Organik asidürilerin tanısında kapiler elektroforez temassız iletkenlik dedektörü uygulamaları: Kemometrik deney tasarımı ile metot optimizasyonu
Capillary electrophoresis with contacless conductivity detection applications for determination of organic acidurias: Method optimization using chemometric experimental design
Yazar: SİRUN ÖZÇELİK
Danışman: DOÇ. DR. NEVİN ÖZTEKİN
İstanbul Teknik Üniversitesi / Lisansüstü Eğitim Enstitüsü / Kimya Ana Bilim Dalı / Kimya Bilim Dalı
Derece: Doktora, 2021
Konu: Kimya = Chemistry

Özet:
Organik asidüri, bir gendeki mutasyon sonucu karbonhidrat, aminoasit ve yağ asidi oksidasyonunun metabolik yolaklarındaki işlev bozuklukları ile oluşan, vücut sıvılarında ve özellikle de idrarda yüksek oranda asit birikimine yol açan kalıtsal metabolik bir hastalıktır. Hastalık teşhisinde basamaklardan biri de idrarda biriken asidin miktarının tayin edilmesidir. Organik asidüri gibi metabolik hastalıklarda çoğunlukla yenidoğanlarda ölümcül olduğundan erken teşhis hayati bir öneme sahiptir. Bu nedenle uzun ön işlemler gerektirmeyen, hızlı ve güvenilir yöntemlere ihtiyaç duyulmaktadır. Kapiler elektroforez (CE) yüksek etkinliği ile küçük iyonlardan kompleks organik bileşiklere kadar çok kapsamlı bir ayırma yelpazesine sahip güçlü bir analitik tekniktir. Analizler kısa sürede ve düşük miktarda numune ile gerçekleştirilebilmektedir. CE ile birleştirilmiş temassız iletkenlik dedektörü (C4D) son yıllarda oldukça fazla ilgi gören bir tekniktir. Temassız iletkenlik dedektörü hassasiyeti yüksek, yapımı kolay olan ve ucuz bir cihazdır. Kemometrik deneysel tasarım çok fazla değişkenin söz konusu olduğu analitik çalışmalarda az sayıda deney ile kimyasal verilerin işlenmesini sağlar. Değişkenlerin, deneysel sonuçları tek tek ve birlikte nasıl etkilediğini matematiksel formüller vasıtasıyla açıklar ve üç boyutlu analizler sunar. Üç bölümden oluşan bu tez çalışmasında amaç farklı organik asidürilerin idrardaki teşhisi için hızlı ve duyarlı bir analitik yöntemi literatüre kazandırmaktır. Küçük organik asitler aromatik grup içermez ve UV dedeksiyon ile tespit edilemezler. İndirekt UV ile dedeksiyonda hassasiyet düşmekte, floresans ile dedeksiyonda ise uzun ön türevlendirme işlemlerine ihtiyaç duyulmaktadır. Temassız iletkenlik dedektörü (CE-C 4D) ile küçük organik asitler direkt olarak belirlenebilmektedir. Bu amaçla hassas ve hızlı uygulanabilen kapiler elektroforez ile birleştirilmiş temassız iletkenlik dedektörü (CE-C4D) metotları geliştirilmiş ve bu yöntemler gerçek idrar örneklerine uygulanmıştır. Metot optimizasyonu kemometrik deneysel tasarım kullanılarak gerçekleştirilmiştir. Çalışmanın ilk basamağında etilmalonik asidürinin teşhisi için idrarda etilmalonik asidin miktarı tayin edilmiş, yöntem etilmalonik asidürili hasta idrarında da uygulanmıştır. İkinci bölümde ise malonik, metilmalonik ve etilmalonik asidin eş zamanlı tayini için analitik bir metot geliştirilmiştir. Asidürisi bulunan hasta idrarlarına yöntem başarı ile uygulanmıştır. Tez çalışmasının son bölümünde ise geliştirilen elektroforetik yöntem idrarda sialik ve mevalonik asidüri tanısı için kullanılabilecektir.

Abstract:
Inherited metabolic diseases (IMD) were first described by Sir Archibald Garrod in 1908. IMD are inborn errors of metabolism and caused by genetic mutation in DNA. This mutation gives rise to defects in production or activity of an enzyme. Organic acidurias are one of the most important classes of inherited metabolic disorders. Organic aciduria is caused by genetic mutation which leads defect in intermediary metabolic pathways such as amino acids, carbohydrate and fatty acid oxidation. This mutation occurs in a single gene (monogenic) and is passed on through generations. Transmission is mostly autosomal recessive. These diseases can affect approximately 1:1,000 to 1:2.500 of newborns. These are rare but life-threatening disorders characterized by abnormal accumulation of acids in body fluids. Determination of the  related acid in urine is an important step for definitive diagnosis of disease. The clinical findings of the diseases in question also vary. The emergence of similar findings with different diseases or the presence of different findings in people with the same disease make diagnosis difficult and sometimes delayed. Most organic acidurias become clinically evident during the neonatal period or early infancy, but there are also milder forms that do not appear until adolescence or adulthood or require medical attention. When it is not detected in newborn, it often goes undiagnosed until symptoms appear. Development delay, cardiomyopathy, abnormal genitalia, hypotonia, brain abnormalities, recurrent vomiting, lethargy, seizures, neurological dysfunction, muscle weakness, liver disease, diarrhea, recurrent petechiae are some of the symptoms for characterization. Even if there is no treatment to avoid or survive permanent neurological effects it is important to diagnose the disease. The incidence of organic acidurias is high in population with high risk of consanguineous marriage and among seriously ill children. Due to the high number of consanguineous marriages in Turkey, the incidence of these diseases is higher than in the world. Early diagnosis 
and accurate determination in newborn patients has a vital importance as it's affecting newborns. Analysis of metabolites in body fluids is one of the most common analyzes in clinical biochemical laboratories. Although many of the most common metabolites can be routinely determined by classical methods, the development of new methods and methods is demanded. One of the important applications of new techniques is metabolite screening for the detection of hereditary diseases. Analysis of diagnostic metabolites found in urine allows for the identification of diseases in clinical studies. Analysis of organic acids in urine is of great importance in diagnosing organic aciduria. Because in these diseases, more than 100 organic acids are excreted in the urine. The methods used in the diagnosis of organic acidurias are GC-MS, GC-MS/MS and LC-MS/MS. These methods include both expensive devices that are not available in every laboratory, and preliminary derivatization steps that require long time and processes. In addition to the rapid diagnosis of such diseases; It is important to develop fast, reliable and economical alternative analysis methods.
Accurate determination of acids in urine of patient has a vital importance to diagnose inborn metabolic errors. Capillary electrophoresis (CE) a newly implemented analytical technique for clinical analysis, which is identified to be highly effective. CE method has advantages such as high separation efficiency, excellent resolution, short 
analysis time and electrolyte and sample consumption. Due to its higher resolving power, CE is especially suitable in the analysis of complex natural matrices. As compared to choromatographic columns, fused silica capillaries increase the efficiency in terms of cost, being washed between each run easily as well as being free of irreversible contamination of the matrix. Therefore, by capillary electrophoresis, biological fluids give an opportunity to be used directly requiring no or simple additional pretreatment step. Capacitively Coupled Contactless Conductivity Detection (C4D) is an alternative technique that has received lots of attention in recent years. It has high sensitivity, easy to construct and inexpensive. Species that do not contain aromatic groups and have poor absorption can usually be detected by indirect UV detection. But the method is less sensitive compared to C4D and has limited linear range. C4D is a suitable detection technique for charged species. This study consists of three parts. In the first part of the thesis the aim was to develop a method for detection of ethylmalonic acid in urine. Concentration of ethylmalonic acid in urine is used for diagnosis of ethylmalonic aciduria. C 4D in CE it is necessary to use a buffer solution of low conductivity in order to obtain good sensitivity and to minimize the electrophoretic current. According to previous studies MES-Tris buffer 
system was chosen as background electrolyte. pH was adjusted with Tris in order to prevent increase in ionic strength of buffer solution. Negatively charged species such as anions of low molecular mass organic acids have high electrophoretic mobilities and long analysis times. Therefore, electroosmotic flow (EOF) is reversed to leads to short analysis times. For this purpose, an EOF modifier such as CTAB was added in the buffer in order to reverse the EOF. In order to optimization of electrophoretic conditions chemometric experimental design was used. The chemometric experimental design enables the processing of chemical data with a small number of experiments in analytical studies where too many variables are involved. The Box-Behnken experimental design, developed by Box and Behnken in 1980, is a useful method for developing second-order response surface models. The CE-C4D method for the determination of ethylmalonic acid in 
urine was optimized by using the Box-Behnken design (BBD) with three variables: buffer concentration, surfactant concentration, and buffer pH. The effects of three experimental variables were investigated on the resolution and height of peak as experimental responses. The optimum conditions were predicated by using a second‐order polynomial model 
fitted to the results obtained by using BBD. Optimum separation conditions were obtained as 50 mM MES-Tris, 0.13 mM CTAB and pH 6.5. Under these conditions, acid was separates between 3-4 min with good peak shape. The analytical parameters 
of method were evaluated such as linearity, precision, detection limit (LOD) andquantification limit (LOQ). Method's intra- and interday precision was <5% relative standard deviations. The linear regressions of the calibration curves indicated good linearity (R2>0.99). LOD (S/N = 3) and LOQ (S/N = 10) values for EMA were about 0.139 mg/L and 0.466 mg/L respectively. The proposed method was successfully applied to determine EMA in urine samples, with very simple preparation procedure of the real samples. The sample of patient urine was provided by the Division of Nutrition and Metabolism in the Department of Pediatrics, Cerrahpaşa Medical Faculty, Istanbul University-Cerrahpaşa. EMA concentration of this urine determined as 245±3 mg/L. The second part of this study a CE-C4D method for the determination of MA, MMA and EMA in urine samples is developed. A three-level Box-Behnken Experimental Design of response surface methodology was used to optimize conditions of determination of malonic acid biomarkers with CE. Chemometric experimental designs are more advantageous than conventional experiments. Fewer experiments are needed for determine optimum conditions. This method provides savings in terms of time and the amount of material. Effective factors were buffer concentration, buffer pH and injection time. Resolution and height of peak were chosen as responces to optimize. Optimum separation conditions were calculated and obtained as 45 mM MES, 0.5 min injection time and pH 6.5. Good linearity was indicated by The linear regressions of the calibration curves (R2>0.99). LOD (S/N = 3) values for MA, MMA and EMA were about 0.222 mg/L, 0.228 mg/L and 0.431 mg/L respectively. LOQ (S/N = 10) values for MA, MMA and EMA were about 0.741 mg/L, 0.761 mg/L and 1.436 mg/L respectively. The method was successfully applied to determine acids in 
urine samples. MMA and EMA concentrations are found in patients who have methylmalonic aciduria as 4158±15 mg/L and 339±5 mg/L respectively. EMA concentration was 251±4 mg/L in patient's urine who has ethylmalonic aciduria. In the last part of the study was to develop a CE-C4D method for simultaneous determination of sialic and mevalonic acid in urine. Fractional factorial design (FFD) was used to screen the experimental factors and for identifying the most significant 
factors. Then Box-Behnken design is used to estimate the optimum electrophoretic conditions. Sample stacking method was applied by injection of large volume of samples. Buffer concentration, buffer pH, surfactant concentration, injection time and pressure were chosen as parameters for FFD. Buffer concentration, injection time and pressure were calculated as effective parameters and BBD was applied. Optimum buffer conditions were 10 mM MA, 0.1 mM CTAB, pH= 6.5, 113 mbar injection pressure ve 33 sec injection time. The linear regressions of the calibration curves indicated good linearity (R2>0.99). LOD (S/N = 3) values for SA and MVA were about 0.223 mg/L and 0.145 mg/L respectively. LOQ (S/N = 10) values for SA and MVA were about 0.743 mg/L and 0.483 mg/L respectively. The method was also successfully applied to determine acids in urine samples. SA acid concentration was found as 1861 22 mg/L and 4726 mg/L in two patients. In this study, a rapid, reliable and simple CE-C4D method for the determination of organic acids in urine samples was developed. There is not any pretreatment step as samples were only diluted with water before injections. The method can be applied 
for clinical analysis of MA, MMA, EMA, MVA and SA in patients