A Fast Quantitative Multi-analyte Method for Growth Promoters in Bovine Meat Using Bead-Disruption, 96-well SPE Clean-up and Narrow-Bore UHPLC-MS/MS Analysis

A new method for detecting low levels of growth promoters in bovine meat was developed with the following goal: easy, fast and sensitive analysis of a wide range of compounds, with reduced consumption of chemicals and disposables. Several classes of growth promoters were included, i.e. resorcylic acid lactones (RALs) and steroids, the latter including corticosteroids and gestagens. For sample treatment, 0.5 g of homogenised bovine meat was simultaneously disrupted and extracted in a bead-ruptor machine. The organic extraction solvent was further processed by solid-phase extraction (SPE) clean-up using 96-Well Oasis® HLB Plates. Six SPE washing steps were applied to remove matrix compounds after which the growth promoters were eluted and analysed using UHPLC-MS/MS. To achieve lower detection levels and to reduce LC-solvent consumption, a narrow-bore column with an internal diameter of 1 mm was used, instead of the conventional 2.1 mm. During analysis, the mass spectrometer was operated in negative and positive ionisation mode (ion switching). The newly developed method was validated according to the Commission Decision 2002/657. The results demonstrate that the method meets the criteria as established in this Commission Decision. The precision of the method for exogenous steroids varies between 85 and 115%, the CCα for the compounds ranges from 0.1–0.9 μg kg−1 and the expanded measurement uncertainty was lower than 36%. Compared to our current in-house methods with analysis times of 2 days for a maximum of 24 samples, the new method offers improved sample throughput (96 samples in less than 24 h) and lower detection limits

A Fast Quantitative Multi-analyte Method for Growth Promoters in Bovine Meat Using Bead-Disruption, 96-well SPE Clean-up and Narrow-Bore UHPLC-MS/MS Analysis

A new method for detecting low levels of growth promoters in bovine meat was developed with the following goal: easy, fast and sensitive analysis of a wide range of compounds, with reduced consumption of chemicals and disposables. Several classes of growth promoters were included, i.e. resorcylic acid lactones (RALs) and steroids, the latter including corticosteroids and gestagens. For sample treatment, 0.5 g of homogenised bovine meat was simultaneously disrupted and extracted in a bead-ruptor machine. The organic extraction solvent was further processed by solid-phase extraction (SPE) clean-up using 96-Well Oasis® HLB Plates. Six SPE washing steps were applied to remove matrix compounds after which the growth promoters were eluted and analysed using UHPLC-MS/MS. To achieve lower detection levels and to reduce LC-solvent consumption, a narrow-bore column with an internal diameter of 1 mm was used, instead of the conventional 2.1 mm. During analysis, the mass spectrometer was operated in negative and positive ionisation mode (ion switching). The newly developed method was validated according to the Commission Decision 2002/657. The results demonstrate that the method meets the criteria as established in this Commission Decision. The precision of the method for exogenous steroids varies between 85 and 115%, the CCα for the compounds ranges from 0.1–0.9 μg kg−1 and the expanded measurement uncertainty was lower than 36%. Compared to our current in-house methods with analysis times of 2 days for a maximum of 24 samples, the new method offers improved sample throughput (96 samples in less than 24 h) and lower detection limits

A global inter-laboratory study to assess acquisition modes for multi-compound confirmatory analysis of veterinary drugs using liquid chromatography coupled to triple quadrupole, time of flight and orbitrap mass spectrometry

According to EU legislation a confirmatory method used for residue analysis should be able to confirm the identity of a compound beyond reasonable doubt. To provide an adequate instrumental set-up, Commission Decision 2002/657/EC introduced the concept of "identification points". A second aspect to assure unequivocal confirmation, is the establishment of ion ratio and retention time criteria. Currently, the gold standard for confirmatory analysis of most veterinary drug residues is liquid chromatography (LC) coupled to tandem mass spectrometry (MS/MS) in selected reaction monitoring (SRM) acquisition mode, isolating one precursor ion and monitoring two a priori selected product ions, yielding 4 identification points. We comprehensively evaluated the use of different low and high resolution LC-MS(/MS) techniques and acquisition modes with respect to the selectivity of 100 veterinary drugs in liver, muscle and urine extracts aiming to critically review the currently established identification points system. A comparison among MS/MS in SRM mode with high resolution mass spectrometry (HRMS) in full scan, all ion fragmentation and targeted MS/MS was made based on a unique inter-laboratory study, which comprises 21 laboratories from four different continents and equipment from all major vendors.In total 186 samples were analysed yielding results for 9282 analyte/matrix combinations. It was observed that the false positive rate approximately doubles if no ion ratio criterion is applied indicating that this criterion is important to prevent false positive results. Full scan HRMS analysis, only monitoring the molecular ion and allowing a ±5 ppm mass tolerance is, in general, less selective than low resolution MS/MS using SRM, and thus full scan alone is considered not sufficient for confirmatory analysis. Furthermore, even though the number of data on all ion fragmentation and targeted MS/MS at high resolution was limited, based on the data obtained, it was observed that the acquisition mode as well as the mass resolution needed, very much depend on the matrix and the compound itself. For complex matrix extracts and non-selective compounds (worst-case situation), only targeted MS/MS, monitoring the precursor ion and a single product ion in HR-MS using a maximum of ±5 ppm mass deviation, leads to comparable selectivity and false positive and negative rate as SRM monitoring two product ions in LR-MS. We conclude that the currently applied identification point system as established in commission decision 2002/657/EC should be revised with respect to the allocation of identification points

A global inter-laboratory study to assess acquisition modes for multi-compound confirmatory analysis of veterinary drugs using liquid chromatography coupled to triple quadrupole, time of flight and orbitrap mass spectrometry

According to EU legislation a confirmatory method used for residue analysis should be able to confirm the identity of a compound beyond reasonable doubt. To provide an adequate instrumental set-up, Commission Decision 2002/657/EC introduced the concept of "identification points". A second aspect to assure unequivocal confirmation, is the establishment of ion ratio and retention time criteria. Currently, the gold standard for confirmatory analysis of most veterinary drug residues is liquid chromatography (LC) coupled to tandem mass spectrometry (MS/MS) in selected reaction monitoring (SRM) acquisition mode, isolating one precursor ion and monitoring two a priori selected product ions, yielding 4 identification points. We comprehensively evaluated the use of different low and high resolution LC-MS(/MS) techniques and acquisition modes with respect to the selectivity of 100 veterinary drugs in liver, muscle and urine extracts aiming to critically review the currently established identification points system. A comparison among MS/MS in SRM mode with high resolution mass spectrometry (HRMS) in full scan, all ion fragmentation and targeted MS/MS was made based on a unique inter-laboratory study, which comprises 21 laboratories from four different continents and equipment from all major vendors.In total 186 samples were analysed yielding results for 9282 analyte/matrix combinations. It was observed that the false positive rate approximately doubles if no ion ratio criterion is applied indicating that this criterion is important to prevent false positive results. Full scan HRMS analysis, only monitoring the molecular ion and allowing a ±5 ppm mass tolerance is, in general, less selective than low resolution MS/MS using SRM, and thus full scan alone is considered not sufficient for confirmatory analysis. Furthermore, even though the number of data on all ion fragmentation and targeted MS/MS at high resolution was limited, based on the data obtained, it was observed that the acquisition mode as well as the mass resolution needed, very much depend on the matrix and the compound itself. For complex matrix extracts and non-selective compounds (worst-case situation), only targeted MS/MS, monitoring the precursor ion and a single product ion in HR-MS using a maximum of ±5 ppm mass deviation, leads to comparable selectivity and false positive and negative rate as SRM monitoring two product ions in LR-MS. We conclude that the currently applied identification point system as established in commission decision 2002/657/EC should be revised with respect to the allocation of identification points.</p

European analytical criteria: past, present, and future

In this paper, the past, present, and (possible) future of the European analytical criteria for residues are described. The elaboration of the revision of Commission Decision 93/256/EC was a long process starting in 1996 and ending with the formation of a European Commission (EC) working group in 1998. This working group took account of developments in scientific and technical knowledge at that time and produced a draft version of the revision within 6 months. The revision, finally published in 2002 (2002/657/EC), includes new ideas on the identification of analytes and the criteria for performance assessment as well as validation procedures. Currently (2009), the evolution in analytical equipment and progress in scientific research, accompanied by recent European regulatory changes, demands an update or revision of the 2002/657/EC

Non-targeted workflow for identification of antimicrobial compounds in animal feed using bioassay-directed screening in combination with liquid chromatography-high resolution mass spectrometry

A non-targeted workflow is reported for the isolation and identification of antimicrobial active compounds using bioassay-directed screening and LC coupled to high-resolution MS. Suspect samples are extracted using a generic protocol and fractionated using two different LC conditions (A and B). The behaviour of the bioactive compound under these different conditions yields information about the physicochemical properties of the compound and introduces variations in co-eluting compounds in the fractions, which is essential for peak picking and identification. The fractions containing the active compound(s) obtained with conditions A and B are selected using a microbiological effect-based bioassay. The selected bioactive fractions from A and B are analysed using LC combined with high-resolution MS. Selection of relevant signals is automatically carried out by selecting all signals present in both bioactive fractions A and B, yielding tremendous data reduction. The method was assessed using two spiked feed samples and subsequently applied to two feed samples containing an unidentified compound showing microbial growth inhibition. In all cases, the identity of the compound causing microbiological inhibition was successfully confirmed

A critical assessment of the performance criteria in confirmatory analysis for veterinary drug residue analysis using mass spectrometric detection in selected reaction monitoring mode

Besides the identification point system to assure adequate set-up of instrumentation, European Commission Decision 2002/657/EC includes performance criteria regarding relative ion abundances in mass spectrometry and chromatographic retention time. In confirmatory analysis, the relative abundance of two product ions, acquired in selected reaction monitoring mode, the ion ratio should be within certain ranges for confirmation of the identity of a substance. The acceptable tolerance of the ion ratio varies with the relative abundance of the two product ions and for retention time, CD 2002/657/EC allows a tolerance of 5%. Because of rapid technical advances in analytical instruments and new approaches applied in the field of contaminant testing in food products (multi-compound and multi-class methods) a critical assessment of these criteria is justified. In this study a large number of representative, though challenging sample extracts were prepared, including muscle, urine, milk and liver, spiked with 100 registered and banned veterinary drugs at levels ranging from 0.5 to 100 µg/kg. These extracts were analysed using SRM mode using different chromatographic conditions and mass spectrometers from different vendors. In the initial study, robust data was collected using four different instrumental set-ups. Based on a unique and highly relevant data set, consisting of over 39 000 data points, the ion ratio and retention time criteria for applicability in confirmatory analysis were assessed. The outcomes were verified based on a collaborative trial including laboratories from all over the world. It was concluded that the ion ratio deviation is not related to the value of the ion ratio, but rather to the intensity of the lowest product ion. Therefore a fixed ion ratio deviation tolerance of 50% (relative) is proposed, which also is applicable for compounds present at sub-ppb levels or having poor ionisation efficiency. Furthermore, it was observed that retention time shifts, when using gradient elution, as is common practice nowadays, are mainly observed for early eluting compounds. Therefore a maximum retention time deviation of 0.2 min (absolute) is proposed. These findings should serve as input for discussions on the revision of currently applied criteria and the establishment of a new, globally accepted, criterion document for confirmatory analysis.</p

Evaluation of a loop-mediated isothermal amplification (LAMP) method for rapid on-site detection of horse meat

Detection of horse DNA by loop-mediated isothermal amplification (LAMP) seems one of the most promising methods to meet the criteria of fast, robust, cost efficient, specific, and sensitive on-site detection. In the present study an assessment of the specificity and sensitivity of the LAMP horse screening assay was made and outcomes were compared with the EURL-AP (European Union Reference laboratory for Animal Proteins in feeding stuffs) qPCR method. The specificity was tested with DNA samples from seven other species. The sensitivity of the LAMP assay was subsequently challenged with different percentages of horse DNA in cattle DNA and different percentages of horse meat in cattle meat. Both qPCR and LAMP were able to reliably detect horse DNA or meat below 0.1%, but LAMP was able to do so in less than 30 min. The DNA of other species did not result in a response in the LAMP horse assay. These features show that the LAMP method is fast, specific, and sensitive. Next, 69 processed meat samples were screened for the presence of horse DNA. The results showed that the LAMP horse assay, combined with a simple and fast on-site DNA extraction method, results in similar outcomes as the EURL-AP qPCR method and is thus a promising screening assay to be used outside the laboratory. Only samples that are screened on-site as suspect in the LAMP horse assay, need to be brought to the laboratory for confirmation with the more laborious EURL-AP qPCR reference method.</p

Evaluation of a loop-mediated isothermal amplification (LAMP) method for rapid on-site detection of horse meat

Detection of horse DNA by loop-mediated isothermal amplification (LAMP) seems one of the most promising methods to meet the criteria of fast, robust, cost efficient, specific, and sensitive on-site detection. In the present study an assessment of the specificity and sensitivity of the LAMP horse screening assay was made and outcomes were compared with the EURL-AP (European Union Reference laboratory for Animal Proteins in feeding stuffs) qPCR method. The specificity was tested with DNA samples from seven other species. The sensitivity of the LAMP assay was subsequently challenged with different percentages of horse DNA in cattle DNA and different percentages of horse meat in cattle meat. Both qPCR and LAMP were able to reliably detect horse DNA or meat below 0.1%, but LAMP was able to do so in less than 30 min. The DNA of other species did not result in a response in the LAMP horse assay. These features show that the LAMP method is fast, specific, and sensitive. Next, 69 processed meat samples were screened for the presence of horse DNA. The results showed that the LAMP horse assay, combined with a simple and fast on-site DNA extraction method, results in similar outcomes as the EURL-AP qPCR method and is thus a promising screening assay to be used outside the laboratory. Only samples that are screened on-site as suspect in the LAMP horse assay, need to be brought to the laboratory for confirmation with the more laborious EURL-AP qPCR reference method.</p