CN111896633B - Analysis system - Google Patents

Analysis system Download PDF

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CN111896633B
CN111896633B CN201910367788.2A CN201910367788A CN111896633B CN 111896633 B CN111896633 B CN 111896633B CN 201910367788 A CN201910367788 A CN 201910367788A CN 111896633 B CN111896633 B CN 111896633B
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supercritical fluid
column
way valve
phase liquid
phase
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CN111896633A (en
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郭彦丽
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Shimadzu Corp
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Shimadzu Corp
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/281—Sorbents specially adapted for preparative, analytical or investigative chromatography
    • B01J20/282—Porous sorbents
    • B01J20/283—Porous sorbents based on silica
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/281—Sorbents specially adapted for preparative, analytical or investigative chromatography
    • B01J20/286—Phases chemically bonded to a substrate, e.g. to silica or to polymers
    • B01J20/287—Non-polar phases; Reversed phases
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/281—Sorbents specially adapted for preparative, analytical or investigative chromatography
    • B01J20/286—Phases chemically bonded to a substrate, e.g. to silica or to polymers
    • B01J20/288—Polar phases
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/16—Injection
    • G01N30/20—Injection using a sampling valve
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/26—Conditioning of the fluid carrier; Flow patterns
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/26—Conditioning of the fluid carrier; Flow patterns
    • G01N30/28—Control of physical parameters of the fluid carrier
    • G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/26—Conditioning of the fluid carrier; Flow patterns
    • G01N30/38—Flow patterns
    • G01N30/46—Flow patterns using more than one column
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/26—Conditioning of the fluid carrier; Flow patterns
    • G01N30/38—Flow patterns
    • G01N30/46—Flow patterns using more than one column
    • G01N30/461—Flow patterns using more than one column with serial coupling of separation columns
    • G01N30/463—Flow patterns using more than one column with serial coupling of separation columns for multidimensional chromatography
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/62—Detectors specially adapted therefor
    • G01N30/72—Mass spectrometers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/62—Detectors specially adapted therefor
    • G01N30/72—Mass spectrometers
    • G01N30/7233—Mass spectrometers interfaced to liquid or supercritical fluid chromatograph
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/62—Detectors specially adapted therefor
    • G01N30/74—Optical detectors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N2030/022—Column chromatography characterised by the kind of separation mechanism
    • G01N2030/027—Liquid chromatography

Abstract

The invention relates to an analysis system, which is suitable for automatic and rapid analysis and detection of fat-soluble components, and comprises the following working paths: the first working channel enables the supercritical fluid chromatographic column to be communicated with the detection instrument or the waste liquid/waste gas outlet through the first multi-way valve group and the second multi-way valve group; a second working path communicating the supercritical fluid chromatography column with one or more reverse phase liquid chromatography columns through the first multi-way valve block; and the one or more reverse phase liquid chromatographic columns are communicated with the detection instrument through the first multi-way valve bank and the second multi-way valve bank, the communication between the one or more reverse phase liquid chromatographic columns and the supercritical fluid chromatographic column is cut off, and the working passages are switched through the switching of the first multi-way valve bank and the second multi-way valve bank.

Description

Analysis system
Technical Field
The invention relates to an analysis system, which is suitable for separating fat-soluble components from grease or biological samples, in particular suitable for separating fat-soluble vitamin components from grease or biological samples, and belongs to the field of detection (qualitative and/or quantitative analysis) of natural substances.
Background
The separation of fat-soluble components from some oils and fats or biological samples has been a hot point of research, and various separation and extraction devices and methods have been studied in the prior art.
For the analysis of fat-soluble components, in general, in view of the relatively complicated situation, for the analysis of a complex sample rich in oil, the prior art still needs to perform tedious and time-consuming pretreatment including saponification, solvent extraction, semi-preparation and the like. For example, cited reference 1 discloses an on-line two-dimensional liquid chromatography method for simultaneously determining vitamin A, D in infant and adult formulas 3 And E, both the first and the two-dimensional separation being based on reverse phase chromatography. After saponification and extraction, the sample is directly subjected to sample injection analysis, and quantitative analysis of each vitamin in the sample can be completed on line by one-time sample injection. However, the process of washing samples extracted from an oily or lipidic matrix using RPLC will result in some being strongly hydrophobicThe fat soluble impurities remain in the RPLC column. Impurities accumulate in the column, which can lead not only to a change in the retention time of the test substance, but also to a reduction in the lifetime of the clean column.
On the other hand, high performance liquid chromatography/ultra high performance liquid chromatography-mass spectrometry (HPLC/UPLC-MS) has been developed for the analysis of fat soluble components in oily vitamin supplements, infant formula or biological fluids using a simpler sample preparation method. However, such methods still typically require an off-line solvent extraction process or a normal phase Solid Phase Extraction (SPE) process to pre-treat or clean up the sample. The citation 2 relates to the establishment of a rapid and sensitive method capable of simultaneously determining fat-soluble vitamins A, D in infant formula milk powder by using UPLC-MS/MS 2 、D 3 And an analysis method of alpha-tocopherol. However, the sample to be tested still needs to be subjected to treatment with an alkaline inorganic substance and extraction with an organic solvent, and there is room for improvement or improvement in the easiness of pretreatment.
Furthermore, in order to obtain a highly efficient automated analysis system, an on-line purification method based on normal phase chromatography is still required. The separated and purified sample can then be analyzed using reverse phase liquid chromatography to provide better resolution. However, due to the mismatch of the two different types of mobile phases, it is difficult to construct a column switching system to include both normal phase and reverse phase liquid chromatography.
In addition, with the development of supercritical fluid technology, extraction or separation of substances using supercritical extraction or supercritical fluid chromatography has also been attempted. Have similar retention times and separation behavior as NPLC in supercritical technology for vitamin D separation and analysis. Reference 3 discloses the separation of vitamin D from mixtures with other components such as dehydrocholesterol, tachysterol and photosterol by column chromatography 3 Or precursor vitamin D 3 The method of (3), wherein the mobile phase in the chromatographic column is supercritical carbon dioxide. But this process only involves isolation or purification after synthesis of the vitamin D product.
It is seen that, in the prior art, improvement in convenience, environmental friendliness, rapidity, and the like of quantitative or qualitative analysis application of a fat-soluble component in a sample having a complex composition is not sufficient.
In addition, there is a demand for versatility and simplicity of the analytical system, for example, although some detection systems can perform on-line purification and separation detection of grease samples containing complex substrates and impurities. However, for oil samples with relatively simple raw materials or matrix compositions with less impurities and less interfering impurities, complicated purification is not usually required, and the oil samples can be directly analyzed under normal phase chromatography. In this case, it may not be necessary to use a complicated separation procedure for the above system, and thus, it is necessary to newly split and combine a complicated analysis system, which is time-consuming and labor-consuming.
Therefore, when analyzing the raw material containing fat-soluble components and the grease sample, the system needs to be modified correspondingly according to the complexity of the sample matrix so as to adapt to different requirements. Thus, not only is the workload increased, but also the equipment and the chromatographic column are worn during repeated disassembly and assembly, which is not favorable for long-term stable use of the equipment.
Cited document 1:
simultaneous determination of vitamin A, D in infant and adult formula nutriment by on-line two-dimensional liquid chromatography 3 And E ", zhang Yanhai, chromatography, 2015 3 months
Cited document 2:
research on simultaneous determination of multiple vitamins in infant formula milk powder by ultra-high performance liquid chromatography/supercritical fluid chromatography-tandem mass spectrometry combined technology, thought of Quansi, master academic thesis of Guangdong pharmaceutical university, 2017
Cited document 3: CN1144782C
Disclosure of Invention
Problems to be solved by the invention
Aiming at the problems of complex pretreatment process, large solvent consumption, lack of convenience for switching between chromatographic analysis and the like of a method for extracting fat-soluble components, particularly fat-soluble components from a system with complex components such as fat or biological samples and the like and an analysis system in the prior art, or the problem of lack of simple operation for processing a system with complex components or simple components in the prior art, the invention provides a method for rapidly analyzing fat-soluble components, particularly fat-soluble vitamins and the like in fat or biological samples and an analysis system by adopting a multidimensional chromatographic system.
Means for solving the problems
In the invention, the following scheme is used for solving the technical problem:
[1] the present invention firstly provides a multimodal analysis system for detecting fat-soluble components in a fat or a biological sample, the multimodal analysis system comprising:
a multi-dimensional chromatography system comprising a supercritical fluid chromatography column, one or more reverse phase liquid chromatography columns;
a supercritical fluid supply pump for supplying a supercritical fluid to the supercritical fluid chromatography column;
a liquid phase pump for delivering a modifying agent to the supercritical fluid chromatography column and a mobile phase to the one or more reverse phase liquid chromatography columns;
the first multi-way valve group and the second multi-way valve group; and a detection instrument for detecting the position of the object,
the analysis system comprises the following working paths:
the first working channel enables the supercritical fluid chromatographic column to be communicated with the detection instrument or the waste liquid/waste gas outlet through the first multi-way valve group and the second multi-way valve group;
a second working path communicating the supercritical fluid chromatography column with one or more reverse phase liquid chromatography columns through the first multi-way valve block;
a third working channel, wherein the one or more reverse phase liquid chromatographic columns are communicated with the detection instrument through the first multi-way valve group and the second multi-way valve group, and the communication between the one or more reverse phase liquid chromatographic columns and the supercritical fluid chromatographic column is cut off,
the working passage is switched by switching the first multi-way valve group and the second multi-way valve group.
[2] The system of [1], wherein the detection instrument is a mass spectrometer.
[3] The system according to [1], wherein a back pressure control unit is arranged between the first multi-way valve group and the second multi-way valve group in the first working channel.
[4] The system according to [1], wherein the modifier is selected from alcohols, nitriles or aqueous solutions thereof.
[5] The system according to [1], wherein the supercritical fluid chromatographic column and one or more reversed phase liquid chromatographic columns in the multidimensional chromatographic system are connected or switched through the first multi-way valve group.
[6] The system of [1], wherein the supercritical fluid chromatography column:
the stationary phase is selected from silica gel modified by polar groups, and the polar groups are selected from hydroxyl, amino or cyano;
the mobile phase comprises a supercritical carbon dioxide fluid.
[7] The system of [1], wherein:
the stationary phase is selected from silica gel modified by hydrophobic groups, and the hydrophobic groups are selected from alkyl;
the mobile phase is a polar organic solvent or an aqueous solution thereof.
[8] The system of [1], placing at least the supercritical fluid chromatography column in a column oven.
[9] The system according to [1], further comprising a pretreatment column for separating a protein component in a sample to be tested.
[10] The system according to [1], wherein the fat-soluble ingredient is a fat-soluble vitamin.
[11] The method for detecting fat-soluble components in the grease or biological sample by using the system in any one of the items [1] to [10], wherein the first working channel is communicated, the nonpolar substance of the sample to be detected is eluted in the supercritical fluid chromatographic column, the weakly polar substance is retained by the stationary phase of the supercritical fluid chromatographic column, and the weakly polar substance is conveyed to a detection instrument for detection after the retention time.
[12] A method for detecting fat-soluble components in grease or biological samples by using the system as in any one of [1] to [10], wherein the first working channel is communicated, nonpolar substances of samples to be detected are eluted in a supercritical fluid chromatographic column, weak polar substances are retained by a stationary phase of the supercritical fluid chromatographic column and then switched to the second working channel, further, the weak polar substances retained by the supercritical fluid chromatographic column are conveyed to an inverse liquid chromatographic column only under the action of the modifier, a supercritical fluid phase flowing out of the supercritical fluid chromatographic column is removed from the inverse liquid chromatographic column, and then the system is switched to the third working channel to separate and detect the weak polar substances.
[13] The method according to [12], wherein the multidimensional chromatography system comprises a reverse phase liquid chromatography column into which the weakly polar substance retained by the supercritical fluid chromatography column is transferred only by the action of the modifier, and in which the supercritical fluid phase flowing out of the supercritical fluid chromatography column is removed, and then the separation of components is performed by the weakly polar substance.
[14] The method according to [12], wherein the multi-dimensional chromatography system comprises two reversed-phase liquid chromatography columns, wherein the weakly polar substance retained by the supercritical fluid chromatography column is transferred to the first reversed-phase liquid chromatography column only by the modifier, and the supercritical fluid phase flowing out from the supercritical fluid chromatography column is removed in the reversed-phase liquid chromatography column,
the less polar substance is then transferred to a second reverse phase liquid chromatography column for separation of the components.
ADVANTAGEOUS EFFECTS OF INVENTION
Through the implementation of the technical scheme of the invention, the following beneficial effects can be obtained:
the multi-way valve group is switched to switch different working paths, so that different working modes can be converted, simple and convenient switching in different working paths is allowed, and convenient and quick switching among different chromatographic columns in one working path is also allowed, so that multiple working modes are realized in one analysis system, samples to be detected with different compositions are dealt with, and the fat-soluble components are quickly and accurately analyzed and detected.
The analysis system provided by the invention can provide a plurality of working paths, and through simple and convenient switching of the working paths, in one system, not only can the detection of a system containing fat-soluble components and more complex interference factors be processed, but also the system containing the fat-soluble components and more simple interference factors can be processed, so that repeated disassembly, assembly and arrangement of devices in the system for dealing with different samples to be detected in the prior art are avoided.
In the present invention, for the analysis and detection of fat-soluble components, particularly the detection of various fat-soluble vitamins from fats and oils or biological samples, there is no need to perform complicated pretreatment of the samples, and therefore, there is no need to extract fat-soluble components using a large amount of solvent as in the conventional pretreatment process.
In the multi-dimensional chromatographic system provided by the invention, the supercritical fluid chromatography and the reversed-phase liquid chromatography can realize direct switching between chromatographic columns of different types, and the defect that the mobile phase is incompatible when the chromatographic columns of different types are combined under the common condition is avoided.
Furthermore, the method or the analysis system provided by the invention can rapidly and accurately realize the analysis and detection of fat-soluble components, especially the components in grease or biological samples, such as fat-soluble vitamins, and particularly can be operated in a highly automated manner, thereby greatly improving the analysis efficiency and reducing the analysis cost.
In addition, when fat-soluble components, particularly vitamin D components are detected according to the method disclosed by the invention, the detection result has a good linear correlation coefficient, and the method is good in reproducibility and high in recovery rate.
Drawings
FIG. 1: a schematic of a vitamin D analysis system in one embodiment of the invention;
FIG. 2 is a schematic diagram: modifier versus CO in SFC process 2 Effect of dosage on retention behavior of different chromatography columns;
FIG. 3: the effect of different contents of methanol in the glycol-based column on retention behavior;
FIG. 4: the effect of different contents of methanol in the C18 column on retention behavior;
fig. 5 and 6: the detection analysis result of the standard sample;
FIG. 7: oily liquid droplet vitamin D 3 The analysis of (2);
FIG. 8: the working channel a1 in the system is used for directly detecting the result of the sample.
Description of the reference numerals
11: supercritical fluids (CO) 2 ) The supply pump 12: methanol liquid phase pump 13: automatic sample injector
14: supercritical fluid chromatography column (diol group) 15: detector (diode array detector)
16: pressure control unit (backpressure control unit)
21: methanol liquid phase pump 22: reversed phase liquid chromatography column (C18)
23: reversed phase liquid chromatography column (C18)
24: detecting instrument (Mass spectrum detector)
41-46: six-way valve group V1
51-56: six-way valve group V2
W: waste liquid/waste gas outlet
Detailed Description
The present invention will be described in detail below. The technical features described below are explained based on typical embodiments and specific examples of the present invention, but the present invention is not limited to these embodiments and specific examples. In the present specification, the numerical range represented by the expression "numerical value a to numerical value B" means a range including the endpoint numerical value A, B.
The use of "%" means volume percent, i.e., "V%" unless otherwise specified.
The term "communicate" as used herein means that a plurality of devices or parts can form a passage through which a mobile phase, a component to be measured, flows.
The switching in the invention refers to that the originally communicated channel is cut off through the switching of the multi-way valve group, and the originally cut-off devices or parts are communicated.
The supercritical fluid chromatography column, the one or more reversed phase liquid chromatography columns, involved in the multi-dimensional chromatography system of the invention are, during operation of the system, in self-evident communication with a pump feeding the mobile phase to provide the respective mobile phase or modifier in the mobile phase in the respective chromatography apparatus.
The invention provides a multi-mode analysis system and a method for rapidly detecting fat-soluble components, in particular fat-soluble components in a grease sample or a biological sample, wherein the multi-mode analysis system comprises:
an optional autosampler device;
a multi-dimensional chromatography system comprising a supercritical fluid chromatography column, one or more reverse phase liquid chromatography columns;
a supercritical fluid supply pump for supplying a supercritical fluid to the supercritical fluid chromatography column;
a liquid phase pump for delivering a modifying agent to the supercritical fluid chromatography column and a mobile phase to the one or more reverse phase liquid chromatography columns;
a first multi-way valve bank and a second multi-way valve bank; and a detection instrument for detecting the position of the object,
the analysis system comprises the following working paths:
the first working channel enables the supercritical fluid chromatographic column to be communicated with the detection instrument or the waste liquid/waste gas outlet through a first multi-way valve bank and a second multi-way valve bank;
a second working path, which enables the supercritical fluid chromatographic column to be communicated with one or more reversed-phase liquid chromatographic columns through a first multi-way valve group;
a third passage which leads the one or more reversed-phase liquid chromatographic columns to be communicated with the detecting instrument through the first multi-way valve group and the second multi-way valve group and cuts off the communication between the one or more reversed-phase liquid chromatographic columns and the supercritical fluid chromatographic column,
the working passage is switched by switching the first multi-way valve group and the second multi-way valve group.
< detection object >
The object to be detected may be various fat-soluble components, and the fat-soluble component to which the present invention is directed is not particularly limited in principle.
In some embodiments of the invention, the fat-soluble ingredients may include various fat-soluble vitamins, such as fat-soluble vitamin a, vitamin D, vitamin E, and/or vitamin K, among others. In some preferred embodiments of the invention, the test subject of the invention may be, inter alia, vitamin D, and the invention is particularly suitable for the testing of dietary supplements or nutraceuticals containing vitamin D, or infant formulas or foods. Therefore, the present invention will be described below with vitamin D as a specific representative of the fat-soluble component.
In some embodiments of the invention, these vitamin D-containing materials may be solid, or may be present in the form of a liquid or oily paste.
In some embodiments of the invention, the test subjects contain an amount of a component that interferes with the lipid-soluble component to be tested, and in other embodiments of the invention, the test subjects are substantially free of a component that interferes with the lipid-soluble component to be tested.
For these samples described above, in some embodiments, optional pre-treatment may or may not be performed. The pretreatment means is not particularly limited as long as the content of vitamin D in the sample to be tested is not impaired. The fine powder may be obtained by drying, grinding or the like to facilitate dissolution of the sample, or a pretreatment such as saponification or enzymatic hydrolysis may be used for milk powder or rice powder, or a simple organic solvent extraction may be directly performed on the sample for some oily components to extract vitamin D and other components therein into an organic solvent.
The sample solution to be measured is prepared by dissolving or diluting an optionally pretreated sample with an organic solvent. In the present invention, the organic solvent may be selected from hydrocarbon solvents, ketone solvents, ether solvents, and the like. In a preferred embodiment, a hydrocarbon solvent such as n-hexane or the like may be used.
The sample solution to be detected obtained after the dissolution is finished can be added into the detection equipment through an automatic sample injector.
In addition, in some other embodiments of the present invention, the protein component in the sample may be removed by chromatography or the like in the sample. For example, since a protein component in a sample is removed in advance by using a pretreatment column, and then a vitamin D component in the sample is separated and detected, the present invention can also detect a biological sample containing vitamin D, for example, blood.
< mode of operation >
The analysis system can realize different working modes in the same system through the switching of the multi-way valve bank. Specifically, different working channels are formed in the analysis system through switching of the first multi-way valve group and the second multi-way valve group, so that analysis of multiple samples to be tested is simply and conveniently realized, and repeated disassembly or reassembly of each testing device or component in the analysis system is not needed. In some specific embodiments of the invention, the first multi-way valve block is designated as multi-way valve block V1 and the second multi-way valve block is designated as multi-way valve block V2.
First working path
In the invention, the first working channel can be used for detecting or preprocessing the sample by switching the multi-way valve groups V1 and V2. Specifically, under the working channel, the automatic sample injection device is communicated with the supercritical fluid chromatographic column, and the supercritical fluid chromatographic column is communicated with the detection instrument or the waste liquid/waste gas outlet through the multi-way valve group V1 and V2. Meanwhile, a back pressure control unit (BPR) is provided between the multi-port valve groups V1 and V2.
As shown in fig. 1, the first working path of the present invention may include a working path a1 and a working path a2.
The working channel a1 communicates the automatic sample injector 13, the supercritical fluid chromatographic column, the multi-way valve group V1 (interfaces 41 and 46), the pressure control unit 16, the multi-way valve group V2 (interfaces 51 and 56) and the detection instrument (mass spectrometer);
the working channel a2 communicates the autosampler 13, the supercritical fluid chromatography column, the manifold block V1 (41 and 46 interfaces), the optional diode array detector 15, the pressure control unit 16, the manifold block V2 (51 and 52 interfaces), and the waste/exhaust outlet.
In some embodiments of the present invention, the working channel a1 can be used to directly detect lipid-soluble components, such as vitamin D, in a sample to be tested. Such a sample to be tested has a relatively simple composition and contains substantially 70 mass% or 80 mass% or 90 mass% or more of such a fat-soluble component. In other embodiments, the content of the component capable of interfering with the testing of the fat-soluble component to be tested in the sample to be tested is less, or such interfering component is substantially absent.
In other specific embodiments of the present invention, when the composition of the sample to be tested is complex and the content of components that can interfere with the testing of the fat-soluble components is significant, the non-polar materials in the sample to be tested are eluted in the working channel a2, and then the working channel a2 is switched to the second working channel and the third working channel for separation and detection through the switching of the multi-way valve sets V1 and V2. Optionally, the degree of elution of the non-polar substance may be detected by the PDA described above, and the working channel a2 is switched to the second working channel before the non-polar substance is eluted and the retention time of the less polar substance is reached.
The second working path
According to the invention, when the vitamin D in the sample to be detected is detected, the content of components which can cause interference to the vitamin D test in the sample to be detected is obvious, and the first working channel is required to be processed and then switched to the second working channel through the multi-way valve bank. In some preferred embodiments, the working channel a2 of the first working channel is switched to the second working channel by means of the group of multi-way valves V1.
As mentioned above, the non-polar substance is eluted in the working channel a2, which is similar to the pretreatment of the sample to be tested, and the low-polar substance is retained on the supercritical fluid chromatographic column at the end of the working channel a2. These weakly polar substances contain not only the fat-soluble components to be measured but also other components that interfere with these components to be measured. And after switching to the second working channel, the weakly polar substance is conveyed to one or more reverse phase chromatographic columns only through the modifier in the supercritical fluid chromatography.
After the weakly polar substance is delivered to the liquid reverse phase chromatography, the mobile phase CO derived from supercritical fluid chromatography in the system may preferably be delivered in the second working path 2 And (5) discharging.
Thus, in some preferred embodiments of the present invention, typically, as shown in fig. 1, the second working channel may be formed by communicating an autosampler 13, a supercritical fluid chromatography column 14, a multi-port set V1 (41 and 42 interfaces), an inverse liquid chromatography column 22, a multi-port set V1 (45 and 46 interfaces), an optional diode array detector 15, a pressure control unit 16, a multi-port set V2 (51 and 52 interfaces), and a waste/exhaust outlet.
The third working path
In the invention, when the weakly polar substances are conveyed to one or more reversed-phase liquid chromatographs through a second working path and CO is removed 2 And then, the communication between the supercritical fluid chromatographic column and the reversed phase liquid chromatogram is cut off by switching the multi-way valve group V1, and the reversed phase liquid chromatographic column is communicated with the detection instrument by switching the multi-way valve group V2.
Therefore, in the present invention, the third working channel can be formed by connecting the reversed phase liquid chromatography column 22, the multi-port set V1 (interfaces 45 and 44), the optional reversed phase liquid chromatography column 23, the multi-port set V2 (interfaces 55 and 56), and the detection instrument (mass spectrometer detector). The mobile phase can be supplied to the third working passage through the multi-port valve group V1 (43 and 42).
The use of the plurality of operation paths will be described below with reference to the description of each part of the multimode analysis system of the invention.
< multidimensional chromatography System >
The multi-dimensional chromatographic system of the invention provides pre-cleaning and separation of the sample solution to be tested. The multi-dimensional chromatography system includes a supercritical fluid chromatography portion, and a reverse phase liquid chromatography portion.
Chromatography part of supercritical fluid
Supercritical Fluid Chromatography (SFC) uses a supercritical fluid as a mobile phase, and relies on the solvating power of the mobile phase to perform a chromatographic process for separation and analysis.
Supercritical fluid chromatography has the characteristics of both gas chromatography and liquid chromatography. The method can analyze samples with high boiling points and low volatility which are not suitable for gas chromatography, and has higher analysis speed and better conditions compared with high performance liquid chromatography. In the invention, the supercritical fluid chromatographic column can be used for pre-cleaning the sample solution, i.e. removing the nonpolar oil component in the solution in the sample to be detected.
The supercritical fluid chromatography column in the present invention is not particularly limited as long as the effects of the present invention can be satisfied, and in some embodiments of the present invention, the supercritical fluid chromatography column may be a packed column or a capillary column, and in other embodiments, packed Column Supercritical Fluid Chromatography (PCSFC) is preferred.
In some embodiments of the invention, the supercritical fluid chromatography column is placed in a column oven during use.
The stationary phase in the supercritical fluid chromatographic column is selected from polar group modified silica gel, and for polar group, hydroxyl, amino or cyano can be selected. In some preferred embodiments, it is preferable to use hydroxyl group as the modification group in consideration of the retention time of vitamin D that is required to be reserved when the sample solution is pre-washed using the supercritical fluid chromatography column, and more particularly, it is preferable to use a diol-based silica gel as the stationary phase in the supercritical fluid chromatography column in the present invention. It is bonded by organic silane containing 1,2-dihydroxypropyl functional group, and the stationary phase is porous spherical silica gel.
Since the mobile phase in the supercritical fluid chromatography column in the present invention can be selected from supercritical fluids, which are a state of a substance under the conditions of the critical temperature and the critical pressure or more, which is between a gas and a liquid. Suitable supercritical fluids may be supercritical carbon dioxide or supercritical ethane, etc. In some embodiments of the invention, the supercritical mobile phase is selected from supercritical carbon dioxide. The operating temperature and pressure are largely determined by the supercritical mobile phase selected. In the present invention, when supercritical carbon dioxide is used as the supercritical fluid phase, the operating temperature is 31 ℃ or higher, preferably 35 ℃ or higher, and the operating pressure is 7.3MPa or higher, preferably 7.5MPa or higher. The operating temperature is preferably 40 to 60 ℃ and the operating pressure is preferably 7.5 to 15MPa from the viewpoint of the solubility of the supercritical fluid in vitamin D, the retention time, and the operability.
In the invention, the modifier is used as a substance for adjusting the polarity of the supercritical carbon dioxide fluid. The modifier may be selected from alcohols or nitriles. As the alcohol substance, various aliphatic alcohols such as methanol and isopropyl alcohol; for nitriles, acetonitrile or the like can be used as a modifier. The amount of modifier (relative flow) may typically be 1% to 5% of the mobile phase. From the aspect of controlling the retention time of vitamin D, the amount of the modifier used is preferably 1.5% to 4%, more preferably 2% to 3%, and most preferably 2% to 2.5%. In addition, from the viewpoint of controlling the retention time, the purity of the modifier is 80% or more, preferably 90% or more, and more preferably 100%.
The mobile phase and the modifier of the invention may be fed to the chromatography column by a liquid phase pump. In some embodiments, the supercritical fluid chromatography column of the present invention can be placed in a column oven. In addition, as shown in FIG. 1, a diode array detector (PDA) and a pressure control unit (e.g., a back pressure control unit BPR) and an independent CO may be further disposed in cooperation with the supercritical fluid chromatography column 2 A supply pump. In some embodiments, a pressure control unit is disposed at an end of the supercritical fluid chromatography section,for removing mobile phase CO 2 The waste liquid can be further discharged through a waste liquid/waste gas outlet to which the multi-way valve block V2 is connected.
The supercritical fluid chromatograph used in the present invention is commercially available, and may be, for example, "Nexera UC" manufactured by shimadzu corporation (kyoto, japan).
After the sample solution is added to the supercritical fluid chromatographic part through the automatic sample feeding device, the supercritical fluid chromatographic part is started, the weak polar substances in the sample, such as vitamin D, are retained on the chromatographic column, and the nonpolar oil is quickly eluted from the chromatographic column.
In this step, the vitamin D in the sample can be directly detected by using supercritical fluid chromatography through the working channel a1 in the first working channel, or the vitamin D in the sample can be detected through the working channel a2+ the second working channel + the third working channel in the first working channel, in this case, the processing of the working channel a2 is equivalent to the completion of the rapid pretreatment or the pre-cleaning of the sample containing the vitamin D component, and the nonpolar oil component can be simply and conveniently removed.
Switching device
In the invention, the switching of the working channels and the connection between different chromatographs in the multi-dimensional chromatographic system are realized through the multi-way valve bank so as to form the first, second or third working channels.
In some embodiments of the present invention, when processing a sample with a high content of interfering components, the analysis system is switched to the second and third working paths through the manifold blocks V1 and V2 after the working path a2 is processed.
The manifold is not particularly limited, and various types of manifold for sample introduction which are conventional in liquid chromatography can be used. Preferably, a six-way or ten-way valve set is used in the present invention, and more preferably a six-way valve set may be used. Different circulation or connection paths can be realized through different switching operations of the multi-way valve group, and the multi-way valve group is particularly suitable for connection and control among various devices.
In the present invention, the supply of the mobile phase to the supercritical fluid chromatography column is stopped at the time of or before the switching from the operation path a2 to the second operation path is performed. In this process, the supply of the modifier is not stopped, and after the switching is completed, the supply rate of the modifier may be increased, for example, to 0.5 to 1mL/min. At this time, the vitamin D-containing weakly polar substance remaining in the supercritical fluid chromatography column can be eluted by only the flow of the modifying agent, so as to be introduced into the reverse phase liquid chromatography section through the multi-way valve block V1 together with the supercritical carbon dioxide remaining in the system.
By the above switching, in the working path a2, the nonpolar ingredient in the sample to be measured is eluted using the supercritical fluid chromatography, and in the second working mode, the weakly polar substance containing vitamin D is transported to the reversed phase liquid chromatography section. When a plurality of reverse phase liquid chromatography columns are used in the system, the weakly polar substance containing vitamin D is transferred into the first reverse phase liquid chromatography column of the reverse phase liquid chromatography section by the switching described above.
Reversed phase liquid chromatography section
In the present invention, the reverse phase liquid chromatography section includes one or more reverse phase liquid chromatography columns. In some embodiments, these reverse phase liquid chromatography columns are placed in a column incubator at the time of use.
The stationary phase in the reversed phase liquid chromatography column of the present invention may be silica gel modified with hydrophobic groups, which may be various hydrocarbon groups, such as C8 group, C18 group, phenyl group, or the like. In some embodiments of the invention, a C18-modified silica gel is used as the stationary phase. As the mobile phase, a polar organic solvent or an aqueous solution thereof, such as an alcohol or nitrile, may be used. As the alcohol, various aliphatic alcohols such as methanol and isopropyl alcohol; for the nitrile substance, acetonitrile or the like can be used. In some embodiments, the mobile phase may be used in the form of an aqueous solution, for example, an aqueous solution using methanol. In the case of using as an aqueous solution, the content of the polar organic solvent in the mobile phase should be 60% or more, preferably 80% or more, more preferably 90% or more, from the viewpoint of shortening the retention time. In some preferred embodiments of the invention, the mobile phase in the reverse phase liquid chromatography is 90% or more methanol or 100% methanol.
In some embodiments of the invention, the reverse phase liquid chromatography portion may comprise only one chromatography column, in which case:
when the system is switched to the second working channel, the supercritical fluid chromatogram is connected in series with the reversed phase liquid chromatogram, and in some preferred embodiments, the reversed phase liquid chromatogram end can be connected with the pressure control unit through the multi-way valve group V1 switching or communicating, so that the mobile phase CO derived from the supercritical fluid chromatogram can be conveniently discharged when needed 2 。
As described above, after the switching of the chromatography column, the weakly polar components remaining in the supercritical fluid chromatography column are continuously washed by the modifier, and in some preferred embodiments, from the viewpoint of improving the elution efficiency, the flow rate of the modifier can be increased, so that the weakly polar components can be eluted from the supercritical fluid chromatography column more rapidly. And then the components and the supercritical fluid phase remained in the system enter a reversed phase liquid chromatographic column together. At this time, since the end of the reverse phase liquid chromatography column is already connected to the pressure control unit, the supercritical fluid phase in the system can be excluded from the system by the operation of the pressure control unit. In such a process, the state of other mobile phases or components is not affected. In this case, in the reversed-phase liquid chromatography, the mobile phase flowing out of the supercritical fluid chromatography is removed.
At the same time or after the supercritical fluid phase is removed, the analytical system can be switched to a third working channel by means of a multi-way valve block V1 and V2. The mobile phase was passed into the reverse phase liquid chromatography column using a liquid pump (shown as 21 in figure 1) for liquid chromatography. In some embodiments of the invention, the mobile phase of the reverse phase liquid chromatography may be the same as or different from the modifier in the supercritical fluid chromatography described above, and in preferred embodiments, both are the same, e.g., an aqueous methanol solution of greater than 80% or greater than 90% purity, or 100% methanol. In the invention, because the supercritical fluid dynamic phase from the supercritical fluid chromatography is removed, and only the weakly polar component to be separated and the modifier with polarity are reserved in the reversed phase liquid chromatography, the polar mobile phase can be directly introduced into the reversed phase liquid chromatography column, thereby solving the problem that the mobile phase is not matched when different types of chromatographic columns are switched.
Under the condition of a third working path, after the reversed phase liquid chromatographic column system is introduced with the polar mobile phase again, the separation of weak polar substances can be carried out, the vitamin D component is separated, and a detection instrument is used for detection and analysis. The vitamin D component herein contains vitamin D 2 And vitamin D 3 And (4) components.
In other embodiments of the invention, more than one column may be included in the reverse phase liquid chromatography portion. In typical embodiments, the reverse phase liquid chromatography portion may comprise two chromatography columns in series communication. In this case:
in the working mode a2, the supercritical fluid chromatography is used for separating components of a sample to be detected, after nonpolar oily substances are removed, the supply of a supercritical fluid dynamic phase is stopped, the modifier is continuously introduced, the multi-way valve group is used for switching the chromatographic columns in a mode of increasing the flow velocity of the introduced modifier, and the weak polar components containing the vitamin D and remaining in the supercritical fluid chromatographic column are conveyed to the first reversed phase liquid chromatographic column. Meanwhile, the mobile phase CO derived from the supercritical fluid chromatography may be discharged by communicating the first reversed phase liquid chromatography column with the pressure control unit, similarly to the case where only one reversed phase liquid chromatography column is used as described above 2 。
Discharge of CO 2 Thereafter, the first reversed-phase liquid chromatography column is disconnected from the pressure control unit and communicated with the second reversed-phase liquid chromatography column by switching through the multi-way valve group V1, and similar to the case of using only one reversed-phase liquid chromatography column as described above, the CO can be removed after the CO is removed 2 Then, a third working path is formed by switching the multi-way valve blocks V1 and V2.Starting a liquid phase pump, conveying a mobile phase to the first reversed phase liquid phase chromatographic column, further washing the weak polarity component retained in the first reversed phase liquid phase chromatographic column into the second reversed phase liquid phase chromatographic column after the first reversed phase liquid phase chromatographic column and the second reversed phase liquid phase chromatographic column are connected, and separating the vitamin D component in the second reversed phase liquid phase chromatographic column.
For the separation of vitamin D in the second reverse phase liquid chromatography, the analysis and detection can be performed using a detection instrument in series with the column, where the series is realized by a manifold V2.
The first and second reversed-phase liquid chromatography columns may be the same or different in terms of stationary phase material and column length. In a preferred embodiment of the invention, the two are different, e.g. the second reverse phase liquid chromatography column is longer than the first reverse phase liquid chromatography column. Such a design makes it easier for less polar substances to be washed in the first reversed phase liquid chromatography column before separation in the second reversed phase liquid chromatography column. Therefore, the first reverse phase liquid chromatography column can be regarded as a pretreatment column of the second reverse phase liquid chromatography column, and the main functions include enriching the weakly polar substance containing vitamin D and removing the supercritical fluid phase.
< detection apparatus >
In the analysis system of the present invention, a detection instrument is used to detect and analyze fat-soluble components, particularly vitamin D, in a sample. The detection device of the present invention is not particularly limited, and a mass spectrometer, a diode array detector, or the like can be used, for example. Preferably, a mass spectrometric detection instrument is used.
In the following, a preferred embodiment of the invention will be described with reference to the accompanying figure 1:
samples of infant vitamin D supplements were analyzed using the protocol of the present invention. An appropriate amount of this oily sample was taken and dissolved in n-hexane. Since the sample contains a large amount of interfering components for vitamin D detection, the detection can be performed by switching between different working pathways according to the present invention.
The sample is placed in the autosampler 13 and the supercritical fluid chromatograph is started to deliver supercritical carbon dioxide and the modifying agent to the chromatographic column 14 via the pump 11 and the liquid pump 12, respectively. At this time, the column 14 is connected to the diode array detector 15, the pressure control unit 16, 51 and 52 of the six-way valve V2, and the waste/off gas outlet through 41 and 46 of the six-way valve V1 in this order to form a working path a2. With the flow of the supercritical fluid phase, the non-polar oily component is washed out in the column 14, and the weakly polar component containing vitamin D is retained on the stationary phase of the column 14, thereby completing the pre-washing of the weakly polar component containing vitamin D. In addition, when the operation path a2 is performed, the reverse phase chromatography columns 22 and 23 may be pre-flushed by the communication of the six-way valve V1 using a liquid phase pump.
Before the retention time of vitamin D is reached, the system is switched using the six-way valve V1 while stopping the supply of the supercritical fluid, but without stopping the supply of the modifying agent and increasing the flow rate of the modifying agent. At the same time, the six-way valve V1 is disconnected between 41 and 46, and is connected between 41 and 42, and is connected between 45 and 46, forming a second working path which in turn contains the column 14, the column 22, the diode array detector 15, the pressure control unit 16, 51 and 52 of the six-way valve V2, and the waste/waste gas outlet. In this path, the flow rate increasing modifier transports the less polar component from the column 14 to the column 22. Further, the supercritical carbon dioxide remaining in the system is discharged through 51-52 in the six-way valve V2 through the waste/off-gas port by the pressure control unit 16. In some preferred embodiments, when the analysis system of the present invention is switched to the second working channel, the liquid pump 21 may be simultaneously connected to the multi-port set V1, 43 and 44, the reverse phase liquid chromatography column 23, and the multi-port set V2 to pre-flush the reverse phase liquid chromatography column 23 for better detection accuracy.
After carbon dioxide removal is completed, 41 and 42, 45 and 46, 42 and 43, 44 and 45, and 55 and 56 in the six-way valve are turned off, and the polar mobile phase is supplied to the column 22 by the liquid phase pump 21. This forms a third working channel containing two reverse phase liquid chromatography columns 22 and 23 in series. The less polar components are flushed from the column 22 through the line to the column 23 and the separation of the vitamin D components is completed in the column 23.
Further, at the end of the third working channel, a chromatography column 23 is connected to a mass spectrometer 24. Thereby realizing the analysis and detection of each component of the vitamin D.
In addition, in some embodiments of the present invention, the system further has a pretreatment column for separating protein components in a sample to be tested, for example, such pretreatment column may be placed before the supercritical fluid chromatography column described above.
In addition, in other embodiments of the present invention, only path a1 may be used in fig. 1 to perform direct detection on a sample to be detected by using supercritical fluid chromatography, and in general, the sample to be detected has a higher content of fat-soluble components, and after the retention time elapses, the component to be detected is delivered to a detection instrument for detection.
< System control >
As described above, the method provided by the present invention allows the simple online detection of different types of samples to be detected by switching different working paths in the same system, and avoids the need of recombining detection equipment and instruments for the detection of different samples to be detected.
The adjustment of various working channels of the invention can be manually adjusted, and can also be automatically controlled by using a method file of workstation software, and after a sample is loaded, the working channels or the combination thereof are automatically controlled according to the property of the sample, thereby realizing an automatic, simple and convenient online detection method. The automatic control software applicable to the system of the present invention is not particularly limited, and a control program commonly used in the art or a program written in situ depending on the actual condition of the device may be used.
Examples
The following examples of the present invention are described, but the present invention is not limited to the following examples.
Example 1
The effectiveness and the analysis accuracy of the analysis system are verified through the embodiment, and the system is used for carrying out quantitative analysis on a system which contains fat-soluble components and is complex in composition.
< chemicals and reagents >
Analytical standard compounds, vitamin D2 and vitamin D 3 Purchased from dr. Vitamin supplements (Baby Ddrops, batch No.: 187759, liquid Vitamin D) 3 400IU per drop, purchased from the Ddrops Company). Methanol (LC-MS grade) and n-hexane (HPLC grade) (SFC grade from Thermo Fisher Scientific). Carbon dioxide (CO) 2 The purity is more than or equal to 99.99 percent, and the purity is Beijing in China).
< Instrument >
The experiment was carried out by coupling the Nexera UC system with LC-MS 8050, manufactured by Shimadzu corporation, kyoto, japan. The UC system consists of a CBM-20A controller, an online DGU-20A degasser, an LC-30AD SF CO 2 Pump, LC-30AD modifier pump, SIL-30AC autosampler (with 5 μ L sample loop), CTO-20AC column oven, SPD M20A diode array detector (with high voltage cell) and an SFC-30A Back Pressure Regulator (BPR). In addition, a high-voltage switch six-way valve is arranged in the column incubator and used for switching the chromatographic columns.
SFC mode inspection of SFC columns (4.6 mm. Times.250 mm; 5 μm) for pre-separation, comprising three materials UC-X Silica, UC-X NH 2 And UC-X Diol (Diol group). Short C18 column (VP-ODS, 4.6 mm. Times.50 mm; 5 μm). A long C18 column (4.6 mm. Times.250 mm; 5 μm) was used to isolate vitamin D. Two reference columns, including a glycol-based column and a C18 column (4.0 mm. Times.10 mm; 5 microns), were examined for vitamin D retention times when different ratios of methanol were used in the mobile phase. All columns were purchased from Shimadzu-GL Sciences laboratory products, inc.
< preparation of Standard solution and sample solution >
Stock solutions of vitamin D standards were prepared in n-hexane as follows: d 3 1mg/mL; d21mg/mL. All stock solutions were stored at-30 ℃. N-hexyl for working standardDiluting the vitamin D standard stock solution to 10-200. Mu.g/L with an alkane.
One drop of Baby Ddrops (about 10 μ g per drop) was diluted with n-hexane, and vitamin D 3 The final concentration of (D) was about 100. Mu.g/L. Extruding vitamin AD drop from a soft capsule container, diluting with n-hexane, and diluting with vitamin D 3 The final concentration of (a) is about 125. Mu.g/L.
< SFC-LC/MS Handover System >
As shown in FIG. 1, the SFC and LC/MS are provided with a high pressure six way valve V1 which changes position between 0 and 1. In one analysis, the valve was changed twice and there were three steps as follows. Note that, the communication relationship of the six-way valve V2 is adaptively adjusted at the time of adjustment of the respective operation passages described below.
Pre-cleaning step with SFC (working channel a 2)
In this step, a vitamin D-containing sample is injected into the system and a mixture of supercritical carbon dioxide fluid and methanol is passed through the supercritical fluid chromatography column, the vitamin D and impurities being retained on the column and separated, respectively. The BPR provides a backpressure of 15MPa. In another aspect, two C18 columns were used for pretreatment and reverse phase separation, respectively, with a flow rate of 1ml/min methanol rinse.
Step of treating supercritical carbon dioxide on pretreatment reverse phase chromatography column (second working channel)
Before vitamin D elution from SFC column, switch valve from position 0 to 1 and stop CO 2 And (6) operating the pump. The mobile phase was left with only 0.5mL/min of methanol flow rate and the BPR back pressure was maintained at 15MPa. Mixing vitamin D and residual supercritical CO 2 A column of pretreated C18 (50 mm in length) was flushed from the SFC column. Vitamin D is retained in the pretreated C18 column, supercritical CO 2 The fluid is eluted from the column and drained. On the other hand, the reversed phase separation C18 column (250 mm long) was still flushed with methanol at a flow rate of 1mL/min.
Step of performing separation using reversed phase separation C18 column (third operation route)
Before the vitamin D is eluted from the pre-treated C18 column, the valve is switched back from position 1 to 0 and the vitamin D is flushed from the pre-treated C18 column into the reverse phase separation C18 column by methanol at a flow rate of 1mL/min. Vitamin D was separated in the reverse phase separation C18 column and then detected by MS/MS in communication with a six-way valve block V2. Data acquisition was performed in positive ion electrospray ionization (ESI) mode, with the following conditions:
capillary voltage: 4 kilovolts; interface temperature: 300 ℃; DL temperature: 250 ℃; temperature of the heating block: 400 ℃; flow rate of spray gas: 3L/min; heating air flow: 10L/min; flow rate of drying gas: 10L/min. Multiple Reaction Monitoring (MRM) means (vitamin D) were used in the process 2 m/z 397.30>69.10，397.30>271.20; vitamin D 3 m/z 385.30>259.20，385.30>367.30). On the other hand, the SFC column was flushed with supercritical fluid, which was the same as step 1, in preparation for the next analysis.
< results and discussion >
Optimization of switching conditions for SFC-LC/MS columns
In order to determine the effect of the type of chromatography column and mobile phase composition on the retention time of vitamin D, the retention behavior of vitamin D on SFC columns with different functional groups and the effect of the mobile phase composition on the retention time were investigated.
Optimization of SFC
Three supercritical fluid chromatographic columns (NH) with functional groups of different polarity 2 Diol and Silica) were used to study the retention behaviour of vitamin D.
As shown in fig. 2, the retention time of vitamin D increased as the proportion of modifier methanol on each column decreased. Under the condition of any methanol proportion, vitamin D 2 And D 3 The retention time of (a) is almost the same. And NH 2 The retention time of vitamin D on the diol-based column was the longest compared to the Silica column. Longer retention times mean that more time is available in the SFC column to wash away the non-polar lipid components without causing the dissolution or elution of the vitamin D components. Finally, a glycol column was selected and the modifier methanol flow was set at 2% (relative to supercritical CO) 2 Flow) was used for washing of vitamin D-containing oil or lipid samples.
CO 2 Effect of flowing phase composition in glycol-based column C18 column after supply stoppage
In CO 2 To determine the effect of the composition of the mobile phase in the glycol-based column and the C18 column after the supply was stopped, the effect of the ratio of methanol to water in the mobile phase on the retention behavior of the glycol-based column and the C18 column was investigated. Guard columns (10 mm long) were used to shorten the analysis time.
It can be seen that in CO 2 Increasing the percentage of methanol in the mobile phase in the supercritical fluid chromatography column after the supply has been stopped reduces vitamin D 3 Retention time on the diol-based column (fig. 3).
Thus, overall, modifier flow is maintained relative to supercritical CO during SFC 2 At lower flow rates and with high methanol levels (90% and 100% methanol) as modifier, longer vitamin D retention times can be achieved during SFC cleaning, while the vitamin D fraction can be eluted from the SFC column more quickly to pretreat the C18 column after the supercritical carbon dioxide supply is stopped.
Further, the retention behavior of the C18 column with different contents of methanol was also investigated. Although in the mobile phase, decreasing the methanol content increased vitamin D 3 But still high content methanol solutions (90% and 100% methanol) can be used as mobile phase in the C18 column, i.e. the modifier of SFC is made the same as the mobile phase composition in the C18 column, since high content of methanol not only enables a fast elution of vitamin D from the glycol based column 3 Can also ensure vitamin D 3 Sufficient retention time on the C18 column.
Thus, from the above analysis, it can be seen that both the modifier in the SFC column and the mobile phase used in the C18 column can be high in methanol.
After the modifier in the SFC column and the mobile phase in the C18 column were determined, the switching time from the SFC column to the pretreated C18 column was set to 8.5 minutes, and the switching time from the pretreated C18 column to the reverse phase separation C18 column was set to 17.5 minutes.
<For vitamin D 2 And D 3 Analysis of the Standard sample>
Contains vitamin D 2 And D 3 Is injected into the column switching system. As shown in FIG. 5, vitamin D 2 And D 3 Eluted from all three columns and detected by MS/MS at 24.9 min and 25.5 min, respectively. Vitamin D 2 And D 3 Are sharp, symmetrical, and are completely separated. A solvent peak was also detected at 20.0 minutes.
Further, five concentrations (20, 50, 100, 150 and 200. Mu.g/L) were used to construct a calibration curve (FIG. 6), and it can be seen that a curve having a good line shape with a linear correlation coefficient of 0.998 or more in the detection range of 20 to 200. Mu.g/L (Table 1) can be obtained. Vitamin D 2 And D 3 The detection limits of the standard samples were 20. Mu.g/L and 16. Mu.g/L, respectively.
TABLE 1 degree of linearity of vitamin D detection in column switching system
Figure BDA0002048792270000241
<Oily liquid droplet vitamin D 3 Analysis of (2)>
As shown in FIG. 7, infant vitamin D supplement Baby Ddrops were directly analyzed using the analysis system of the present invention. After dilution of the oily sample with n-hexane, direct injection into the analytical system gave good reproducibility (n =6, RSD =1.47%, where n is the number of tests and RSD is the standard deviation). And at the same time, a recovery rate of around 100% is obtained.
Example 2
In this example, analysis was performed on a supplement containing vitamin D, baby Ddrops, which has a simple composition, and this example relates only to the working channel a1 (SFC-MS) in the system, as compared with example 1.
In the step, a sample containing vitamin D is injected into the system, and a mixture of supercritical carbon dioxide fluid and methanol is introduced, so that the vitamin D sample passes through the supercritical fluid chromatographic column and is detected by a mass spectrometry detection instrument communicated with the working passage.
Figure 8 shows the results of the mass spectrometer instrument.
Industrial applicability
The detection method and the detection device can be used for fat-soluble component analysis, especially vitamin D component analysis in industrial production.

Claims (14)

1. A multimodal analysis system for detecting fat soluble components in a fat or biological sample, the multimodal analysis system comprising:
a multi-dimensional chromatography system comprising a supercritical fluid chromatography column, one or more reverse phase liquid chromatography columns;
a supercritical fluid supply pump for supplying a supercritical fluid to the supercritical fluid chromatography column;
a liquid phase pump for delivering a modifying agent to the supercritical fluid chromatography column and a mobile phase to the one or more reverse phase liquid chromatography columns;
the first multi-way valve group and the second multi-way valve group; and a detection instrument for detecting the position of the object,
the analysis system comprises the following working paths:
the first working channel enables the supercritical fluid chromatographic column to be communicated with the detection instrument or the waste liquid/waste gas outlet through the first multi-way valve group and the second multi-way valve group;
a second working path communicating the supercritical fluid chromatography column with one or more reverse phase liquid chromatography columns through the first multi-way valve block;
a third working channel, wherein the one or more reversed-phase liquid chromatographic columns are communicated with the detection instrument through the first multiway valve group and the second multiway valve group, and the communication between the one or more reversed-phase liquid chromatographic columns and the supercritical fluid chromatographic column is cut off,
the working passage is switched by switching the first multi-way valve group and the second multi-way valve group.
2. The system of claim 1, wherein the detection instrument is a mass spectrometer detector.
3. The system of claim 1, further comprising a back pressure control unit disposed in the first working channel between the first and second banks of multiport valves.
4. The system of claim 1, wherein the modifier is selected from the group consisting of alcohols, nitriles, and aqueous solutions thereof.
5. The system of claim 1, wherein the connection or switching of connections between the supercritical fluid chromatography column and the one or more reverse phase liquid chromatography columns in the multi-dimensional chromatography system is performed through the first multi-port valve bank.
6. The system of claim 1, wherein the supercritical fluid chromatography column comprises:
the stationary phase is selected from silica gel modified by polar groups, and the polar groups are selected from hydroxyl, amino or cyano;
the mobile phase comprises a supercritical carbon dioxide fluid.
7. The system of claim 1, wherein in the reverse phase liquid chromatography column:
the stationary phase is selected from silica gel modified by hydrophobic groups, and the hydrophobic groups are selected from alkyl;
the mobile phase is a polar organic solvent or an aqueous solution thereof.
8. The system of claim 1, wherein at least the supercritical fluid chromatography column is placed in a column oven.
9. The system of claim 1, further comprising a pre-treatment column for separating protein components from the sample to be tested.
10. The system of claim 1, wherein the fat-soluble ingredient is a fat-soluble vitamin.
11. A method for detecting fat-soluble components in a lipid or biological sample by using the system of any one of claims 1-10, wherein the first working channel is connected, non-polar substances of the sample to be detected are eluted in the supercritical fluid chromatographic column, weakly polar substances are retained by the stationary phase of the supercritical fluid chromatographic column, and the weakly polar substances are conveyed to the detection instrument for detection after the retention time.
12. A method for detecting fat-soluble components in a lipid or biological sample by using the system of any one of claims 1 to 10, wherein the first working channel is communicated, nonpolar substances of a sample to be detected are eluted in the supercritical fluid chromatographic column, and the weakly polar substances are switched to the second working channel after being retained by the stationary phase of the supercritical fluid chromatographic column, and further, the weakly polar substances retained by the supercritical fluid chromatographic column are transferred to the reversed phase liquid chromatographic column only under the action of the modifier, and the supercritical fluid phase flowing out of the supercritical fluid chromatographic column is removed in the reversed phase liquid chromatographic column, and then the system is switched to the third working channel, and separation and detection of the weakly polar substances are performed.
13. The method of claim 12, wherein the multi-dimensional chromatography system comprises a reverse phase liquid chromatography column into which the weakly polar substance retained by the supercritical fluid chromatography column is transferred only by the modifier, and in which the supercritical fluid phase flowing out of the supercritical fluid chromatography column is removed, and in which the separation of components is performed by the weakly polar substance.
14. The method of claim 12, wherein the multi-dimensional chromatography system comprises two reversed-phase liquid chromatography columns, wherein the weakly polar substance retained by the supercritical fluid chromatography column is transferred to the first reversed-phase liquid chromatography column only by the modifier, and the supercritical fluid phase flowing out of the supercritical fluid chromatography column is removed in the reversed-phase liquid chromatography column,
the less polar substance is then transferred to a second reverse phase liquid chromatography column for separation of the components.
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