RU2532237C1 - Method for chromatographic analysis of parabens (4-hydroxybenzoic acid esters) in food products, cosmetics, pharmaceutical preparations and biologically active additives - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention relates to analytical chemistry and can be sued in chemical, cosmetic, pharmaceutical and other industries when analysing parabens by high-performance liquid chromatography (HPLC). The invention enables identification and quantitative analysis of parabens using spectrophotometric or (and) diode-array detectors. The method includes procedures for preparing samples and conditions for chromatographic separation and detection. A starting sample of a food product, cosmetic product, pharmaceutical preparation or biologically active additive is prepared according to one of sample preparation procedures. The prepared sample then undergoes separation on a chromatographic column. At the output, each fraction is detected by measuring absorption of emitted light according to the Bouguer-Lambert-Beer law. Parabens are identified from retention times. An additional identification criterion can be signal ratios of peak heights or areas obtained at different wavelengths or (and) electronic spectra of the compounds of interest. Quantitative determination of concentration of parabens is carried out by an external standard method, taking into account the linear range of the relationship between the output signal and concentration or mass of parabens in standard solutions.

EFFECT: avoiding the need to obtain derivatives, relatively fast sample preparation and chromatographic analysis, relatively low cost of analysis, identical chromatographic analysis conditions for all types of the analysed products, which shortens the time for preparing the system in between analyses, enabling use of additional criteria for identifying parabens (signal rations or (and) electronic spectra).

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Description

The invention relates to the field of analytical chemistry and can be used in chemical, cosmetic, pharmaceutical and other industries for the analysis of parabens by high performance liquid chromatography (HPLC).

The constantly growing needs of mankind are leading to an increase in the pace of creation and consumption of food products, pharmaceuticals, and cosmetic products. This necessitates the use in such a production of a wide range of chemical additives to ensure their long-term safety. One of these additives are parabens. The main advantage of parabens in comparison with other preservatives, such as benzoic, salicylic, sorbic acids, is the independence of the degree of preservative effect on the pH of the product in which they are used. A characteristic feature of food products, pharmaceuticals, cosmetics is their close contact with various tissues and organs of the human body (gastrointestinal tract, skin, blood, etc.), which facilitates the penetration of undesirable chemical compounds into the body. Studies show that parabens can have a cytotoxic and carcinogenic effect on human tissues (Sony MG, Carabin IG, Burdock GA Safety assessment of esters of 4-hydroxibenzoic acid (parabens). Food and Chem. Toxicol., 2005, vol. 43 , P.985-1015).

The invention allows the identification and quantitative analysis of parabens using spectrophotometric or (and) diode array detectors. The method includes sample preparation procedures and chromatographic separation and detection conditions. The initial sample of a food product, cosmetic product, pharmaceutical preparation or dietary supplement is pre-prepared according to one of the sample preparation procedures (procedures 1-4) and then subjected to chromatographic separation on a liquid chromatograph.

A known method for the determination of parabens in soybean by capillary gas chromatography CN 102323367 (A) - 2012-01-18, which includes weighing the sample, adding an internal standard solution to the sample, acidification, ether extraction, dehydration and gas chromatographic analysis. The disadvantages of this method is a narrow circle of objects (soybean samples), for which this approach is applicable.

Canosa et al. Proposed a method for gas chromatographic analysis of parabens using a mass selective detector (Canosa P., Rodriguez I., Rubi E., and Cela R. .. Determination of Parabens and Triclosan in Indoor Dust Using Matrix Solid-Phase Dispersion and Gas Chromatography with Tandem Mass Spectrometry, Anal. Chem., Vol. 79, 2007, P.1675-1681). Along with high sensitivity and selectivity, the method requires a procedure for the preparation of derivatives (derivatization) with N-methyl-N- (tert-butyldimethylsilyl) trifluoroacetamide, and therefore it becomes necessary to control the completeness of derivative production. Moreover, the use of derivatizing agents and a mass spectrometric detector significantly increase the cost of analysis, which complicates its implementation in industrial practice.

A known method for the determination of parabens by HPLC, proposed by Borremans M. et al. (Borremans M., Loco J. Van., Roos P., Goeyens L. Validation of HPLC Analysis of 2-Phenoxyethanol, 1-Phenoxypropan-2-ol, Methyl, Ethyl, Propyl, Butyl and Benzyl 4-Hydroxybenzoate (Parabens) in Cosmetic Products, with Emphasis on Decision Limit and Detection Capability. Chromatographia, 2004, vol. 49, P.47-53). The method offers the determination of parabens in cosmetic products using high performance liquid chromatography after extraction with a water-ethanol solution and ultrasonic treatment. The disadvantages of this approach are the impossibility of determining methylparaben in the presence of sorbic acid in the sample, as well as a rather narrow range of objects of analysis (cosmetic products).

Proposed by Kulikov et al. (Kulikov AU, Verushkin AG Simultaneous Determination of Paracetamol, Caffeine, Guaifenesin and Preservatives in Syrups by Micellar LC. Chromatographia, 2008, vol. 67, P.347-355), the ion-pair method selected as a prototype ( micellar) HPLC makes it possible to determine only methyl and propyl paraben, while analysis of ethyl paraben is not described.

This invention allows in one analysis to carry out a qualitative and quantitative determination of four compounds of interest: methyl, ethyl, propyl and butyl esters of 4-hydroxybenzoic acid (parabens) using the method of high performance liquid chromatography on a reversed-phase sorbent using spectrophotometric or (and) diode matrix detectors in samples of food products, cosmetics, pharmaceuticals and biologically active additives with preliminary sample preparation by trade.

The proposed method is distinguished by: the absence of the need to obtain derivatives, relatively quick sample preparation and chromatographic analysis, relatively low cost of analysis, the identity of the conditions of chromatographic analysis for all types of test products, which reduces the system preparation time between analyzes, the possibility of applying additional criteria for identifying parabens (signal ratios or (i) electronic spectra).

The technical result is achieved due to the fact that in the method of chromatographic analysis of parabens (4-hydroxybenzoic acid esters) in food products, cosmetics, pharmaceuticals and biologically active additives, sample preparation of the test sample is carried out, the use of high performance liquid chromatography on reverse phase sorbent using spectrophotometric detector or (and) diode-matrix detector, calculating the concentration of parabens, based on the received signal value in according to the invention for food products, sample preparation includes weighing, extraction with an aqueous-organic mixture of solvents, quantitative transfer of the sample to measuring dishes, precipitation of interfering compounds, filtration, dilution of the resulting filtrate; A quantitative calculation of the concentration of parabens is carried out by the external standard method, taking into account the linear range of the dependence of the output signal on the concentration or mass of standard solutions.

When analyzing samples of cosmetics and fat-based pharmaceuticals, sample preparation includes weighing the sample, extracting with ethyl acetate, column cleaning with silica gel, evaporating the solvent under vacuum, dissolving the dry residue in the mobile phase.

In the analysis of solid pharmaceutical preparations, substances, and solid and free-flowing biologically active additives, sample preparation includes sample weighing, extraction with ethyl acetate, filtration, dehydration of the filtrate, evaporation of the solvent under vacuum, dissolution of the dry residue in the mobile phase.

In the analysis of liquid and suspension pharmaceuticals and liquid dietary supplements, sample preparation includes sample dilution and filtration.

The figures show the results of applying the method to various types of the studied object:

Figure 1. Chromatogram of the pharmaceutical preparation "Maalox".

Figure 2. Chromatogram of Proteskin cosmetic cream.

1, 1-methylparaben, 2-propylparaben are indicated. 2, 1-methylparaben is indicated.

The method is as follows. Before chromatographic separation, samples are prepared according to the procedures below.

Procedure 1. For food samples, the following sample preparation procedure applies.

Product weighing. 2 g of product up to the third digit, extraction of 50 cm ^{3 of} water-acetonitrile mixture (50:50 vol.%), Quantitative transfer of the sample to volumetric containers with a capacity of 100 cm ³ , precipitation of interfering compounds with Karreza solutions, bringing water to the mark, filtering, tenfold dilution of the obtained filtrate. The final filtrate is passed through a membrane filter.

Procedure 2. For samples of cosmetics and fat-based pharmaceuticals.

Product weighing. 2 g of the product to the third digit, homogenization, triple extraction of 25 cm ^{3 of} ethyl acetate, purification on a column containing 10 g of silica gel, evaporation of the solvent under vacuum, dissolution of the dry residue in 2 cm ^{3 of the} mobile phase, additional purification from hydrophobic compounds by double extraction with hexane 1 cm ³ .

Procedure 3. For solid pharmaceuticals, substances, and solid and bulk dietary supplements.

Grinding the sample in a mortar, weighing the product. 2 g of the product to the third digit, extraction of 15 cm ^{3 of} ethyl acetate three times, dehydration of the extract, filtration, evaporation of the organic solvent in vacuo, dissolution of the dry residue in 4 cm ^{3 of the} mobile phase, dilution to the intended concentration included in the calibration range.

Procedure 4. For liquid and suspension pharmaceuticals and liquid dietary supplements.

Dilution of 1-5 g of the sample to the expected concentration falling into the calibration range, filtering the diluted sample to remove suspended particles through a membrane filter.

The prepared sample is subjected to chromatographic separation under the following conditions:

Pre-column: 2.1 × 10 mm 120, C18, 5 μm, 120 Å;

Column: 2.1 × 150 mm, C18, 3 μm, 120 Å;

Mobile phase: Acetonitrile (25 vol.%) - water (75 vol.%), Gradient elution mode (table 1).

Table 1 Gradient Elution Conditions Time min Acetonitrile, vol.% Water, vol.% 0 25 75 16 25 75 16 40 60 thirty 40 60 thirty 25 75 45 25 75

The flow rate of the eluent: 0.2 cm ³ / min;

Column thermostat temperature: 30.0 ° C;

Injected volume: 10 μl - cosmetics, pharmaceuticals, 50 μl - food products;

Detection: absorption at λ ₁ = 254 nm, control at λ ₂ = 230 nm.

It is allowed to use other conditions of chromatographic analysis that do not impair the resolution of analyte signals.

At the outlet of the column, each fraction is detected by measuring the absorption of the emitted light according to the Bouguer-Lambert-Beer law. Parabens are identified by retention times. As an additional identification criterion, it is possible to use signal ratios of heights or peak areas obtained at different wavelengths or (and) electronic spectra of the compounds of interest. The magnitude of the signal relations is determined by the formula:

, $$R=\frac{A_{\lambda \mathrm{one}}}{A_{\mathrm{<figure-callout\; id="2"\; label="\lambda "\; filenames="00000006.png"\; state="\{\{state\}\}">\lambda </figure-callout>}2}}$$

,

where A _λ1 is the magnitude of the signal measured at a radiation wavelength of λ1;

A _λ2 is the magnitude of the signal measured at a radiation wavelength of λ2.

Quantitative calculation of paraben concentrations (C, mg / kg) is carried out by the external standard method, taking into account the linear range of the dependence of the output signal on the concentration or mass of standard solutions.

Calculation 1. When analyzing food products (procedure 1), use the following formula:

, $$C=\frac{A\ast K\ast V_{\mathrm{one}}\ast V_2}{m}$$

,

where A is the magnitude of the received signal (peak area or height);

K is the calibration coefficient found from the calibration graph;

V ₁ is the volume of the volumetric flask into which the sample is transferred, cm ³ ;

V ₂ - volumetric volumetric flask for final dilution, cm ³ ;

m is the mass of the sample, g.

Calculation 2. When analyzing samples of cosmetics, fat-based pharmaceuticals, as well as solid pharmaceuticals, substances and dietary supplements (procedure 2-3), use the following formula:

, $$C=\frac{A\ast K\ast V_{\mathrm{one}}\ast V_2}{m}$$

,

where A is the magnitude of the received signal (peak area or height);

K is the calibration coefficient found from the calibration graph;

V ₁ - the volume of the mobile phase in which the precipitate is dissolved after evaporation, cm ³ ;

V ₂ - volumetric volumetric flask for final dilution, cm ³ ;

m is the mass of the sample, g.

Calculation 3. For liquid and suspension pharmaceutical preparations (procedure 4), the calculation of the concentration of parabens is carried out according to the following formula:

, $$C=\frac{A\ast K\ast V_{\mathrm{one}}}{m}$$

,

where A is the magnitude of the received signal (peak area or height);

K is the calibration coefficient found from the calibration graph;

V ₁ - volumetric volumetric flask for final dilution, cm ³ ;

m is the mass of the sample taken for dilution, g

Claims (1)

The method of chromatographic analysis of parabens (4-hydroxybenzoic acid esters) in food, cosmetics, pharmaceuticals and biologically active additives, which consists in carrying out sample preparation of the test sample, using high-performance liquid chromatography on reverse phase sorbent using a spectrophotometric detector, and calculating the concentration of parabens by the obtained value of the signals, characterized in that the sample preparation includes weighing the sample and extraction, when for foodstuffs, extraction is carried out with an aqueous-organic mixture of solvents, and sample preparation additionally includes transferring the sample to measuring dishes, precipitating interfering compounds, filtering, diluting the filtrate obtained; A quantitative calculation of the concentration of parabens is carried out using the external standard method, taking into account the linear range of the dependence of the output signal on the concentration or mass of standard solutions. When analyzing parabens in cosmetics and pharmaceutical preparations based on fat, extraction is carried out with ethyl acetate, and sample preparation additionally includes purification on a column of silica gel and evaporation of the solvent under vacuum, dissolution of the dry residue in the mobile phase, in the analysis of parabens in solid pharmaceutical preparations In tach, substances, and solid and granular biologically active additives, extraction is carried out with ethyl acetate, and sample preparation additionally includes filtration, dehydration of the filtrate, evaporation of the solvent under vacuum, and dissolution of the dry residue in the mobile phase.

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