Method for detecting Cu-pyropheophytin a in oil samples

Abstract

The present invention relates to a method for rapidly, efficiently, and accurately detecting Cu-pyropheophytin a in an oil sample, comprising: separating the components of an oil sample using a supercritical fluid chromatograph; removing non-polar components in the oil sample so that Cu-pyropheophytin a remains in the column of the supercritical fluid chromatograph; introducing the components containing Cu-pyropheophytin a eluted from the column of the supercritical fluid chromatograph into a reversed-phase liquid chromatograph; separating Cu-pyropheophytin a using the reversed-phase liquid chromatograph and detecting it with a mass spectrometer, wherein the mobile phase in the supercritical fluid chromatograph comprises a supercritical fluid and a modifying agent, the modifying agent adjusts the polarity of the supercritical fluid, and the scan mode of the mass spectrometer is multiple reaction monitoring (MRM) mode.

Description

Technical Field

[0001] The present invention belongs to the field of detection of food additives, and specifically relates to a method for detecting Cu-pyropheophytin a in an oil sample, a method for determining whether copper chlorophyll is contained in an oil sample such as olive oil using the method, and a method for identifying adulterated olive oil.

Background Art

[0002] The main physiological function of oil is to store and supply energy. The energy supplied by metabolism is about twice that of energy from sugar and protein, and it is one of the important energy sources for the human body. Olive oil is a kind of edible vegetable oil extracted from olive fruits, rich in monounsaturated fatty acids such as oleic acid, and polyunsaturated fatty acids such as linoleic acid and linolenic acid, and the ratio is exactly the ratio required by the human body. Unsaturated fatty acids can adjust the ratio of high-density lipoprotein cholesterol and low-density lipoprotein cholesterol in human plasma and prevent excessive cholesterol and cardiovascular diseases in the body. Therefore, olive oil is considered to be the most suitable oil for human nutrition among the oils discovered so far. The International Olive Oil Council classifies olive oil into two categories: Virgin Olive Oil and Refined Olive Oil. Virgin olive oil contains chlorophyll, chlorophyll derivatives, and lutein, so it shows a yellow-green color. Therefore, consumers often judge the authenticity and quality of olive oil by its color.

[0003] Copper chlorophyll (CNS number 08.009) is a lipid-soluble copper chlorophyll derivative in which the magnesium ion in the metal center of chlorophyll is replaced with a copper ion. Regarding widely used food colorings, China's national standard GB2760-2014 "National Standard for Food Safety - Standards for the Use of Food Additives" specifies that copper chlorophyll can be used as a coloring agent in creams, candies, and bakery products, with a maximum usage amount to be appropriate according to production needs. Copper chlorophyllin sodium and copper chlorophyllin potassium (CNS number 08.009) can be used in foods such as frozen drinks and canned vegetables, with a maximum usage amount of 0.5 g / kg, but it does not specify that they can be used in edible oils. However, in reality, unscrupulous merchants add copper chlorophyll to edible oils to disguise them as olive oil, or add copper chlorophyll to refined olive oil to disguise it as virgin olive oil, in order to make huge profits. The amount of copper chlorophyll added to counterfeit olive oil ranges from 0.09 to 0.16 mg / L. Therefore, there is a growing demand for detecting copper chlorophyll in olive oil to identify and eliminate counterfeit and substandard products, protect consumer rights, and ensure food safety.

[0004] China's national standard GB5009.260-2016, "National Standard for Food Safety - Measurement of Sodium Copper Chlorophyllin in Food," specifies a method for measuring sodium copper chlorophyllin in food using spectrophotometric method, with a measurement wavelength of 405 nm, a limit of detection (LOD) of 0.001 g / kg, and a limit of quantification (LOQ) of 0.0051 g / kg. However, currently, there are no standards for detecting copper chlorophyll in edible oils.

[0005] Methods developed to date for detecting copper chlorophyll in vegetable oils primarily involve spectroscopy and chromatography using ultraviolet absorption detectors (UV) and photodiode array detectors (PDA). Reference 1 discloses a method for measuring the copper chlorophyll content in olive oil by ultraviolet-visible spectroscopy, but this method calculates the copper chlorophyll content using complexed copper and requires many steps and manual labor. Reference 2 discloses a method for detecting copper chlorophyll by ultraviolet spectroscopy, but the sample needs to be pre-treated with light to eliminate interference from chlorophyll and pheophytin on the absorption spectrum of copper chlorophyll. Reference 3 reports a method for detecting and screening copper chlorophyll in olive oil using surface-enhanced Raman spectroscopy, but this method could not accurately detect copper chlorophyll in samples with high chlorophyll content. Reference 4 reports the separation and detection of lipid-soluble copper chlorophyll in counterfeit olive oil using HPLC-DAD. This method requires pretreatment steps such as solvent extraction and vacuum rotary evaporation, and furthermore, the low response signal of the DAD detector and high background noise limit its ability to detect low-concentration samples.

[0006] Cu-pyropheophytin a (Copper pyropheophytin a or Cu(II)pyropheophytin a, CAS number: 105791-89-7) is a major derivative of lipid-soluble copper chlorophyll and can be used as an indicator component for copper chlorophyll addition to olive oil. Reference 5 reports the detection of the presence or absence of Cu-pyropheophytin a in olive oil using ultrafast liquid chromatography-quadrupole time-of-flight tandem mass spectrometry (UPLC-MS / MS), but this method requires complex solid-phase extraction (SPE) as a pretreatment, and quantitative detection has not yet been achieved. [Prior art document] References: Cited document 1: CN107389572B Cited document 2:TW201818062A Citation 3: "Rapid detection of copper chlorophyll in vegetable oils based on surface-enhanced Raman spectroscopy", LIAN WN, SHIUE J, et al., Food Additives & Contaminants Part A Chemistry Analysis Control Exposure & Risk Assessment, 2015, 32(5), 627-634 Citation 4: “Control of Olive Oil Adulteration with Copper-Chlorophyll Derivatives”, ROCA M, GALLARDOGUERRERO L, et al., Journal of Agricultural&Food Chemistry, 2010, 58(1), 51-56 Reference 5: "Screening and Verification of Copper Chlorophyll in Olive Oil," Song Shufeng, Wang Dan, Yang Jie, et al., Chinese Journal of Preventive Medicine, 2015, 49(1), 45-49. [Overview of the project] [Problems that the invention aims to solve]

[0007] As mentioned above, conventional methods for detecting copper chlorophyll have problems such as limited sample types, inability to accurately quantify the substance, and the need for complex sample preparation. Furthermore, although techniques for detecting Cu-pyropheophytin a as an indicator component of copper chlorophyll have been reported, only qualitative detection has been reported, and complex solid-phase extraction preparation is required. Currently, there is no method for rapidly, efficiently, and accurately detecting Cu-pyropheophytin a.

[0008] Therefore, the object of the present invention is to provide a method for detecting Cu-pyropheophytin a in an oil sample more efficiently and accurately without complicated pretreatment, a method for determining whether or not copper chlorophyll is present in an oil sample such as olive oil by detecting Cu-pyropheophytin a, and a method for identifying counterfeit olive oil. [Means for solving the problem]

[0009] To solve the above problems, the inventors conducted extensive research and found that Cu-pyropheophytin a can be effectively separated from an oil sample by using supercritical fluid chromatography and reversed-phase liquid chromatography in combination, and that quantitative detection with high sensitivity, good specificity, reproducibility, and accuracy is possible by mass spectrometry based on multiple reaction monitoring (MRM).

[0010] Specifically, the present invention includes the following embodiments. One aspect of the present invention is, The components of the oil sample are separated by supercritical fluid chromatography, and nonpolar components in the oil sample are removed so that Cu-pyropheophytin a remains in the column of the supercritical fluid chromatography. A method for detecting Cu-pyropheophytin a in an oil sample, comprising introducing a component containing Cu-pyropheophytin a eluted from the column of the supercritical fluid chromatograph into a reversed-phase liquid chromatograph, separating Cu-pyropheophytin a by the reversed-phase liquid chromatograph, and detecting it with a mass spectrometer, wherein The mobile phase in the supercritical fluid chromatograph comprises a supercritical fluid and a modifier. The aforementioned modifying agent adjusts the polarity of the supercritical fluid. The scanning mode of the mass spectrometer is a multiple reaction monitoring mode. In some embodiments, preferably, in the above method of the present invention, the supercritical fluid comprises supercritical carbon dioxide, and the modifier comprises an alcohol-based substance and / or a nitrile-based substance. In some embodiments, preferably, in the method of the present invention, the stationary phase in the supercritical fluid chromatograph is selected from silica gel modified with a polar group selected from a diol group, an amino group, or a cyano group. In some embodiments, preferably, in the method of the present invention, the mobile phase in the reversed-phase liquid chromatograph is selected from an alcohol-based substance, a nitrile-based substance, or an aqueous solution thereof. In some embodiments, preferably, in the above method of the present invention, the supercritical fluid chromatograph comprises one supercritical fluid column, and the reversed-phase liquid chromatograph comprises one or more reversed-phase liquid columns. In some embodiments, preferably, in the above method of the present invention, Cu-pyropheophytin a remaining in the column of the supercritical fluid chromatograph is sent to a reversed-phase liquid chromatograph under the action of the denaturant, and the supercritical fluid from the supercritical fluid chromatograph is removed in the reversed-phase liquid chromatograph. In some embodiments, preferably, in the method of the present invention, the reversed-phase liquid chromatograph includes a first reversed-phase liquid column as a capture column and a second reversed-phase liquid column positioned at the rear end of the first reversed-phase liquid column as an analysis column, wherein the Cu-pyropheophytin a is detected after passing through the analysis column. More preferably, the Cu-pyropheophytin a remaining in the column of the supercritical fluid chromatograph is sent to the capture column under the action of the denaturant, and the supercritical fluid from the supercritical fluid chromatograph is removed in the capture column. In some embodiments, preferably, in the method of the present invention, the ion source of the mass spectrometer is an atmospheric pressure chemical ionization ion source or an electrospray ion source. More preferably, the ion source is an atmospheric pressure chemical ionization ion source. In some embodiments, preferably, in the above method of the present invention, the oil sample is olive oil. In some embodiments, preferably, the above method of the present invention further includes a step of diluting the oil sample before separating the components of the oil sample by supercritical fluid chromatography. Another aspect of the present invention is a method for determining the content of copper chlorophyll in olive oil, comprising detecting the content of Cu-pyropheophytin a in an olive oil sample by the method for detecting Cu-pyropheophytin a in an oil sample according to the present invention. Another aspect of the present invention is a method for identifying counterfeit olive oil, comprising detecting the content of Cu-pyropheophytin a in an olive oil sample by the method for detecting Cu-pyropheophytin a in the oil sample of the present invention. [Effects of the Invention]

[0011] The present invention can achieve the following effects by implementing the above-described solution. (1) The present invention provides a method for detecting Cu-pyropheophytin a, which solves the problem of optical detectors being interfered with by chlorophyll and its derivatives by using a mass spectrometer, enabling rapid detection of samples with low Cu-pyropheophytin a content and complex samples containing interfering components such as chlorophyll and its derivatives, thereby significantly improving detection sensitivity and lowering the detection limit. (2) The detection method provided by the present invention does not require complicated pretreatment of oil samples, and oil samples can be easily diluted and then directly subjected to analysis. Since sample purification is performed online using supercritical fluid chromatography separation technology with carbon dioxide as the mobile phase, consumption of organic solvents and pretreatment consumables is reduced, the automation and convenience of detection are improved, labor and materials are saved, it is environmentally friendly, costs are reduced, and time is saved. (3) When detecting Cu-pyropheophytin a in oil by the method of the present invention, the detection results have a good linear correlation coefficient, good reproducibility, and high recovery rate. Compared with the prior art, the detection accuracy and sensitivity are improved, and an effective criterion for food safety detection is provided. (4) By using a supercritical fluid chromatograph and a reversed-phase liquid chromatograph in combination, the present invention can achieve rapid and automatic detection, greatly improve the detection efficiency, and significantly shorten the time required for detection compared with the prior art. Generally, the total detection time including the sample preparation time in the present invention can be controlled within 30 minutes. (5) According to the method of the present invention, the presence or content of Cu-pyropheophytin a in olive oil can be accurately detected, and it can be determined whether copper chlorophyll is contained in the olive oil, and thus the authenticity of the olive oil can be reliably determined.

Brief Description of the Drawings

[0012] [Figure 1] Schematic diagram of the analysis system of the present invention (first operation path). [Figure 2] Schematic diagram of the analysis system of the present invention (second operation path). [Figure 3] Schematic diagram of the analysis system of the present invention (third operation path). [Figure 4] Analysis results of a sample containing chlorophyll A and Cu-pyropheophytin a. The upper part of FIG. 4 is by a method that does not use a reversed-phase liquid chromatograph, and the lower part of FIG. 4 is by the method of the present invention. [Figure 5] Standard curve of Cu-pyropheophytin a by the detection method of the present invention.

Embodiments for Carrying out the Invention

[0013] The content of the present invention will be described in detail below. The technical features described below are explained based on representative embodiments and specific examples of the present invention, but the present invention is not limited to these embodiments and specific examples. Unless otherwise defined, it is as follows.

[0014] In this specification, "copper chlorophyll", "copper chlorophyll" and "(copper chlorophyll)" refer to the same substance and have the same meaning.

[0015] In this specification, "Cu-pheophytin a" refers to the substance represented by the following chemical formula and having a CAS number of 105791-89-7.

Chemical formula

[0016] The numerical range represented by "numerical value A to numerical value B" used in this specification means a range including the limit values A and B.

[0017] The "%" used refers to volume percentage, that is, "V%".

[0018] In this specification, "communicate" and "connect" mean that a plurality of devices or members can form a path for the flow of the mobile phase, the test component, and / or waste gas.

[0019] The "switching" described in the present invention means, for example, by switching a multi-way valve group, blocking a path that was originally communicated, and communicating a device or member that was originally blocked.

[0020] Although it is self-evident, the supercritical fluid column and one or more reversed-phase liquid columns according to the multidimensional chromatography system of the present invention are connected to a pump for supplying the mobile phase in order to supply the corresponding mobile phase and / or modifier in the mobile phase in each chromatograph device during the operation of the system.

[0021] In this specification, the use of "may" includes both the meaning of performing some action and not performing any action.

[0022] In this specification, the use of “optional” or “optional” indicates whether or not certain elements, such as a particular substance, component, execution step, or application condition, are used or not used.

[0023] All unit names used in this specification are International Standard Unit Names.

[0024] In this specification, unless otherwise specified, "multiple (items / types)" means two or more.

[0025] In this specification, references to “several specific / preferred embodiments,” “another specific / preferred embodiment,” “embodiment,” etc., mean that certain elements (e.g., configuration, structure, properties, and / or characteristics) described in relation to such embodiments are included in at least one embodiment described herein and may or may not be present in other embodiments. Furthermore, it should be understood that these elements can be combined in any preferred form in various embodiments.

[0026] The present invention provides a method for detecting Cu-pyropheophytin a in an oil sample.

[0027] oil The oils suitable for the present invention are not particularly limited and may include mixed oils obtained by mixing one or more types of vegetable oils and animal oils.

[0028] In some specific embodiments, detectable vegetable oils include one or more of the following: rice bran oil, sunflower oil, palm oil, palm kernel oil, peanut oil, rapeseed oil, soybean oil, linseed oil, cottonseed oil, safflower oil, perilla oil, camellia seed oil, hemp seed oil, jojoba oil, olive oil, cocoa oil, black oak oil, almond oil, apricot kernel oil, tung oil, rubber seed oil, corn germ oil, wheat germ oil, sesame seed oil, evening primrose oil, hazelnut oil, pumpkin seed oil, walnut oil, grape seed oil, linseed oil, borage seed oil, sea buckthorn oil, canola oil, tomato seed oil, macadamia oil, and coconut oil.

[0029] In specific embodiments, detectable animal oils include one or more of the following: beef tallow, lard, mutton tallow, chicken tallow, fish oil, seal oil, whale oil, dolphin oil, oyster oil, and lanolin.

[0030] Furthermore, any of the above types of oils or blended oils may be fresh oils obtained by chemical impregnation or pressing, or stored oils.

[0031] From the standpoint of ease of detection, the detection method of the present invention is particularly suitable for detecting copper chlorophyll in vegetable oil or a blend of multiple vegetable oils.

[0032] In some preferred embodiments, the oil of the present invention is preferably olive oil, grape seed oil, and more preferably olive oil.

[0033] Pre-treatment The present invention is not particularly limited with respect to the pretreatment of oil samples before detection, and for example, several pretreatment methods for removing interfering components disclosed in the art can be used. Of course, as stated above, the method provided by the present invention can provide reliable detection results while improving detection efficiency even without these pretreatments.

[0034] In another specific embodiment, the oil sample of the present invention can be subjected to simple pretreatment, such as diluting the oil sample to reduce its viscosity and facilitate detection. There are no particular limitations on the solvent that can be used for dilution; one or more hydrocarbon solvents, ester solvents, ether solvents, or ketone solvents can be used. Preferably, hydrocarbon solvents such as n-hexane and n-heptane can be used. Furthermore, there are no particular limitations on the amount of diluent used, and it can be adjusted according to the physical properties of different oils, the characteristics of the detector, and the quantification method. In some preferred embodiments of the present invention, from the viewpoint of detection convenience, economy, and detection reliability, the dilution ratio can be 1:1 to 1:100 in terms of the ratio of the mass (g) of the oil sample to the volume (ml) of the diluent. Furthermore, from the viewpoint of improving the recovery rate, the ratio of the mass (g) of the oil sample to the volume (ml) of the diluent is preferably 1:25 to 1:70, and more preferably 1:35 to 1:60. The diluted oil sample can be directly detected according to the method of the present invention.

[0035] The procedure of the present invention and the analytical system used will be described below. Analysis System The method of the present invention includes separating the components of an oil sample by supercritical fluid chromatography and removing non-polar components from the oil sample such that Cu-pyropheophytin a remains in the column of the supercritical fluid chromatography. First, the supercritical fluid chromatography will be described.

[0036] (Supercritical fluid chromatography) Supercritical fluid chromatography (SFC) is a type of chromatography that uses a supercritical fluid as the mobile phase.

[0037] Supercritical fluids possess properties intermediate between gases and liquids. Compared to conventional liquid chromatography (LC) and gas chromatography (GC), SFC combines the advantages of both and offers exceptional separation selectivity. Furthermore, the CO2 mobile phase is mild, non-toxic, chemically inert, readily available, and environmentally friendly. In this invention, a supercritical fluid column is used to pre-treat the sample and remove non-polar components from the sample. Here, the non-polar components in this invention refer to components in oils and fats that are less polar than Cu-pyropheophytin a, particularly components that affect the detection of Cu-pyropheophytin a.

[0038] The column used in the supercritical fluid chromatograph of the present invention (i.e., the supercritical fluid column) may be a packed column, and in some embodiments, it is preferable to use a diol column as the SFC column. In some embodiments, the inner diameter of the SFC column is preferably 1 to 10 mm, more preferably 3 to 6 mm, and the length is preferably 5 to 30 cm, more preferably 15 to 30 cm.

[0039] The stationary phase in the supercritical fluid column is selected from silica gel modified with one or more polar groups selected from diol groups, amino groups, or cyano groups. In some preferred embodiments, it is preferable to use a diol group as the modifying group, considering that it is necessary to ensure an appropriate retention time for Cu-pyropheophytin a when pre-washing the sample solution using the supercritical fluid column. More specifically, the stationary phase in the supercritical fluid column of the present invention can preferably be diol-grouped silica gel, and more preferably diol-grouped silica gel to which an organosilane containing a 1,2-dihydroxypropyl functional group is bonded can be used. Alternatively, the stationary phase may be porous spherical silica gel.

[0040] The mobile phase in the supercritical fluid column of the present invention includes a supercritical fluid. The supercritical fluid is a state of matter under conditions above critical temperature and critical pressure, referring to an intermediate state between gas and liquid. Applicable supercritical fluids may be supercritical carbon dioxide or supercritical ethane, etc. In some preferred embodiments of the present invention, the supercritical fluid is selected from supercritical carbon dioxide. The operating temperature and pressure are mainly determined by the selected supercritical fluid. In the present invention, when supercritical carbon dioxide is used as the supercritical fluid, the operating temperature is 31°C or higher, preferably 35°C or higher, and the operating pressure is 7.3 MPa or higher, preferably 7.5 MPa or higher. From the viewpoint of the degree of solvation of the supercritical fluid to Cu-pyropheophytin a, retention time and handling, the operating temperature is preferably 40 to 60°C, more preferably 45 to 55°C, and the operating pressure is preferably 8.5 to 15 MPa, more preferably 9 to 12 MPa.

[0041] In this invention, a denaturing agent is used as a substance to adjust the polarity of the supercritical fluid. The denaturing agent may contain an organic solvent such as an alcohol-based substance or a nitrile-based substance, and is preferably an alcohol-based substance or a nitrile-based substance. As the alcohol-based substance, various fatty alcohols such as methanol and isopropanol can be used. As the nitrile-based substance, acetonitrile can be used as a denaturing agent. From the viewpoint of controlling the retention time of Cu-pyropheophytin a, the denaturing agent is preferably isopropanol. Compared to the case where other denaturing agents such as methanol are used, using isopropanol makes it easier for Cu-pyropheophytin a to elute, and the detection efficiency and accuracy can be further improved.

[0042] The amount of modifying agent used (relative flow rate) can usually be 1% to 30% of the supercritical fluid. From the viewpoint of controlling the retention time of Cu-pyropheophytin a, the amount of modifying agent used in the present invention is preferably 25% or less of the supercritical fluid, more preferably 20% or less, specifically 15%, 14%, 13%, and 12%. Furthermore, from the viewpoint of controlling the retention time, a modifying agent solution with a modifying agent content of 80% or more, preferably 90% or more, and more preferably 100%, can be used. Therefore, when 100% isopropanol is used as the modifying agent, the amount used is preferably 10% to 20% of the supercritical fluid, more preferably 12% to 17%.

[0043] The supercritical fluid and modifying agent of the present invention can be supplied to the column by a pump, preferably by an independent CO2 pump. In some specific embodiments of the present invention, the modifying agent can be supplied by a single liquid-phase pump equipped with a flow control valve, or by a plurality of independent liquid-phase component supply pumps. Furthermore, in some specific embodiments, the supercritical fluid column of the present invention can be installed in a column oven.

[0044] Furthermore, when performing certain operating modes, the supercritical fluid chromatograph can be connected to or communicate with auxiliary detectors and pressure control units via a connecting device. In some embodiments, the pressure control unit is located at the end of the supercritical fluid chromatograph section. The auxiliary detector is not particularly limited and may be located before or after the pressure control unit, and can detect various components eluted from the column, and in particular, the retention time of Cu-pyropheophytin a in the oil sample can be determined by detecting the elution signal of any Cu-pyropheophytin a. The auxiliary detector can be used without limitation as long as it can qualitatively detect Cu-pyropheophytin a, for example, a voltage-resistant diode array detector or a mass spectrometry (MS) detector can be used, preferably a voltage-resistant diode array detector.

[0045] The supercritical fluid chromatography system used in this invention is commercially available, and for example, the "Nexera UC" supercritical fluid chromatography system manufactured by Shimadzu Corporation can be used.

[0046] After the oil sample to be tested is placed in a supercritical fluid chromatograph via an automated sample injection device, the supercritical fluid chromatograph is started, and the mobile phase composition and flow rate are adjusted to rapidly elute components in the oil that hinder the detection of Cu-pyropheophytin a from the column, leaving Cu-pyropheophytin a in the column.

[0047] In this process, oil samples were rapidly pre-treated using supercritical fluid chromatography.

[0048] (Connection device) In this invention, a supercritical fluid chromatograph is connected to a reverse-phase liquid chromatograph, mass spectrometer, etc., using a connecting device, and different operating modes or operating paths can be formed by setting the connection means of the connecting device.

[0049] In the present invention, a preferred connection device may be a multi-way valve group. After supercritical fluid chromatography treatment and removal of oily components, the analysis system is switched to reverse-phase liquid chromatography before the (fastest) retention time of Cu-pyropheophytin a is reached.

[0050] The multi-way valve group is not particularly limited, but various injection multi-way valve groups commonly used in liquid chromatographs can be used. In the present invention, a 6-way valve group or a 10-way valve group is preferably used, and a 6-way valve group is more preferably used. Various switching operations of the multi-way valve group can realize various flow paths or operating paths, and are particularly suitable for connecting and controlling multiple devices.

[0051] (Reverse-phase liquid chromatography) The method of the present invention further includes introducing a component containing Cu-pyropheophytin a eluted from the column of the supercritical fluid chromatograph into a reversed-phase liquid chromatograph, separating Cu-pyropheophytin a using the reversed-phase liquid chromatograph, and detecting it with a mass spectrometer. The reversed-phase liquid chromatograph will be described below.

[0052] In the present invention, the reversed-phase liquid chromatograph section includes one or more reversed-phase liquid columns. In some embodiments, these reversed-phase liquid columns are placed in a column oven during use. In some preferred embodiments of the present invention, the length of the reversed-phase liquid column is 1 to 35 cm and the inner diameter is 0.6 to 15 mm, in order to improve the linear range of detection and detection capability (limit of quantification LOQ, limit of detection LOD). When multiple reversed-phase liquid columns are used, the length of the reversed-phase liquid column that captures Cu-pyropheophytin a in the preceding stage (i.e., the capture column) is preferably 1 to 12 cm, more preferably 2 to 8 cm, and the inner diameter is preferably 1 mm to 10 mm, more preferably 2.5 to 7.5 mm. The length of the reversed-phase liquid column for the final separation of Cu-pyropheophytin a (i.e., the analysis column) is preferably 5 to 30 cm, more preferably 8 to 22 cm, and the inner diameter is preferably 1 mm to 4 mm, more preferably 1.7 to 2.4 mm.

[0053] The stationary phase in the reversed-phase liquid column of the present invention may be silica gel modified with a hydrophobic group, and the hydrophobic group may be various hydrocarbon groups such as a C8 group, a C18 group, or a phenyl group. In some embodiments of the present invention, C18-modified silica gel is used as the stationary phase. In some embodiments of the present invention, preferably, both the capture column and the analysis column are C18 reversed-phase columns. A polar organic solvent or an aqueous solution thereof can be used as the mobile phase, preferably a polar organic solvent such as an alcoholic substance or a nitrile substance. In the case of an alcoholic substance, various aliphatic alcohols such as methanol and isopropanol can be used, and in the case of a nitrile substance, acetonitrile can be used. In some embodiments, the mobile phase can be used as an aqueous solution, for example, an aqueous solution of isopropanol or acetonitrile is used.

[0054] When used as an aqueous solution, the content of polar organic solvents in the mobile phase must be 60% or more, preferably 80% or more, and more preferably 90% or more, from the viewpoint of shortening the retention time. In some preferred embodiments of the present invention, from the viewpoint of improving the linear range to be detected and the detection capability (limit of quantification LOQ, limit of detection LOD), the mobile phase in the reversed-phase liquid chromatograph preferably contains 70% to 100% isopropanol, and more preferably 100% isopropanol. When 100% isopropanol is used as the mobile phase, the flow rate can be 0.05 to 2.5 ml / min, and from the viewpoint of balancing separation efficiency and analysis time, it is preferably 0.15 to 1.2 ml / min, and more preferably 0.2 to 0.6 ml / min. Furthermore, in some specific embodiments of the present invention, the use of one or more mobile phases in the reversed-phase liquid chromatograph described above can be performed by gradient elution. For example, the gradient elution method is configured according to the actual detection or test conditions and is supplied by a single liquid-phase pump with a flow control valve or multiple independent liquid-phase component supply pumps.

[0055] In some embodiments of the present invention, the reverse-phase liquid chromatograph section may include only one chromatograph column. As described above, after supercritical fluid chromatography, the multi-way valve group is switched to connect the supercritical fluid chromatograph in series with the reverse-phase liquid chromatograph (after supercritical fluid chromatography, the liquid outlet of the supercritical fluid column is disconnected from the auxiliary detector and pressure control unit described above, and the liquid outlet of the supercritical fluid column is connected to the liquid inlet of the reverse-phase liquid chromatograph via the multi-way valve group). At this time, the end of the reverse-phase liquid chromatograph can also be switched or connected to the pressure control unit described above.

[0056] After the column is switched, the Cu-pyropheophytin a that originally remained in the supercritical fluid column is further washed away by the denaturing agent. In some preferred embodiments, from the viewpoint of improving elution efficiency, the flow rate of the denaturing agent can be increased at this time to allow the strongly retained pyropheophytin a to elute more quickly from the supercritical fluid column. For example, the flow rate of the denaturing agent can be increased to 0.4 to 2 mL / min, preferably 0.6 to 1.4 mL / min, and more preferably 0.8 to 1.2 mL / min. In this case, the Cu-pyropheophytin a remaining in the supercritical fluid column can be eluted by the flow of the denaturing agent (alone), and therefore, together with the supercritical fluid remaining in the system, it passes through the multi-way valve group and enters the reverse-phase liquid chromatograph section.

[0057] At this point, since the end of the reversed-phase liquid column is already connected to the pressure control unit, the supercritical fluid can be separated in its supercritical state using the pressure control unit. This process does not affect the state of other mobile phases or components. In other words, the reversed-phase liquid chromatograph removes the mobile phase that has leaked out of the supercritical fluid chromatograph.

[0058] Simultaneously with or after the removal of the supercritical fluid, the mobile phase is introduced into the reversed-phase liquid column using a liquid-phase pump. In this invention, the supercritical mobile phase is removed from the supercritical fluid chromatograph, and only the polar modifier and the component to be separated remain in the reversed-phase liquid chromatograph. At this time, the polar mobile phase can be directly introduced into the reversed-phase liquid column, solving the problem of mobile phase incompatibility that occurs when switching between different types of chromatographic columns. Furthermore, in some embodiments, it is preferable to use the same substance as the modifier in the SFC column as the mobile phase of the reversed-phase liquid column. This further solves the problem of mobile phase incompatibility that occurs when switching between different types of chromatographic columns.

[0059] After reintroducing the polar mobile phase into the reversed-phase liquid column system, the strongly retained Cu-pyropheophytin a can be separated and detected. In other words, by further introducing the mobile phase in the reversed-phase liquid chromatograph described above, substances present in the reversed-phase liquid chromatograph can be separated, and the separated components can be obtained.

[0060] Furthermore, when performing the above processing, in particular, after completely removing the supercritical mobile phase from the supercritical fluid chromatograph using the pressure control unit as described above, the reversed-phase liquid chromatograph can be disconnected from the pressure control unit and connected to the detector by switching the multi-way valve group as described above. At this time, the copper chlorophyll component obtained by the above separation can be detected and analyzed by the detector.

[0061] In another embodiment of the present invention, the reversed-phase liquid chromatograph section may comprise one or more columns. In a typical embodiment, the reversed-phase liquid chromatograph section may comprise two columns that can be connected in series.

[0062] When separating components of a sample by supercritical fluid chromatography using two reversed-phase chromatography columns connected in series, after removing nonpolar oily substances, the supply of the supercritical mobile phase is stopped and the denaturing agent is continuously introduced. By increasing the flow rate of the denaturing agent as described above, the columns can be switched via a multi-way valve group, and Cu-pyropheophytin a, which is strongly retained in the supercritical fluid column, can be sent to the first reversed-phase liquid column. Furthermore, the end of the first reversed-phase liquid column is connected to a pressure control unit to remove the supercritical mobile phase delivered into the column. Furthermore, the multi-way valve group is switched again, disconnecting the first reversed-phase liquid column from the pressure control unit and connecting it to the second reversed-phase liquid column.

[0063] Here, when performing the above process, simultaneously with or after the removal of the supercritical mobile phase, the liquid phase pump is started to send the polar mobile phase to the first reversed-phase liquid column, the first and second reversed-phase liquid columns are connected, and Cu-pyropheophytin a held in the first reversed-phase liquid column is further eluted into the second reversed-phase liquid column, where Cu-pyropheophytin a is separated and detected.

[0064] Cu-pyropheophytin a, separated in the second reversed-phase liquid chromatograph, can be analyzed and detected by a mass spectrometer connected in series with the column.

[0065] The first reversed-phase liquid column and the second reversed-phase liquid column described above may be the same or different. In a preferred embodiment of the present invention, they may be different, for example, the second reversed-phase liquid column being longer than the first reversed-phase liquid column. With such a design, substances strongly retained in the first reversed-phase liquid column are more easily eluted, improving the separation accuracy in the second reversed-phase liquid column. Therefore, the first reversed-phase liquid column corresponds to a pretreatment column for the second reversed-phase liquid column, primarily serving to concentrate Cu-pyropheophytin a, remove the supercritical mobile phase, and improve the stability and accuracy of detection by the latter column.

[0066] (mass spectrometer) In this invention, Cu-pyropheophytin a, separated by reverse-phase liquid chromatography, is detected by a mass spectrometer, and qualitative or quantitative analysis is performed. From the viewpoint of improving detection accuracy and detection limit, in some specific embodiments of this invention, the mass spectrometer uses a multiple reaction detection mode. In other words, the mass spectrometer is also called a tandem mass spectrometer, and typically a triple quadrupole mass spectrometer or a quadrupole time-of-flight (Q-TOF) mass spectrometer can be used.

[0067] In the present invention, the mass spectrometer detects Cu-pyrofeophytin a in MRM mode, and from the viewpoint of analytical accuracy, it is preferable to use m / z values ​​of 873.4 > 594.2 and 873.4 > 522.3 for detection.

[0068] Furthermore, detection using the MRM mode with m / z A > B is a measurement in which a precursor ion with m / z A is selected in the pre-stage mass separator, and product ions with m / z B derived from that precursor ion with m / z A are detected. Therefore, if the tandem mass spectrometer performing this MRM measurement is a triple quadrupole mass spectrometer, the precursor ion with m / z A is selectively passed through the pre-stage quadrupole mass filter, and the product ions with m / z B derived from that precursor ion with m / z A are selectively passed through the post-stage quadrupole mass filter for detection. Also, if the tandem mass spectrometer performing this MRM measurement is a Q-TOF mass spectrometer, the precursor ion with m / z A is selectively passed through the pre-stage quadrupole mass filter (Q), and product ions containing m / z B from among the product ions derived from that precursor ion with m / z A are detected by the post-stage time-of-flight mass separator (TOF) and ion detector.

[0069] In the above embodiment of the present invention, it is preferable to use m / z 873.4 > 594.2 and 873.4 > 522.3 for detection. An example will be given where m / z 873.4 > 594.2 is used for detection. That is, a precursor ion having m / z 873.4 is selected in the pre-mass separator, and a product ion with m / z 594.2 derived from that precursor ion with m / z 873.4 is detected. Here, the actual mass-to-charge ratio of the ions selected by the mass separator, such as a quadrupole mass filter, naturally depends on the performance of the equipment used. Therefore, in this specification, when m / z 873.4 is specified, for example, the most intended mass-to-charge ratio value of the ion is m / z 873.4, but ions within the ranges of m / z 873.4±0.1, m / z 873.4±0.2, m / z 873.4±0.3, or m / z 873.4±0.4 may also be included as intended ions. The same applies to m / z 594.2.

[0070] Furthermore, the mass spectrometer can use an electrospray ion source (ESI) or an atmospheric pressure chemical ionization (APCI) source. From the viewpoint of efficiently ionizing Cu-pyropheophytin a and improving the accuracy of MRM detection, APCI is preferably used.

[0071] (Other auxiliary devices) In this invention, in addition to the apparatus described above, other auxiliary devices as needed in the industry can be used without limitation.

[0072] Typically, the auxiliary equipment may include containers for supercritical fluid, denaturants, polar solvents, etc., a power supply (typically a pump) for supplying these reagents to other detection devices, and a sample injection device, which may typically be an autosampler. Regarding the autosampler capacity, from the viewpoint of improving the linear range and detection capability (limit of quantification LOQ and limit of detection LOD) detected by the present invention, the autosampler capacity should be greater than 10 μL, preferably 15 μL or more, for example, 20 μL, 25 μL, or 30 μL. There is no particular upper limit to the autosampler capacity, but from the viewpoint of handling, it is preferably 100 μL or less.

[0073] Furthermore, as mentioned above, auxiliary detectors and pressure control units are optionally used when performing supercritical fluid chromatography. While suitable auxiliary detectors may include voltage-resistant diode array detectors or mass spectrometers, using a voltage-resistant diode array detector is more preferable from the viewpoint of improving detection efficiency. The pressure control unit is not particularly limited, but is mainly used to assist in controlling the supercritical fluid conditions. In some preferred embodiments, the pressure control unit can be selected from back pressure control units (BPRs).

[0074] Furthermore, the method for detecting Cu-pyrofeophytin a according to the present invention can be performed manually, or it can be performed using a semi-automatic or fully automatic method.

[0075] In semi-automatic or fully automatic systems, automatic control is performed using method files in workstation software. After a sample is loaded, the operating path or a combination thereof is automatically controlled according to the properties of the sample, thereby realizing an automatic and simple automatic (online) detection method. There are no particular limitations on the automatic control software applicable to the system of the present invention; a general control program in the industry or a program created on-site based on the actual conditions of the device can be used.

[0076] Operating mode The typical operating modes and pathways that appear in the detection method of the present invention will be described in detail below with reference to the drawings. In particular, the operating modes when using two reversed-phase liquid columns will be described with reference to Figures 1 to 3.

[0077] Specifically, Figures 1 to 3 show that the detection system of the present invention forms different operating paths in the analysis system by switching the multi-way valve V, and furthermore, that the analysis and detection of Cu-pyropheophytin a in oil samples can be easily achieved.

[0078] (First operating path) In this invention, oil samples can be pretreated using a first operating path by switching a multi-way valve V. Specifically, in this operating path, an automatic sample injection device and a supercritical fluid column are connected, and the supercritical fluid column is further connected to a detector, a pressure control unit, and a waste liquid / exhaust gas outlet via the multi-way valve V.

[0079] As shown in Figure 1, the first operating path of the present invention may include connecting an autosampler 13, a supercritical fluid column 14, a multiway valve V (3 and 2 are connected), a back pressure control device 15, and a waste liquid / exhaust gas outlet, with liquid phase pumps 11 and 12 supplying carbon dioxide and a modifying agent, respectively. Here, the liquid phase pump 12 is equipped with a flow control valve (not shown) for adjusting the composition of the modifying agent, and of course, in some specific embodiments, two liquid phase pumps supplying water and a modifying agent, respectively, can be used instead of the liquid phase pump 12.

[0080] In this invention, nonpolar components in the oil sample are eluted using a first operating pathway, thereby preventing these components from inhibiting the detection of Cu-pyropheophytin a.

[0081] Before reaching the retention time for Cu-pyrofeophytin a, the first operating pathway ends, and the second operating pathway begins due to the switching of the multi-way valve.

[0082] (Second operating path) In this invention, after the first operating pathway is completed, the system switches to a second operating pathway, and the Cu-pyropheophytin a component is delivered to a reversed-phase chromatography column by a denaturing agent in a supercritical fluid chromatograph.

[0083] After the components containing Cu-pyropheophytin a are sent to the reverse-phase liquid chromatograph, preferably in a second operating path, supercritical CO2 is removed from the supercritical fluid chromatograph in the system (discharged from the waste liquid / exhaust outlet by the pressure control unit).

[0084] Therefore, in some preferred embodiments of the present invention, typically as shown in Figure 2, the second operating path can connect the autosampler 13, the supercritical fluid column 14, the multiway valve group V (3 and 4 are connected), the reversed-phase liquid column 21 (i.e., the capture column), the multiway valve V (1 and 2 are connected), the back pressure control device 15, and the waste liquid / exhaust gas outlet.

[0085] (Third operating path) In the present invention, a third operating path can be formed by modifying the second operating path (see Figure 3), that is, by disconnecting the communication between 3 and 4 of the multi-way valve V, and also by disconnecting the communication between 1 and 2 of the multi-way valve V. At the same time, a path is formed by connecting the liquid-phase pump 20, the multi-way valve V (5 and 4 are connected), the reverse-phase liquid column 21, the multi-way valve V (1 and 6 are connected), the reverse-phase liquid column 22 (i.e., the analytical column), and the mass spectrometer 23.

[0086] Through a third operating pathway, Cu-pyropheophytin a is transferred from reversed-phase liquid column 22 to reversed-phase liquid column 23, and subsequently separated by the action of the mobile phase, and introduced into a mass spectrometer for detection.

[0087] When the detection method of the present invention is used to detect Cu-pyropheophytin a in olive oil, it is possible to determine whether or not copper chlorophyll has been added to the olive oil, since Cu-pyropheophytin a is a characteristic component contained in copper chlorophyll.

[0088] Therefore, the present invention further, The present invention provides a method for determining the copper chlorophyll content in olive oil, which includes detecting the content of Cu-pyropheophytin a in an olive oil sample.

[0089] In the above method, the criteria for determination are not particularly limited and may be international standards, national standards, or industry standards. If such standards do not exist, the criteria may be standards established by persons skilled in the art based on experience. Alternatively, the quantification limit of the method for detecting Cu-pyropheophytin a in an olive oil sample according to the present invention may be used as the criterion. For example, in some embodiments, the above method may further include a determination step in which it is determined that the olive oil contains copper chlorophyll if the detected Cu-pyropheophytin a content is 6 μg / kg or more.

[0090] Furthermore, the present invention provides a method for identifying counterfeit olive oil, comprising detecting the content of Cu-pyropheophytin a in an olive oil sample by the method of the present invention.

[0091] In the above method, the criteria for judgment are not particularly limited and may be international standards, national standards, or industry standards. If such standards do not exist, the criteria may be standards established by persons skilled in the art based on experience. Alternatively, the criterion may be the quantitative limit of the method for detecting Cu-pyropheophytin a in an olive oil sample according to the present invention. For example, in some embodiments, the above method may further include a determination step in which, if the content of Cu-pyropheophytin a measured in the above detection step is 6 ug / kg or more, the olive oil is determined to be counterfeit olive oil. In this specification, "counterfeit olive oil" includes, for example, counterfeit or inferior olive oils such as olive oil that has been disguised as olive oil by adding copper chlorophyll to an oil other than olive oil, olive oil mixed with an oil other than olive oil and olive oil that has been disguised as pure olive oil by adding copper chlorophyll, virgin olive oil that has been disguised as virgin olive oil by adding copper chlorophyll to refined olive oil or mixed olive oil, or olive oil that has been expired or deteriorated that has been disguised as fresh olive oil by adding copper chlorophyll.

[0092] The detection method of the present invention offers extremely excellent convenience and reliability in detecting Cu-pyropheophytin a, and allows for rapid and reliable qualitative and quantitative detection, which is significantly superior to conventional detection methods. Furthermore, it provides a viable detection method for determining whether or not copper chlorophyll is present in olive oil and for determining the authenticity of olive oil. [Examples]

[0093] The effectiveness of the detection method of the present invention and the accuracy of the analysis have been verified through examples, and several olive oils, including conventional products, were quantitatively analyzed using the method of the present invention.

[0094] <Chemicals and Reagents> Isopropanol (LC-MS grade) and n-hexane (HPLC grade) (HPLC grade from Thermo Fisher Scientific). Carbon dioxide (CO2, purity ≥ 99.99%, Beijing, China). Chlorophyll A (standard product) Cu-Pyrofeophytin a (standard product) Three commercially available olive oil brands: Olive Oil AL, Olive Oil PL, Olive Oil FQ

[0095] <Device> The experiment used the Nexera UC system, manufactured by Shimadzu Corporation (Kyoto Prefecture, Japan). The chromatograph section consists of an LC-30AD SF CO2 pump, an LC-30AD pump, a SIL-30AC autosampler (20 μL), a CTO-20AC column oven, columns, and one SFC-30A back pressure regulator (BPR). The column oven is also equipped with a high-pressure six-way valve for switching columns. Here, the following columns were used: SFC column (4.6 mm × 250 mm; 5 micrometers), packing material UC-X Diol (diol group); C18 short column (VP-ODS, 4.6 mm × 50 mm; 5 micrometers); C18 long column (2.1 mm × 150 mm; 5 micrometers). All columns were purchased from Shimadzu (Shanghai) Laboratory Equipment Co., Ltd. Mass spectrometer: Triple quadrupole rod mass spectrometer LCMS-8060, Shimadzu Corporation (Kyoto Prefecture, Japan). Operating conditions were: Ion source: APCI, Scan mode: Multiple reaction monitoring (MRM).

[0096] <Preparation of standard samples> To compare the analytical results with and without the use of reversed-phase liquid chromatography, appropriate amounts of chlorophyll A standard and Cu-pyropheophytin a standard were dissolved in n-hexane to prepare solutions containing 5 ppb of chlorophyll A and 5 ppb of Cu-pyropheophytin a. An appropriate amount of Cu-pyropheophytin a standard was dissolved in n-hexane to prepare the mother liquor. This mother liquor was then diluted with n-hexane to obtain standard samples with concentrations of 0.2 ppb, 1 ppb, 5 ppb, 10 ppb, and 20 ppb, respectively, which were used to create standard curves.

[0097] <Preparation of the sample to be analyzed> Olive oil AL, olive oil PL, and olive oil FQ were weighed and diluted with n-hexane to prepare the analyte samples. The dilution ratio was 0.2 g of olive oil to 10 mL of n-hexane. Although the diluted samples were measured, the sample mass and concentration listed in the measurement results below are the values ​​converted to olive oil AL, olive oil PL, and olive oil FQ.

[0098] <Measurement results> The solution containing 5 ppb of chlorophyll A and 5 ppb of Cu-pyropheophytin a was detected without separation by reversed-phase liquid chromatography (i.e., MS analysis was performed immediately after the first working pathway), using the system shown in Figures 1 to 3 (passing through the first, second, and third working pathways in order). The detection results are shown in the upper and lower parts of Figure 4, respectively. Among these, the characteristic MRM transitions for Cu-pyropheophytin a were 873.4 > 594.2 and 873.4 > 522.3, while the characteristic peak for chlorophyll was 892.5 > 613.4, etc. As can be seen in Figure 4, pretreatment using an SFC column effectively separated Cu-pyropheophytin a from other interfering components such as chlorophyll, particularly chlorophyll A. Cu-pyropheophytin a could then be qualitatively and quantitatively detected by MRM measurements at 873.4 > 594.2 and 873.4 > 522.3, yielding highly accurate detection results.

[0099] <Examples of application> Using the system shown in Figures 1 to 3 (passing through the first, second, and third operating paths in sequence), a standard sample was detected and processed to obtain the standard curve shown in Figure 5. The linear equation was y = 18186.6x, the linear range was 10 to 1000 μg / kg, the limit of quantification (LOQ) was 6 μg / kg, and the limit of detection (LOD) was 2 μg / kg. Here, 873.4 > 594.2 and 873.4 > 522.3 were used for MRM measurement. Thus, it was found that the detection method of the present invention not only significantly simplifies the pretreatment means and shortens the detection time to within 30 minutes, but also achieves a detection limit comparable to or exceeding that of conventional detection methods, enabling highly sensitive and accurate quantitative analysis.

[0100] Example 1 Using the standard curve described above, the Cu-pyropheophytin a content in olive oil AL, olive oil PL, and olive oil FQ was measured, and the results are shown in Table 1. According to Table 1, the Cu-pyropheophytin a content in olive oil AL is very low, and it can be concluded that copper chlorophyll has not been artificially added, suggesting that it is likely genuine olive oil. On the other hand, the concentration of Cu-pyropheophytin a in olive oil PL and olive oil FQ is relatively high, suggesting that copper chlorophyll has been added, and thus they can be identified as counterfeit olive oil.

[0101] [Table 1]

[0102] Example 2 Olive oil PL samples were measured repeatedly, and the measurement results are shown in Table 2 below. The method of the present invention was found to have good reproducibility.

[0103] [Table 2]

[0104] Example 3 The results of the spike test using olive oil AL as a blank sample are shown in Table 3 below. The method of the present invention was found to have a good recovery rate.

[0105] [Table 3]

[0106] Furthermore, the results of a spike test using olive oil AL as a blank sample are shown in Table 2 below. The method of the present invention was found to have a good recovery rate.

[0107] Examples 1 to 3 above demonstrate that the present invention can rapidly detect Cu-pyropheophytin a in oil samples, and that the detection results have good accuracy and reliability. [Industrial applicability]

[0108] The detection method of the present invention can achieve rapid and accurate qualitative and quantitative analysis of Cu-pyropheophytin a in oil samples, and can be used industrially as a detection method for oil samples. Furthermore, it can be used industrially and commercially as a method for determining the presence or absence of copper chlorophyll in oil samples such as olive oil, and as a method for identifying counterfeit olive oil. [Explanation of symbols]

[0109] 11: Liquid-phase pump (supplies carbon dioxide); 12: Liquid-phase pump (supplies denaturant); 13: Autosampler; 14: Supercritical fluid column; 15: Back pressure control device; V: Hexagonal valve; 20: Liquid-phase pump (supplies the mobile phase for the liquid chromatograph); 21: Liquid-phase column (capture column); 22: Liquid-phase column (analytical column); 23:Mass spectrometer

Claims

1. The components of the oil sample are separated by supercritical fluid chromatography, and nonpolar components in the oil sample are removed so that Cu-pyropheophytin a remains in the column of the supercritical fluid chromatography. The process includes introducing a component containing Cu-pyropheophytin a eluted from the column of the supercritical fluid chromatograph into a reversed-phase liquid chromatograph, separating Cu-pyropheophytin a using the reversed-phase liquid chromatograph, and detecting it with a mass spectrometer. A method for detecting Cu-pyropheophytin a in an oil sample, The mobile phase in the supercritical fluid chromatograph comprises a supercritical fluid and a modifier. The aforementioned modifying agent adjusts the polarity of the supercritical fluid. The scan mode of the mass spectrometer is the multiple reaction monitoring mode. A method characterized by the following:

2. The method according to claim 1, characterized in that the supercritical fluid contains supercritical carbon dioxide, and the modifying agent contains an alcohol-based substance and / or a nitrile-based substance.

3. The method according to 1 or 2, characterized in that the stationary phase in the supercritical fluid chromatograph is selected from silica gel modified with a polar group selected from a diol group, an amino group, or a cyano group.

4. The method according to 1 or 2, characterized in that the mobile phase in the reversed-phase liquid chromatograph is selected from an alcohol-based substance, a nitrile-based substance, or an aqueous solution thereof.

5. The method according to 1 or 2, characterized in that the supercritical fluid chromatograph includes one supercritical fluid column, and the reversed-phase liquid chromatograph includes one or more reversed-phase liquid columns.

6. The method according to 1 or 2, characterized in that the Cu-pyropheophytin a remaining in the column of the supercritical fluid chromatograph is sent to a reversed-phase liquid chromatograph under the action of the denaturing agent, and the supercritical fluid from the supercritical fluid chromatograph is removed in the reversed-phase liquid chromatograph.

7. The method according to 1 or 2, wherein the reversed-phase liquid chromatograph includes a first reversed-phase liquid column as a capture column and a second reversed-phase liquid column positioned at the trailing end of the first reversed-phase liquid column as an analysis column, and the Cu-pyropheophytin a is detected after passing through the analysis column.

8. The method according to 1 or 2, characterized in that the ion source of the mass spectrometer is an atmospheric pressure chemical ionization ion source or an electrospray ion source.

9. The method according to 1 or 2, characterized in that the oil sample is olive oil.

10. The method according to 1 or 2, further comprising the step of diluting the oil sample before separating the components of the oil sample by supercritical fluid chromatography.

11. A method for determining the copper chlorophyll content in olive oil, characterized by comprising detecting the content of Cu-pyropheophytin a in an olive oil sample by the method according to any one of claims 1 to 10.

12. A method for identifying counterfeit olive oil, characterized by comprising detecting the content of Cu-pyropheophytin a in an olive oil sample by the method described in any one of claims 1 to 10.

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