Vitamin D on HPLC

[kristind858]

I am currently working on the Vit D3 Method 1 from the USP. Here are the analytical conditions:

Column: Spherisorb NH2 (150 mm x 4.6 mm, 3 um particle size), Packing L8
Mobile Phase: Hexane, IPA (99:1)
Flow rate: 1.0 ml/min
Column temp: 25 deg C
Detection: UV; 265 nm
Sample volume: 100 ul

When I inject my working standard (2 ug/ml) of Vit D3, I get two peaks. One smaller peak with an RT of ~3.2 (7-dehydrocholestrol) and a much larger peak with an RT ~4.2 (cholecalciferol~Vit D3). The resolution solution is made by heating a portion of the working standard for 1 hour at 60 deg C in a waterbath. Once injected, the peak area of the Vit D3 decreases and the peak area of it's precursor, 7-dehydrocholestrol increases. I used Vit D2 as an internal standard but with the analytical conditions set forth, the Vit D2 only added to the area of Vit D3. So I will have to find another internal standard Here are a few questions I have:


Has anyone tried this analytical condition? If so , does this retention time seem too quick off the column? I see other posts mention 15-20 min retention time but that is with different packings I think. I let each sample injection run time go for 6 minutes.

The method calls for n-hexane but the HPLC hexane is only 60% n-hexane, but is >99% total hexanes. This is still of high enough purity to use since the HPLC column will not seperate these isomers unless using chiral HPLC..Is this correct?

[kristind858]

Hello all,
I now would like to see if my calculation for the above method is correct:

I transfer the equivalent of 5 capsules to a glass bottle and record the weight in ug. I perform the method extraction and inject my samples. I average the areas of my vitamin D3 samples and get the rsd for my intra-assay precision. I calculate the concentration of my standard and make the correction for purity. The nominal concentration of Vit D3 that should be in the sample is then calculated by:

(Weight of sample transferred/Net fill calculated per cap) X (the nominal label claim)
100 ml

I divide by 100 ml since my sample is filled to 100 ml volume. Is this the correct way to calculate the nominal concentration of Vit. D3 that should be in my sample? I use the USP formula to calculate the formula % of Vitamin D3. This % is then multplied by the conc. of my vit D3 standard (since this is considered 100% right ??) to give me the Vit D3 conc in ug/ml of my sample. The ug/ml is then converted into IU/ml. The calculated IU/ml is then divided by the weight of sample which was transferred to the glass bottle to get the IU/ug. To get IU/cap, I multiply (IU/ug) x (ug/cap) (which was calculated by obtaining the net weight of the capsule). Please let me know if you see anything that is incorrect. Thanks so much !!!!

[kristind858]

The chromatogram above is of an oil based formulation using the conditions previously posted above. I was originally weighing out my oil mixture equivalent to 5 capsules and dissolving it in n-hexane. I then added 10 ml of DMSO. The method that was being followed was the USP 35/NF 30 Vol. 1, Method 1. But with this method, I was not seperating the lipids from the Vitamin D3. To increase the resolution of the Vitamin D3 peak, should I be doing a saponification to turn the triglycerides into soap and glycerine? The soap and glycerine will then be soluble in the DMSO layer, leaving mainly Vitamin D3 in the n-hexane layer. Please any suggestions will help Thanks !!!

[kristind858]

Can anyone think of a suitable internal standard that I can use on a normal phase column? Vitamin D2 coelutes with D3. Thanks !!!

[kyleb]

Hello,

Don't know if this will help, but honestly for Vitamin D2/D3/Fat-Soluble vitamins I think you would have much better luck with a simple "reverse phase" C-18 column instead of normal phase. Use some of the less polar solvent such as: 100% Methanol, 100% Acetonitrile, or 95% ACN/5% Water. You can even try different ratios of Methanol and Acetonitrile (i.e. 75/25 MeOH/ACN).

If you are stuck with the normal phase, try Ergosterol for internal std. It should elute later than Vitamin D3.

What kind of samples do you anlayze?

I've done a lot of work with Vitamin D2 (from irradiated yeast cell wall) so maybe I can help?

[kristind858]

Hello Kyle !
Our samples are fish oil capsules which already contain endogenous Vit D3, flavoring, and vitamin E. I would have picked the AOAC method which involves saponification, clean up on normal phase, followed by seperation on reverse phase. The aminopropylsilane column was already purchased to follow the USP method and that is why I am trying to make this work.The method calls for adding 10 mL of DMSO to the oil with 15 mL of n-hexane, then heat at ca 60 deg C for 45 min. The hexane extracts are collected and analyzed. Do you know what is actually going into the DMSO layer ? I would think the triglycerides and other non polar compounds would remain in the hexane layer. I would think saponification would work best by turning the triglycerides into soap thereby making it more soluble in DMSO. Leaving the vitamin D3 and a few other non-polar compounds in the hexane layer. Any advice would be great Thank you !!!

[kyleb]

Hello,
I have personally not worked with DMSO, I do know that it can dissolve both polar and non polar compounds. So maybe the hexane/DMSO layer could be the issue? Are you aggitating the samples (gently shaking, inverting) periodically through out the saponifaction?

When I do a saponification with yeast cell wall (liquid of dried), I'm using 2 g of sample, with 2 mL DI H20 and ~1.35 g KOH in a 100C bath for 2 hrs. So there is no "extraction" taking place with a solvent. I tried a method that sounds similar to what you are attempting with diethyl ether, and did not have very good results.

I hope this helps!

-Kyle

[kristind858]

Hi Kyle,
Thanks for all your help ! I am not adding any base so there is no saponification. I would think that if I did do a saponification step that this would clean up my sample a bit more. I am using sonication to agitate the sample with occasional shaking. I am just not seeing the purpose on the DMSO I think I will try getting the ergosterol

[kyleb]

Hello,
I'm not sure about the DMSO either . I know that Hexane is good for extracting oils from water/environmental samples because I've done that in soil/water testing.

Yes, Ergosterol (or provitamin D) should work nicely for you!

Also, you may just want to try a saponification (like the one I mentioned) without any sort of extraction with solvents/organics. Once the sample is saponified, I just dilute with MeOH (or in your case, Hexane), and filter through 0.2 um syringe filter.

Good luck!

-Kyle

[kristind858]

Hi Kyle,
I think the ergosterol may coelute with the precusor to Vitamin D3, 7-dehydrocholesterol. They are very similiar in structure. Ergosterol has one less hydrogen than 7-dehydrocholesterol We partially convert our Vitamin D3 to 7-dehydrocholesterol by heating at ca 60 deg C for 1 hour. The peak elutes before Vitamin D3. Thanks for all your help !!! I think I will try saponifying sample.

[kyleb]

Whoops! I should have read your previous posts more carefully. You will indeed have some issues with coeluting.

Good luck!

Kyle

[dp297]

Dear kristind858,

are u still struggling with VitD? Are u measuring VitD or 25OHD? I am developing also a method, so we could discuss some issues!

[ph/Amr Tarek]

this method i applied it on a syrup containing vit D3 and gave posotive results :
mobile phase : 250 ml methanol + 750 ml acetonitrile
column : hypersil BDS C18 150 × 4.6 mm
Detector : UV at 265 nm
Flow rate : 2ml/min
inj vol : 40 micro litre
Resolution standard : vitamin E acetate
Use isopropanol to dissolve both vit D3 and resolution standard

[kristind858]

Hi dp297,
I am trying to quantitate cholecalciferol using the USP method. I have created some blank placebo samples and then spiked them with Vitamin D3 standard. Our formulation contains fish oil, Vitamin E, and some flavoring. My recovery for the low end was 95 %, mid range was 101%, and high end was 100.7% . This sounds pretty good but there is a close eluting peak near cholecalciferol, which can be seen in the blank. Sometimes when analyzing a sample, the peaks coelute together. I have tried adjusting the mobile phase (n-hexane, IPA) but that only makes both peaks elute close together with a different retention time. Maybe I need to equilibrate the column longer as well. How is your Vitamin D going?