Improvement of methods and techniques for identification and determination at nanoscale of steroid structures contaminants in food, CONTALIM

• To elaborate modern methods which use high performance analytical techniques as: High performance liquid chromatography coupled with mass spectrometry (LC-MS) or LC-MS/MS, gas chromatography coupled with mass spectrometry (GC-MS) to determine the contaminants with steroid structure from different complex matrices, at nanoscale level;
• To identify and determinate the metabolites that can be formed due to the interactions of steroid compounds with the animal or human organism;
• To use (micro)extraction techniques in solid phase SP(M)E;
• To use purifying and concentrating techniques before chromatographic determination;
• To create an integrated database and to correlate it with international quality standards.

• to rise the research capacity of ICIA;
• to facilitate the integration of ICIA research in „food quality control” field of the international collaboration system;
• to achieve values exchange at European level
• to learn and apply modern, high performance investigations methods;
• to facilitate a faster communication between both partners;
• to find out the results in the qualitative and quantitative determinations of steroids structure veterinary drug residues in animal originated food;
• to extend the collaboration to other fields, including analysis quality assurance field, by performing inter-laboratories comparisons;
• to extend the knowledge about modern analysis methods of contaminants steroids structures in human consumption products.

• Publications:

1. internal and external scientific journals,
2. in international conferences proceedings;

• Monitoring reports;
• Database.

Stage I
Stage duration: June 2008 – Nov. 2008

I.1 Official trip to Science University Eötvös, Budapest
Purpose: establishing the chemical compounds which contaminate the food.
Results:

I.1 Official trip to Science University Eötvös, Budapest Purpose: establishing the chemical compounds which contaminate the food. Results:

• evaluation of ICIA’s technology;
• analysis of method’s characteristics;
• identification of international trends in analysis methods of food contaminants;
• establishment of the contaminant chemical compounds;
• establishment of optimal methods with quantification at nanoscale;
• method’s characteristics;
• instruction course for ICIA’s personnel in ELTE University: course of liquid chromatography coupled with mass spectrometry (LC-MS) with triple quadrupol and time of flight, presented by EKOL Group – Budapest;
• technical-economical report.

I.2 Receiving of the official call from the Science University Eötvös Loránd, Budapest
Purpose: elaboration of methods for qualitative and quantitative determination of chemical residues in food.
Results:

• an official visit at S.C. Jidvei S.R.L. – Jidvei, jud. Alba;
• sampling and samples transportation from S.C. Jidvei S.R.L. – Jidvei, jud. Alba at ICIA Laboratory for analysis.

I.3 Activities foreseen in the research program of the young researcher
Purpose: elaboration of methods for samples preparation, before chromatographic analysis: extractions.
Results:

• obtaining extracts from food (various types of extraction methods);
  * training course for ICIA’s personnel in ELTE University: course for the use of Twister (magnetic stirrer coated with polymer) – Gerstel (Agilent Technologies) (extractions SPDE, SPME).

Stage II
Stage duration: Dec.2008 – Nov. 2009

II.1 Official trip Eötvs Loránd University, Budapest
Purpose: Qualitative determination of chemical contaminants
Results:

• qualitative determination of chemical compounds with steroid structure: estrone, β-estradiol, cholesterol in aqueous sample matrices by SPE-GC-MS/MS;
• qualitative determination of compounds with steroid structures: cholic acids (cholic acid, acid litocholic, chenodeoxicholic acid, ursodeoxicholic acid, 3-hydroxy 7-ketocolanic acid, dehidrocholic acid), from aqueous samples by derivatized in two stages, then detection by gas chromatography coupled with mass spectrometry with ion trap (GC-MS/MS);
• qualitative determination of organochloride pesticides (OPC) and polychlorinated biphenyls (PCBs) by injecting standards and samples under the same chromatographic conditions and comparing the retention times of pure standards with those of sample analytes separated by column chromatography;
• techno-economic report stage.

II.2 Receiving visit from the Eötvös Loránd University, Budapest
Purpose: quantitative determination of chemical contaminants (etalons, calibrations)
Results:

• quantitative determination of organochloride pesticides (OPC) and polychlorinated biphenyls (PCBs) in milk and grapes;
• official visit to S.C. Lech Lacto – Lechinta, Bistrita-Nasaud;
• sampling and transportation of samples from S.C Lech Lacto – Lechinta, Bistrita-Nasaud to ICIA lab for analysis.

II.3 Activities foreseen in the research program of the young researcher
Purspose: Elaboration of sample preparation methods, prior to chromatographic analysis: purifications, concentrations
Results:

• elaboration of methodologies for sample preparation prior to chromatographic determination, by performing the purification and concentration using: on solid phase extraction (SPE), solid phase microextraction (SPME), liquid-liquid extraction and application their different tests for determining the structure of compounds contaminated with steroid (estrone, estradiol) and organochloride pesticides and polychlorinated biphenyls;
• determination of steroid hormones in aqueous and organic matrices complex (serum) by the technique of GC/MS, after microextraction and derivatization on fiber.

Final report

Collaboration between the two teams has led to the accomplishment of the Hungarian-Romanian project objectives. Research activity in the analysis of steroid structure of food contamination led to the development and improvement of methods for identification and detection of contaminating steroids in aqueous and biological matrices. Determination of contaminants in food was performed using chromatographic techniques by the application of advanced extraction methods, sensitive and accurate. Degree of achievement of objectives The first stage of the project led to goals accomplishment, namely:

• Romanian partner was trained to the ELTE University, Budapest, in high resolution chromatographic determination;
• Romanian and Hungarian partners have proposed methods for analysis of food (different types of grapes) for identification and quantification of organochloride pesticides and PCB in grapes;
• developed modern methods, cutting-edge, using advanced analytical techniques;
• was hired a young scientist;

The second stage of the project led to goals accomplishment, namely:

• Romanian team with partner team members have made a qualitative determination of chemical compounds with steroid structure: ♦ estrone, ♦ β-estradiol, ♦ cholesterol in aqueous sample matrix, by the presentation of fragmentation modes of complete derivatizated species of steroid compounds and their derivatives trimethylsilyl ether / ester (oxime) with GC-MS/MS software tool (with ion trap);
• was performed the quality determination of pesticides and polychlorinated biphenyls organoclorurate by injecting pure samples and samples under the same chromatographic conditions and comparing the retention times of pure standards with those of sample analytes separated by column chromatography;
• was performed quantitative determination of compounds in samples of food contaminants;
• were determined organochorinated pesticides and polychlorinated biphenyls in food samples (milk and grape). Organochlorined pesticides (OCPs) and polychlorinated biphenyls (PCBs) were determined from samples of varieties grape: Muscat Ottonel (MO), Sauvignon Blanc (SB) and Italian Riesling (RI), harvested from the vineyard Jidvei, the 2009 harvest, method of analysis is the extraction fluid -liquid, followed by gas chromatographic analysis;
• was visited on dairy Lech-Lacto Lechinta;

Program young researcher hired chem. Lacrimioara Senila consisted of:

• developing a methodology for sample preparation prior to chromatographic determination, by performing the purification and concentration by:
  • solide phase extraction (SPE);
  • solide phase microextraction (SPME);
• methods were applied in an analysis to determine the structure of compounds with steroid contaminants and organochlorined pesticides (OCPs) and polychlorinated biphenyls (PCBs);
• results were disseminated through publication and communication.

It have been implemented in the REZALIM – ICIA laboratory the following methods developed in this project:

1. Method of solid phase extraction (SPE) that allows simultaneous separation and concentration of samples;
2. Method for multiresidual, qualitative and quantitative determination of contaminants (Chlorinated pesticides, Polychlorinated biphenyls and Chlorobenzenes) in various foods by liquid-liquid extraction and GC-ECD detection;
3. Qualitative determination of steroid hormones (estrone, β-estradiol, cholesterol) in aqueous matrix by solid phase extraction, derivatization in two stages (oxymation and silylation) and detection by gas chromatography coupled to linear ion trap mass spectrometry (SPE-GC -MS/MS);
4. Qualitative determination of organochlorinated pesticides and polychlorinated biphenyls in milk and grapes samples;
5. Quantitative determination of organochlorinated pesticides and polychlorinated biphenyls in milk and grapes samples by liquid-liquid extraction and detection by gas chromatography with electron capture detection (LLE-GC-ECD);
6. Method for the determination of steroid hormones in aqueous matrix by GC-MS, after solid phase microextractions (SPME) and fiber derivatization;
7. Method for the determination of steroid hormones in biological matrix by GC-MS after solid phase microextraction (SPME) and fiber derivatization.

The obtained results can be the basis for national or European project proposals in the further development and validation methods;
The proposed methods for the determination of chemical contaminants in different matrices (water, food) can be used to monitor drinking water quality and different food samples during the technological flow (from raw materials to finished products, both for producers and for distributors);
Method for the determination of steroid hormones (estrone, β-estradiol, cholesterol) in liquid matrices by SPE-GC-MS/MS with double-derivatization (oxymation and silylation) and Method for the determination of steroid hormones (estrone and β-estradiol) in samples with complex liquid matrix (waste water and blood serum) by solid phase microextraction (SPME) with head-space on fiber derivatization with MSTFA and analysis by gas chromatography coupled with mass spectrometry can be patented.

The obtained results were disseminated, certifying their quality:

1. Lacrimioara Senila, Mirela Miclean, Cecilia Roman, Cornelia Majdik, Gyula Záray, Determination of steroid hormones in Somes River water by Solid Phase Microextraction and Gas Chromatography – Mass Spectrometry, Studia Universitatis Babes-Bolyai, seria CHEMIA, 2009. ISSN 1224-7154 (Category A, ISI)
2. Mirela Miclean, Lacrimioara Senila, Adriana Gog, Cecilia Roman, Ioan Groza, Multicomponent determination of organochlorinated compounds in milk by gas chromatography with electron-capture detection after liquid-liquid extraction, Bulletin UASVM 2009. (Category B+)
3. Mirela Miclean, Adriana Gog, Anca Naghiu, Cecilia Roman, Multirezidual analysis of organochlorine pesticides, chlorobenzenes and polychlorobiphenyls in different varieties of Romanian grapes, Environment and Progress, vol. 13, 2009. ISSN 1584-6733 (Category C, CNCSIS Code 697/2008)
4. Mirela Miclean, Lacrimioara Senila, Cecilia Roman, Erika Levei, Gyula Zaray, Ioan Groza, Comparison of DI-SPME-GC-MS and SPE-GC-MS with derivatization for analysis of steroid hormones in river water, Revista de Chimie, ISSN 0034-7752, (Category A, ISI). (sent to publication)
5. Senila Lacrimioara, Miclean Mirela, Levei Erika, Roman Marius, Roman Cecilia, Determination of steroid hormones by solid phase microextraction and mass spectrometry in poultry serum, 16th Symposium on Analytical and Environmental Problems, SZAB Szeged, Hungary, 28 September 2009, POSTER + ARTICLE (in proceeding)
6. Lacrimioara Senila, Mirela Miclean, Erika Levei, Cecilia Roman, Cornelia Majdik, Ana Maria Incze, DI-SPME-GC-MS analysis of steroid structures in poultry serum, International Conference on Chemistry, 15th Edition, 12-15 November, 2009, Targu-Mures, Romania, POSTER + ARTICLE (in proceeding).

There have been proposed two doctoral theses on the project, which run at the USAMV Cluj-Napoca (Anca Naghiu – Research on the incidence and hygienic significance of polycyclic aromatic hydrocarbons in food chain, and Mirela Miclean – Methods at nanoscale level for endocrine disruptor determination in environmental samples and biological fluids);

• There has been made a master on project theme (Anca Naghiu – at USAMV Cluj-Napoca – Chromatographic analysis of red wines quality from Romania);
• USAMV Cluj-Napoca shown great interest in the results because the food chain is a reality and quality of life is inextricably linked to food quality.

CS II Dr. Cecilia Roman
Tel. 0264-420.590, 0755038705
Fax. 0264-420.667
Email. icia@icia.ro, mailto:cecilia.roman@icia.ro