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Nitrofuran residues in food, particularly intensively farmed prawns, were identified as an important trade and health issue. No certified reference materials were available to allow laboratories to demonstrate satisfactory performance of their analytical methods. Freeze-dried prawn reference materials MX012A and MX012B were prepared at the National Measurement Institute, Australia, from blank and contaminated prawn tissue. A high-accuracy, exact-matching isotope dilution mass spectrometry method was developed for the analysis of two nitrofuran metabolites in the prawn materials. Residues were extracted by acid hydrolysis and derivatisation with 2-nitrobenzaldehyde, extracts were purified by liquid–liquid extraction followed by solid-phase extraction, and instrumental analysis was by ultra-high-pressure liquid chromatography with electrospray tandem mass spectrometry (UHPLC-ESI-MSMS). The method was used to certify the reference materials for mass fraction of 3-amino-2-oxazolidinone (AOZ) and semicarbazide (SEM), including the preparation of detailed measurement uncertainty budgets. MX012A contains AOZ at (137.5 ± 8.5) ng/g of freeze-dried material, and MX012B contains AOZ at (30.2 ± 1.8) ng/g and SEM (fortified) at (70.3 ± 3.1) ng/g. When reconstituted as directed on the certificate, the materials contain AOZ at (20.6 ± 1.3) ng/g and (4.53 ± 0.27) ng/g (MX012A and MX012B, respectively) and SEM at (10.5 ± 0.5) ng/g (MX012B). The individual components contributing to the measurement uncertainty estimates were the mass fractions assigned to calibration standards, gravimetric mass measurements, precision of the analytical method, reproducibility, long-term storage stability of the material at −20 °C, stability of the material during transportation, and potential bias due to extraction efficiency of the incurred analyte and matrix interference.

Most methods reported in the literature for determination of nitrofuran metabolites use the same extraction and derivatisation conditions to hydrolyse and extract the protein-bound residues from animal tissue. While undertaking certification of reference materials for nitrofuran metabolites in freeze-dried prawn, it was found that these conditions are satisfactory for recovery of spiked residues; however, extraction efficiencies of incurred furazolidinone could be substantially increased by further optimisation of the extraction conditions. The availability of a suitable certified reference material allows laboratories to ensure that their method is optimised for incurred residues.

Within the Organic Analysis Working Group (OAWG) of the Comité Consultatif pour la Quantitè de Matiére (CCQM), key comparison CCQM-K141 and associated pilot study CCQM-P178 were coordinated by the National Research Council Canada (NRC). This comparison was a Track A key comparison that formed part of the OAWG 10-year strategic plan. The comparison demonstrated capabilities for measuring high-polarity analytes in a high-fat and high-protein matrix. The measurand chosen as the model system was enrofloxacin and sulfadiazine in bovine tissue. Thirteen National Metrology Institutes or Designated Institutes participated in the CCQM-K141, while two National Metrology Institutes participated in CCQM-P178. The bovine muscle tissue study material was derived from a single live animal that was administered with chemical based pharmaceutical agents prior to processing. Therefore, the study material was naturally incurred, providing a true test of extraction procedures relative to more commonly encountered spiked materials. NRC confirmed excellent homogeneity and stability of the material prior to shipping. Three 10 g bottles of freeze dried powdered muscle tissue were supplied. NRC also provided isotopically labelled solutions of the two measurands, 13C6-sulfadiazine and enrofloxacin-d5 (HI Salt), to those interested in using isotope dilution mass spectrometry (IDMS) methodologies. Procurement and purity assignment with appropriate metrological traceability of native calibrants were the responsibility of individual participants. The study required extraction, clean-up, analytical separation, and selective detection of the analytes. The level of agreement was reasonable given the measurands and matrix were new for most laboratories. The KCRV values and their uncertainties at the 95% confidence level of 57.81 ± 2.57 μg/kg for enrofloxacin and 2285 ± 68 μg/kg for sulfadiazine were calculated using the DSL means. While one participant's value was voluntarily excluded from the KCRV calculations for enrofloxacin, all other participants demonstrated equivalence for both measurands. Significant effort was undertaken post-study to identify the major sources of variability between results. In particular, the various extraction conditions used by participants were investigated thoroughly. While there appeared to be a correlation between highly acidic conditions and higher recovery, this was not definitive and could not be confirmed. The form of standards employed (i.e. free base vs salts) and potential differential solubility between forms was also a suspected source of variability. Biases could also have been introduced with the choice of solvents used for standard preparation, with some solvents better able to minimize adsorption of the analytes to glass surfaces. KEY WORDS FOR SEARCH enrofloxacin, sulfadiazine, bovine tissue, incurred, key comparison Main text To reach the main text of this paper, click on Final Report. Note that this text is that which appears in Appendix B of the BIPM key comparison database kcdb.bipm.org/. The final report has been peer-reviewed and approved for publication by the CCQM, according to the provisions of the CIPM Mutual Recognition Arrangement (CIPM MRA).

A reversed-phase high-performance liquid chromatographic method with tandem mass-spectrometric detection was developed and validated for the simultaneous analysis of eight quinolones and fluoroquinolones (oxolinic acid, flumequine, piromidic acid, enrofloxacin, ciprofloxacin, danofloxacin, sarafloxacin and orbifloxacin) in trout tissue, prawns and abalone. The analytes were extracted from homogenised tissue using acetonitrile and the extracts subjected to an automated two-stage solid-phase extraction process involving polymeric reversed-phase and anion-exchange cartridges. Good recoveries were obtained for all analytes and the limit of quantification was 5 microg/kg (10 microg/kg for ciprofloxacin). The limit of detection was 1-3 microg/kg, depending on the analyte and matrix. Confirmation of the identity of a residue was achieved by further tandem mass-spectrometric analysis. A procedure for estimating the uncertainty associated with the measurement is presented.