LC-MS—A Vital Tool for Food Safety Laboratories

---

Introduction
Food safety is a growing concern worldwide. Citizens expect their governments to ensure the food they eat is safe and wholesome. Food importers expect that the products they import will meet regulatory guidelines, and exporters know that they will lose markets if they don’t fulfill the same requirements. One important area of food safety is the detection and analysis of veterinary drug residues in animal products. Drugs, such as antibiotics, are administered to livestock for disease prevention and treatment, improved reproductive performance and growth promotion. These drugs should not be present in animal-based food products above regulated amounts known as Maximum Residue Levels (MRLs). High-performance analytical techniques such as liquid chromatography/mass spectrometry (LC-MS) and liquid chromatography/tandem mass spectrometry (LC-MS/MS) provide a means of accurately measuring these residues in agri-food products.
The Food Safety Division (FSD) of Alberta Agriculture, Food and Rural Development (a department of the Government of Alberta) is at the forefront of LC-MS/MS detection of veterinary drug residues in agri-food products. The FSD’s chemistry section provides science-based surveillance data on chemical residues found in Alberta agri-food products. FSD’s laboratories are accredited with the Standards Council of Canada to ISO 17025 guidelines.

Solution to an Analytical Problem
Analysis of chemical residues in food and agriculture products requires both sensitive and specific techniques. Techniques used must be sensitive enough to detect and quantify residues at — or preferably, below — the compound’s MRL in a given sample matrix. In order to ensure accurate quantitation at this action level, the FSD’s chemistry section aims for method detection limits that are lower than the MLR by a factor of 10.
Specificity is a parameter that describes the degree of certainty in the identity of a compound, and freedom of the method from interferences. These are obviously important factors when dealing with chemical residues in complex matrices such as food. Based on stringent sensitivity and specificity criteria, LC-MS/MS was selected as the analytical tool of choice for detection of veterinary drug residues.
The FSD chemistry lab has been using LC-MS technology since 1996 and LC-MS/MS since 2002. During this time, quantitative multi-residue methods that detect compounds from several different classes of drugs simultaneously have been developed. These methods require fewer sample preparation steps, often combining extraction, clean up and concentration. This translates into a significant reduction in turnaround time and reagent volume. In some instances, screening tests have been eliminated, with tandem mass spectrometry providing simultaneous screening and confirmatory sample analysis.
The analysis of raw milk for antibiotic residues serves as an example. There are more than 700 milk producers in the Alberta dairy industry, producing more than 50 million litres of milk each month. Specialized dairy tanker trucks, carrying approximately 28,000 litres per truck, collect the milk and deliver it to dairy plants. Upon delivery, every truckload is screened for drug residues. All dairy plants use an approved rapid screening procedure to test for beta-lactam antibiotics, and some plants also screen for sulfonamides.
In addition to the screening carried out by the plants, the FSD tests for a comprehensive range of antimicrobials at levels well below the MRLs. An LC-MS/MS method developed on a Waters® Alliance® LC and Quattro Ultima Pt™ mass spectrometer is used to simultaneously screen and confirm residues of 17 antibiotics in raw milk: nine sulfonamides, three tetracyclines, three macrolides and two lincosaminides (see Figure 1 for an example chromatogram). Sample preparation required for this method is minimal — only a simple combined de-proteination/extraction step is needed. Matrix standards are used for quantitation, and detection limits range from one to 20 ppb. Run time for this sensitive and specific method is 22 minutes from injection to injection.
LC coupled with tandem mass spectrometry has also proved beneficial for the analysis of antibiotics in honey. Honey producers use antibiotics to combat bacterial diseases that can infect and harm honeybees, such as American foulbrood (AFB), which is caused by the bacteria Paenibacillus larvae. In the past, honey producers successfully treated this disease with oxytetracycline (OTC). However, antibiotic-resistant strains of AFB have recently appeared in Alberta and worldwide. Although OTC is the only antibiotic registered for use against AFB in Canada, honey producers are now searching for other antibiotics to combat the resistant strain, and may resort to using sulfonamides, which were used in the past.
The FSD chemistry lab has developed an LC-MS/MS method for the automated analysis of sulfonamides in honey samples. On-line solid phase extraction (SPE) is incorporated in the analysis performed on a tandem MS. The hardware is configured to perform the SPE clean up automatically, with all steps handled by the system operating software. Diluted honey samples are injected onto an HLB on-line column, and a solvent gradient program is employed to separate analytes from the sample matrix. Using the built-in six-port valve on the MS, the bulk of the sample matrix is diverted to waste, and only components of interest are eluted into the MS for detection.
This method reduces sample handling, analyst workload and keeps the MS source clean. The run time is short (21 minutes, injection to injection) and the method is capable of detecting seven different sulfonamides at levels of 5 ppb. Since the SPE column exhibits enough chromatographic resolution to reduce sample background interference, as well as separate the analytes of interest, no analytical column is necessary (Figure 2). The longevity of the SPE column has proved to be very good, with many hundreds of samples processed and no significant change in retention behavior.

Conclusion
By using LC coupled with tandem mass spectrometry, the chemistry section of the FSD has been able to perform high-throughput antibiotic residue testing on a variety of agri-food sample matrices. Turnaround time has improved by using simultaneous screening and confirmation for multiple residues, allowing the elimination of screening tests. Staff can process more samples due to simplified sample preparation protocol. Without LC-MS/MS, it would not be possible to detect certain chemical residues at concentrations that are relevant in terms of food safety.

Jane Calvert is the research officer for the Agri-Food Laboratories Branch of the Food Safety Division, and Tom Thompson, PhD is an organic residue chemist. The chemistry laboratory, managed by Don Noot, is located in Edmonton, Alta.