JP2016138841A - Global analysis method of fat - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for analyzing globally an inclusion fat, concerning a sample containing various fats such as a horny layer.SOLUTION: A global analysis method of a fat in a sample includes steps for: subjecting the sample to a reverse-phase liquid chromatography; subjecting the sample subjected to the chromatography to ionization by a multiple ion source inducing both of electrospray ionization and atmospheric pressure chemical ionization; and subjecting the sample subjected to the ionization to mass analysis, to thereby detect ceramide, free fatty acid, cholesterol and cholesterol sulfate.SELECTED DRAWING: None

Description

  The present invention relates to a method for comprehensively analyzing various lipids in a sample.

  The stratum corneum is located on the outermost surface of the epidermis and contributes to the skin barrier function and moisturizing function. Between the stratum corneum cells that make up the stratum corneum, intercellular lipids exist, and the intercellular lipid layer and the water molecule layer form a “lamellar structure” that alternately and regularly overlaps the stratum corneum. Supports the work of the layer. The intercellular lipid in the human stratum corneum is mainly composed of ceramide, cholesterol, free fatty acid, cholesterol sulfate and the like. Ceramide of the human stratum corneum has been reported as a main factor related to the skin barrier function and moisturizing function. Intercellular lipids other than ceramide have also been reported to be associated with skin function.

  In Patent Document 1, the ceramide molecules in the skin stratum corneum of the subject are comprehensively analyzed by liquid chromatography (LC) -mass spectrometry (MS), and the skin quality of the subject is evaluated based on the information obtained by the analysis. A method is disclosed. Patent Document 2 discloses a method for judging skin quality such as atopic dermatitis tendency from information on ceramide composition in a subject obtained by LC-MS method or the like.

  Non-Patent Documents 1 and 2 disclose that ceramide molecules in the skin stratum corneum of a subject were comprehensively analyzed by normal phase liquid chromatography (NPLC) -electrospray ionization mass spectrometry (ESI-MS). Non-patent documents 3 to 4 disclose that the ceramide molecular profile of the human skin stratum corneum obtained by the NPLC-ESI-MS method has a correlation with atopic dermatitis and the state of skin function. Has been. Non-Patent Document 5 discloses that hair ceramide was quantified by a reverse phase liquid chromatography (RPLC) -ESI-MS method.

  Non-Patent Document 6 discloses that stratum corneum sphingolipids such as ceramide were detected by RPLC-atmospheric pressure photoionization mass spectrometry (APPI-MS) method. Non-Patent Document 7 discloses that ceramide in the stratum corneum was comprehensively analyzed by NPLC-atmospheric pressure chemical ionization mass spectrometry (APCI-MS). Non-patent document 8 discloses that the stratum corneum lipids were comprehensively analyzed by combining detection of ceramide and cholesterol by the NPLC-APCI-MS method and detection of free fatty acids by the RPLC-APCI-MS method. Has been.

  In recent years, ion sources that induce ionization by ESI and APCI simultaneously or almost simultaneously (hereinafter also referred to as multi-ion sources) have been developed (for example, Non-Patent Document 9). However, the detection sensitivity when using such a multi-ion source having both ESI and APCI principles is generally inferior to that when each ion source is used exclusively.

  In general, NPLC exhibits separation behavior according to hydrophilic interaction (polar group and basic structure skeleton), so it is not easily affected by impurities, but is not suitable for separation of ionic compounds. Is difficult to apply. On the other hand, RPLC generally exhibits separation behavior according to hydrophobic interaction (hydrophobic group), so it can be applied to the separation of various lipids having hydrophobic groups, but may be affected by contaminant components. Is expensive. For this reason, in RPLC, a complicated pretreatment is usually required to remove contaminant components from the sample.

  Therefore, in the conventional high performance liquid chromatography (LC) -mass spectrometry (MS) method, LC separation by reversed phase or normal phase and MS detection by ESI or APCI are performed depending on the structure or chemical characteristics of the component to be analyzed. Are generally used in appropriate combinations. For example, when analyzing free fatty acids, cholesterol sulfate, cholesterol, and ceramide, which are the main components of stratum corneum lipids, the ionization conditions for LC separation and MS detection are different, and other stratum corneum components and adhesive components for stratum corneum peeling tape Therefore, it is extremely difficult to perform LC-MS analysis on all of them at the same time. In the conventional analysis of stratum corneum lipids, as disclosed in Non-Patent Document 8, LC separation and ionization were performed under different conditions for each analysis target component.

JP 2007-108060 JP 2008-261741 A

Journal of Lipid Research, 2008, 49: 1466-1476. Journal of Lipid Research, 2009, 50: 1708-1718. Journal of Investigative Dermatology, 2010, 130: 2511-2514. Archives of Dermatological Research, 2013, 305: 151-162. Lipids, 2007, 42: 275-290. Journal of Chromatography A, 2006, 1133: 58-68. Journal of Lipid Research, 2011, 52: 1211-1221. Biochimica et Biophysica Acta, 2014, 1841: 70-79. Chromatography, 2006, 27: 81-84.

  The present invention relates to a method for comprehensive analysis of lipids, which can detect lipids contained in a sample containing various lipids such as skin and hair, for example, ceramide, free fatty acid, cholesterol, cholesterol sulfate, etc. at a time.

  As a result of various investigations, the present inventors have separated samples containing lipids together by reverse phase liquid chromatography, and then applied them to a mass spectrometer equipped with a multi-ion source that induces ionization by ESI and APCI. It has been found that various lipids conventionally detected by separate LC-MS analysis such as ceramide, free fatty acid, cholesterol and cholesterol sulfate in the sample can be detected at a time.

That is, the present invention is a comprehensive analysis method for lipids in a sample,
Subjecting the sample to reverse phase liquid chromatography;
Subjecting the chromatographed sample to ionization with a multi-ion source that induces both electrospray ionization and atmospheric pressure chemical ionization; and
Subjecting the sample subjected to ionization to mass spectrometry to detect ceramide, free fatty acid, cholesterol and cholesterol sulfate;
A method comprising:

  According to the present invention, it is possible to comprehensively analyze lipids in a sample containing various lipids by a single LC-MS analysis. In particular, according to the present invention, lipids such as ceramide, free fatty acids, cholesterol and cholesterol sulfate, which have conventionally been detected by separate LC-MS analysis, can be analyzed in a single LC-MS analysis. Can be detected together. The method of the present invention is suitable for skin and hair lipid analysis. The analysis result obtained by the method of the present invention can be applied to the evaluation of the condition of skin quality and hair quality.

The block diagram which shows roughly an example of the comprehensive analysis system of the lipid based on the comprehensive analysis method of the lipid of this invention. Multistage extracted ion chromatogram of stratum corneum sample. Aa: negative ion mode detection of forearm sample, Ab: positive ion mode detection of forearm sample, Ba: negative ion mode detection of cheek sample, Bb: positive ion mode detection of cheek sample. CERs: Ceramide, FFAs: Free fatty acid, ChSO4: Cholesterol sulfate, COL: Cholesterol.

  The present invention provides a comprehensive analysis method for lipids in a sample. The sample applied to the method of the present invention is not particularly limited as long as it may contain a lipid targeted for analysis. Preferred examples of the sample include skin, skin stratum corneum, hair, organ, blood, body fluid, and cells and tissues reconstituted from them collected from living organisms and corpses of humans and animals. Examples include skin stratum corneum and hair removed, scraped or peeled from a living body, and more preferably skin stratum corneum collected by a tape stripping method. The adhesive component of the stratum corneum peeling tape used for the tape stripping method is not particularly limited, but is preferably an acrylic polymer.

  The sample is subjected to solvent extraction prior to chromatographic separation and prepared into a solvent extract containing lipids. As the solvent for solvent extraction, a solvent that has high solubility of the target lipid and preferably does not dissolve the tape used for tape stripping or its adhesive component is used. Examples of preferred solvents include methanol, ethanol, isopropanol and the like. Furthermore, if the lipid analysis may be affected by contaminant components, a pretreatment for removing contaminants may be applied to the sample. Alternatively, as a method for preparing a solvent extract containing lipid from the sample, a known method for separating and purifying a lipid component from a biological sample, such as the Bligh and Dyer method or the Folch method, can be applied. However, in the method of the present invention, the above pretreatment and complicated separation and purification procedures are not necessary, and sufficiently accurate data can be obtained even if the solvent extract is directly subjected to reverse phase liquid chromatography.

  In the comprehensive analysis method for lipids of the present invention, the sample prepared by the above procedure is chromatographed. More specifically, the solvent extract prepared by the above procedure is subjected to reverse phase liquid chromatography (RPLC). In the method of the present invention, unlike the conventional analysis method, it is not necessary to divide a sample for each target lipid, or to subject them separately to NPLC and RPLC, and all the samples may be chromatographed under the same conditions.

  Examples of the filler for RPLC include a filler in which a hydrocarbon chain is bonded to silica gel or a polymer gel base material. Preferably, a filler (also referred to as ODS or C18 filler) in which an octadecyl group is bonded to a silica gel base material is used.

  The chromatographic eluent is preferably one that can hold the target lipid appropriately and can be separated into molecular types and does not contain a high concentration of non-volatile acids and salts. For example, the eluent a is a methanol / water mixture, the eluent b is isopropanol, and a gradient of 100: 0 to 0: 100 (volume ratio) can be applied between the eluents a and b.

Next, the sample subjected to the chromatography is subjected to ionization for mass spectrometry. Preferably, the ionization is performed in the presence of an ionization promoter. As the ionization accelerator, [M + H] ⁺ , [M + H−H ₂ O] ^{+ and the} like are detected with high sensitivity in the positive ion mode of mass spectrometry, and [M−H] ⁻ , [M + HCOO] ⁻ , [ M + CH ₃ COO] ^{− and the} like are substances that improve the ionization efficiency of the target lipid during the ionization so that they can be detected with high sensitivity. Examples of the ionization accelerator include at least one selected from the group consisting of formic acid, acetic acid, and salts thereof (for example, ammonium formate, ammonium acetate). The ionization accelerator may be added to the eluent obtained by the above chromatographic separation, or may be added to the above-described chromatographic eluent.

  In the method of the present invention, the sample subjected to the chromatography is subjected to ionization of both electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) at a time. Thereby, the target lipid molecule in the sample can be ionized regardless of its polarity or structure specificity. In order to subject a sample to both ESI and APCI at once, the sample may be subjected to ionization by an ion source that induces both ESI and APCI simultaneously or nearly simultaneously (ie, a multi-ion source). For example, a sample may be introduced into a mass spectrometer equipped with the multi ion source. In this specification, “an ion source that induces ESI and APCI simultaneously or substantially simultaneously” or “multi-ion source” means that a composite ionization method that simultaneously performs ESI and APCI can be performed, or ESI and APCI are respectively The ion source can ionize the sample so that the ions generated by ESI and APCI can be detected simultaneously in one mass analysis by switching the single ionization method without dead time. Means. Such a multi-ion source is called a multi-mode ion source, a dual ion source, an ESCi multi-mode ion source, a duo spray ion source, a hybrid ion source, or the like. Agilent Technologies, Shimadzu Corporation, Waters Corporation, AB Sciex, Inc. It is sold or disclosed by Hitachi High-Technologies Corporation (for example, US Pat. No. 6,646,257, International Publication No. 2003/102537, International Publication No. 2007/032088, International Publication No. 2014/084015).

  Lipid molecules in the sample subjected to both ESI and APCI in the above procedure and subjected to ionization are subjected to mass spectrometry. For mass spectrometry, a quadrupole mass spectrometer (Q-MS), a time-of-flight mass spectrometer (TOF-MS), an ion trap mass spectrometer (IT-MS), a Fourier transform mass spectrometer (FT) -MS) and other known masses such as single-type mass spectrometers, hybrid mass spectrometers such as Q-TOF and IT-TOF, and tandem mass spectrometers such as triple quadrupole type (MS / MS, etc.) An analytical device can be used. Detection of ionized lipid molecules in mass spectrometry may be performed in either the positive ion mode or the negative ion mode, and the positive or negative ion mode may be selected depending on the target lipid molecular species. For example, it is preferable to detect free fatty acid and cholesterol sulfate in negative ion mode and cholesterol in positive ion mode. Ceramide can be detected in either positive or negative ion mode, but detection in negative ion mode is preferred in order to avoid the influence of impurities.

  In the case of a molecule that can be detected in either positive or negative ion mode, such as ceramide, a positive ion mode that is superior in terms of sensitivity is generally used. However, in order to avoid the influence of impurities, detection in the negative ion mode is desirable. In order to detect with high sensitivity in the negative ion mode, it is desirable to improve the ionization efficiency of the target molecule in the negative ion mode by using the ionization accelerator described above.

  Lipids in the sample are comprehensively detected by ionization and mass spectrometry using ESI and APCI. More specifically, according to the method of the present invention, both polar and nonpolar lipids in a sample can be detected by a single mass analysis. Preferable examples of target lipids for the method for comprehensive analysis of lipids of the present invention include intercellular lipids in the stratum corneum and endogenous lipids in hair. More preferably, the target lipid can be at least ceramide, free fatty acid, cholesterol and cholesterol sulfate.

  The ionized lipid molecules subjected to mass spectrometry are separated and detected by mass number / charge (m / z). Although the detected molecular species can be identified based on m / z, it is preferably identified based on the technique disclosed in Patent Document 1. In summary, this technique is as follows: First, a lipid-containing sample is chromatographically separated, ionized and detected by the mass spectrometer, and then the chromatographic peak is obtained from the data output from the mass spectrometer. Multi-stage extraction ion chromatograms developed in three axes based on the retention time, m / z and ion intensity measured by mass spectrometry. Next, by searching a database for the retention time and m / z of the target lipid peak prepared in advance, the lipid molecular species that form each peak on the multistage extracted ion chromatogram are identified.

  When quantitatively analyzing the identified lipid, the peak area on the extracted ion chromatogram of each lipid may be measured and compared. For more accurate quantitative analysis, it is preferable to calibrate the retention time of the peak in chromatography and the data of m / z or ionic strength measured by mass spectrometry using an internal standard. For example, each lipid can be quantified by adding an internal standard substance having a known concentration to the solvent extract of the sample described above and determining the ratio of the peak area of each lipid to the peak area of the internal standard substance. Examples of internal standard substances include N-Heptadecanyl-D-erythro-Sphingosine (C17: 0-Ceramide) for ceramide, Arachidic Acid-d39 for free fatty acid, Chholesterol-d7 for cholesterol, and Sodium eichoyl for cholesterol sulfate. A sulfate can be mentioned.

  If more precise quantification is required, an absolute calibration curve for each lipid molecular species can be generated. Based on the calibration curve, the absolute amount of the corresponding lipid molecular species can be calculated. For example, when ceramide is quantified, prepare standard products of all ceramide molecules, create their absolute calibration curve, and calculate the absolute amount of various ceramide molecules contained in the sample based on the absolute calibration curve Can do.

  Furthermore, the amount of lipid quantified is divided by the amount of sample collected, for example, the area or weight of the skin, stratum corneum or hair, the number of cells, the amount of protein, etc. The amount of each lipid to be calculated can be calculated.

  Information on the lipid composition (type, amount, etc.) in a sample obtained by the comprehensive analysis method for lipids according to the present invention can be used to evaluate the state of the sample and the organism from which it is derived. For example, when the sample is human skin or hair, the human skin quality or hair quality can be evaluated from the lipid composition in the sample. As a more detailed example, the procedure of skin quality evaluation will be described below. First, a lipid composition (type and amount) such as ceramide, free fatty acid, cholesterol, cholesterol sulfate in the stratum corneum of skin is obtained from a human population by the method of the present invention, and the lipid composition and their skin quality (for example, Normal skin, dry skin, oily skin, skin with poor moisture retention, skin with poor barrier function, skin with acne, skin with scaling, skin with erythema, psoriasis skin, atopic dermatitis Create association data with patient skin quality, etc.). Subsequently, the lipid composition (type and amount) of ceramide, free fatty acid, cholesterol, cholesterol sulfate and the like in the stratum corneum of the skin of the subject is obtained by the method of the present invention. Based on the association data between the lipid composition and the skin quality obtained from the human population, the skin quality can be evaluated from the lipid composition obtained from the subject. Preferably, in addition to the lipid composition obtained by the method of the present invention, other skin properties (transdermal moisture transpiration, moisture content, appearance, etc.) are also referred to as necessary. This makes it possible to more accurately evaluate the skin quality. In the same procedure, it is possible to evaluate the hair quality (for example, the degree of damage caused by chemical treatment).

  Hereinafter, an embodiment of a method for comprehensive analysis of lipids according to the present invention will be described with reference to the drawings.

  FIG. 1 is a block diagram schematically showing an example of an analysis system applied to the method for comprehensive analysis of lipids of the present invention. The system 1 includes a liquid chromatograph 10, a mass spectrometer 20, and a calculation device 30.

  The liquid chromatograph 10 includes gradient pumps 11a and 11b for feeding eluents a and b, an autoinjector 12 for introducing a sample solution c, and a separation column 13. As the sample solution c, the above-described sample solvent extract is preferably used. The eluents a and b are as described above, and the pumps 11a and 11b adjust their concentration gradients. The separation column 13 is packed with the above-described chromatography filler.

  With the liquid chromatograph 10, the target lipid in the sample solution c can be separated. The target lipid separated by the liquid chromatograph 10 is introduced into the subsequent mass spectrometer 20.

  The mass spectrometer 20 includes an ionizer 21 and a mass separation detector 22. The target lipid separated by the liquid chromatograph 10 is introduced into the ionization device 21, where lipid molecules in the mixed solution are ionized.

  The ionizer 21 is a multi-ion source (so-called multi-mode ion source, dual ion source, ESCi multi-mode ion source, duo spray ion source, hybrid ion source, etc.) that induces both ESI and APCI simultaneously or almost simultaneously. Prepare. In the ionizer 21, the lipid in the mixed solution is subjected to ionization of both ESI and APCI at a time and ionized.

  The mass separation detection device 22 separates and detects ions generated by the ionization device 21 for each m / z. The mass separation detection device 22 may be the above-described single-type, hybrid-type, or tandem-type mass spectrometer.

  As said liquid chromatograph 10 and mass spectrometer 20, the commercially available liquid chromatography-mass spectrometer which united these can be used.

  The computing device 30 includes computing means for creating an extracted ion chromatogram based on output data from the mass spectrometer 20. Preferably, the computing device 30 creates a multistage extracted ion chromatogram by developing the retention time of the peak in the liquid chromatograph 10 and the m / z and ion intensity detected by the mass spectrometer 20 on three axes. Have means. Preferably, the arithmetic unit 30 can access a database (not shown in FIG. 1) relating to the retention time and m / z of each target lipid molecular species, and the multistage extracted ions created by the arithmetic means. A comparison calculation means for identifying the lipid molecular species forming each peak on the chromatogram based on the database is provided. Furthermore, it is preferable that the calculation device 30 includes a display unit that outputs and displays the extracted ion chromatogram created by the calculation unit and / or the lipid molecular species identified by the comparison calculation unit in a desired format.

  For example, the calculation device 30 is installed with software for executing calculations by the calculation means and / or the comparison calculation means, and also stores a memory for storing output data and calculation results from the mass spectrometer 20, and calculation results. It is a computer provided with the display which displays. The database may be always stored in the computing device 30, but is usually stored in a storage medium or hard disk outside the computing device, and may be called up via a network as necessary.

  As exemplary embodiments of the present invention, the following compositions, production methods, uses or methods are further disclosed herein. However, the present invention is not limited to these embodiments.

<1> A comprehensive analysis method for lipids in a sample,
Subjecting the sample to reverse phase liquid chromatography;
Subjecting the chromatographed sample to ionization with a multi-ion source that induces both electrospray ionization and atmospheric pressure chemical ionization; and
Subjecting the sample subjected to ionization to mass spectrometry to detect ceramide, free fatty acid, cholesterol and cholesterol sulfate;
Including a method.

<2> The method according to <1>, wherein the sample is preferably a skin stratum corneum or hair cut, scraped or peeled from a living body, and more preferably a skin stratum corneum obtained by a tape stripping method.

<3> The method according to <2>, wherein the adhesive component of the stratum corneum peeling tape used in the tape stripping method is preferably an acrylic polymer.

<4> The method according to any one of <1> to <3>, wherein the sample is preferably subjected to solvent extraction before being subjected to the chromatography, and prepared into a solvent extract containing lipids. .

  <5> The method according to <4>, wherein the solvent is preferably methanol, ethanol, or isopropanol.

<6> Preferably, the ionization is performed in the presence of at least one ionization accelerator selected from the group consisting of acetic acid, ammonium acetate, formic acid and ammonium formate, and any one of <1> to <5> The method described in the paragraph.

<7> The method according to <6>, wherein the ionization accelerator is preferably added to the eluent of the chromatography.

<8> Preferably, the ionization is performed by a mass spectrometer equipped with a multi-ion source that induces ionization by ESI and APCI simultaneously or substantially simultaneously, <1> to <7> Method.

<9> Preferably, the detection by mass spectrometry is performed in positive ion mode and negative ion mode, more preferably, cholesterol is detected in positive ion mode, and free fatty acid, cholesterol sulfate and ceramide are detected in negative ion mode. The method according to any one of <1> to <8>.

<10> The method according to any one of <1> to <9>, wherein the intercellular lipid in the stratum corneum is preferably detected by the mass spectrometry.

  EXAMPLES Hereinafter, an Example is shown and this invention is demonstrated more concretely.

(1) Extraction of skin stratum corneum and lipid extraction Film masking tape (Teraoka Seisakusho / 465 # 40, 2.5 cm width × 3 cm length) was pressed for 10 seconds on the forearm and cheek of a healthy male. The skin stratum corneum was sampled by repeating this operation three times continuously at the same location (collected area = 7.5 cm ² × 3).

(2) Preparation of sample solution The tape from which the stratum corneum was collected was immersed in 1.9 mL of methanol solution in a 5 mL screw tube (trade name: Maruemu / No. 2), and sonicated at room temperature for 10 minutes to extract lipids. did. Next, an internal standard substance mixed solution [C17: 0-Ceramide (Avanti Polar Lipids, Inc.) as an internal standard substance of the ceramide group, and Aracidic Acid-d39 (Cambridge Island Laboratories as an internal standard substance of the free fatty acid group) was added to the screw tube. , Inc.), Cholesterol-d7 (Avanti Polar Lipids, Inc.) as an internal standard substance of cholesterol, and 100 μL of a mixed solution of Sodium eosysulfate (Sigma-Aldrich) as an internal standard substance of cholesterol sulfate] Was prepared.

(3) LC-MS analysis The sample solution prepared in (2) was subjected to LC-MS analysis under the following conditions, and various lipids (ceramide, free fatty acid, cholesterol and cholesterol sulfate) were detected and quantified.
(apparatus)
LC / 1100 series, mass spectrometer / G1956B single quadrupole (Agilent Technologies)
(Chromatographic separation)
Separation column: L-column ODS 2.1 mm inner diameter × 150 mm, particle size 5 μm (40 ° C.)
Eluent a: 5 mmol / L acetic acid · 10 mmol / L ammonium acetate-containing methanol / water = 1/1 (v / v)
Eluent b: 2-propanol containing 5 mmol / L acetic acid and 10 mmol / L ammonium acetate Gradient: Eluent b 20% (0 min) → 20% (1 min) → 60% (2 min) → 100% (30 min) → 100% ( 35 min)
Mobile phase flow rate: 0.2 mL / min
Equilibration time: 10 min
Injection volume: 20 μL
(Mass spectrometry)
Ionization method: Multi-mode ion source (ESI / APCI)
Polarity: Positive ion and negative ion Charging voltage: 2000V
Capillary voltage: 2000V (positive ion), 4000V (negative ion)
Vaporizer temperature: 200 ° C
Nebulizer pressure: 60 psig
Drying gas temperature: 350 ° C
Dry gas flow rate: 4L / min
(Detection mode)
Ceramide: SIM detection of molecules ([M + CH ₃ COO] ⁻ ) to which acetate ions have been added in negative ion mode Free fatty acid: SIM detection of deprotonated molecules ([M−H] ⁻ ) in negative ion mode Cholesterol sulfate: metal Ion or proton ion desorbed molecule (eg [M-Na] ⁻ ) in negative ion mode with SIM detection Cholesterol: protonated and dehydrated molecule ([M + H—H ₂ O] ⁺ ) in positive ion mode with SIM detection

(4) Data analysis The obtained data was developed into a multistage extracted ion chromatogram having three axes of retention time, m / z, and ionic strength. As shown in the multistage extracted ion chromatogram of FIG. 2, ceramide, free fatty acid, cholesterol and cholesterol sulfate were detected by the analysis method of the present invention.

DESCRIPTION OF SYMBOLS 1 Analysis system 10 Liquid chromatograph 11a, 11b Gradient pump 12 Autoinjector 13 Separation column 20 Mass spectrometer 21 Ionizer 22 Mass separation detector 30 Arithmetic device a, b Eluent c Sample solution

Claims (5)

A comprehensive analysis method of lipids in a sample,
Subjecting the sample to reverse phase liquid chromatography;
Subjecting the chromatographed sample to ionization with a multi-ion source that induces both electrospray ionization and atmospheric pressure chemical ionization; and
Subjecting the sample subjected to ionization to mass spectrometry to detect ceramide, free fatty acid, cholesterol and cholesterol sulfate;
Including a method.   The method according to claim 1, wherein the detection of ceramide by mass spectrometry is performed in a negative ion mode.   The method according to claim 2, wherein the detection of free fatty acid and cholesterol sulfate by mass spectrometry is performed in negative ion mode, and the detection of cholesterol is performed in positive ion mode.   The method according to claim 1, wherein the sample is a solvent extract of a skin stratum corneum obtained by a tape stripping method.   The method according to claim 1, wherein the ionization is performed in the presence of at least one ionization accelerator selected from the group consisting of acetic acid, ammonium acetate, formic acid, and ammonium formate.