Determination of alendronate using FMOC as derivatizing agen

[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.

[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.

[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.

[HW Mueller]

Did you first check the derivatization with an ideal standard solution? How did it look regarding sensitivity?

[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?