method for hydroxocobalamin & mecoblamin

[ramiaqu]

hi

I am working with "mecobalamine" I am looking for HPLC Related substance method that elute the " hydroxocobalamin" after the mecobalamine since i found severals method but all of them gives me rapid elution of hydroxocobalmine (approx. 2min). Can any one gives me some ideas about it?

thank you

[Uwe Neue]

It is normal for a reversed-phase method that a compound with a hydroxy group will elute before the same compound without such a group. To inverse the elution order, the best bet is to do HILIC = hydrophilic interaction chromatography.

[ramiaqu]

what do you mean by HILIC = hydrophilic interaction chromatography , do you have a method, and if anyone have please help me.

[Uwe Neue]

I do not have a method, but if you want to invert the elution order seen in RP between a compound and its hydroxylated metabolite, you will not be successfull with straight reversed-phase chromatography. However, if you can run your compound in HILIC, the principle is the inverse of RP, and your hydroxylated metabolite will elute last.

[ramiaqu]

but still i dont know what is the HILIC = hydrophilic interaction chromatography ? and how can i use it

[Uwe Neue]

You use a silica column, equilibrate it in 95% acetonitrile, 5% water, then run a gradient to 50% acetonitrile, 50% water. Further refinements are possible by using a buffer.

It is in principle a partitioning of the analyte into the water-rich layer on the surface of a very hydrophilic packing.

More in my book on "HPLC Columns".

[ramiaqu]

how can i get your book? where can i found it?

[Patrik Appelblad]

If you're interested in learning more about HILIC, you should get the SeQuant "Practical Guide to HILIC". It is a comprehensive booklet that gives a thorough introduction to HILIC as well as it expemplifies areas of use via a selection of applications. Please visit www.sequant.com, leave your contact details, and it will be sent to you for free.

[Uwe Neue]

My book on "HPLC Columns" can be purchased through Amazon.com, or from Waters, part number WAT038216.