Anion exchange polymeric sorbent coupled to high-performance liquid chromatography with UV diode array detection for the determination of ten N-nitrosamines in meat products: a validated approach

Among mechanisms and molecules presumably involved in the carcinogenicity induced by meat consumption, the N-nitrosamines (N-NAs) are a class of compounds characteristic of processed meats. In this work, an analytical method for the determination of ten N-nitrosamines (N-nitrosomorpholine, N-nitrosomethylethylamine, N-nitrosopyrrolidine, N-nitrosodiethylamine, N-nitrosopiperidine, N-nitrosodipropylamine, N-nitrosodimethylaniline, N-nitrosodibutylamine, N-nitrosodiphenylamine and N-nitrosodibenzylamine) in fresh meats and meat products was developed, optimised and validated. The method is based on optimised sample purification by solid-phase extraction (anion exchange polymeric sorbent) and separation/detection by high-performance liquid chromatography coupled to UV diode array detection. The validation procedure allowed to ascertaining good analytical performances in terms of sensitivity, selectivity towards interfering compounds, accuracy and robustness. The values obtained for precision (range: 4.3–14.4%) and recovery percentages (range: 80.8–95.1%) were compared to reference values indicated in the Decision No. 657/2002/EC, resulting as compliant. The measurement uncertainty (lower than 14.6%) was satisfactory for each N-NA as well.     

New phenylenediamine antiozonants for commodities based on natural and synthetic rubber

For protection of elastomeric materials against ageing, antioxidants such as UV-stabilizers and antiozonants are used. Although historically N-phenyl-N'-(1,3-dimethylbutyl)-p-phenylenediamine (6PPD) was the only approved antiozonant in Germany, a range of other phenylene diamine antiozonants (excluding 6PPD) are permitted for use in rubber articles intended for repeat food-contact use in the US (FDA regulations chapter 21 Part 177.2600). The biggest disadvantage of 6PPD is its partial decomposition during the vulcanization leading to the formation of toxic primary aromatic amines (PAA), such as aniline and secondary aromatic amines (SAA). A number of new PPDs have been developed and patented, that due to their chemical structures, are far less soluble in aqueous solutions but a lot more soluble within the rubber matrix. They therefore show significantly less migration of PAA and SAA. These new antiozonants were investigated and compared to 6PPD using commercial rubber materials with a certain content of antiozonant with regard to their migration of PAA and SAA into three different food simulants. The lowest concentration of PAA and SAA in all food simulants was measured in the RU 997 stabilized elastomer. Due to this fact RU 997 was permitted as a new antiozonant for commodities based on rubber according to the Recommendation XXI 'Articles based on natural and synthetic rubber' of the Federal Institute for Risk Assessment (BfR). RU 997 therefore represents an alternative for 6PPD with less migration of aromatic amines.     

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.     

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).     

Liquid chromatography-tandem mass spectrometry determination of N-nitrosamines released from rubber or elastomer teats and soothers

A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method as an alternative to a gas chromatography-thermal energy analyser (GC-TEA) method recommended by the European Committee on Standardization (CEN) was validated for the simultaneous determination of eight N-nitrosamines released into artificial saliva from rubber or elastomer teats and soothers. N-nitroso-dipropylamine-d₁₄ (NDPA-d₁₄) was used as internal standard for accurate quantification. The method was validated with relatively good analytical results, including sufficiently low limits of detection (0.1–2?µg?kg^?1 of sample) and good linearity (r ^2?>?0.99) throughout the studied concentration ranges. Intra- and inter-day precisions expressed with the relative standard deviation (RSD, %) were 3.4–8.0% and 4.4–11.3%, which were below the performance criteria based on one-half of the value derived from the Horwitz value. It was also found that the LC-MS/MS method is sufficiently rugged and successfully applicable to its routine analysis for the compliance test of Commission Directive 93/11/EEC.     

Simultaneous analysis and exposure assessment of migrated bisphenol analogues,phenol, and p-tert-butylphenol from food contact materials

ABSTRACT

A simple, rapid, and novel liquid chromatography tandem-mass spectrometry (LC-MS/MS) method was developed and validated to determine levels of eight bisphenol analogues (A, S, F, B, P, AF, AP, and Z), phenol, and p-tert-butylphenol migrated from food contact material (FCM) into food simulants. Method validation showed acceptable values in terms of linearity, precision, and accuracy. The limits of detection and quantification were 0.53–29.6 and 1.77–29.6 μg L^?1, respectively. Water, 4% acetic acid, 50% ethanol, and n-heptane were employed as food simulants for the migration tests, and the proposed method was applied to 234 articles of 11 FCMs including polycarbonate, polyethersulfone, polypropylene, and polyethyleneterephthalate, obtained from domestic markets and manufacturers in Korea. Only phenol was found in the FCMs poly(cyclophexane-1,4-dimethylene terephthalate), polylactide, and thermoplastic polyurethane. Eight bisphenol analogues and p-tert-butyl phenol were not found in any samples. Using the obtained migration results, the estimated daily intake (EDI) of phenol was calculated. Exposure assessments were carried out to compare the EDI with the tolerable daily intake (TDI), showing a low percentage (0.18%) of the TDI reported. This is the first study to examine eight bisphenol analogues and two phenols simultaneously in FCMs using the LC-MS/MS.     

HS-GC/MS method development and exposure assessment of volatile organic compounds from food packaging into food simulants

ABSTRACT

A simultaneous headspace-gas chromatography/mass spectrometry (HS-GC/MS) method was developed and validated to determine the migration of 12 volatile organic compounds (methanol, acetone, methylethylketone, ethylacetate, isopropylalcohol, benzene, toluene, ethylbenzene, xylene, cumene, propylbenzene, and styrene) from food contact materials into food simulants (water, 4% acetic acid, 50% ethanol, and n-heptane). The limits of detection and quantification were 0.007–0.201 mg L^?1 and 0.023–0.668 mg L^?1, respectively. The method was applied to 205 samples of paper/paperboard, polyethylene, polypropylene, polystyrene, and polyethylene terephthalate. The estimated daily intake (EDI) was calculated using the migration results. Exposure assessments were carried out to compare the EDI to the tolerable daily intake (TDI); the results indicated that the EDI of styrene represented only a small percentage (8.0%) of the TDI. This analytical method will be a useful tool to examine levels of various volatile compounds migrating from food packaging to food simulants using HS-GC/MS method.     

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.     

Determination of the overall migration from silicone baking moulds into simulants and food using 1H-NMR techniques

Different silicone baking moulds (37 samples) were characterized with respect to potential migrating substances using ¹H-NMR, RP-HPLC–UV/ELSD and GC techniques. In all cases cyclic organosiloxane oligomers with the formula [Si(CH₃)₂–O] _n were identified (n = 6 … 50). Additionally, linear, partly hydroxyl-terminated organosiloxanes HO–[Si(CH₃)₂–O] _n –H (n = 7 … 20) were found in 13 samples. No substances other than siloxanes could be detected, meaning the migrants mainly consist of organopolysiloxanes. Based on this knowledge, a ¹H-NMR quantification method for siloxanes was established for the analysis of both simulants and foodstuffs. Validation of the ¹H-NMR method gave suitable performance characteristics: limit of detection 8.7 mg kg^–1 oil, coefficient of variation 7.8% (at a level of 1.0 mg kg^–1 food). Migration studies were carried out with simulants (olive oil, isooctane, ethanol (95%), Tenax) as well as preparation of different cakes. From the 1st to 10th experiment, siloxane migration into cakes only slightly decreased, with a significant dependence on fat content. Migration never exceeded a level of 21 mg kg^–1 (3 mg dm^–2) and was, therefore, well below the overall migration limit of 60 mg kg^–1 (10 mg dm^–2). However, migration behaviour into simulants differed completely from these results.     

The formation and analysis of n-nitrosamines

The assessment of the potential carcinogenic risk to human health of the small quantities of N-nitrosamines formed in some foodstuffs depends on the availability of information on the routes by which they can be formed and of analytical methods having adequate sensitivity and selectivity for their determination. The present state of knowledge on these two aspects is reviewed in this paper.     

N-nitrosamines and residual nitrite in cured meats from the Dutch market

Summary A total of 140 samples of 16 kinds of cured meats were analyzed for contents of residual nitrite andN-nitrosamines. Nitrite was determined by reaction with sulfanilamide/naphthylethylenediamine and colorimetric measurement.N-nitrosamines were isolated from the samples by vacuum distillation and determined by gas-chromatography with chemiluminescence detection (GC-TEA). In six samples no nitrite was detectable (< 1 mg NaNO₂/kg), the remaining samples contained 1–140 mg NaNO₂/kg, median value 6.8 mg/kg. In 46 samples (33%) noN-nitrosamines were detected, i.e. less than 0.1–0.5 g/kg of the individual nitrosamines, depending upon their structure.N-nitrosodimethylamine (NDMA) was the nitrosamine present most frequently, in 75 samples, contents were 0.1–0.9 g/kg, mean 0.3 g/kg. OtherN-nitrosamines found were:N-nitrosopiperidine (NPIP), 10 times, 0.3-25 g/kg;N-nitrosodiethylamine (NDEA), three times, 0.2–0.9 g/kg;N-nitrosopyrrolidine (NPYR), three times, 1.3–4.2 g/kg;N-nitrosomorpholine, once, 0.7 g/kg andN-nitrosothiazolidine (NTHZ), 36 times, 0.5–91 g/kg, mean 5.7 g/kg. NTHZ was found most often and with the highest contents in smoked products. Frying of bacon and cured, smoked pork bellies led to substantially increased levels of NPYR in both products, and for the pork bellies also of NTHZ. In five samples of cured, smoked pork bellies after frying NTHZ-contents of 3.6–490 g/kg (mean 179) were found. No correlation between residual nitrite levels andN-nitrosamine contents could be established. Investigations during the nineteen seventies gave much higher levels for NDMA, NDEA, NPIP and NPYR in Dutch cured meats than now found; at that time NTHZ was not measured.

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N-Nitrosamine und Restnitrit in holländischen Fleischerzeugnissen

Zusammenfassung Insgesamt wurden 140 Proben von 16 verschiedenen Fleischerzeugnissen auf ihren Gehalt an Restnitrit und flüchtigenN-Nitrosamine geprüft Nitrit wurde nach Reaktion mit Sulfanilamid/Naphthylethylendiamin colorimetrisch bestimmt.N-Nitrosamine wurden mittels Vakuumdestillation aus den Proben isoliert und mit Gaschromatographie-Chemiluminescenzdetektion (GC-TEA) gemessen. In 6 Proben war kein Nitrit nachweisbar (< 1 mg NaN0₂/kg), die Nitritgehalte der übrigen Proben lagen zwischen 1 und 140 mg NaNO₂/kg, Medianwert 6,8 mg/kg. In 46 Proben (33%) waren keineN-Nitrosamine nachweisbar d.h. weniger als 0,1–0,5 g/kg.N-Nitrosodimethylamin (NDMA) kam am häufigsten vor, in 75 Proben mit Gehalten von 0,1–0,9 g/kg, Mittelwert 0,3 g/kg. Von den sonstigen Nitrosaminen waren nachweisbar:N-Nitrosopiperidin (NPIP), 0,3–25 g/kg (zehnmal);N-Nitrosodiethylamin (NDEA), 0,2–91 g/kg (dreimal),N-Nitrosopyrrolidin (NPYR), 1,3–4,2 g/kg (dreimal),N-Nitrosomorpholin (NMOR), 0,7 g/kg (einmal) undN-Nitrosothiazolidin (NTHZ), 0,5–91 g/kg (36mal), Mittelwert 5,7 g/kg. NTHZ wurde am häufigsten und mit den höchsten Mengen in geräucherten Produkten gefunden. Braten von Bacon und Frühstückspeck erhöhte die Gehalte an NPYR erheblich in beiden Produkten und bei Frühstückspeck auch den Gehalt an NTHZ. In 5 Proben Frühstückspeck wurden nach dem Braten NTHZ-Gehalte von 3,6–490 g/kg (Mittelwert 179) gemessen. Es konnte keine Beziehung zwischen den Gehalten an Restnitrit undN-Nitrosaminen festgestellt werden. Untersuchungen in den siebziger Jahren ergaben viel höhere Gehalte an NDMA, NDEA, NPIP und NPYR in holländischen Fleischwaren als die jetzt gefundenen; auf NTHZ wurde damals nicht analysiert.

     

Time‐ and temperature‐dependent migration studies of Irganox 1076 from plastics into foods and food simulants

The study provides an exhaustive set of migration data for octadecyl 3‐(3,5‐di‐tert‐butyl‐4‐hydroxyphenyl)propionate (Irganox 1076) from low‐density polyethylene (LDPE) in several food matrices. Irganox 1076 was used as a model migrant because it represents one of the typical substances used as an antioxidant in food packaging polymers. Kinetic (time‐dependent) migration studies of Irganox 1076 were performed for selected foodstuffs chosen with different physical–chemical properties and in relation to the actual European food consumption market. The effect of fat content and of the temperature of storage on the migration from plastic packaging was evaluated. The results show that migration increased with fat content and storage temperature. All data obtained from real foods were also compared with data obtained from simulants tested in the same conditions. In all studied cases, the kinetics in simulants were higher than those in foodstuffs. The work provides data valuable for the extension of the validation of migration model developed on simulants to foodstuffs themselves.     

Determination of the veterinary drug maduramicin in food by fluorescence polarisation immunoassay

A fluorescence polarisation immunoassay (FPIA) using a specific polyclonal antiserum for the detection of maduramicin (MD) was developed and optimised. The polyclonal antiserum was produced against MD linked to bovine serum albumin. Fluorescein‐labelled MD (tracer) was synthesised by N‐hydroxysuccinimide active ester method and purified using thin layer chromatography. The developed FPIA for MD had a dynamic range from 0.01 to 5.6 μg mL^?1 with an IC₅₀ value of 0.16 μg mL^?1 and a limit of detection of 0.002 μg mL^?1. Recoveries from chicken muscle, fat and egg samples spiked at 0.25, 5 and 10 μg g^?1 levels were 82–130%. The FPIA results from analysis of incurred residues in chicken muscle samples showed that the simple procedure is viable.     

Study of the partition coefficients Kp/f of seven model migrants from LDPE polymer in contact with food simulants

This study evaluated the influence of parameters such as temperature and type of low-density polyethylene (LDPE) film on the log K_p/f values of seven model migrants in food simulants. Two different types of LDPE films contaminated by extrusion and immersion were placed in contact with three food simulants including 20% ethanol, 50% ethanol and olive oil under several time–temperature conditions. Results suggest that most log K_p/f values are little affected by these parameters in this study. In addition, the relation between log K_p/f and log P_o/w was established for each food simulant and regression lines, as well as correlation coefficients, were calculated. Correlations were compared with data from real foodstuffs. Data presented in this study could be valuable in assigning certain foods to particular food simulants as well as predicting the mass transfer of potential migrants into different types of food or food simulants, avoiding tedious and expensive laboratory analysis. The results could be especially useful for regulatory agencies as well as for the food industry.     

Single-laboratory validation of a saponification method for the determination of four polycyclic aromatic hydrocarbons in edible oils by HPLC-fluorescence detection

An analytical method is reported for the determination of four polycyclic aromatic hydrocarbons (benzo[a]pyrene (BaP), benz[a]anthracene (BaA), benzo[b]fluoranthene (BbF) and chrysene (CHR)) in edible oils (sesame, maize, sunflower and olive oil) by high-performance liquid chromatography. Sample preparation is based on three steps including saponification, liquid–liquid partitioning and, finally, clean-up by solid phase extraction on 2 g of silica. Guidance on single-laboratory validation of the proposed analysis method was taken from the second edition of the Eurachem guide on method validation. The lower level of the working range of the method was determined by the limits of quantification of the individual analytes, and the upper level was equal to 5.0 µg kg^?1. The limits of detection and quantification of the four PAHs ranged from 0.06 to 0.12 µg kg^?1 and from 0.13 to 0.24 µg kg^?1. Recoveries of more than 84.8% were achieved for all four PAHs at two concentration levels (2.5 and 5.0 µg kg^?1), and expanded relative measurement uncertainties were below 20%. The performance of the validated method was in all aspects compliant with provisions set in European Union legislation for the performance of analytical methods employed in the official control of food. The applicability of the method to routine samples was evaluated based on a limited number of commercial edible oil samples.     

Determination of Total Nitrosamines in Vegetables Cultivated Organic and Conventional Using Diamond Electrode

Vegetables were analyzed for total N-nitrosamines (NAs) and the influence of disinfection processes was assessed. Differences in NAs found in cabbage, spinach, and broccoli were determined by square wave voltammetry using a boron-doped diamond electrode. Analysis of samples showed that all samples contained detectable levels of NAs but the results indicated that organic contained less than conventionally grown products. The sum of the total NAs was higher in the cabbage samples, ranging between 2.8–3.1 ppb and lower in broccoli samples at 0.2–1.1 ppb. The method described is simple, rapid, selective, and sensitive. The results suggested that the disinfection process affects the level of NAs, in this manner affecting the level of human exposure to NAs.     

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.     

Optimisation of an analytical method and results from the inter-laboratory comparison of the migration of regulated substances from food packaging into the new mandatory European Union simulant for dry foodstuffs

This paper presents the outcome of the development, optimisation and validation at European Union level of an analytical method for using poly(2,6-diphenyl phenylene oxide – PPPO), which is stipulated in Regulation (EU) No. 10/2011, as food simulant E for testing specific migration from plastics into dry foodstuffs. Two methods for fortifying respectively PPPO and a low-density polyethylene (LDPE) film with surrogate substances that are relevant to food contact were developed. A protocol for cleaning the PPPO and an efficient analytical method were developed for the quantification of butylhydroxytoluene (BHT), benzophenone (BP), diisobutylphthalate (DiBP), bis(2-ethylhexyl) adipate (DEHA) and 1,2-cyclohexanedicarboxylic acid, diisononyl ester (DINCH) from PPPO. A protocol for a migration test from plastics using small migration cells was also developed. The method was validated by an inter-laboratory comparison (ILC) with 16 national reference laboratories for food contact materials in the European Union. This allowed for the first time data to be obtained on the precision and laboratory performance of both migration and quantification. The results showed that the validation ILC was successful even when taking into account the complexity of the exercise. The results showed that the method performance was 7–9% repeatability standard deviation (rSD) for most substances (regardless of concentration), with 12% rSD for the high level of BHT and for DiBP at very low levels. The reproducibility standard deviation results for the 16 European Union laboratories were in the range of 20–30% for the quantification from PPPO (for the three levels of concentrations of the five substances) and 15–40% from migration experiments from the fortified plastic at 60°C for 10 days and subsequent quantification. Considering the lack of data previously available in the literature, this work has demonstrated that the validation of a method is possible both for migration from a film and for quantification into a corresponding simulant for specific migration.     

Perfluorinated compounds in food simulants after migration from fluorocarbon resin-coated frying pans,baking utensils,and non-stick baking papers on the Korean market

ABSTRACT

Perfluorinated compounds (PFCs) are used in manufacturing food contact materials, including non-stick cookware coatings and oil- and moisture-resistant paper coatings. The chemical stability of PFCs poses an issue for human safety, as they do not degrade well naturally and hence may accumulate in the body. In terms of food safety, since dietary intake is thought to be a major source of exposure to PFCs, it is necessary to assess the migration of PFCs from food packaging articles to food under typical cooking and storage conditions. An analytical method was developed for assessing the migration of 16 PFCs from food contact materials to food simulants using liquid chromatography–tandem mass spectrometry. The applicability of the method for regular inspection was assessed by monitoring 312 samples. Based on the results of the exposure assessment, all food contact materials deemed to be safe for use, which evaluated migrated concentrations and dietary food intake.     

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.