Staying one jump ahead: Screening antibiotics in frog legs by accurate mass spectrometry

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  • Published: Jun 1, 2012
  • Author: Steve Down
  • Channels: Base Peak
thumbnail image: Staying one jump ahead: Screening antibiotics in frog legs by accurate mass spectrometry

Fast food

Veterinary antibiotics have been detected in frog legs and other aquacultured fish by an LC/MS method using high resolution mass spectrometry, confirming that most frog legs imported into the US have residues above regulatory action levels.

Frog legs are associated with France but you might be surprised to know that the vast majority of them are farmed in the Far East and exported to European countries, including France, as well as the USA. Unfortunately for the importing countries, the industry is not particularly well controlled. Salmonella contamination of the imported food is a particular problem along with the residual presence of veterinary antibiotics above acceptable levels.

In the USA, this has been taken so seriously that the FDA has issued two Import Alerts, allowing consignments of frog legs and aquacultured products from certain Asian countries to be "automatically detained by FDA without the added step of FDA conducting an inspection, examination or sampling of the product."

Unacceptable levels of several antibiotics, including some fluoroquinolones and quinolones, have been detected by several analytical methods, including HPLC with fluorescence detection, and LC/MS. However, scientists from the FDA in Denver, CO, think that these methods are limited by the fact that they look for a particular set of compounds. What would be better, they say, is a broader method that will find any contaminants present without specifying them first.

So, this FDA team, led by Sherri Turnipseed from the Animal Drugs Research Center, took a look at an LC/MS method incorporating a hybrid quadrupole-time-of-flight mass spectrometer. With this high-resolution instrument, data can be collected in full-scan mode at very high accuracy, so that compounds can be identified from the accurate mass measurements of their molecular and fragment ions by searching a database that also contains the retention times for further confirmation.

Accurate masses

In the first instance, several veterinary drugs were selected as target analytes based on their regular occurrence in frog legs and their known use in aquaculture in general. These comprised two fluoroquinolones (ciprofloxacin and enrofloxacin), three quinolones (oxolinic acid, nakidixic acid and flumequine), trimethoprim, chloramphenicol and sulfamethoxazole.

They were added in known amounts to the homogenised flesh of frog legs that had been purchased in a local store. Extraction with a mixture of aqueous acetic acid, acetonitrile and sodium chloride led to an organic extract that was cleaned up with a molecular weight cutoff filter to remove proteins before analysis.

The final extracts were analysed by LC/MS with electrospray ionisation in positive mode for all of the target analytes except chloramphenicol, which was detected in negative ion mode. The accurate protonated or deprotonated molecular ions were searched against an in-house database of more than 200 veterinary drugs for identification and the abundances of the peaks were used to estimate the concentrations.

These drugs are not approved for use in aquaculture in the USA, so there are no established tolerance levels. The researchers set “levels of interest” which were based on the US action levels of 1 ng/g for chloramphenicol, 5 ng/g for the fluoroquinolones and 10 ng/g for the remainder.

The method detection limits were in the range 0.2-3.9 ng/g, all of the values being below the level of interest for the particular drug. Tandem mass spectrometry was also used to identify the compounds from some of the fragment ions.

All frog legs contaminated

All of the target drugs were detected in fortified frog legs at the levels of interest, as well as at concentrations 50% below those values, demonstrating the sensitivity of the procedure. There were some interferences from co-extracted components that caused ion suppression and other effects, especially for sulfamethoxazole, chloramphenicol and enrofloxacin. However, the main purpose of the project was to screen for these drugs rather than measure them, so the researchers did not attempt to improve the extraction procedure as it might lead to the elimination of other drugs from the extract.

When the optimised method was applied to imported frog legs, the two fluoroquinolones were detected frequently at levels up to 40 ng/g, the values agreeing with those determined by HPLC with fluorescence detection. However, more than one drug was found in all of the 11 samples tested.

The most common finding was trimethoprim which was found in all of the samples at levels up to 98 ng/g, way above the level of interest. This drug is often added in combination with sulphonamides like sulfamethoxazole to increase their effectiveness. Sulfamethoxazole itself was found in only three samples at levels which were generally lower than those of trimethoprim.

Chloramphenicol was found in four samples at up to 80 ng/g. This drug can cause aplastic anaemia, so the scientific community has agreed that it should be detectable below ng/g levels in food. The detection limit in this new method is 0.2 ng/g.

The researchers said that “it is uncommon to detect residues from different chemical classes in the same sample. This underscores the importance of broad spectrum residue monitoring, as the indiscriminate use of chemotherapeutics in the farming of frogs is clearly evident.”

All of the antibiotics were also detected in other types of aquacultured fish like shrimp, tilapia and catfish that were purchased from the store.

The high-resolution mass spectrometry method was also used to look for non-target analytes, finding several other drugs like norfloxacin, crystal violet and florfenicol. They were confirmed from their retention times and MS/MS spectra. Further compounds were found by comparing the data to the in-house database and to a commercial database of more than 6000 compounds.

"The presence of these additional drug residues, in addition to the fluoroquinolone residues targeted in the established method, would not have been detected with a MS procedure that limits data acquisition," said Turnipseed, emphasing the importance of the full scan method.

Related Links

Journal of Agricultural and Food Chemistry 2012 (Article in Press): "Analysis of veterinary drug residues in frog legs and other aquacultured species using liquid chromatography quadrupole time-of-flight mass spectrometry"

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.

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