Dairy cows are regularly given antibiotics to protect against a series of bacterial infections such as mastitis, chronic bronchitis and pneumonia. One of the most common products is the sulphonamide sulfamethoxazole which is often applied in a 5:1 combination with trimethoprim to increase the overall antibiotic activity. Within this medication, known as co-trimoxazole or SMX-TMP, each drug inhibits a different step in the tetrahydrofolate synthetic pathway, to break the supply of folic acid to bacteria.

As with many animal drugs, there are always fears that some residues might remain in the animal. For dairy cows, the principal concern is tainting of the milk which would lead to inadvertent human consumption of the two compounds. Good analytical methods for measuring the concentrations of SMX and TMP in milk already exist, some using HPLC generally in conjunction with UV-vis detection. Included in these successful applications are some that use restricted access media (RAM) for online cleanup before direction to the analytical column.

Scientists in Brazil have now broadened the methods by using two-dimensional HPLC, with RAM and analytical columns in series, in conjunction with electrochemical detection. Quezia Cass and colleagues from the Federal University of Sao Carlos were attracted by the high selectivity and sensitivity that electrochemical detectors can bring.

The sensitivity can be modified by changing the ionic strength and pH of the electrolyte as well as the buffer type. However, use of this type of electrode is not without its drawbacks. The oxidation/reduction reactions taking place in solution can attack the electrode surface as well as bring about deactivation by the adsorption of oxidation/reduction products.

The researchers decided to evaluate a novel kind of electrode, the boron-doped diamond electrode (BDD), which has been reported to withstand deactivation during electrochemical detection of several compounds, including sulphonamides. They used a lab.-built BDD electrode to detect and measure SMX-TMP in spiked bovine milk samples. The electrochemical cell was fitted with a stainless steel counter electrode, a silver/silver chloride reference electrode and the BDD electrode which has an 8000 ppm B-in-diamond film.

Skimmed cow's milk was centrifuged to give a thin fatty upper layer, a middle aqueous layer and a small cell pellet as the lower layer. This was the only sample preparation step. The middle layer was injected into the 2D HPLC system onto an immobilised bovine serum albumin RAM column to trap the analytes and elute the milk proteins to waste with a solution of potassium dihydrogen phosphate in acetonitrile. As this step was being carried out, the analytical column was conditioned.

The analytes were then eluted from the column and transferred to the analytical octylsilica column and eluted with potassium dihydrogen phosphate in acetonitrile to the detector. During elution from the analytical column, the RAM column was cleaned and conditioned.

Using a UV-vis detector, it was confirmed that the milk proteins were eluted from the RAM column within five minutes, followed by the two drugs in a narrow band after roughly 12 minutes. Subsequent separation on the analytical column proved to be interference-free with good separation between TMP and SMX.

The optimum BDD electrode potential, inferred from hydrodynamic voltammograms of the analytes in aqueous solution, was 1.25 V vs. the silver/silver chloride electrode and the resultant chromatograms confirmed that the drugs were well separated.

No fouling of the analytical column or the BDD electrode was observed with over 100 sample injections totalling more than 20 mL during several hours of experimentation.

Under these conditions, recoveries were 98-105%, linear concentration ranges were 50-800 and 25-400 µg/L and the other analytical data were all acceptable. The detection limits were 25.0 and 15.0 µg/L for SMX and TMP, respectively, which are lower than the maximum residue limits set within the USA and Europe.

This is one of only a few reported 2D LC methods that use electrochemical detection and the first, say the researchers, involving the analysis of SMX-TMP in milk. The minimal sample preparation renders the method amenable to automation and its good performance and low electrode fouling capacity suggest that it could be useful for the routine analysis of bovine milk.

Related links:

• Analytica Chimica Acta 2009 (Article in Press): "A multidimensional high performance liquid chromatography method coupled with amperometric detection using a boron-doped diamond electrode for the simultaneous determination of sulfamethoxazole and trimethoprim in bovine milk"

Article by Steve Down

The views represented in this article are solely those of the author and do not necessarily represent those of John Wiley and Sons, Ltd.