Question

Fenbendazole is a weakly basic, hydrophobic drug used to treat cattle. The detection and determination of residual fenbendazole in milk and milk products is therefore important. Since milk and milk products cannot be injected directly into a chromatograph, various liquid-liquid extraction (LLE) procedures were investigated using spiked buffer solutions and milk samples. The table below shows the %recovery obtained when performing LLE on various samples at different pH values with either dichloromethane or ethyl acetate as the extracting organic solvent:

	Dichloromethane	Ethyl acetate
pH	Buffer Milk	Buffer Milk
4.5	98 55	99 65
6.5	100 57	101 60
8.0	99 60	97 57
10.0	101 62	100 70

(a) Assuming equal volumes of sample and solvent, and a single extraction step, calculate the value of Dm (DIffusion Coefficient in mobile phase) for fenbendazole between:

(i) buffer at pH 8.0 and dichloromethane

(ii) milk and dichloromethane

(iii) buffer at pH 8.0 and ethyl acetate, and

(iv) milk and ethyl acetate.

(b) Draw the structure of fenbendazole. What is the likely reason that the recovery of fenbendazole from spiked milk samples is systematically lower than that from aqueous buffer samples?

(c) In subsequent studies, it was found that acetonitrile was a good solvent for the initial extraction, since it did not require the sample to be buffered, but co-extracted lipids (i.e. fat) had to be removed prior to analysis of the acetonitrile–aqueous sample extract. Suggest a solvent that would be suitable for removing the lipid content of this extract, and explain your choice.

(d) In the final method, the fenbendazole extract was chromatographed on a reverse-phase C18 column using a mobile phase consisting of 50% acetonitrile and 50% 0.01 M phosphoric acid, with the addition of octanesulphonic acid (10 mM) to act as an ion-pairing reagent. What is an ion-pairing reagent, and why might it have been necessary for this sample?