By Anonymous on Wednesday, March 24, 2004 - 04:09 am:
I am developing HPLC method for measuring netilmicin and glucosamine drug in serum.
Both drugs have no chromophore (as I know). I am trying to develop condition for derivatization of netilmicin first. I choose FMOC as an derivatizing agent because it is said to be more stable than OPA. I use C18 column (Luna 5u c18(2) 250x4.60mm), acetronitrile/water as a mobile phase (have tried %acetronitrile as 90% and 70%) and ex260nm em315nm for fluorescence detector.
Condition for derivatization have FMOC, boric acid and glycine (the ratio and concentration are on the developing process).
In chromatographic profile, I can find a peak of FMOC reagent and this peak is decreased when there is netilmicin or gentamicin (internal standard) in standard sample, so it means the derivaization process occured. And the FMOC peak is almost disappeared when I increase concentration of glycine. The problem is there is no peak for netilmicin-FMOC derivative or gentamicin-FMOC derivative. Now I can’t guess where it has gone?
Please help me for FMOC derivatization condition or any suggestion. Thanks anyway.
By Uwe Neue on Wednesday, March 24, 2004 - 03:17 pm:
I don't know what netilmycin is, but it sounds like a polypeptide. You may want to add a buffer to your mobile phase. Standard stuff would be 0.1% formic acid or ammonium formate, pH 3.7.
By Anonymous on Thursday, March 25, 2004 - 06:17 am:
Netilmicin (C21 H41 N5 O7) is one kind of aminoglycoside group. It has one benzene ring connected with two cyclohexane rings. There are 3 free -NH2 group in netilmicin.
Thanks Uwe for your advice.
Sittiporn
By Anonymous on Friday, March 26, 2004 - 12:35 am:
Is it possible that the excitation and emission
wavelengths have changed after derivatizaion?