Chromatography Forum

Hi
We are trying to analyse Ivermectin by fluoresence following derivitisation with TFAA in the presence of NMIM. The TFAA and ACN is kept on molecular sieve and the TFAA is added through the vial septum to minimise moisture interactions. We are using a C18 coloumn and 5% water in methanol.
The first problem we are finding is that the ivermectin peak does not always appear on the first injection but may appears on subsequent injections whilst a large peak at 2 mins (presumably the excess TFAA) will always appear???
Secondly large shifts in Rt occur day to day (12 mins to 15mins)???
Any help would be much appreciated
Thanks
Dr Mark Bateman
Coventry UNiversity

What are your sample types, premix, animal feed or tissue?

Hi
We are looking at cattle faeces with a lengthy extraction process following the only two published methods that we can find (both in Jou Agric Food Chem). The problem though has been with straight standards in attempts to get consistent HPLC peaks after the derivitisation reaction. Logical explanation fails me as there is no obvious source of error!
Cheers
Mark Bateman

Hi Mark!
I detemine ivermectin in milk and liver with the same analytical technique and way of derivatisation as you. My chromatographic conditions are: moblile phase:ACN: THF: H2O (96:3:1), flow rate: 0.8ml/min, column temp: 40 C, column: (Phenomenex) Luna 150x4.6, 3 um. Retention time of ivermectin is about 12 min and is very stable. The peak shape is perfect.
I add 200 ul of methanol to sample after derivatisation and I wait 15 min (some authors wrote about some reaction prodycts, which can give bad shepe of peaks)
About RT: I am affraid that you problem is your HPLC system or column.