Development and performance evaluation of a microbiological method for screening and LC-MS/MS for conformation of sulfonamides in animal-derived foods.

This study developed a highly sensitive microbiological method utilizing a novel microtiter plate to screen 10 sulfonamides in chicken muscles, eggs, and prawns. This plate was fabricated from agar incorporating trimethoprim and spread with Bacillus megaterium. After residue detection by bioassay, the same test solutions were analyzed by LC-MS/MS for accurate identification and quantification. It also proved eco-friendly compared to using other quantitative methods. The residual drugs were extracted with McIlvaine buffer and purified using an Oasis® MCX cartridge. A triethylamine/methanol/water (0.5:75:24.5, v/v/v) mixture was used as the eluate. The obtained LOD values of the bioassay ranged from 5 to 25 µg kg-1 allowing the detection of the target drugs at the MRLs established in Japan. Adhering to ISO/IEC 17025 standards, the performance of the bioassay was evaluated. Based on the inhibition zone size in bioassay results, quality control yielded a Z score within ±2, indicating reasonable control over the screening process. Proficiency testing of a chicken muscle sample spiked with sulfadimidine demonstrated the inhibition zone detection of the bioassay and quantified value alignment of LC-MS/MS with reference values. In a surveillance study of 91 samples, sulfamethoxazole was detected in one prawn sample.

Monitoring of residual antibacterial agents in animal and fishery products in Tokyo from 2003 to 2019: application and verification of a screening strategy based on microbiological methods.

Residual antibacterial agents in 5909 animal and fishery products in Tokyo, Japan, were investigated over 17 consecutive years (2003-2019). Monitoring of 32 antibacterial agents (lincosamides, macrolides, penicillins, quinorones and tetracyclines) per product was accomplished via two steps: screening (by microbiological methods) and confirmation (by instrumental methods). Microbiological screening methods identified presumptive groups and determined semi-quantitative values. The instrumental methods quantified 81 residues of 11 different antibacterial agents in 72 samples. The screening strategy based on microbiological methods demonstrated the following: (i) the majority of the samples (over 99%) met Japanese regulations, (ii) using multiple methods provided a reliable inspection system with accurate quantitative values and (iii) there was a constant presence of tetracyclines and unexpected residues (lincomycin and norfloxacin) in various products. Thus, this long-term monitoring and screening strategy provided evidence that the frequencies and trends of residual antibacterial agents not only enhance food safety but also help to prevent antimicrobial resistance.

[Determination of Antibacterial Agents for Animals in Swine Muscles by Microbiological Screening and LC-MS/MS].

The determination of antibacterial agents for animals in swine muscles was improved by microbiological screening and liquid chromatography-mass spectrometry (LC-MS/MS) analyses. In the first instance, the residual drugs were extracted from the samples using the Na2EDTA-McIlvaine buffer (pH 6.0). Subsequently, the agents were purified utilizing a PLS-3 cartridge and extracted with acetonitrile. Considering the microbiological methods, the sensitivities of the investigated drugs were higher and the test plate conditions were improved using a new reference organism Geobacillus stearothermophilus. As a result, a microbiological screening approach able to detect 33 drugs at MRL was developed in Japan. Remarkable, no false positives were detected. Moreover, the same preparation method enabled rapid and reliable microbiological screening, resulting in efficient screening with no undeterminable results.

Development of an alternative approach for detecting botulinum neurotoxin type A in honey: Analysis of non-toxic peptides with a reference labelled protein via liquid chromatography-tandem mass spectrometry.

In this study, we developed a reference labelled protein containing the partial amino acid sequence of botulinum neurotoxin type A (BoNTA). We also applied it as an internal standard to detect specific and non-toxic peptides originated from BoNTA in honey with the use of liquid chromatography-tandem mass spectrometry (LC-MS/MS). Original proteins in the honey sample were collected through a two-step process that included solubilisation and trichloroacetic acid (TCA) precipitation. Solubilisation by adding water enabled processing of proteins in honey. TCA precipitation collected proteins without specific binding. The combination of protein alkylation and an appropriate enzyme-to-protein ratio ensured feasibility of tryptic digestion. A desalting process eliminated a large amount of salts and other tryptic peptides in the honey sample. The use of the reference labelled protein enabled compensation for tryptic digestion efficiency and electrospray ionisation efficiency based on LC-MS/MS measurement. After the peptide selection and protein BlastP analysis, five unique peptides were chosen. The non-toxic peptides originating from BoNTA were reliably detected using LC-MS/MS based on a multiple-reaction monitoring mode. Detection of several peptides ensured screening of BoNTA in honey samples. Based on the responses, the proteotypic peptide LYGIAINPNR was selected as the quantitative peptide. Due to maintaining the relative ion ratios, the selective transition completely identified the non-toxic peptides. The intensity of the transitions established a detection limit of BoNTA estimated to be 9.4 ng mL-1. Although extraction efficiency was not evaluated using the BoNTA standard, the results suggested this method may be used for quantification of BoNTA in honey. The method was applied to 19 honey samples purchased in Tokyo; none of them was found to contain the target toxin. Overall, the method is expected to accelerate BoNTA monitoring for food safety.

[Determination of Antifungal Drugs in Fish and Livestock Products Using LC-MS/MS].

We developed an analytical method for determining 15 antifungal drugs, 2 antiparasitic drugs, and 3 veterinary drugs in fish and livestock products using LC-MS/MS. First, 50% ethanol was added to their products, and the mixture was homogenized to reduce drug degradation. Thereafter, 20 drugs were extracted from the pretreated sample mixture using acetonitrile. Cleanup was performed using an alumina-N SPE cartridge. Finally, chromatographic separation was performed using a fully porous octadecyl silanized silica column. The new method is applicable to fish in which the matrix hampers accurate analysis. It was validated on 8 fish and livestock products. Drug recovery rates ranged from 70.2 to 109.3%, RSDs of repeatability were <18.0%, and RSDs of within-laboratory reproducibility were <18.7%. It fulfills the Japanese guideline criteria. The limits of quantification were estimated as 3 ng/g.

Quantification of staphylococcal enterotoxin type A in cow milk by using a stable isotope-labelled peptide via liquid chromatography-tandem mass spectrometry.

In this study, the staphylococcal enterotoxin type A (SEA) contaminant was quantified in cow milk by liquid chromatography-tandem mass spectrometry (LC-MS/MS) with the use of a stable isotope-labelled peptide of SEA as an internal standard. SEA was cleaned up in a two-step process that included pH control and trichloroacetic acid (TCA) precipitation. The pH control phase eliminated other proteins. TCA precipitation cleaned up SEA without special equipment. An appropriate enzyme-to-protein ratio maximised tryptic digestion. A desalting process guaranteed the stable retention of SEA-digested peptides. The coverage of amino-acid sequences (>10%) clearly identified the toxin's presence. SEA was accurately quantified using LC-MS/MS based on a multiple-reaction monitoring mode. The developed method was validated based on spiked recovery tests at 50 and 100 µg kg-1 conducted with two samples collected on a daily basis for five days based on Japanese validation guidelines. The new method exhibited good accuracy which ranged from 80% to 82%. The relative standard deviations of repeatability were 13-14% and the relative standard deviations of within-laboratory reproducibility were 13-18%. These standard deviations satisfied the criteria of the Japanese validation guidelines. The quantification limit was estimated to be 10 µg kg-1.

Simultaneous determination of nine acaricides and two metabolites in comb honey by LC/MS/MS.

We developed a method for the simultaneous determination of acaricides in comb honey using LC/MS/MS. Because methods for honey analysis had not previously been applied to comb honey, we modified three techniques for sample preparation and LC/MS/MS conditions. First, we used a modified QuEChERS method that changed the extraction solution from ethyl acetate to acetonitrile. Second, we replaced the InertSep® MA-1 (30 mg, 1 ml) clean-up cartridge with an Oasis® HLB (60 mg, 3 ml). Third, we changed the ionisation mode from ESI to atmospheric pressure chemical ionisation (APCI). With these modifications, sample matrices had no effect on the identification and quantification of analytes, using an external solvent calibration curve. We verified this new method with nine acaricides and two metabolites on comb honey and honey samples from three different honey origins. The trueness ranged from 74.0 to 99.4%. The relative standard deviation of repeatability (RSDr) ranged from 0.8 to 14.8% and that of within-laboratory reproducibility (RSDWR) ranged from 1.3 to 14.8%. All criteria met Japanese validation guidelines. The LOQ was 1.0 µg kg-1 for all analytes. We applied this method to 10 comb honey and 31 honey samples commercially available in Tokyo. From the results of the analysis of 41 samples, we observed that amitraz remained as N-(2,4-dimethylphenyl)-N-methylformamidine (DMPF) in 9 comb honey and 23 honey samples and that their residual concentrations were less than 20 µg kg-1. Using this new method, we improved recovery and precision, which enabled precise quantitative determination. Furthermore, the residual amitraz value in honey determined by both this new and the previous method were in good agreement.

Identification and quantification of cereulide in cow's milk using liquid chromatography-tandem mass spectrometry.

In this study, the presence of cereulide in cow's milk was identified and quantified using our validated method with liquid chromatography-tandem mass spectrometry. Cereulide was concentrated using protein acid-precipitation and extracted from the precipitate by using acetonitrile twice. The combination of protein acid-precipitation and extraction sufficiently eliminated the matrix compounds from the milk and a further clean-up step utilising solid-phase extraction could be omitted. For robustly measuring the samples and keeping the MS devices clean, the extraction solution was diluted 10-fold using methanol. Owing to the minimisation of the interferences caused by fragmentation patterns, multiple reaction monitoring information-dependent acquisition-enhanced product ion spectra enabled the characterisation and identification of cereulide. Besides the matrix effect (-4%), an external solvent calibration curve was adapted for accurate quantification. The method was validated using fortified recovery tests, at two concentrations (10 and 50 µg kg-1), using three samples daily on five different days based on the Japanese guidelines. This new method exhibited good accuracy ranging from 91% to 94%. The relative standard deviations of repeatability ranged from 2% to 5%, and the relative standard deviation of within-laboratory reproducibility ranged from 5% to 6%. These standard deviations satisfied the criteria for the Japanese validation guidelines. The limit of quantification (LOQ) was estimated to be 2 µg kg-1. On the product ion spectra at the LOQ level, the library match was satisfactory with a purity fit value of >70%. The method was applied to 14 raw milk and three milk samples pasteurised using the low-temperature, long-time process and collected in Tokyo. None of the samples was found to contain the target toxin.

[Determination of Cyromazine in Livestock Products by LC-MS/MS on an Anion-Cation Exchange Mode ODS Column].

Cyromazine in livestock products was determined using a validated LC-MS/MS method. There are three key points in our methods. First, the extraction was performed with two solutions, methanol and pH 3.0 McIlvaine buffer. The process was optimized for each type of sample. Secondly, cleanup was performed using a reversed-phase and strong cation exchange mixed-mode cartridge. The cartridge was washed with 0.14％ ammonium solution. Thirdly, the chromatographic separation was performed on an anion-cation exchange mode ODS column. There was no matrix effect on the extraction and determination for five livestock products. The quantification was carried out using an external standard calibration curve. This new method satisfies the Japanese guideline criteria. Recovery ranged from 77.2 to 92.1％, the relative standard deviation of repeatability （RSDr） was under 2.2％, and RSDwr was under 6.1％. Residual cyromazine was detected in raw milks and eggs.

Simultaneous determination of multi-class veterinary drugs in chicken processed foods and muscle using solid-supported liquid extraction clean-up.

We developed a simultaneous determination method for 37 veterinary drugs in two chicken processed foods (deep-fried chicken and non-fried chicken cutlet) and muscle via liquid chromatography-mass spectrometry. The veterinary drugs belong to 7 different classes, including 4 antifolics, 4 benzimidazoles, 5 macrolides, 7 polyethers, 2 quinolones, 7 sulfonamides, and 8 other classes. The samples were extracted with ethyl acetate followed by acetonitrile with salt and buffers extraction. The two-step extraction enabled analyte extraction from highly lipid samples. The clean-up procedure, a solid-supported liquid extraction clean-up using a diatomaceous earth mini-cartridge, eliminated lipid co-extraction. The prepared sample matrix did not have an effect on the 36 analytes. The method was validated in accordance with the requirements of Japanese validation guidelines. Almost all targeted veterinary drugs successfully satisfied the guideline criteria in the three types of food matrices. The method exhibited recoveries of 70-105%, and the precision of repeatability and within-laboratory reproducibility ranged from 1 to 11% and 1 to 15%, respectively. The limits of quantification were estimated to range from 0.2 to 1.0µg/kg. Applying this method to samples commercially available in Tokyo, residues were detected in 3 out of 26 deep-fried chickens, 5 out of 20 non-fried chicken cutlets, and 17 out of 39 chicken muscles.

Simultaneous determination of amantadine, rimantadine, and memantine in processed products, chicken tissues, and eggs by liquid chromatography with tandem mass spectrometry.

A simultaneous determination of amantadine, rimantadine, and memantine in processed products (deep-fried chicken, fried chicken, fried quail egg, and grilled chicken) with liquid chromatography tandem mass spectrometry (LC-MS/MS) was developed. This new method was also applicable for chicken tissue (muscle, liver, and gizzard) and eggs. The chromatographic separation was performed on a Kinetex® XB-C18 core-shell technology column using a mobile phase of acetonitrile and 0.1% formic acid in a 10mmol/L ammonium formate solution, resulting in the complete separation of isomers (rimantadine and memantine) and any other obstructive peaks from the sample matrices. Sample preparation was performed by a modified QuEChERS method using acetonitrile and a 0.1% acetic acid extraction solution and cleaned using an Oasis® MCX cartridge. The sample matrix had no effect on the identification of the compounds. For quantification, an external solvent calibration curve was used. This new method exhibited good accuracy ranging from 79.9% to 91.5%. The relative standard deviation of repeatability (RSDr) ranged from 1.2% to 3.6% and the relative standard deviation of within-laboratory reproducibility (RSDWR) ranged from 1.3% to 6.0%. These standard deviations satisfied the criteria for Japanese validation guidelines. The limit of quantification (LOQ) was 1.0µg/kg for all samples. Analyte residues were not detected in 55 samples using the validated method.