How is the drug detected?

The new CFIA method employs a neat trick to detect the drug. Its called post-column reaction. Here's how it works:

As we discussed about, the drug, internal standard and other components of the sample are separated on the HPLC column. Once they come through the column they are exposed to a coloured chemical that reacts with sulfamethazine, sulfamerazine and other related sulfa drugs such as sulfathiazole. Generally, most other components of the sample don't react and are invisible to the detector.

The coloured material passes through an ultraviolet detector. The detector shines light having a wavelength of 450nm (blue in colour) through a photocell, through which the sample passes. Any material that has reacted with the coloured material will absorb the light. Photodiodes register the loss of light and convert it into an electrical signal. These changes in light intensity are recorded as a series of peaks called a chromatogram.

Connected to the detector are an integrator and a printer. The printer record the chromatogram. The integrator calculates the area under each peak and the time each component leaves the HPLC column (also know as the retention time).

How do we know which peak is sulfamethazine?

We use pure standards, purchased from a reputable chemical supplier, which we dilute at different concentrations. These standards are injected into the HPLC for comparison with the actual samples.

How do we know how much sulfamethazine is in your sample?

Using the pure standards, we perform a calibration to convert from peak area to the actual amount of sulfamethazine in your sample. From the standards, a graph of concentration versus area is produced.

By comparing the ratio of drug peak area to internal standard peak area in your sample the ratio for the standards used to prepare the calibration graph, we can calculate how much drug is in your sample.

How good are these tests?

The new CFIA method can detect as low as 5ppm in feed and up to high percent levels in premixes. We use another HPLC method to see below 5ppm for trace level analysis.

The result obtained by the CFIA method is usually within +/- 5% of the true value. We check this each time we analyze a batch of samples by including a feed sample which contains a known amount of sulfamethazine. We also include a feed sample which doesn't contain the drug, called a blank. If the test is working properly, no sulfamethazine should be detected in the blank.

How quickly can the test be done?

Although HPLC tests can often be done the same day, they generally need 2-5 days.

How do we know that the test is actually measuring the right drug?

This is an important question. With HPLC analyses, there is generally little doubt about the identity of the drug. To be detected by the CFIA method, the drug must elute from the HPLC at the expected time and react with the post column reagent. There aren't many other molecules that will do that. In trace level work, though, a positive HPLC result can easily be cross checked using a different HPLC test. There are also many other ways to confirm the identity by HPLC.

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Click here for a listing of the veterinary drugs ISI can test for.