RU2796599C1 - Method for determination of alkaloids in thermopsis extract - Google Patents

Abstract

FIELD: analytical chemistry.

SUBSTANCE: invention can be used in the pharmaceutical industry and related industries. The method for determining the alkaloids of lanceolate thermopsis is characterized by the use of high-performance thin-layer chromatography — densitometry (HPTLC-densitometry). Extraction is carried out with a composition consisting of chloroform, water, 32% ammonia solution in a ratio of 20:1:1, anhydrous sodium sulfate is added, the composition is mixed and filtered. The extract is extracted two more times with chloroform and filtered, evaporated to a dry residue, which is dissolved in methanol and separated on a chromatographic plate with a layer of silica gel with a fluorescent indicator in a chamber with a mobile phase consisting of chloroform, methanol, ammonia solution of 35 v/v.% in the ratio of 20:3:0.5. Then the plate is treated with a solution containing a composition of a 0.48% solution of basic bismuth nitrate in glacial acetic acid and a 60% aqueous solution of potassium iodide in a ratio of 25:5, with methanol added to it in a ratio of 1:7.33. The plate is scanned on a spectrodensitometer at a wavelength of 520 nm.

EFFECT: development of a technical solution in the form of a method characterized by selectivity, sensitivity and accuracy.

8 cl, 12 dwg, 7 tbl

Description

This technical solution relates to the field of chemistry, pharmacy, medicine, and can be used in the pharmaceutical industry and related industries to determine active biological substances in medicinal herbal raw materials and / or in medicinal herbal preparations, dietary supplements, using high performance thin layer chromatography - densitometry (HPTLC - densitometry).

State of the art

High performance thin layer chromatography - densitometry (HPTLC - densitometry) is currently actively used in the pharmaceutical industry and research activities. HPTLC - densitometry is based on measuring the absorption of electromagnetic radiation by the adsorption zone of the analyte on a chromatographic plate after separation of the sample components with or without derivatization.

HPTLC densitometry has a high separation efficiency, low detection limit of analytes (up to 1 picogram per adsorption zone), high reproducibility (standard deviation less than 3%), but the method is simple to perform, currently highly automated, and takes a small amount of time , in contrast to high-performance liquid chromatography or gas chromatography, as it allows chromatography of up to 40 tracks on one chromatographic plate with subsequent scanning.

HPTLC - densitometry can be used in the analysis of various biologically active substances of plant origin, such as alkaloids, phenolic compounds, steroid compounds, terpenoids, vitamins and many other substances. HPTLC - densitometry allows you to determine the content of an individual biologically active substance or a group of biologically active substances in herbal raw materials or medicines / biologically active additives based on them, while minimizing the effect of ballast substances, which are a serious problem in the analysis of herbal medicines, since chemical the composition of such preparations is extremely complex and may include many different substances.

For herbal medicines, there is an urgent need for sensitive, selective and accurate methods of analysis, as currently the analysis uses methods that do not have the above characteristics, or very complex and time-consuming methods of analysis.

The following were chosen as objects of study: dry extract of thermopsis, standardized by the amount of alkaloids in terms of thermopsin, and bearberry leaves, standardized by the content of phenol glycoside - arbutin.

The prior art method of quantitative determination of the amount of alkaloids in terms of thermopsin by titrimetry [1]. The disadvantages of this method are: low selectivity. The method allows to determine the amount of bases in the extract from the drug, which include not only alkaloids, but also a number of amino acids, some vitamins, decarboxylated derivatives of amino acids and other organic and inorganic bases. This method does not allow to determine the content of individual alkaloids of lanceolate thermopsis, as well as their ratio. Possibility of error due to the high subjectivity of the determination of the end point of the titration. The test solution has a different color: from pale yellow to brownish-yellow, while the indicator color transition ranges from green to blue-violet.

From the prior art there is another method for the determination of alkaloids in thermopsis lancet by HPLC. This method has high accuracy, sensitivity and selectivity, but it has disadvantages: it requires very expensive equipment, takes a lot of time, does not allow to determine the amount of alkaloids, since only the peak of thermopsin can be identified in the chromatogram of the test solution compared to the standard solution of thermopsin, identification the remaining peaks cannot be traced due to the lack of standard samples of other thermopsis alkaloids [2].

The method for quantitative determination of the amount of alkaloids by gravimetry is described in [3] and has very low selectivity and accuracy, since a large amount of ballast substances enter the extraction from the herbal preparation.

The method for quantitative determination of the amount of alkaloids of Macleay cordate by densitometry is described in [4]. This method has high sensitivity and selectivity. However, this method does not allow to determine the content of specific alkaloids and their ratio and has a high measurement error.

The prior art method for the quantitative determination of arbutin by spectrophotometry using a standard sample of arbutin or using the specific absorption index [5]. However, this method has a low selectivity and degree of accuracy, the results of the quantitative determination of arbutin using this method can reach more than 25% in terms of dry raw materials.

In the monograph of the European Pharmacopoeia "BEARBERRY LEAF", 07/2013:1054 [6], the HPLC method, which has high selectivity and accuracy, is used for the quantitative determination of arbutin in bearberry leaves. The disadvantages include the high duration and cost of the analysis, insufficient resolution between the arbutin peak and the peaks of related substances when using chromatographic columns from individual manufacturers.

A method for the quantitative determination of arbutin using HPTLC - densitometry was described in the source [7]. However, the presented method has disadvantages: due to the lack of purification of the test solution, a strong baseline noise is observed during scanning, extraction conditions are not indicated, the arbutin peaks are asymmetrical, which does not allow a reliable assessment of the results.

In the TLC method - densitometry, disclosed in the source [8], TLC plates are used, which leads to an increase in the analysis time, a single extraction is used, which cannot guarantee the complete extraction of arbutin from the herbal drug; purification of methanol extract from a medicinal herbal preparation from tannins is carried out with lead acetate, which is a toxic, carcinogenic compound (hazard class 3 according to NFPA 704), while the resulting amorphous precipitate is difficult to filter, it is preferable to use centrifugation; the use of the internal standard method complicates the calculation of the arbutin content.

The technical task is to develop a method for determining biologically active substances in medicinal plant raw materials and / or herbal medicinal preparations, using HPTLC - densitometry, which has sensitivity, selectivity and accuracy, as well as allowing to determine its authenticity and the content of the main biologically active substances in herbal medicinal products. origin or in dietary supplements.

Disclosure of the essence of the invention

The technical task is to develop a method for determining biologically active substances in medicinal plant materials and medicinal plant preparations using HPTLC - densitometry: in one case, a method for determining the amount of alkaloids in a thermopsis extract, in another case, a method for determining arbutin in bearberry leaves. The developed method is characterized by sensitivity, selectivity, accuracy and allows you to determine its authenticity and the content of the main active substances in a herbal medicinal product.

The technical result is the development of a technical solution in the form of a method characterized by selectivity, sensitivity and accuracy.

In one case, the determination of alkaloids, the technical result of the claimed technical solution is achieved by:

1) extraction with a composition (mixture of solutions) consisting of chloroform, ammonia solution 32%, water, followed by washing with chloroform;

2) standard samples of cytisine and thermopsin;

3) the method of absolute graduation;

4) high-performance chromatographic plates with a layer of silica gel and a mobile phase consisting of chloroform, methanol, ammonia solution 32%;

5) a solution for processing the plate, including a composition of a solution of bismuth nitrate basic in acetic glacial acid and an aqueous solution of potassium iodide and methanol added to it.

In another case, the definition of arbutin, the technical result of the claimed technical solution is achieved by:

1) triple extraction (extraction) with ethanol 70% when heated in a boiling water bath;

2) elution of the resulting filter (extraction) with ethanol 70% on a preparative chromatographic column with basic aluminum oxide;

3) a standard sample of arbutin;

4) the method of absolute graduation;

5) high-performance chromatographic plates with a layer of silica gel with a fluorescent indicator and a mobile phase consisting of chloroform, methanol and glacial acetic acid;

6) use for processing a chromatographic plate of a composition (solution) consisting of concentrated sulfuric acid and ethanol 96% in a ratio of 2:8.

Terms and definitions used

Baseline - section of the chromatogram corresponding to the detector signal from a mobile phase that does not contain substances to be separated.

High performance liquid chromatography (hereinafter - HPLC).

High performance thin layer chromatography - densitometry (hereinafter referred to as HPTLC - densitometry).

Polynomial regression in statistics is a form of regression analysis in which the relationship between the independent variable x and the dependent variable y is modeled as an nth degree polynomial in x . Polynomial regression corresponds to a non-linear relationship between the x value and the corresponding conditional mean y , denoted by E(y|x). Although polynomial regression fits a non-linear model to the data, as a statistical estimation problem, it is linear in the sense that the regression function E(y|x) is linear in the unknown parameters that are estimated from the data. For this reason, polynomial regression is considered a special case of multiple linear regression [10].

The absolute calibration method for quantitative determination of the content of components in the analyzed sample consists in constructing a calibration curve for the dependence of the area or height of the chromatographic peak on the content of this substance in the analyzed sample [11].

Implementation of the invention

In the field of quality assurance of medicinal products obtained from medicinal plant materials, an urgent task is the development of modern, high-precision methods of analysis that can reliably determine the content of active substances in medicinal plant materials.

In contrast to the Dragendorff reagent and the modified Dragendorff reagent used in HPTLC - densitometry, for vegetable raw materials containing alkaloids, solutions were developed with improved rheological characteristics that ensure rapid evaporation from the surface of the plate, the absence of streaks, smudges and other defects on the chromatographic plate, while , evenly coloring the silica gel. Processing of chromatographic plates was carried out by detection, by immersing them in a solution for processing the plate.

In one case, a dry extract of thermopsis was used as an object of research for the determination of alkaloids in medicinal plant materials.

It is known [9] that the main alkaloids contained in the herb lanceolate thermopsis are:

thermopsin having the following structural formula:

;

;

cytisine having the following structural formula:

;

;

N-methylcytisine having the following structural formula:

;

;

Spartein (Pachycarpine), having the following structural formula:

All alkaloids of lanceolate thermopsis have a tertiary amino group, while cytisine has a tertiary and secondary amino group responsible for the ionization of the alkaloid molecule.

A method for determining the alkaloids of lanceolate thermopsis, characterized in that high-performance thin-layer chromatography - densitometry (HPTLC-densitometry) is used, extraction is carried out with a composition consisting of chloroform, water, 32% ammonia solution in a ratio of 20:1:1, anhydrous sodium sulfate is added, stirred and filtered, the extract is extracted two more times with chloroform and filtered, evaporated to a dry residue, which is dissolved in methanol, then separated on a chromatographic plate with a layer of silica gel with a fluorescent indicator in a chamber with a mobile phase consisting of chloroform, methanol, ammonia solution of 35% vol. ./about. in a ratio of 20:3:0.5, then the plate is treated with a solution containing a composition of a 0.48% solution of bismuth nitrate basic in glacial acetic acid and a 60% aqueous solution of potassium iodide in a ratio of 25:5, with methanol added to it in a ratio 1:7.33, scanned on a spectrodensitometer at a wavelength of 520 nm.

The method is further characterized in that the thermopsis alkaloids are cytisine and thermopsin.

A method further characterized in that the cell is saturated with the mobile phase for 1 hour.

A method additionally characterized in that a standard solution of 2 µl, 4 µl, 8 µl, 12 µl, 16 µl, 20 µl was applied to the plate in the form of strips 8 mm wide at a distance of 1 cm from the bottom edge of the plate.

A method further characterized in that 15 µl of the test solution was applied to the plate in three parallels, two applications in the form of stripes 8 mm wide at a distance of 1 cm from the bottom edge of the plate.

A method further characterized in that the standard solution is a solution of 0.05% cytisine and 0.025% thermopsin in methanol.

The method is additionally characterized in that it is filtered through a paper filter previously washed with chloroform, with anhydrous sodium sulfate placed on it.

A method further characterized in that the length of the passage of the mobile phase is 4 cm from the start line.

A method further characterized in that the chromatographic plate is highly efficient.

Conducting sample preparation.

From the dry thermopsis extract, alkaloids are extracted, in the form of bases, with a composition (mixture of solutions) consisting of chloroform, ammonia solution 32% and water, taken in a ratio of 20:1:1, respectively, and mixed. Anhydrous sodium sulfate is added to the obtained extract (extract), mixed and filtered.

Next, chloroform was added to the anhydrous sodium sulfate extract, extracted, and then filtered. Repeat twice.

Filtration is carried out through a paper filter pre-washed with chloroform with anhydrous sodium sulfate placed on it.

The resulting filtrate is evaporated at a temperature of 40°C to a dry residue, which is then dissolved in methanol. Apply the test solutions to a high performance chromatographic plate.

As a standard solution, a solution of cytisine 0.05% and thermopsin 0.025% in methanol is used.

In the claimed method, a measuring container or any other laboratory measuring glassware known to a person skilled in the art is used.

The upward elution is carried out in a chamber pre-saturated with a mobile phase of the following composition: chloroform, methanol and ammonia solution 32% in a ratio of 20:3:0.5, respectively.

The distance of passage of the mobile phase front from the start line is 4 cm when applying sample solutions to a height of 1 cm from the lower edge of the plate.

As a solution for processing a chromatographic plate, a solution is used that includes a composition of a 0.48% solution of bismuth nitrate basic in glacial acetic acid and a 60% aqueous solution of potassium iodide in a ratio of 25:5, with methanol added to it in a ratio of 1:7.33 .

The chromatographic plate is immersed for 1 second in the chromatographic plate processing solution.

Chromatography is carried out under the following conditions:

- absolute graduation, where a standard solution of 2 µl, 4 µl, 8 µl, 12 µl, 16 µl, 20 µl is applied to the plate in the form of a strip 8 mm wide at a distance of 1 cm from the bottom edge; 15 µl of the test solution in three parallels, two applications;

- saturate the chamber with the mobile phase for 1 hour;

- passage of the front of the mobile phase 4 cm long from the start line;

- scanning on a spectrodensitometer at a wavelength of 520 reflection, micro slit 6×0.1.

In another case, bearberry leaves were used as an object of research. The main active ingredient of bearberry leaves is phenol glycoside - arbutin, which is a β-D-glucopyranoside of hydroquinone, characterized by the structural formula:

A method for determining arbutin in bearberry leaves, characterized in that high-performance thin-layer chromatography - densitometry (HPTLC-densitometry) is used, sample preparation is carried out, where it is extracted with 70% ethanol in a boiling water bath for 15 minutes, cooled, filtered, extraction is repeated twice for 10 minutes, then the filtrate was placed on a preparative column and eluted with ethanol 70%, further separated on a high performance chromatographic plate with a layer of silica gel with a fluorescent indicator using a mobile phase consisting of chloroform, methanol, glacial acetic acid in a ratio of 11:3.5:1 and then the plate treated with a composition consisting of concentrated sulfuric acid and ethanol 96% in a ratio of 2:8, dried, scanned on a spectrodensitometer at a wavelength of 285 nm.

The method is further characterized in that the chamber was saturated with the mobile phase for 30 minutes.

The method is additionally characterized by the fact that a standard solution of 1 µl, 2 µl, 4 µl, 6 µl, 8 µl, 10 µl is applied to the plate in the form of a strip 8 mm wide at a distance of 1 cm from the bottom edge.

The method is additionally characterized by the fact that 5 μl of the test solution is applied to the plate in three parallels, two applications at a distance of 1 cm from the bottom edge.

The method is further characterized by the use of a standard solution of 0.1% arbutin in 70% ethanol.

The method is additionally characterized by the fact that the length of the passage of the mobile phase is 4 cm from the start line.

The method is additionally characterized by the fact that bearberry leaves are used, crushed in the form of a powder.

Ethanol 70% is added to crushed bearberry leaves and extracted in a boiling water bath for 15 minutes. The resulting extract is cooled to room temperature, filtered. The extraction procedure in a boiling water bath followed by filtration is repeated, where the re-extraction is carried out for 10 minutes. The filtrate was placed on a preparative column and eluted with 70% ethanol, made up to the mark with 70% ethanol and stirred.

Filtration is carried out through a paper filter, pre-washed with 70% ethanol.

A 0.1% standard solution of arbutin in ethanol 70% is used as a standard solution.

The separation is carried out on a high-performance chromatographic plate using a mobile phase (composition) of the following composition: chloroform, methanol, glacial acetic acid (at least 98.8%) in a ratio of 11:3.5:1, respectively.

The distance of passage of the front of the mobile phase is 4 cm, when applying sample solutions to a height of 1 cm from the bottom edge of the plate.

As a solution for processing a chromatographic plate, a composition consisting of concentrated sulfuric acid (93.56-95.68%) and ethanol 96% in a ratio of 2:8, respectively, is used.

A preparative column (height 20 cm and diameter 1.5 cm) washed with ethanol 70% and filled with basic aluminum oxide is used.

The chromatographic plate is immersed for 1 second in the chromatographic plate processing solution.

Chromatography is carried out under the condition:

- preparation of a high-performance chromatographic plate with a layer of silica gel with a fluorescent indicator by chromatography in the mobile phase of ethanol 96% to a height of 5 cm from the bottom edge of the plate, heating to 103±2°C, and then cooling to room temperature;

- absolute graduation, where a solution of a standard sample of arbutin is applied to the plate in 1 µl, 2 µl, 4 µl, 6 µl, 8 µl, 10 µl in the form of a strip 8 mm wide at a distance of 1 cm from the bottom edge; 5 µl of the test solution in three parallels, two applications;

- saturation of the chamber with the mobile phase for 30 minutes;

- the length of the passage of the front of the mobile phase is 4 cm from the start line;

- scanning on a spectrodensitometer at a wavelength of 285 nm, micro slit 6×0.1.

Brief description of drawings and other materials (Appendices 1-12).

Fig. 1. Visible light chromatogram after development with plate processing solution. 1.1 - SS solution of Cytisine and Thermopsin, 2 µl; 1.2 - solution of SS Cytisine and Thermopsin 4, µl; 1.3 - solution of SS Cytisine and Thermopsin, 8 µl; 1.4 - SS solution of Cytisine and Thermopsin, 12 µl; 1.5 - SS solution of Cytisine and Thermopsin, 16 µl; 1.6 - SS solution of Cytisine and Thermopsin, 20 µl; 1.7-1.8 - test solution No. 1, 15 µl; 1.9-1.10 - test solution No. 2, 15 µl; 1.11-1.12 - test solution No. 3, 15 µl.

Fig. 2. Chromatogram of the test solution when scanned at a wavelength of 520 nm.

Fig. 3. Chromatogram of standard solutions (applying 8 µl) when scanning at a wavelength of 520 nm.

Fig. Fig. 4. Spectra of cytisine adsorption zones (at the top is the spectrum of the standard solution, at the bottom of the test solution).

Fig. Fig. 5. Spectra of thermopsin adsorption zones (at the top is the spectrum of the standard solution, at the bottom of the test solution).

Fig. 6. Calibration curve of standard solutions of cytisine.

Fig. 7. Calibration curve for thermopsin standard solutions.

Fig. 8. Chromatogram of the development with a solution for processing the plate at 254 nm:

2.1 - SS solution of arbutin, 1 µl; 2.2 - SO solution of arbutin, 2 µl; 2.3 - SO solution of arbutin, 4 µl; 2.4 - SO solution of arbutin, 6 µl; 2.5 - SO solution of arbutin, 8 µl; 2.6 - arbutin SS solution, 10 µl, 2.7-2.8 - test solution No. 1, 5 µl; 2.9-2.10 - test solution No. 2, 5 µl; 2.11-2.12 - test solution No. 3, 5 µl.

Fig. 9. Chromatogram of the test solution when scanned at a wavelength of 285 nm.

Fig. Fig. 10. Spectrum of the arbutin adsorption zone of the 1 mg/ml arbutin standard solution (top), spectrum of the arbutin adsorption zone of the test sample solution (bottom).

Fig. 11. Chromatogram in visible light after development with a solution for processing the plate: 2.1 - solution of SS arbutin, 1 µl; 2.2 - SO solution of arbutin, 2 µl; 2.3 - SO solution of arbutin, 4 µl; 2.4 - SO solution of arbutin, 6 µl; 2.5 - SO solution of arbutin, 8 µl; 2.6 - arbutin SS solution, 10 µl, 2.7-2.8 - test solution No. 1, 5 µl; 2.9-2.10 - test solution No. 2, 5 µl; 2.11-2.12 - test solution No. 3, 5 µl.

Fig. 12. Dependence of the peak area (S) of arbutin on the concentration (C) of arbutin in the standard solution.

The possibility of implementing the claimed method is disclosed in the following examples.

Example 1. Determination of the authenticity and quantitative content of the total alkaloids in terms of thermopsin in dry extract of thermopsis.

Chloroform, 32% ammonia solution, water are added to the dry thermopsis extract (1 g), in a ratio of 20:1:1, respectively. The resulting extract is stirred on an orbital shaker for 20 minutes. Anhydrous sodium sulfate (2 g) was then added to the extract, stirred again, and then filtered.

Next, 10 ml of chloroform was added to the extract with anhydrous sodium sulfate, extracted and then filtered. Repeat twice.

Filtration is carried out through the "black tape" ash filter with anhydrous sodium sulfate (3 g) placed on it, previously washed with chloroform.

The resulting filtrate is evaporated to dryness on a rotary evaporator at 40°C, and then the residue is dissolved in methanol (10 ml).

As a standard solution, a solution of cytisine 0.05% and thermopsin 0.025% in methanol is used.

In the claimed method, a measuring container with a thin section with a tight-fitting stopper/lid or any other container or container known to a person skilled in the art that allows extraction/extraction is used.

On a high-performance chromatographic plate with a layer of silica gel measuring 20 × 10 cm, 1 cm from the bottom edge of the plate, a standard solution is applied in the form of a strip 8 mm long, 2 μl, 4 μl, 8 μl, 12 μl, 16 μl, 20 μl, 15 μl of the test solution in three parallels, two applications.

The plate is placed in a chromatography chamber pre-saturated with mobile phase for one hour.

The mobile phase is a mixture of solutions (composition) of chloroform, methanol, ammonia solution 32% (v/v) in a ratio of 20:3:0.5, respectively.

After the mobile phase front has passed 4 cm from the start line, the plate is removed from the chromatographic chamber and dried in a fume hood until traces of solvents disappear. The plate is immersed for 1 second in the solution for processing the chromatographic plate. The plate is dried in a stream of hot air for 2-3 minutes and then scanned on a spectrodensitometer.

As a composition (solution) for processing a chromatographic plate, a solution obtained by mixing a 0.48% solution of bismuth nitrate basic in glacial acetic acid (at least 98.8%) and an aqueous solution of potassium iodide 60%, taken in a ratio of 25:5, is used, respectively, where methanol is added to the resulting composition (mixture of solutions) in a ratio of 1:7.33, respectively.

Scan conditions:

Wavelength: 520 nm;

Mode: absorption/reflection.

Slit: micro 6×0.1mm

According to the test results, 7 alkaloid adsorption zones are found on the chromatograms of the test solution. The adsorption zone with Rf≈0.45 corresponds to cytisine, the adsorption zone with Rf≈0.8 corresponds to thermopsin (Fig. 1, 2). On the chromatograms of the standard solution, two adsorption zones are found, corresponding to thermopsin and cytisine (Fig. 1,3).

The scanning wavelength of 520 nm corresponds to the maximum absorption of the spectra of thermopsin and cytisine (Fig. 4,5).

Build calibration curves for standard samples of cytisine and thermopsin on the area of the peak. The curve formula uses the polynomial regression method.

The calculation of the cytisine content is carried out by the method of absolute calibration based on the results of the analysis of six different concentrations of the cytisine standard (according to the peak area).

The calculation of the thermopsin content was carried out by the method of absolute calibration based on the results of the analysis of six different concentrations of the thermopsin standard (according to the peak area).

The calculation is carried out according to the formula (1):

,

,

where A is the content of the analyte in mg, (calculated);

a - weighed portion of the preparation, mg;

W - moisture content in the preparation,%.

The content of the sum of alkaloids in terms of thermopsin is calculated by the formula (2):

,

,

where C is the content of cytisine in mg in a sample of the preparation.

T is the content of thermopsin in mg in a sample of the drug.

- сумма площадей всех пиков;

- the sum of the areas of all peaks;

SC - peak area of cytisine;

ST is the area of the thermopsin peak;

a - weighed portion of the preparation, mg;

W - moisture content in the preparation,%.

During the analysis, it was found that thermopsin, cytisine, and an unidentified alkaloid with Rf ≈ 0.25 are the dominant alkaloids, the sum of their peak areas is about 80% of the sum of the areas of all alkaloid peaks (Table 1).

Table 1 . Peak areas in the chromatogram of the test solution. Peak No. RF Area, A Square, % Sample name 1 0.1086 0.0022 9.17 unidentified 2 0.2505 0.00704 29.05 unidentified 3 0.355 0.00085 3.49 unidentified 4 0.555 0.00521 21.51 cytisine 5 0.716 0.00037 1.51 unidentified 6 0.763 0.00131 2.10 unidentified 7 0.830 0.00724 29.87 thermopsin

The peak area of thermopsin is 29.9% of the sum of the areas of all peaks, the peak area of cytisine is 21.5% of the sum of the areas of all peaks.

Table 2. Results of analyzes of the content of the total alkaloids in the dry extract of Thermopsis for series 1 Try 1 2 3 4 5 6 7 8 9 Humidity,% - 3.7
The amount of alkaloids in terms of thermopsin by titration,% - 0.98±0.061 RSD=3.59% The content of thermopsin, % 0.157±
0.009 0.147±
0.018 0.146±0.023 0.143±0.012 0.149±0.019 0.142±0.012 0.149±0.005 0.145±0.005 0.149±
0.006 Xav.%=0.147±0.034 RSD%=3.00 Cytisine content, % 0.232±
0.012 0.230±0.016 0.222±
0.027 0.224±
0.020 0.234±
0.020 0.240±
0.027 0.235±
0.016 0.227±
0.004 0.239±
0.011 Xav.%=0.230±0.0045 RSD%=2.54 The amount of alkaloids in terms of
thermopsin, % 0.666±
0.146 0.670±
0.032 0.690±
0.133 0.655±
0.011 0.697±
0.019 0.676±
0.045 0.697±0.143 0.716±0.111 0.699
±0.098 Xav.%=0.685±0.015 RSD%=2.89

Table 3. Results of analyzes of the content of the total alkaloids in dry thermopsis extract for series 2 Try 1 2 3 4 5 6 7 8 9 Humidity,% - 2.1
The amount of alkaloids in terms of thermopsin by titration,% - 0.98±0.056 RSD=3.15% The content of thermopsin, % 0.180±0.031 0.173±0.009 0.170±0.002 0.168±0.036 0.169±0.013 0.168±0.023 0.168±0.011 0.171±0.025 0.179±0.033 Xav.%=0.171±0.030 RSD%=2.73 Cytisine content, % 0.253±0.022 0.244±0.002 0.248±0.014 0.240±0.038 0.235±0.050 0.246±0.012 0.240±0.021 0.238±0.005 0.253±0.004 Xav.%=0.244±0.005 RSD%=2.63 The amount of alkaloids in terms of
thermopsin, % 0.748±0.060 0.723±0.085 0.719±0.055 0.740±0.074 0.695±0.074 0.750±0.043 0.745±0.035 0.715±0.010 0.696±0.055 Xav.%=0.726±0.016 RSD%=2.95

The content of alkaloids was determined using a device known to a person skilled in the art - a spectrodensitometer.

The content of cytisine in the dry extract of thermopsis is approximately 1.5 times higher than that of thermopsin (Tables 2, 3).

The calculation of unidentified alkaloids of dry thermopsis extract was calculated on thermopsin [6].

The developed method was validated, confirming its suitability for the quantitative determination of the amount of alkaloids in terms of thermopsin in dry thermopsis extract. The results of the validation are presented in Table 4. To confirm the specificity of the method, a model mixture was used, consisting of 23.46 mg of cytisine, 15.06 mg of thermopsin and 9.98 g of sucrose. 1 gram of the model mixture was analyzed according to the above method. The authenticity of the drug is confirmed by visual inspection of the plate based on the correspondence of the color and Rf values of the adsorption zones on the chromatogram of the test solution to the adsorption zones of cytisine and thermopsin on the chromatogram of the standard solution.

Table 4. Results of validation of the method for determining the amount of alkaloids in terms of thermopsin Characteristic Requirements Result Specificity The presence of concomitant components does not unintentionally affect the result of the analysis 1) The spectrum of the cytisine adsorption zone of the test solution completely coincides in terms of the position of absorption maxima and minima with the spectrum of the cytisine adsorption zone of the standard solution.
The spectrum of the thermopsin adsorption zone of the test solution completely coincides in terms of the position of absorption maxima and minima with the spectrum of the thermopsin adsorption zone of the standard solution.
2) The true values of the content of cytisine ( 0.234 mg/g ) and thermopsin ( 0.151 mg/g ) in the model mixture lie within the boundaries of the confidence intervals of the results of the analysis of the content of cytisine and thermopsin in the model mixture. Linearity The results of measurements of analytical signals of 6 samples with different contents of cytisine were processed by the least squares method using a linear model Correlation coefficient
r=0.9903 Equation of a straight line: Y = 0.0015 x + 0.001
Correlation coefficient: r = 0.9903 The results of measurements of analytical signals of 6 samples with different contents of thermopsin were processed by the least squares method using a linear model
Correlation coefficient
r=0.9939 Equation of a straight line: Y = 0.0039 x - 0.0008
Correlation coefficient: r = 0.9939 Analytical area The technique is applicable in the range from 80% to 120% of the nominal value of the content of cytisine and thermopsin. Compliant based on linearity study data Right The free term of the equation is less than its confidence interval
Δ a \u003d t (0.05, n - 2) × s _a
a ≤ ∆ a Based on data from the study of linearity
Calculation for cytisine:
a = 0.001
sa ₌ 0.00461
t (0.05, n - 2) = 2.78
Δa = 2.78 × 0.00461 = 0.01282
a < Δa Calculation for thermopsin:
a = 0.0008
sa = _0.00038
t (0.05, n - 2) = 2.78
Δa = 2.78 × 0.00038 = 0.00106
a < Δa Precision Repeatability RSD of the results of 9 determinations of the quantitative content of the total alkaloids in terms of thermopsin should be no more than 3.0% X _{series 1} = 0.685±0.015%
RSD = 2.89%
X _{series 2} = 0.726±0.016%
RSD = 2.95% Inside
laboratory precision The resulting value of the Fisher criterion, calculated from the results of testing by different performers on different equipment, should be less than the tabular value F _pract = s 1 2 / s 2 2 ≤ F _teor F _practice = 0.00030 / 0.00003 = 1.11
F _teor (0.05; 2; 2) = 19.00 F _practice < F _teor Note. t(0.95; n - 2) - Student's coefficient, where 0.95 - probability, f - number of degrees of freedom; F _pract , F _teor (0.05; 2; 2) - derived and tabular Fisher criteria, respectively, where 0.05 - significance level, 2 and 2 - the number of degrees of freedom; s - standard deviation; s1, s2 - greater and lesser standard deviations of the obtained results, respectively; a - free term of the linear dependence, sa - standard deviation of the free term of the linear dependence, RSD - relative standard deviation (%).

In series 1, the content of thermopsin in % was 0.147% ±0.034 (RSD %=3.00), the content of cytisine in % was 0.230±0.0045 (RSD %=2.54),content the sum of alkaloids in terms of thermopsin, % was 0.685±0.015 (RSD %=2.89).

In series 2, the content of thermopsin in % was 0.171±0.030 (RSD %=2.73), the content of cytisine in % was 0.244±0.005 (RSD %=2.63),content the sum of alkaloids in terms of thermopsin was 0.726±0.016 (RSD %=2.95) (Tables 2, 3). This example confirms the possibility of determining the authenticity and content of the amount of alkaloids by the claimed method.

The claimed method has selectivity, sensitivity, accuracy in determining the content of cytisine, thermopsin and unidentified alkaloids in medicinal plant materials and/or drugs based on them.

Example 2. The linearity of the method in the analytical region is confirmed by the linear dependence of the peak area of the test substance on its concentration in solution. In the developed method, the analytical range of cytisine content corresponds to the range of their concentrations from 1 µg to 8 µg, the analytical range of thermopsin content corresponds to the range of their concentrations from 0.5 µg to 4 µg. The obtained data were processed by the method of polynomial regression.

As a result of calculations, the correlation coefficients were 0.999 for the calibration curve of cytisine (Fig. 6) and 0.996 for the calibration curve of thermopsin (Fig. 7), which indicates a high correlation between the estimated parameters.

Example 3. Determination of the authenticity and content of arbutin in bearberry leaves.

Ethanol 70% (20 ml) is added to crushed leaves of bearberry (1.0 g) and extracted (extracted) in a boiling water bath for 15 minutes. The resulting extract (extract) is cooled to room temperature, filtered.

The extraction procedure in a boiling water bath followed by filtration is repeated, where the re-extraction is carried out for 10 minutes. The resulting filtrate was brought to the mark with 70% ethanol and stirred.

Filtration is carried out through a paper filter, previously washed with a 70% ethanol solution.

In the claimed method, a measuring container with a thin section with a tight-fitting stopper/lid or any other container or container known to a person skilled in the art that allows extraction/extraction is used.

The standard solution is a 0.1% arbutin solution in 70% ethanol.

A preparative column (height 20 cm and diameter 1.5 cm) washed with ethanol 70% and filled with basic aluminum oxide is used.

The resulting extract (10.0 ml) is transferred to the preparative column and eluted at a rate of 1-2 drops/sec. Ethanol 70% (5 ml) was added to the column and eluted in the same way. The operation is repeated. Bring the volume of the solution with the same solvent (ethanol 70%) to the mark and mix (test solution).

Composition (solution) for processing the plate, consisting of concentrated sulfuric acid and 96% ethanol, taken in a ratio of 2:8, respectively, cooled to room temperature.

Chromatography plate preparation: A high-performance chromatographic plate with a layer of silica gel with a fluorescent indicator is placed in a chamber with 96% ethanol and chromatographed to a height of 5 cm from the bottom edge of the plate, then heated to (103 ± 2) ° C for five minutes and then cooled to room temperature. temperature.

On the start line (10 mm from the lower edge) of the prepared high-performance chromatographic plate, a solution of a standard arbutin sample is sequentially applied: 1 µl, 2 µl, 4 µl, 6 µl, 8 µl, 10 µl with a strip of 8 mm. Two times 5 µl of the test solution. Leave until traces of solvent are removed. The plate is placed in a chromatographic chamber saturated for 30 minutes with a mobile phase (composition) consisting of: chloroform, methanol, glacial acetic acid in a ratio of 11:3.5:1, respectively, and chromatographed in an ascending manner.

When the solvent front passes 4 cm from the start line, the plate is removed from the chamber, dried in a stream of warm air under draft until the solvent traces disappear. The plate is scanned on a spectrodensitometer at a wavelength of 285 nm, the slit size is 6×0.1 micro, the calculation is based on the peak area, the curve type is polynomial regression.

The plate is then treated with a plate treatment solution and heated on a plate heater for 2-3 minutes at (103±2)°C. Chromatograms of the test solution should show orange-brown to dark brown adsorption zones with an Rf of about 0.4 (arbutin).

According to the test results, on the chromatograms of standard and test solutions in ultraviolet light at 254 nm, an adsorption zone of arbutin with an Rf of about 0.4 is detected (Fig. 8).

On the chromatograms of the test solutions after scanning at a wavelength of 285 nm, a clear, symmetrical peak of arbutin is detected, separated from the rest of the peaks. Baseline noise does not interfere with chromatogram processing (FIG. 9).

The content of arbutin according to pharmacopoeial articles should be at least 6.0% in accordance with the State Pharmacopoeia of the Russian Federation, at least 7.0% - in accordance with the Pharmacopoeia of the European Union.

The content of arbutin in bearberry leaves is calculated by the formula:

C - the content of arbutin in a sample, mg;

a - weighed portion of the drug, mg;

W is the moisture content of the herbal preparation, %.

The results of the quantitative determination and validation of the developed method, confirming its suitability for the quantitative determination of arbutin in the leaves of bearberry, are shown in tables 5 and 6.

Table 5 - Results of Quantitative Determination of Arbutin in Bearberry Leaves by HPTLC Try 1 2 3 4 5 6 7 8 9 10 Result, % 7.64 7.69 7.64 7.67 7.82 7.93 7.88 7.90 7.98 7.83 Statistical data n=10
t(p, f)=2.26 mean=7.79%
S STD = 0.09155 RSD% = 1.64
Confidence interval (Р=95%, α=0.05) ±0.09155
upper bound CI 7.88863
lower limit CI 7.70554

Table 6 - Results of the validation of the method for determining arbutin in the leaves of bearberry by HPTLC Characteristic Requirements Result Specificity The presence of concomitant components does not unintentionally affect the result of the analysis The UV spectrum of the adsorption zone of arbutin completely coincides in terms of the positions of the absorption maxima and minima with the UV spectrum of the adsorption zone of the standard sample of arbutin. Linearity The data of experimental measurements of analytical signals of 6 samples with different content of arbutin were processed by the least squares method using a linear model
Correlation coefficient r ≥ 0.99 Straight line equation: Y=0.0029x+0.0046
Correlation coefficient: R=0.9964 Analytical area The method is applicable in the range from 80% to 120% of the nominal value of the arbutin content Compliant based on linearity study data Right The value taken as true lies within the confidence interval of the average results of the analysis obtained experimentally using this method Conforms based on comparison of results obtained using a validated method and the exemplary method of the European Pharmacopoeia Precision Repeatability The RSD of 10 arbutin quantification results must be less than 3.0% average value of arbutin content = 7.74%
RSD% = 2.04 Interlaboratory Precision The resulting value of the Fisher criterion, calculated from the results of testing by different performers on different equipment, should be less than the table value Fpract. = s1 2 /s2 2 ≤ Fteor Fpract.= 0.00910 /0.00252= 3.61
Fteor.(0.05; 2; 2) = 19.00 Fpract. <Fteor . Note. t(0.95; n-2) - Student's coefficient, where 0.95 - probability, n-2 - number of degrees of freedom; Fpract. , Fteor. (0.05; 2; 2) - obtained and tabular Fisher criteria, respectively, where 0.05 - significance level, 2 and 2 - the number of degrees of freedom; s - standard deviation; s1 , s2 - greater and lesser standard deviations of the obtained results, respectively; sa is the standard deviation of the intercept, RSD is the relative standard deviation (%)

The spectra of the arbutin adsorption zone of the test solution and the adsorption zone of the standard arbutin solution are identical and have maxima at 224 nm and 286 nm (Fig. 10).

The authenticity of the herbal medicinal product is confirmed by detection by treating the chromatographic plate with a composition (solution) consisting of concentrated sulfuric acid and 96% ethanol, taken in a ratio of 2:8, respectively.

The disadvantages of existing analogues include lower sensitivity (4 μg of arbutin for a solution of sodium phosphomolybdate 10% in accordance with the State Pharmacopoeia of the Russian Federation); the complexity of preparing an anhydrous sodium carbonate reagent and a solution of 2,6-dichloroquinone-4-chlorimide in methanol in accordance with the European Pharmacopoeia; insufficient specificity of detection in UV - light at a wavelength of 254 nm (Fig. 11).

This example demonstrates the possibility of determining the authenticity and content of arbutin by the claimed method. The content of arbutin was 7.8%, which meets the requirements of the pharmacopoeial documents.

Example 4

The linearity of the method in the analytical region is confirmed by the linear dependence of the peak area of the test substance on its concentration in solution (Fig. 12). In the developed method, the analytical range of arbutin content corresponds to the range of its concentrations of 1-6 μg.

The obtained data were processed by the least squares method, which consists in finding the coefficients a, b and the correlation coefficient r of the linear model:

Y \u003d b * x + a,

where x is the arbutin concentration, y is the arbutin peak area, b is the model slope, and a is the free term.

As a result of calculations, the correlation coefficient was 0.996, which proves the linearity of the claimed methodology according to the pharmacopoeial article.

Example 5. To confirm the possibility of industrial application of the proposed invention, its correctness was proved by comparing the obtained results with the HPLC method of the monograph 07/2013:1054 of the European Pharmacopoeia (Table 7), which had previously passed the validation process.

Table 7. The results of testing the quantitative determination of arbutin in the bearberry leaves, obtained by HPLC and HPTLC for five different batches of the medicinal herbal preparation "bearberry leaves". Series 1 2 3 4 5 Arbutin content %, HPLC method 7.79±0.09 7.61±0.17 7.14±0.21 8.02±0.11 7.94±0.20 Arbutin content %, HPTLC method 7.74±0.26 7.53±0.23 6.99±0.34 7.94±0.28 7.98±0.35

The results obtained using HPTLC - densitometry are comparable with the results obtained using HPLC (Table 7). The results obtained demonstrate the possibility of identifying arbutin in the leaves of bearberry with its subsequent quantitative determination.

The presented examples do not limit the scope of the claims of the present invention and serve only for the purpose of illustration.

Industrial Applicability

All the above examples confirm the selectivity, sensitivity, accuracy of the claimed method.

Thus, the set technical problem, namely, the development of selectivity and sensitivity, the claimed method is achieved, as evidenced by the examples.

The application of the claimed method is possible in pharmacy, medicine, chemistry and related industries, which is confirmed by the relevant examples.

Bibliography:

1. State Pharmacopoeia of the Russian Federation XIV Edition Volume 4 / Ministry of Health of the Russian Federation. URL https://femb.ru/femb/pharmacopea.php

2. Pat. CN102349940A. A method for obtaining alkaloids of lanceolate thermopsis and a number of high-purity medicinal substances.

3. M.T. Kurbanova, I.Zh. Zhalolov. Dynamics of accumulation of alkaloids and elements in different organs of the plant glaucium elegans. Science and innovation international scientific journal, 2022. N 1. C. 192-200. https://doi.org/10.5281/zenodo.6496948

4. A.A. Pogotskaya, G.N. Buzuk. Application of a scanner and computer programs for digital image processing for the quantitative determination of alkaloids in the leaves of Macleay cordate. Bulletin of Pharmacy, 2009. No. 4 (46). pp. 32-38.

5. State Pharmacopoeia of the Russian Federation XIV Edition Volume 3 / Ministry of Health of the Russian Federation. URL https://femb.ru/femb/pharmacopea.php (accessed 12/15/2022).

6. European Pharmacopoeia. 11th ed. URL https://www.edqm.eu/en/european-pharmacopoeia.

7. P. Alamab, S.I. Alqasoumia, F. Shakeelc and others. HPTLC densitometric analysis of arbutin in bulk drug and methanolic extracts of Arctostaphylos uva-ursi. natural product research. 2011, 25(17). P. 1671-1675. .http://dx.doi.org/10.1080/14786419.2010.529447.

8. A. Pyka, K. Bober, A. Stolarczyk. Densitometrick determination of arbutine cowberry leaves. Acta Poloniae Pharmaceutica - Drug Research. 2007.63(5). P.395-400.

9. I.P. Tsypysheva, E.G. Galkin, A.S. Erastov. O.A. Karimova, I.P. Baikova., A.V. Kovalskaya, I.U. Khalilova, L.M. Abramova, L.S. Yunusov. Plant sources of quinolizidine alkaloids on the territory of the Republic of Bashkortostan. I. alkaloids Thermopsis schischkinii and Thermopsis lanceolata ssp. Sibirica (Fabaceae) under conditions of introduction. Chemistry of plant raw materials, 2014. No. 4. pp. 181-186.

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Claims (9)

1. A method for determining the alkaloids of lanceolate thermopsis, characterized in that high-performance thin-layer chromatography - densitometry (HPTLC-densitometry) is used, extraction is carried out with a composition consisting of chloroform, water, 32% ammonia solution in a ratio of 20: 1: 1, anhydrous sodium sulfate is added , stirred and filtered, the extract was extracted two more times with chloroform and filtered, evaporated to a dry residue, which was dissolved in methanol, then separated on a chromatographic plate with a layer of silica gel with a fluorescent indicator in a chamber with a mobile phase consisting of chloroform, methanol, ammonia solution 35 % v/v in a ratio of 20:3:0.5, then the plate is treated with a solution containing a composition of a 0.48% solution of bismuth nitrate basic in glacial acetic acid and a 60% aqueous solution of potassium iodide in a ratio of 25:5, with methanol added to it in a ratio 1:7.33, scanned on a spectrodensitometer at a wavelength of 520 nm. 2. The method according to claim 1, further characterized in that the thermopsis alkaloids are cytisine and thermopsin. 3. The method according to p. 1, further characterized in that the cell was saturated with the mobile phase for 1 hour. 4. The method according to claim 1, additionally characterized in that a standard solution of 2 μl, 4 μl, 8 μl, 12 μl, 16 μl, 20 μl was applied to the plate in the form of strips 8 mm wide at a distance of 1 cm from the lower edge of the plate . 5. The method according to claim 1, additionally characterized in that 15 μl of the test solution was applied to the plate in three parallels, two applications in the form of strips 8 mm wide at a distance of 1 cm from the bottom edge of the plate. 6. The method according to p. 5, additionally characterized in that the standard solution, which is a solution of cytisine 0.05% and thermopsin 0.025% in methanol. 7. The method according to p. 1, additionally characterized in that it is filtered through a paper filter previously washed with chloroform, with anhydrous sodium sulfate placed on it. 8. The method according to p. 1, additionally characterized in that the length of the passage of the mobile phase is 4 cm from the start line. 9. The method of claim. 1, further characterized in that the chromatographic plate is highly efficient.

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