Vitamin A and D by HPLC (USP Method)

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Hi all!

I was wondering if anyone has ever tried the vitamine A and vitamin D method (HPLC) as per USP.

We're trying it for the first time and we're having an hard time here to obtain results that makes sense...

Any feedback, precautions or advice?

Column used, modification of the method required?

Thanks a lot and nice forum btw!


What are the column dimensions suggested by the USP?

Thought I had a usp at home, seems that I don't have it anymore, I will get back to you with this information next week I will be on vacation for the next couple of days. I should have write more information in first post...

Never ran it, sorry, but I do have a USP here at work with me. Here are the USP conditions for Vitamin A Assay, Chromatographic Method - The liquid chromatograph is equipped with a 325-nm detector and a 4.6-mm × 15-cm column that contains packing L8. The flow rate is about 1 mL per minute. 40 µL. n-hexane as mobile phase and diluent.

I don't see anything about vitamin D...?

Hope that helps, sorry if its not what you were looking for.


I run Vitamin A and D all the time, what kind of problems are you having with it? What solvent/mobile phase/column are you using? What are your run conditions? What are you using as your standard?

Also, in what form do you have the vitamin D/A? Is it pure, or within some sort of matrix? If it is pure, you can usually dissolve directly into hexane, and run normal phase with n-hexane mobile phase. If it's in a matrix, you will have to do an extraction because it won't dissolve directly into hexane in that form.

The reason you are most likely having problems is because the USP general method assumes that you have the pure form. The USP method is useless if you are not using a pure form of vitamin A or D. So you would have to make modifications and validate your own method.

Let's start with vitamin D, try this:

Mobile Phase: n-hexane (you can shorten your RT with the addition of some IPA, but no more than 0.15 mL per liter of hexane.)

Column: The column used in the USP method should work fine. You also might need to heat your column, or else your run time will be VERY long.

Sample: Thoroughly dissolve sample in a solution ( 15-20 mL) of Citric Acid in DMSO, and extract with about 10 mL of Hexane. (one wash should be enough, just make sure you shake for at least 10 minutes) Inject a portion of the hexane layer.

Let me know what kind of results you get.

Below are similar conditions on Unison UK-Amino: ... TI330E.pdf

Just curious, why citric acid is added in DMSO?

To be perfectly honest, I have no clue. We have an in-house method that was developed a long time ago. And since I am in QC, I just test things, and I never cared enough to ask :-) If I had to guess, I would imagine that the raw material is soluble in the DMSO, and the Citric Acid aids in the phase transfer.

Thanks for the reply.

We're testing the Vitamine A and D in Cod Liver Oil USP. It's a new product for us. I would assume that we're having matrix effect.

For now we're running in the USP conditions for the mobile phase and solvent.


For the vitamine D we use the following column :
Chromatographic system—Use a chromatograph, operated at room temperature, fitted with an UV detector that monitors absorption at 265 nm; a 25-cm × 4.6-mm stainless steel cleanup column packed with column packing L10 and using Mobile phase A; (Zorbax CN) and a 15-cm × 4.6-mm stainless steel analytical column with 5-µm packing L1 ( Supelcolsil LC-18 ), and using Mobile phase B. Chromatograph five injections of the Standard preparation, and measure the peak responses as directed for Procedure: the resolution, R, between cholecalciferol and ergocalciferol is not less than 1.4; and the relative standard deviation for the cholecalciferol peak response is not more than 2.0%.


For the vitamine A we use the following column : Zorbax NH2 / 5um 4.6um * 150mm

We've been able to performs the Vitamin A analysis but we still have a lot of peak shifting, certainly in relation with the normal phase used for the system.

Any advice? We're using HP1100

Yeah peak shifting is a common problem with these materials. I've found this testing to be very sensitive to even the slightest variations in column temp. If you aren't using a column heater to keep your column a constant temp, you should try that. You just need to make sure you equilibrate your column thoroughly. How long are your run times?

Hopes this helps.

Vitamin A :
We do not use the heater column, but we will for the future analysises. Thanks for the tips.

the peaks for vitamine A have a RT of 30min.


Vitamin D

I would be curious of the column used in general by the industry if you can provide me such informations.

Thanks for your answers, they are very appreciated.

Yeah an RT of 30 minutes sounds about right at room temp. We typically get RTs between 15-20 minutes. But we are using a column heater which shortens the retention time.

Vitamin D columns just vary by application. We use two different columns for our Vitamin D analysis. The are both NH2 columns, but one is Supelco NH2-NP 250 mm, and the other is Zorbax NH2 15 mm in length. I read over the USP method for cod liver oil, it's pretty involved. I am not sure I understand the setup, are these columns attached to each other in tandem or do you have a dual column setup going to the same detector?
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 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?
please I'm working on vitamin d3 assay by reversed phase HPLC with uv detector and thermo hypersil column 250 4.5 and i had no results at all by the usp method or the british any help will be appreciated
I am facing same problem when analysing vitamin A and D in palm oil sample. When palm oil sample contains BHT and TBHQ how to seperate vitamin A using HPLC? Can any one give an proper method for analysis vitamin A and D in oil?
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