facebook
twitter
vk
instagram
linkedin
google+
tumblr
akademia
youtube
skype
mendeley
Wiki

THE USAGE OF LIQUID CHROMATOGRAPHY FOR CHEMICAL-TOXICOLOGICAL ANALYSIS OF METFORMIN

Автор Доклада: 
V. Moskalenko, S. Merzlikin, I. Zhuravel
Награда: 
THE USAGE OF LIQUID CHROMATOGRAPHY FOR CHEMICAL-TOXICOLOGICAL ANALYSIS OF METFORMIN

UDC: 615.272.3:543.544:54.061

THE USAGE OF LIQUID CHROMATOGRAPHY FOR CHEMICAL-TOXICOLOGICAL ANALYSIS OF METFORMIN

Valeriya Moskalenko, aspirant
Sergij Merzlikin, doctor of pharmacy, professor
Iryna Zhuravel`, doctor of chemistry, docent
The National University of Pharmacy


Conditions of the identification and quantitative determination methodic of metformin by HPLC method were offered. The availability of method for aims of chemical-toxicological analysis was stipulated with a relative error of the quantitative determination of metformin, which is ±5,15%.
Key words: antidiabetic drugs, metformin, high-performance liquid chromatography, chemical-toxicological analysis

Запропоновано умови виявлення та кількісного визначення метформіну методом ВЕРХ. Прийнятність методики для цілей хіміко-токсикологічного аналізу обумовлена відносною невизначеністю середнього результату при кількісному визначенні досліджуваної речовини, яка складає ±5,15%.
Ключові слова: протидіабетичні засоби, метформін, високоефективна рідинна хроматографія, хіміко-токсикологічний аналіз

The derivate of biguanide metformin hydrochloride (Siofor) is a basic antidiabetic drug of many therapeutic schemes in treatment of diabetes mellitus 2 type [1–3]. However it was known, that acute poisoning, registered in many countries, are consequences of overdosing with metformin. This fact grounds the development of conditions of methodology of chemical-toxicological research of biological material on the content of this drug [4–9].
In decision of question of implementation the chemical–toxicological analysis on poisoning drug the main condition was the usage of modern analytical methods, such as HPLC [10, 11]. However, the use of known HPLC-techniques in determination of metformin [12–15] in a case of undirectional chemical-toxicological analysis of unknown substance requires large amounts of experiments and materials. We think that the decision of this scientific problem is possible at the expense of HPLC-analyzer, developed by closed company “EcoNova”, city Novosibirsk, Russian Federation [16], which was successfully applied in chemical-toxicological analysis of drugs [17–20].
The aim of our research was definition of possibility of use HPLC-analyzer (“EcoNova”, city Novosibirsk, Russian Federation) for identification and quantitative determination of metformin with HPLC-method, acceptable for aims of chemical-toxicological analysis.
MATERIALS AND METHODS
Research was conducted with help of chromatograph “Milichrom A-02” (“EcoNova”, city Novosibirsk, Russian Federation) in scientific-manufacturing company “Analytic” (Kharkiv) in such conditions: reverse-phase column № 000000 ProntoSIL – 120 – 5 – С18 – AQ with size Ø2 × 75 mm, grain 5 mcm («Bischoff Analysetechnik und Geräte GmbH», Germany). Gradient elution was performed with a mixture of 2 eluents: eluent A consists of mixture of 0,2 M LiClO4 – 0,005 M HClO4 and water in a ratio of 5:95; eluent B was acetonitril 100 % grade “for HPLC”. The linear gradient constituted from 2 % to 100 % of acetonitril during 40 min. and then 100 % acetonitril during 3 min.; flow rate – 100 mcl/min.; temperature of column 40°C, maximal pressure 4,0 MPa. Volume of sample of injector – 4,0 mcl. Shown eluents have high clarity in short-wave area of UV-range; they haven’t any UV-absorbing impurities. These conditions provide the clarity of analysis. The UV-detection was spending at wavelengths 210, 220, 230, 240, 250, 260, 280, 300 nm, which was reflected in chromatogram with appropriate specters with the same retention time, but with different spectral characteristics. The purity of solvent was periodically controlled with shown methodic. The validity of methodic was controlled with chromatographing of standard solution of bromide-ion, uridine, caffeine, proserine, m-nitroaniline and triftazin [16]. Chromatograms were analyzed and processed using the program “Analytica-Chrom”, which was developed by scientific-manufacturing company “Analytic”.
The methodic of the construction of the calibration plot for metformin hydrochloride
To build the calibration plot 100,0 mg accurately weighed of metformin hydrochloride, inserted into volumetric flask 100 ml, dissolved with purified redistilled water and volume was adjusted to the mark with same solvent (solution 1, concentration 1000 mcg/ml). Then 40,0 ml of standard solution 1 was inserted in volumetric flask 100 ml and its volume was adjusted to the mark with purified redistilled water (solution 2, concentration 400 mcg/ml). In two volumetric flasks 100 ml inserted 50,0 ml and 25,0 ml of solution 2 and adjusted the volume to the mark with purified redistilled water (solutions 3 and 4, concentrations 200 and 100 mcg/ml respectively). In volumetric flask 100 ml inserted 25,0 ml of solution 3 and adjusted the volume to the mark with purified redistilled water (solution 5, concentration 50 mcg/ml). In volumetric flask 100 ml inserted 10,0 ml of solution 4 and adjusted the volume to the mark with purified redistilled water (solution 6, concentration 10 mcg/ml). In volumetric flask 100 ml inserted 10,0 ml of solution 6 and adjusted the volume to the mark with purified redistilled water (solution 7, concentration 1 mcg/ml). All solution prepared 10 minutes before analysis at room temperature. Aqueous solutions of metformin hydrochloride 3, 4, 5, 6 and 7 chromatographed in shown conditions thrice, volume of sample was 4 mcl.
The methodic of determination of metformin hydrochloride in model solutions
Model solutions of metformin hydrochloride with concentrations 10, 50, 100 and 200 mcg/ml were prepared according to shown methodic. Prepared model solutions of metformin hydrochloride were chromatographed in shown conditions, volume of sample was 4 mcl. To calculate the content of metformin hydrochloride of the calibration plot was used.
RESULTS AND DISCUSSION
During the research it was ascertained, that maximal area of peak of metformin hydrochloride on chromatogram (R(Sλ/S210)=1,000) observe at the wavelength 210 nm, which was selected like an analytical one. The retention time of search substance composed 2,4 min. Basic chromatographic characteristics of metformin hydrochloride presented in table 1 and table 2, and its chromatogram – at the picture 1.
                                  optical units

The chromatogram of aqueous solution of metformin hydrochloride


 time, min
Pict. 1. The chromatogram of aqueous solution of metformin hydrochloride (C=200 mcg/ml, λ=210 nm, volume of sample 4 mcl)

Table 1
Basic chromatographic parameters of metformin hydrochloride at determination
 

Dependence of area of peak of solutions of metformin hydrochloride of its concentrations

Table 2
Dependence of area of peak of solutions of metformin hydrochloride of its concentrations (λ=210 nm, volume of sample 4 mcl)

Dependence of area of peak of solutions of metformin hydrochloride of its concentrations

The calibration plot was build, which corresponds to the equation of a line, such as y = bx + a [10]:
S = (2,50±0,02)•10-3•C (1)
where, S – area of peak;
С – concentration of substance, mcg/ml.
Metrological characteristics of received calibration dependence present in the table 3.
Table 3
Metrological characteristics of calibration dependence of peak area of content of substance
(y = bx + a)

Metrological characteristics of calibration dependence of peak area of content of substance

It was established, that peak areas of solution of metformin hydrochloride depend linearly of concentration of metformin hydrochloride and lie in the interval 1-200 mcg/ml, what corresponds to the content substance in the sample from 2 to 400 ng respectively.
The equation of the line (1) was used for calculation of concentration of search substance in model solutions. Results of this research present in table 4.

Table 4
Results of quantitative determination of metformin hydrochloride in model solutions (λ=210 nm, volume of sample 4 mcl)

Results of quantitative determination of metformin hydrochloride in model solutions

CONCLUSIONS
In unified conditions of implementation of HPLC-analysis it was developed methodic of identification and quantitative determination of metformin hydrochloride, acceptable for purposes of chemical-toxicological research.

References:
1. Уильямз Г. Руководство по диабету / Г. Уильямз, Д. Пикап. – 2-е изд. – М.: МЕДпресс-информ, 2003. – 248 с.
2. Аметов А. С. Метформин – основа терапии пациентов с метаболическим синдромом / А. С. Аметов, Л. В. Кондратьева // Русский медицинский журнал – Эндокринология. – 2006. – Т. 14, № 26. – C. 1905.
3. Зилов А. В. Метформин – 50 лет в клинической практике / А. В. Зилов, А. Л. Терехова // Лечащий врач. – 2008. – № 3. – С. 16-19.
4. Carnicer J. O. Pancreatitis aguda, fallo renal agudo, intoxicación por metformina y adenoma velloso rectal / Carnicer J. O., Ambrós Checa A., Diarte de Miguel J. // Medicina Intensiva. – 2006. – V. 30. – P. 409-410.
5. Carnicer J. O. Overdose of metformin secondary to acute renal insufficiency: a report of six cases / Carnicer J. O. // Medicina Intensiva. – 2007. – V. 31, № 9. – P. 521-525.
6. Holanda P. Severe lactic acidosis associated to metformin intoxication / P. Holanda // Nutricion hospitalaria: organo oficial de la Sociedad Espanola de Nutricion Parenteral y Enteral. – 2007. – V. 22, № 1. – P. 124-125.
7. De Pont A. C. Fatal autointoxication with metformin / A. C. De Pont // Nederlands tijdschrift voor geneeskunde. – 2007. – V. 151, № 17. – P. 981-984.
8. Gambaro V. A case of fatal intoxication from metformin / V. Gambaro // Journal of forensic sciences. – 2007. – V. 52, № 4. – P. 988-991.
9. Мерзлікін С. І. Інформаційний огляд щодо обґрунтування хіміко-токсикологічного дослідження на метформін / С. І. Мерзлікін, В. Ю. Москаленко // Фармакологія та лікарська токсикологія. – 2010. – № 1-2 (14-15). – С. 3-10.
10. Державна Фармакопея України / Державне підприємство «Науково-експертний Фармакопейний центр». – 1-е вид. – Харків: РІРЕГ. – Доповнення 2. – 2008. – 620 с.
11. Clarke’s Analysis of Drugs and Poisons: 3-d edition. – London: Pharmaceutical Press, electronic version, 2005.
12. Tache F. Specificity of an analytical HPLC assay method of metformin hydrochloride / F. Tache, M. Albu // Review of roumanian chimistry. – 2007. – V. 052, № 6. – P. 603-609.
13. HPLC-UV determination of metformin in human plasma for application in pharmacokinetics and bioequivalence studies / V. Porta, S. G. Schramm, E. K. Kano, E. E. Koono et al. // Journal of Pharmaceutical and Biomedical Analysis. – 2008. – V. 46, № 1. – P. 143-147.
14. Amini Hossein. Determination of metformin in human plasma by high-performance liquid chromatography / Amini Hossein, Ahmadiani Abolhassan, Gazerani Parisa // Journal of Chromatography B. – 2005. – V. 824, № 1-2. – P. 319-322.
15. Arayne M. S. Development and validation of RP-HPLC method for the analysis of metformin / M. S. Arayne, N. Sultana, M. H. Zuberi // Journal of Pharmaceutical Science. – 2006. – V. 19. – P. 231-235.
16. Барам Г. И. Высокоеффективная жидкостная хроматография в контроле качества лекарственных средств / Г. И. Барам, Д. В. Рейхарт, Е. Д. Гольдберг, Б. Н. Изотов, М. О. Родинко, В. А. Хазанов // Бюллетень експериментальной биологии и медицины. – 2003. – Т. 135, № 1. – С. 75-79.
17. Шовкова З. В. Хіміко-токсикологічний аналіз біологічного матеріалу на каптоприл. Методичні рекомендації / З.В. Шовкова. – Х.: Вид-во НФаУ, 1998. – 23 с.
18. Шовкова З. В. Високоефективна рідинна хроматографія в аналізі каптоприлу / З.В. Шовкова, С.І. Мерзлікін, В.В. Болотов // Вісник фармації.– 2006. – № 3(47). – С. 31-34.
19. Болотов В. В. Застосування методу високоефективної рідинної хроматографії для кількісного визначення каптоприлу в хіміко-токсикологічному аналізі / В.В. Болотов, З.В. Шовкова, С.І. Мерзлікін // Науково-технічний прогрес і оптимізація технологічних процесів створення лікарських препаратів: матер. І Міжнар. наук.-практ. конф., м. Тернопіль, 6-7 квіт. 2006 р. – Тернопіль: Укрмедкнига, 2006. – С. 86-87.
20. Вивчення зопіклону за допомогою методу високоефективної рідинної хроматографії / В. В. Болотов, Л. Ю. Клименко, В. Ф. Першин, С. М. Кузнєцова та ін. // Матер. наук.-практ. конф. з міжнар.участю «Стан, перспективи судово-токсикологічної служби та наукових досліджень». 9-10 листопада 2005 р. – Харків. – С. 25-26.

9.57143
Your rating: None Average: 9.6 (7 votes)

Очень актуальная статья.

Очень актуальная статья. Авторы освоили и доступно изложили сложную методику определения концентрации метформина. Кроме того, затронута тема токсичности метформина из-за его кумуляции в организме. Введение подобной методики в диагностику позволит своевременно поставить диагноз возможной передозировки.

Спасибо за высокую оценку

Спасибо за оценку статьи.

Здоровье

В статье предложена методика определения химического вещества. Авторам статьи удалось предложить и доступно изложить методику выявления количества метформина. Борьба с сахарным диабетом - важнейшее направление современной терапии, фармакологии и химии. Научно доказанные результаты, представленные в статье, возможно, будут способствовать успешному лечению указанного заболевания. Статья отличается научностью, актуальностью и новаторским решением.

Благодарим за оценку статьи.

Благодарим за высокую оценку статьи.

PARTNERS
 
 
image
image
image
image
image
image
image
image
image
image
image
image
image
image
image
image
image
image
image
image
image
image
image
image
image
image
image
image
image
image
image
image
image
image
image
image
image
image
image
image
image
image
image
image
image
image
image
image
image
image
image
image
image
image
image
image
image
image
image
image
Would you like to know all the news about GISAP project and be up to date of all news from GISAP? Register for free news right now and you will be receiving them on your e-mail right away as soon as they are published on GISAP portal.