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George Orwell

mendel

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Isolation of biogenic amines using paramagnetic microparticles off-line coupled with ion exchange liquid chromatography

Biogenic amines are formed as products of physiological metabolic activities of microorganisms, plants and animals [1]. They are produced by amino acid decarboxylase, which contain pyridoxal phosphate as the cofactor [2], in the enzymatic driven process called decarboxylation. The decarboxylase cleaves the carboxyl to form basic amine [3], whereas some of these amines. The produced polyamines spermine (Spm) and spermidine (Spd) and diamines putrescine (Put) and cadaverine (Cad) are constituents of eukaryotic and prokaryotic cells, having multiple functions in living organisms [4]. On the other hand, increased levels of these substances above physiological concentration are toxic to cells and can facilitate cell death based on the enhanced oxidative stress [5, 6]. Thus their pathological presence could be utilized for doing diagnostic more precisely. In particular, in urine there was previously shown the increased levels of biogenic amines associated with various types of tumors [7, 8], and recovery of urinary diacetylpolyamines to the normal levels, accompanied with remission of cancer was described [9]. For diagnostic, namely putrescine, spermidine, spermine and cadaverine could be interesting, because of their presence in many malignant diseases [10]. On the other hand, histamine can affect the numerous processes including anaphylaxis, dermatosis, phlogistic reactions, rheumatoid arthritis and many others [11]. Moreover biogenic amines dopamine (Da), norepinephrine (Ne), and serotonin (5-HT) are important neurotransmitters in central and peripheral nervous system [12]. Concentrations of norepinephrine and dopamine in urine specimens above 80 and 400 µg per 24 h were reported to be linked with pheochromocytoma [13].

In order to determine the concentrations of biogenic amines in biological matrices like urine, techniques providing high resolution and sensitivity are demanded. In our study, we decided to use ion-exchange liquid chromatography (IEC) with post-column ninhydrin derivatization and Vis detection for biogenic amines separation and detection, which minimize the sample pre-cleaning requirement [14]. The main aim of the present study consisted of synthesis and characterization of two types of paramagnetic microparticles (PMPs), composed of nanomaghemite (?-Fe2O3) core, modified with chitosan, or tetraethyl orthosilicate (TEOS) covered with Dowex. Subsequently, our prepared microparticles were tested for their ability to establish a binding with biogenic amines (spermine, spermidine, putrescine, cadaverine, histamine, and tyramine), and finally they were applied as a first part of our 2D separation approach, consisting of PMPs and IEC for isolation of biogenic amines in a sample without time-consuming pre-treatment. Determination of biogenic amines is not easy task regarding to demands on the sensitivity and accuracy of measurements and the influence of the matrix on the sample pre-treatment steps. Determination of biogenic amines is usually carried out using thin-layer chromatography [21], gas chromatography, capillary zone electrophoresis [22], ion exchange liquid chromatography [23], enzyme immunoassay [24] and/or high-performance liquid chromatography (HPLC) [25, 26]. In this study, we attempted to combine advantages of paramagnetic particles based isolation of the amines with their subsequent analysis using IEC. Primarily, we had to optimize the conditions for biogenic amines separation and detection using IEC with ninhydrin postcolumn derivatization and dual channel VIS detection (? = 570 nm, and 440 nm). As representatives of biogenic amines, we selected the most commonly occurred biogenic amines as spermine, spermidine, putrescine, cadaverine, histamine and tyramine. Besides their abundance, they have been found as the potential cancer biomarkers [7, 8]. Based on the optimization of separation of biogenic amines, the conditions were as follows: elution of biogenic amines was performed using gradient elution using buffers of different ionic strength and pH, as well as using temperature gradient. Finally two elution buffers were used (composition is mentioned in chapter Materials and Methods – Ion-exchange Liquid Chromatography) and biogenic amines were eluted under the following the program 0 – 60 minutes elution with buffer A, and 60 – 86 minutes elution with buffer B.

After separation, the column was regenerated using 0.2 mol.L-1 NaOH for 15 minutes, and stabilized for 19 minutes using buffer A. The column temperature was set to 76 °C. Typical IE chromatograms of various concentrations of cadaverine, spermidine, tyramine, spermine, histamine, and putrescine are shown. In insets of these figures, calibration curves measured under the optimal conditions are shown too. Correlation coefficients equal to or greater than 0.995 were obtained for peak area based calibration curves indicating strictly linear dependencies. LODs were shown to be within the range of 90 – 140 ng.mL-1 for the studied biogenic amines. These values were comparable with other reported UV and fluorescence detection levels obtained using various derivatization agents [27, 28] or with derivatization electrochemical method [29].

Práce je spojená s projektem SIX CZ.1.05/2.1.00/03.0072.

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