RU2769094C1 - Method for identification and quantitative determination of 20e-ecdysteroids in food raw materials and extracts from it - Google Patents

Abstract

FIELD: analytical chemistry.

SUBSTANCE: invention relates to analytical chemistry and can be used for identification and quantitative determination of ecdysteroids in food plant raw material or extract from it by the main ecdysteroid 20-hydroxyecdysone (20E). A method for the identification and quantitative determination of 20E-ecdysteroids in food plant raw materials and extracts from it includes multiple extraction of raw materials, followed by high-performance liquid chromatography of the obtained extract with elution in a gradient mode, identification and quantitative determination using a detector, while food raw materials are extracted three times with a 70% solution ethanol in a water bath at 80°C for 4 hours, then chromatography is carried out with elution in a gradient mode with a mixture of 0.1% formic acid in water and acetonitrile - eluent B in the following mode: 0 min - 5% eluent B, 5 min - 27% eluent B, 5.5 min - 90% eluent B, 8.5 min - 90% eluent B, 9.5 min - 5% eluent B, 13.5 min - 5% eluent B, at a flow rate of 0.4 ml/min, and the identification and quantification of ecdysteroids is carried out on a triple quadrupole mass detector with an electrospray ionization source with a mass transition in MS/MS.

EFFECT: expanding the arsenal of technical means for the identification and quantitative determination of 20E-ecdysteroids in food plant materials and extracts from it, reducing the analysis time, increasing the accuracy and reproducibility of the analysis results.

2 cl, 1 dwg, 1 tbl

Description

Field of invention

The invention relates to analytical chemistry and can be used to identify and quantify ecdysteroids in food plant materials or an extract from it by the main ecdysteroid 20-hydroxyecdysone (20E), data on which are necessary for calculating doses when using the extract as a biologically active food supplement , raw material for dietary supplements for food or food ingredient for food enrichment.

Prior Art

The most well-studied representative of phytoecdysteroids is the low-toxic compound 20-hydroxyecdysone. Numerous clinical studies of ecdysteroids in restorative sports medicine, in asthenic and astheno-depressive conditions, in the treatment of infectious diseases, neuroses and hypotension have confirmed their high efficiency. A particularly promising direction is the use of 20-hydroxyecdysone as part of dietary supplements for food and specialized products for the nutrition of athletes.

For the detection of ecdysteroids in plant food raw materials and the subsequent quantitative assessment of their content, it is necessary to use a variety of analytical techniques.

A known method for the detection and quantification of ecdysteroids in plant objects, disclosed in patent RU2082168 [1], publ. 06/20/1997, including carrying out thin-layer high-performance liquid or gas-liquid chromatography of seeds of flowering plants. The disadvantages of the method are: the duration and complexity of the analysis, high consumption of reagents (especially in the case of thin layer chromatography) and non-selective identification of the results obtained by UV detection at a wavelength of 254 nm, since many biologically active substances have a maximum absorption spectrum in this range.

Also, quite close to the proposed invention is the method described in the work "Phytoecdysteroids of the aerial part of Serratulla Centauroudes growing in the Baikal region", D.N. Oleinikov and N.I. Kashchenko // Chemistry of plant raw materials, 2018, No. 2, p. 37-44 [2]. As a result of chromatographic separation using HPLC chromatography and gradient elution from the aerial part of S. centauroides, nine compounds were isolated, identified on the basis of UV, NMR spectroscopy and mass spectrometry. The disadvantages of this method are: the duration of the chromatographic analysis is 45 minutes, the analysis on HPLC-MS includes only qualitative characteristics, and the quantitative analysis was carried out by another method: microcolumn HPLC-UV, which also affects the time of the analysis, the study was carried out only for the presence of various phytoecdysteroids on various parts of Serratula centauroides L.

Closest to the claimed method is the method disclosed in the work "Method for rapid analysis of 20-hydroxyecdysone in plants and ferns using solid-phase extraction on polyamide and microcolumn HPLC-UV", D.N. Olennikov, N.I. Kashchenko // Chemistry of plant raw materials, 2018, No. 3, p. 41-52 [3], which describes in detail the qualitative and quantitative analysis of 20E by microcolumn HPLC-UV using solid phase extraction. The method includes preparation of samples of food plant raw materials: drying, grinding of raw materials, multi-stage ultrasonic extraction with sampling. The combined extract was subjected to solid phase extraction on a polyamide cartridge, eluted with water, filtered through a PTFE filter and further analyzed by HPLC with UV detection. The method has the following disadvantages: extraction does not allow obtaining a maximum recovery of 20E; multi-stage dilution using 3-dimensional flasks will lead to inevitable losses and a large analysis error; extraction, the duration of the solid-phase purification of the sample, a higher limit of detection and lower reliability of identification.

The choice of the substance to be determined is due to the fact that out of the whole variety of exidesteroids, only 20E is included in the list of permitted biologically active substances for food enrichment and as a raw material for dietary supplements for food.

The present invention is based on the task of reliably identifying and quantifying the main 20-hydroxyecdysone (20E) in food raw material and/or food plant extract according to four main features that were found based on the structure of 20E.

Disclosure of invention

The technical result, which is achieved by using the proposed method, is to expand the arsenal of technical means for the identification and quantification of 20E-ecdysteroids (20E) in food plant materials and extracts from it, as well as to reduce the analysis time, reduce the consumption of organic solvents required for analysis, while improving the accuracy and reproducibility of the analysis results.

As a result of the method, the main features of the absolute identification of 20E were determined: two mass transitions (481.3 m/z -> 445.4 m/z and 481.3 m/z -> 371.4 m/z), ratios between ions (481 .3/445.4 m/z and 481.3/371.4 m/z) at the chosen collision energies (8 eV and 12 eV) and the exit time of the main 20E. This makes it possible to accurately identify and quantify 20E and to standardize the obtained extract and/or food plant material against it.

The claimed technical result is achieved by the proposed method for the identification and quantification of 20E-ecdysteroids in food plant materials and extracts from it, including three extractions with 70% ethanol solution in a water bath at 80°C for 4 hours, followed by high-performance liquid chromatography of the obtained extract with elution in gradient mode with a mixture of 0.1% solution of formic acid in water and acetonitrile (eluent B) in the following mode: 0 min - 5% eluent B, 5 min - 27% eluent B, 5.5 min - 90% eluent B, 8, 5 min - 90% eluent B, 9.5 min - 5% eluent B, 13.5 min - 5% eluent B, at a flow rate of 0.4 ml / min, and the identification and quantification of ecdysteroids is carried out on a triple quadrupole mass detector with an electrospray ionization source with a mass transition in MS/MS.

In this case, the identification and quantitative determination of 20E-ecdysteroids is carried out with the mass transition in the MS/MS mode: 481.3 m/z -> 371.4 m/z with an impact energy of 12 eV and 481.3 m/z -> 445.4 m/z with an impact energy of 8 eV, respectively.

Implementation of the invention

The method is carried out as follows.

Preparation of samples of finished extracts.

To 20 μl of an aqueous or alcoholic extract was added 980 μl of a 50% solution of methanol in water. Vortexed for 10 seconds and centrifuged with a relative centrifugation force of 18407g for 10 minutes. In the study of dry purified extracts, 4 ml of a 50% solution of methanol in water was added to 10 mg of the extract and vortexed until dissolved, then the sample was prepared as for liquid extracts.

Preparation of samples of food plant raw materials.

5 ml of 70% ethanol was added to a 1 g sample of pre-crushed food plant raw materials, mixed and placed in a water bath at a temperature of 80°C for 4 hours, then cooled and centrifuged for 5 minutes at 2000 g, 4 ml of the obtained extract was taken. Further, the extraction was repeated two more times on an orbital shaker for 10 minutes, taking 5 ml of the extract, which made it possible to carry out a complete extraction of 20E from the raw material. The obtained extracts were combined, mixed, centrifuged at 18407g for 10 minutes. Then a 1 ml sample was taken into a vial and analyzed by HPLC-MS/MS under the conditions described below.

Identification and quantification on HPLC-MS/MS.

For identification, an Agilent Technologies 1100 chromatographic system with an Agilent Technologies 6410 mass detector is used; an Agilent Technologies Poroshell 120 EC-C18 3.0*50 mm, 2.7 µm column and the following reagents and standards: ultrapure water (resistivity 18.6 MΩcm), acetonitrile, methanol (HPLC grades) , 20-hydroxyecdysone (≥93%, Sigma).

Gradient elution is carried out with a mixture of a 0.1% solution of formic acid in water (eluent A) and acetonitrile (eluent B): 0 min - 5% eluent B, 5 min - 27% eluent B, 5.5 min - 90% eluent B, 8.5 min. - 90% eluent B, 9.5 min. - 5% eluent B, 13.5 min. - 5% eluent B. The flow rate was 0.4 ml/min.

Triple quadrupole mass selective detector with electrospray ionization source with the following parameters: spray gas pressure: 2.8 bar, drying gas temperature: 350°C, drying gas flow rate: 10 l/min, positive polarity. The fragmentator voltage was 98 V. The following mass transitions were recorded in the MS/MS mode: 481.3 m/z -> 445.4 m/z with an impact energy of 8 eV (used for quantitative analysis), 481.3 m/z ->371.4 m/z with an impact energy of 12 eV (used for qualitative confirmation).

The linearity range for determination by HPLC-MS/MS was 0.01 - 5 µg/ml. Table 1 shows a comparative analysis of analogues with this invention.

Spinach and quinoa food raw materials and extracts were tested under the conditions described above. The following results were obtained: the content of 20E in dried spinach leaves was 70 μg/g, in quinoa grains: 255 μg/g. On fig. 1 shows a chromatogram of spinach extract (peak - 20E). The content of 20E in quinoa grains was 37.2±1.9 mg/g.

Claims (2)

1. A method for the identification and quantitative determination of 20E-ecdysteroids in food plant raw materials and extracts from it, including multiple extraction of raw materials, followed by high-performance liquid chromatography of the obtained extract with elution in a gradient mode, identification and quantification using a detector, characterized in that food raw materials extracted three times with 70% ethanol solution on a water bath at 80°C for 4 hours, then chromatography is carried out with elution in a gradient mode with a mixture of 0.1% formic acid in water and acetonitrile - eluent B in the following mode: 0 min - 5% eluent B, 5 min - 27% eluent B, 5.5 min - 90% eluent B, 8.5 min - 90% eluent B, 9.5 min - 5% eluent B, 13.5 min - 5% eluent B , at a flow rate of 0.4 ml/min, and the identification and quantification of ecdysteroids is carried out on a triple quadrupole mass detector with an electrospray ionization source with mass transfer to MS/MS. 2. The method according to claim 1, characterized in that the identification and quantification of 20E-ecdysteroids is carried out with a mass transition in the MS / MS mode: 481.3 m / z - > 371.4 m / z with an impact energy of 12 eV and 481 .3 m/z - >445.4 m/z with an impact energy of 8 eV, respectively.

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