N-nitrosamines migrating from food contact materials into food simulants: analysis and quantification by means of HPLC-APCI-MS/MS

It was the aim of the work described here to develop a validated analytical method for the determination of N-nitrosamines in food simulants. Here, we present the validation of a liquid chromatography–atmospheric pressure chemical ionisation–tandem mass spectrometry method for the determination of 13 N-nitrosamines in the food simulants deionised water, 3% acetic acid and 10% ethanol. Method validation encompassed linearity, LOD, LOQ, recovery, precision and stability of the N-nitrosamines. The method was found to be sufficiently rugged and suitable for routine analysis. In addition, the developed method is on average 10 times more sensitive than the gas chromatography–thermal energy analyser (GC-TEA) method that is currently stipulated in Recommendation XXI for commodities based on natural and synthetic rubber issued by the German Federal Institute for Risk Assessment (BfR). The developed method was applied to determine the N-nitrosamine contents in migration solutions of 12 elastomer samples covering a range of different elastomer types. In 10 out of 12 samples, N-nitrosamines were determined. In most samples, the guidance value of 1 µg/dm² specified in BfR Recommendation XXI was not exceeded. In conclusion, the analytical method presented here offers a useful alternative to the GC-TEA method currently stipulated in BfR Recommendation XXI. This is of wider relevance, since BfR recommendations are used for compliance assessment of Article 3, paragraph 1 a of the Regulation (EC) No. 1935/2004 with respect to their health safety.     

Literature compilation of volatile N-nitrosamines in processed meat and poultry products - an update

ABSTRACT

A compilation of volatile N-nitrosamine levels in processed (e.g., cured, canned, smoked) meat and poultry products is presented. Over 1800 samples of processed meat products including bacon, ham, salami, sausage, and various other processed meat and poultry products have been examined for the presence of eight volatile N-nitrosamines. The database compiled from the literature is based on 25 references published for the period of 1985 to 2018 from 14 countries. N-nitrosodimethylamine (NDMA), N-nitrosopiperidine (NPIP), and N-nitrosopyrrolidine (NPYR), are the most frequently identified volatile N-nitrosamines occurring in processed meat and poultry products. N-nitrosodiethylamine (NDEA), and N-nitrosodibutylamine (NDBA) are also frequently observed to a lesser extent. The processed meat and poultry products with the highest levels of volatile N-nitrosamines were pork (fried, fat only eaten), poultry (fried), poultry (spiced, grilled), and bacon (fried).     

Volatile N-nitrosamines in meat products: Potential precursors,influence of processing,and mitigation strategies

Meat products can be contaminated with carcinogenic N-nitrosamines, which is ascribed to the reaction between a nitrosating agent, originating from nitrite or smoke, and a secondary amine, derived from protein and lipid degradation. Although in model systems it is demonstrated that many amine containing compounds can be converted to N-nitrosamines, the yield is dependent of reaction conditions (e.g., low pH and high temperature). In this article, the influence of the composition of the meat products (e.g., pH, a_w, spices) and processing (e.g., ageing, ripening, fermentation, smoking, heat treatment and storage) on the presence and availability of the amine precursors and the N-nitrosamine formation mechanism is discussed. In addition, this article explores the current N-nitrosamine mitigation strategies in order to obtain healthier and more natural meat products.     

Studies on the occurrence and formation of 2-(hydroxymethyl)-N-nitrosothiazolidine-4-carboxylic acid (HMNTCA) and 2-(hydroxymethyl)-N-nitrosothiazolidine (HMNTHZ) in various cured smoked meats,fish and cheese

Abstract : Because of the carcinogenic nature of many N-nitroso compounds, there is concern about their presence in foods. This paper presents some data on the levels of two non-volatile compounds (2-(hydroxymethyl)-N-nitrosothiazolidine-4-carboxylic acid (HMNTCA) and 2-(hydroxymethyl)-N-nitrosothiazolidine (HMNTHZ)) in various smoked foods such as 25 cured meats, eight smoked fish and seafoods, 15 smoked poultry products, and 17 smoked cheeses. All samples were negative for HMNTHZ, but 11 of the cured meats, six poultry products and two smoked fish contained HMNTCA at levels of 10–260 μg kg^?1. Upon frying, appreciable levels of HMNTHZ were formed in only one of 10 samples of cured meat products. The smoked cheeses were all negative for HMNTCA.     

Analysis of fumonisin mycotoxins with capillary electrophoresis – mass spectrometry

ABSTRACT

This paper demonstrates the development of an analytical method based on CE coupled to ESI-MS for the identification and quantification of fumonisin mycotoxins. Separation and detection parameters (pH of background electrolyte (BGE), organic modifier content, sheath liquid (SL) composition, MS mode and nebuliser pressure) were optimised. Ammonium formate/ammonia (pH = 9.5) with 10% ACN modifier was found the most suitable BGE. Positive mode MS was used for detection by scanning the m/z range of 400–1200. Separation was highly affected by the nebuliser pressure, a 25% improvement in peak resolution was achieved by applying the optimised parameters. The ‘dilute and shoot’ approach was applied to overcome disturbing effects caused by the matrix of fungi supernatant samples. The available sample volume affected the reproducibility of the measurements greatly: the scattering of peak intensities were between 4 and 11 RSD% instead of 27–195 RSD% for fumonisin B₁ and fumonisin B₂ when the available volume was ~200 µL instead of ~20 µL. Quantitative determinations were carried out in Fusarium verticillioides and Fusarium proliferatum culture supernatant (raw) and mycelium (cleaned up) samples. The optimised method enabled the detection of 11 fumonisins in Fusarium proliferatum inoculated rice samples; 2 of them were quantified based on external calibration and 4 other compounds with fumonisin-like formulas were detected.     

Application of inductively coupled plasma–mass spectrometry for trace element characterisation of equine meats

A validated and accredited analytical method of inductively coupled plasma–mass spectrometry was used to determine the levels of 22 trace elements in 52 equine meat samples collected during 2015. Greater amounts of Zn, Fe, and Ca were found with mean values of over 25 µg g^?1. Levels of non-essential trace elements, that is, Pb, Cd, Hg, and As, were generally low (mean values lower than 11.3 ng g^?1). Equine gender and geographic origin of meats (Italy and Poland) were compared, with no significant differences being found, whereas equine meats could be differentiated from bovine through a multivariate approach. As regards trace element accumulation, evaluated considering slaughter age, Zn, Fe, Ca, and Cr showed greater increase. Finally, a good correlation was obtained between two pairs of trace elements, Zn/Fe (r = 0.82) and Ca/Fe (r = 0.87).     

Influence of putrescine,cadaverine, spermidine or spermine on the formation of N-nitrosamine in heated cured pork meat

The influence of biogenic amines (i.e. putrescine, cadaverine, spermidine and spermine) on the N-nitrosamine formation in heated cured lean meat was studied in the presence or absence of sodium nitrite and at different meat processing temperatures. Experimental evidence was produced using gas chromatography with thermal energy analysis detection (GC–TEA). Concentration of N-nitrosamines was modelled as a function of the temperature and the nitrite concentration for two situations, i.e. presence or absence of added biogenic amines to the meat. The significance of the influence of the changing parameters was evaluated by ANOVA (Analysis of Variance).     

Determination of Seven <Emphasis Type="Italic">N</Emphasis>-nitrosamines in Agricultural Food Matrices Using GC-PCI-MS/MS

The quantitative analytical methods for seven N-nitrosamines including N-nitrosodimethylamine (NDMA), N-nitrosomethylethylamine (NMEA), N-nitrosodiethylamine (NDEA), N-nitrosodibutylamine (NDBA), N-nitrosopiperidine (NPIP), N-nitrosopyrrolidine (NPYR), and N-nitrosomorpholine (NMOR) were established for agricultural food matrices. Four food matrices were used for the method development: rice soup as a fatless solid matrix, apple juice as a fatless liquid matrix, corn oil as a fat-rich liquid matrix, and 20 % alcohol as an alcohol matrix. A combination of solid-supported liquid-liquid extraction (SLLE) using Extrelut NT and a solid phase extraction (SPE) using Florisil was employed for fatless matrices. For an alcohol matrix, only SLLE was used without SPE, and liquid-liquid extraction (LLE) was established for a fat-rich matrix. The extract was analyzed by gas chromatography-positive chemical ionization-tandem mass spectrometry (GC-PCI-MS/MS) using ammonia gas as an ion source. Linearity, recovery, repeatability, inter-day precision, reproducibility, and uncertainty were evaluated for method validation using four matrices. Method detection limits for all of the investigated N-nitrosamines were ranged from 0.10 to 0.18 μg/kg for the rice soup, from 0.10 to 0.19 μg/kg for the apple juice, 0.10 μg/kg for the corn oil, and from 0.10 to 0.25 μg/kg for 20 % alcohol, depending on N-nitrosamines. Established methods were applied to determine seven N-nitrosamines in some agricultural food products.     

The Water Activity of Canned Foods

This work reports the a_w values of a diversity of commercially canned foods (54 samples), including fruits and vegetables and cured and uncured meat products. The a_w of canned fruit products (pieces in syrup, purees and juices) ranges between 0.950 and 0.992. Canned cured meats (i.e. deviled ham, meat and liver pastes, corned beef, cooked ham) have a_w values in the range 0.970–0.984, while uncured canned meats display a_w values above 0.982.     

Authentication of a traditional game meat sausage (Alheira) by species-specific PCR assays to detect hare,rabbit, red deer,pork and cow meats

Alheira is a traditional meat product that is typical from the Northeast region of Portugal and much appreciated. It is a sort of sausage produced industrially or by small artisanal producers, having wheat bread and meats as main ingredients. Game meat Alheira (Alheira de caça) is considered one of the most attractive products since it should include different game meats. The aim of the present work was to identify the species of origin of meats added to game meat Alheira samples to verify their compliance with labelling. Species-specific PCR assays targeting mitochondrial genes of rabbit, hare, red deer, cow and pork were optimised and applied to industrial and artisanal samples. The assays revealed adequate specificity for each of the targeted species, with sensitivities of 0.01–0.1%. Results of the evaluation of 18 commercial samples identified several inconsistencies with labelling, namely the absence of declared game species (red deer, hare and rabbit) in ten samples and the presence of undeclared cow species in nine of the analysed samples. These findings indicate the occurrence of misleading labelling, suggesting the adulteration by substitution of game meats by cow meat to reduce production costs and the need to protect and valorise this kind of traditional food product.     

Quality changes during the storage of dehydrated chicken kebab mix

The level of binders (starch, refined wheat flour and milk powder) was optimised with respect to sensory quality of ready‐to‐eat kebab made from a dehydrated mix using the experiment based on the Box–Behnken design. The optimised levels (as % of cooked meat) were 4.5% starch, 9.5% refined wheat flour and 2% milk powder. Dehydrated chicken kebab mix prepared with optimised quantities of binders and spices was packed in metalised polyester pouches, stored at ambient temperature (27 ± 2 °C) for 6 months and sampled periodically for quality evaluation. A gradual decrease (P ≤ 0.05) in pH from 5.8 to 5.5, increases (P ≤ 0.05) in a_w from 0.31% to 0.42%, in moisture from 5.4% to 6.2%, in free fatty acid values from 0.99 to 1.74 as per cent oleic acid and in thiobarbituric acid numbers from 2.9 to 5.3 mg malonaldehyde kg^?1 were observed during storage. Hunter colour a values increased (P ≤ 0.05) from 0.30 to 1.29, whereas changes in Hunter L (51.8–53.4) and in Hunter b (19.0–19.5) values were marginal (P ≥ 0.05) during storage. The chicken kebab mix was microbiologically safe as indicated by low bacterial counts and absence of coliforms throughout the storage period of 6 months. Fried chicken kebabs prepared from mix stored for up to 6 months were acceptable.     

The World Cancer Research Fund report 2007: A challenge for the meat processing industry

One of the 10 universal guidelines for healthy nutrition in a report of the World Cancer Research Fund released at the end of 2007 is to “limit intake of red meat and avoid processed meat”, as a result of the “convincing evidence” for an association with an increased risk of colorectal cancer development. In the present paper, the scientific evidence for the association between processed meats intake and colorectal cancer development is explored and the most probable hypothesis on the mechanism underlying this relationship formulated.It seems that the present state of knowledge is not well understood but relates to a combination of haem iron, oxidative stress, formation of N-nitroso compounds and related residues in the digestive tract as the causal factors. Although criticisms of the inaccurate definition of processed meats and the insufficient accounting for the large variability in composition of meat products have been expressed, it is clear that the report urges proper action by the meat and nutrition research community and the meat industry.Research items that in our view should be addressed are discussed. They include: (1) evaluating the health risks associated with processed meats intake within the context of the supply of beneficial nutrients and other nutrition associated health risks; (2) definition of the role of nitrites and nitrates in meat processing; (3) investigating the role of red and processed meats on the endogenous formation of N-nitroso compounds in the digestive tract; and (4) developing improved processed meats using new ingredients.     

Role of proline and hydroxyproline in N-nitrosamine formation during heating in cured meat

N-Nitrosamines are formed in a multi-step reaction of nitrite with free amino acids and amines in the meat products. The aim of this study was to determine the role of proline and hydroxyproline in N-nitrosamines formation during heating of cured meat. A lean meat model was used with different nitrite concentrations (0, 120, and 480 mg/kg), and addition of proline and hydroxyproline (1000 mg/kg), followed by heating at different temperatures. Volatile nitrosamines were analyzed with GC-TEA.     

Antimicrobial and Antioxidative Strategies to Reduce Pathogens and Extend the Shelf Life of Fresh Red Meats

Abstract: The shelf life of packaged fresh red meats is most frequently determined by the activity of microorganisms, which results in the development of off‐odors, gas, and slime, but it is also influenced by biochemical factors such as lipid radical chain and pigment oxidation causing undesirable flavors and surface discoloration. The predominant bacteria associated with spoilage of refrigerated meats are Pseudomonas, Acinetobacter/Moraxella (Psychrobacter), Shewanella putrefaciens, lactic acid bacteria, Enterobacteriaceae, and Brochothrix thermosphacta. The spoilage potential of these organisms and factors influencing their impact on meat quality are discussed. High O₂‐modified atmosphere (80% O₂+ 20% CO₂) packaging (MAP) is commonly used for meat retail display but vacuum packaging remains the major MAP method used for meat distribution. Two‐step master packaging (outer anoxic‐20% CO₂+ 80% N₂/inner gas‐permeable film) is used for centralized MAP distribution, but CO use (0.4%) in low O₂ packaging systems is limited by consumer uncertainty that CO may mask spoilage. Active packaging where the film contributes more than a gas/physical barrier is an important technology and has been studied widely. Its application in combination with MAP is very promising but impediments remain to its widespread industrial use. The influence of processing technologies including modified atmospheres on lipid oxidation and discoloration of meats are analyzed. Because both organic acids and antioxidants have been evaluated for their effects on microorganism growth, in concert with the prevention of lipid oxidation, work in this area is examined.     

2-(Hydroxymethyl)-N-nitrosothiazolidine-4-carboxylic Acid in Smoked Meats and Bacon and Conversion to 2-(Hydroxymethyl)-N-Nitrosothiazolidine During High-Heat Cooking

Concentrations of 2-(hydroxymethyl)-N-nitrosothiazolidine-4-carbox-ylic acid (HMNTCA) in a variety of smoked meats and bacon were determined by a newly developed method based on derivatization of HMNTCA with diazomethane and analysis by high performance liquid chromatography-thermal energy analyzer. Of samples analyzed, 4/8 raw bacon, 6/8 fried bacon, and 7/14 miscellaneous cured meat products contained 10–540 ppb of HMNTCA. Evidence suggested 2-(hydroxyrnethyl)-N-nitrosofhiazolidine in fried bacon likely originated by heat-induced decarboxylation of HMNTCA. Identity of HMNTCA in selected samples was confirmed by mass spectrometry.     

A survey of fish products for volatile N-nitrosamines

A study was undertaken to determine the levels of nitrate, nitrite and volatile N-nitrosamines of various fish products (halibut, salmon, cod, sole, ocean perch, scallops). The samples were analysed for five volatile nitrosamines, namely dimethyl-nitrosamine (DMN), diethylnitrosamine (DEN), dibutylnitrosamine (DBN), nitrosopyrrolidine (NPyr) and nitrosopiperidine (NPip), all of which are potent carcinogens. Both gas–liquid chromatographic (Coulson electrolytic conductivity detector, pyrolytic mode) and thin-layer chromatographic methods were used for estimating the levels of nitrosamines. The average level of nitrate was 8 part/10⁶ (range 0–94 parts/10⁶), and nitrite was present in traces only. Traces of DMN (3–12 parts/10⁹) were found in some uncooked (raw) fish. Increased levels of DMN (3–18 parts/10⁹) and traces of DEN (4–14 parts/10⁹) were observed after baking and frying, indicating formation of these compounds during cooking. None of the samples contained DBN, NPyr or NPip. In 10 out of 15 samples the identities and levels of DMN and DEN were confirmed by gas liquid chromatography–high resolution mass spectrometry.     

A PCR assay to detect trace amounts of soybean in meat sausages

Soybean proteins are the most widely used source vegetable proteins in the meat industry because of several interesting characteristics. As soybean is included in the group of ingredients potentially allergenic, if not declared, it can be considered a hidden allergen, representing a potential risk to sensitised individuals. The aim of this work was to optimise and apply DNA‐based techniques for soybean detection in meat products, as alternative to the currently used protein‐based methods. The optimised polymerase chain reaction (PCR) protocol targeting the soybean lectin gene enabled the detection of the addition of 0.1% and 0.5% of hydrated textured protein, which corresponded to 0.01% and 0.06% (w/w) of soybean protein in unprocessed and heat‐processed pork meats, respectively. The established PCR technique, when applied to commercial meat sausages (eighteen samples), confirmed the presence of soybean declared in nine samples and indicated the presence of soybean in four samples with no labelled information about soybean. Additionally, the event‐specific PCR detection of Roundup Ready^® soybean was also performed, enabling the detection of transgenic DNA in three samples.     

Application of magnetic immuno-polymerase chain reaction assay for detection of <Emphasis Type="Italic">Salmonella</Emphasis> spp. in chicken meats

Since contaminated chicken meats have been the principal foodborne source of the contamination of Salmonella to human beings and cultural detection methods are labor-intensive and time-consuming, a study evaluating the performance of the combination of two techniques that are immunomagnetic separation (IMS) and polymerase chain reaction (PCR) for the detection of Salmonella in chicken meats was conducted. The IMS and PCR assay combines selective extraction of Salmonella by specific antibodies with primer-specific (primer pair based on the sequence of invA gene) PCR amplification. Initially chicken meat samples, in which no Salmonella contamination had been determined by using ISO 6579 reference method, were inoculated with Salmonella Enteritidis culture and subsequently the shortest non-selective pre-enrichment time, that had been needed for the detection of approximately 1 or 10 CFU/mL chicken meat levels of target bacteria by magnetic immuno-PCR assay, was found by using 14, 12, 10 and 8-h periods. In conclusion, it was found that magnetic immuno-PCR assay was able to detect 1–10 CFU Salmonella/25 g chicken meat, after only incorporating a non-selective pre-enrichment period of 12 h. Therefore, an overall 16-h (magnetic immuno-PCR assay in conjunction with 12-h non-selective pre-enrichment) magnetic immuno-PCR assay statistically evaluated as sufficient (p = 0.182 > 0.05) for rapid and sensitive detection of approximately 1–10 CFU Salmonella from 25 g chicken meat samples. Accordingly, 16-h magnetic immuno-PCR assay can be promising for routine use in the detection of Salmonella in chicken meat samples, and it consequently may prevent the risk of Salmonella infections in regard to chicken meats.     

Growth/survival of natural flora and Aeromonas hydrophila on refrigerated uncooked pork and turkey packaged in modified atmospheres

To evaluate the growth/survival of natural flora and Aeromonas hydrophila on refrigerated normal low (pork) and high (turkey) pH meats packaged in modified atmospheres, A. hydrophila was inoculated onto fresh pork and turkey meat slices. Inoculated and control samples were packaged in modified atmospheres (100% N₂, 20/80 and 40/60 CO₂/O₂) or in air in plastic bags and kept at 1 and 7°C. Samples packaged in air showed a similar microbiological pattern to that usually observed in fresh meat stored aerobically. Packaging in modified atmosphere produced a strong inhibition of bacterial growth at 1°C, particularly in samples stored in CO₂/O₂enriched atmospheres. Aeromonas hydrophila grew on turkey and pork meat stored in 100% N₂at 1 and 7°C. Likewise, growth of this bacterium was detected on turkey stored in 20/80 CO₂/O₂at 7°C. No growth was observed in 40/60 CO₂/O₂in any meat at both temperatures assayed.     

Nitrate and nitrite quantification from cured meat and vegetables and their estimated dietary intake in Australians

High dietary nitrate and nitrite intake may increase the risk of gastro-intestinal cancers due to the in vivo formation of carcinogenic chemicals known as N-nitroso compounds. Water and leafy vegetables are natural sources of dietary nitrate, whereas cured meats are the major sources of dietary nitrite. This paper describes a simple and fast analytical method for determining nitrate and nitrite contents in vegetables and meat, using reversed-phase HPLC-UV. The linearity R² value was >0.998 for the anions. The limits of quantification for nitrite and nitrate were 5.0 and 2.5 mg/kg, respectively. This method is applicable for both leafy vegetable and meat samples. A range of vegetables was tested, which contained <23 mg/kg nitrite, but as much as 5000 mg/kg of nitrate. In cured and fresh meat samples, nitrate content ranged from 3.7 to 139.5 mg/kg, and nitrite content ranged from 3.7 to 86.7 mg/kg. These were below the regulatory limits set by food standards Australia and New Zealand (FSANZ). Based on the average consumption of these vegetables and cured meat in Australia, the estimated dietary intake for nitrate and nitrite for Australians were 267 and 5.3 mg/adult/day, respectively.