How the Veterinary Drug Sulfamethazine is Measured in Animal Feeds.

An Inside Look at High Pressure Liquid Chromatography (HPLC)

Sulfamethazine, shown in the picture, is a synthetic antibiotic used widely in animal feeds to treat infections in farm animals. A trace of the drug in the meat of animals, easily detected by quick test kits used by inspectors, is enough to cause the animals to be rejected, meaning lost income for the farmer.

Occurrences such as these generally kick-off a follow-up investigation into the source of the drug. Feeds are sampled and analyzed to determine where the drug originated. In such cases, the laboratory is looking for traces of the drug where it is not supposed to be. We call this trace level or residue analysis.

Manufacturers of medicated feeds, including pharmaceutical firms, feed mills and farmers, periodically need to assure themselves that the correct level of sulfamethazine is present in their feed products. Typically, the feed or premix should contain within +/- 20% of the expected amount, according to current Canadian Food Inspection Agency (CFIA) guidelines. In our lab we refer to this type of test as a verification analysis.

In late 1999 the CFIA issued their new assay for sulfamethazine in feed products. The method is based on the use of High Pressure Liquid Chromatography; HPLC for short. In this article we explore how the test works.

Preparing the sample

We start by grinding the sample so that we can take a sub-sample (usually 5 grams) for analyses. We use a rotary grain grinder and mortar and pestle to transform the entire sample into a fine powder. An analytical balance is then used to weigh 5g of sample into a 250mL Erlenmeyer flask. The sub-sample is now ready to be extracted.

Extracting the sample

An extraction is how we separate the drug from the sample. First, an internal standard is added to all samples. This is a chemical very similar to sulfamethazine called sulfamerazine. Because of its chemical similarity, the internal standard behaves in the same fashion as sulfamethazine. It serves as an indicator of extraction efficiency and will correct the results should the extraction of sulfamethazine be poor.

Next, we add a mixture of methanol, water, diethylamine and hydrochloric acid. This solvent will extract the drug. The solution is put on a mechanical shaker for one hour and allowed to settle for 10 minutes. After settling, there will be a clear liquid at the top and grains at the bottom.

We carefully pour the supernatant into a 10mL glass syringe which is connected to a syringe filter. The filtrate, which contains the sulfamethazine from your sample along with the internal standard, is collected in a small vial. The filtered extract is now ready for analysis.

Analysis by HPLC

A small sample of the extract is now injected into a high pressure liquid chromatograph (HPLC). HPLC is an analytical instrument which causes the components of the sample to be separated and detected. The separation happens by a distribution of the sample components between two different types of material called phases.

One phase is a set of specially coated particles held in a steel column. The other phase is a solvent that is pumped at high pressure through the column. In this case, the solvent of mobile phase, is a mixture of acetonitrile and water containing acetic acid (much like vinegar).

As the sample extract travels through the column, the components will distribute differently between the liquid and the solid phases according to their chemical characteristics. For example, sulfamethazine and the internal standard sulfamerazine differ by only one carbon atom. Sulfamerazine is the smaller of the two. On the analytical column we use, sulfamerazine comes out first, followed soon after by sulfamethazine.

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