Rapid detection and specific identification of offals within minced beef samples utilising ambient mass spectrometry.

The morphological transformation of beef tissues after various processing treatments facilitates the addition of cheap offal products. Undetectable to the naked eye, analytical techniques are required to identify such scenarios within minced and processed products. DNA methodologies are ill-equipped to detect adulteration of offal cuts from the same species and vibrational spectroscopic studies, although rapid and non-destructive, have proved inconclusive as to whether the specific adulterant can be identified. For the first time we present a mass spectrometric approach employing an ambient ionisation process to eliminate sample preparation and provide near-instantaneous results. Rapid evaporative ionisation mass spectrometry (REIMS) was used to assess its capabilities of detecting minced beef adulteration with beef brain, heart, kidney, large intestine and liver tissues and chemometric analysis enabled unique or significant markers to be identified. The adulteration levels detected with the REIMS technology when analysing raw adulterated beef burgers were; brain (5%); heart (1-10%); kidney (1-5%); large intestine (1-10%) and liver (5-10%). For boiled adulterated samples; brain (5-10%); heart (1-10%); kidney (1-5%); large intestine (1-10%) and liver (5-10%). REIMS allows rapid and specific identification of offal cuts within adulterated beef burgers and could provide a paradigm shift across many authenticity applications.

Meat Vulnerabilities to Economic Food Adulteration Require New Analytical Solutions.

Meat has been identified as one of the food categories at most risk of food fraud. Meat species substitution has been in the spotlight with the European horse meat scandal in 2013. Analysis of cases reported on the web shows that incidents of meat substitution are still recurring worldwide. Altogether these cases highlight significant weaknesses in the supply chain transparency and traceability of raw meat materials. This has triggered recent progress from the food industry to apply new software tools enabling the mapping of meat supply chains. Nevertheless, a meat vulnerability assessment showed that meat and derivatives are highly susceptible to many fraudulent malpractices. Therefore, more effective measures are needed to manage the risk and new analytical solutions are required to increase the deterrence of meat adulteration and rapid detection of fraud. DNA-based methods have evolved rapidly as shown with the application of the new LCD array and Next Generation Sequencing (NGS) in order to detect broad meat species adulteration. Moreover, new technologies such as NGS together with the Rapid Evaporative Ionization Mass Spectrometry (REIMS) are emerging as a really promising association of analytical approaches for rapid detection of several malpractices.

Distribution of serum levels of persistent organic pollutants, heterocyclic aromatic amine theoretical intake and nutritional cofactors in a semi-rural island population.

Persistent organic pollutant (POP) exposure is strongly associated with negative health effects in humans. Heterocyclic aromatic amines (HAAs) are formed during high temperature cooking of foods (i.e. meat and fish). Human exposure to HAA is through food consumption and from similar food groups to POPs. A study of serum samples for POPs in a non-occupational exposed population (n = 149, age range 18-80 years, recruited in 2012) and comparison with estimated HAA daily intake calculations based on food diaries were undertaken. Three different age groups (group 1, 18-29 years; group 2, 30-44 years; and group 3, 45-80 years) were used to explore possible relationships between POP levels present in blood, HAA intake and nutritional cofactors. Significant differences (p < 0.05) between groups (1 and 3) for POP levels were found for p,p'-DDE, polychlorinated biphenyl (PCB) 153, PCB 138 and the sum of PCBs. A similar trend was found between groups 2 and 3 for PCB 153 and sum of PCBs. Significant differences were found between groups 1 and 3 and groups 2 and 3 for HAA intake., i.e. HAA intake was lowest in those of middle age, which may well reflect a different pathway of human exposure between HAA and POPs through the diet preferences.

Acid-labile protein-adducted heterocyclic aromatic amines in human blood are not viable biomarkers of dietary exposure: A systematic study.

Heterocyclic aromatic amines (HCA) are carcinogenic mutagens formed during cooking of protein-rich foods. HCA residues adducted to blood proteins have been postulated as biomarkers of HCA exposure. However, the viability of quantifying HCAs following hydrolytic release from adducts in vivo and correlation with dietary intake are unproven. To definitively assess the potential of labile HCA-protein adducts as biomarkers, a highly sensitive UPLC-MS/MS method was validated for four major HCAs: 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline (4,8-DiMeIQx) and 2-amino-3,7,8-trimethylimidazo[4,5-f]quinoxaline (7,8-DiMeIQx). Limits of detection were 1-5 pg/ml plasma and recoveries 91-115%. Efficacy of hydrolysis was demonstrated by HCA-protein adducts synthesised in vitro. Plasma and 7-day food diaries were collected from 122 fasting adults consuming their habitual diets. Estimated HCA intakes ranged from 0 to 2.5 mg/day. An extensive range of hydrolysis conditions was examined for release of adducted HCAs in plasma. HCA was detected in only one sample (PhIP, 9.7 pg/ml), demonstrating conclusively for the first time that acid-labile HCA adducts do not reflect dietary HCA intake and are present at such low concentrations that they are not feasible biomarkers of exposure. Identification of biomarkers remains important. The search should concentrate on stabilised HCA-peptide markers and use of untargeted proteomic and metabolomic approaches.

Determination of the persistence of dimetridazole, metronidazole and ronidazole residues in black tiger shrimp (Penaeus monodon) tissue and their stability during cooking.

The depletion of three banned nitroimidazole drugs - dimetridazole (DMZ), metronidazole (MNZ) and ronidazole (RNZ) - was investigated in black tiger shrimp (Penaeus monodon) following in-water medication. The highest concentrations of residues were measured immediately after the 24-h immersion (d0). At this time, MNZ and MNZ-OH residues were measured in shrimp tissue samples at concentrations ranging from 361 to 4189 and from 0.28 to 6.6 µg kg(-1), respectively. DMZ and its metabolites HMMNI ranged in concentration between 31,509 and 37,780 and between 15.0 and 31.9 µg kg(-1), respectively. RNZ and HMMNI concentrations ranged from 14,530 to 24,206 and from 25.0 to 55 µg kg(-1), respectively. MNZ, DMZ and RNZ were the more persistent marker residues and can be detected for at least 8 days post-treatment. MNZ-OH was only detectable on d0 following treatment with MNZ. HMMNI residues were only detectable up to d1 (0.97-3.2 µg kg(-1)) or d2 (1.2-4.5 µg kg(-1)) following DMZ and RNZ treatment, respectively. The parent drugs MNZ, DMZ and RNZ were still measureable on d8 at 0.12-1.0, 40.5-55 and 8.8-18.7 µg kg(-1), respectively. The study also investigated the stability of nitroimidazole residues under various cooking procedures (frying, grilling, boiling, and boiling followed by microwaving). The experiments were carried out in shrimp muscle tissue containing both high and low concentrations of these residues. Different cooking procedures showed the impact on nitroimidazole residue concentration in shrimp tissue. Residue concentration depleted significantly, but partially, by boiling and/or microwaving, but the compounds were largely resistant to conventional grilling or frying. Cooking cannot therefore be considered as a safeguard against harmful nitroimidazole residues in shrimp.

Optimised extraction of heterocyclic aromatic amines from blood using hollow fibre membrane liquid-phase microextraction and triple quadrupole mass spectrometry.

Heterocyclic aromatic amines (HCA) are carcinogenic mutagens formed during cooking of proteinaceous foods, particularly meat. To assist in the ongoing search for biomarkers of HCA exposure in blood, a method is described for the extraction from human plasma of the most abundant HCAs: 2-amino-1-methyl-6-phenylimidazo(4,5-b)pyridine (PhIP), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) and 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline (4,8-DiMeIQx) (and its isomer 7,8-DiMeIQx), using hollow fibre membrane liquid-phase microextraction. This technique employs 2.5cm lengths of porous polypropylene fibres impregnated with organic solvent to facilitate simultaneous extraction from an alkaline aqueous sample into a low volume acidic acceptor phase. This low cost protocol is extensively optimised for fibre length, extraction time, sample pH and volume. Detection is by UPLC-MS/MS using positive mode electrospray ionisation with a 3.4min runtime, with optimum peak shape, sensitivity and baseline separation being achieved at pH 9.5. To our knowledge this is the first description of HCA chromatography under alkaline conditions. Application of fixed ion ratio tolerances for confirmation of analyte identity is discussed. Assay precision is between 4.5 and 8.8% while lower limits of detection between 2 and 5pg/mL are below the concentrations postulated for acid-labile HCA-protein adducts in blood.

BRCA1 deficiency exacerbates estrogen-induced DNA damage and genomic instability.

Germline mutations in BRCA1 predispose carriers to a high incidence of breast and ovarian cancers. BRCA1 functions to maintain genomic stability through critical roles in DNA repair, cell-cycle arrest, and transcriptional control. A major question has been why BRCA1 loss or mutation leads to tumors mainly in estrogen-regulated tissues, given that BRCA1 has essential functions in all cell types. Here, we report that estrogen and estrogen metabolites can cause DNA double-strand breaks (DSB) in estrogen receptor-α-negative breast cells and that BRCA1 is required to repair these DSBs to prevent metabolite-induced genomic instability. We found that BRCA1 also regulates estrogen metabolism and metabolite-mediated DNA damage by repressing the transcription of estrogen-metabolizing enzymes, such as CYP1A1, in breast cells. Finally, we used a knock-in human cell model with a heterozygous BRCA1 pathogenic mutation to show how BRCA1 haploinsufficiency affects these processes. Our findings provide pivotal new insights into why BRCA1 mutation drives the formation of tumors in estrogen-regulated tissues, despite the general role of BRCA1 in DNA repair in all cell types.

Maximum residue level validation of triclabendazole marker residues in bovine liver, muscle and milk matrices by ultra high pressure liquid chromatography tandem mass spectrometry.

Triclabendazole is the only anthelmintic drug, which is active against immature, mature and adult stages of fluke. The objective of this work was to develop an analytical method to quantify and confirm the presence of triclabendazole residues around the MRL. In this work, a new analytical method was developed, which extended dynamic range to 1-100 and 5-1000 µg kg(-1) for milk and tissue, respectively. This was achieved using a mobile phase containing trifluoroacetic acid (pK(a) of 0.3), which resulted in the formation of the protonated pseudomolecular ions, [M+H](+), of triclabendazole metabolites. Insufficient ionisation of common mobile phase additives due to low pK(a) values (<2) was identified as the cause of poor linearity. The new mobile phase conditions allowed the analysis of triclabendazole residues in liver, muscle and milk encompassing their EU maximum residue levels (MRL) (250, 225 and 10 µg kg(-1) respectively). Triclabendazole residues were extracted using a modified QuEChERS method and analysed by positive electrospray ionisation mass spectrometry with all analytes eluted by 2.23 min. The method was validated at the MRL according to Commission Decision (CD) 2002/657/EC criteria. The decision limit (CCα) of the method was in the range of 250.8-287.2, 2554.9-290.8 and 10.9-12.1 µg kg(-1) for liver, muscle and milk, respectively. The performance of the method was successfully verified for triclabendazole in muscle by participating in a proficiency study, the method was also applied to incurred liver, muscle and milk samples.

Comparison of sample preparation methods, validation of an UPLC-MS/MS procedure for the quantification of tetrodotoxin present in marine gastropods and analysis of pufferfish.

Tetrodotoxin (TTX) is one of the most potent marine neurotoxins reported. The global distribution of this toxin is spreading with the European Atlantic coastline now being affected. Climate change and increasing pollution have been suggested as underlying causes for this. In the present study, two different sample preparation techniques were used to extract TTX from Trumpet shells and pufferfish samples. Both extraction procedures (accelerated solvent extraction (ASE) and a simple solvent extraction) were shown to provide good recoveries (80-92%). A UPLC-MS/MS method was developed for the analysis of TTX and validated following the guidelines contained in the Commission Decision 2002/657/EC for chemical contaminant analysis. The performance of this procedure was demonstrated to be fit for purpose. This study is the first report on the use of ASE as a mean for TTX extraction, the use of UPLC-MS/MS for TTX analysis, and the validation of this method for TTX in gastropods.

Enzyme immunoassay for semicarbazide--the nitrofuran metabolite and food contaminant.

Semicarbazide (SEM), the marker residue for the banned nitrofuran veterinary antibiotic nitrofurazone (NFZ), has been detected regularly in foods (47% of recent nitrofuran EU Rapid Alerts involve SEM). However, the validity of SEM as a definitive marker for NFZ has been undermined by SEM arising from other sources including azodicarbonamide, a plastics blowing agent and flour treatment additive. An inexpensive screening test for SEM in food matrices is needed--all SEM testing currently uses expensive LC-MS/MS instrumentation. We now report the first production of antibodies against derivatised SEM. A novel carboxyphenyl SEM derivative was used to raise a polyclonal antibody that has been incorporated into a semi-quantitative microtitre plate ELISA, validated according to the criteria set out in Commission Decision 2002/657/EC, for use with chicken muscle. The antibody is highly specific for derivatised SEM, cross-reactivity being 1.7% with NFZ and negligible with a wide range of other nitrofurans and poultry drugs. Samples are derivatised with o-nitrobenzaldehyde and simultaneously protease digested before extraction by cation exchange SPE. The ELISA has a SEM detection capability (CCbeta) of 0.25 microg kg(-1) when a threshold of 0.21 microg kg(-1) is applied to the selection of samples for confirmation (lowest observed 0.25 microg kg(-1) fortified sample, n=20), thus satisfying the EU nitrofurans' minimum required performance limit of 1 microg kg(-1). NFZ-incurred muscles (12) containing SEM at 0.5-5.0 microg kg(-1) by LC-MS/MS, all screened positive by this ELISA protocol which is also applicable to egg and chicken liver.

Nitrofurazone accumulates in avian eyes--a replacement for semicarbazide as a marker of abuse.

Intact nitrofurazone is present in whole eyes of chickens fed varying levels of this banned antibiotic and may therefore be used as an alternative to the controversial marker residue, semicarbazide, to monitor for abuse of this drug in primary production.

Nitrofuran antibiotic metabolites detected at parts per million concentrations in retina of pigs--a new matrix for enhanced monitoring of nitrofuran abuse.

Nitrofuran metabolite residues AOZ, AMOZ, AHD and SEM were detected at parts per million concentrations in retina of pigs fed therapeutic doses of nitrofuran antibiotics. Discovery of this residue depot may allow widespread technology transfer to laboratories lacking LC-MS/MS thus improving global monitoring of these drugs.

A specificity-enhanced time-resolved fluoroimmunoassay for zeranol employing the dry reagent all-in-one-well principle.

A simple dry chemistry time-resolved fluorescence immunoassay (TR-FIA) method was developed for the measurement of zeranol in bovine urine samples. The samples were purified by immunoaffinity chromatography and a specificity-enhanced zeranol antibody was employed in the immunoassay. This resulted in a highly selective method, which had only negligible reactivity with Fusarium spp. toxins. The all-in-one-well dry chemistry concept made the assay very simple to use because all the assay-specific reagents were already present in the reaction wells in dry form. Only the addition of diluted sample extract was required to perform the competitive one-step TR-FIA and the results were available in less than 1 h. The analytical limit of detection (mean + 3s) for the immunoassay was 0.16 ng ml(-1) (n = 12) and the functional limit of detection for the whole method, estimated by the analysis of zeranol-free samples, was 1.3 ng ml(-1) (n = 20). The recovery of zeranol at the level of 2 ng ml(-1) was 99% (n = 18) and the within-assay variation ranged between 4.5 and 9.0%.