JP2612652B2 - Analysis of trace amounts of amino group-containing phospholipids - Google Patents
Analysis of trace amounts of amino group-containing phospholipidsInfo
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- JP2612652B2 JP2612652B2 JP3264104A JP26410491A JP2612652B2 JP 2612652 B2 JP2612652 B2 JP 2612652B2 JP 3264104 A JP3264104 A JP 3264104A JP 26410491 A JP26410491 A JP 26410491A JP 2612652 B2 JP2612652 B2 JP 2612652B2
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- Prior art keywords
- amino group
- containing phospholipids
- trace amount
- analyzing
- phospholipids
- Prior art date
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Description
【０００１】[0001]
【産業上の利用分野】本発明は、微量のアミノ基含有リ
ン脂質の分析法、詳しくは、高速液体クロマトグラフ法
（ＨＰＬＣ）により、微量のアミノ基含有リン脂質類を
選択的に分析するアミノ基含有リン脂質の分析法、更に
詳しくは、多量のホスファチジン酸及びトリグリセリド
を含有する試料中の微量のアミノ基含有リン脂質類をＨ
ＰＬＣにより高感度で簡便に分析するアミノ基含有リン
脂質の分析法に関する。BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for analyzing a trace amount of a phospholipid containing an amino group, more specifically, an amino acid for selectively analyzing a trace amount of a phospholipid containing an amino group by high performance liquid chromatography (HPLC). A method for analyzing group-containing phospholipids, more specifically, a method for analyzing trace amounts of amino group-containing phospholipids in a sample containing a large amount of phosphatidic acid and triglyceride,
The present invention relates to a method for analyzing amino group-containing phospholipids which is easily and simply analyzed by PLC.
【０００２】[0002]
【従来の技術及び発明が解決しようとする課題】従来の
アミノ基含有リン脂質類の分析法としては、例えば、Ｔ
ＬＣフルオレサミン法、ＨＰＬＣ紫外検出法、あるいは
ＨＰＬＣプレラベル蛍光検出法などが知られている。2. Description of the Related Art Conventional methods for analyzing amino group-containing phospholipids include, for example, T
The LC fluorescamine method, the HPLC ultraviolet detection method, the HPLC prelabel fluorescence detection method, and the like are known.
【０００３】しかしながら、ＴＬＣフルオレサミン法
は、ＴＬＣの操作が煩雑で共存成分による妨害があって
微量のアミノ基含有リン脂質類を定量する場合には精度
に課題があった。また、ＨＰＬＣ紫外検出法は操作は簡
便であるものの微量のアミノ基含有リン脂質類を定量す
ることが困難であった。また、従来のＨＰＬＣプレラベ
ル蛍光検出法は、微量のアミノ基含有リン脂質を高感度
に検出することができるが、操作が煩雑であり、かつ試
料中に共存成分が含まれる場合はこの共存成分の影響に
より絶対検量線法で定量することができない。例えば、
多量のホスファチジン酸及びトリグリセリドを含有する
試料中の微量のアミノ基含有リン脂質類を定量すること
ができないという問題点を有している。[0003] However, the TLC fluorescamine method has a problem in precision when quantifying a trace amount of amino group-containing phospholipids due to complicated TLC operation and interference by coexisting components. In addition, although the HPLC ultraviolet detection method is simple in operation, it was difficult to quantify a trace amount of amino group-containing phospholipids. Further, the conventional HPLC prelabel fluorescence detection method can detect a trace amount of an amino group-containing phospholipid with high sensitivity, but the operation is complicated, and when a coexisting component is contained in the sample, the coexisting component is not detected. It cannot be quantified by the absolute calibration method due to the influence. For example,
There is a problem that it is not possible to quantify a trace amount of amino group-containing phospholipids in a sample containing a large amount of phosphatidic acid and triglyceride.
【０００４】従って、本発明の目的は、多量のホスファ
チジン酸及びトリグリセリドを含有する試料中の微量の
アミノ基含有リン脂質類を簡便且つ高感度に定量するこ
とができる、ＨＰＬＣプレラベル蛍光検出法による微量
のアミノ基含有リン脂質の分析法を提供することにあ
る。[0004] Accordingly, an object of the present invention is to provide a method for detecting trace amounts of amino group-containing phospholipids in a sample containing a large amount of phosphatidic acid and triglyceride by a HPLC pre-labeled fluorescence detection method, which is capable of simple and highly sensitive quantification. To provide a method for analyzing amino group-containing phospholipids.
【０００５】[0005]
【課題を解決するための手段】本発明者らは、ＨＰＬＣ
による微量のアミノ基含有リン脂質の分析法について種
々検討した結果、微量のアミノ基含有リン脂質と特定の
内部標準物質を特定のラベル化剤で選択的にプレラベル
化しそれぞれの蛍光誘導体とした後、ＨＰＬＣでそれぞ
れの蛍光誘導体を分離検出し、内部標準法を用いること
によって各蛍光誘導体を定量することができ、もって上
記目的を達成し得ることを知見した。Means for Solving the Problems The present inventors have developed HPLC.
As a result of various studies on the analysis method of a trace amount of amino group-containing phospholipids, a trace amount of amino group-containing phospholipid and a specific internal standard substance were selectively pre-labeled with a specific labeling agent to obtain respective fluorescent derivatives, It has been found that each fluorescent derivative can be separated and detected by HPLC, and each fluorescent derivative can be quantified by using an internal standard method, thereby achieving the above object.
【０００６】本発明は、上記知見に基づいてなされたも
ので、高速液体クロマトグラフ法により、微量のアミノ
基含有リン脂質を分析する方法であって、多量のホスフ
ァチジン酸及びトリグリセリドを含有する試料中の微量
のアミノ基含有リン脂質類を内部標準物質であるグリシ
ンと共に７−フルオロ−４−ニトロベンゾ−２−オキサ
−１，３−ジアゾールによって選択的にラベル化してそ
れぞれの蛍光誘導体とし、蛍光誘導体それぞれを高速液
体クロマトグラフ法で分離検出した後、分離された蛍光
誘導体を内部標準法によってそれぞれ定量することを特
徴とする微量のアミノ基含有リン脂質の分析法を提供す
るものである。The present invention has been made based on the above findings, and is a method for analyzing a trace amount of an amino group-containing phospholipid by high performance liquid chromatography, wherein the method comprises the steps of: analyzing a sample containing a large amount of phosphatidic acid and triglyceride; glycine the traces amino group-containing phospholipids of the internal standard substance
The respective fluorescent derivative was selectively labeled by 7-fluoro-4-nitrobenzo-2-oxa-1,3-diazole with down, after separating detect each fluorescent derivative by high performance liquid chromatography, separated An object of the present invention is to provide a method for analyzing a trace amount of a phospholipid containing an amino group, characterized by quantifying a fluorescent derivative by an internal standard method.
【０００７】[0007]
【作用】本発明によれば、多量のホスファチジン酸及び
トリグリセリドを含有する試料中の微量のアミノ基含有
リン脂質類を内部標準物質であるグリシンと共に７−フ
ルオロ−４−ニトロベンゾ−２−オキサ−１，３−ジア
ゾール（ＮＢＤ−Ｆ）によって選択的にラベル化してそ
れぞれの蛍光誘導体とし、蛍光誘導体それぞれをＨＰＬ
Ｃで分離検出した後、内部標準法によって分離された蛍
光誘導体をそれぞれ定量することができ、すなわち、ア
ミノ基含有リン脂質類を定量することができる。According to the present invention, a trace amount of amino group-containing phospholipids in a sample containing a large amount of phosphatidic acid and triglyceride is converted into 7-fluoro-4-nitrobenzo-2-oxa-1 together with glycine as an internal standard. , 3-diazole (NBD-F) to selectively label each fluorescent derivative,
After separation and detection in C, the fluorescent derivatives separated by the internal standard method can be quantified, that is, the amino group-containing phospholipids can be quantified.
【０００８】[0008]
【実施例】以下、図１、図２を参照しながら本発明のＨ
ＰＬＣによる微量のアミノ基含有リン脂質の分析法につ
いて説明する。尚、各図中、図１は標準ホスファチジル
エタノールアミン、標準リゾホスファチジルエタノール
アミン、標準ホスファチジルセリン及びグリシンをＮＢ
Ｄ−Ｆによってプレラベル化した蛍光誘導体それぞれの
クロマトグラムと本ＨＰＬＣで用いられた移動相のグラ
ジエントパターン（破線）を示す図、図２は本発明の一
実施態様による多量のホスファチジン酸及びトリグリセ
リドを含有する試料中の微量のアミノ基含有リン脂質類
のクロマトグラムを示す図である。DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Referring to FIGS.
A method for analyzing a trace amount of an amino group-containing phospholipid by PLC will be described. In each figure, FIG. 1 shows that standard phosphatidylethanolamine, standard lysophosphatidylethanolamine, standard phosphatidylserine and glycine were NB.
FIG. 2 shows a chromatogram of each of the fluorescent derivatives prelabeled by DF and a gradient pattern (broken line) of a mobile phase used in the present HPLC. FIG. 2 contains a large amount of phosphatidic acid and triglyceride according to one embodiment of the present invention. FIG. 3 is a view showing a chromatogram of a trace amount of amino group-containing phospholipids in a sample.
【０００９】本発明の一実施態様では、予め、図１に示
すような、標準試料と内部標準物質の蛍光誘導体のクロ
マトグラムを本発明の下記実施態様に従って作成してお
く。然る後、未知試料の分析を行ない、上記クロマトグ
ラムにおける保持時間を指標としてピークの帰属を行
う。In one embodiment of the present invention, a chromatogram of a standard sample and a fluorescent derivative of an internal standard as shown in FIG. 1 is prepared in advance according to the following embodiment of the present invention. Thereafter, the unknown sample is analyzed, and peak assignment is performed using the retention time in the chromatogram as an index.
【００１０】そのためには、まず、多量のホスファチジ
ン酸及びトリグリセリドを含有する試料中の微量のアミ
ノ基含有リン脂質類を内部標準物質であるグリシンと共
に７−フルオロ−４−ニトロベンゾ−２−オキサ−１，
３−ジアゾール（ＮＢＤ−Ｆ）によって選択的に下記化
１のようにラベル化してそれぞれの蛍光誘導体を得る。
ここで、アミノ基含有リン脂質類としては、例えば、ホ
スファチジルエタノールアミン、リゾホスファチジルエ
タノールアミン、ホスファチジルセリンを挙げることが
できる。但し、下記化１において、Ｒ−ＮＨ2 はアミノ
基含有リン脂質類の一般式を表わす。For this purpose, first, a trace amount of amino group-containing phospholipids in a sample containing a large amount of phosphatidic acid and triglyceride is combined with 7-fluoro-4-nitrobenzo with an internal standard substance , glycine. -2-oxa-1,
Each fluorescent derivative is obtained by selectively labeling with 3-diazole (NBD-F) as shown below.
Here, examples of the amino group-containing phospholipids include phosphatidylethanolamine, lysophosphatidylethanolamine, and phosphatidylserine.
I can . However, in the following Chemical Formula 1, R-NH 2 represents a general formula of amino group-containing phospholipids.
【００１１】[0011]
【化１】 Embedded image
【００１２】そこで、プレラベル化反応について詳述す
ると、まず、多量のホスファチジン酸及びトリグリセリ
ドを含有する試料を１ｍｇ程度を採取し、採取した試料
中に約１０ｐｐｍのグリシン溶液を１００μｌ添加す
る。得られた溶液の溶媒を窒素雰囲気下で留去した後、
０．５Ｍのトリエチルアミン溶液〔トルエン：エタノー
ル＝１：１（体積比）溶媒〕を２０μｌ添加し、更に、
ラベル化剤として５ｍＭのＮＢＤ−Ｆ溶液〔トルエン：
エタノール＝１：１（体積比）溶媒〕を１００μｌ添加
し、４５℃で３０分間インキュベーションして微量の上
記アミノ基含有リン脂質類をグリシンと共にラベル化し
てそれぞれの蛍光誘導体を合成する。その後、遮光下で
室温になるまで静置する。然る後、０．０２Ｎ 塩酸溶
液〔トルエン：エタノール＝４：１（体積比）〕を５．
０ｍｌ添加して希釈する。このようにインキュベーショ
ン後、遮光下で冷却した後塩酸のトルエンとエタノール
との混合溶液を添加することによって溶液中の各蛍光誘
導体を少なくとも５時間は安定な状態で存在させること
ができ、従って、オートサンプラーを用いて後述の分
離、検出を自動的且つ安定的に精度良く行なうことがで
きる。Therefore, the prelabeling reaction will be described in detail. First, about 1 mg of a sample containing a large amount of phosphatidic acid and triglyceride is collected, and 100 μl of a glycine solution of about 10 ppm is added to the collected sample. After distilling off the solvent of the obtained solution under a nitrogen atmosphere,
Triethyl amine solution of 0.5M [toluene: ethanol = 1: 1 (volume ratio) solvent] was 20μl added, further,
A 5 mM NBD-F solution [toluene:
Ethanol = 1: 1 (volume ratio)], and incubate at 45 ° C. for 30 minutes to label a trace amount of the above-mentioned amino group-containing phospholipids with glycine to synthesize respective fluorescent derivatives. After that, it is allowed to stand at room temperature under light shielding. Thereafter, a 0.02 N hydrochloric acid solution [toluene: ethanol = 4: 1 (volume ratio)] was added to the mixture.
Add 0 ml and dilute. After such incubation, each fluorescent derivative in the solution can be allowed to exist in a stable state for at least 5 hours by adding a mixed solution of hydrochloric acid in toluene and ethanol after cooling under light shielding and then adding the solution. Separation and detection, which will be described later, can be performed automatically, stably, and accurately using a sampler.
【００１３】然る後、蛍光誘導体それぞれをＨＰＬＣで
分離検出した後、分離された蛍光誘導体を内部標準法に
よってそれぞれ定量する。即ち、上記希釈溶液の調製後
５時間以内に、希釈溶液２０μｌを高速液体クロマトグ
ラフ装置に注入しそれぞれの蛍光誘導体を室温のカラム
で下記の〔分離条件〕により分離し、図１に示すクロマ
トグラムを得た。次いで、分離された各蛍光誘導体を蛍
光検出器によって検出して図２に示す各アミノ基含有リ
ン脂質類およびグリシンの蛍光誘導体、即ち、ＮＢＤ−
ＰＥ，ＮＢＤ−ＬＰＥ，ＮＢＤ−ＰＳ及びＮＢＤ−Ｇｌ
ｙのクロマトグラムを得、各アミノ基含有リン脂質類か
ら得られた蛍光誘導体の図２に示すピーク面積をそれぞ
れ求めることによって内部標準法によりホスファチジル
エタノールアミン、リゾホスファチジルエタノールアミ
ン及びホスファチジルセリンを定量した。〔定量値 ：
ホスファチジルエタノールアミン ２．２８％，リゾ
ホスファチジルエタノールアミン ０．３４％，ホスフ
ァチジルセリン０．３７％〕 本実施態様に用いられるＨＰＬＣには、固定相と移動相
が使用される。上記固定相としては、例えばシリカゲル
等が好ましい。また、移動相としては、２種以上の有機
溶媒の混合比を漸次変化させたもの（グラジエント）が
使用される。上記移動相には、例えばアンモニア水、ト
リエチルアミンなどの塩基性物質を添加することが好ま
しい。上記有機溶媒としては、クロロホルム，ジクロロ
メタン，エタノール，メタノール等が好ましい。Thereafter, each of the fluorescent derivatives is separated and detected by HPLC, and then the separated fluorescent derivatives are quantified by the internal standard method. That is, within 5 hours after the preparation of the diluted solution, 20 μl of the diluted solution was injected into a high performance liquid chromatograph, and each fluorescent derivative was separated by a column at room temperature according to the following [separation conditions], and the chromatogram shown in FIG. I got Next, each of the separated fluorescent derivatives was detected by a fluorescence detector, and the fluorescent derivatives of each of the amino group-containing phospholipids and glycine shown in FIG. 2, that is, NBD-
PE, NBD-LPE, NBD-PS and NBD-Gl
The chromatogram of y was obtained, and the phosphatidylethanolamine, lysophosphatidylethanolamine, and phosphatidylserine were quantified by the internal standard method by determining the peak areas shown in FIG. 2 of the fluorescent derivatives obtained from the respective amino group-containing phospholipids. . [Quantitative value:
2.28% of phosphatidylethanolamine, 0.34% of lysophosphatidylethanolamine, 0.37% of phosphatidylserine] A stationary phase and a mobile phase are used for HPLC used in the present embodiment. As the stationary phase, for example, silica gel or the like is preferable. Further, as the mobile phase, one in which the mixing ratio of two or more organic solvents is gradually changed (gradient) is used. It is preferable to add a basic substance such as aqueous ammonia or triethylamine to the mobile phase. As the above organic solvent, chloroform, dichloromethane, ethanol, methanol and the like are preferable.
【００１４】また、本実施態様で実行される検出器の条
件としては、励起波長４３０〜４９０ｎｍ，蛍光波長５
００〜５４０ｎｍが好ましく、より好ましくは励起波長
４６５ｎｍ，蛍光波長５２０ｎｍである。 〔分離条件〕 溶離液 Ａ；ジクロロメタン：メタノール：アンモニア水＝９
０：７：０．５ Ｂ；ジクロロメタン：メタノール：アンモニア水＝９
０：２０：０．５ 流速 １．５ｍｌ／ｍｉｎ カラム ＬｉＣｈｒｏｓｐｈｅｒ Ｓｉ６０ １２５ｍｍ×４ｍ
ｍφ（メルク社製） 室温 蛍光検出器 励起波長４６５ｎｍ 蛍光波長５２０ｎｍThe conditions of the detector executed in the present embodiment include an excitation wavelength of 430 to 490 nm and a fluorescence wavelength of 5
It is preferably from 00 to 540 nm, more preferably an excitation wavelength of 465 nm and a fluorescence wavelength of 520 nm. [Separation conditions] Eluent A; dichloromethane: methanol: aqueous ammonia = 9
0: 7: 0.5 B; dichloromethane: methanol: aqueous ammonia = 9
0: 20: 0.5 Flow rate 1.5 ml / min Column LiChrospher Si60 125 mm × 4 m
mφ (Merck) Room temperature Fluorescence detector Excitation wavelength 465 nm Fluorescence wavelength 520 nm
【００１５】以上説明したように本実施態様によれば、
多量のホスファチジン酸及びトリグリセリドを含有する
試料中の微量のアミノ基含有リン脂質類を高感度に検出
して簡便且つ高精度に分析することができるため、例え
ば、ホスファチジン酸中に不純物として存在する微量の
アミノ基含有リン脂質類の含有量を分析することがで
き、もって各種食油の原料となるホスファチジン酸の製
造条件を検討することができ、製造工程における品質管
理を行なうことができ、更に、各種食油の開発研究にも
有効に利用することができる。As described above, according to this embodiment,
Since a very small amount of amino group-containing phospholipids in a sample containing a large amount of phosphatidic acid and triglyceride can be detected with high sensitivity and easily and accurately analyzed, for example, trace amounts of impurities present in phosphatidic acid as impurities Can be analyzed for the content of amino group-containing phospholipids, so that the production conditions of phosphatidic acid, which is a raw material of various edible oils, can be examined, and quality control in the production process can be performed. It can also be used effectively for research on the development of cooking oil.
【００１６】尚、本発明は、上記実施例に何等制限され
るものではない。The present invention is not limited to the above embodiment.
【００１７】[0017]
【発明の効果】本発明によれば、多量のホスファチジン
酸及びトリグリセリドを含有する試料中の微量のアミノ
基含有リン脂質類を高感度に且つ簡便に分析することが
できる。According to the present invention, a trace amount of amino group-containing phospholipids in a sample containing a large amount of phosphatidic acid and triglyceride can be easily analyzed with high sensitivity.
【図１】標準ホスファチジルエタノールアミン、標準リ
ゾホスファチジルエタノールアミン、標準ホスファチジ
ルセリン及びグリシンをＮＢＤ−Ｆによってプレラベル
化した蛍光誘導体それぞれのクロマトグラムと本ＨＰＬ
Ｃで用いられた移動相のグラジエントパターン（破線）
を示す図である。FIG. 1: Chromatograms of standard phosphatidylethanolamine, standard lysophosphatidylethanolamine, standard phosphatidylserine, and fluorescent derivatives obtained by prelabeling glycine with NBD-F and the present HPL
Gradient pattern of the mobile phase used in C (dashed line)
FIG.
【図２】本発明の一実施態様による多量のホスファチジ
ン酸及びトリグリセリドを含有する試料中の微量のアミ
ノ基含有リン脂質類を分析したクロマトグラムを示す図
である。FIG. 2 shows a chromatogram of a trace amount of amino group-containing phospholipids in a sample containing a large amount of phosphatidic acid and triglyceride according to one embodiment of the present invention.
Claims (2)
のアミノ基含有リン脂質を分析する方法であって、多量
のホスファチジン酸及びトリグリセリドを含有する試料
中の微量のアミノ基含有リン脂質類を内部標準物質であ
るグリシンと共に７−フルオロ−４−ニトロベンゾ−２
−オキサ−１，３−ジアゾールによって選択的にラベル
化してそれぞれの蛍光誘導体とし、蛍光誘導体それぞれ
を高速液体クロマトグラフ法で分離検出した後、分離さ
れた蛍光誘導体を内部標準法によってそれぞれ定量する
ことを特徴とする微量のアミノ基含有リン脂質の分析
法。1. A method for analyzing a trace amount of an amino group-containing phospholipid by high performance liquid chromatography, wherein a minute amount of an amino group-containing phospholipid in a sample containing a large amount of phosphatidic acid and triglyceride is used as an internal standard. substance der
7-Fluoro-4-nitrobenzo-2 with glycine
-Selectively labeling with -oxa-1,3-diazole to obtain each fluorescent derivative, separating and detecting each fluorescent derivative by high performance liquid chromatography, and then quantifying the separated fluorescent derivative by an internal standard method A method for analyzing a trace amount of an amino group-containing phospholipid, characterized by the following.
ァチジルエタノールアミン、リゾホスファチジルエタノ
ールアミン、ホスファチジルセリンである、請求項１記
載の微量のアミノ基含有リン脂質の分析法。2. The method according to claim 1, wherein said amino group-containing phospholipids are phosphatidylethanolamine, lysophosphatidylethanolamine, and phosphatidylserine.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP3264104A JP2612652B2 (en) | 1991-10-11 | 1991-10-11 | Analysis of trace amounts of amino group-containing phospholipids |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP3264104A JP2612652B2 (en) | 1991-10-11 | 1991-10-11 | Analysis of trace amounts of amino group-containing phospholipids |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH0599913A JPH0599913A (en) | 1993-04-23 |
| JP2612652B2 true JP2612652B2 (en) | 1997-05-21 |
Family
ID=17398571
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP3264104A Expired - Lifetime JP2612652B2 (en) | 1991-10-11 | 1991-10-11 | Analysis of trace amounts of amino group-containing phospholipids |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP2612652B2 (en) |
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| CN109358134B (en) * | 2018-12-19 | 2021-06-01 | 武汉绿剑可瑞信科技有限公司 | Pretreatment method and quantitative detection method for endogenous phosphosaccharide compounds in plant sample |
-
1991
- 1991-10-11 JP JP3264104A patent/JP2612652B2/en not_active Expired - Lifetime
Non-Patent Citations (2)
| Title |
|---|
| Ａｎａｌ．Ｃｈｉｍ．Ａｃｔａ，２２３（１９８９），Ｐ．２２９−３０８ |
| Ｊ．Ｃｈｒｏｍａｔｏｇ．，４２０（１９８７），Ｐ．１４１−１４５ |
Also Published As
| Publication number | Publication date |
|---|---|
| JPH0599913A (en) | 1993-04-23 |
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