RU2747370C1 - Method for determination of parabens in medicinal drugs by glc method - Google Patents

Abstract

FIELD: analytical chemistry.

SUBSTANCE: invention relates to the field of analytical chemistry, it can be used for the determination of parabens in medicinal drugs. The method for the determination of parabens in medicinal drugs by capillary gas-liquid chromatography includes the preparation of a standard solution of parabens in chloroform at a concentration corresponding to the expected concentration in the analyzed medicinal drug, the extraction of 4 ml of the test solution of the medicinal drug with the addition of 5 ml of chloroform, subsequent mixing on an orbital shaker for 10 minutes at 400 rpm and subsequent chromatography of the prepared samples on a capillary column under selected chromatographic conditions using a flame ionization detector, identification of parabens by retention times and quantitative calculation of paraben concentrations by an external standard method.

EFFECT: ability to elute parabens faster and obtain peaks with better chromatographic parameters on chromatograms.

5 cl, 6 dwg, 2 tbl

Description

The invention relates to the field of analytical chemistry and can be used in pharmaceutical, chemical and other industries in the determination of parabens (4-hydroxybenzoic acid esters) by capillary gas-liquid chromatography (GLC) in drugs using a flame ionization detector.

A known analogue of the invention is a method for the chromatographic determination of parabens in liquid and suspension pharmaceutical preparations and liquid biologically active additives (RF patent No. 2532237). This invention allows in one analysis to carry out the qualitative and quantitative determination of methyl-, ethyl-, propyl- and butylparaben by high-performance liquid chromatography on a reversed-phase sorbent using spectrophotometric and / or diode-matrix detectors in samples of liquid and suspension pharmaceutical preparations and biologically active additives with preliminary sample preparation (dilution of the sample and filtration through a membrane filter with a pore diameter of no more than 0.5 μm). The disadvantages of this analogue include the duration of the chromatographic analysis (more than 30 min) and the relatively high cost of the analysis due to the use of a guard column and reagents for preparing the mobile phase.

Also known is an analogue of the invention - gas chromatographic determination of parabens using a mass spectrometric detector (Bajaj A., John C., Choudhury J., Tripathi RM Development and Validation of Gas Chromatography / Mass Spectrometry Method for the Simultaneous Determination of Some Preservatives in Personal Care, Cosmetics and Pharmaceutical Formulations. Research J. Science and Tech. 2017; 9 (3): 308-312). It includes dissolving samples in a NaCl solution (pH 2), aging in an ultrasonic bath for 20 min, filtration, triple extraction with ethyl acetate, dehydration, subsequent extraction with methanol and filtration followed by gas chromatographic analysis. The disadvantages of this determination of parabens include lengthy sample preparation, as well as the use of a mass spectrometric detector, which significantly increase the cost of analysis, which complicates its implementation into industrial practice as a routine analysis.

There is another method of gas chromatographic determination of parabens using a mass spectrometric detector (Canosa P., Rodriguez I., Rubi E., Cela R. Determination of Parabens and Triclosan in Indoor Dust Using Matrix Solid-Phase Dispersion and Gas Chromatography with Tandem Mass Spectrometry. Anal. Chem. 2007; 79: 1675-1681). Its essence lies in the preparation of derivatives (derivatization) with N-methyl-N- (tert-butyldimethylsilyl) trifluoroacetamide and subsequent chromatographic determination. The disadvantages of this analogue include the need to control the completeness of obtaining derivatives, as well as an increase in the cost of analysis due to the use of derivatizing agents and mass spectrometric detection.

As a prototype, a method for the quantitative determination of methylparaben and propylparaben in the drug "Loma Lux Psoriasis" was chosen, the essence of which consists in the extraction of parabens with chloroform followed by gas chromatographic determination using a packed column filled with particles of a diatomite carrier "Chromosorb AW-HMCS" (base - silica) modified with 3% polyethylene glycol adipate (PEGA) and 1% phosphoric acid. The disadvantages of the prototype include low sensitivity and resolution of the chromatographic method, as well as a relatively long analysis time.

A technical problem is the need for a derivatization process to obtain volatile derivatives of parabens, low resolution and sensitivity of the chromatographic method, long analysis time.

The technical result of the invention is to improve the accuracy and sensitivity of determination in a mixture of individual parabens (esters of 4-hydroxybenzoic acid) with high resolution by capillary GLC in medicinal preparations in dosage forms "homeopathic oral solution", "gel for external use", "ointment for external use ”,“ aerosol for external use ”.

The achievement of the technical result is ensured thanks to the following technical solutions:

- the use of a capillary column modified with a thin film of hydrophobic polydimethylsiloxane, which makes it possible to register peaks with good chromatographic parameters on chromatograms (peak asymmetry is 1.0, high column efficiency and resolution between peaks) and, accordingly, increases the accuracy of the quantitative determination results;

- the choice of chromatographic conditions with high temperatures of various elements of the chromatographic system and the carrier gas flow rate, which are possible only in a system with a capillary column, which excludes the stage of obtaining volatile derivatives of parabens and, as a consequence, reduces the analysis time and labor costs;

- the use of the carrier gas flow division system allows to improve the symmetry of peaks, increase the efficiency of the chromatographic column and reduce the noise level, which leads to an increase in the sensitivity of the determination of parabens.

In the claimed invention, a method for the determination of parabens (4-hydroxybenzoic acid esters) by capillary GLC in medicinal preparations in dosage forms "homeopathic solution for oral administration", "gel for external use", "ointment for external use", "aerosol for external use" is implemented as follows.

Sample preparation

To prepare standard solutions, accurate weighed portions of standard samples of methylparaben and propylparaben (about 0.02 g each) are placed in a 20 ml volumetric flask, dissolved in chloroform and the volume of the solution is brought to the mark in order to obtain a concentration corresponding to the nominal amount of parabens in the drug, in which should determine their content.

Sample preparation of the tested drug solution is carried out by means of extraction. For this, 5 ml of chloroform is added to 4 ml of the previously obtained test solution of the drug and stirred on an orbital shaker for 10 minutes at 400 rpm to increase the completeness of the extraction of parabens into chloroform.

The preparation of the test solution of a medicinal product depends on its dosage form:

1. In the case of the "homeopathic oral solution" dosage form, the drug itself is used for extraction directly (4 ml as described above).

2. The test solution of the drug in the "gel for external use" dosage form is prepared by dissolving a sample of about 0.3 g of the gel in 5 ml of water on a vortex for 2 minutes at a speed of 1250 rpm. 4 ml of the resulting solution is taken and sample preparation is carried out in accordance with the description above.

3. The test solution of the drug in the dosage form "ointment for external use" is prepared by placing about 0.6 g of the ointment in 10 ml of water, heating at 37 ° C in a water bath for 10 minutes, vortexing for 5 minutes at a speed of 2000 rpm, treatment in an ultrasonic bath for 30 min and filtration of the resulting solution through a porous membrane filter made of regenerated cellulose with a pore diameter of 0.45 μm. 4 ml of the resulting solution is taken and sample preparation is carried out in accordance with the description above. Then another 4 ml of the resulting drug solution is taken and a test solution is prepared with the addition by extraction in 5 ml of a standard solution of methylparaben and propylparaben in chloroform.

4. The test solution of the medicinal product in the dosage form "aerosol for external use" is prepared as follows: the mass of the medicinal product is removed from the vial under pressure, a sample of about 0.5 g is placed in a volumetric flask with a capacity of 50 ml, dissolved in 35 ml of water on a vortex for 2 minutes at a speed of 1500 rpm, then in an ultrasonic bath for 30 minutes, then stored at 4 ° C to prevent foaming of the solution for 1 hour. The resulting solution volume at room temperature is brought to a volume of 50 ml with water. 4 ml of the resulting solution is taken and sample preparation is carried out in accordance with the description above. Then another 4 ml of the resulting drug solution is taken and a test solution is prepared with the addition by extraction in 5 ml of a standard solution of methylparaben in chloroform.

The quantitative determination of methylparaben and propylparaben is carried out on an Agilent 7890B gas chromatograph with a flame ionization detector equipped with an Agilent G4513A autosampler (Agilent Technologies, USA). Chromatogram registration and results processing is carried out using Open Lab software version 2.2.

Column (stationary phase): DB-1 (30 m × 0.32 mm, 3.0 μm), which is a quartz capillary, the inner walls of which are modified with 100% polydimethylsiloxane (Agilent Technologies, USA).

Carrier gas (mobile phase): helium.

Chromatographic conditions are presented in Table 1.

Table 1

Chromatographic system parameter Value chromatographic column 100% polydimethylsiloxane
(30 m × 0.32 mm, 3.0 μm) column temperature 210 ° C carrier gas helium carrier gas flow rate 60 cm / s detector temperature 220 ° C injector temperature 220 ° C split ratio 1:40 injection volume 1 μl

In the case of dosage forms "homeopathic solution for oral administration" and "gel for external use", the standard and test solutions are sequentially injected using an autosampler syringe into the injector, where the sample is evaporated. For dosage forms "aerosol for external use" and "ointment for external use", the test solution of the drug and the same solution of the drug with the added known amount of the determined paraben (with the "additive") are injected. Parabens are identified by retention times. The retention time of methylparaben is about 4.0 minutes, the retention time of propylparaben is about 6.4 minutes.

The analytical range is 0.005–1.450 mg / ml for methylparaben and propylparaben in chloroform, the correlation coefficients of the obtained dependences of the analytical signal on the concentration of parabens for methylparaben are 0.9997, for propylparaben - 0.9991.

The quantitative calculation of the concentration of parabens is carried out by the method of an external standard for hydrophilic dosage forms ("homeopathic solution for oral administration", "gel for external use"), using the calibration dependence of the peak area of the paraben on its concentration in the standard solution. The paraben content is calculated from the measured analytical signal (peak area) using an inverse function.

For dosage forms with a more complex matrix ("ointment for external use", "aerosol for external use"), the additive method is used in order to take into account the effect of the matrix on the assessment of the result. The calculation is made according to the following formula:

C _x is the concentration of the determined paraben in the test solution, mg / ml;

С _add - concentration of paraben additive, mg / ml;

y _x is the area of the peak of the determined paraben on the chromatogram of the test solution;

y _add is the area of the peak on the chromatogram of the test solution with the "additive".

The essential distinguishing features of the claimed invention are:

- reduction of the elution time of parabens due to the use of a capillary column modified with a thin film of hydrophobic polydimethylsiloxane, instead of using a packed column filled with particles of a more hydrophilic sorbent, which is a diatomite carrier "Chromosorb AW-HMCS" (base - silica), modified with 3% polyethylene glyphenate (PEGA) and 1% orthophosphoric acid (the time of chromatographic analysis by the claimed method is 7 minutes);

- increasing the accuracy of the obtained results of quantitative determination by integrating the peaks with the best parameters on the chromatograms (peak asymmetry, efficiency, resolution between peaks);

- reduction of analysis time and labor costs by eliminating the stage of obtaining volatile derivatives of parabens by selecting chromatographic conditions with high temperatures of various elements of the chromatographic system and the carrier gas flow rate, which are possible only in a system with a capillary column;

- increasing the sensitivity of the determination, by reducing the volume of the liquid sample when using the specific property of the capillary GLC system - the separation of the carrier gas flow.

Brief description of drawings and other materials:

FIG. 1. Chromatograms of a standard solution of methylparaben (1) and propylparaben (2), obtained on a capillary (a) column by the claimed method and on a packed column (b) by the prototype method.

FIG. 2. Chromatograms of a sample of the drug "Loma Lux Psoriasis" in the dosage form "homeopathic solution for oral administration" (a) and standard solution (b), obtained by the claimed method: methylparaben (1) and propylparaben (2).

FIG. 3. Chromatograms for the determination of methylparaben in the sample of the drug "Dalatsin" in the dosage form "gel for external use" (a) and standard solution (b), obtained by the claimed method.

FIG. 4. Chromatograms for the determination of methylparaben in a sample of the drug "Panthenol" in the dosage form "aerosol for external use" (a) and a sample of the same drug with the addition of methylparaben (b), obtained by the claimed method.

FIG. 5. Chromatograms of a sample of the drug "Oflomelid" in the dosage form "ointment for external use" (a) and a sample of the same drug with the addition of methylparaben and propylparaben (b), obtained by the claimed method: methylparaben (1) and propylparaben (2).

FIG. 6. Chromatogram of the test mixture of methylparaben (1), ethylparaben (2), propylparaben (3) and butylparaben (4).

The possibility of implementing the claimed invention is shown by the following examples.

Example 1. Comparison of the chromatographic parameters of the peaks of methylparaben and propylparaben on chromatograms and the resolution between the peaks obtained by the claimed method and the prototype method (Table 2).

table 2

Determination of parabens by the claimed method Determination of parabens
prototype way 1. Factors of asymmetry of peaks for both peaks 1.0; column efficiency - up to 57,500 theoretical plates (Fig. 1) 1. Factors of asymmetry of peaks 4.4 and 3.1; column efficiency - up to 900 theoretical plates 2. Resolution between peaks 27.3 (Fig. 1) 2. Resolution between peaks 3.4 3. Limit of quantitative determination of methylparaben and propylparaben - 0.005 mg / ml 3. Limit of quantitative determination of methylparaben and propylparaben - 0.020 mg / ml

The preparation of standard solutions with a concentration of each determined paraben of 1.2 mg / ml is carried out in accordance with the description of the invention.

The resulting standard solution is subjected to chromatography under the conditions of the proposed method shown in Table 1.

As seen in FIG. 1, the efficiency and resolution of gas chromatographic separation of parabens in the case of using a packed column is significantly lower (900 theoretical plates, resolution between peaks 3.4) than in the claimed method (57,500 theoretical plates, resolution between peaks 27.3). The asymmetry factors for methylparaben and propylparaben in the case of using the prototype method are 4.4 and 3.1, respectively, when using the proposed method - 1.0 for both peaks.

The sensitivity of the chromatographic system is lower when using the prototype column. The peak heights are comparable to those for a capillary column with the same injection volume of 1 μL (but in the latter case, 1/40 μL of solution falls into the column).

At comparable heights, the chromatographic peaks are much wider and less symmetrical when using the prototype column. In the inventive method, it is possible to reduce the amount of injected sample and obtain symmetrical peaks, which can still be accurately integrated and obtain a reliable result. The blurred broad peaks obtained on the prototype column do not allow this, and therefore more sample injection is required, i.e. the prototype method has a lower sensitivity compared to the claimed method.

This example confirms the increase in efficiency and sensitivity of the chromatographic system, due to the resolution between the peaks and the improvement of their symmetry.

Example 2. Quantitative determination of methylparaben and propylparaben in the drug "Loma Lux Psoriasis" in the dosage form "homeopathic oral solution" by the claimed method (Fig. 2).

The preparation of a standard solution with a concentration of 1.4 mg / ml of each determined paraben is carried out in accordance with the description of the invention.

Sample preparation of the tested drug solution is carried out in accordance with the description of the invention.

The quantitative determination of methylparaben and propylparaben, as well as the processing of the results are carried out in accordance with the description of the invention. The standard and test solutions are injected sequentially, respectively.

The content of methylparaben was found to be 1.38 mg / ml - 91.7% of the nominal (relative standard deviation - RSD - was 1.6%), the content of propylparaben - 0.30 mg / ml - 100.3% of the nominal (RSD - 0 ,nine %).

Example 3. Quantitative determination of methylparaben in the drug "Dalatsin" in the dosage form "gel for external use" by the claimed method (Fig. 3).

The preparation of a standard solution with a methylparaben concentration of 0.1 mg / ml is carried out in accordance with the description of the invention.

Sample preparation of the tested drug solution is carried out in accordance with the description of the invention.

The quantitative determination of methylparaben, as well as the processing of the results are carried out in accordance with the description of the invention. The standard and test solutions are injected sequentially, respectively.

The content of methylparaben was found to be 2.82 mg / g - 94.0% of the nominal (RSD - 1.7%).

Example 4. Quantitative determination of methylparaben and propylparaben in the drug "Oflomelid" in the dosage form "ointment for external use" by the claimed method (Fig. 4).

The preparation of a standard solution with a concentration of methylparaben 0.05 mg / ml and propylparaben 0.02 mg / ml is carried out in accordance with the description of the invention.

Sample preparation of the tested drug solution is carried out in accordance with the description of the invention.

To prepare a solution with the addition of 4 ml of the resulting solution, 5 ml of a solution of methylparaben and propylparaben in chloroform with a concentration of 0.05 mg / ml and 0.02 mg / ml, respectively, are extracted. The calculation of the content of parabens is carried out according to the formula of the additive method in accordance with the description of the invention.

The content of methylparaben was found to be 0.73 mg / g - 91.3% of the nominal (RSD - 1.9%), propylparaben 0.18 mg / g - (RSD - 2.3%).

Example 5. Quantitative determination of methylparaben in the drug "Panthenol" in the dosage form "aerosol for topical use" by the claimed method (Fig. 5).

The preparation of a standard solution with a methylparaben concentration of 0.02 mg / ml is carried out in accordance with the description of the invention.

Sample preparation of the tested drug solution is carried out in accordance with the description of the invention.

To prepare a solution with the addition of 4 ml of the resulting solution, 5 ml of a solution of methylparaben in chloroform with a concentration of 0.02 mg / ml are extracted. The calculation of the methylparaben content is carried out according to the formula of the additive method in accordance with the description of the invention.

The content of methylparaben was found to be 1.82 mg / g - 91.0% of the nominal (RSD - 1.3%).

Thus, examples 2-5 demonstrate the possibility of using the proposed method in the determination of parabens in a variety of drugs in different dosage forms.

Example 6. Application of the proposed method for the separation of a multicomponent mixture of parabens.

A test mixture is prepared from methyl-, ethyl-, propyl- and butylparaben in chloroform (the concentration of each compound was 1.25 mg / ml).

The mixture is subjected to chromatography under the conditions of the proposed method. Parabens are separated with a resolution of up to 18.1 in 9 minutes (Fig. 6), while the asymmetry factor of the peaks is 1.0, and the system efficiency is up to 58,900 theoretical plates.

This example demonstrates the separation of a multicomponent mixture from a larger amount of parabens by the claimed method.

The examples presented are not intended to limit the scope of the claimed invention and are for illustration purposes only.

Claims (5)

1. A method for the determination of parabens in medicinal products by capillary gas-liquid chromatography, including the preparation of a standard solution of parabens in chloroform at a concentration corresponding to the expected concentration in the analyzed medicinal product, extraction of 4 ml of the test solution of the medicinal product by adding 5 ml of chloroform, followed by mixing on an orbital shaker for 10 min at 400 rpm and subsequent chromatography of the prepared samples on a capillary column under selected chromatographic conditions using a flame ionization detector, identification of parabens by retention times and quantitative calculation of paraben concentrations by the external standard method. 2. The method according to claim 1, characterized in that the test solution of the drug in the dosage form "gel for external use" is prepared by dissolving a sample of about 0.3 g of gel in 5 ml of water on a vortex for 2 minutes at a speed of 1250 rpm min. 3. The method according to claim 1, characterized in that the test solution of the drug in the dosage form "ointment for external use" is prepared by placing about 0.6 g of the ointment in 10 ml of water, heating at 37 ° C in a water bath for 10 min, vortexing for 5 min at a speed of 2000 rpm, processing in an ultrasonic bath for 30 min, filtration of the resulting solution through a porous membrane filter made of regenerated cellulose with a pore diameter of 0.45 μm, followed by a quantitative calculation of the parabens content by the addition method. 4. The method according to claim 1, characterized in that the test solution of the medicinal product in the dosage form "aerosol for external use" is prepared by extracting the mass of the medicinal product from the vial under pressure, placing a sample of about 0.5 g in a 50 ml volumetric flask, dissolving in 35 ml of water on a vortex for 2 minutes at a speed of 1500 rpm, then in an ultrasonic bath for 30 minutes, storage at 4 ° C to prevent foaming of the solution for 1 hour, bringing the resulting volume of solution at room temperature with water to a volume of 50 ml, followed by a quantitative calculation of the content of parabens by the addition method. 5. The method according to claim 1, characterized in that the capillary column is modified with 100% polydimethylsiloxane, with parameters 30 m × 0.32 mm, and a modifying film thickness of 3 µm.

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