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QUANTITATIVE DETERMINATION OF ETHACYSINE IN TABLETS BY SPECTROFLUOROMETRY AS ITS SULFONE

QUANTITATIVE DETERMINATION OF ETHACYSINE IN TABLETS BY SPECTROFLUOROMETRY AS ITS SULFONE
Владислав Евтухов, студент

Андрей Дорошенко, заведующий кафедрой, доктор химических наук, профессор

Харьковский Национальный Университет им. В.Н.Каразина, Украина

Микола Блажеєвський, профессор, доктор химических наук, профессор

Харьковский национальный фармацевтический университет, Украина

Участник первенства: Национальное первенство по научной аналитике - "Украина";

Открытое Европейско-Азиатское первенство по научной аналитике;

The new method was elaborated for quantitative determination of ethacysine hydrochloride (the diethylamino analog of ethmozine) (ЕТ) in the form of corresponding sulfonic derivative obtained with the use of potassium hydrogenperoxomonosulphate, through the spectrofluorometry ex= 264 nm/ λеm= 380 nm). Linear concentration dependence was preserved in the concentrations interval (1-8)∙10-6 mol/lЕТ, lgI=97047c– 0.003 (r=0,999).LOQ=1.1∙10-6 mol/l. It was showed that in the determination of ET in the tablets 50 mg (Olainfarm, Latvia) using the developed methodRSD=1.7% (accuracy, δ=- 0.2%).

Keywordskinetic, potassium hydrogenperoxomonosulfate, ethacysine, spectrofluorometryquantitative determination

IntroductionEthacysine (sin. Aethacizin; Etacizin; Ethacizin; Ethacyzin; EZ-55; NIK-244) – ethyl N-[10-[3-(diethylamino)propanoyl]phenothiazin-2-yl]carbamate hydrochloride (ET) – belongs to 10-acyl derivatives of phenothiazine (the diethylamino analog of ethmozine) and is used in medicine as the antiarrhythmic agent [1] (fig. 1). It is produced in the form of 2.5% solution for injections in 2 ml ampoules, and also 0.05 g tablets (manufactured by Olainfarm, Latvia).

Despite the wide application of ET in medical practice, analytical method of quantitative determination of this pharmaceutical preparation  has not been investigated enough. 

For quantitative determination of ET  in medical preparations and biological fluids the ВЕРХ method was suggested [2,3] of direct ultraviolet spectrophotometry [4], photoelectrocolorymetry in the form of  oxydative-hydrolitic decomposition product in the sulfuric acid environment [5]. For the purpose of detecting the falsified medicines (identity clarification) the methods TLC, UV, and IR-spectroscopy were suggested [6].

Despite the wide application of ET in medical practice, analytical method of quantitative determination of this pharmaceutical preparation  has not been investigated enough. 

Fig. 1. Ethacysine hydrochloride structure

Besides, in the literature a number of original articles were found describing the highly-sensitive spectrofluorometric methods of identification and quantitative determination of the phenothiazine derivatives in different medicines [7-9]. However, the ET  fluorescent characteristics have not been studied before, and there appeared to be no methods.

The aim of this article is to provide a detailed investigation of the kinetics of ET oxidation with the potassium hydrogenperoxomonosulfate, and fluorescence spectrums of ET and its oxidation products for development of the unified highly-sensitive and selective method of quantitative ET determination in the pharmaceutical preparations.

For quantitative determination of ET  in medical preparations and biological fluids the ВЕРХ method was suggested [2,3] of direct ultraviolet spectrophotometry [4], photoelectrocolorymetry in the form of  oxydative-hydrolitic decomposition product in the sulfuric acid environment [5]. For the purpose of detecting the falsified medicines (identity clarification) the methods TLC, UV, and IR-spectroscopy were suggested [6].
Despite the wide application of ET in medical practice, analytical method of quantitative determination of this pharmaceutical preparation  has not been investigated enough. 
For quantitative determination of ET  in medical preparations and biological fluids the ВЕРХ method was suggested [2,3] of direct ultraviolet spectrophotometry [4], photoelectrocolorymetry in the form of  oxydative-hydrolitic decomposition product in the sulfuric acid environment [5]. For the purpose of detecting the falsified medicines (identity clarification) the methods TLC, UV, and IR-spectroscopy were suggested [6].
Besides, in the literature a number of original articles were found describing the highly-sensitive spectrofluorometric methods of identification and quantitative determination of the phenothiazine derivatives in different medicines [7-9]. However, the ET  fluorescent characteristics have not been studied before, and there appeared to be no methods.
The aim of this article is to provide a detailed investigation of the kinetics of ET oxidation with the potassium hydrogenperoxomonosulfate, and fluorescence spectrums of ET and its oxidation products for development of the unified highly-sensitive and selective method of quantitative ET determination in the pharmaceutical preparations.
Besides, in the literature a number of original articles were found describing the highly-sensitive spectrofluorometric methods of identification and quantitative determination of the phenothiazine derivatives in different medicines [7-9]. However, the ET  fluorescent characteristics have not been studied before, and there appeared to be no methods.

The aim of this article is to provide a detailed investigation of the kinetics of ET oxidation with the potassium hydrogenperoxomonosulfate, and fluorescence spectrums of ET and its oxidation products for development of the unified highly-sensitive and selective method of quantitative ET determination in the pharmaceutical preparations.

Experimental section

Instruments, matrials, reagentsand methods

Ethacysine hydrochloride, substance-powder, manufactured by FSUC State Research Center of Organic Products and Colorants (NIOPIK, Russia) complying with the ND 42-8072-97.

Ethacysine tablets 0.05 g produced by AS Olainfarm, Latvia (ser. 280615). Film-coated tablets: tablet core: active substance: Ethacysine hydrochloride (ethyl N- [10- [3- (diethylamino) propanoyl] phenothiazin-2-yl] carbamate hydrochloride) 50 mg of additive agents: potato starch – 9.57 mg; sucrose - 19.3 mg; microcrystalline methylcellulose – 0.33 mg; calcium stearate – 0.8 mg shell: sucrose – 37.695 mg; povidone - 0.753 mg; quinoline yellow dye ( E104 ) – 0.025 mg; dye "sunset" yellow FCF (E110) – 0.003 mg; calcium carbonate - 6.308 mg; magnesium hydroxycarbonate main – 3.678 mg; titanium dioxide ( E171 ) - 0.665 mg; silica dioxide – 0.827 mg; wax Carnuba Wax – 0.046 mg.

Oxone®, monopersulfate (2КНSO5∙ КНSO4∙ К2SO4) (SIGMA-ALDRICH), CAS: 70693-62-8 (further as oxone), Active oxygen (AO)  4.5 % w/w.

For preparation of 4∙10-2 mol/l of the initial solution ofpotassium hydrogenperoxomonosulfate (КНSO5) the sample weight 0.615 g oxone was diluted in 50 ml double-distilled water. Solutions were kept for a week at the room temperature. The solution with the concentration of 2.2∙10-3 mol/l was received through the corresponding dilution of double-distilled water.

The standard ET solutions were prepared at the exact sample weights of preparation substance on thedouble-distilled water. The working standard solutions of ET were prepared out of the initial solutions through the corresponding dilution with double-distilled water. All solutions were kept at the room temperature in the dark cool place.

The absorption and fluorescence spectrums were recorded at the temperature of 20ºС on the fluorescent spectrophotometer MPF-4 «Hitachi», equipped with the specialized MPF computer (612-0655). The gauge and recording of the fluorescence spectrums of the researched ET oxidized derivatives were conducted at least 5 times,  averaged and deducted the averaged specter of base solution (without the determined derivative: potassium hydrogenperoxosulphate taking into account the oxidation stoichiometry).

Oxone solution standardization procedure. The composition of active oxygen in the oxone samples and concentration of potassium hydrogenperoxosulphate solutions were determined using the iodometric titration method: precisely weighted amount of oxone is diluted in 10-15 ml of double-distilled water, acidified with 1-2 ml 0.1 М dipping acid solution, added 1 ml potassium iodide solution 5% and free iodine was titrated with 0.02 М of standard sodium thiosulphatesolution using 10 ml microburette. The amount of standard test reagent was measured with the accuracy of ±0.01 ml.

Standard sodium thiosulphate solution was prepared of the standard titre fixanal ampoule on the double-distilled water. Titrated 0.02 М thiosulphate solution was prepared through the corresponding dilution of the initial solution in the newly boiled double-distilled water with the addition of chemically pure sodium carbonate [10].

The solutions рН were prepared using the electrometric compensation method on the laboratory ion-meter И-130 with the glass electrode ЭСЛ-43-07 together with "SSCE" (sat. Silver/Silver Chloride Electrode).

The necessary environment acidity was maintained using the buffer solutions prepared on КН2РО4 and К2НРО4 according to Green [11]. The S-oxidation kinetics of phenothiazine derivatives was studied using the methods of samples selection according to the discharge of potassium hydrogenperoxosulphate (iodometric titration of oxidant residue).

Methodology of the reaction kinetics studying using the iodometric titration method. Into 100ml measuring flask 20-30 ml buffer solution, 20. 0 ml 1∙10-2 mol/l potassium hydrogen peroxomonosulphate and 5.0 ml 1∙10-2  mol/l ET solution were sequentially poured (the stopwatch started), shaking the solution in the flask immediately the volume to was brought to the mark, corked and thoroughly mixed turning the flask. Then after some time using the 10 ml pipette reaction mixture was selected and while mixing poured into the conic flask with 1 ml 5% potassium iodide and 5 ml 0.1 mol/l dipping acid solution. The released iodine was titrated with 0.02 mol/l solution of sodium thiosulphate measuring the volume with an accuracy of ±0,01 ml.

Spectrums of fluorescence of ET solutions of concentration (solution рН) for the maximum excitation band (lex, 264 nm) position of maximum emission band, lem, 392 nm: ЕТО (ET sulfoxide) 1∙10-5 mol/l (рН 5.6; 0.02 mol/l КH2PO4 and К2HPO4) (264) 380. ЕТО2 (ET sulfone) 1∙10-5М(9.2, 0.02 mol/l К2HPO4) (264) 380 (fig. 2 and 3).

 

Fig. 2. Electronic absorption spectrum ЕТО2 с(ЕТО2)=2.2∙10-5 mol/l; рН=9.2

Fig. 3. Fluorescence spectrum of  ЕТО2 с(ЕТО2)=2.2∙10-5 mol/l;  рН=9.2

Kinetics of ETO oxidation reaction was also studied spectrofluorimetrically according to the formed oxidation product (ЕТО2) at 380 nm, the cell thickness l = 1 sm, for the solutions mixing the Budarin’s reactor was used [12], the time was recorded using the stopwatch from the moment of solutions mixing. Before draining the solutions were thermostated in the thermostat UTU-2 (Zeamit, Horizont Krakow-Poland) at 20 ±0.5° С. The reactions constants (kеф) were found by the slope ratio of the initial sections of kinetic time curves lnIfl.

Results and discussion

The kinetics studying results showed that at с(КHSO5)=1.77∙10-3 mol/l; с(ЕТ)=3.1∙10-4 mol/l ET oxidation takes place quantitatively and stoichiometrically with the formation of corresponding sulfoxide of ET (ЕТО) and sulfone of ET (ЕТО2)  etacisin derivative: in acid medium (рН 5.6-6.5) per 1 mol ЕТ 1 mol КНSO5 (formation of ETO) is spent, and in the alkaline medium (рН 8.5-9.2) – 2 mol КНSO5 (ЕТО2 formation). Stoichiometric ETO formation is achieved practically immediately (observation period 1min); ЕТО2 is quantitatively formed during the period not exceeding 15 min(fig. 4).

Fig. 4. Kinetic curves of ЕT oxidation using potassium hydrogenperoxomonosulphate с(КHSO5)=1.77∙10-3 моль/л; с(ЕТ)=3.1∙10-4 моль/л; рН : 1 – 5.6; 2 – 8.5-9.2.

The fig. 5 provides the general scheme of reaction of ET S-oxidation using potassium hydrogenperoxomonosulphate.

R2S + KHSO5 →  R2SO + KНSO4 (pH<7)

R2SO+ KHSO5 → R2SO2+KНSO4  (pH≥7)

Fig. 5. Scheme of ET oxidation using potassium hydrogenperoxomonosulphate

It was determined that reaction of ETO oxidation into ЕТО2 is biomolecular, first-order with two reagents. Under the conditions of pseudo-first reaction behavior order (КНSO5 surplus) the kef were calculated. The ЕТ oxidation reaction equation was derived which looks as follows:

- d[ЕТ]/dt = k1[ЕТО],  d[ЕТО]/dt = k1[ЕТ] – k2[ЕТO], d[ЕТO2]/dt = -k2[ЕТO],  k1>>k2.

The quantitative sulfone of ET formation was achieved for 15 min in the presence of oxidant surplus at рН≥8,5. Under the comparative conditions the fluorescence of ET sulfonic derivative is next stronger than that of unoxidized ET or partially oxidized derivative (ET sulfoxide).  The highest fluorescence was observed in the alkali water solution with рН 9.2. Based on the results received the relatively simple and quite sensitive method was developed of spectrofluorimetric ET determination in the coated 0.05 g tablets (manufactured by «Olainfarm», Latvia). The method was based on the formation of intensely fluorescent S-oxidation product, formed at the interaction of ET with potassium  hydrogenperoxomonosulfate in the alkali medium (рН 9.2).

Into the measuring flask 25 ml of thoroughly filtered through the paper filter (blue bond) analyzed pills solution (or working standard sample) ET was poured, the oxone(surplus) solution was added as well as the buffer mixture solution, and, thus the volume was brought up to the mark with double-distilled water and thoroughly mixed. After 15 min of storage the fluorescence of the received oxidation product was measured (λex = 264 nm/ λеm = 380 nm). Thepreparation composition was determined using the standard method, taking into account the dilution.


Fig. 6. lg Іfl dependence from concentration of EТO2 (рН 9.2)

Linear concentration dependence was preserved within the concentrations range (1-8)∙10-6 mol/l ЕТ, lgI=(97.0±7.9)∙103c, where с in mol/l  (r=0.999) (Fig. 6). Using the method of «introduced (μ)- found ()» the analysis results correctness was verified, δ < RSD, where   (n=5, P=0.95). It was showed that when determining the ET in the tablets 50 mg manufactured by Olainfarm using the researched method RSD =1.7% (δ =- 0.2%, as compared with the certificate data). LOQ =1.1∙10-6 mol/l. The content of the active pharmaceutical ingredient (API) was 50.3 mg (at admission 47.5- 52.5 mg) to one tablet.

Conclusions

  • 1. The kinetics was studied of the reaction of ethacysine S-oxidation using the potassium hydrogenperoxomonosulphate in the acid and alkali medium under the conditions of oxidant surplus. The oxidation products identification was conducted.
  • 2. The study was conducted of the simple, selective, and sensitive method of the quantitative ethacysine determination in the form of corresponding sulfonic derivative (ethacysine sulfone) using the spectrofluorometry method in the tablets 0.05 g.

 

References:

  • 1. Kovalenko V.N. Compendium 2014 – medicine drugs. K. Morion. 2014 – 2448 p.(Russian)
  • 2. Prokof'eva V. I., Chernova S. V., Kashtanova V., Shavratskii V. Kh., Gneushev E. I. Use of high-performance liquid chromatography in the evaluation of purity of ethacizine// Pharm. Chem. J. – 1990. –Vol. 24, № 4 . – P. 306-307.
  • 3. Beloborodov V.L., Zalesskaya M.A., Tyukavkina N.A. Quantitative Determination of Metacizine Components in Biological Fluids// Khimiko-Farmatsevticheskii Zhurnal [Pharmaceutical Chemistry Journal]. – 2000. – V. 34, № 12. – S. 41-44. (Russian)
  • 4. Kubrak Z. V., Popova V. I. Quantitative determination of ethacizine by UV spectrophotometry. Farmatsevtychnyi Zhurnal (Kiev). –1992, №1. – P. 79-80.(Ukrainian)
  • 5. Kubrak Z.V., Byeikin S.H. Ethacizine determination in biological fluids //Farmatsevtychnyi Zhurnal (Kiev). –1989. – №2. – S. 69-70. (Ukrainian)
  • 6. Kuvyrchenkova I.S. Methods of analysis of phenothiazine derivatives // Pharmacy. – 2006. – No. 6. – P. 18–21. (Russian)
  • 7. Mohamed F.A. Spectrofluorimetric determination of chlorpromazine hydrochloride and thioridazine hydrochloride// Anal. Lett. – 1995. – V. 28, №14. – P. 2491-2501.
  • 8. Yang G.J., Qu X.L., Shen M., Qu Q.S., Wang C.Y., Zhu A.P., Hu X.Y. Trace measurement of phenothiazine drugs in tablets by micellar-enhanced fluorophotometric method// Fluoresc. – 2007. – V. 17, № 2. – P. 119-126.
  • 9. Shlyusar O.I., Blazheyevs’kyy M.Ye., Aleksandrova D.I. Kil’kisne vyznachennya tryfluoperazyn v likars’kykh preparatakh metodom spektrofluorymetriyi u vyhlyadi S-oksydu// Medychna khimiya. –2012.–T. 14, No 2(51). – S. 39-43.(Ukrainian)
  • 10. Suslennikova V.M., Kiseleva E.K. Rukovodstvo po prigotovleniyu titrovannykh rastvorov. – L.: Khimiya, 1978. – 184 s. (Russian)
  • 11. Spravochnik khimika-analitika/ A.I. Lazarev, I.P. Kharlamov, P.Ya.Yakovlev, Ye.F. Yakovlev – Moskow.: Metallurgiya, 1976. – 184 s. (Russian)
  • 12. Yatsimirskiy K.B. Kineticheskie metody analiza [Kinetic methods of the analysis]., Khimiya Publ., 1967. – 200 s. (Russian)
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Ваша оценка: Нет (2 голоса)
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Симонян Геворг Саркисович

Уважаемые коллеги Андрей Олегович, Никола Евстафьевич и Владислав Александрович интересная, аналитическая и практическая работа. Работа является результатом кооперации двух школ – кафедр физико-органической химии (определения биологически активных компонентов в продуктах растительного происхождения) и физической и коллоидной химии (кинетика и механизмы реакций окисления органических веществ; разработка кинетических методов анализа лекарственных веществ).Вы приводите, что кинетикие исследование показали, что при С (КHSO5 ) = 1,77 ∙ 10-3 моль/л, с (ЕТ) = 4,7∙10 -4 моль/л ET окисление происходит количественно и стехиометрически с образованием соответствующего сульфоксида ЭТ (ЕТО) и сульфон ЕТ (ЕТО 2 ) , в кислой среде (рН 5.6-6.5) на 1 моль ЕТ 1 моль КНSO 5 (образование ЕТО) расходуется, так и в щелочной среде (рН 8.5-9.2) - 2 моль КНSO 5 (ЕТО 2 образование). Однако из рисунка 4 выдно, что окисление не идет количественно. При условии (ЕТ) = 4,7 ∙10 -4 моль/л и рН 5.6-6.5 расход КHSO5 должен был также 4,7 ∙10-4 моль/л, а не 2,8 ∙10 -4 моль/л. Владислав Александрович желаю успехов в учебе. С уважением к.х.н., доцент Геворг Саркисович.

Евтухов Владислав Александрович

Уважаемый Геворг Саркисович! Вы совершенно правы! Из-за ошибочно неверно наведенной нами конечной 4,7 на 10-4 моль/л концентрации этацизина в растворе под рис. 4 может сложиться впечатление о нестехиометричности реакции. В действительности концентрация этацизина была 3,1 на 10-4 моль/л. Расход окислителя при образовании сульфонового производного в пределах ошибки был точно в два раза большим от такового, израсходованого на образование сульфоксида, что свидетельствовало о четкой стехиометрии реакции при изменении рН. Дополнительно хотим сообщить, что после 45 мин в щелочной среде мы наблюдали некоторый перерасход окислителя, что, вероятно, связано с образованием аминоксида сульфонового производного.Просим прощения за эту неточность и благодарим за внимательное прочтение нашей работы. С уважением авторы.

Подлипная Марина Петровна

Уважаемые Микола Блажеєвський, Андрей Дорошенко, Владислав Евтухов Ваше исследование очень актуальное. Спасибо за очень интересный и познавательный доклад. Желаю Вам успехов и практического внедрения в дальнейшем. С уважением Подлипная Марина.

Евтухов Владислав Александрович

Уважаемая Марина Петровна! Большое спасибо за высокую оценку нашей совместной работы! Очень надеемся, что разработанная нами методика в будущем сможет быть использована для колличественного контроля в данном лекарственном препарате!
Комментарии: 4

Симонян Геворг Саркисович

Уважаемые коллеги Андрей Олегович, Никола Евстафьевич и Владислав Александрович интересная, аналитическая и практическая работа. Работа является результатом кооперации двух школ – кафедр физико-органической химии (определения биологически активных компонентов в продуктах растительного происхождения) и физической и коллоидной химии (кинетика и механизмы реакций окисления органических веществ; разработка кинетических методов анализа лекарственных веществ).Вы приводите, что кинетикие исследование показали, что при С (КHSO5 ) = 1,77 ∙ 10-3 моль/л, с (ЕТ) = 4,7∙10 -4 моль/л ET окисление происходит количественно и стехиометрически с образованием соответствующего сульфоксида ЭТ (ЕТО) и сульфон ЕТ (ЕТО 2 ) , в кислой среде (рН 5.6-6.5) на 1 моль ЕТ 1 моль КНSO 5 (образование ЕТО) расходуется, так и в щелочной среде (рН 8.5-9.2) - 2 моль КНSO 5 (ЕТО 2 образование). Однако из рисунка 4 выдно, что окисление не идет количественно. При условии (ЕТ) = 4,7 ∙10 -4 моль/л и рН 5.6-6.5 расход КHSO5 должен был также 4,7 ∙10-4 моль/л, а не 2,8 ∙10 -4 моль/л. Владислав Александрович желаю успехов в учебе. С уважением к.х.н., доцент Геворг Саркисович.

Евтухов Владислав Александрович

Уважаемый Геворг Саркисович! Вы совершенно правы! Из-за ошибочно неверно наведенной нами конечной 4,7 на 10-4 моль/л концентрации этацизина в растворе под рис. 4 может сложиться впечатление о нестехиометричности реакции. В действительности концентрация этацизина была 3,1 на 10-4 моль/л. Расход окислителя при образовании сульфонового производного в пределах ошибки был точно в два раза большим от такового, израсходованого на образование сульфоксида, что свидетельствовало о четкой стехиометрии реакции при изменении рН. Дополнительно хотим сообщить, что после 45 мин в щелочной среде мы наблюдали некоторый перерасход окислителя, что, вероятно, связано с образованием аминоксида сульфонового производного.Просим прощения за эту неточность и благодарим за внимательное прочтение нашей работы. С уважением авторы.

Подлипная Марина Петровна

Уважаемые Микола Блажеєвський, Андрей Дорошенко, Владислав Евтухов Ваше исследование очень актуальное. Спасибо за очень интересный и познавательный доклад. Желаю Вам успехов и практического внедрения в дальнейшем. С уважением Подлипная Марина.

Евтухов Владислав Александрович

Уважаемая Марина Петровна! Большое спасибо за высокую оценку нашей совместной работы! Очень надеемся, что разработанная нами методика в будущем сможет быть использована для колличественного контроля в данном лекарственном препарате!
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