removing H2O in serum sample for HILIC chromatography

[Kazimierz]

Dear all,

I'm looking for method for removing water from acetonitril extract of serum
samples. 100 ul blood serum + 400 ul acetonitril = 20%H2O.

In my mobile phase is 12%H2O (Silica column 25x4.6, 5 um, SF). Separation is much more excellent when injected samples are in pure acetonitrile.

I'm looking for method which could be more easy than eveporation step or solid-phase extraction or desalting.

[Ann]

Is this just a problem of having something in your extract that coelutes with your analyte of interest? You say that the separation is much better with your sample in 100% CH3CN - I presume that your sample in 100% CH3CN is your standard solution? Have you injected a blank plasma extract? Is there anything in the blank extract that interferes with your analyte?

All the best, Ann

[HW Mueller]

Why not use considerably more ACN in the extraction? That way your water content tends to 0% and your protein precipitation will be vastly improved.

[HW Mueller]

Also: Adding 2,2-dimethoxy propane (someone please correct me if my memory fails here) is another trick. You will have to acidify (maybe in form of an ion exchanger), the products are methanol and acetone which may be injected directly (assuming you are talking about NP HPLC).
(Stoichiometry is 1+1, dimethoxy...+H2O)

[Kazimierz]

Dear Ann,
The method is for ganciclovir. In blank of the serum generally interferences are absent in the drug region. However, in the IS region (acyclovir) is interference. After including evaporation step and use 100% acetonitril for sample extract, all peaks are much more sharp and higher, and IS could be also used. Actually, method works excellent without IS. Peak wide=0.09 min when Rt=8.03 min. The pH of the mobile phase is 2.2 and 88% acetonitril.

Dear Hans,
Thank you for your reply and valuable concepts. I will able to dilute sample with more acetonitril. But your concept of 2,2-dimethoxy propane method for H2O remowing from sample before HILIC chromatography could be very interesting. I will verify it after ordering this chemical from SIGMA (100 ml = 13.3 euro only). As I remember, GC-chromatographers used some chemical methods for drying samples for gas-chromatography analyses.

[Ann]

Glad you got it sorted

Best wishes Ann