DEVELOPMENT OF METHODS OF PRE-COLUMNAR DERIVATIZATION OF GLUTATHIONES RECOVERED BY 4-METHOXY-2-NITROPHENYL- ISOTHIOTOCIONATE FOR DETERMINATION BY METHOD OF HIGH-EFFECTIVE LIQUID CHROMATOGRAPHY

В настоящее время активно исследуется фармакологическая роль глутатиона в терапии канцерогенеза, нервно-дегенеративных и глазных болезней, заболеваний сердца, иммунной системы и старения организма. Поэтому для разработки фармацевтических объектов на его основе необходимо создание оптимальной аналитической базы. Целью настоящего исследования является разработка методики анализа глутатиона восстановленного с помощью предколоночной дериватизации 4-метокси-2-нитрофенил-изотиоцианатом. Материалы и методы. Поскольку глутатион не имеет необходимых спектральных характеристик для непосредственного анализа, то исходя из этого, разработана методика определения глутатиона с помощью предколоночной дериватизации 4-метокси-2-нитрофенил-изотиоцианатом методом обращенно-фазной высокоэффективной хроматографии (ОФ ВЭЖХ). Детекцию образовавшегося деривата проводили по поглощению в УФ-свете с помощью диодно-матричного детектора. Результаты и обсуждение. В ходе описанного эксперимента, были получены хроматограммы деривата глулатиона с 4-метокси-2-нитрофенил-изотиоцианатом. Данную методику также оценивали на возможность количественного определения глутатиона. Чувствительность методики составила 0,01% или 3,1*10-1 моль. Прямолинейная зависимость между аналитическим сигналом (площадь пика) и концентрацией наблюдалась в диапазоне 0,01–0,08%, коэффициент корреляции – 0,995. Заключение. В ходе проведённых исследований разработана методика определения глутатиона с помощью предколоночной дериватизации 4-метокси-2-нитрофенил-изотиоцианатом методом ОФ ВЭЖХ. При этом образуется дериват со временем удерживания 22,3 мин и максимумом поглощения 398 нм. Также данная методика позволяет оценить количественное содержание исследуемого объекта. Ключевые слова: глутатион восстановленный, 4-метокси-2-нитрофенил-изотиоцианат, обращенно-фазная высокоэффективная жидкостная хроматография, дериватизация

for the development of pharmaceutical medical forms on its basis, it is necessary to create an optimal analytical base. The aim of this study is to develop a methodology for the analysis of glutathione recovered by pre-columnar derivatization of 4-methoxy-2-nitrophenyl isothiocyanate. Materials and methods. Since glutathione does not have the necessary spectral characteristics for its direct analysis, a methodology for the determination of glutathione with the use of pre-columnar derivatization of 4-methoxy-2-nitrophenyl-isothiocyanate by reversed-phase high-performance chromatography (RP HPLC) has been developed on that basis. Detection of the resulting derivative has been carried out by absorption in UV light using a diode array detector. Results and discussion. In the course of the experiment described, chromatograms of a glulathione derivative with 4-methoxy-2-nitrophenyl isothiocyanate were obtained. This technique was also evaluated for the possibility of quantitative determination of glutathione. The sensitivity of the methods was 0.01% or 3.1*10 -1 mol. The linear relationship between the analytical signal (peak area) and concentration was observed within the range of 0.01-0.08% and the correlation coefficient of 0.995. Conclusion. In the course of the studies, a methodology for the determination of glutathione has been developed with the use of pre-columnar derivatization of 4-methoxy-2-nitrophenyl-isothiocyanate by RP HPLC. In this case, the derivative is formed with the retention time of 22.3 minutes and the absorption maximum of 398 nm. This method also allows estimating the quantitative content of the object under study.

INTRODUCTION.
Glutathione is a tripeptide formed by three amino acids: glutamic acid, cysteine and glycine. This molecule takes part in maintaining the redox potential of cells, in the process of neutralizing xenobiotics of various origin, participating both as a direct conjugating agent and a co-factor of a number of enzyme biotransformation systems [1]. The importance of this tripeptide for humans is due to the fact that changes in any homeostatic parameters of the body -age, activation of immune processes, the emergence of virtually all acute and chronic diseases -are accompanied by shifts in the synthesis of glutathione and as a consequence of transformation of the oxidative status [2]. The physiological function of glutathione is realized in several ways: neutralization of toxic electrophilic particles, by direct contact with active forms of oxygen and activation of biotransformation enzymes, i. e. glutathione peroxidase and glutathione transferase [3,4].
Glutathione is a stabilizer of the thiol status of proteins, being a receptacle of cysteine. He also coordinates DNA synthesis and immunity [5,6], helps maintain a constant level of nitric oxide (NO) [7], converts protein activity through S-glutathionylation [8] and neurotrans-mitter receptors [9]. In mitochondria, glutathione regulates apoptosis, inhibiting necrotic processes, and in the nucleus it is a regulator of proliferation [10].
The described biological effects of glutathione underscore its potential therapeutic activity in the correction of numerous nosologies and indicate the promise of creating drugs based on it. To develop optimal pharmaceutical forms of glutathione, it is necessary to develop an effective method for its analysis.
A number of analytical methods for the analysis of glutathione have been described in the scientific literature.
The currently known methods are as follows. Spectrophotometric method for the determination of glutathione, based on the interaction of 5.5'-dithiobis-2-nitrobenzoic acid (DTNB) with the SH group GSH. As a result, the reaction forms the chromophore 5-thio-2-nitrobenzoic acid (TNB) with a maximum optical density at 412 nm [11].
Spectrofluorimetric method for the determination of glutathione. The method is based on the interaction of o-phthalaldehyde (OPT) with the GS-SH group, resulting in the formation of a fluorescent conjugate [12,13]. DOI: 10.19163/2307-9266-2018-6-3-229-240 Glutathione determination by spectrophotometric method with Ellman reagent. Nowadays, the Ellman reagent method is one of the most used approaches for the detection of glutathione, despite the fact that it provides an approximate estimate of the glutathione content [14].
Definitions of glutathione by liquid chromatography. The method is based on the use of liquid chromatography with various detectors [15].
To identify glutathione conjugated to proteins, liquid chromatography in combination with mass spectrometry and tandem mass spectrometry is used to detect it in bound or free forms in organs and cells [16].
As prechromatographic derivatization reagents for HPLC analysis of glutathione, ethacrynic acid and its methyl ester have been proposed in biological samples [17].
Since glutathione is a marker of oxidative stress, in blood and other tissues it is determined by derivatization with the alkylating agent N-ethylmaleimide, which prevents auto-oxidation of glutathione. Conjugate is then determined by HPLC [18] or by liquid chromatography-tandem mass spectrometry [19]. This approach is also used to determine glutathione in the striatum of the brain of Wistar rats [20].
There is a technique of glutathione joint analysis and its precursors such as cysteine, cysteinilglycine and homocysteine in the saliva, which consists in restoring disulfides in thiols, derivatization of the 2-S-quinoline with 2-chloro-1-metilhinoliniytetraftorborata and quantitative determination by high performance liquid chromatography [21,22].
A direct electrochemical determination of recovered glutathione and glutathione has been proposed [23,24].
A number of described glutathione analysis techniques is primarily concerned with its immediate determination in biological objects, but for certain reasons they are practically unsuitable for pharmaceutical analysis.
Based on the foregoing, THE AIM of the present study is to develop the optimal glutathione detection methods allowing to assess its quality in medicaments.
Chromatographic separation was carried out on the equipment "Agilent Technologies 1200 Infinity" (USA). Electronic spectra were recorded with the use of an Agilent 1200 diode array detector.
The separation was carried out on a steel column Ascentis express C182.7μm × 100 mm × 4.6 mm.
The composition of the mobile phase included (A) -1% aqueous solution of acid formic, (B) -ethyl alcohol. The elution was carried out under the following conditions: Flow rate: 0.5 ml / min Column temperature: 35°C Detection: 398 nm Volume of sample: 1 μl The elution was carried out in the gradient mode shown in Table 1.

Table 1 -Conditions for the gradient elution of glutathione derivatives
Time, min А, % В, % 0 100 10 60 0 100 Glutathione is a molecule that does not have significant chromophore fragments, suitable for the investigation by UV spectroscopy and HPLC with diode-matrix detection. Due to the lack of intrinsic absorption in the ultraviolet region of the spectrum, a glutathione analysis by RP HPLC by its chemical modification has been developed. The popular reagent that supports the production of chromophores is phenylisothiocyanate. However, for derivatization 4-methoxy-2-nitrophenyl-isothiocyanate was used, which is a phenylisothiocyanate derivative with more active sorption properties for UV light. The technique is in the use of pre-columnar derivatization of the molecule under study by 4-methoxy-2-nitrophenyl-isothiocyanate. In this case, a direct chemical reaction of glutathione with 4-methoxy-2-nitrophenyl isothiocyanate takes place in an alkaline medium, since the reaction proceeds according to the type of electrophilic substitution. As a result, the structure of glutathione undergoes a chemical transformation and acquires a chromophore label the signal of which can be detected by a diode array detector during HPLC analysis.
Solvents such as acetone, acetonitrile and ethanol were used to prepare a solution of 4-methoxy-2-nitrophenyl isothiocyanate. The highest solubility of 4-methoxy-2-nitrophenyl isothiocyanate was shown in ethanol, therefore, further on ethyl alcohol was used as a solvent.
Thus, to prepare a solution of 4-methoxy-2-nitrophenyl isothiocyanate, about 0.025 g of the reagent was transferred to a 25 ml volumetric flask, 10 ml of ethanol was added, shaken until completely dissolved, the volume of the solution was labeled with the same solvent and mixed.
Since the interaction of amino acids with 4-methoxy-2-nitrophenyl-isothiocyanate occurs as an electrophilic substitution, an alkaline reaction of the medium is required to proceed with this reaction. Therefore, 0.05 N sodium tetraborate solution was used to prepare the glutathione solution.
0.025 g of the test glutathione sample was placed in a volumetric flask, 10 ml of a 0.05N sodium tetraborate solution was poured into 25 ml of a volume, stirred until the substance was completely dissolved, and the solution was brought to the mark with the same solvent, and mixed.

Фармацевтическая и токсикологическая химия Pharmaceutical and toxicological chemistry
2 ml of the prepared glutathione solution was placed in a 25 ml volumetric flask, 2 ml of a 0.1% solution of 4-methoxy-2-nitrophenyl isothiocyanate was added and agitated. The resulting solution was brought to the mark with ethanol, stirred and heated in an oven at the temperature of 80°C for 30 minutes. After the time was over, the solution was cooled down to the room temperature and chromatographed under the above conditions. RESULTS AND DISCUSSION. In the course of the described experiment, chromatograms of glulathione derivative with 4-methoxy-2-nitrophenyl isothiocyanate were obtained. The 4-methoxy-2-nitrophenyl-isothiocyanate reagent is characterized by the presence of a single peak with the retention time of 33.97 minutes. The UV spectrum of 4-methoxy-2-nitrophenyl isothiocyanate is observed at the wavelength at λ max = 410 nm (Fig. 1).

Fig. 3 -UV-spectrum of glutathione derivate with 4-methoxy-2-nitrophenyl-rhodinide
Calculations of the suitability parameters of the used chromatographic system are given in Table 2. The results of calculating the suitability criteria listed in Table 2 (N> 5000, R s > 1.5, T f <2) fit into the recommended suitability parameters of the EF [25]. Thus, the present chromatographic system can be recognized as acceptable for the determination of glutathione derivatives.
To assess the possibility of using this method for the quantitative determination of glutathione, a direct relationship was established between the concentration of glutathione derivative and the analytical signal (peak area). To do this, a series of 6 calibration solutions of glutathione derivatives were prepared in the concentration range of 0.01-0.08%. The resulting calibration solutions were chromatographed under the conditions given above. Based on the results of chromatography, a calibration graph was constructed for the dependence of the concentration of glutathione derivative on the peak area (Fig. 4).
The detectable minimum for determination of glutathione derivative equals to 0.01%.
In the indicated range of concentrations, the calibration dependence was rectilinear. The regression equation looked like this: y = 8571.8x -3.3753 The correlation coefficient was 0.9998 which indicates the presence of a linear relationship between the peak area and the glutathione derivative content.
Biologically active peptides including glutathione are a fairly complex object for analysis, since they cannot absorb the light. Therefore, the complexity of recording glutathione during the most important in the pharmaceutical analysis of the UV spectrophotometric method is due to the fact that it does not have chromophores.
However, high-performance liquid chromatography with diode-matrix detection makes it possible to analyze glutathione due to its preliminary derivatization with the acquisition of the chromophore label before direct separation on the column. Several methods have been developed for the derivatization of glutathione, where a number of conventional reagents are used as derivatizing agents, for example 2,4-dinitrochlorobenzene, 5,5'-dithiobis-2-nitrobenzoic acid, o-phthalaldehyde, Ellman's reagent. We first proposed a procedure for the derivatization of glutathione with 4-methoxy-2phenyl-rhodanide followed by chromatographic separation using liquid chromotorphy. Among the merits of this technique in comparison with those described in the literature there are the following ones: z only one derivate is produced; z a derivate and a derivative agent are well-separated in a chromatogram; z a derivate and a derivative agent have clearly distinguishable absorption bands; z reaction of derivatization goes fast enough; Фармацевтическая и токсикологическая химия Pharmaceutical and toxicological chemistry z the product is stable; z the reaction is sensitive; z there is a straight-line correlation between the analytical signal and the concentration of the sample. The the above mentioned advantages allow us to recommend the derivatization of glutathione with 4-methoxy-2 phenyl-thiocyanate for the identification and quantitative estimation of glutathione in the framework of development of pharmaceutical compositions and biopharmaceutical studies.

CONCLUSION.
In the course of the research, a methodology for the determination of glutathione has been developed with the use of pre-columnar derivatization of 4-methoxy-2-nitrophenyl-isothiocyanate by RP HPLC. In this case, the derivative is formed with the retention time of 22.3 minutes and the absorption maximum of 398 nm. This method also allows estimating the quantitative content of the object under study. The sensitivity of the determination was 0.01%. The linear relationship between the analytical signal (peak area) and the concentration was observed in the range of 0.01-0.08%.