This Method Helps to Identify Antibiotics in Vegetables

A study analyzing 60 samples of vegetables obtained from local markets in China, including cabbage, cucumber, cauliflower, leek, and other commonly consumed vegetables, found that a 33 percent of the samples contained detectable levels of antibiotics (Food Analytical Methods. 2018;11:2857–2864). The vegetables are likely to have absorbed the antibiotics from soil contaminated by antibiotics.

Antibiotics are still routinely added to animal feed to prevent or treat microbial infections, as well as promote animal growth in livestock production. Most (50 to 90 percent) antibiotics and their primary metabolites are rapidly excreted and ultimately end up in sewage and manure. Some of this is then spread on agricultural fields as fertilizer for growing crops. Vegetables elsewhere, including corn, potatoes, and lettuce, have also been found to contain antibiotic residues. Worryingly, there are currently no regulations to check and monitor for antibiotics in food products. Moreover, antibiotics have been detected in groundwater leading to concerns over their entry into food chain. Antibiotic residue levels should be monitored in fertilizer, the soil, and vegetables for risk assessment and control (Environ Pollut. 2006;143:565–571, Scientific American. January 2006).

Analysis

Despite efforts to curtail the use of antibiotics in the era of antibiotic-resistant microorganisms, antibiotics are still widely used to treat human and animal diseases. Antibiotic resistance poses a global threat to public health; antibiotic resistance is responsible for 25,000 annual deaths in the European Union and 23,000 annual deaths in the U.S. There are numerous causes of antibiotic resistance, including over-prescribing, patients not taking antibiotics as prescribed, poor infection control in hospitals, poor hygiene and sanitation practices, lack of rapid laboratory tests, and unnecessary antibiotic use in agriculture.

The analysis to detect the antibiotics in the vegetables used a novel highly sensitive method devised to detect 49 target antibiotics, which fall into different classes, including sulfonamides, quinolones, macrolides, beta-lactams, and tetracyclines. Of these 49 antibiotics, five were most commonly detected across 20 samples: oxytetracycline, doxycycline, sulfamethoxazole, enrofloxacin, and chlortetracycline.

The highest concentration was of oxytetracycline in cabbage, found to be 126 μg/kg and roughly 1% of the usual daily dose (1000 mg) for an adult. While this does not sound like much, it could become substantial if exposure is chronic. Oxytetracycline is a broad-spectrum antibiotic and is associated with gastrointestinal and skin-sensitivity side effects. It is contraindicated in pregnancy because it can cross the placenta and may have toxic effects on fetal tissues (Natl Health Stat Report. 2018;122:1–16). Although lower compared with the oxytetracycline, doxycycline, sulfamethoxazole, enrofloxacin, and chlortetracycline were also detected, at concentrations ranging between 2.0 and 12.8 μg/kg in the vegetables (Food Analytical Methods 2018;11:2857–2864).

Method for Detecting Antibiotics in Vegetables

The method used to detect and identify this wide range of antibiotics in vegetable samples is a relatively new one, involving the quick, easy, cheap, effective, rugged, and safe (so-called QuEChERS) procedure to prepare the sample for liquid chromatography and mass spectroscopic analysis using SCIEX ExionLC and QTRAP 4500 systems (Food Analytical Methods. 2018;11:2857–2864). The QuEChERS technique is a simple, rapid, and cost-efficient method of extracting and preparing the sample for liquid chromatography tandem mass spectrometry (LC-MS/MS) (Annal Chem. 2012;84(13):5677–5684). It requires less time and solvent than other methods to detect antibiotics, including solid-phase extraction (SPE) after ultrasonic, vortex, or vibration extraction. For the LC-MS/MS analysis of multiple antibiotic residues in different vegetable samples, the extraction timing and buffer system, dispersive solid-phase extraction (d-SPE) clean-up, and other parameters, such as those controlling for matrix effects, were also optimized (see Figure 1).

Along with the improved extraction procedure, the research team also optimized the LC-MS/MS technique. It is common practice to use LC to separate out the analytes in the sample, and then transfer them into a triple quadrupole-based mass spectrometer (triple-quad) to further separate and scan the discrete analytes using a multiple reaction monitoring (MRM). However, using the triple-quad approach to detect and identify multiclass antibiotics can result in type I errors (false positives) due to interferences that have MRM transition signatures that coincide with those of the antibiotics. Type II errors (false negatives) may also occur, should the antibiotic analyte be present at a very low concentration, thus producing a weak response in the second transition (Food Analytical Methods. 2018;11:2857–2864; Annal Chem. 2012;84(13):5677–5684). Therefore, the team used a quadrupole linear ion trap mass spectrometer, which combines the rapid, multiple scanning functionality of a triple-quad with the sensitivity of a linear ion trap mass spectrometer (Food Analytical Methods 2018;11:2857–2864; Annal Chem. 2007;79(24):9372–9384). With such an advanced hybrid system, the SCIEX QTRAP 4500, coupled with the SCIEX ExionLC ultra-high performance LC system, the team were able to develop and validate their method to simply and reliably detect and identify multiple antibiotic residues from different classes (Food Analytical Methods 2018;11:2857–2864).