Development of a determination method for atropine and scopolamine in various human foods (Bachelor thesis)

Dalampira, Maria

This graduation thesis is about the development of a determination method for the tropane alkaloids, atropine and scopolamine and the analysis of various food categories for the presence of these compounds. Atropine and scopolamine are two of the most known tropane alkaloids. They originate from edible plants or from plants that are accidentally collected with the crops. They can be highly toxic to humans and deadly to infants and children. Analysis of atropine and scopolamine by LC-MS/MS is one of the methods recommended by EFSA for the monitoring of tropane alkaloids. However, a few detailed analytical procedures have been reported. The method combines great sensitivity and selectivity along with fast and accurate analysis. After experiments with different extraction solvents, such as acetonitrile instead of methanol and with or without the addition of formic acid, the following extraction solvent was used: CH3OH:H2O (3:2)+HCOOH 0.2%+ACN 0.2%. It showed good recovery results, efficiency and repeatability and great abundance of the peaks of atropine and scopolamine. Experiments with two different food samples, flour from the Italian market and imported noodles, were performed and the flour was chosen as the matrix for the preparation of the calibration curve. The calibration curves that were prepared with flour had very good linearity results, as R2 was both for atropine’s and scopolamine’s calibration curves equal to 0.9999. The samples that were checked for contamination belonged to seven different food categories: flour, pasta, tea/herbs, spices, cake, unknown samples and gallettes with rice. A total of sixty-two samples were analyzed. The contamination level was considered to be equal to 1ppb. Eighteen samples were found contaminated. Most of them were flour made from buckwheat of different Italian brands. The majority of the contaminated samples showed contamination of atropine and no contamination of scopolamine, while the samples that were contaminated with both alkaloids showed higher contamination in atropine than in scopolamine. The results can be explained by the ratio of these substances in the original plants from where these three samples were originated. A variety of other tropane alkaloids were found and identified in buckwheat, seeds (semi datura) and pulverized leaves (foglie datura) from the plant Datura stramonium. In the other food categories, a spice (fieno greco) was contaminated with scopolamine. Three out of ten unknown samples (one brand of baby food and two brands of flour) were contaminated with atropine and scopolamine.
Institution and School/Department of submitter: Σχολή Τεχνολόγων Γεωπόνων και Τεχνολογίας Τροφίμων και Διατροφής/Τμήμα Τεχνολογίας Τροφίμων
Subject classification: Food additives
Πρόσθετα τροφίμων
Food contamination
Μόλυνση τροφίμων
Keywords: Scopolamine;Σκοπολαμίνη;Atropine;Ατροπίνη;Human foods;Ανθρώπινες τροφές
Description: Μεταπτυχιακή εργασία--Σχολή Τεχνολόγων Γεωπόνων και Τεχνολογίας Τροφίμων και Διατροφής--Τμήμα Τεχνολογίας Τροφίμων,2015--7058
Table of contents: INTRODUCTION 1. TROPANE ALKALOIDS 8 1.1 Information about tropane alkaloids and their importance 8 1.2 Historical background 9 1.2.1 The main plants from which atropine and scopolamine derive (Datura Stramonium, Atropa Belladonna and Mandrake) 9 Datura Stramonium 9 Atropa Belladonna 11 Mandragora Officinarum (mandrake) 13 1.3 Atropine and Scopolamine Characteristics 14 1.3.1. Atropine 15 1.3.2. Scopolamine 16 1.4. Contamination-Poisoning Cases 17 1.4.1. Contamination 17 1.4.2.Occurrence in food and food poisoning cases 17 A selection of cases of human poisonings by atropine and scopolamine 18 2. AIM OF THE RESEARCH 20 3.LIQUID CHROMATOGRAPHY-MASS SPECTROMETRY 21 3.1.Liquid Chromatography. 21 3.1.2. The parts of an HPLC instrument 21 3.1.3. Types of elution 22 3.1.4. The reason why the chromatography is usually reverse-phase. 22 3.1.5 The basis of the substance’s separation in LC 23 3.1.6. Column categories. 23 3.1.7. Retention time and its importance in LC 23 3.1.8. The importance of mass spectrometry’s advantages 23 3.1.9. Mass spectrometry 24 3.1.10. The functionality of a mass spectrometer 24 3.1.11. Tandem (MS/MS) mass spectrometers. 25 3.1.12. Εlectrospray Ionization. 25 3.1.13. Positive and negative ionization. 27 3.1.14. The analysis of tropane alkaloids with LC-MS/MS. 27 4. MATERIALS AND METHODS 28 4.1. Food Samples 28 4.1.1. Classified food samples list 28 4.1.2. Chemicals and Reagents 30 4.1.3. Instruments and materials 30 Instruments 30 Materials 31 4.1.4. Preparation procedures 31 Preparation procedure of standards solutions atropine and scopolamine 31 Extraction procedure of the food samples for the analysis 31 4.2. Instrumentation of LC-MS/MS system 32 4.3 Determination of LOD and LOQ 33 5. Development of the chromatographic and mass spectrometric method 33 5.1. Atropine and Scopolamine 33 5.2. Gradient profile, Diagrams, Tables and development of the method 34 5.3. Examination of the best extraction solvent with flour samples 39 5.3.1. First extraction and sample preparation 40 5.3.2. Second extraction samples-experiments with acetonitrile 42 Repeatability results 43 5.3.3. Third extraction experiments – Recovery results of the extraction solvents in four different food samples 47 6. Analysis of the samples 52 6.1. Preparation of the food samples 52 6.1.1. Selection of the food sample for the matrix of the calibration curve. 53 6.2 Analysis of natural and spiked samples 55 6.2.1. Preparation of the food samples 55 6.2.2. Table of contents for the spiked samples-Recovery results of the food samples according to the two different matrixes used for the preparation of the calibration curve. 56 6.2.3 Preparation of the following samples for analysis 59 6.3 Results of the chromatographic analysis of all the natural samples in the research conducted for tropane alkaloids 63 6.3.1. Results of the samples that were analysed and discussion of the results 63 6.4 Mass scan results of Semi Datura, Foglie Datura and Buckwheat. 66 7. Discussion 68 8. References 70
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