Akrilamid ve biyojenik aminler için gıda örneklerine yönelik kapiler elektroforetik analiz yöntemlerinin geliştirilmesi
Development of capillary electrophoretic analysis methods for acrylamide and biogenic amines in food samples
Yazar: SELDA BAŞKAN KAHRAMAN
Danışman: PROF. DR. F. BEDİA ERİM BERKER
İstanbul Teknik Üniversitesi / Fen Bilimleri Enstitüsü / Kimya Ana Bilim Dalı
Derece: Doktora, 2012
Konu: Kimya = Chemistry
Dizin: Akrilamitler = Acrylamides ; Biyojenik aminler = Biogenic amines

Özet:
Gıda ürünlerinin hazırlanış basamaklarında gıda ürünleri içinde doğal olarak bulunan bazı bileşenler yeni kimyasallara dönüşebilmektedir. Gıda son ürününün hazırlanması sırasında oluşan bu yeni bileşiklerin bir kısmı toksik, alerjen ve/veya kanserojen özellikler gösterebilmektedir. Son yıllarda insan sağlığını olumsuz yönde etkileyecek gıda bileşenlerinin analizleri için yeni ve etkin analiz yöntemlerinin geliştirilmesi önem kazanmıştır. Bu çalışmada gıdanın kendi içinde doğal olarak bulunan bileşenlerin gıda işlemi esnasında kimyasal reaksiyonlar sonucu oluşturdukları toksik gıda bileşenlerinin analizleri için yeni kapiler elektroforetik analiz yöntemleri geliştirilmesi hedeflenmiştir.Akrilamid uzun yıllardan beri çoğunlukla poliakrilamid polimeri eldesi için ticari olarak üretilmektedir. Hayvanlar üzerinde yapılan deneyler sonucunda akrilamidin memeliler için genetik toksik ve mutajenik olduğu kanıtlanmıştır. Akrilamid Uluslararası Kanser Araştırma Kurumu (IARC) tarafından ?İnsanlar için olası kanserojen madde? olarak sınıflandırmıştır.Akrilamid'in ilk kez 2002 yılında ısıl işlem gören gıda ürünlerinde oluştuğu keşfedilmiştir. Bu tarihten bu yana uluslararası gıda kuruluşları her yıl marketteki gıda ürünlerinin akrilamid içeriklerini web sitelerinde yayınlamaktadır. Özellikle patates kızartması ve bisküvi, kek ve ekmek gibi çocukların çok tükettiği ürünlerde akrilamid oluşumu konuyu güncel ve önemli kılmaktadır.Biyojenik aminler özellikle proteince zengin gıdalarda ilgili aminoasitlerin dekarboksilasyonu sonucu oluşan bileşiklerdir. İnsan ve hayvanların biyolojik fonksiyonlarında önemli rollere sahip olmalarına rağmen, yüksek dozlarda alımı insanlar üzerinde alerjen ve toksik özellikler göstermektedir. Bu gruptan histamin alerjik etkisi en çok araştırılan biyojenik amindir. Putresin ve kadaverin gibi biyojenik aminler ortamda bulunan nitrat iyonları ile reaksiyon vererek kanserojen nitrosaminlere dönüşebilmektedir.Kapiler elektroforez (CE) analiz yöntemleri ile en küçük anorganik iyonlardan, DNA gibi en büyük büyük moleküllere kadar geniş spektrumdaki bileşiklerin yüksek elektrik alan altında ayrılması ve tayini mümkündür. CE yüksek ayırma gücü nedeniyle özellikle karmaşık yapıdaki matriksler için uygun bir yöntemdir. Son yıllarda gıda analizlerinde kapiler elektroforetik yöntemlere olan ilgi giderek artmaktadır. Bu amaçla hızlı, uygulanması kolay ve düşük tayin sınırlarında yeni kapiler elektroforez yöntemleri geliştirilmektedir.Bu tez çalışmasının ilk adımında akrilamidin, bir gıda matriksi içinden yüksek elektrik alan altında ayrılarak aynı anda nicel ve nitel tayini için yeni bir susuz ortam kapiler elektroforez (NACE) yöntemi geliştirilmiştir. Analiz yöntemi, sulu ortamda yüksüz olan akrilamid molekülünün, organik çözücü içerisinde asit-baz karakterinin değişmesine ve pozitif yükle yüklenmesine dayanmaktadır. Yüklü akrilamid molekülü, yüksek elektrik alan altında yürütülerek gıda matriksinin diğer bileşenlerinden ayrılarak UV detektör ile 200 nm dalga boyunda tespit edilmiştir. Akrilamid piki 3,5 dakika gibi kısa bir sürede görülmüştür. Yöntemin akrilamid için belirleme sınırı 0,041 µg/mL ve tayin sınır 0,137 µg/mL olarak bulunmuştur. Yöntem kızarmış patates ve patates cipsi örneklerinde akrilamid tayinine başarı ile uygulanmıştır.Çalışmanın ikinci basamağında, biyojenik aminlerin nicel ve nitel tayini için lazer indüklenmiş floresans dedektörle birleştirilmiş, yeni bir yüksüz misel elektrokinetik kromatografik analiz yöntemi geliştirilmiştir. Floresan bir boya ile türevlendirilen altı biyojenik aminin aynı anda ayrılması ve tayini 10 dakikadan kısa sürede gerçekleştirilmiştir. Kullanılan yüzey aktif madde (Brij 35), türevlendirilmiş biyojenik amin moleküllerinin floresans duyarlığını arttırmış ve bu şekilde literatürde rapor edilen tayin sınırlarının altına inilmiştir. Yöntemin belirleme sınırı 0,067- 0,11 ng/mL arasında, tayin sınırı 0,22-0,37 ng/mL arasında tespit edilmiştir. Yöntem salamura ve tuzlu balık örneklerinde biyojenik aminlerin tayinine başarı ile uygulanmıştır.

Abstract:
During food production steps, some components which naturally exist in food products may form new chemicals. These new chemicals formed during food preparation may be toxic, allergen and/or carcinogenic. In recent years, development of new and effective analysis methods have become crucial for analysis of food components which affect human health negatively. In this study, it is aimed to develop new capillary electrophoretic methods for the analysis of toxic food ingredients which are formed during food processes as a result of chemical reactions of naturally existing food components.Acrylamide has been produced commercially for many years mainly for the production of polyacrylamide polymer. Experiments on animals have proven that acrylamide is possibly a genetic toxic, mutagenic and carcinogenic for mammals. Acrylamide has been classifed as ?probable carcinogen compound for humans? by the International Agency for Research on Cancer (IARC).Acrylamide was first detected in heat-treated foods in 2002. It is supposed that acrylamide in food is largely derived from heat-induced reactions between the amino group of the free amino acid asparagine and the carbonyl group of reducing sugars such as glucose during baking and frying, by the so-called Maillard reaction, though the production mechanism of acrylamide in food has not been exactly explained. Since that date, international food organizations have been publishing annual acrylamide contents of food products sold in markets on their web sites. Intensive activity began examining the many different types of food and thousands of analyses have been undertaken worldwide. Formation of acrylamide in products like fried potato and biscuit,cake and bread which are consumed by children, makes the issue important and actual.Biogenic amines are compounds formed especially in protein-rich foods, such as fish and meat, by the decarboxylation of the related amino acids. Although they have important roles in biological functions of humans and animals, high doses cause allergenic and toxic effects on humans. Histamine, belonging to this group, is the biogenic amine of which allergenic effect has been researched mostly. Moreover, putrescine and cadaverine have been suggested to potentiate histamine toxicity. Biogenic amines like putrescine and cadaverine may transform to carcinogen nitrosamines by reacting with nitrate ions in the medium. High amounts of biogenic amines in foods are considered indicators of spoilage. For this reason, it has great importance of quantify the individual biogenic amines in food samples by reliable separation and detection methods.Separation and determination of a wide spectrum of components from smallest inorganic ions to biggest molecules like DNA is possible under high electrical field with capillary electrophoresis (CE) methods. CE is a suitable method, especially for complex matrices; due to its high separation power. Capillary electrophoresis method has been increasingly popular for food analysis in recent years.In the first step of this thesis study, a new nonaqueous capillary electrophoresis (NACE) method was developed for simultaneous qualitative and quantitative determination of acrylamide. The analysis method is based on the fact that, acrylamide molecule which is a non-ionic molecule in aqueous medium, changes its acid-base character in an organic solvent and can be positively charged by low concentrations of a strong acid. Ionic acrylamide molecule was separated from other constituents of food matrix under high electrical field and determined at 200 nm wavelength with UV detector. The electrophoretic behavior of acrylamide together with N, N-dimethylformamide and benzamide was tested in three different HClO4 concentrations, i.e., 10, 20, and 30 mmol/l, respectively. The runnig electrolyte was chosen as 30 mmol/l HClO4 and 218 mmol/L CH3COOH in in acetonitrile for nonaqueous separation. The separation was performed at 25 kV. The temperature was set at 25º C. Injections were made at 40 mbar for 6 s. The total length of the capillary was 52.5 cm and the length to the detector was 44 cm. The detection limit of the method for acrylamide was found as 0.041 µg/mL and the limit of quantification was found as 0.137 µg/mL. The run-to-run and day-to-day precisions with respect to the corrected peak areas were found as 1.65% and 3.90% respectively. The method was successfully applied to acrylamide determination in fried potato and potato chips samples. Potato chips and French fries were ground in a blender and 2 g portions from fried samples and 4 g portions from the chips were weighed into a 50 mL centrifuge tube and 10 mL of ACN was added. These samples were shaken for 1 hour, and then 5 mL hexane was added, defattting was done three times. After the suspension was centrifuged at 4000 rpm for 2 min, it was filtered from 0.2 mm micro filter and directly injected. The acrylamide content in the tested french fried sample was determined as 0.187 ± 0.019 mg/kg for three independent extractions of the same sample. The acrylamide amount of tested chip sample was found as 2.95 ± 0.11 mg/kg for three separate extractions. The recovery values of the spiked samples at different concentrations are found between 90 and 102%. Acrylamide contents in food samples investigated in this study are in agreement with the reported values. Published values for potato chips vary between 0.17? 3.7 mg/kg and for French fries vary between 0.2?12 mg/kg. The presence of acrylamide in foods is by now certain. However, it is seen that its quantity is very different in even similar food samples. It is suggested from our present study that the CE method presented here is a method that can be easily used by many laboratories especially for the rapid and economical comparison of different food processing.In the second step of the study, a new nonionic micellar electrokinetic chromatographic method combined with laser-induced fluorescence detector was developed the analysis of biogenic amines. Because of the fact that biogenic amines show poor sensitivity of UV absorption detection , the combination of LIF detector with capillary electrophoretic separations provided a remarkable improvement in detection limits. The separation and determination of six biogenic amines derivatized with a fluorescent dye was achieved in less than 10 minutes. We used FITC as derivative agent which is a common dye used in the CE-LIF analysis of biogenic amines. The runnig electrolyte was chosen 10 mM Brij 35 in 75mM borate buffer at pH 9.7 . As a result of preliminary optimization studies, fivefold molar excess of total biogenic amine concentration was found sufficient for FITC I isomer concentration. In order to derivatization step the mixture vial was capped and stand in the dark at 40 0C for 4 h.This temperature and time were selected optimal by monitoring the conditions at which the peak areas reached the maximum. This solution was diluted 1000-fold in two steps with distilled water and injected into the capillary column. Brij 35, as a nonionic surfactant, was the first time tested for the separation of biogenic amines. Complete resolution of six biogenic amines ? FITC derivatives ? was achieved in less than 10 min, employing 10mM Brij 35 in 75mM borate buffer and at pH 9.7 as the running electrolyte. Relative fluorescence intensities of biogenic amines enhanced considerably and separation time decreased considerably when Brij 35 was substituted for SDS in the same buffer. The used non-ionic surfactant, Brij 35, increased fluorescence intensity of derivatized biogenic amine molecules and by this way it has been achieved to low detection limits for biogenic amines compared to those reported in literature.. The separation was performed at 25 kV. The temperature was set at 250C. Injections were made at 50 mbar for 6 s. The total length of the capillary was 63 cm and the length to the detector was 47 cm.For the precision of the method, the biogenic amine mixture was injected five times in 1 day. For the day-to-day reproducibility, the same solution was injected five times in three different non-consecutive days. In the same day, precisions of the corrected peak areas (%RSD) were between 1.98 and 3.24. Between days, precision values were lower than 5.6. Detection limits of the method for six biogenic amines were determined between 0.067 and 0.11 ng/mL.; the limits of quantification were determined beetween 0.22 and 0.37 ng/mL. The method has been applied successfully to the determination of biogenic amines in brined and dry-salted fish samples. Two traditionally processed fish products, brined Atlantic bonito (Sarda sarda) and dry-salted sardine (Sardina pilchardus), were obtained from a local fish processing company in Trabzon, Turkey. Fish samples were homogenized in a hand blender. Five gram of the blended fish samples was extracted twice with 10 mL portions of 6% TCA using a vortex mixer for 2 min. The supernatants were combined after centrifugation (3500 rpm) for 10 min and filtered through a Whatman 41 filter paper and then through a 0.45-mm membrane filter. The pH of solution was arranged to pH 7 with NaOH solution and diluted to 25 mL with deionized water. Since amino acids in the fish content also react with FITC, the optimal concentration of FITC in the initial derivatization mixture was selected by gradually increasing FITC concentration, monitoring the increases in peak heights of biogenic amines in the samples, and observing the excess FITC peak in electropherograms to ensure complete derivatization. The derivatized sample solutions were kept in the dark at 40ºC for 4 h, this solution was diluted with distilled water between 20- and 1000-fold for experiments and injected into the capillary column. The Lakerda sample includes a considerable amount of tyramine, histamine, and putrescine. In the sardine sample, cadaverine and histamine are the main amines and small amount of tyramine was also detected.The biogenic amine type and amount are depended on the type of fish as well as processing conditions. It is suggested from our present study that the MEKC-LIF method presented here is a method that can be easily used by many laboratories especially for the rapid and economical comparison of different food processing. The high sensitivity of the method leads to the analysis of biogenic amines in low concentrations.