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Determination of alendronate using FMOC as derivatizing agen
Posted: Tue Oct 27, 2009 9:08 am
by csk1982
Hi, I am currently determining alendronate from human plasma using FMOC as derivatizing agent by HPLC/fluorescence. I am facing problem which my sensitivity is quite low. A concentration of 62.5ng/ml of alendronate only given area value ~20000. How to increase the sensitivity? I know FMOC is very pH dependent. I've ensure my final pH is ~12 before adding in the FMOC. I have increased my plasma volume from 1ml to 1.5ml. But still it didn't help a lot. Another thing is my result isn't reproducible. How to overcome this? Please advise. Thank you.
Posted: Sun Nov 01, 2009 2:18 am
by sassman
Can you purify the compound from the plasma before derivatizing? Precipitate proteins with acetonitrile or use ion exchange cleanup to isolate the analyte? Then you will get better derivatization efficiency.
Posted: Thu Nov 05, 2009 3:13 am
by csk1982
I've deproteinized my plasma with perchloric acid before proceed to ion exchange cleanup using DEA cartridge. Then finally only derivatized with FMOC. I am facing sensitivity problem. My sample preparation is similar to this paper, M.-H. Yun, K. Kwon / Journal of Pharmaceutical and Biomedical Analysis 40 (2006) 168–172.
Posted: Thu Nov 05, 2009 8:59 am
by HW Mueller
Did you first check the derivatization with an ideal standard solution? How did it look regarding sensitivity?
Posted: Thu Nov 05, 2009 9:13 am
by csk1982
Yes, I've done that and the sensitivity is good which resulted extraction recovery is poor. The result is not reproducible too. Which step in the sample preparation should I look into?