a while we had stable results that worked for both cereal and tablets.
The past is there to guide us into the future, not to dwell in.
The standard is 2 months old. purity : 99,4%.
expiry date: 23rd October 2021
moisture is not written but i can check it.

by the way, my raw material in the premix is: thiamine mononitrate and the rate is: 1,26%
and if my standard is thiamine HCl, when I check my premix which contains thiamine mononitrate, I got 1,02% as a result(instead of 1,26).
but if my standard is thiamine mononitrate too, I got 1,43% as thiamine mononitrate result (instead of 1,26).

I am really confused.

Rndirk wrote:
Let's call it an educated guess, but it's not the only source of error I suspect (see my first post). Did you check the purity, age and moisture of your B1 analytical standard? This should be on the certificate of the standard.

Is there a reason to think that there is something eluting beneath the B1 peak in your samples? For example, is the peak shape different than in the standards?
pnrrdl wrote:
The standard is 2 months old. purity : 99,4%.
expiry date: 23rd October 2021
moisture is not written but i can check it.

by the way, my raw material in the premix is: thiamine mononitrate and the rate is: 1,26%
and if my standard is thiamine HCl, when I check my premix which contains thiamine mononitrate, I got 1,02% as a result(instead of 1,26).
but if my standard is thiamine mononitrate too, I got 1,43% as thiamine mononitrate result (instead of 1,26).

I am really confused.

Rndirk wrote:
Let's call it an educated guess, but it's not the only source of error I suspect (see my first post). Did you check the purity, age and moisture of your B1 analytical standard? This should be on the certificate of the standard.

Is there a reason to think that there is something eluting beneath the B1 peak in your samples? For example, is the peak shape different than in the standards?


Are any components calculated as the free thiamine or as the salt? I had that problem with diaquat once where one standard was calculated as diaquat and the other was diaquat dibromide. Depending on which we used as primary for calibration the secondary check would either be very high or very low.

The instrument will measure thiamine, so calculate the weight to use when making standards as thiamine, so that you will use 1g+weight of salt so that you have 1g of thiamine in the solution, then it won't matter what salt was used in the samples you will have thiamine calculated accurately. If whoever made the sample did not correct it will not give what they might expect but at least you will know your results are correct.
The past is there to guide us into the future, not to dwell in.
Hi,

I was outside of the country so i could not check the forum for a while. Anyway thanks for all the interest finally we solved the problem but do not ask me how? :) because there is no reason at all, just suddenly we can get the results.

Thank you.
pnrrdl wrote:
Hi,

I was outside of the country so i could not check the forum for a while. Anyway thanks for all the interest finally we solved the problem but do not ask me how? :) because there is no reason at all, just suddenly we can get the results.

Thank you.

can you suggest me your method because i'm working on the estimation of vitamin-B complex.
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