Bebek mamalarındaki nitrat ve nitrit iyonlarının kapiler elektroforez (CE) yöntemi ile tayini
Determination of nitrate and nitrite ions in baby foods by capilar electrophores (CE) method
Yazar: NIGAR KAMILOVA
Danışman: PROF. DR. AYŞEGÜL GÖLCÜ ; ÖĞR. GÖR. ZEYNEP KALAYCIOĞLU
İstanbul Teknik Üniversitesi / Lisansüstü Eğitim Enstitüsü / Kimya Ana Bilim Dalı / Kimya Bilim Dalı
Derece: Yüksek Lisans, 2022
Konu: Kimya = Chemistry

Özet:
Uzun yıllar boyunca yapılan çalışmalar, sağlıklı beslenme ile insan sağlığı arasındaki ilişkinin doğru orantılı olduğunu göstermiştir. İnsan sağlığı üzerindeki olumsuz etkilerinden dolayı "nitrat ve nitrit" içerikli maddelerin, belirli dozların üstünde gıdalarda bulunmaması gerekmektedir. Bu nedenle, gıdalarda nitrat ve nitrit analizi büyük önem taşımaktadır.
Nitrat ve nitrit iyonları insan vücuduna  mineral ve vitamin bakımından zengin olan sebzelerin yanı sıra, su ve hazır gıdalar ile  girmektedir. Alınan nitratın % 80'ni vücuttan atılırken, kalan kısmı ağızda bulunan bakteriler vasıtası ile nitrite indirgenmekte ve yutma yolu ile mideye taşınmaktadır. Nitrit iyonlarının sağlığımız  üzerindeki negatif etkilerinden birincisi, nitritin nitrozamin oluşumuna neden olmasıdır. Nitrozaminler, hücre DNA'sının yapısına etki eden  kanserojen bileşiklerdir. İkinci olumsuz etkisi ise nitritin  vücudumuzda oksijen taşıma görevi yapan hemoglobin ile birleşerek methemoglobin oluşturmasıdır. Bu durum kanın  dokulara oksijen taşıma fonksiyonunu bozmakta veya tamamen durdurmaktadır. 'Methemoglobinemi' olarak bilinen bu hastalık yetişkinlerde nadiren görülse de  çocukluk döneminde daha sık görülmekte ve 'mavi bebek sendromu' olarak da adlandırılmaktadır. 
Bebek mamalarındaki nitrat ve nitrit miktarı, mamanın içeriğine göre değişmektedir. Sebze bazlı bebek mamalarındaki nitrat ve nitrit miktarının sabit değerde olmamasının sebebi, sebzelerin çeşitliliği ve yetiştirilme koşullarının farklı olmasıdır. Tarım zamanı toprağa gereğinden fazla gübrenin verilmesi, hasat zamanının düzgün planlanmaması sebzelerde nitrat birikimine yol açmaktadır. Nitrat birikimi olan sebzelerden üretilen gıdaların bebek sağlığı üzerindeki olumsuz etkilerinden dolayı, nitrat ve nitrit iyonlarının tayini önemli bir gıda kontrol konusu olmuştur.
Kapiler elektroforez uygulanan yüksek  elektrik alan altında yüklü taneciklerin göç etmesi prensibine dayanan, ayırmanın silika kapiler kolonda gerçekleştiği, etkin bir ayırma ve tayin etme metodudur. Katyonların, anyonların, karbohidratların, vitaminlerin, ilaçların, polinükleotidlerin ve aminoasitlerin ayrılması gibi çok geniş çalışma alanına sahiptir. Az miktarda örnek ve çözücü kullanımı, analiz süresinin kısa olması, yüksek ayırma hızı, analiz sırasında kullanılan silika kolonun ucuz olması  kapiler elektroforezin diğer kromatografik yöntemlere göre avantajlarıdır.
Bu tez çalışmasında, kapiler elektroforez ile  bebek beslenmesinde büyük tehlike arz eden, "nitrat ve nitrit"in kalitatif ve kantitatif tayinini yapmak için hassas, güvenilir metod geliştirilmesi hedeflenmiştir. Kapiler elektroforez cihazı ile UV dedektör kullanılarak bebek mamalarındaki nitrat ve nitrit miktarının kantitatif tayini için hassas bir yöntem geliştirilmesi üzerinde çalışılmıştır. Çalışma hassasiyetinin artırılması amacıyla örnek sıkıştırma metodu uygulanmış, asidik çalışma ortamı (pH=4.00) seçilmiştir.  Ayırma tamponu olarak, 30 mM formik asit ve 30 mM sodyum sülfat (pH=4.00) kullanılmıştır.

Abstract:
Studies conducted for many years have shown that the relationship between healthy nutrition and human health is directly proportional. Due to their negative effects on human health, nitrate and nitrite are undesirable substances to be found in foods above certain doses. For this reason, nitrate and nitrite analysis in foods is of great importance.
Nitrate and nitrite ions are inorganic ions that are easily soluble in water. It can enter the human body with water and ready-made foods, as well as vegetables that are nutritious in terms of minerals and vitamins. While 80% of the nitrate taken is excreted from the body, the remaining part is reduced to nitrite by the bacteria in the mouth and carried to the stomach by swallowing. The first of the negative effects on human health is that nitrite causes the formation of nitrosamines. Nitrosamines are carcinogenic compounds that affect the structure of cell DNA. The second negative effect is that nitrite combines with hemoglobin, which carries oxygen in our body, to form methemoglobin. This situation impairs or completely stops the oxygen-carrying function of the blood to the tissues. This disease, known as 'methemoglobinemia', is rarely seen in adults, it is more common in childhood and is also called 'blue baby syndrome'.
The amount of nitrate and nitrite in baby food varies according to the content of the food. The reason why nitrate and nitrite amounts in vegetable-based baby foods are not constant is due to the variety of vegetables and different growing conditions. Giving too much fertilizer to the soil during agriculture and not planning the harvest time properly cause nitrate accumulation in vegetables. The determination of nitrate and nitrite ions is an important food control issue due to the negative effects of foods produced from vegetables with nitrate accumulation on infant health.
In the literature, there are studies based on chromatographic methods such as HPLC, IC, GC for the analysis of nitrite and nitrate anions in various foods. However, it has been accepted by the AOAC International Official Analysis Methods that nitrate and nitrite analysis be carried out in food control laboratories with a spectrophotometric method based on the reduction of nitrate to nitrite with cadmium. This accepted method does not allow for the simultaneous determination of nitrate and nitrite ions, as well as long sample preparation processes. In this study, a new method has been developed for the combined determination of nitrate and nitrite ions in baby foods by capillary electrophoresis.
In capillary electrophoresis, all species, regardless of their charge, move towards the cathode and the detector placed in this direction, thanks to the electroosmotic flow created under a very high electric field in the silica column. They are detected by separating according to differences in electrophoretic mobility. However, since the electrophoretic mobility of small anions such as nitrite and nitrate is very high, they have difficulty in being dragged towards the negative electrode with the electroosmotic flow. Some modifications must be made to separate and detect these ions. 
In this study, by lowering the pH of the separation electrolyte, the electroosmotic flow rate was greatly reduced and the anions moved to the detector in the positive pole direction against the electroosmotic flow with their own electrophoretic mobility. In this method, the injection was made from the negative pole. 
In the literature, there are studies in which nitrate and nitrite are separated by this method. However, nitrate and nitrite ions could not be determined in baby foods. After the literature review, it was decided to apply the sample stacking method.  By using the sample stacking method in capillary electrophoresis, the injection volume used in normal capillary electrophoresis can be increased. On the other hand, some changes are made in the conductivity of the separation electrolyte and the sample zone, so that the analyte is concentrated between the sample zone and the buffer zone. Thus, steep peaks are obtained and the detection limits are significantly reduced. When the normal capillary electrophoresis mode is compared with the sample compression mode, it was seen that the detection sensitivity for both ions increased by 30 times.
Acetonitrile is a low-conductivity compound. In this thesis study, In this thesis work, how acetonitrile affects the sample stacking method was investigated. As a result of the study, it was seen that acetonitrile caused nitrate and nitrite peaks to appear steeper. Excellent separation efficiency was observed when the sample solution contained 10.0 % (v/v) ACN. 
Nitrate nad nitrite anions were determined in 8 baby food sample using the optimized CE- sample stacking method. After weighing 0.1 grams, the baby foods were extracted with 10 ml of 60 degrees deionized water. Then, the suspensions we obtained were kept in a magnetic stirrer at 60 degrees for 15 minutes and in an ultrasonic bath for 30 minutes. After passing through the 0.45 μm micro filter, the determined amount of ACN was added and it was made ready for weighing.
In this study, Agilent capillary electrophoresis (Waldbornn, Germany) device was used. Detection was done at 210 nm by means of a UV detector. The separation voltage was -25 kV, and measurements were performed at 25 0C. The column where ion migration takes place is Polymicro Technologies (Phoenix, AZ/America) brand and has a total length of 65 cm, an effective length of 57 cm, and an inner diameter of 50 μm. Agilent ChemStation program was used to analyze the data.Samples, standards and buffer solutions were given to the device through 1.5 mL vials. Injections were made in 160 seconds by applying 50 mbar pressure, and the separation was carried out at 25℃ by applying -25kV.
Calibration curves showed linearity in the concentration range of 1.5-50 µmol/L for nitrate ion and 1.5-30 µmol/L for nitrite ion. Correlation coefficients were determined as 0.998 for both ions. %RSD values for both anions are lower than 5.93. The LOD value for nitrate and nitrite was 0.45 µmol/L and the LOQ value was 1.5 µmol/L, respectively.
The nitrate anion has been found in all infant formulas in amounts ranging from 16.1 mg/kg to 285 mg/kg. The baby food sample with the highest nitrate content includes pumpkin, banana and carrot. This example was followed by broccoli soup with bone broth with a nitrate content of 235 mg/kg. The samples with the lowest nitrate content are organic cereal-based supplementary food (13.7 mg/kg) and a baby food with red fruits (16.1 mg/kg). The nitrite anion is below the limit of detection (LOQ) in all baby food samples.