RU2335769C1 - Method of determining phosphatidylcholine content in phospholipide complexes - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: method involves drying of phospholipide concentrate obtained from examined samples in vacuum and dissolution in chloroform, application of chloroform phospholipide solution samples on chromatographic paper soaked in citrate buffer solution, performance of zonal high-voltage electropherography at 600 V for 60 minutes, after electropherography the paper is dried, dry paper strips are processed in 5% phosphorous molybdenum acid solution in ethanol and dried at room temperature, electrophoretic phosphatidylcholine track length is measured, and phosphatidylcholine content is determined by graduation scheme obtained for standard phosphatidylcholine solution with phosphatidylcholine content (mg/cm³) and its electrophoretic track length (mm) as coordinates.

EFFECT: simplified method; increased precision of determination and reduced cost.

1 ex, 2 dwg

Description

The invention relates to the field of analysis of materials by electrical means, namely by electrophoresis, and can be used to determine the quantitative content of phosphatidylcholine in phospholipid complexes obtained from animals, food or plant objects, during analytical and scientific research.

Known methods for determining the phospholipid composition of the lipid mixture using chromatographic methods. These methods include extraction of the lipid mixture from food, plant or animal objects, chromatographic separation of the lipid mixture into separate fractions and detection of chromatographic zones with specific reagents. The types of chromatographic separation used are different; the most commonly used are thin layer chromatography and column chromatography. The reagents used also vary.

These methods are aimed at obtaining information mainly about the qualitative composition of the phospholipid component in the analyzed lipid mixture. Interpretation of the quantitative results of chromatographic separation requires additional analysis methods, for example, photocolorimetry, densitometry, enzymatic methods, specific qualitative reactions, and weighing of eluates, which leads to a number of preliminary sample preparation operations for further quantitative detection, and, as a result, increases quantification error. In addition, this significantly increases the time of quantitative analysis, the amount of equipment and reagents used, and the cost of the analysis.

A known method for determining the content of phospholipids in butter, involving the extraction of phospholipids, thin-layer chromatography of the selected phospholipids, the development of chromatograms, and to determine the content of the sum and individual fractions of phospholipids carry out photodensitometry (AS USSR No. 567133, IPC G01N 33/04, 1977). A known method for determining the content of phospholipids in vegetable oil, including sample preparation, constructing a calibration curve in coordinates, the content of phospholipids (%) is the conductivity value, determining the conductivity of the sample and calculating using the formula based on the data of the calibration curve (RF patent No. 2170428, IPC 7 G01N 33/03, 2001).

All of these methods have the above disadvantages.

The most common biologically active phospholipid is phosphatidylcholine (FC). The unique surface-active properties of PF make it possible to restore damaged portions of membranes when taking preparations containing it, and thus prevent the development of structural pathologies of cells. Since this phospholipid has a hepatoprotective effect, liver cells quickly restore lost functions. Currently, there are many preparations containing phosphatidylcholine (Lezithin, Multipower, LIVOLIN-FORTE, Medicap, Essentiale, Ron-Pulenk Rohrer, Phosphoglyph, Russia).

The objective of the invention is to create an express method for determining the quantitative content of an individual phospholipid - phosphatidylcholine - in phospholipid complexes obtained from plant, food and animal objects.

The technical result consists in simplifying the method, increasing the accuracy of determination and reducing material costs in determining the quantitative content of phosphatidylcholine.

The technical result is achieved in that a method for determining the quantitative content of phosphatidylcholine in phospholipid complexes is that the phospholipid complex obtained from the studied objects is dried at 30-35 ° C under vacuum, then dissolved in chloroform to 1% concentration, samples of a chloroform solution are applied phospholipids on a chromatographic paper impregnated with citrate buffer solution in a volume of 1-2 μl, conduct high-voltage zonal electrophoresis at 600 V for 60 min, after electrophoresis is completed the magician is dried at 110 ° C for 20-30 minutes, the dried strips are treated with a 5% solution of phosphoromolybdenum acid in ethanol, dried at room temperature, the electrophoretic path length of phosphatidylcholine is measured, and the quantitative content of phosphatidylcholine is determined using a calibration curve obtained for a standard solution phosphatidylcholine in coordinates the content of phosphatidylcholine (mg / cm ³ ) - the value of the length of its electrophoretic path (mm).

Zone high-voltage electrophoresis is carried out in a citrate buffer solution prepared according to a standard recipe: 1 l of distilled water + 17.5 g of citric acid + 8.2 g of NaOH.

The proposed method for determining the quantitative content of FC in phospholipid complexes can significantly simplify the analysis process, reduce its time.

Figure 1 presents the electrophoregram of standard solutions of PF (in citrate buffer solution, reagent-detector - 5% solution of phosphoromolybdic acid in ethanol) with concentrations:

1) 39.38 mg / cm ³ ;

2) 58.08 mg / cm ³ ;

3) 78.76 mg / cm ³ ;

4) 98.72 mg / cm ³ .

Figure 2 presents a calibration graph for determining PF in a nitrate buffer solution, built on the basis of experimental data using standard PF solutions, and as a detection reagent, a 5% solution of phosphoromolybdic acid in ethanol (H is the height of the electrophoretic zone).

The development of the proposed method was carried out on standard solutions of phospholipids of the companies "Sigma", "ICN Biomedical" in chloroform. In this case, various buffer solutions, detection agents, volumes of applied samples of standard phospholipid solutions, exposure time and voltage were used. It was found that:

1. The volume of the applied sample of standard chloroform solutions of phospholipids significantly affects the separation process and the quality of electrophoretic zones. A large amount of the applied sample significantly worsens the separation pattern, and the bulk of the phospholipids remains at the start. Therefore, we experimentally established the optimal volumes of applied samples of a chloroform solution of PC, since its amount in any phospholipid complex dominates, which amounted to 1-2 μl.

2. When using a citrate buffer solution (prepared according to a standard recipe) as a conductive liquid, the best separation and quality of electrophoretic zones was achieved.

3. The most preferred was GOST 10395-63 chromatographic paper and a density of 85. When choosing a grade of chromatographic paper, we were guided by the fact that different brands of paper have unequal absorbency, which determines its electrical conductivity in the wet state. In our case, the most preferred was chromatographic paper with the highest absorbency, and hence electrical conductivity in the wet state. Based on these considerations, a chromatographic paper of the above GOST and density was selected.

4. The exposure time and the optimal voltage were established experimentally - they were 60 minutes and 600 V, respectively, when using a citrate buffer solution.

5. In the nitrate buffer solution, the electrophoregram of FC has the form of “candles”, and the height of such candles linearly depends on the concentration of FC in the region of 30-100 mg / cm ³ . This fact was used to build a calibration graph in the coordinates of the content of phosphatidylcholine (mg / cm ³ ) - the value of the length of its electrophoretic path (mm).

The proposed method is implemented as follows.

A start line was applied to chromatographic paper measuring 25 × 10 cm in the center and wetted with citrate buffer solution over the entire surface of the paper, except for a 1.5 cm strip on both sides of the start line (to prevent erosion of the analyzed sample).

The phospholipid complex obtained from food, animal or plant objects, and intended for determination, was dried in a Petri dish under vacuum at a temperature of 30-35 ° C, then dissolved in chloroform to a 1% concentration.

The chloroform solution of phospholipids thus prepared was applied with a MSH-10 microsyringe (Russia) to the starting line at a distance of at least 1 cm from the edge of the paper. The volume of applied samples was 1 and 2 μl, the distance between them was at least 2.5 cm from each other. The paper was dried and placed on an electrophoretic table so that the ends of the paper were immersed 1 cm in cuvettes with citrate buffer solution. To prevent excessive evaporation of the citrate buffer solution, which can lead to a decrease in its concentration and a change in pH, the entire system was placed in a closed chamber and electrophoresis was performed. The electrode cuvettes were filled with citrate buffer solution and the cuvettes were connected with strips of chromatographic paper.

Electrophoresis was performed at a voltage of 600 V for 60 min.

After the completion of the electrophoretic process, the paper was transferred to a thermostat and dried at a temperature of 110 ° C for 20-30 minutes.

Detection of electrophoretic zones after separation was carried out as follows. Dried strips of paper were placed in dry cuvettes and treated with 5% PMA solution. Then, strips of paper were hung on a frame and dried at room temperature. The electrophoretogram measured the length of the electrophoretic path of the FC — the “candle” —and, using a previously constructed calibration graph, the quantitative content of the PC in the studied phospholipid complex was determined.

Example.

0.05-0.07 g of a phospholipid complex obtained from amaranth seed oil was transferred quantitatively to a 25 ml volumetric flask and the solution volume was adjusted to the mark with chloroform. Received a chloroform solution of the studied phospholipids with a concentration of 2-3 mg / cm ³ .

An exact weighed sample of a standard Sigma PF weighing 2 g was placed in a 50 ml beaker, 10 ml of chloroform was added and mixed until the PF was completely dissolved. Then the contents of the beaker were quantitatively transferred to a 25 ml volumetric flask and adjusted to the mark with chloroform. The result was a standard solution of PF with a concentration of 80 mg / cm ³ . Then, by serial dilution, a series of standard FC solutions was prepared with concentrations of 60, 40, and 20 mg / cm ³ .

On the starting line of chromatographic paper of 25 × 10 cm in size, 2 μl of the studied chloroform PF solution and 2 μl of standard PF solutions with a concentration of 20, 40, 60 and 80 mg / cm ³ were applied using a microsyringe. Electrophoretic separation was carried out at 600 V in a citrate buffer solution. Detection of electrophoretic zones was carried out with a 5% solution of phosphoromolybdic acid in ethanol. Then, a calibration graph was built on the basis of the obtained experimental data using standard FC solutions and the desired content of FC in the studied phospholipid complex was calculated using it.

Claims (1)

A method for determining the quantitative content of phosphatidylcholine in phospholipid complexes, which consists in the fact that the phospholipid complex obtained from the studied objects is dried at 30-35 ° C under vacuum, then dissolved in chloroform to 1% concentration, samples of a chloroform solution of phospholipids are applied to the impregnated citrate buffer a solution of chromatographic paper in a volume of 1-2 μl, zonal high-voltage electrophoresis is carried out at 600 V for 60 minutes, after completion of electrophoresis, the paper is dried at 110 ° C for 20-30 minutes , the dried strips are treated with a 5% solution of phosphoromolybdenum acid in ethanol, dried at room temperature, the electrophoretic path length of phosphatidylcholine is measured and the quantitative content of phosphatidylcholine is determined using the calibration graph obtained for a standard solution of phosphatidylcholine in coordinates the content of phosphatidylcholine (mg / cm ³ - the value of the length of its electrophoretic shoot (mm).

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