Method for identification and quantitative determination of metformin hydrochloride and taurine in combined form by high-performance thin-layer chromatography

Abstract

FIELD: pharmaceutical compositions.SUBSTANCE: method for the quantitative determination of metformin hydrochloride and taurine in a pharmaceutical composition by high-performance thin-layer chromatography, comprising: preparing solutions of a sample and standard solutions in water; applying the resulting solutions to a chromatographic plate with a fixed layer of silica gel on an aluminum substrate measuring 20±10 cm by spray method, while 15 tracks are applied to the plate, three tracks of which are standard solutions with volumes of 3, 4 and 5 μl for plotting the calibration curve, and the remaining 12 tracks are the test samples, applied in a volume of 4 μl each; elution of the samples in the elution chamber, pre-saturated for 20 min with vapors of the solvent system ethanol-chloroform-15% ammonium acetate solution in a ratio of 6:3:2 (vol. / vol. / vol.), to a distance of 70 mm of the eluent front from the lower edge of the plate, with subsequent drying of the plate in a stream of cold air for 5 min; quantitative determination of metformin hydrochloride using a scanning densitometer at a wavelength of 239 nm; subsequent derivatization of the plate by immersion in a 0.01% solution of ninhydrin in 96% ethanol, followed by drying and heating at a temperature of 75±5  C for 10 min to develop chromatographic zones of taurine; quantitative determination of taurine by scanning the developed zones using a scanning densitometer at a wavelength of 487 nm; calculation of the content of metformin hydrochloride and taurine by constructing multi-point calibration dependencies using standard solutions.EFFECT: solution to the problem of quantitative determination of metformin hydrochloride and taurine in their joint presence in various matrices (single- and multi-component medicinal products, dietary supplements, food and chemical industry products, cosmetics), which would combine the simplicity of sample preparation, the possibility of joint analysis of both components in the presence of standards under identical conditions and with simple sample preparation.1 cl, 4 dwg, 1 ex

Description

[0001] The invention relates to the chemical-pharmaceutical industry, namely to a method for detecting, confirming the authenticity and standardizing active pharmaceutical substances, medicinal products, chemical compounds and cosmetic products containing metformin hydrochloride and taurine.

[0002] Methods for individual quantitative determination of metformin hydrochloride and taurine are known. Thus, for taurine, a method using high-performance liquid chromatography (HPLC) is known, however, it requires complex multi-stage sample preparation with centrifugation, the use of buffer solutions and stop reagents, which makes the analysis labor-intensive and time-consuming (Poluyanov A.M., Kochug A., Mitrofanova L.S., Nikitin I.D., Vergasov O.Yu., Shokhin I.E., Fisher E.N. Development, validation and testing of an analytical method for the quantitative determination of taurine by HPLC-UV as part of a comparative dissolution kinetics test. Development and registration of drugs. 2023; 12 (2): 62-72. https: / / doi.org / 10.33380 / 2305-2066-2023-12-2-62-72).

[0003] A method for determining taurine and another substance (allantoin) using HPLC is also known, disclosed in patent RU 2643312 C1, FGBOU VO "VSU", dated January 31, 2018. This method also uses pre-column derivatization with 2,4-dinitrochlorobenzene with heating and subsequent sample manipulation to determine taurine, while direct detection under different chromatographic conditions is used for allantoin. Thus, the known method effectively consists of two separate analyses, conducted sequentially under different chromatographic conditions, which prevents their simultaneous and rapid determination in a single sample.

[0004] A method for determining metformin concentrations using high-performance liquid chromatography is known, with the linear range of metformin determination being from 5 to 1600 ng / mL. (RU2705363 C1, A.I. Evdokimov Moscow State Medical University of the Ministry of Health of the Russian Federation, November 7, 2019). However, this method has several drawbacks that limit its use to clinical diagnostics and make it unsuitable for the combined determination of metformin and taurine. These include: the inability to simultaneously analyze multicomponent mixtures, limitation to biological fluids, complex and time-consuming sample preparation, and the impossibility of using it for standard quality control procedures such as the Dissolution Test.

[0005] At the same time, the following publications are known: "JUSTIFICATION FOR THE DEVELOPMENT OF A COMBINED ANTI-DIABETIC DRUG CONTAINING METFORMIN AND TAURINE" by Bakulin K.S., Ogay M.A. In the collection: Young scientists in solving current scientific problems. Proceedings of the XIII International Scientific and Practical Conference. Vladikavkaz, 2023. Pp. 116-120. Such a combination will require the development of a methodology for the quantitative determination of both components in various matrices (from finished dosage forms and substances to biological environments) for quality control, pharmacokinetic and biopharmaceutical studies.

[0006] The present invention is aimed at solving the problem of quantitative determination of metformin hydrochloride and taurine in their joint presence in various matrices (single- and multi-component drugs, dietary supplements, food and chemical industry products, cosmetics), which would combine the simplicity of sample preparation, the possibility of joint analysis of both components in the presence of standards under identical conditions and with simple sample preparation, which is the technical result of the invention.

[0007] The patented method for the quantitative determination of metformin hydrochloride and taurine contained in a mixture in a pharmaceutical composition by the HPTLC method includes the following operations:

[0008] - preparation of sample solutions and standard solutions in water;

[0009] - spray application of solutions onto a Merck Silicagel 60 F254 HPTLC plate on an aluminum backing measuring 20×10 cm using an applicator. Fifteen tracks are applied to each plate, three of which are standard solutions with volumes of 3, 4, and 5 μl for plotting the calibration curve, and the remaining 12 tracks are test samples, applied in a volume of 4 μl each in triplicate;

[0010] - elution in the solvent system ethanol-chloroform-ammonium acetate 15% (6:3:2 v / v / v) to a distance of 70 mm from the lower edge of the plate in a vertical glass chamber with preliminary saturation for 20 min with filter paper, followed by drying the plate in a stream of cold air for 5 min;

[0011] -Detection of metformin hydrochloride at a wavelength of 239 nm using a TLC Scanner 4 scanning densitometer (CAMAG, Switzerland);

[0012] - derivatization of the plate was carried out by immersion in a 0.01% solution of ninhydrin in 96% ethanol for 1 second, followed by drying and heating at a temperature of 75 ± 5°C for 10 min with visual control;

[0013] Detection of taurine at a wavelength of 487 nm using a TLC Scanner 4 scanning densitometer (CAMAG, Switzerland);

[0014] - calculation of the content of metformin hydrochloride and taurine by constructing multi-point calibration dependencies using standard solutions.

[0015] This solution makes it possible to identify and quantify metformin hydrochloride and taurine when they are present together in samples with minimal time and reagent costs.

[0016] The HPTLC method used provides high productivity (analysis of up to 4 samples simultaneously), simplified sample preparation, reduced solvent consumption, environmental friendliness, and high reproducibility (RSD < 2%).

[0017] The essence of the invention is explained in the figures, where:

[0018] Fig. 1. Scheme of applying tracks on a chromatographic plate;

[0019] Fig. 2. Track characteristics on the chromatographic plate;

[0020] Fig. 3. Scanning of metformin hydrochloride zones;

[0021] Fig. 4. Scanning of taurine zones.

[0022] Example of implementation. Determination of the identity and quantitative content of metformin hydrochloride and taurine in a solid combination dosage form for four samples.

[0023] 1. Sample preparation (for each of the four samples). Place an accurately weighed amount of powdered capsule contents, corresponding to 500 mg of metformin hydrochloride and 500 mg of taurine, into a 50 ml volumetric flask. Add 40 ml of purified water, sonicate for 20 min, and cool to room temperature. Dilute the solution to the mark with water, mix, and filter through a 0.45 μm membrane filter. Place 1.0 ml of the resulting solution into a 100 ml volumetric flask and dilute to the mark with purified water. Use the solution freshly prepared.

[0024] 2. Preparation of standard solutions (reference solutions). The standard solution of metformin hydrochloride is prepared as follows. Approximately 10 mg (accurately weighed) of the standard sample of metformin hydrochloride is placed in a 100 ml volumetric flask, dissolved in 80 ml of purified water, and the solution is made up to the mark with water. The standard solution of taurine is prepared in a similar manner.

[0025] 3 Arrangement and application of samples onto the chromatographic plate (see Figs. 1, 2). Merck Silicagel 60 F254 HPTLC chromatographic plates measuring 20x10 cm on an aluminum backing were used. Using an Automatic TLC Sampler 4 (C AM AG, Switzerland), tracks 1, 8 and 15 are applied - standard solutions of metformin hydrochloride and taurine, applied sequentially to the same tracks in volumes of 3; 4 and 5 μl of each standard solution. Tracks 2-7 and 9-14: 12 tracks of prepared solutions to be analyzed (four samples, each in triplicate) with a volume of 4 μl each. Application parameters: track width - 8 mm; left and right indents - 20 mm; bottom indent - 10 mm; distance between tracks - 11.4 mm.

[0026] 4. Chromatography. The plate is placed in the ADC2 automatic elution chamber (CAMAG, Switzerland), in which the saturation time (20 min), front distance (70 mm), and drying time (5 min) are set. A 15% ethanol-chloroform-ammonium acetate (6:3:2 v / v / v) solution is poured into both funnels. The ADC2 automatic chamber is started, and upon completion, the plate is removed and further dried in a TLC Plate Heater 3 (CAMAG, Switzerland) at 75°C for 10 minutes.

[0027] 5. Detection of metformin hydrochloride. The dried plate is scanned using a TLC Scanner 4 densitometer (CAMAG, Switzerland) at a wavelength of 239 nm. Densitograms are recorded and then integrated; the resulting peaks are identified by specified parameters (retention factor R f ) (see Fig. 3). The ordinate axis shows the intensity of light absorption at a wavelength of 239 nm, and the abscissa axis shows the distance from the application site to the peak center.

[0028] The obtained data are calibrated using peak height (metformin hydrochloride r=0.9923; sdv=2.85%). Calibration is performed using peak area (metformin hydrochloride r=0.98965; sdv=3.61%).

[0029] 6. Derivatization and detection of taurine. The plate is derivatized using a Chromatogram Immersion Device 3 (CAMAG, Switzerland) by automatically immersing it in a 0.01% solution of reagent grade ninhydrin in 96% ethanol. The plate is dried on a TLC Plate Heater 3 at 75°C for 10 minutes. Taurine is detected by scanning at a wavelength of 487 nm (Fig. 4). Based on the data obtained, calibration is performed through the peak height (taurine g = 0.92260; sdv = 3.98%), calibration through the peak area (taurine g = 0.98946; sdv = 2.75%).

[0030] 7. Quantitative calculation. The resulting chromatograms are processed using [winCATS] software. Multipoint calibration curves are constructed for metformin hydrochloride and taurine separately based on peak height and centerline at three standard points (2.0, 2.5, and 3.0 µl). For calculations, the value that provides the maximum correlation coefficient of variation (CV) for each component is selected. The program automatically calculates the content in the analyzed samples.

[0031] Validation characteristics of the developed method:

[0032] Specificity: No peak interference Linearity:

[0033] R 2 > 0.999 in the range of 0.1-1.0 mg / ml

[0034] Accuracy: 98.5-101.5%

[0035] Precision: RSD < 2%

[0036] Limit of quantification: 31.25 μg / mL

[0037] Robustness: RSD < 2% with varying parameters

[0038] Stability of solutions: 24 hours at room temperature

[0039] This method enables the simultaneous determination of the identity, purity, and quantitative content of metformin hydrochloride and taurine when present together in different matrices on a single chromatography plate without interference. Advantages include the ability to simultaneously analyze up to four samples on a single plate, which triples productivity and is environmentally friendly. Multipoint calibration with peak parameter optimization ensures high reliability of results (RSD < 2%).

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