RU2647451C1 - Method of the vitamin k1 content determining in products of plant origin - Google Patents

Abstract

FIELD: measurement technology; biotechnology.

SUBSTANCE: invention relates to methods of the biologically active substances quantitative determination in plant raw material and food products derived therefrom, namely to a method of the vitamin K₁ content determining in the products of vegetable origin, and can be used in the chemical, pharmaceutical and food industries, medicine, including food hygiene. Method of the vitamin K₁ content determining in products of vegetable origin includes the vitamin K₁ (phylloquinone) quantitative extraction from the plant products with a propanol-2 and hexane mixture, separation of the obtained extract, elimination of the matrix components effect by filtering of the extract through a hydrophobic membrane, evaporation of the extract in a stream of nitrogen, the sample re-dissolution in the eluent and analysis by high-performance liquid chromatography with chromatography data mathematical processing, which allows to establish the absolute value of the phylloquinone concentration in 100 g of a plant origin product.

EFFECT: method allows to determine the vitamin K₁ concentration both without concentration process, and with 60-fold concentration with the use of a minimum amount of reagents and the minimum configuration of the analytical system, as well as in any of the plant origin products.

1 cl, 2 dwg, 8 tbl, 2 ex

Description

FIELD OF THE INVENTION

The invention relates to methods for the quantitative determination of biologically active substances in plant materials and food products obtained on its basis, and in particular to a method for determining the content of vitamin K ₁ in products of plant origin, and can be used in chemical, pharmaceutical and food industries, medicine, including including food hygiene.

State of the art

Currently, there are methods for determining vitamins in various objects, including products and plant materials, which allow controlling micro- and macroconcentrations of biologically active substances and are based on the use of chromatographic methods in most studies.

As analogues to the method for determining the content of vitamin K ₁ (phylloquinone) in plant products based on the requirements based on the principles of economy, rationality and effectiveness of achieving the result, the following can be considered:

- “Determination of vitamin K ₁ by high performance liquid chromatography” (Interstate standard GOST EN 14148-2015). The standard establishes a method for determining vitamin K ₁ in food products using high performance liquid chromatography (HPLC). The method is applicable to the analysis of milk, infant formula and other food products. Determination of vitamin K _{1 is} carried out by separating it in a sample solution by HPLC, subsequent restoration of the vitamin in a post-column reactor and determining the reduced form by fluorimetric method. For this, 15/5 ml of water with a temperature of 40 ° C is added to 1 g of powdered / 10 g of liquid sample and mixed; add 5 ml of phosphate buffer with a pH of 7.9-8.0 and mix; add 1.0 g of lipase and mix; close the cork and shake for 2-3 minutes; then the mixture is kept at a temperature of (37 ± 2) ° C for 2 hours (shaking manually at regular intervals); the mixture is cooled to room temperature, add 10 ml of a mixture of ethanol: methanol and 1.0 g of potassium carbonate, mix; add 30 ml of n-hexane and shake; left in the dark until phase separation or centrifuged at 2000 rpm for 10 min; an aliquot of the hexane extract is transferred to a vial, the solvent is removed in a stream of nitrogen and the residue is dissolved in 1 ml of methanol; 20 μl of the resulting solution is injected into the chromatographic system, including a column with a stationary phase C18, 5 μm, 150 × 3.9 mm, a column for derivatization of stainless steel 20 × 4 mm, filled with zinc powder, and a fluorimeter. A mixture of dichloromethane: methanol: zinc chloride-acetate (9.95: 89.55: 0.50 vol.%) Is used as an eluent.

The disadvantages of this method are: the use of a wide range of reagents, as well as the inclusion in the analytical system of an additional element in the form of a post-column reactor, due to the fact that the identification and quantification of vitamin K _{1 is} carried out by measuring its restored form, all of the above leads to an increase in analysis time, costs for reagents and materials.

- “A method for the determination of fat-soluble vitamins A, D ₂ , E and β-carotene in the joint presence by thin layer chromatography” (RU 2530620 C1, 07/30/2013). The invention relates to methods for standardizing biologically active additives, premixes, herbal raw materials, vegetable oils, oil extracts, food products, chemical and cosmetic industries for the content of the main fat-soluble vitamins A, D ₂ , E and β-carotene when combined in one and multicomponent preparations. The method includes the isolation of fat-soluble vitamins A, D ₂ , E and β-carotene from a substance by extraction with 96% ethanol, separation of the alcoholic extract of vitamins using a separatory funnel, sequential chromatography using Sorbfil PTSX-P-A silica gel plates on a polymer substrate with the participation of two eluents with different mileage (saturation time of the chamber with vapors of the eluent — 20 min, elution time — 55 min, eluent 1 — hexane: chloroform (19: 1), eluent 2 — hexane: chloroform (3: 1)), drying at a temperature not m less than 80 ° C plates in the thermostat for 3-5 minutes, the processing of the plate by the developer (5% alcohol solution of phosphoromolybdic acid).

The disadvantages of this method are: the method involves the determination of a number of fat-soluble vitamins in objects with a complex matrix by analyzing by the method of front thin layer chromatography using silica gel plates, which, after chromatography, are required to be dried in a thermostat at a temperature of at least 80 ° C. A similar approach cannot be applied to the determination of vitamin K ₁ , since this substance excludes the possibility of any heat treatment due to its instability when exposed to high temperatures.

- "Nettle dioecious leaves" (FS.2.5.0019-15, 01/01/2016). The normative and technical document establishes requirements for the quality of medicinal plant materials - dioecious nettle leaves - and methods for its control. One of the main biologically active substances, the content of which is required to be controlled according to this document, is vitamin K ₁ . The concentration of vitamin K _{1 is} regulated to be assessed as follows: about 1 g of raw material, crushed to a particle size of not more than 0.25 mm, is placed in a flat-bottomed conical flask with a capacity of 50 ml, 10 ml of hexane are added and mixed on a mechanical shaker for 3 hours; the resulting suspension is filtered through a paper filter, the solvent is distilled off on a rotary evaporator at a water bath temperature of not higher than 45 ° to a volume of 2-3 ml (test solution); 100 μl of the test solution and 10 μl of a standard solution of vitamin K ₁ are applied to the start line of the analytical chromatographic plate with a silica gel layer with a fluorescent indicator on an aluminum substrate measuring 10 × 15 cm; the sample plate is dried in air at room temperature for 5-10 minutes, placed in a chamber pre-saturated for 30 minutes with the upper layer of hexane-chloroform mixture, and chromatographed in an ascending manner; when the solvent front passes about 80-90% of the length of the plate from the start line, it is removed from the chamber, dried to remove traces of solvents and viewed in UV light at a wavelength of 365 nm. On the chromatogram of a standard solution of vitamin K ₁ , an adsorption zone with fluorescence of yellow-green color should be detected, and the test solution should have the same zone at the level of the vitamin K ₁ adsorption zone.

The disadvantages of this method are: according to the instructions of the authors themselves, vitamin K ₁ in the analyzed sample may have adsorption zones different from the standard, which indicates the ambiguity of identification of the compound being determined; the technique is based on thin-layer chromatography, the analytical capabilities of which depend on difficult to control external conditions, as well as the subjective factor during quantitative determination, which leads to an increase in measurement error.

The fundamental difference between the present invention and analogues is that a method has been developed that allows you to: identify, quantitatively extract vitamin K ₁ from plant products, eliminate the influence of matrix components and determine the content of vitamin K ₁ in a wide range of concentrations: from 1.6 to 6400 μg / 100 g, with a detection limit of 0.53 μg / 100 g with a measurement error of not more than 10%.

The closest to the technical solution of the tasks of the considered analogues is "Determination of vitamin K ₁ by high performance liquid chromatography" (Interstate standard GOST EN 14148-2015), which was adopted as a prototype. The disadvantages noted for the prototype are taken into account and are absent in the proposed method.

Disclosure of the invention

By the beginning of the XXI century, due to changes in the nature of human activity, the nutrition of various population groups underwent significant changes. Studies conducted in the regions of the Russian Federation showed that the diet of the inhabitants of our country is characterized by a high consumption of simple carbohydrates and animal fats. At the same time, the level of consumption of polyunsaturated fatty acids, dietary fiber, vitamins, micro and macro elements (nutrients) remains low.

In this regard, in the framework of the implementation of the Law of the Russian Federation “On Quality and Safety of Food Products”, adopted on January 2, 2000, and the second stage of the “Concept of State Policy in the Field of Healthy Nutrition of the Population of the Russian Federation for the Period Until 2005”, Methodological recommendations were developed “Recommended levels of consumption of food and biologically active substances” [1]. In recent years, a number of measures have been taken aimed at improving the nutrition of the population: the production of food products enriched with biologically active substances has been established, methods have been developed to control the content of various nutrients in food products, and Methodological recommendations “Norms of physiological requirements for energy and nutrients have been approved for various groups of the population of the Russian Federation ”[2]. Despite the measures taken, the nutrition of many residents of our country remains irrational, which entails the occurrence of diseases associated with a deficiency of various nutrients.

To preserve and strengthen public health and prevent diseases caused by unbalanced nutrition, the Government of the Russian Federation approved the “Fundamentals of the state policy of the Russian Federation in the field of healthy nutrition for the period up to 2020”. In this regard, the task of developing ways to control the quality of food remains as relevant and insufficiently resolved.

One of the most important biologically active substances necessary for the human body are lipophilic vitamins of group K, which participate in the normalization of blood coagulation by ensuring the biosynthesis of prothrombin and factors VII, IX and X. In addition, vitamins of group K increase intestinal motility and secretion, relieve the symptoms of dyskinesia intestines are involved in the processes of oxidative phosphorylation [3, 4]. Deficiency of these nutrients leads to a decrease in the carboxylation of osteocalcin, the main structural protein of the bone, which, in turn, causes a violation of the structure of bone tissue, increasing the risk of bone fractures [5]. Recently, new functions of group K vitamins have been discovered, which make it possible to consider these nutrients as a neuroprotective drug [6].

By the chemical structure, vitamins of this group are derivatives of 2-methyl-1,4-naphthoquinone, which in position 3 have wick, diheranyl, dipharnesyl, farnesyldigranyl, dipharnesyl geranyl or solanesil as a substituent [7].

There are two natural forms of coagulation vitamins. One of them is the group of menaquinones (vitamin K ₂ , 2-methyl-1,4-naphthoquinone with a hydrogen atom substituted in position 3 by a chain of 2-13 prenyl groups), which are products of the synthesis of bacteria of the intestinal microflora. Also, the source of vitamin K ₂ is food of animal origin (meat, milk, etc.) containing small amounts of menaquinone-4, which is a product of tissue-specific transformation of phylloquinone, another form of K-group vitamins [8].

Phylloquinone (vitamin K ₁ , 2-methyl-3-wick-1,4-naphthoquinone) is involved in photosynthesis and, therefore, is found in large quantities in green leafy vegetables, fruits, berries, root crops and other plant foods. Due to the high concentration in plant foods, vitamin K ₁ is the main food source of nutrients in this group.

It should be noted that due to the widespread prevalence of phylloquine in food products in healthy people, conditions of K-hypovitaminosis or K-vitamin deficiency are extremely rare. However, K-vitamin deficiency can occur with obstructive jaundice, gallbladder fistulas and other diseases of the digestive system. In addition, the development of a deficiency of this nutrient is facilitated by the use of antibiotics that inhibit the formation of vitamin K by intestinal bacteria, and anticoagulants - antagonists of vitamin K. In connection with the above, it is extremely important to maintain health control of the content of this nutrient in the body and timely adjustment of the human diet. However, such an adjustment is not possible without determining the content of vitamin K in foods used in food.

Currently, in the field of ensuring a balanced diet, active research is being conducted on the development of methods for determining vitamin K ₁ in various objects: dietary supplements and premixes, pharmacopoeial raw materials, and various food products [9-12].

To date, a lot of work has been done, which shows that the analysis of phylloquinone in plant materials is possible in various ways. One of the first is a method for determining vitamin K ₁ using a photoelectric colorimeter, based on preliminary separation of the extract on calcined alumina [13]. However, this method is very time-consuming and time-consuming, therefore, its use for routine analysis is impractical.

Researchers from Tomsk State University and Tomsk Polytechnic University have shown the possibility of gas chromatographic determination of vitamin K ₁ in infusions of green tea leaves with preliminary sorption concentration [14]. The disadvantage of this method is the low detection limit of 5 μg / ml, which makes it impossible to use the described method to determine the content of vitamin K ₁ in plant products with its low content.

In recent years, the method of high performance liquid chromatography (HPLC) has become widespread. It allows you to avoid the destruction of unstable vitamin K ₁ during the experiment by analysis at temperatures close to room temperature, and to exclude the interaction of the defined compounds with air. A variety of technical equipment of liquid chromatography systems allows the identification and quantification of phylloquinone using various detectors.

In foreign practice, to solve this problem, the most frequently used method is liquid chromatography with mass spectrometric detection, the capabilities of which allow determining trace amounts of phylloquinone [12, 15, 16]. The advantages of this method include greater selectivity and high speed analysis, however, the mass spectrometric detector is very expensive, which necessitates the use of other detection methods.

Another option for determining vitamin K ₁ in food is high performance liquid chromatography with spectrophotometric detection. Studies have shown that the present method is sensitive and suitable for the analysis of pharmacopoeial raw materials [10, 17].

Another method for measuring the mass concentration of phylloquinone in plant objects is HPLC with a fluorimetric detector [18], based on measuring the fluorescence emission of light absorbed by a vitamin K ₁ derivative. To carry out this method, the chromatographic system must be equipped with an additional device for post-column derivatization of the target component.

It should be noted that in our country, to control the content of vitamin K ₁ in food products, an interstate standard identical to the European one was introduced [19]. He establishes a method for analyzing phylloquinone in food using HPLC with fluorimetric detection. This methodology has been validated for milk and baby food, however, it is reported that some laboratories have shown the possibility of using the methodology for other products. Nevertheless, there remains a need to develop a domestic highly sensitive, selective, least expensive and technically affordable way to determine phylloquinone in food products.

The objective of the present invention is to develop and justify a method for the quantitative determination of vitamin K ₁ in products of plant origin, including the extraction of vitamin K ₁ from the products and eliminating the influence of the components of the matrix by choosing the optimal analysis conditions with a minimum configuration of the analytical system.

The problem is solved by the proposed method, which is based on the dependence of the concentration of vitamin K ₁ in the analyzed product in μg / 100 g on the height of the analytical peak in mV obtained by HPLC.

The technical result of the present invention is the development of an analysis algorithm that allows you to quantitatively extract vitamin K ₁ from products of plant origin, eliminate the influence of matrix components and determine the content of vitamin K ₁ in a wide range of concentrations with the possibility of reducing the detection limit.

The proposed method in comparison with the prototype has a simpler, less expensive and affordable technical base, fewer reagents, which allows to reduce the preparation time of the solutions necessary for the analysis, that is, it reduces the time and material costs of the study; equally effective in determining vitamin K ₁ in various products of plant origin, since it allows the concentration of phylloquinone, thereby reducing the limit of determination and expanding the range of determined concentrations.

The achievement of the technical result is ensured by the fact that in the method, the following stages of the analysis algorithm are conditionally isolated and performed:

1) a physical method is used to homogenize a product sample;

2) carry out the process of extraction of vitamin K ₁ from the analyzed sample;

3) carry out the filtration of the obtained extract;

4) vitamin K _{1 is} redissolved in the eluent;

5) carry out chromatography of a solution of vitamin K ₁ and mathematical processing of chromatographic data;

6) in case of too low a signal, the procedure of concentration of vitamin K ₁ in the eluent is carried out.

The present invention is fundamentally different from analogues and prototype in that an original method of direct determination of vitamin K _{1 is created} , which allows to establish the concentration of this vitamin both without concentration and with a 60-fold concentration; using a minimum number of reagents, a minimum configuration of the HPLC system and ensuring high sensitivity and study efficiency.

The implementation of the invention

The development of the proposed method was carried out in the research process of dried leaves of green tea. The implementation of the method and the evaluation of the results provided for the following activities (studies), which are considered by example.

1. The rationale for the choice of extractant

Due to the solubility of vitamin K ₁ in organic solvents, hexane, ethanol, petroleum ether, trichloromethane, 1,2-dichloroethane, propanol-2 and their mixtures in a 1: 1 ratio were studied as extractants. A study of the absorption spectra of the obtained extracts showed that only ten of them have maxima that characterize the absorption spectrum of phylloquinone (Fig. 1) [7].

FIG. 1 - Absorption spectra of extracts of vitamin K ₁ from green tea leaves with organic solvents and their mixtures in a ratio of 1: 1 No. 1 - hexane; No. 2 - ethanol; No. 3 - propanol-2; No. 4 - propanol-2: 1,2-dichloroethane; No. 5 - propanol-2: hexane; No. 6 - trichloromethane: hexane; No. 7 - ethanol: trichloromethane; No. 8 - ethanol: propanol-2; No. 9 - trichloromethane: petroleum ether; No. 10 - petroleum ether: propanol-2

Chromatographic analysis of these extracts of vitamin K ₁ showed that the greatest response of the analytical signal is observed when using ethanol, propanol-2 and their mixture in the ratio 1: 1 as extractants, and the smallest response is when using hexane and mixtures of trichloromethane with hexane and petroleum ether .

The decisive criterion for choosing the most optimal extractants for extracting vitamin K ₁ from plant materials was the completeness of the separation of the analyzed sample: only those extractants were selected whose chromatographic analysis of the extracts did not interfere with the determination of the components of the sample matrix. Thus, ethanol, propanol-2, and mixtures of propanol-2 with hexane, 1,2-dichloroethane, and petroleum ether in a ratio of 1: 1 were selected for further research.

To eliminate the false-positive result of the identification of vitamin K ₁ , an additional analysis of samples of extracts with the addition of a standard was performed, standard solutions of vitamin K ₁ were prepared in the concentration range of 0.3-2.1 μg / ml. The peak of vitamin K ₁ in all samples with the addition of the standard was close to the Gaussian curve, had no kinks and peak riders. According to the results obtained (Table 1), the relative error in determining vitamin K ₁ was 13% in the case of extraction with a mixture of propanol-2: petroleum ether and did not exceed 4% when using other studied extractants. Thus, all the studied organic solvents and their mixtures are acceptable for the extraction of vitamin K ₁ from plant materials, however, in the case of ethanol and propanol-2, a slight drift of the baseline is observed, which can distort the results of quantitative analysis of vitamin K ₁ at its low concentrations.

The next stage of the study was the determination of the volumetric ratio of the components of the extractants, in which the extraction of the target compound from plant products is maximum. Bicomponent extractants in an organic solvent ratio of 1: 1 were studied; 1: 2; 1: 3; 1: 4; 1: 5; 2: 1; 3: 1; 4: 1; 5: 1.

A mixture of propanol-2 with 1,2-dichloroethane and hexane with compositions of 1: 1 and 3: 1, respectively, had an optimal ratio of the degree of extraction of vitamin K ₁ from plant materials and the convergence of parallel studies (Table 2).

Further study of the extraction ability of extractants of this composition showed that when using a mixture of propanol-2: hexane, a higher convergence of the results is observed - the range of concentrations does not exceed 1.9% (Table 3). In addition, when using the extractant composition propanol-2: hexane (3: 1), a more complete extraction of vitamin K ₁ is observed.

2. The rationale for the selection of the ratio of the mass of the product to the volume of extractant

The following ratios of the mass of the plant product to the volume of the extracting mixture were studied: 1: 4, 1: 5, 1: 6, 1: 7, 1: 8, 1: 9, 1:10. The results indicate that to obtain the optimal extraction of vitamin K _{1 it is} enough to use an extractant with a volume 4 times the mass of the product (table. 4).

3. The rationale for the multiplicity of extraction

In order to study the degree of extraction of vitamin K ₁ from the product, it was repeatedly extracted with an extractant of the selected composition and the concentration of vitamin K ₁ in each subsequent fraction was estimated. The results are presented in table. 5.

According to the data obtained, during a 4-fold extraction, a 94% extraction of vitamin K ₁ from the product is observed, which is an acceptable value. Carrying out the 5th extraction process will significantly increase the time spent on sample preparation without increasing the quality of the analysis, that is, this extraction can be neglected.

4. The rationale for the choice of the composition of the eluent

During the experiment, it was found that a three-component eluent of the composition acetonitrile: methanol: methylene chloride in a ratio of 50:40:10 separates the analyzed sample of the extract, while the retention time of vitamin K ₁ is 15-16 minutes. In addition, methanol and acetonitrile in the eluotropic row are located close to each other. In this regard, to optimize the separation, it was decided to use a two-component mobile phase containing a large volume fraction of acetonitrile and a lower volume fraction of less polar solvents. The main criteria for assessing the possibility of using a particular eluent for the task were considered to be the resolution of vitamin K ₁ relative to the nearest components of the extract matrix and the time of separation of the sample.

First, the mobile phase of acetonitrile (AcN) was studied: methylene chloride (CH ₂ Cl ₂ ) in the following volume ratios: 95: 5, 90:10, 85:15, 80:20, 75:25 and 70:30 (table 6) .

From the results it follows that a sufficient resolution (R≥1) is achieved using mixtures of 90:10, 85:15 and 75:25. It should be noted that when using an eluent of the composition acetonitrile: methylene chloride 90:10, 96 minutes are required to separate one sample of vitamin K ₁ extract from plant products. This significantly increases the time spent.

It is known that for symmetrical peaks at a resolution equal to one, about 2% of the areas of two peaks overlap. For mobile phases No. 3 and No. 5, the resolution is close to unity. This situation is considered acceptable for quantitative determination, however, by changing the composition of the eluent, it is possible to achieve a better separation of the peaks, and hence a more accurate analysis result.

At the next stage of the study, chromatographic separation of samples of vitamin K ₁ extract was carried out using a mobile phase based on acetonitrile with the addition of 10, 15, 20 and 25 vol. % chloroform (CHCl ₃ ) (table. 7).

It can be seen from the data presented in the table that the eluent of the composition acetonitrile: chloroform allows one to separate the components of the sample, i.e. exclude the influence of the matrix on the determination of vitamin K ₁ , only with a ratio of 85:15.

Analysis of the chromatographic data obtained using eluents of composition No. 3, No. 5 and No. 8 showed that the addition of chloroform to acetonitrile provides an improvement in the resolution of the eluent as compared with a mixture of acetonitrile: methylene chloride. In this regard, the eluent of the composition acetonitrile: chloroform in the ratio 85:15 was adopted as the most optimal eluent, which allows achieving the best resolution, that is, eliminating the overlapping of the peaks of the matrix components on the peak of vitamin K ₁ , which means improving the quality of the analysis.

Study sequence

1. Before conducting the study, the following solutions are prepared:

Chromatography eluent was acetonitrile: trichloromethane (85:15 vol%). In a flask with a capacity of 500 cm ^3, 425 cm ^{3 of} acetonitrile and 75 cm ^{3 of} trichloromethane are placed. The solution is thoroughly mixed and degassed using an automatic degassing and solvent filtration unit.

Vitamin K ₁ Solutions:

Solution A - basic solution with a mass concentration of vitamin K ₁ 500 .mu.g / cm ^3. About 1/3 of the volume of the eluent is introduced into a volumetric flask with a capacity of 10 cm ³ and weighed, then a drop (about 0.005 g) of vitamin K _{1 is} introduced with a needle and weighed again. Thoroughly mixed until the vitamin is completely dissolved, adjusted to the mark with the eluent and mixed again.

Solution B - a working solution with a mass concentration of vitamin K ₁ 20 μg / cm ³ . In a volumetric flask with a capacity of 10 cm ³ using a dispenser make 400 mm ³ solution A, bring the eluent to the mark and mix thoroughly.

Graduation solutions. In accordance with table 8 in volumetric flasks with a capacity of 10 cm ³ using a dispenser make a certain volume of solution B, bring the eluent to the mark and mix thoroughly.

2. Before performing measurements, a chromatographic column is prepared: a cartridge protective system, an analytical chromatographic column are sequentially installed on the chromatograph, then the mobile phase is fed into the system at a speed of 1 cm ³ / min until equilibrium is established in the column, which is determined by the stability of the zero line of the detector.

3. The calibration characteristic is established by measuring the heights of the peaks of vitamin K ₁ in calibration solutions. Each of the calibration solutions in an amount of 0.1 cm ³ is injected with a syringe into the chromatographic system. Using a loop dispenser, 0.02 cm ^{3 is} cut off. The analysis of the introduced solution is carried out under the following conditions:

Mobile phase flow rate 1 cm ³ / min Spectrophotometric Wavelength 248 nm Vitamin K ₁ retention time 14-15 minutes

For each calibration solution, 5 parallel measurements are performed. Using the arithmetic mean values of the five results of measuring the peak heights in the chromatographic data collection and processing program, a calibration graph is constructed in the coordinates: "vitamin K ₁ concentration (C _ol ), μg / cm ³ " - "vitamin peak height (N), mV" ( Fig. 2).

FIG. 2 - Calibration dependence

4. The products, if necessary, are cleaned of contaminants, the inedible part is separated, crushed, homogenized using a laboratory mill, the crushed sample is additionally wiped through a sieve with a mesh diameter of 0.1 mm.

5. Spend the extraction of vitamin K ₁ from the studied objects. Into a dark glass bottle add 1 g of a homogenized product, add 3 cm ^{3 of} propanol-2 and 1 cm ^{3 of} hexane. The vial is placed in an ultrasonic bath filled with ice water and extraction is carried out for 25 minutes. Then the resulting extract is carefully poured into another dark glass vial. The extraction procedure is carried out four times.

After the fourth extraction, the contents of the vial together with the homogenized product are quantitatively transferred to a plastic centrifuge tube and centrifuged for 3 min at 3000 rpm. The extract is carefully poured to the previously obtained extracts of vitamin K ₁ .

1 cm ^{3 of the} extract is taken into an insulin syringe, a filter nozzle with a hydrophobic membrane is mounted on the syringe, and the selected extract is pumped through the installed filter. The syringe is used once.

An aliquot of the filtered extract in an amount of 0.5 cm ^{3 is} transferred into an Eppendorf type tube and evaporated to dryness in a stream of nitrogen. The dry residue is dissolved in 0.5 cm ^{3 of} eluent. For each sample, two parallel measurements are performed.

In the case of an unenriched sample, concentration is carried out. For this, 6 cm ^{3 of the} extract is filtered off and, after drying, it is redissolved in 0.1 cm ^{3 of} eluent.

6. After the chromatograph enters the regime, 0.1 cm ^{3 of a} calibration solution with a concentration lying in the middle of the operating range is introduced into the chromatographic system and analyzed under the conditions specified in paragraph 3. At the end of the chromatographic analysis, the retention time and peak height of the vitamin are determined K ₁ . In order to adjust the calibration characteristic, the value of the obtained height is applied to the calibration graph.

7. 0.1 cm ^{3 of the} analyzed solution is introduced into the chromatographic system and the analysis is carried out under the conditions indicated above. At the end of the analysis, manual marking and identification of the detected peaks is performed. Vitamin K ₁ is determined by retention time. The coincidence of the retention time of vitamin K ₁ in the sample with the retention time of vitamin K ₁ in the calibration solution (with an error of no more than 5%) indicates the correct identification.

8. Using a program for the collection and processing of chromatographic data, a quantitative calculation of the mass concentration of vitamin K ₁ is carried out. In this case, the absolute calibration method is used. The concentration of vitamin K ₁ (C _ol , μg / cm ³ ) in the sample is calculated automatically.

9. The arithmetic average of two parallel measurements C _CR1 , C _{CR2 is calculated} , the difference between them should not exceed 5% of the average value:

With _pr - the mass concentration of vitamin K ₁ in the extract, μg / cm ³ .

9.1. In the enriched product, the mass concentration of vitamin K _{1 is} calculated by the formula:

C is the concentration of vitamin K ₁ in the product, mcg / 100 g;

With _pr - the mass concentration of vitamin K ₁ in the extract, μg / cm ³ ;

16 - the volume of the extract, cm ³ ;

1 - product mass, g;

100 - conversion factor.

9.2. In the case of an unenriched product and preliminary concentration of the sample, the mass concentration of vitamin K _{1 is} calculated by the formula:

C is the concentration of vitamin K ₁ in the product, mcg / 100 g;

With _pr - the mass concentration of vitamin K ₁ in the extract, μg / cm ³ ;

60 - coefficient taking into account 60-fold concentration of the sample;

16 - the volume of the extract, cm ³ ;

1 - product mass, g;

100 - conversion factor.

Example No. 1. Determination of vitamin K ₁ without concentration on the example of a plant product - leaf black tea

In the case of an allegedly high vitamin content in plant materials, phylloquinone is analyzed without concentration.

Suppose that you want to determine the content of vitamin K ₁ in black leaf tea. It is known that in tea vitamin K _{1 is} contained in large quantities, therefore, the analysis can be carried out without concentration.

Before analysis, leaf black tea is homogenized using a laboratory mill and additionally sieved through a sieve with a mesh diameter of 0.1 mm.

In a bottle of dark glass we add 1 g of homogenized tea leaves, add 3 cm ^{3 of} propanol-2 and 1 cm ^{3 of} hexane. We place the bottle in an ultrasonic bath filled with ice water and carry out the extraction for 25 minutes. Then, the resulting extract is carefully poured into another dark glass bottle and the extraction procedure is repeated one more time.

After the last extraction, the contents of the vial together with the homogenized sample are quantitatively transferred to a plastic centrifuge tube and centrifuged for 3 min at 3000 rpm. The extract is carefully drained to the previously obtained extracts of vitamin K ₁ .

We take 1 cm ^{3 of the} extract into an insulin syringe, install a filter nozzle with a hydrophobic membrane on the syringe and pump the selected extract through the installed filter.

An aliquot of the filtered extract in an amount of 0.5 cm ^{3 is} transferred into an Eppendorf type tube and evaporated to dryness in a stream of nitrogen. The dry residue is dissolved in 0.5 cm ^{3 of} eluent. We carry out two parallel measurements.

Introduce 0.1 cm ^{3 of the} analyzed solution into the chromatographic system and carry out the analysis. We identify vitamin K ₁ by the retention time of a standard solution:

T _beats (vit. K ₁ in a standard solution) = 14.310 min;

T _beats (vit. K ₁ ) = 14.435 min.

The difference in retention times does not exceed 0.9%, therefore, the vitamin can be considered identified.

Quantitative calculation of the mass concentration of vitamin K _{1 is} carried out using a program for the collection and processing of chromatographic data. In this case, we use the absolute calibration method. The concentration of vitamin K ₁ (C _ol , μg / cm ³ ) in the sample is calculated automatically and is:

C _pr1 = 0.595 μg / ml;

With _pr2 = 0.607 μg / ml.

The discrepancy between the parallel results is 2%. We calculate the arithmetic mean of two parallel measurements: C _av = 0.601 μg / ml. We calculate the mass concentration of vitamin K ₁ in black leaf tea according to the formula:

C (Vit. K ₁ ) = 1600-0.601 mcg / ml = 961.6 mcg / 100 g.

Example No. 2. Determination of vitamin K ₁ with concentration on the example of a plant product - cranberries

In the case of an alleged low vitamin content in plant materials, phylloquinone analysis with concentration is carried out.

Suppose you want to determine the vitamin K ₁ content in cranberries. It is known that in cranberry berries vitamin K _{1 is} contained in low quantities, therefore, before the analysis, concentration of the sample is necessary.

Thoroughly wash the cranberries, drain them with a paper towel, homogenize them using a laboratory mill and additionally wipe them through a sieve with a mesh diameter of 0.1 mm.

In a bottle of dark glass we add 1 g of a homogenized mass, add 3 cm ^{3 of} propanol-2 and 1 cm ^{3 of} hexane. We place the bottle in an ultrasonic bath filled with ice water and carry out the extraction for 25 minutes. Then, the resulting extract is carefully poured into another dark glass bottle and the extraction procedure is repeated one more time.

After the last extraction, the contents of the vial together with the homogenized sample are quantitatively transferred to a plastic centrifuge tube and centrifuged for 3 min at 3000 rpm. The extract is carefully drained to the previously obtained extracts of vitamin K ₁ .

We take 6 cm ^{3 of the} extract into a 10 cm ³ syringe, install a filter nozzle with a hydrophobic membrane on the syringe and pump the selected extract through the installed filter.

The filtered extract is quantitatively transferred into an Eppendorf type tube by gradual addition and evaporated to dryness in a stream of nitrogen. The dry residue is dissolved in 0.1 cm ^{3 of} eluent. We carry out two parallel measurements.

Introduce 0.1 cm ^{3 of the} analyzed solution into the chromatographic system and carry out the analysis. We identify vitamin K ₁ by the retention time of a standard solution:

T _beats (vit. K ₁ in standard solution) = 14.217 min;

T _beats (vit. K ₁ ) = 14.128 min.

The difference in retention times does not exceed 0.6%, therefore, the vitamin can be considered identified.

Quantitative calculation of the mass concentration of vitamin K _{1 is} carried out using a program for the collection and processing of chromatographic data. In this case, we use the absolute calibration method. The concentration of vitamin K ₁ (C _ol , μg / cm ³ ) in the sample is calculated automatically and is:

With _pr1 = 0.064 μg / ml;

With _pr2 = 0.061 μg / ml.

The discrepancy between the parallel results is 4.8%. We calculate the arithmetic average of two parallel measurements: C _av = 0.0625 μg / ml. We calculate the mass concentration of vitamin K ₁ in cranberry berries by the formula:

C (Vit. K ₁ ) = 26.67-0.0625 μg / ml = 1.67 μg / 100 g.

Thus, the materials of the above examples demonstrate the possibility of using this method for the quantitative determination of vitamin K ₁ in any plant-derived products.

Information sources

1. Recommended levels of intake of food and biologically active substances. Guidelines MP 2.3.1.1915-04 / Federal Service for Supervision of Consumer Rights Protection and Human Well-Being. - M., 2004 .-- 25 p.

2. Norms of physiological requirements for energy and nutrients for various groups of the population of the Russian Federation. Guidelines MP 2.3.1.2432-08 / Federal Service for Supervision of Consumer Rights Protection and Human Well-Being. - M., 2008 .-- 25 p.

3. Bremener S.M. Vitamins and their clinical use. - M .: Medicine, 1966. - 420 p.

4. Ovchinnikov Yu.A. Bioorganic chemistry. - M .: Education, 1987 .-- 815 p.

5. Gromova OA, Torshin. I.Yu., Limanova O.A. The multifaceted role of macro- and microelements in the construction of bone tissue // Gynecology, 2014. - No. 2. - S. 50-56.

6. Povarova OV, Medvedev O.S. Neuroprotective effect of vitamin K // Experimental and Clinical Pharmacology, 2015. - T. 78. - No. 10. - S. 40-44.

7. Berezovsky V.M. Chemistry of vitamins. Ed. 2nd. - M.: Food Industry, 1973. - 632 p.

8. Booth S.L. Vitamin K: food composition and dietary intakes // Food & Nutrition Research. 2012. - V. 56. - 5505 - DOI: 10.3402 / fnr.v56i0.5505.

9. Dietary supplements and premixes. Measurement of the mass concentration of vitamins K ₁ (phylloquinone) and D ₃ (cholecalciferol). MUK No. certification certificate 18 / 143-01.00259-2013 / 2014

10. Luksha E.A., Pogodin I.S., Kalinkina G.I., Kolomiyets N.E., Velichko G.N. Development of a method for the quantitative determination of phylloquinone (vitamin K ₁ ) in plant objects // Modern problems of science and education. 2014. - No. 3. http://www.science-education.ru/ru/article/view?id=13736 (date of access: 05.20.2016).

11. Park J.-S., Ryu E.-C., Park J.-W., Kim H.-S. Determination of Vitamin K1 in infant formula in Korean domestic market // Journal of preventive veterinary medicine. 2016 .-- V. 40 (3). - P. 90-95.

R.B., Jakobsen J. Analysis of vitamin K1 in fruits and vegetables using accelerated solvent extraction and liquid chromatography tandem mass spectrometry with atmospheric pressure chemical ionization. 2016. - V. 192. - P. 402-408.12.

RB, Jakobsen J. Analysis of vitamin K1 in fruits and vegetables using accelerated solvent extraction and liquid chromatography tandem mass spectrometry with atmospheric pressure chemical ionization. 2016. - V. 192. - P. 402-408.

13. Ermakov A.I. Methods of biochemical research of plants. Ed. 2nd, rev. and add. - L .: Kolos. Leningra. Department, 1972.- 456 p.

14. Gavrilenko M.A., Slizhov Yu.G., Burmetyeva M.S., Bilyalov A.A., Gavrilenko N.A. Concentration of phyloquinone on copper dipropyldithiocarbamate // Basic research. 2013. - No. 8. - S. 575-579.

15. Huang B., Wang Z., Yao J., Ke X., Xu J., Pan X.-D., Xu X., Lu M., Ren Y. Quantitative analysis of vitamin K1 in fruits and vegetables by isotope dilution LS-MS / MS // Analytical Methods. 2016. - No. 8. - P. 5707-5711.

16. Gentili A., Miccheli A., Tomai P., Baldassarre M.E., Curini R., Perez-Fernandez V. Liquid chromatography - tandem mass spectrometry method for the determination of Vitamin K homologues in human milk after overnight cold saponification. 2016. - V. 47. - P. 21-30.

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19. GOST EN 14148-2015. Food products. Determination of vitamin K ₁ by high performance liquid chromatography.

Claims (1)

A method for determining the content of vitamin K ₁ in products of plant origin, which includes the following steps of the analysis algorithm: quantitatively extract vitamin K ₁ by 4 times processing 1 g of a homogenized product with a particle size of not more than 0.1 mm with a mixture of propanol-2: hexane (3: 1 ) in an ultrasonic bath during cooling; combine the obtained extracts; eliminate the influence of matrix components by filtering 1 cm ^{3 of the} extract during analysis without concentration and 6 cm ^{3 of the} extract in the case of an unenriched product through a filter nozzle with a hydrophobic membrane; evaporate 0.5 cm ^{3 of the} extract during analysis without concentration and 6 cm ^{3 of the} extract in the case of an unenriched product in a stream of nitrogen; redissolved the dried residue in 0.5 cm ^{3 of an} eluent of the composition acetonitrile: trichloromethane (85:15 vol.%) during analysis without concentration and 0.1 cm ^{3 of the} eluent of the same composition in the case of an unenriched product; prepare a series of calibration solutions of vitamin K ₁ by dissolving a certain mass of pure substance in the eluent; establish a calibration dependence by measuring the heights of the peaks of vitamin K ₁ in calibration solutions and constructing a calibration graph in the coordinates: "concentration of vitamin K ₁ (C _pr ), μg / cm ³ " - "height of the peak of vitamin (N), mV"; 0.02 cm ^{3 of the} test sample is injected into the chromatographic system and analyzed by high performance liquid chromatography with spectrophotometric detection at a wavelength of 248 nm; identify vitamin K ₁ by the retention time of the component and conduct quantitative processing by absolute calibration; perform mathematical processing of chromatographic data, which allows to establish the absolute value of vitamin K ₁ in 100 g of plant material.

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