JP2003028849A - Simultaneous autoanalyzer for lipid compound - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an autoanalyzer by which the chemical structure of a molecular species belonging to the main molecular group of a lipid is identified by a method wherein the main molecular group is separated by high-performance liquid chromatography so as to be introduced on-line into an electrospray ionization mass spectrometer. SOLUTION: A solvent which promotes the ionization of a neutral lipid is fed by a pump (4) in a holding time zone in which the neutral lipid is eluted, it is mixed with an eluant from a separation column via a tee (12), and the ionization efficiency of the neutral lipid is increased so as to be capable of being detected by the mass spectrometer.

Description

Description: BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a lipid group extracted from a body fluid or a tissue of any living organism by a single high-performance liquid chromatography in a single molecule, ie, a neutral lipid (glyceride). , Fatty acids and their oxidized derivatives, ceramides), glycolipids, and phospholipids. An automated device that separates the molecules and introduces them into an electrospray ionization mass spectrometer online to identify the chemical structure of the molecular species belonging to the molecules. About. 2. Description of the Related Art Crude extracted lipids are injected into a high performance liquid chromatograph, and major separated lipid molecules are introduced on-line into an electrospray ionization mass spectrometer to analyze the molecular species belonging to these molecules. Identifying the chemical structure
Although possible in principle, an automated and practical device has not yet been proposed. Heretofore, there has been reported in the literature a measurement method in which the analysis target is limited to phospholipids or ceramides. [0003] Normal phase chromatography is effective for separating major lipid molecular groups into individual molecular groups. Neutral lipids having low polarity are eluted first under low-polarity solvent conditions, and phospholipids having a phosphate group are eluted later under solvent conditions having higher polarity. In order to ionize molecules by the electrospray ionization method, it is necessary that the solvent has a certain degree of polarity, and the ionization efficiency is extremely poor under low-polarity solvent conditions where neutral lipids elute. There was a point. According to the present invention, a major group of lipid molecules is baseline-separated by normal phase chromatography, all the separated lipid groups are ionized with high efficiency by an electrospray ionization method, and are separated into the molecular groups by a mass spectrometer. An object of the present invention is to provide an automated lipid composition analyzer for identifying the chemical structure of a molecular species to which it belongs. [0005] In order to achieve the above object, in the automated lipid composition analyzer of the present invention, ionization is carried out by a pump (4) during a retention time zone in which neutral lipids elute. The enhancing solvent is pumped and mixed with the eluent from the separation column via the tee (12) to increase the ionization efficiency of the neutral lipid and make it detectable by the mass spectrometer. DESCRIPTION OF THE PREFERRED EMBODIMENTS Selection of column and eluent: Normal phase chromatography using a column packed with silica gel is effective as a method for separating major lipid molecules into individual molecules. However, the solvent conditions vary greatly depending on the method for producing silica gel. In the practice of the present invention, a stable supply of LiChrompher
e (R) Si100 (5 μm particle size) is 1 × 150 mm stainless steel tube or 0.1 × 200 mm PEEKsi
The tube was packed and used, but the size of the column is not limited to this. For higher sensitivity, a capillary column having an inner diameter of 0.1 mm or less is desirable.
For the column, stainless steel, fused silica capillary, PEEK resin, PEEKsil (fused silica capillary whose outside is reinforced with PEEK resin) tube, or the like can be used. In addition, silica gel, D
Chemically bonded silica gels such as iol, CN, NH _{2 and the} like are also used. The particle size of the gel is desirably as small as possible due to the back pressure of the column in order to improve the resolving power.
A μm particle size is appropriate. In normal phase chromatography, a substance adsorbed on a column equilibrated with a low-polarity solvent is generally eluted by increasing the polarity of the solvent. Many low-polarity solvents used in these applications have high toxicity, such as chloroform, but in the practice of the present invention, low-toxicity hexane is used as a basic solvent to adjust the polarity, and low-toxicity solvents are used. Isopropanol, ethanol and water were added. However, a solvent such as chloroform can be used if a function of protecting other low-toxic solvents and toxic solvents is added to the analyzer. It is desirable to add a volatile salt such as ammonium formate to the solvent in order to improve the peak shape and recovery rate of eluted lipids, especially acidic lipids. In order to increase the ionization efficiency in the electrospray ionization method, it is necessary to add these salts. The specific solvent composition used in Examples 1 and 2 is as follows. Solvent a: hexane / isopropanol / formic acid; 100:
0.5: 0.1 (v / v / v) Solvent b: hexane / isopropanol / ethanol /
0.1 M ammonium formate methanol solution; 40: 5
0: 10: 1 (v / v / v / v) Solvent c: hexane / isopropanol / water / 1M aqueous ammonium formate; 40: 60: 9.76: 2.24
(V / v / v / v) Selection of the solvent to be mixed at the outlet of the column in order to increase the ionization efficiency of neutral lipids in the electrospray ionization method: The following factors must be considered.
To obtain good ionization by electrospray, it is necessary to add a highly polar solvent such as alcohol. In addition, glyceride is cationized as an ammonia adduct, and ceramide is efficiently ionized in the presence of ammonium formate or ammonium acetate having a final concentration of about 5 mM. Therefore, it is preferable to add these volatile salts. Isopropanol containing a final concentration of 10 mM ammonium formate as a solvent that satisfies these factors and further satisfies the condition that the eluted hexane is well mixed with the main solvent and can maintain the solubility of the eluted lipid and volatile salt /methanol;
1: 1 (v / v) is preferred. However, ethanol may be used instead of methanol. When the final concentration of ammonium formate exceeds 20 mM, the ionization efficiency does not change much but the S / N ratio decreases. In consideration of maintaining the solubility of the eluted lipid and the volatile salt, this ionizing solvent was fed from the pump (4) at a flow rate of の of the flow rate of the chromatography and mixed with the eluate by the tee (12). The pump (4) was controlled from the controller (15) so that the solution was fed only during the retention time period when the neutral lipid was eluted. Embodiment 1 An embodiment will be described with reference to FIG. Lipid standard samples (triolein, diolein, ceramide, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, phosphatidylcholine,
(Sphingomyelin) Each 10 pmol mixture was injected from an autosampler (6), and at the same time, a start signal was sent to a liquid chromatograph controller (15) and a mass analyzer (14) to start analysis. Immediately after that, the pump (4) was started to be driven from the controller (15), and the ionizing solvent (d) was fed at a flow rate of 0.025 ml / min for 40 minutes. Column is 1x100mm LiChrospher
Flow rate of 0.05 ml / min using e (R) Si100
It is. First, the valves (8) and (9) are set to flow paths indicated by solid lines, and the lipid is retained in the trap column (10) equilibrated with the solvent a and the flow is discarded. After one minute, valve (9)
Was switched to a flow path indicated by a broken line so that lipids could be sent from the trap column (10) to the separation column (11). Triolein and diolein, which have low retention, are sent to the separation column to be separated, and the other standard lipids are stored in the trap column (10).
Was adsorbed. After 10 minutes, the valve (8) is switched to the flow path indicated by the broken line to reverse the flow direction in the trap column (10), and the lipid adsorbed on the column end is directly separated from the column (1).
Introduced in 1). Thereby, an improvement in column separation ability can be obtained. A lipid standard sample was separated using the following solvent gradient. The solvent gradient is not limited to this, and further optimization is preferable when analyzing a multi-component complex sample. After the completion of one gradient, the column was equilibrated with the solvent (a) for 20 minutes, and immediately thereafter, the valves (8) and (9) were switched to the initial state (solid line flow path) to prepare for the next analysis. In this embodiment, an ion trap type mass spectrometer (14) equipped with an electrospray ion source (13) is used. However, other quadrupole triple stage type and quadrupole / time-of-flight mixing are used. Type, Fourier transform ion cyclotron type, etc. mass spectrometer can also be used. At present, an apparatus capable of measuring a tandem mass spectrum (MS / MS) is desirable. Ion trap mass spectrometer (1
4) is easy to set MS / MS measurement conditions and is advantageous for online measurement of MS / MS spectra of multicomponent lipids. However, if a database of the retention time of chromatography under certain conditions and the mass spectrum of eluted lipids accumulates, it will be possible to respond by confirming only unknown substances by the MS / MS method. . Therefore, it is presumed that the MS / MS function is not necessarily required and an inexpensive system can be constructed. In this example, the neutral lipid region (the first 40
Min), only cations were detected, and then for 40 minutes (phospholipid region), cations and anions were detected alternately. In addition, a positive ion MS / MS spectrum was measured for the largest ion peak depending on the data taken in. As can be seen from the cation chromatogram in FIG. 2, by mixing the solvent for ionization from the pump (4) through the tea (12), the triolein, diolein, and ceramide are almost equal to or higher than the phospholipid. It is efficiently cationized and the injected total lipids are well separated.
In anion detection, monovalent anions in which one H ⁺ is dissociated from phosphatidylethanolamine, phosphatidylinositol, and phosphatidylserine are abundantly detected. In addition, phosphatidylcholine and sphingomyelin, which tend to generate monovalent cations easily, were rich in formate anion-added monovalent anions. Detecting both ions in a single analysis advantageously increases the amount of information for lipid identification. In this embodiment, a column having a diameter of 1 mm is used. However, a column having an inner diameter of 0.1 mm or less can be used to increase the sensitivity. For this purpose, a splitter must be inserted between the mixer (5) and the autosampler (6) to reduce the flow rate to 1 μl / min or less, and to convert the pipe downstream from the splitter into a PEEKsil pipe having a diameter of about 50 μm. Example 2 In the stratum corneum of the skin, there are ceramides having a complex composition characteristic of the stratum corneum. To demonstrate that neutral lipids can also be efficiently analyzed by the present invention, lipids extracted from mouse stratum corneum were used to focus on the ceramide region and analyzed in the same manner as in Example 1. The corneal lipid is hexane /
Extracted with isopropanol (3: 2 v / v), 6.67
It was used after washing with an aqueous sodium sulfate solution. FIG. 3 shows a cation chromatogram of the ceramide region. The mass spectra of peaks (1) and (2) are shown in FIGS. 5 and 4, respectively. Peak (1) is m / z = 1066.6 in FIG.
From the MS / MS spectrum of the C.I. ion, ceramide 1 having an omega hydroxy fatty acid moiety of C34: 1 as a main component, and the C / A fatty acid moiety of C26: 0 from the MS / MS spectrum of the m / z = 678.3 ion in FIG. It was found to be ceramide 2 as the main component.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 Automated lipid composition simultaneous analyzer [Description of symbols] 1 pump 2 pump 3 pump 4 ionizing solvent pump 5 mixer 6 autosampler 7 manual injector 8 four-way switching valve 9 four-way switching valve Reference Signs List 10 trap column 11 separation column 12 tea 13 electrospray ion source 14 mass spectrometer ad solvent bottle [FIG. 2] cation chromatogram of standard lipid TG, triolein; DG, diolein; PE, phosphatidylethanolamine; PI, Phosphatidylinositol; PS, phosphatidylserine; PC, phosphatidylcholine; SM, sphingomyelin. [FIG. 3] Total ion chromatogram of mouse horny layer ceramide region. De 1 Mass spectrum of 2 Ceramide 2 [4] [3] (2) Mass spectrum [5] [3] (1)

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[Procedure amendment] [Submission date] October 26, 2001 (2001.10.
26) [Procedure amendment 1] [Document name to be amended] Description [Item name to be amended] Brief explanation of drawings [Amendment method] Change [Content of amendment] [Brief explanation of drawings] [Fig. 1] Simultaneous automated lipid composition FIG. 2 is a plan view showing the outline of an analyzer. FIG. 2 is a cation chromatogram of a lipid standard substance measured using the apparatus shown in FIG. 1. FIG. 3 is a diagram showing a lipid extracted from the stratum corneum of a mouse skin measured using the apparatus shown in FIG. Total ion chromatogram of the ceramide region at the time of [Figure 4] Mass spectrum of the peak of Fig. 3 (2) [Fig. 5] Mass spectrum of the peak of Fig. 3 (1) [Explanation of symbols] [Fig. Solvent feed pump 4 Ionization promoting solvent feed pump 5 Mixer 6 Autosampler 7 Manual injector 8 Four-way switching valve 9 Four-way switching valve 10 Trap column 11 Separation column 12 Tee 13 Electro Spray ion source 14 Mass spectrometer 15 Controller ad Solvent bottle [FIG. 2] TG triolein DG diolein PE phosphatidylethanolamine PI phosphatidylinositol PS phosphatidylserine PC phosphatidylcholine SM Sphingomyelin The number attached to each peak indicates the retention time. . FIG. 3 1 Elution peak 2 of ceramide 1 Elution peak of ceramide 2 The number attached to each peak represents the retention time. FIG. 4 shows the m / z value of the number attached to each peak. FIG. 5: Numbers attached to each peak represent m / z values.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) G01N 30/72 G01N 30/72 G

Claims (1)

Claims: 1. A method for analyzing lipids, comprising separating lipids by normal phase column chromatography. 2. A method of mixing the neutral lipid separated by the method of claim 1 with a solvent mixed at an outlet of a column in order to efficiently ionize the neutral lipid by an electrospray ionization method. 3. A method for analyzing lipids, comprising analyzing the structure of lipids ionized by electrospray ionization using a mass spectrometer. 4. A method for analyzing lipids, comprising separating lipids after adsorbing them on a trap column. 5. An automatic lipid analysis method using the analysis method according to claim 1, 3 or 4. 6. An automatic lipid analyzer using the analysis method according to claim 1, 3 or 4. 7. An analysis method wherein the solvent used in the analysis method according to claim 1, 3, or 4 comprises only a low-toxicity solvent. 8. An analysis method according to claim 1, wherein the solvent used in the analysis method according to claim 1, 2 or 4 is at least three or more.