Determination of related impurities in glimepiride tablets by the HPLC method

Glimepiride is one of the modern and effective drugs that counteract diabetes mellitus type 2. Like other drugs glimepiride must comply with quality standards set by relevant government departments. Aim . To determine the content of related impurities in glimepiride tablets in order to assess the drug quality. Materials and methods . The study was performed by the reversed-phase HPLC with a spectrophotometric detector in the isocratic mode, in the UV-region of the spectrum. To determine impurities the method of comparison with the solution having the known concentration of impurities and the active substance was used. Results and discussion . It has been shown that the drug contains ≤ 0.18 % of identified and unidentified impurities. Conclusions . The purity of the drug complies with the requirements of normative documents.

Выводы. Показано, что по проверенному показателю таблетки глимепирида соответствуют требованиям нормативных документов. Diabetes is a chronic disease of the endocrine nature. It is a disorder of all types of metabolism of the human body [1]. Diabetes is a heavy social and economic burden, cause many complications and pathologies that reduce the efficiency and the life expectancy of the population of the average and preretirement age [2,3].
Currently, more than 250 million people in the world have diabetes. The prevalence of diabetes in the population is 3-4 %, and it reaches 10-16 % among middleaged adults in the Near East [1,2].
There is a persistent trend of increasing morbidity, which has an epidemic character. It is believed that due to the aging population, the number of patients over the next two decades will double [3].
In Ukraine diabetes was diagnosed for 2.8 % of the population in 2014 [4]. Annually aproximately 100 000 new cases of the disease are recorded.
The state projects for reducing the diabetes negative impact exist in many countries. In particular, the state target program "Diabetes mellitus" was functioned in Ukraine in 2009-2013 (CMU Resolution No. 877 from 19.09.2009) [5].
An insufficient production of insulin by the pancreas is the cause of the pathogenic effect of diabetes type 1, whereas expressed insulin inactivation or destruction of insulin receptors on membranes of the body cells is observed in the diabetes type 2.
One of the modern and effective drug to control diabetes type 2 is glimepiride, which triggers the insulin production by the pancreas, increases the number of insulin-sensitive receptors and inhibits gluconeogenesis [7,8].
All medicines in Ukraine must meet quality standards, which are regulated by the State Pharmacopoeia of Ukraine, the documents of the State Expert Center of the MHP of Ukraine and the State Service of Ukraine on medicines and drug control. The quality control of medicines is carried out in the authorized analytical laboratories, which have the necessary equipment and the staff of the qualified professionals, certified and accredited in the relevant institutions [9]. Scheme 1 of the glimepiride synthesis based on the use of 5-dihydro-3-ethyl-4-methylpyrolin-2-one 1 as the starting reagent contains 5 stages [10].
Taking into account the multi-stage synthesis and possible deviations from the optimal storage conditions the substance and the finished dosage forms of glimepiride may contain compounds that are byproducts of formation and partial decomposition of the active substance (related impurities). They are pollutants because their content is strictly limited and requires mandatory control.
The aim of this work is to study the content of related impurities in glimepiride by HPLC for compliance with the requirements of normative documents.

Materials and methods
For the tests glimepiride tablets of foreign production were used.
The study was performed by the reversed-phase HPLC on an Agilent 1200 chromatograph with a spectrophotometric detector in the isocratic mode, in the UV-region of the spectrum using an Inertsil CAS-3V column (with the length of 250 mm, the diameter of 4.6 mm, the particle size of 5 mm). The column temperature was 25 °C, the rate of the mobile phase -1 ml/min, λ = 228 nm, the injection volume -20 μl, the mobile phase -phosphate buffer: acetonitrile.
Sulfonamide solution: add 50 ml of methanol to 39.92 mg of the sulfonamide standard in a 100.0 mL volumetric flask, shake to dissolve and dilute to the volume with the same solvent.
Carbamate solution: add 50 ml of methanol to 4.20 mg of the carbamate standard in a 100.0 ml volumetric flask, shake to dissolve and dilute to the volume with the same solvent.
Glimepiride solution: add 50 ml of methanol to 4.12 mg of the glimepiride standard in a 100.0 mL volumetric flask, shake to dissolve and dilute to the volume with the same solvent. The results of the chromatographic study of the reference and test solutions are shown in Tab. 1 and 2, respectively.
Statistical analysis of the results obtained and estimated uncertainty were carried out in accordance with the pharmacopoeial requirements (SPhU and Ph. Eur.) [11,12].
The chromatographic system suitability test corresponded to the requirements specified (the mean value of the symmetry factor for 5 parallel injections was within 1.04-1.05; the relative standard deviation of peak areas of the reference solution was less than 2.0 %, and the number of theoretical plates was ≥ 5000).

Results and discussion
According to the data of the European Pharmacopoeia [12] the glimepiride substance can have not more than 0.9 % of impurities, in particular identified compounds A-J (Scheme 2).
Compounds B (sulfonamide) and C (carbamate) are formed at the last stages of the glimepiride synthesis. They have similar solubility. These impurities are basic in the substance and medicines of glimepiride.
For glimepiride tablets under study the following requirements were set by manufacturer: the content of related impurities should be not more than 1.0 % (for sulfonamide and carbamate -not more than 0.6 and 0.1 %, respectively).
For the quantitative determination of impurities the method of comparison with the standard solution containing the known concentration of impurities and the active substance was used The reference and test solutions were injected, and peak areas on the chromatograms were integrated, after that the content of impurities was calculated.
The test results are given in Tab Table 2 The results of the chromatographic study of the test solution of glimepiride peak area of sulfonamide on the chromatogram of the standard solution; m samp -is the weight of the sulfonamide standard (mg); m gl -is the weight of the glimepiride sample (599.43 mg); Аv -is the average weight of tablets (359.85 mg); D -is the labeled amount of glimepiride per tablet, Р -is the purity of the standard sample of sulfonamide (0.991).
Calculation using digital data from Tab. 1, 2 and information from the experimental part shows that the sulfonamide content is 0.12 %.
The content of carbamate (%) was calculated using the formula: the average weight of tablets (359.85 mg); D -is the labeled amount of glimepiride per tablet; Р -is the purity of the standard sample of carbamate (0.969).
Calculation using data from Tab. 1, 2 and information from the experimental part indicates that the carbamate content is 0.01 %.
Calculation of the content of unidentified impurities was performed using the formula: where: S inv -is the average peak area of the unidentified impurity on the chromatogram of the test solution; S gl -is the average peak area of glimepiride on the chromatogram of the standard solution; m st -is the weight of the glimepiride standard (mg); m samp -is the weight of the glimepiride sample (599.43 mg); Аv -is the average weight of tablets (359.85 mg); D -is the labeled amount of glimepiride per tablet; Р -is the purity of the standard sample of glimepiride (0.995).
Calculation using data from the experimental part and Tab. 1.2 shows that the content of unidentified impurities 1-3 is 0.01, 0.03 and 0.01 % (0.05 % of the total amount), respectively.
The total content of identified and unidentified impurities 1-3 is 0.18 %. This is significantly less than the permitted value of 1.0 %. CONCLUSIONS Testing of glimepiride (tablets) on the content of related impurities by the HPLC method has shown that the drug meets the requirements of normative documents and is safe for consumers.
Conflicts of Interest: authors have no conflict of interest to declare.