Separation of biogenic amines by capillary electrophoresis coupled with contactless conductivity detection and determination in fermented foods

Foods and food products are frequently investigated for the determination of their contents with the intentions of to be ensure about the safety of the food and to be aware of their potential toxicity and the quality of raw matarails and food processing technolgy. Biogenic amines are biologically active, nitrogen based, low molecular mass substances and they consider as quality and toxicity indicators of foods. The amount of biogenic amines inidcates also the freshness of the food products. These compounds form during production or storage of protein rich foods under unhygenic conditions. Although these compounds form in metabolic processes, their levels should not exceeded the limits which they are poses a risk for health. Histamine, tyramine, tyrptamine, cadaverine and putrescine are some of these biogenic amines and histamine is the most well-known biogenic amine due to its high toxicity. The adverse effects of excess intake of biogenic amines mainly by histamine and tyramine from foods such as fish and cheese cause food poisoning. Some of these symptoms are known as headache, vomiting, nausea and gastrointestinal problems and more severe cases they can cause even deaths. Biogenic amines mostly found in fish, meat and meat products and diary products which are rich in protein. Decarboxylation of free amino acids results the formation of desired biogenic amines in foods. Moreover, these biogenic amines are naturaly occurs in some vegetables and fruits; which were also detected in wines and cause changing in sensory properties of the product. These amines are usually formed in protein rich foods by the act of microorganisms which has decarboxylase activity under inappropriate technological processing conditions especially while fermantation processes. Fermented foods such as cheese, yoghurt and traditional foods such as kefir contain high amount of protein and these foods are widely consume in our country and these foods can be either fermented by industrially or by home made processes which can be result by the formation of biogenic amines during production. Milk is a great source of protein and it is the raw material of all types of fermented foods thus it is conveinent for the formation of biogenic amines. These circumstances outweigh the importance of controlling factors affected the biogenic amine formation during processing and storage. Development of rapid, reliable, repeatable and highly effective methods in order to determine of these compounds in fermented foods is a significant research area. Capillary electrophoresis (CE) is a novel technique for seperation and quantification of analytes ranged from small ions to large DNA molecules, gained great importance in last decades and widely used due to its numerous advantages over other chromatographic techniques. CE has a high seperation power as a result of the applied high voltages through narrow capillaries filled with buffer solution where seperation takes place. This technique offers short analysis time, low chemical consumption, high resolution power and feasibility of determination of analytes in complex matrices; due to the capillary tubes which are less expensive and can be regenerated easily. Capillary electrophoresis has several modes of application to perform an electrophoretic separation. Among these modes capillary zone electrophoresis is the simplest and widely using mode. During the separation process, all charged particles moves in a buffer solution, toward to the cathode pole where detector is positioned and all analytes separeted in bands related with their different charge-mass ratios. The movement of all ionic species in one direction is related with the bulk flow called elecroosmotic flow (EOF) result of the negatively charged inner capillary wall influced from high electrical field. Formation of an electrical double layer results with the movement of solvent with the ionic species solvated in that buffer soluton. All charged particles can be analysed by CZE without any modifications. All sample ions in CE are injected from anodic side and detected from cathodic side by the detector in the order of their own electrophoretic mobilities under EOF. The small and highly charged cations are moving at faster rates while small and highly negative charged particles are reached detector finally. In this thesis study, a novel, simple and rapid CZE method was developed for the determination of histamine, putrescine, cadaverine and spermidine in fermented foods: cheese, yoghurt which were purchased from local markets, yoghurt and kefir which were fermeted from milk by not using industrially. Determination of biogenic amine contents of various foods and beverages have been investigating with the aid of CE for several decedes and the detection of these compounds usually achieved by indirect UV detection or flourescence detection. Flourescence detection of biogenic amines can only achieved after derivatization which results long reaction times. Direct detection of biogenic amines with high resolution and sensitivity has been performed by using condutometric techniques recently as a new method of detection is introduced. Contactless conductivity detection (C4D) is a new technique which has many advantages compared to the other conductometric detectors. While C4D coupled with CE, electrodes is not directly contact with the solution thus prevents the coupling problems caused by contact of the solution with electrodes. This technique offers fast and direct detection of all charged ions during separation according the difference in their conductivity from background electrolyte which is buffer solution itself. The cell of C4D composed of 2 tubular electrodes around capillary, positioned apart from few milimeters from each other. After AC is applied to the first electrode, current passes through the cell and collected from second electrode and than the signal is amplified and indicates the difference in conductivity between buffer solution and analytes. Biogenic amines has high pKa values and in acidic contiditions they can be determined as cations and they were detected by C4D directly. The optimum buffer solution composition was selected 15 mM HIBA and %0,01 (w/v) HPMC. The pH was adjusted to 3,57 with Tris solution in order to prevent increase in conductivity and joule heating. The addition of HPMC was affected EOF by decreasing the rate to improve resolution between analytes. Proposed method for determination of biogenic amines and combined with direct detection was successfuly applied to food samples. The separation procedure was carried out in a narrow bore capillary tube with 50 µm inner diameter. The effective length of capillary tube was 65 cm. The voltage applied was 28 kV. The sample preparation procedure was simple, the food samples were homogenized and weighned. Extraction procedure was performed by adding % 6 (w/v) TCA solution and using a vortex mixer for 2 minutes and than centrifuged for 15 minutes. The solution was filtered and injected to the capillary after dilution and filtration through microfilters. Except spermidine, histamine, putrescine and cadevrine were found in all samples. Calibration curves of these amines were showed lineer range between 0,2 - 6,0 ppm and correlation coefficients were between 0,9981-0,9996. The method introduced ensures a very simple sample pretreatment process with very short analysis time with a good precision and reproducibility. Mixture of standard solution included four biogenic amines was injected 5 times in a day and intraday repeatability were calculated. % RSD of migration time of the amines were ranged between 0,24 and 0,28 and for corrected peak areas % RSD values were ranged between 2,35 and 4,25. Interday repeatability for migration times and corrected peak areas were also calculated as % RSD and results ensures good repeatability. The limit of detections (LOD) for biogenic amines were improved and found (S/N=3) in between 22,79 - 31,60 ng/mL and limit of quantifications (LOQ) were found (S/N=10) in between 75,98 - 105,34 ng/mL. Recoveries of four biogenic amines were calculated by adding standart mixtures of biogenic amines on fermented food samples and the obtained results were in the good range in between % 88,73 and 104,65. There are only limited number of studies in literature investigated the biogenic amine contents of foods by CE coupled contactless conductometric detection and it can be concluded that the study is one of the first report which was investigated biogenic amines in fermented foods such as cheese, yoghurt and kefir with CE-C4D.