Easy and rapid method for the quantitative determination of pyrrolizidine alkaloids in honey by ultra performance liquid chromatography-mass spectrometry: An evaluation in commercial honey

Pyrrolizidine alkaloids (PAs) are toxins biosynthesised by plants and they are known to be present in approximately 6000 plant species, about 3% of all flowering plants. PAs are probably the most widely distributed natural toxins and represent a potential risk to human health, since poisoning caused by these toxins is associated with acute and chronic liver damage and may lead to death. One of the most common sources of PAs exposure in humans is honey consumption. We have developed a quick and easy method to quantify nine different PAs (echimidine, heliotrine, intermedine, lycopsamine, lasiocarpine, retrorsine, seneciphylline, senecionine, senkirkine) in honey based on QuEChERS sample extraction and ultra fast liquid chromatography coupled with mass spectrometry detection. We performed a validation study of the method and it resulted in good precision and accuracy, high recoveries, and good linear calibrations. The limit of detection ranged from 0.021 to 1.39 μg Kg⁻¹ and the limit of quantification from 0.081 to 4.35 μg Kg⁻¹. This new approach was applied to the quantification of PAs in retail honeys purchased in local supermarkets, classified by their country of origin: Italian honeys, blends of honey of European countries and blends of honey of European and non-European countries. The concentrations detected ranged from 1 to 169 μg PAs/kg⁻¹ with higher concentrations in blends of European and non-European honeys. This study reveals that many samples tested would exceed the tolerable daily intake suggested for these substances and they could be a hazard to human health.     

Determination of methenamine residues in edible animal tissues by HPLC-MS/MS using a modified QuEChERS method: Validation and pilot survey in actual samples

A modified QuEChERS (quick, easy, cheap, effective, rugged, and safe) method was developed for the determination of methenamine in edible animal tissues by high performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS). Samples were extracted with acetonitrile, and cleaned with anhydrous sodium sulfate and primary secondary amine (PSA) sorbent. An isotope dilution mass spectrometry technique was applied to compensate for matrix effect. The separation was performed on a HILIC column, and the mobile phase composed of acetonitrile-0.1% of formic acid and 5.0 mmol/L of ammonium acetate in water. The method showed a linear relationship in the range of 1.0–20.0 μg/L for methenamine, and the determination coefficients (R²) ranged from 0.9939 to 0.9995. The limit of detection (LOD) and quantification (LOQ) for methenamine in animal tissues sample was 1.5 μg/kg and 5.0 μg/kg, respectively. The average recoveries were 86.7–109.5% at spiked levels of 5.0, 25.0, 100.0 μg/kg. The intra-day precision ranged from 2.6 to 7.0%, and the inter-day precision ranged from 4.9 to 11.3%. The validated method was successfully applied to determination of methenamine in swinish muscle, kidney and liver.     

Development of an aptasensor for the fast detection of Versicolorin A

Aflatoxins (AFs) are secondary metabolites produced by Aspergillus flavus and Aspergillus parasiticus. The molds may contribute to pre-harvest aflatoxin contamination of susceptible crops. For the customer and food producer, a predictive model for aflatoxin detection is very desirable. Versicolorin A (VerA), which is the first precursor in the pathway of aflatoxin B₁ (AFB₁) biosynthesis, shares similar toxic group with the furofuran structure in aflatoxin B₁. VerA exhibits a much lower teratogenic toxicity than AFB₁ and may be used as a predictive indicator for aflatoxin B₁ contamination of storage crops. Therefore, the development of a fast detection method for VerA is important. One of the randomly computer-generated aptamers of VerA was confirmed by isothermal titration calorimetry with K_d = 9.26 × 10⁻⁶ mol l⁻¹. In addition, a simple and sensitive label-free aptasensor was developed for the electrochemical detection of VerA. According to the results from differential pulse voltammetry (DPV), a linear relationship existed between the log conc. of VerA (ranged from 0.01 to 100 ng ml⁻¹) and the current (△I_p) with a limit of detection (LOD) of 10 pg ml⁻¹. The resulting aptasensor exhibited good reproducibility for detecting VerA and stability after storage for 15 days at 4 °C with acceptable anti-interference against ZEN, OTA, DON, and FB₁. When used in corn samples, the recoveries of VerA were determined to be in the range of 81.3%–104.4 %. Although with some intercross, result suggests that the obtained aptamer for VerA is potentially used as a sorbent for the preparation of solid-phase-extraction procedure to clean up food samples in conjunction with high-performance liquid chromatography analysis.     

Development and in-house validation of a competitive ELISA for the quantitative detection of gluten in food

Wheat gluten contains peptide sequences, which activate specific T cells causing a chronic inflammation of the small intestine in celiac disease patients. It is well established that next to wheat gluten, the gluten-like proteins in barley and rye are similarly harmful to celiac disease patients whereas oat is generally considered safe. This study focuses on the development of an ELISA method for the detection of native and processed gluten proteins. The developed test utilizes a monoclonal antibody specific for the DQ2.5-glia-α3 T cell epitope present in the α-gliadins, which are part of wheat gluten that triggers celiac disease. The developed competitive ELISA uses a synthetic DQ2.5-glia-α3 peptide standard for calibration. The conversion from the measured DQ2.5-glia-α3 peptide concentration to gliadin content is achieved by using the experimentally determined multiplication factor of 250. The gluten content can be then calculated by multiplying the gliadin concentration by a factor of 2. A simple sample preparation method with 60% ethanol is used to extract the disease-causing proteins from cereals and processed foods. The assay was found to be specific for the detection of gluten from wheat, barley and rye with no cross-reaction with 8 tested oat varieties. The LOD and LOQ for gliadin were calculated based on the results obtained for 60 blank oats samples and they were 2.9 and 3.6 ppm, respectively. The assay could detect as little as 0.01% wheat gluten and gluten-like proteins from rye and barley in oats. The ELISA was also found to be applicable to the analysis of a range of processed food such as sauces, beers, soups and bread. In conclusion, the developed assay is a sensitive, specific and cost-effective tool for screening cereals and processed foods for the presence of harmful wheat gluten and gluten-like proteins from barley and rye.     

Potential use of carvacrol and citral to inactivate biofilm cells and eliminate biofouling

Biofouling (i.e., accumulation of microorganisms on wetted surfaces) represents a major problem in the food industries, since bacterial biofilms are common sources of persistent infections due to their resilience to cleaning and disinfection treatments. Therefore, alternative treatments based on the use of essential oils or their individual compounds against this bacterial adaptation phenomenon are currently being studied. This work presents a quantitative comparison of the disinfectant potential of 500–2000 μL/L of carvacrol or citral against mature biofilms of Staphylococcus aureus SC-01, Listeria monocytogenes EGD-e or Escherichia coli MG1655. Treatments with 1000 ppm of carvacrol or citral at 45 °C for 60 min were capable of reducing more than 5 logarithmic cycles of the sessile cells forming part of mature biofilms of all the three species. Furthermore, the synergism observed between carvacrol and heat allowed for the physical removal of biofilms by treatments simulating in situ wash conditions (80 °C/60 s). These results demonstrate the great potential of the essential oils’ constituents citral and carvacrol in the eradication of biofilms formed by foodborne pathogenic microorganisms.     

Determination of aflatoxins in walnut sujuk and Turkish delight by HPLC-FLD method

This study aims to assess the risk of aflatoxins (AFs) in traditional confectionery products (walnut sujuk and Turkish delight) of Turkey. A high performance liquid chromatography coupled with fluorescence detection (HPLC-FLD) method was used for the determination of AFs. Evaluation of the method showed good selectivity, linearity, recovery and precision. The limit of quantification (LOQ) ranged from 0.106 to 0.374 μg kg⁻¹. The expanded measurement uncertainty was less than 40% for all target analytes. The validated method was successfully applied to the determination of AFs in 112 traditional confectionery products containing nuts (hazelnuts and walnuts). AFs were detected in 43.8% of walnuts and 60.9% of hazelnuts used as ingredients in walnut sujuk and Turkish delight and at levels ranging from 0.58 to 15.2 μg kg⁻¹ and 0.43–63.4 μg kg⁻¹, respectively. This means that AFs levels in walnut sujuk and Turkish delight were up to levels of 6.1 and 9.5 μg kg⁻¹, respectively. Six walnut samples and twenty-one hazelnut samples were above the EU maximum limits (MLs) of 2 and 5 μg kg⁻¹ for aflatoxin B₁ (AFB₁), respectively.     

Highly enhanced bactericidal effects of medium chain fatty acids (caprylic,capric, and lauric acid) combined with edible plant essential oils (carvacrol,eugenol, β-resorcylic acid,trans-cinnamaldehyde,thymol, and vanillin) against Escherichia coli O157:H7

Medium chain fatty acids (MCFAs) and essential oils (EOs) are known to be natural antimicrobials, but their combined effects have not been fully investigated. The objective of the present study was to examine the bactericidal effects of various combined treatments of MCFAs [caprylic (CA), capric (CPA), and lauric acid (LRA)] and EOs [carvacrol (CAR), eugenol (EUG), β-resorcylic acid (RA), trans-cinnamaldehyde (TC), thymol (TM), and vanillin (VNL)]. Escherichia coli O157:H7, was treated with 1) control (2% ethanol), 2) MCFA alone, 3) EO alone, and 4) different combinations of MCFAs and EOs at 37 °C for 5 and 10 min. Synergistic bactericidal effects were observed with combined treatments; the log reduction in viable bacteria in response to the combined treatments was much greater than the sum of the effects of the two compounds applied individually. For example, individual treatment with 0.2 mM CPA (0.004%) and 0.4 mM RA (0.006%) for 5 min resulted in a negligible reduction in bacterial load (0.25 and 0.21 log reduction, respectively), whereas combined treatment at the same concentrations and for the same time reduced the bacterial population in the test sample to an undetectable level (initial population: 7.51 log CFU/ml; detection limit: 1 CFU/ml). The ranking of EOs showing the highest bacterial killing activities when combined with MCFAs was generally RA, CAR, TM > EUG > TC > VNL. All the antimicrobials used in this study are natural compounds that have been widely used in industry, so they are both consumer- and user-friendly. Combined treatment can overcome the disadvantages of MCFAs and EOs such as unpleasant odor and high cost because the required concentrations can be reduced. Our results indicate that the combined treatments used here could be successfully used to eliminate foodborne pathogens, significantly improving the microbiological safety of foods.     

Real-time pixel based early apple bruise detection using short wave infrared hyperspectral imaging in combination with calibration and glare correction techniques

High speed data processing for online food quality inspection using hyperspectral imaging (HSI) is challenging as over hundred spectral images have to be analyzed simultaneously. In this study, a real-time pixel based early apple bruise detection system based on HSI in the shortwave infrared (SWIR) range has been developed. This systems consists of a novel, homogeneous SWIR illumination unit and a line scan camera. The system performance was tested on Jonagold apples bruised less than two hours before scanning. Partial least squares-discriminant analysis was used to discriminate bruised pixel spectra from sound pixel spectra. As the glossiness of many fruit and vegetables limits the accuracy in the detection of defects, several reflectance calibrations and pre-processing techniques were compared for glare correction and maximizing the signal to noise ratio. With the best combination of first derivative and mean centering, followed by image post-processing, this system was able to detect fresh bruises in thirty apples with 98% accuracy at the pixel level with a processing time per apple below 200 ms.     

Moiety and linker site heterologies for highly sensitive immunoanalysis of cyprodinil in fermented alcoholic drinks

Cyprodinil is a new-generation anilinopyrimidine fungicide widely used in crop protection and frequently found in fruits. In this study, novel derivatives of cyprodinil with linker site heterologies were synthesized and employed in order to produce antibodies with enhanced affinity. Moreover, moiety-heterologous haptens were designed and prepared for assay sensitivity improvement. Two competitive enzyme-linked immunosorbent assays for the analysis of this active substance were developed using direct and indirect formats, achieving IC₅₀ values around 0.15 μg/L. Analytical figures of merit and usability of the optimized assays were evaluated with wine and cider as model food processed matrices. The obtained recoveries were from 90% to 120%, and the limit of quantification was in the 1–5 μg/L range. Finally, a monitoring study (n = 150) was performed to estimate the occurrence and the concentration of cyprodinil in commercial wine and cider products from different origins. We found that 28% of the analysed wine samples contained cyprodinil residues at levels higher than 5 μg/L.     

Bioaccessibility and changes on cylindrospermopsin concentration in edible mussels with storage and processing time

The alkaloid cylindrospermopsin has been recognized of increased concern due to the global expansion of its main producer, Cylindrospermopsis raciborskii. Previous studies have shown that bivalves can accumulate high levels of cylindrospermopsin. Based on the potential for human health risks, a provisional tolerable daily intake of 0.03 μg/kg-body weight has been recommended. However, the human exposure assessment has been based on the cylindrospermopsin concentration in raw food items. Thus, this study aimed to assess the changes on cylindrospermopsin concentration in edible mussels with storage and processing time as well as cylindrospermopsin bioaccessibility. Mussels, (Mytilus galloprovincialis) fed cylindrospermopsin-producing C. raciborskii, were subjected to the treatments and then analyzed by LC-MS/MS. Mussels stored frozen allowed a significantly higher recovery of cylindrospermopsin (52.5% in 48 h and 57.7% in one week). The cooking treatments did not produce significant differences in cylindrospermopsin concentration in the mussel matrices (flesh), however, cylindrospermopsin was found in the cooking water, suggesting that heat processing can be used to reduce the availability of cylindrospermopsin. The in vitro digestion considerably decreased the cylindrospermopsin availability in uncooked and steamed mussels, highlighting the importance in integrating the bioaccessibility of cylindrospermopsinin in the human health risk assessment.     

An improved method for microbiological testing of paper-based laminates used in food packaging

Food packaging materials fundamentally contribute to food quality and safety, as they protect the packaged food against external influences. In this context, the determination of the hygiene status of the packaging material is of great importance. However, European legislation neither sets any microbiological criteria nor provides any approved standard for the microbiological testing of food packaging materials. Nevertheless, reliable routine control is essential for guaranteeing high hygienic quality of packagings.With the aim to achieve a maximum recovery rate at low contamination levels, an improved experimental design was developed for the enumeration of the total colony count, yeasts and molds and Enterobacteriaceae on the surface of roll stock packaging materials. For this purpose, two different types of paper laminates were selected and exemplarily used as objects of investigation. Moreover, the performance of different growth media was compared for each microbiological parameter. This approach was followed by method validation using a selection of quantitative reference materials of representative microorganisms, including resistant forms of microbes such as bacterial endospores and fungal spores.     

Co-occurrence of aflatoxins and ochratoxin A in cereal flours commercialised in Turkey

In this study, we aim to determine co-occurrence of aflatoxins (AFs) and ochratoxin A (OTA) in cereal flours commercialised in Corum, Turkey. One hundred cereal flours were checked for target fungal metabolites between the years 2011 and 2013. The samples were analysed by high-performance liquid chromatography with fluorescence detection (HPLC-FLD) after immunoaffinity column (IAC) clean-up procedure. The method was successfully validated in accordance to European Union guidelines acceptance criteria for specificity, linearity, sensitivity, trueness and repeatability. All the results are well below the maximum limits specified in the EU legislation. AFs were detected neither wheat flour nor rice flour samples, while 66.7% of maize flours contained AFs with maximum concentration of 1.12 μg kg⁻¹. OTA was present in 26.7% of wheat flour, 41.7% of maize flour and 18.8% of rice flour samples, with mean levels of 0.247, 0.218 and 0.154 μg kg⁻¹, respectively. The co-occurence of AFs and OTA was found in 9 maize flour samples.     

A reliable and sensitive time-resolved fluorescent immunochromatographic assay (TRFICA) for ochratoxin A in agro-products

Immunochromatographic assays (ICAs) are considered as a suitable diagnostic tool for the detection of mycotoxins. Mycotoxins and especially, ochratoxin A are analytes with more demanding sensitivity requirements. To enhance the sensitivity of current immunochromatographic assays for ochratoxin A (OTA), a novel sensitive ICA was developed in this study. In the assay, microspheres enclosing fluorescent europium (III) [Eu(III)] nanoparticles (EuNPs) were used as a label for OTA monoclonal antibody (OTA-mAb) conjugation. Accordingly, assay was called time-resolved fluorescent immunochromatographic assay (TRFICA). The test strip was composed of three parts: a sample pad, nitrocellulose membrane and an absorbent pad. As for detection, a proper concentration of conjugated microspheres was pipetted into the microtube and sample extract was added to it. Then the strip was inserted into the tube and the fluid flow along the strip. The TRFICA results were obtained in 8 min and read by a portable TRFICA strip reader. The established method allows quantitative determination of OTA with limit of detection as low as 1.0 μg kg⁻¹ in the samples. For validation, spiked samples including wheat, maize, soybean and rice were respectively assayed by TRFICA and a standard high performance liquid chromatography equipped with a fluorescence detector (HPLC-FLD), and good agreement of results was obtained between two methods.     

Presence of mycotoxins in animal milk: A review

Mycotoxins can cause toxicity when ingested by humans and animals. Although the rumen is supposed to be a barrier against mycotoxins, some studies demonstrate that carry-over of mycotoxins to milk is possible. Different studies have found mycotoxin levels in animal milk, mainly related to contaminated feed for ruminants. Aflatoxin M1 is the most studied mycotoxin in milk and levels exceeding the EU maximum level for this mycotoxin in this matrix (0.050 μg/kg) have been found. Maximum levels in milk for other mycotoxins have not been established; however ochratoxin A, aflatoxins G1, G2, B1, B2 and M2, fumonisin B1, cyclopiazonic acid, zearalenone and its metabolites and deepoxy-deoxynivalenol have also been found in milk samples. Taking into account that multi-exposure to mycotoxins is the most likely scenario and co-occurrence of mycotoxins could affect their toxicological effects in humans and animals, there is a need to determine the co-occurrence of mycotoxins in milk.     

Aflatoxins and ochratoxin A in dried eggplant and green bell pepper

In this study, 50 dried eggplant and 50 dried green bell pepper samples were analyzed in terms of their aflatoxin and ochratoxin A (OTA) content. Aflatoxins G₂, G₁, B₂, and B₁, and OTA contents were analyzed using high-performance liquid chromatography with a flame ionization detector (HPLC–FID). Total aflatoxin and, as well as aflatoxin G₂, G₁, B₂, and B₁ content in dried eggplant samples were ranged between 0.82 and 2.58, 0.10–0.23, 0.32–1.35, 0.12–0.67, and 0.17–0.71 μg kg⁻¹, respectively. Total aflatoxin and, as well as aflatoxin G₂, G₁, B₂ and B₁ content in dried green bell pepper samples were 0.81–2.42, 0.11–0.22, 0.32–1.38, 0.13–0.66, and 0.18–0.91 μg kg⁻¹, respectively. OTA content was varied from 8.88 to 21.35 μg kg⁻¹ in eggplant samples, and from 15.38 to 24.70 μg kg⁻¹ in dried green bell pepper samples. Of the dried eggplant samples and dried green bell pepper samples, 36% and 24% of them, respectively, had aflatoxin B₁ values which were below the minimum limit of detection (LOD) of 0.05 μg kg⁻¹. None of the analyzed samples exceeded the legal limit values of 10 μg kg⁻¹ for total aflatoxin content, and 5 μg kg⁻¹ for aflatoxin B₁ content. However, 80% of the dried eggplant samples and 100% of the dried green bell pepper samples exceeded the legal limit value for OTA content (15 μg kg⁻¹). According to the results, it was concluded that dried vegetables should be examined in terms of their aflatoxins. It is essential to analyze OTA content more thoroughly, as it has the potential to pose a risk for public health, as well as for the economy.     

Determination of water-soluble vitamins in energy and sport drinks by micellar electrokinetic capillary chromatography

A method for the determination of water-soluble vitamins in several energy and sport drinks by micellar electrokinetic chromatography (MEKC) has been developed in this work. The separation of vitamins was studied in terms of background electrolyte composition (borate content, pH, surfactant type and content) and in other MEKC parameters. A study of the possible compounds found in the vitamin-enriched drinks that could interfere in vitamin determination was also performed, and a modified procedure with enhanced resolution was developed. The proposed method was successfully applied to the analysis of water-soluble vitamins in a variety of energy and sport drinks and also in fruit nectars. The method implies minimal sample preparation and reagent consumption, being environmentally sustainable. Thus, the proposed methodology could be useful for quality control purposes in the soft drink industry.     

Species determination of pine nuts in commercial samples causing pine nut syndrome

Consumption of pine nuts from the species of Pinus armandii has been reported to cause dysgeusia, commonly known as pine mouth, or pine nut syndrome (PNS). However, the number of reports on pine nut consumptions of the different species and PNS is limited. This leaves open the possibility that other pine species than P. armandii could be involved in PNS as well. This study investigated 18 samples involved in PNS and received at the Danish Veterinary and Food Administration in 2011 through 2012. Samples were subjected to gas chromatographic analysis of fatty acids. The content of 11 individual fatty acids was used together with the diagnostic index and the sum of Δ5-fatty acids as diagnostic parameters. Diagnostic parameters from samples were then compared to reference material and literature data to determine the species. In a limited number of samples, the diagnostic parameters matched neither our reference materials nor literature data. However, the morphology, the fatty acid analysis, and externally obtained DNA sequencing data suggest a P. armandii subspecies or a variety. With these possible P. armandii subspecies, P. armandii was identified in all analyzed samples. The application of principal component analysis (PCA) to the data set showed a satisfactory separation of the majority of the 13 pine species included in the study.     

Determination of multi-mycotoxin occurrence in maize based porridges from selected regions of Tanzania by liquid chromatography tandem mass spectrometry (LC-MS/MS), a longitudinal study

Residents of certain areas of Tanzania are exposed to mycotoxins through the consumption of contaminated maize based foods. In this study, 101 maize based porridge samples were collected from villages of Nyabula, Kikelelwa and Kigwa located in different agro-ecological zones of Tanzania. The samples were collected at three time points (time point 1, during maize harvest; time point 2, 6 months after harvest; time point 3, 12 months after harvest) over a 1-year period. Ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) was used to detect and quantify 9 mycotoxins: aflatoxin B₁ (AFB₁), aflatoxin B₂ (AFB₂), aflatoxin G₁ (AFG₁), aflatoxin G₂ (AFG₂), fumonisin B₁ (FB₁), fumonisin B₂ (FB₂), deoxynivalenol (DON), ochratoxin A (OTA) and zearaleneone (ZEN) in the samples following a QuEChERS extraction method. Eighty two percent of samples were co-contaminated with more than one group of mycotoxins. Fumonisins (FB₁ + FB₂) had the highest percentage occurrence in all 101 samples (100%) whereas OTA had the lowest (5%). For all three villages the mean concentration of FB₁ was lowest in samples taken from time point 2. Conversely, In Kigwa village there was a distinct trend that AFB₁ mean concentration was highest in samples taken from time point 2. DON concentration did not differ greatly between time points but the percentage occurrence varied between villages, most notably in Kigwa where 0% of samples tested positive. ZEN occurrence and mean concentration was highest in Kikelelwa. The results suggest that mycotoxin contamination in maize can vary based on season and agro-ecological zones. The high occurrence of multiple mycotoxins found in maize porridge, a common weaning food in Tanzania, presents a potential increase in the risk of exposure and significant health implications in children.     

A structural modeling on food safety knowledge,attitude, and behaviour among Bum Bum Island community of Semporna,Sabah

The objective of this study was to evaluate the relationship among food safety knowledge, attitude and behavior in Bum Bum Island community, Semporna, Sabah. Proportional stratified sampling method was used in this survey. A total of 250 respondents were selected randomly from ten villages in Bum Bum Island. Face-to-face interview was conducted to complete the questionnaire. In general, respondents exhibited average food safety knowledge level especially in their awareness of personal hygiene and kitchenware hygiene. Food safety attitude of the community was found strongly affected their food safety behavior in positive way, which was proven by the highest standard β among variables tested (β₁ = 0.885, p < 0.05). However, food safety knowledge was negatively affected the food safety behavior of the respondents (β₁ = −0.128, p < 0.05). Our result confirmed that Structural Equation Modeling (SEM) was successfully used to model the relationship among food safety knowledge, attitude and behavior.     

Mass spectrometry quantification of beef and pork meat in highly processed food: Application on Bolognese sauce

Food frauds have become a very important issue in the field of food quality and safety. The risk of food adulteration is higher in highly processed food and mainly affects high added value foodstuff. The methods currently available to face this issue, PCR and ELISA, are very sensitive and specific, but they have some limitations. In the present work, tandem mass spectrometry is presented as an emerging approach to detect beef and pork meat in very complex and highly processed food matrices, such as Bolognese sauce, both in qualitative than in quantitative way. The detection is achieved using two different marker peptides, specific for beef and pork meat, both deriving from α2-collagen chain. Then, a calibration curve is set up using real sauces made by different percentages of pork and beef meat in a working range from 0 to 100%. The method here developed allows to quantify beef and pork meat in a complex product such as Bolognese sauce.