Chromatography Forum

I'm hoping for some help from some vitamin experts, specifically from food chemists.

I'm working with an AOAC method 2002.05 that I have modified to analyze both D2 and D3 simutaneously. In the method, I believe D2 is used as an internal standard to analyze D3. The cleanup/extraction is very complicated and I am getting approx. 70% recovery, so I know I need an internal standard.

What else can I use besides D2 as an internal standard? During the semiprepative phase, I need the int. std to be included in my fraction collected.

Thanks!
Natalie

What detector you are using? If MS is used, you may consider using deuterated Vitamin D3 which is commerically available, albeit it is rather expensive.

The Semipreparative step is to extract your D2/D3 from the food matrix, so you can do your quantation of D3 in the analytical HPLC run.

To my understanding, the recovery is not important when using internal standards as your recovery of D2 would be same as your D3 recovery. And you are quantifing D3 concentration base on your D2 amount added relative to the D3 in sample (D2/D3 ratio).

i.e. If you have 70% recovery on D2, you should have 70% recovery on your D3, but your relative ratio between D2/D3 is same rather it's 70% or 100% recovery.

What detector you are using? If MS is used, you may consider using deuterated Vitamin D3 which is commerically available, albeit it is rather expensive.

I WISH I was using a MS. That would make my life so much easier. Unfortunately, I am using HPLC-UV.

The Semipreparative step is to extract your D2/D3 from the food matrix, so you can do your quantation of D3 in the analytical HPLC run.

To my understanding, the recovery is not important when using internal standards as your recovery of D2 would be same as your D3 recovery. And you are quantifing D3 concentration base on your D2 amount added relative to the D3 in sample (D2/D3 ratio).

i.e. If you have 70% recovery on D2, you should have 70% recovery on your D3, but your relative ratio between D2/D3 is same rather it's 70% or 100% recovery.

But what if I want to quantify D2 and D3? My sample has both. Is there another uncommon analyte in food that comes out close to D2/D3 that I could use?

You can try to use some other pro-vitamin D such as 7-Dehydrocholesterol or lumisterol.

Otherwise, I think you can still calculate your respective D2 and D3 from using standard addition.

You can use the method and calculation described in BP or USP for vitamin D3 test in cod-liver oil.

Thanks!

Dear All,

I'm trying to qualify the USP assay method I for Oil Soluble Vitamins in Dietary Supplements, for the vitamin D3 (cholecalciferol) and I would like to have your advice.

My problem is with the accuracy (recovery) of the method and the correction factor of 1.09 (for the amount of previtamin D in samples). I get recoverys between 109 and 115 % and it seems that the correction factor doesn't work for freshly spiked reconstitued sample. The sample extraction do not gives significant amount of previtamin D, and it seems that the previtamin D is only present in real tablet samples.

Does someone have more explanations about this correction factor of 1.09?