Quality Control and Food Safety

Innovative Analytical Methodologies Control
of Quality and Food Safety

Research Lines


The objectives of this program represent a higher step in the research line which began in the year 2005, in part because new researchers have joined the original groups but mainly because the experience obtained in the past consortium. The good scientific results, the analytical platform now available, the scientific and technical knowledge developed, and the positive interactions among the participants will allow developing new analytical methodologies to be applied, broadening our research field, to new analytes, new foods and new analytical problems. Our final objective is to be able to establish an effective control of food quality and safety, from the production line to the consumer.


In the new program, new methodologies will be developed to deal with new aspects of safety control, -spanning a broader spectrum of toxic compounds in foods-, and of food quality control, covering research lines related to food adulteration, characterization and composition, and to the changes produced through the food processing.


With these objectives, advanced methodologies will be developed (or previous methodologies will be adapted) in order to include the detection of new exogenous toxic contaminants, introduced unintentionally in the alimentary chain, such as pharmaceuticals (antibiotics, anti-inflammatory and antiparasitic), estrogens and emerging toxic contaminants (chiral PCBs, PXBs, PCBs metabolites and new flame retardants (HBCDs), compounds (PFCs).


Related with quality control, its three most important aspects are included in the objectives: adulteration by addition of non-authorized products (such as azoic colorants) or by illegal changes (which can be or not intentional) in the composition, as in the case of chiral aminoacids: determination of natural components (iminosugars, inositols, oligosaccharides) and authorized ingredients and determination of Se species in enriched foods. Finally, methods to detect and to control undesirable changes during food processing, such as products derived from phenols and acrylamide, from triglyceride oxidation, from degradation of terpenes and polyphenols, and the D-aminoacids produced through fermentation processes.



OBJECTIVE 1: New methodologies for the detection of emerging toxic food.


New methodologies were developed for the detection of toxic and persistent pollutants (POPs) from industrial activities, agriculture (pesticides of new generation), livestock (antibiotics, antiparasitic, anti-inflammatory and anti-estrogens), and toxics generated during food processing (products from acrylamide ).


Description of activities including the research to be undertaken and the methodology.

1.1     Chemical pollutants from industrial activities. New methodologies for the detection of methylsulfone polychlorinated biphenyls derivates (MeSO2-PCBs), polychloro-brominated biphenyls (PXBs), compounds (PFCs) and brominated flame retardants (BFRs) such as hexabrominated cyclododecane (HBCDs), 1,2-bis-(2,4,6-tribrominated-phenoxy)-ethane (BTBPE) and decabrominated diphenyl ethane (DBDPE), all considered emerging contaminants, toxic and persistent of industrial origin, for which there are currently no methods of analysis in food. Also Hg compounds (inorganic mercury and methylmercury) will be studied due to their high toxicity. Both, sample preparation (mainly based on PLE and MSPD) and instrumental techniques based on GC, GC x GC and HPLC coupled to MS in different modes (MS / MS; ToF, QMS, QqQ) will be studied. Analytical methodologies developed will be validated and applied to food of animal origin.


1.2     Pollutants from agricultural activities: New methodologies for the detection of the latest generation of pesticides (triazine, organophosphates and pyrethroids) will be developed. These pesticides, commonly used on fruits, which are considered toxic contaminants from agricultural sources, will be detected to levels below the maximum limits allowed by current legislation. Miniaturized simple preparation methods will be developed. They will be mainly based on pressurized liquid extraction (PLE), dispersion of the solid phase matrix (SPMD) and cartridge solid phase extraction method using molecular imprinted polymers, enabling the extraction and simultaneous purification in different types of fruit. The instrumental determination will be carried out using GC and GC x GC with ECD and MS and detection systems. The analytical methodologies developed will be validated and applied to samples of plant foods: fruits, extracts, etc.


1.3     Pollutants from livestock: It is proposed to extend the spectrum of drugs studied in the previous program (Analisyc), developing new methodologies based on the development of molecular imprinting polymers (MIPS) to estrogens (estriol, estrone, -estradiol and ethinylestradiol) anti-inflammatory (diclofenac) and antiparasitic (albendazole, fuberidazol, Rabeprazole, thiabendazole, benomyl). Synthesis of selective MIPs , characterization and analytical evaluation of the polymers targeted to prepare the sample in a single step will be studied. Their use as selective sorbents in SPE, or as stationary phases in HPLC and CE, will provide a new tool for the rapid determination of those toxics in meat, dairy products, fruit and water. On the other hand, we also seek to prepare and characterize new mesoporous silica for the preparation of extraction columns for sample preparation prior to the determination of the mentioned compounds. The instrumental determination will be carried out by HPLC with DAD and fluorescence and MS detectors.


1.4     Toxic Pollutants (acrylamide) generated during food processing. In the previous program (Analysic) we developed new analytical methods to detect the formation of acrylamide during the frying process in cereals (forest chips) and potatoes, as well as rapid methods for coupled on-line in the production chain. Those studies found the existence of various factors affecting the formation of acrylamide as well as various strategies to minimize their formation. Some mitigation strategies are based on the use of specific antioxidants and phenolic compounds, so the program aims to develop specific analytical methodologies for those new structures. To this end, the analytical techniques used in the analytical platform created by the consortium will be used. The analytical methods developed in sections 1.1 to 1.4 will be validated and applied to foodstuffs samples, some of them provided by commercial supermarket and other by industries associated with the program


The milestones that can be use to measure its progress.


1.1.    Development of miniaturized sample preparation for the analysis, based on PLE and MSPD of emergent persistent toxic pollutants and pesticides.


1.2.    Developing methods for simultaneous sample preparation techniques based on MISPE and MSPD for the selective extraction of estrogens and veterinary drugs.


1.3.    Preparation and characterization of stationary phases based on mesoporous silica hybridized to prepare samples for analysis of estrogens, drugs and Hg.


1.4.    Application of the methodologies MSPD and PLE developed for the determination of emerging POPs and pesticides based using GC-MS and HPLC-MS in dairy products and fruits, respectively.


1.5.    Application of the methodologies PLE and MSPD developed, as well as mesoporous stationary phases for the determination of estrogens and pharmaceuticals using HPLC-DAD, HPLC-fluorescence and HPLC-MS in milk products and drinking water.


1.6.    Development of sensitive methods for detecting the reaction products of acrylamide-phenols


Role of each partner (research groups, laboratories and/or associated partners).


The firms Pascual and Agilent Technologies will participate in all the activities. In the activity 1.1. will participate CSIC-AI, CSIC-IQAB, UNED-QA y UAH-QA; in the activity 1.2 CSIC-AI, URO520, UAH-QA and UNED-QA; in the activity 1.3. will participate UNED-QA, UAH-QA y UCM-QA, and URO520 (REDLAB) In the activity 1.4. will CSIC-AA and Laboratory 160 (REDLAB)


OBJECTIVE 2: New analytical methodologies for the detection of food adulterations.


Description of the activities including the research to be performed and methodology.


2.1 Determination of azoic colorants: Sudan I-IV and Para-Red in sauces and tomato preparations.Recently the presence of some red dyes, namely Sudan I-IV, which is prohibited in the EU, has been detected on food imported from third countries (China, Mexico, Nigeria, etc). The Para-Red is an allowed dye, although it is expected that it will be prohibited in the near future. The determination of these compounds in complex mixtures is laborious, which makes it necessary to develop simple and robust new analytical method. The proposed methodology is based on the use of molecular imprinting polymers (MIPS), including synthesis, characterization and analytical assessment of their potential for these analytes, and the MPSD method for the extraction step, “on-line” coupling with chromatographic techniques (HPLC-DAD). Finally, the methods developed will be validated


2.2 Determination of non protein amino acids in foods (olives, soybean or sunflower seeds, olive oil, seed oils, infant formulas, dietary foods, sport supplements, etc.). There exist a great number of amino acids which are not constituents of proteins (non protein amino acids) and that have scarcely been investigated although they can have a high potential for the detection of adulterations in foods. The two following cases will be investigated: i) The determination under achiiral conditions of a non protein amino acid characteristic of a given seed or seed oil in other vegetable oil (i.e., olive) would allow to propose such amino acid as quality marker due to its potential for detecting adulterations. ii) Different non protein amino acids are being employed frequently for the elaboration of different foods for human consumption: infant formulas, sport supplements, dietary foods, etc. (i.e., carnitine). Most of them are chiral and due to the different biological activity of their enantiomers, there exist regulations controling the use of one of these enantiomers for the preparation of foods forbidding the use of the racímate. The enantiomeric determination of non protein amino acids would enable the detection of adulterations in foods due to the use of the racemate instead of the corresponding pure enantiomer. In both cases, the determination of non protein amino acids has a high potential in the control of the food quality and safety. For this reason, the development of new strategies enabling the development of analytical methodologies under achiral and chiral conditions and being rapid, sensitive, and with low economic and environmental cost is proposed in this project. Optical and mass spectrometric detection will be employed and the developed methodologies will be applied to the analysis of commercial foods.


2.3 Analysis of the protein fraction of vegetable foods. Innovative and rapid analytical methodologies will be developed for the separation and determination of vegetable proteins using HPLC and Capillary Electrophoresis techniques with optical and mass spectrometry detection. With this aim, sample preparation will be optimized in order to obtain the protein extract to be analyzed by the separation techniques. New protein markers will be investigated for the detection of possible adulterations in foods.


The milestones that can be use to measure its progress.


2.1 Development of an analytical methodology based on HPLC-DAD coupled on-line to an extraction system based on MISPE cartridges for the determination of azoic colorants (Sudan I-IV and Para Red) in commercial tomato sauces.


2.2 Development of achiral analytical methodologies based on the use of Capillary Electrophoresis for the determination of non protein amino acids in fruits (olive), seeds (soybean, sunflower), and vegetable oils.


2.3 Development of achiral analytical methodologies based on the use of Capillary Electrophoresis for the enantiomeric determination of non protein amino acids in infant formulas, dietary foods, sport supplements, etc.


2.4 Development of analytical methodologies for the analysis of the protein fraction of vegetable foods in order to investigate new markers for adulterations.


Functions of each partner (research groups, laboratories and/or associated).


The firms Pascual, Enantiosep, Campofrio Alimentación and Agilent Technologies will participate in all the activities UNED-QA (activity 2.1 and hit 2.1) and UAH-QA and URO 520 (activities 2.2, 2.3 y 2.4 and hits 2.2, 2.3 y 2.4) are involved in this objective.


OBJECTIVE 3: New analytical methodologies for the determination of natural components and/or allowed ingredients in foods, including legume and cereal cultivars or their derivatives, fruits, vegetable oils, enriched foods, food extracts and food formulas.


Description of the activities including the research to be performed and methodology.


3.1 Characterization of carbohydrates with biological activity (iminosugars, inositole derivatives, and oligosaccharides) in vegetable extracts, including samples prepared in the laboratory and commercial products. New advances methodologies based on the use of separation techniques (GC y HPLC) coupled to mass spectrometry (MS) will be developed. Samples will be provided by Puleva-Biotech, prepared in our laboratory or acquired in specialized shops. Different fractionation techniques will be assayed in order to optimize the concentration of the compounds studied. In general, PLE and solvent extraction will be employed (including the possibility of using extraction with ionic liquids, ILE), while for the separation of the target compounds from other with similar characteristics, adsorption or partial fermentation will be used. The determination of non polar compounds with low molecular mass being apolar (volatile) or polar (iminosugars or inositole compounds using previous derivatization) will be achieved using GC-MS. The determination of compounds with a high molecular mass (oligosaccharides) will be accomplished by LC-MS and direct infusion in the ESI-MS system.


3.2 Analysis of Se enriched foods and evaluation of their healthy characteristics Antioxidant and anticarcinogenic characteristics of Se are well known. For this reason, in this project, the preparation of enriched Se products such as fermented dairy products, vegetables and fish will be prepared. Se species in fermented dairy products, fish, and plants will be investigated. The compounds resulting of the fermentation and accumulation processes will be determined and their antioxidant and anticarcinogenic characteristics will be established. ICP-MS and ESI-MS coupled to chromatographic separation techniques will be employed for their determination.


3.3 Quantitative determination of bioactive peptides in legumes, cereals, and comercial formulas. The research activities in the field of bioactive peptides are increasing considerably as shown by the development of functional foods and of pharmaceutical formulations containing them. Although a significant number of studies have been performed in order to investigate the presence of bioactive peptides, the research aimed to develop analytical methodologies for the quantitative determination of bioactive peptides in foods is very scarce. In this project, advances analytical methodologies will be developed enabling the quantitative determination of bioactive peptides in legume (soybean) and cereal (barley, maize) cultivars as well as in their food derivatives. With this aim, HPLC or micro/nano-HPLC with optical or mass spectrometry detection will be employed. Moreover, a second objective of this project will be to study the bioactivity of commercial formulas such as infant formulas prepared from soybean hydrolizates. Peptides contained in these formulas will be separated and isolated and their bioactivity will be investigated. Those peptides with the highest bioactivity will be identified and quantified in the commercial formulas studied.


3.4 Food characterization based on the study of the protein fraction. Innovative and rapid analytical methodologies will be developed for the separation and determination of vegetable proteins using HPLC and Capillary Electrophoresis techniques with optical and mass spectrometry detection. With this aim, sample preparation step will be optimized previously in order to obtain the protein extract to be analyzed by means of the above-mentioned separation techniques. The developed methodologies will be applied to the characterization of fruits (olive) and seeds (soybean, sunflower) as well as their oils in order to achieve the characterization and differentiation of genotypes with important implications in their quality and cost.


The milestones that can be use to measure its progress.


3.1 Development of analytical methodologies based on GC-MS enabling the control of the concentrations of target compounds in natural extracts as well as for the characterization of other compounds with a possible biological effect.


3.2 Characterization of the antioxidant and anticarcinogenic activities of Se products.


3.3 Identification and quantification of Se species.


3.4 Development of analytical methodologies based on HPLC and Capillary Electrophoresis for the

analysis of the protein fraction of vegetable foods.


3.5 Development of analytical methodologies based on HPLC and micro/nano-HPLC for the separation and quantification of bioactive peptides in foods.


Functions of each partner (research groups, laboratories and/or associated).


Agilent Technologies will be involved in all activities. CSIC-AI, AR-GAMA, UCLM-OENNAT y PUEXCAL will participate in activity 3.1, UCM-QA and Ganaderias Priegola in the activity 3.2, and UAH-QA, URO520, Novozymes, Pascual, Enantiosep, Novozymes andCampofrio Alimentación in the activities 3.3 y 3.4.


OBJECTIVE 4: New methodologies for determining the effect of processing on the composition, quality and security of foodstuffs.


Description of the activities, including the research and methodological plans.


Research activities will focus on the evaluation of the effects of a variety of processes, including frying, boiling, fermentation, etc on the quality and security of selected foodstuffs. To achieve this goal, both the compounds formed during processing and their possible interactions among them and with other food components will be evaluated.


4.1 On the basis of the knowledge achieved through the development of the ANALISYC program, new analytical methodologies will be developed to investigate and characterise the chemical structures formed due to the interaction between acrylamide and certain phenols in model system and foodstuffs. The experimental conditions involved in these studies will reproduce those of backing and frying. The analytical instrumental techniques required for these investigations, i.e. HPLC with either DAD, MS or qToF, are available at CSIC-AA and CSIC-USTA (REDLAB#166). This activity is close related to objectives 1 and 3.


4.2 Compounds formed by oxidation of the lipids (epoxy- ceto- and hydroxy-triglecerides and polymerisation compounds). Many of these compounds have a profound effect on food quality and are potentially toxic. Their identification of the main analytes formed by the oxidation of the lipids require the use of selective chromatographic techniques, such as GC-MS, GC-MS/MS, HPLC-MS, HPSEC-RID and HPLC-ELSD. This activity is also close related to objectives 1 and 3. 4.3 Study of the volatile compounds formed by the Maillard reaction. The volatile compounds will be extracted by direct thermal desorption (OPTIC), head-space and SPME, and will be determined by GC-MS and GCxGC-MS. Achieving this goal will require the optimisation of the extraction conditions as well as the careful selection of the column combinations and chromatographic parameters (modulation time and temperatures, etc). The optimisation of the analytical methods will be done first on the base of model mixtures containing aminoacids and hydrocarbons; later on, more complex systems will be evaluated. The possible relationship among the formation of certain pirazines and that of acrylamide will also be investigated.


4.4 Effect of processing on the composition of the volatile and phenolic fractions for processed juice and vegetables. The composition of the volatile fraction of these processed foodstuffs will be evaluated by simultaneous distillation-extraction (SDE) using polar and non-polar solvents. Environmental friendly extraction techniques, i.e. SPME and SFE will also be evaluated. The obtained extracts will be analysed by GC-MS. When required because of the extremely high of the generated extracts, multidimensional separation techniques based on the direct coupling RPLC-GI via the interface TOTAD will be used. Both chiral and non-chiral columns will be tested for as complete as possible characterization of the investigated foodstuffs. The phenolic fraction of the foodstuffs will be isolated by using SFE and the obtained extracts will be analysed by HPLC, HPLC-MS and RPLC-GC with the TOTAD interface.


4.5 Determination of selenium species and chiral aminoacids in fermented foodstuffs. The possible biotransformation of Se (IV) by either lactic fermentation (yogurt, cheese, etc) or bioaccumulation in plants and fishes will be evaluated and the generated species of Se in each case will be identified by ICP-MS ESI-MS coupled to chromatographic systems. Capillary electrophoresis will be used for the characterisation of derivatised non-protein aminoacid enantiomers in the same fermented foodstuffs.


Milestones which can be used in order to monitor the progress in the objective activities.


4.1 Characterization of products of the reaction between acrylamide and phenols, and development of advanced analytical methods for its control in processed food.


4.2 Development of analytical methodologies aimed to the characterization of products of lipidic oxidation, both in foods and in samples simulating gastrointestinal digestion.


4.3 New methodology related to the analysis of volatile compounds from Maillard reaction.


4.4 Development of analytical methods based in multidimensional techniques for the quality control of turmeric, strawberries and raspberries.


4.5 New analytical methods designed for the determination of non-protein chiral aminoacid enantiomers in fermented foods


4.6 New analytical methods designed for the determination of selenium species in fermented lactic foods.


Functions of each research partner (research groups, laboratories and associated groups).


The following groups will take part in this objective: CSIC-AA and 160 (REDLAB) (activities described in 4.1 and 4.2), CSIC-AA, UCLM-OENNAT and CSIC-AI (activity 4.3), CSIC-AI andUCLM-OENNAT (activity 4.4), UCM-QA, UAH-QA, Novozymes, Ganaderias Priegola, Enantiosep, Pascual y Campofrio (activity 4.5)


TRANSVERSAL OBJECTIVE.  Development of new techniques for sample preparation and fractionation; advances in the validation and in the optimization of analytical data; integration of instrumental techniques through the implementation of a shared instrumental platform.


Description of the activities, including the research work to be carried out and its methodology.


This transversal objective covers the advances to be carried out in methodologies shared by all the research groups, such as sample preparation and data processing, included in several activities described under the objectives 1 to 4. Both these methodologies and the equipment included in the proposed platform are of common interest to all the participating groups. Although the development of the related techniques will basically correspond to one or two groups, their shared use by all research groups is expected.


5.1 Volatile component fractionation. Determination of sample volatile components requires adapting fractionation techniques, such as SPME, DTD, SDE, etc., to the characteristics of the different samples and components. Volatile components to be determined include those produced during Maillard reaction or through food processing


5.2 Compound fractionation from their polarity. In several objectives, a global determination of components sharing common chemical characteristics is considered. In these cases, techniques to be optimized include organic solvent extraction, PLE, SFE and ILE, studying the effect of the different operation variables relevant in each case.


5.3 Sorptive fractionation. Sorptive fractionation will be carried out by SPE extraction using new stationary phases, including those based on hybrid mesoporous silica. The methodology will be applied to different sample types, for the separation of antibiotics, hormones, heavy metals, etc.


5.4 Specific fractionations. This methodology includes the preparation, characterization and evaluation of new molecular imprinted polymers (MIPs) and materials for matrix solid-phase dispersion (MSPD) which will be used in selective separations. They will be packed in SPE (MISPE) cartridges, or as stationary phases in separation techniques, to be applied to benzimidazols, antibiotics, estrogens and food artificial colourings.


5.5 Chemical reactions. This section includes new strategies for chemical transformation (derivatization, hydrolisis) as a previous step to their chromatographic or electrophoretic analysis, to be applied to proteins, aminoacids, carbohydrates, etc. These strategies will be addressed to a reduction of the time required for protein hydrolysis or for aminoacid derivatization, using techniques such as the focused ultrasound probe.


5.6 Technique optimization. It will be carried out from experimental variable ranges defined in previous assays, and by usin statistical techniques. Optimization will be based on the increase of both precision and recovery for the fractionation technique and relevant compounds, and it will take into account the total cost and the environmental impact. A similar strategy will be followed when the optimization of other steps of the method will be required.


5.7 Data processing. The methodologies which are currently applied for validation support (advances in accuracy and precision) and for optimization (estimation of the chromatographic response) will be improved.


5.8 Instrumental platform. It will include the instrumental equipment, both currently available and to be purchased in the near future, of shared used between the participants. Among them are fractionation systems and analytical instruments (GCs, HPLCs, CEs, MS, hyphenated techniques (GC x GC, RPHPLC x GC, etc,), assigned to the different groups as described under objectives 1-4. The shared use of these instruments will be regulated by specific protocols, taking into account the good results of the last four years in the past program.


The milestones that can be use to measure its progress.


No specific milestones can be pointed out for this objective, since its results will be techniques integrated into analytical method which will be validated as a whole.


Functions of each partner (research groups, laboratories, associated)


One or two research groups will carry out the development of each method or technique listed for the described activities (objectives 1-4),. However, probably all research groups will use the sample preparation or the data processing techniques, since they can be used in the method implementation for most of the above mentioned activities.