JP2661210B2 - Determination of lysophosphatidylcholine - Google Patents

Description

The present invention relates to the determination of small amounts of lysophosphatidylcholine present in natural lecithin, such as soy lecithin and egg yolk lecithin, and in synthetic phospholipids. Used for management and quality control analysis.

[Prior Art] Phospholipids used as raw materials for liposomes, which have attracted attention as drug carriers and as supplementary elements for diagnostic agents, need to be of high purity, and it is particularly undesirable to include a lyso-form having a hemolytic action. . Therefore, a method for accurately and quickly analyzing lyso-forms in phospholipids is extremely important for process control and quality control of products.

Conventionally, as a method for quantifying lysophosphatidylcholine in phospholipids, two-dimensional thin-layer chromatography separates spots, decomposes lipid phosphorus into inorganic phosphorus, and performs absorption analysis as a phosphomolybdate complex [Oil Chemistry, 35
Vol. P1018 (1986)], a method for analyzing a phospholipid mixture with thin-layer chromatography-flame ionization detector [Oil Chemistry, Vol. 26, p454 (1977)], and a method for detecting spots separated by thin-layer chromatography with a minimum detection amount method Analysis method. However, each of these methods has advantages and disadvantages,
In addition, it has drawbacks such as frequent operations and inferior analysis accuracy, and has poor sensitivity.

[Problems to be Solved by the Invention] As described above, a method for accurately and quickly analyzing a lyso-form has not been found so far, and therefore, a standard value of a product cannot be set. It hindered management analysis, and the development of an analytical method was desired.

On the other hand, with the recent spread of high-performance liquid chromatography, separation and analysis techniques have remarkably advanced. However, there is no universal type in the detector, for example, the main component in natural soybean and egg yolk phospholipids is detected by an ultraviolet detector, but the content is small like lysophosphatidylcholine and mainly has a saturated alkyl group Things can hardly be detected. Therefore,
In such a case, derivatization that can be detected by an ultraviolet detector is required.

In addition, lysophosphatidylcholine used as a standard for high-performance liquid chromatography requires high purity, but its purification is complicated and takes a long time. Although it is not preferable, the internal standard method is better, but it requires the search for an appropriate internal standard.

[Means for Solving the Problems] As a result of intensive studies to solve these problems, the inventors have found that lysophosphatidylcholine having a saturated alkyl group is labeled with high sensitivity to an ultraviolet detector. Focusing on the need to react the reagents to prepare their derivatives and to increase the sensitivity, such labeling agents include aromatic nuclei such as phthalic anhydride and maleic anhydride or acids having a double bond. The anhydride was found to be optimal. That is, the sample is 75-80 in tetrahydrofuran.
By reacting phthalic anhydride, maleic anhydride or benzoic anhydride at ℃ and esterifying it into a derivative that is highly sensitive to ultraviolet detectors, it is possible to analyze samples containing about 0.1% lysophosphatidylcholine. became.

On the other hand, in the case of lysophosphatidylcholine quantification by high-performance liquid chromatography, the internal standard method is superior to the absolute calibration method, and if the internal standard is properly selected, the variation due to the separation parameter can be reduced. Focusing on the advantage that it can be corrected, the present inventor
Using a silicic acid column, acetonitrile / methanol /
As a result of examining an internal standard substance when a phosphate-based mobile phase solvent was used, it was found that a benzyl-type quaternary ammonium salt which is mutually separated from various phospholipids was the most suitable compound. This compound can be easily synthesized,
Also, it is suitable as an internal standard substance because it can be easily purified to obtain a high-purity product.

Examples of the acid anhydride having an aromatic nucleus or a double bond used as an esterifying agent for lysophosphatidylcholine in the present invention include phthalic anhydride, maleic anhydride and benzoic anhydride.

As the benzyl-type quaternary ammonium salt used as the internal standard, stearyldimethylbenzylammonium chloride is most suitable.

One example of a method for analyzing lysophosphatidylcholine according to the present invention is as follows. First, a sample is esterified by reacting phthalic anhydride, maleic anhydride or benzoic anhydride in tetrahydrofuran at 75 to 80 ° C., and then the above-described method. Stearyl dimethylbenzylammonium chloride was added as an internal standard to the esterified product prepared in (1), and the column was made of a silica-based column (Polygosil 60-7,4 ^mm
× 300 ^mm , manufactured by Chemco), high-performance liquid chromatography using acetonitrile / methanol / 75% phosphoric acid (78/1/1) as the mobile phase solvent, and 210 n with a UV detector
It is detected at a wavelength of m and quantified by the internal standard method.

The chromatogram obtained by this method is shown in FIG.
As can be seen from the figure, the sensitivity of lysophosphatidylcholine could be increased, and the separation from the main component phosphatidylcholine was good.

In the method of the present invention, the content of lysophosphatidylcholine can be determined by the following equation.

Where La: peak area of lysophosphatidylcholine to be analyzed Iw: amount of internal standard (mg) Ip: purity of internal standard (%) K: correction factor Ia: peak area of internal standard W: amount of sample ( mg) However, the correction coefficient K was obtained by performing the method of the present invention on lysophosphatidylcholine (standard product) and obtaining the following formula from the obtained results.

Where K: correction coefficient Sw: weight of lysophosphatidylcholine (standard) (mg) Ia: peak area of internal standard substance Sp: purity of lysophosphatidylcholine (standard) (%) Iw: weight of internal standard (mg) Sa: Area of lysophosphatidylcholine (standard) Ip: Purity of internal standard substance (%) Table 1 shows the results of determining the correction coefficient. According to Table 1, when phthalic anhydride is used as the labeling agent in this analysis method, the response to the internal standard substance hardly changes in both the natural system and the synthetic system. However, when maleic anhydride is used, there is no significant difference in the correction coefficient between the synthetic systems, but a difference between the natural system and the natural system.

[Examples] Next, the present invention will be described with reference to Examples and Comparative Examples.

Example 1 L-α-phosphatidylcholine (Sigma, purity: about 99%) and L-α-lysophosphatidylcholine (Sigma, purity: about 99%) separated and purified from egg yolk in proportions shown in Table 2 About 50 mg of the mixture is weighed correctly in a test tube (15 ^mm x 150 ^mm ) with rubbing, 2 ml of a 1% solution of phthalic anhydride in tetrahydrofuran is added, and the reaction is carried out at 75 to 80 ° C for 2 hours with a cooling tube attached. . After the reaction, tetrahydrofuran was distilled off, the residue was dissolved in a small amount of ethanol and transferred to a 5 ml volumetric flask, and 0.5% ethanol solution of 0.1% stearyldimethylbenzylammonium chloride was added.
After adding, adjust to the marked line with ethanol. High-performance liquid chromatography analysis of the obtained sample was performed under the following conditions, and a chromatogram as shown in FIG. 1 was obtained.

Column: Polygosil 60-7 (manufactured by Chemco, 4 ^mm x 300 ^mm ) Mobile phase: acetonitrile / methanol / 75% phosphoric acid (78 /
1/1) Flow rate: 2 ml / min Detection: 210 nm (ultraviolet ray detector) From this chromatogram, the content of lysophosphatidylcholine was determined by the above-mentioned calculation formula. The results are shown in Table 2.

Example 2 Table 3 shows the results of analyzing commercially available egg yolk lecithin under the conditions of Example 1 and determining the content of lysophosphatidylcholine.

Example 3 L-α-dimyristoyl phosphatidylcholine [manufactured by NOF Corporation, purity: 99.9%] and L-α-monomyristoyl phosphatidylcholine (Avanti Polar-Lipid, Inc.
(Purity: 99%) in the proportions shown in Table 4
And a chromatogram as shown in FIG. 2 was obtained. Table 4 shows the results of the determination of the content of lysophosphatidylcholine.

Comparative Example 1 Using the mixed sample of Example 1, the content of lysophosphatidylcholine was determined by two-dimensional thin-layer chromatography-phosphorus determination. The results are shown in Table 2.

Comparative Example 2 Table 3 shows the results of analyzing the commercially available egg yolk lecithin used in Example 2 by the minimum detection amount method of thin layer chromatography.

Comparative Example 3 Table 4 shows the results of analysis of the mixed sample of Example 3 by thin-layer chromatography-flame flame ionization detection method (Yatron Scan manufactured by Yatron).

As shown in Tables 2 and 4, the analysis results of Example 1 and Example 3 by the method of the present invention were in good agreement with the blending ratio of the standard product, and were compared with Comparative Examples 1 and 3. High analytical accuracy. The analysis operation is simple and can be performed in a relatively short time.

[Effects of the Invention] The method for quantifying lysophosphatidylcholine in the phospholipid of the present invention is performed by using a labeling agent such as phthalic anhydride or maleic anhydride to form a compound that is highly sensitive to an ultraviolet detector by 0.1%. The lyso form can be analyzed to a certain degree, and the benzyl quaternary ammonium salt can be used as an internal standard for quantification by the internal standard method. Applicable to quality control of phospholipid products as raw materials.

[Brief description of the drawings]

FIG. 1 shows that a mixture of 96.88% of L-α-phosphatidylcholine obtained from egg yolk and 3.12% of L-α-lysophosphatidylcholine also from egg yolk (stearyl dimethylbenzylammonium chloride as an internal standard) was used in Example 1. It is a chromatogram obtained by performing high-performance liquid chromatography analysis under the conditions. FIG. 2 shows that a mixture of 95.12% of synthetic L-α-dimyristoylphosphatidylcholine and 4.85% of L-α-monomyristoylphosphatidylcholine (stearyldimethylbenzylammonium chloride as an internal standard) was analyzed under the conditions of Example 3. FIG.

Claims (1)

(57) [Claims] 1. After esterifying lysophosphatidylcholine with an acid anhydride having an aromatic nucleus or a double bond, the internal standard is determined by high performance liquid chromatography using a benzyl quaternary ammonium salt as an internal standard and an ultraviolet ray detector. A method for quantifying lysophosphatidylcholine, characterized by quantification by a method.