Chromatography Forum

I have devloped Reverse phase chromatography Assay method in Soft geletin capsules which is made from Captex 355 oil (MCT oil), and my capsules strength is 0.5 mcg, chromatographic condition are below,

Mobile phase :Methanol : Water(85:15)
Column : C18, 100x4.6mm, 2.6µ
Column Temp: 40°C
RT: about 8 min.

My problem is, In my sample prepration oil is not miscible with ACN and MEOH but miscible with Ethanol and my drug also soluble with ethanol but when I am using the 100% Ethanol (Organic) and inject 100µl injcetion volume my peak is split and getting broad peak due to the 100% organic in sample prep. because I can not add aqueous in my sample prep due to oil is not miscible with water as well as not extract 100% drug.

so If you have any idea about column which will give a better and sharp peak shape when inject 100µl injection volume or any (in sample pre.) organic solvent which will dissolve my drug and oil.

Thanks
Hitesh P

We do not kno what the drug is. Sometimes a HILIC or normal-phase approach would be better, since the oils is soluble in normal-phase solvents.

Do you need to inject 100 microL?

At-column dilution might also work, but you will need a different instrument setup with an additional pump.

Do you really need to be able to get the oil into solution?
What is you standrd dissolved in?
Could you keep the oil out of solution and use an internal standard?
Could increase the concentration of the active in your sample solution and decrease the injection volume?

We do not kno what the drug is.Vitamin D Sometimes a HILIC or normal-phase approach would be better, since the oils is soluble in normal-phase solvents. My chromatography is Reversed phase and I already used Hilic Column.

Do you need to inject 100 microL? Yes because I can't increase my concentration due to the lower dose of my capsules i.e 0.5 mcg

At-column dilution might also work, but you will need a different instrument setup with an additional pump.

HILIC won't work for vitamin D.
With RP, you will have the trouble that you have.
Buy a silica column and do normal-phase chromatography, and all the current troubles will go away. You will need to brush up on the workings of normal-phase chromatography by reading a book, such as my book on "HPLC Columns".

Hey, did you solve your problem? I have exactly the same issue with wit D3 in oil formulation.. I'll appreciate any advice