JPH02132372A - Method for determining lysophosphatidyl cholin - Google Patents
Method for determining lysophosphatidyl cholinInfo
- Publication number
- JPH02132372A JPH02132372A JP28577688A JP28577688A JPH02132372A JP H02132372 A JPH02132372 A JP H02132372A JP 28577688 A JP28577688 A JP 28577688A JP 28577688 A JP28577688 A JP 28577688A JP H02132372 A JPH02132372 A JP H02132372A
- Authority
- JP
- Japan
- Prior art keywords
- internal standard
- anhydride
- lysophosphatidylcholine
- analysis
- performance liquid
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 238000000034 method Methods 0.000 title claims description 20
- 235000008979 vitamin B4 Nutrition 0.000 title 1
- 239000011579 vitamin B4 Substances 0.000 title 1
- 238000004128 high performance liquid chromatography Methods 0.000 claims abstract description 9
- 238000010813 internal standard method Methods 0.000 claims abstract description 5
- 150000008065 acid anhydrides Chemical class 0.000 claims abstract description 4
- 150000003242 quaternary ammonium salts Chemical class 0.000 claims abstract description 3
- RYCNUMLMNKHWPZ-SNVBAGLBSA-N 1-acetyl-sn-glycero-3-phosphocholine Chemical compound CC(=O)OC[C@@H](O)COP([O-])(=O)OCC[N+](C)(C)C RYCNUMLMNKHWPZ-SNVBAGLBSA-N 0.000 claims description 18
- 239000000126 substance Substances 0.000 claims description 10
- 125000003118 aryl group Chemical group 0.000 claims description 3
- 238000011002 quantification Methods 0.000 claims 1
- 238000004458 analytical method Methods 0.000 abstract description 19
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 abstract description 12
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 abstract description 12
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 abstract description 8
- LGRFSURHDFAFJT-UHFFFAOYSA-N Phthalic anhydride Natural products C1=CC=C2C(=O)OC(=O)C2=C1 LGRFSURHDFAFJT-UHFFFAOYSA-N 0.000 abstract description 7
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 abstract description 7
- JHIWVOJDXOSYLW-UHFFFAOYSA-N butyl 2,2-difluorocyclopropane-1-carboxylate Chemical compound CCCCOC(=O)C1CC1(F)F JHIWVOJDXOSYLW-UHFFFAOYSA-N 0.000 abstract description 7
- FPYJFEHAWHCUMM-UHFFFAOYSA-N maleic anhydride Chemical compound O=C1OC(=O)C=C1 FPYJFEHAWHCUMM-UHFFFAOYSA-N 0.000 abstract description 7
- SFVFIFLLYFPGHH-UHFFFAOYSA-M stearalkonium chloride Chemical compound [Cl-].CCCCCCCCCCCCCCCCCC[N+](C)(C)CC1=CC=CC=C1 SFVFIFLLYFPGHH-UHFFFAOYSA-M 0.000 abstract description 6
- 238000001514 detection method Methods 0.000 abstract description 5
- NOGFHTGYPKWWRX-UHFFFAOYSA-N 2,2,6,6-tetramethyloxan-4-one Chemical compound CC1(C)CC(=O)CC(C)(C)O1 NOGFHTGYPKWWRX-UHFFFAOYSA-N 0.000 abstract description 4
- 229910000147 aluminium phosphate Inorganic materials 0.000 abstract description 4
- RMAQACBXLXPBSY-UHFFFAOYSA-N silicic acid Chemical compound O[Si](O)(O)O RMAQACBXLXPBSY-UHFFFAOYSA-N 0.000 abstract description 4
- 235000012239 silicon dioxide Nutrition 0.000 abstract description 4
- 239000002904 solvent Substances 0.000 abstract description 3
- 238000006243 chemical reaction Methods 0.000 abstract description 2
- 239000010422 internal standard material Substances 0.000 abstract 2
- 125000001797 benzyl group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])* 0.000 abstract 1
- 238000000825 ultraviolet detection Methods 0.000 abstract 1
- 150000003904 phospholipids Chemical class 0.000 description 8
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 6
- 238000012937 correction Methods 0.000 description 6
- 239000003795 chemical substances by application Substances 0.000 description 5
- 230000000052 comparative effect Effects 0.000 description 5
- 238000004809 thin layer chromatography Methods 0.000 description 5
- 102000002322 Egg Proteins Human genes 0.000 description 4
- 108010000912 Egg Proteins Proteins 0.000 description 4
- 235000013345 egg yolk Nutrition 0.000 description 4
- 210000002969 egg yolk Anatomy 0.000 description 4
- 238000002372 labelling Methods 0.000 description 4
- 150000001875 compounds Chemical class 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 229910052698 phosphorus Inorganic materials 0.000 description 3
- 239000011574 phosphorus Substances 0.000 description 3
- 238000003908 quality control method Methods 0.000 description 3
- 238000000926 separation method Methods 0.000 description 3
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 description 3
- JQWAHKMIYCERGA-UHFFFAOYSA-N (2-nonanoyloxy-3-octadeca-9,12-dienoyloxypropoxy)-[2-(trimethylazaniumyl)ethyl]phosphinate Chemical compound CCCCCCCCC(=O)OC(COP([O-])(=O)CC[N+](C)(C)C)COC(=O)CCCCCCCC=CCC=CCCCCC JQWAHKMIYCERGA-UHFFFAOYSA-N 0.000 description 2
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 2
- 125000000217 alkyl group Chemical group 0.000 description 2
- -1 benzylic quaternary ammonium salt Chemical class 0.000 description 2
- 238000011088 calibration curve Methods 0.000 description 2
- WTJKGGKOPKCXLL-RRHRGVEJSA-N phosphatidylcholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCCC=CCCCCCCCC WTJKGGKOPKCXLL-RRHRGVEJSA-N 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- JLPULHDHAOZNQI-ZTIMHPMXSA-N 1-hexadecanoyl-2-(9Z,12Z-octadecadienoyl)-sn-glycero-3-phosphocholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCC\C=C/C\C=C/CCCCC JLPULHDHAOZNQI-ZTIMHPMXSA-N 0.000 description 1
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 1
- 235000010469 Glycine max Nutrition 0.000 description 1
- 244000068988 Glycine max Species 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 238000001212 derivatisation Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 229940039227 diagnostic agent Drugs 0.000 description 1
- 239000000032 diagnostic agent Substances 0.000 description 1
- CITHEXJVPOWHKC-UHFFFAOYSA-N dimyristoyl phosphatidylcholine Chemical compound CCCCCCCCCCCCCC(=O)OCC(COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCCCCCCCC CITHEXJVPOWHKC-UHFFFAOYSA-N 0.000 description 1
- 229960003724 dimyristoylphosphatidylcholine Drugs 0.000 description 1
- 239000003937 drug carrier Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 239000008344 egg yolk phospholipid Substances 0.000 description 1
- 229940068998 egg yolk phospholipid Drugs 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000002949 hemolytic effect Effects 0.000 description 1
- 239000000787 lecithin Substances 0.000 description 1
- 235000010445 lecithin Nutrition 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 239000002502 liposome Substances 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 239000000546 pharmaceutical excipient Substances 0.000 description 1
- DHRLEVQXOMLTIM-UHFFFAOYSA-N phosphoric acid;trioxomolybdenum Chemical compound O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.OP(O)(O)=O DHRLEVQXOMLTIM-UHFFFAOYSA-N 0.000 description 1
- 238000004886 process control Methods 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 239000011347 resin Substances 0.000 description 1
- 229920005989 resin Polymers 0.000 description 1
- 229940083466 soybean lecithin Drugs 0.000 description 1
- 239000008347 soybean phospholipid Substances 0.000 description 1
- 239000010421 standard material Substances 0.000 description 1
Abstract
Description
【発明の詳細な説明】
［産業上の利用分野］
本発明は、大豆レシチンや卵黄レシチンのような天然レ
シチン及び合成リン脂質中に存在する少量のリゾホスフ
ァチジルコリンの定量に関するもので、これら製品の工
程管理や品質管理分析に利用される。Detailed Description of the Invention [Industrial Application Field] The present invention relates to the determination of small amounts of lysophosphatidylcholine present in natural lecithins and synthetic phospholipids, such as soybean lecithin and egg yolk lecithin, and to Used for management and quality control analysis.
［従来の技術］
ドラッグキャリャーや診断薬の増副素子として注目を集
めているリポソームの原料として用いられるリン脂質は
、高純度のものを必要とし、特に溶血作用のあるリゾ体
の含有は好ましくない。そのためリン脂質中のリゾ体を
正確、かつ迅速に分析する方法は製品の工程管理や品質
管理上きわめて重要である。[Prior Art] Phospholipids used as raw materials for liposomes, which are attracting attention as drug carriers and additives for diagnostic agents, need to be of high purity, and it is particularly preferable to contain lysoforms that have hemolytic effects. do not have. Therefore, a method for accurately and quickly analyzing lyso forms in phospholipids is extremely important for product process control and quality control.
従来、リン脂質中のリゾホスファチジルコリンの定量法
としては、二次元薄層クロマトグラフィーで分離後スポ
ットをかきとり、脂質リンを無機リンに分解し、リンモ
リブデン酸錯体として吸光分析する方法［油化学、３５
巻ｐｌｏ１８（１９８６）］、リン脂質混合物を薄層ク
ロマトグラフィ一一水素炎イオン化検出器で分析する方
法［油化学、２６巻ｐ４５４ （１９７７）］及び薄層
クロマトグラフィーで分離したスポットを最小検出量法
で分析する方法などがある。しかし、これらの方法は何
れも一長一短があり、また操作が繁雑であったり、分析
精度が劣るなどの欠点を有し、また感度もよくない。Conventionally, the method for quantifying lysophosphatidylcholine in phospholipids is to scrape off the spot after separation using two-dimensional thin layer chromatography, decompose the lipid phosphorus into inorganic phosphorus, and perform absorption analysis as a phosphomolybdic acid complex [Oil Chemistry, 35
vol. plo 18 (1986)], a method for analyzing phospholipid mixtures using thin-layer chromatography and a flame ionization detector [Oil Chemistry, vol. 26, p. 454 (1977)], and a method for analyzing spots separated by thin-layer chromatography using a minimum detectable amount method. There are ways to analyze it. However, all of these methods have advantages and disadvantages, and they also have disadvantages such as complicated operations and poor analysis accuracy, and are also not sensitive.
［発明が解決しようとする問題点］
以上の如《、リゾ体を正確、かつ迅速に分析する方法は
現在までのところ見当らず、従って製品の規格値も設定
できないので、製品の工程管理や品質管理分析に支障を
来たしており、分析法の開発が望まれていた。[Problems to be solved by the invention] As mentioned above, there is currently no method for accurately and quickly analyzing lyso-bodies, and therefore it is not possible to set standard values for products. This has caused problems in management analysis, and the development of an analytical method has been desired.
一方、最近高速液体クロマトグラフィーの普及により、
分離分析技術が著しく進歩した。しかし、検出器に万能
型のものがなく、例えば天然の大豆や卵黄リン脂質中の
主成分は紫外線検出器で検出されるが、リゾホスファチ
ジルコリンのように含有量が少な《、主として飽和アル
キル基を有するものは殆ど検出できない。従って、この
ような場合、紫外線検出器で検出できるような誘導体化
が必要となる。On the other hand, with the recent spread of high-performance liquid chromatography,
Separation and analysis technology has made significant progress. However, there is no universal detector. For example, the main components in natural soybean and egg yolk phospholipids can be detected with an ultraviolet detector, but those with low content (mainly saturated alkyl groups) such as lysophosphatidylcholine Those that do have it are almost undetectable. Therefore, in such cases, derivatization that can be detected with an ultraviolet detector is required.
また、高速液体クロマトグラフィーの標準として使用す
るリゾホスファチジルコリンは高純度のものを必要とす
るが、その精製は煩雑で長時間を要するために分析の度
毎に標準物質を使用する絶対検量線法は好まし《なく、
むしろ内部標準法の方が優れているが、そのためには適
当な内部標準物質の探索が必要である。In addition, the lysophosphatidylcholine used as a standard for high-performance liquid chromatography must be of high purity, but its purification is complicated and takes a long time, so the absolute calibration curve method, which uses a standard material for each analysis, is difficult. Not preferable,
The internal standard method is actually better, but it requires searching for a suitable internal standard substance.
あることに着目し、本発明者は、ケイ酸系力ラムを用い
、アセトニトリル／メタノール／リン酸系の移動相溶媒
を用いた場合の内部標準物質について検討した結果、各
種リン脂質と相互分離するベンジル型第４級アンモニウ
ム塩が最適の化合物であることを見出した。この化合物
は、容易に合成することができ、また容易に精製して高
純度品を得ることができるので内部標準物質として適し
ている。Focusing on one thing, the present inventor investigated an internal standard substance when using a silicic acid-based ram and an acetonitrile/methanol/phosphoric acid-based mobile phase solvent, and found that it mutually separates from various phospholipids. It has been found that a benzylic quaternary ammonium salt is the most suitable compound. This compound is suitable as an internal standard substance because it can be easily synthesized and purified to obtain a highly pure product.
本発明においてリゾホスファチジルコリンのエステル化
剤として用いられる芳香族核または二重結合を有する酸
無水物としては無水フタル酸、無水マレイン酸あるいは
無水安息香酸などをあげることができる。In the present invention, examples of the acid anhydride having an aromatic nucleus or double bond used as an esterifying agent for lysophosphatidylcholine include phthalic anhydride, maleic anhydride, and benzoic anhydride.
内部標準物質として使用するベンジル型第４級アンモニ
ウム塩としてはステアリルジメチルベンジルアンモニウ
ムクロリドが最適である。Stearyldimethylbenzylammonium chloride is most suitable as the benzylic quaternary ammonium salt used as an internal standard substance.
本発明に従いリゾホスファチジルコリンの分析を行なう
方法の一例を示すと、先ず試料をテトラヒド口フラン中
、７５〜８０℃において無水フタ［課題を解決するため
の手段］
発明者らはこれらの問題点を解決するために鋭意検討し
た結果、飽和アルキル基を有するリゾホスファヂジルコ
リンに対しては、紫外線検出器に対して高感度なラベル
化剤を反応させてその誘導体を調製し、感度アップを計
る必要があることに着目し、このようなラベル化剤とし
ては無水フタル酸、無水マレイン酸などの芳香族核また
は二重結合を有する酸無水物が最適であることを見出し
た。すなわち、試料なテ１・ラヒドロフラン中、７５〜
８０℃において無水フタル酸、無水マレイン酸あるいは
無水安息香酸を反応させてエステル化し、紫外線検出器
に対して高感度な誘導体とすることで、０．１％程度の
りゾホスファヂジルコリンを含有する試料の分析が可能
となった。An example of a method for analyzing lysophosphatidylcholine according to the present invention is as follows: First, a sample is heated in tetrahydrofuran at 75 to 80°C with an anhydrous lid [Means for Solving the Problems] The inventors have solved these problems. As a result of extensive research, we decided to increase the sensitivity of lysophosphadidylcholine, which has a saturated alkyl group, by reacting a highly sensitive labeling agent with a UV detector to prepare a derivative thereof. We focused on this need and found that acid anhydrides having aromatic nuclei or double bonds, such as phthalic anhydride and maleic anhydride, are optimal as such labeling agents. That is, in the sample Te1-rahydrofuran, 75~
By reacting and esterifying phthalic anhydride, maleic anhydride, or benzoic anhydride at 80°C to create a derivative that is highly sensitive to ultraviolet detectors, it contains approximately 0.1% of sophosphadylcholine. It is now possible to analyze samples that
方、高速液体クロマトグラフィーによりリゾホスファチ
ジルコリンの定量法の場合、絶対検量線法よりも内部標
準法の法がすぐれており、内部標準法は、内部標準物質
を適正に選べば、分離パラメーターによる変動を補正で
きるという利点がル酸、無水マレイン酸あるいは無水安
息香酸を反応させてエステル化し、次に前記の方法で調
製したエステル化物にステアリルジメチルベンジルアン
モニウムクロリドを内部標準物質として加え、カラムに
はケイ酸系カラム（Ｐｏｌｙｇｏｓｉｌ６０−７． ４
１ＩＩＩＸ３００ｆｆｌｌｌ′，ケムコ製）、移動相溶
媒としてはアセトニトリル／メタノール／７５％リン酸
（７８／１／１）を用いた高速液体クロマトグラフィー
を用い、紫外線検出器により２１０ｎｍの波長で検出し
、内部標準法で定量する。On the other hand, when determining lysophosphatidylcholine using high-performance liquid chromatography, the internal standard method is superior to the absolute calibration curve method. The advantage of being able to correct this is to react and esterify silicic acid, maleic anhydride, or benzoic anhydride, then add stearyldimethylbenzylammonium chloride as an internal standard to the esterified product prepared by the above method, and add silicic acid to the column. system column (Polygosil60-7.4
1IIIX300ffllll', manufactured by Chemco), high performance liquid chromatography using acetonitrile/methanol/75% phosphoric acid (78/1/1) as the mobile phase solvent, detection at a wavelength of 210 nm with an ultraviolet detector, and internal standard Quantify by method.
この方法で得られるクロマトグラムは、第１図と第２図
に見られるようにリゾホスファチジルコリンの感度アッ
プが可能となり、また主成分のホスファチジルコリンと
の分離も良好であった。In the chromatogram obtained by this method, as shown in FIGS. 1 and 2, it was possible to increase the sensitivity of lysophosphatidylcholine, and the separation from the main component phosphatidylcholine was also good.
本発明の方法においてはリゾホスファチジルコリンの含
有量は次式により求めることができる。In the method of the present invention, the content of lysophosphatidylcholine can be determined by the following formula.
ここに
Ｌａ：分析対象のリゾホスファチジルコリンのビーク面
積
エｗ：内部標準物質の量（ｍｇ）
工ｐ：内部標準物質の純度（％）
Ｋ：補正係数
１ａ：内部標準物質のピーク面積
Ｗ：試料の量（ｍｇ）
但し、補正係数Ｋは、リゾホスファチジルコリン（標準
品）について本発明方法を実施し、得られた結果から次
式により求めたものである。where La: peak area of lysophosphatidylcholine to be analyzed w: amount of internal standard (mg) p: purity of internal standard (%) K: correction factor 1a: peak area of internal standard W: amount of sample Amount (mg) However, the correction coefficient K was determined by the following formula from the results obtained by implementing the method of the present invention on lysophosphatidylcholine (standard product).
Ｋ：補正係数
Ｓｗニリゾホスファチジルコリン（標準品）の重量（ｍ
ｇ）
■ａ：内部標準物質のピーク面積
Ｓｐ：リゾホスファ．チジルコリン（標準品）の純度（
％）
■ｗ：内部標準物質の重量（ｍｇ）
無水フタル酸のテトラヒドロフラン溶液を２ｍｌ加え、
冷却管を付けて７５〜８０℃で２時間反応させる。反応
後、テトラヒド口フランを留去し、残渣は少量のエタノ
ールに溶解して５ｍｌメスフラスコに移し、０．１％ス
テアリルジメチルベンジルアンモニウムクロリドのエタ
ノール溶液を０．５ｍ℃加えてからエタノールで標線に
合わせる。得られた試料を下記の条件で高速液体クロマ
ト分析を行ない、第１図の如きクロマトグラムが得られ
た。K: Correction coefficient Sw Weight of nyrisophosphatidylcholine (standard product) (m
g) ■a: Peak area of internal standard substance Sp: Lysophospha. Purity of tidylcholine (standard product) (
%) ■w: Weight of internal standard substance (mg) Add 2 ml of a solution of phthalic anhydride in tetrahydrofuran,
Attach a cooling tube and react at 75 to 80°C for 2 hours. After the reaction, the tetrahydrofuran was distilled off, and the residue was dissolved in a small amount of ethanol and transferred to a 5 ml volumetric flask, and an ethanol solution of 0.1% stearyldimethylbenzylammonium chloride was added at 0.5 m℃, and then the marked line was diluted with ethanol. Match. The obtained sample was subjected to high performance liquid chromatography analysis under the following conditions, and a chromatogram as shown in FIG. 1 was obtained.
カラム：Ｐｏｌｙｇｏｓｉｌ ６０−７ （ケムコ製
、４”Ｘ ３ ０ ０”）
移動相：アセトニトリル／メタノール／７５％リン酸（
７８／ｌ／１）
流速：２ｍβ／ｍｉｎ
検出：２１０ｎｍ（紫外線検出器）
このクロマトグラムから前述した計算式によりリゾホス
ファチジルコリンの含有量を求めた。Column: Polygosil 60-7 (manufactured by Chemco, 4" x 300") Mobile phase: Acetonitrile/methanol/75% phosphoric acid (
78/l/1) Flow rate: 2 mβ/min Detection: 210 nm (ultraviolet detector) From this chromatogram, the content of lysophosphatidylcholine was determined using the above-mentioned formula.
結果を第２表に示す。The results are shown in Table 2.
実施例ス
Ｓａ：リゾホスファチジルコリン（標準品）の面積
工ｐ：内部標準物質の純度（％）
第１表に補正係数を求めた結果を示す。第１表によれば
、本分析法においては、ラベル化剤として無水フタル酸
を用いた場合は、天然系も合成系も内部標準物質に対す
るレスポンスは殆ど変わらない。しかし、無水マレイン
酸を用いた場合は、補正係数は合成系相互の間に大きな
差は認められないが、天然系との間には差が認められる
。Example Sa: Area of lysophosphatidylcholine (standard product) p: Purity (%) of internal standard substance Table 1 shows the results of determining the correction coefficient. According to Table 1, in this analytical method, when phthalic anhydride is used as a labeling agent, there is almost no difference in response to the internal standard substance in both natural and synthetic systems. However, when maleic anhydride is used, there is no significant difference in the correction coefficient between the synthetic systems, but there is a difference between the natural system and the correction coefficient.
［実施例］ 次に本発明を実施例及び比較例により説明する。[Example] Next, the present invention will be explained with reference to Examples and Comparative Examples.
夾施伝１
卵黄より分離精製されたし−α−ホスファチジルコリン
（Ｓｉ ｇｍａ社製、純度 約９９％）とＬ一α−リゾ
ホスファチジルコリン（Ｓｉｇｍａ社製、純度 約９９
％）を第２表に示した割合に配合し、その約５０ｍｇを
すり合わせ付き試験管（１ ５ｆｆｌｆｆｉｘ １ ５
０″″）に正しく量りとり、１％の市販の卵黄レシチ
ンを実施例１の条件で分析し、リゾホスファチジルコリ
ンの含有量を求めた結果を第３表に示す。Koseden 1 Lys-α-phosphatidylcholine (manufactured by Sigma, purity approximately 99%) and L-α-lysophosphatidylcholine (manufactured by Sigma, purity approximately 99%) isolated and purified from egg yolk.
%) in the proportions shown in Table 2, and about 50 mg of it was added to a test tube with a test tube (1 5ffffix 1 5
0''''), 1% commercially available egg yolk lecithin was analyzed under the conditions of Example 1, and the content of lysophosphatidylcholine was determined. Table 3 shows the results.
去施例ｌ
Ｌ−α−ジミリストイルホスファチジルコリン［日本油
脂■製、純度９９．９％］とＬ一α−モノミリストイル
ホスファチジルコリン（Ａｖａｎｔｉ Ｐｏｌａｒ−
Ｌｉｐｉｄ，Ｉｎｃ．製純度９９％）を第４表に示した
割合に配合し、実施例１の条件で分析し、第２図の如き
クロマトグラムが得られた。これよつりゾフォスファチ
ジルコリンの含有量を求めた結果を第４表に示す。Example 1 L-α-dimyristoyl phosphatidylcholine [manufactured by NOF ■, purity 99.9%] and L-α-monomyristoyl phosphatidylcholine (Avanti Polar-
Lipid, Inc. (purity: 99%) were blended in the proportions shown in Table 4 and analyzed under the conditions of Example 1 to obtain a chromatogram as shown in FIG. Table 4 shows the results of determining the content of zophosphatidylcholine.
比較例↓
実施例１の混合試料を用い、二次元薄層クロマトグラフ
ィー−リン定量法によりリゾホスファチジルコリンの含
有量を求めた。結果を第２表に示す。Comparative Example↓ Using the mixed sample of Example 1, the content of lysophosphatidylcholine was determined by two-dimensional thin layer chromatography-phosphorus quantitative method. The results are shown in Table 2.
比較例λ
実施例２で用いた市販の卵黄レシヂンを薄層クロマトグ
ラフィーの最小検出量法で分析した結果を第３表に示す
。Comparative Example λ Table 3 shows the results of analyzing the commercially available egg yolk resin used in Example 2 by the minimum detection amount method of thin layer chromatography.
比較泗且
実施例３の混合試料を用い、薄層クロマトグラフィ一一
水素炎イオン化検出法（ヤトロン社製ヤトロスキャン）
で分析しな結果を第４表に示す。Comparative Example 3 Using the mixed sample of Example 3, thin layer chromatography - flame ionization detection method (Yatroscan manufactured by Yatron)
The results of the analysis are shown in Table 4.
第２表および第４表に示したように、本発明の方法によ
る実施例１および実施例３の分析結果は、標準品の配合
割合と良く一致しており、比較例１および３に比較して
分析精度が高い。また分析操作も簡便で比較的短時間で
分析できる。As shown in Tables 2 and 4, the analysis results of Examples 1 and 3 by the method of the present invention are in good agreement with the blending ratio of the standard product, and compared with Comparative Examples 1 and 3. The analysis accuracy is high. In addition, the analysis operation is simple and analysis can be performed in a relatively short time.
以下余白
［発明の効果］
本発明のリン脂質中のリゾホスファチジルコリンの定量
法は、無水フタル酸や無水マレイン酸のようなラベル化
剤により紫外線検出器に対して高感度な化合物を生成さ
せることにより０． １％程度のリゾ体の分析が可能
となり、またベンジル型第四級アンモニウム塩を内部標
準物質として用いることにより内部標準法で定量するこ
とができるので、操作が簡単で精度の高い分析法であり
、医薬品原料としてのリン脂質製品の品質管理に適用で
きる。The following margin [Effects of the Invention] The method for quantifying lysophosphatidylcholine in phospholipids of the present invention is performed by generating a compound that is highly sensitive to ultraviolet light detectors using a labeling agent such as phthalic anhydride or maleic anhydride. 0. It is possible to analyze about 1% of the lyso form, and it can be quantified using the internal standard method by using a benzyl-type quaternary ammonium salt as an internal standard substance, so it is an easy-to-operate and highly accurate analytical method. , can be applied to quality control of phospholipid products as pharmaceutical raw materials.
第１図は、卵黄より得られたＬ−α−ホスファチジルコ
リン９６．８８％と、同じく卵黄よりのし一α−リゾホ
スファチジルコリン３．１２％の混合物（ステアリルジ
メチルベンジルアンモニウムクロリドを内部標準とする
）を実施例１の条件で高速液体クロマト分析を行ない、
得られたクロマトグラムである。
第２図は、合成のし−α−ジミリストイルホスファチジ
ルコリン９５．１２％とＬ−α−モノミ゛）リストイル
ホスファチジルコリン４．８５％の混合物（ステアリル
ジメチルベンジルアンモニウムクロリドを内部標準とす
る。）を実施例３の条件で分析して得られたクロマトグ
ラムである。Figure 1 shows a mixture of 96.88% L-α-phosphatidylcholine obtained from egg yolk and 3.12% L-α-lysophosphatidylcholine obtained also from egg yolk (using stearyldimethylbenzylammonium chloride as an internal standard). High performance liquid chromatography analysis was performed under the conditions of Example 1,
This is the obtained chromatogram. Figure 2 shows a mixture of 95.12% of synthesized -α-dimyristoyl phosphatidylcholine and 4.85% of L-α-monomiyristoylphosphatidylcholine (stearyldimethylbenzylammonium chloride is used as an internal standard). This is a chromatogram obtained by analysis under the conditions of Example 3.
Claims (1)
合を有する酸無水物とエステル化した後、ベンジル型第
四級アンモニウム塩を内部標準物質として紫外線検出器
を用いる高速液体クロマトグラフィーにより内部標準法
で定量することを特徴とするリゾホスファチジルコリン
の定量法。1. After esterifying lysophosphatidylcholine with an acid anhydride having an aromatic nucleus or a double bond, quantification is performed using an internal standard method using high-performance liquid chromatography using a benzyl-type quaternary ammonium salt as an internal standard substance and an ultraviolet detector. A method for quantifying lysophosphatidylcholine.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP28577688A JP2661210B2 (en) | 1988-11-14 | 1988-11-14 | Determination of lysophosphatidylcholine |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP28577688A JP2661210B2 (en) | 1988-11-14 | 1988-11-14 | Determination of lysophosphatidylcholine |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH02132372A true JPH02132372A (en) | 1990-05-21 |
| JP2661210B2 JP2661210B2 (en) | 1997-10-08 |
Family
ID=17695919
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP28577688A Expired - Fee Related JP2661210B2 (en) | 1988-11-14 | 1988-11-14 | Determination of lysophosphatidylcholine |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP2661210B2 (en) |
-
1988
- 1988-11-14 JP JP28577688A patent/JP2661210B2/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| JP2661210B2 (en) | 1997-10-08 |
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