Analytical method for steroid by liquid chromatography-mass spectrometry
Patent Information
- Application Number
- JP2023015154
- Authority / Receiving Office
- JP · JP
- Patent Type
- Applications
- Current Assignee / Owner
- Filing Date
- 2023-02-03
- Publication Date
- 2025-10-30
AI Technical Summary
Existing methods for analyzing steroid compounds in samples using liquid chromatography mass spectrometry face challenges in efficiently handling samples with varying steroid concentrations and require lengthy analysis times, especially when dealing with trace amounts.
A method involving fractionation of a sample into multiple liquid fractions, followed by different treatments on each fraction, mixing these fractions to form a combined sample solution, and analyzing it using liquid chromatography mass spectrometry, which includes using organic acid or salt buffers and appropriate solvents like ammonium formate and acetonitrile.
This approach reduces analysis time by allowing for targeted treatments of different steroid compounds, enhancing sensitivity and accuracy, particularly for trace compounds like estradiol, while minimizing interference from unreacted derivatization reagents.
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Abstract
Description
[Technical field]
[0001] The present invention relates to a method for analyzing a steroid compound in a sample using a liquid chromatography mass spectrometer. [Background technology]
[0002] Steroid compounds are a general term for compounds that have a structure called a steroid skeleton, which consists of three hexagonal rings and one pentagonal ring, and are known to exhibit various physiological activities depending on the type of side chain. There are various natural steroids in the body, such as sex hormones, steroid hormones such as adrenal steroids, bile acids, and cholesterol, which is an important lipid in the composition of cell membranes. In addition, many synthetic steroid drugs have been developed, such as dihydrotestosterone, a banned drug that is often used illegally in sports competitions.
[0003] Quantitative and qualitative measurements of steroids in the body have been conducted for clinical diagnosis, pathological analysis, and for the testing of prohibited drugs. Liquid chromatography-mass spectrometry (LC / MS or LC / MS / MS) has been widely used for steroid analysis because it can specifically and simultaneously analyze various steroids present in the body.
[0004] The amount of steroids present in the body varies widely, and for example, only trace amounts of estrogens (female hormones) such as estradiol are present in the bodies of children and postmenopausal women. In order to measure even such trace amounts of steroid compounds with high sensitivity, a method has been proposed in which only steroid compounds having a specific structure among the steroid compounds in a sample are derivatized and then analyzed (Patent Document 1).
[0005] The method disclosed in Patent Document 1 uses a derivatization reagent that specifically reacts with a steroid compound having one hydroxyl group to introduce an N-alkylpyridinium group into the hydroxyl group of the steroid compound to derivatize the steroid compound. When a sample contains a steroid compound having one hydroxyl group and a steroid compound having two or more hydroxyl groups, some of the steroid compounds are not derivatized even if the derivatization reagent is added to the sample. Since the derivatization reagent is an unnecessary component for LC / MS analysis of a steroid compound having two or more hydroxyl groups, in the past, a sample was divided into two, one sample was pretreated by adding the derivatization reagent, and the other sample was pretreated without adding the derivatization reagent, and each sample was subjected to LC / MS analysis.
[0006] However, when the amount of a sample is small, it is difficult to divide the sample into two or more pieces, and increasing the amount of the sample places a burden on the subject from whom the sample is taken. In addition, performing LC / MS analysis on each sample increases the time required for analysis. [Prior art documents] [Patent documents]
[0007] [Patent Document 1] JP 2003-161726 A Summary of the Invention [Problem to be solved by the invention]
[0008] The problem to be solved by the present invention is to shorten the time required for analyzing steroid compounds contained in a sample. It is. [Means for solving the problem]
[0009] The present invention, which has been made to solve the above problems, is a method for analyzing a steroid compound in a sample, comprising the steps of: A fractionation step of fractionating a sample containing a steroid compound into a plurality of liquid fractions; a processing step in which each of the plurality of liquid fractions is subjected to a different processing; a mixing step of mixing the plurality of liquid fractions after the treatment to prepare one mixed sample solution; an analysis step of analyzing the steroid compounds contained in the mixed sample solution by a liquid chromatograph mass spectrometer; It includes. Effect of the Invention
[0010] According to the present invention, when a single sample contains multiple types of steroid compounds, the steroid compounds can be fractionated into multiple liquid fractions, and the steroid compounds contained in each liquid fraction can be subjected to different processing, and the liquid fractions containing the processed steroid compounds can be combined into one and subjected to liquid chromatography mass spectrometry, thereby shortening the time required for analysis. [Brief description of the drawings]
[0011] [Figure 1] FIG. 1 is a schematic diagram of an embodiment of a high performance liquid chromatograph mass spectrometer for carrying out a steroid compound analysis method according to the present invention. [Diagram 2] FIG. 1 is a diagram showing a derivatization reaction of estradiol. [Diagram 3] FIG. 4 is a diagram showing a procedure for preparing a mixed sample solution. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0012] In the method of the present invention for analyzing steroid compounds in a sample, a sample containing steroid compounds is first fractionated into a plurality of liquid fractions, and each of the plurality of liquid fractions is subjected to a different treatment, after which the plurality of treated liquid fractions are mixed to prepare a mixed sample solution, and the steroid compounds contained in the mixed sample solution are analyzed by a liquid chromatograph mass spectrometer.
[0013] The samples to be analyzed in the above-mentioned analytical method include tissues, blood, urine, and other biological components suspended or diluted in water, an organic solvent, or a mixture thereof. The biological components can be collected from patients with a certain disease or from healthy individuals. The steroid compounds contained in the sample include natural steroids produced in vivo and synthetic steroids artificially synthesized, and either type of steroid compound can be the subject of analysis in the present invention. The steroid compounds contained in the mixed sample solution are substantially the same as the steroid compounds contained in the sample, but also include steroid compounds that have been derivatized by a specified treatment.
[0014] In the fractionation step of the above analytical method, it is ideal to fractionate a single sample so that each of the multiple liquid fractions contains a steroid compound; however, as a result of fractionation, there may be cases where some of the multiple liquid fractions do not contain steroid compounds. In the processing step, each of the multiple liquid fractions obtained in the fractionation step is subjected to a treatment appropriate for the steroid compounds expected to be contained in each of the liquid fractions.
[0015] Typically, in the fractionation step, the sample is fractionated into two liquid fractions, and the treatment performed on one liquid fraction includes a derivatization treatment of a steroid compound, while the treatment performed on the other liquid fraction does not include said derivatization treatment. In this case, therefore, a single sample is fractionated under conditions such that steroid compounds requiring derivatization and steroid compounds not requiring derivatization are contained in separate liquid fractions.
[0016] In the mixing step, when the multiple liquid fractions after the treatment are mixed to prepare one mixed sample solution, it is preferable that the mixed sample solution contains an organic acid or an organic acid salt buffer as a solvent.
[0017] Generally, organic acids refer to carboxylic acids including acetic acid, formic acid, oxalic acid, lactic acid, tartaric acid, citric acid, trifluoroacetic acid, etc., and as the organic acid salt buffer, for example, ammonium formate buffer or ammonium acetate buffer can be used. The organic solvent is not particularly limited, but typically acetonitrile can be used.
[0018] That is, in one embodiment of the present invention, the mobile phase may be a mixture of an ammonium formate buffer and acetonitrile, and in that case, it is preferable to perform a gradient analysis in which the concentration of ammonium formate is increased over time.
[0019] For example, ammonium formate contains positively charged ammonium ions and negatively charged formate ions, and contributes to the retention of oxyhalogen acid ions in the sample by the ion exchange function of the stationary phase in the column of a liquid chromatograph. In addition, since organic acid salt buffers such as ammonium formate are volatile salts, they are unlikely to cause problems such as precipitation even when introduced into, for example, the ESI ion source of a mass spectrometer. On the other hand, organic solvents such as acetonitrile are polar solvents, and contribute to the hydrophobic interaction of the stationary phase in the column (i.e., the function of the reversed phase), and further contribute to efficient ionization of sample molecules in, for example, the ESI source of a mass spectrometer.
[0020] FIG. 1 is a schematic diagram of a high performance liquid chromatograph mass spectrometer (HPLC / MS), which is an example of a mass spectrometer used in the analysis step of a steroid compound analysis method. In Fig. 1, a first liquid delivery pump 2 draws in mobile phase A from a first mobile phase container 1 and delivers it at a predetermined flow rate, and a second liquid delivery pump 4 draws in mobile phase B from a second mobile phase container 3 and delivers it at a predetermined flow rate. Mobile phase A and mobile phase B are mixed in a mixer 5 and delivered to a column 7 via an injector 6. In the injector 6, a liquid sample to be analyzed is injected into the mobile phase using a microsyringe or the like, and the liquid sample is carried along with the flow of the mobile phase and delivered to a column 71 in a column oven 7. Various components in the sample are separated as they pass through the column 71 and are eluted from the outlet of the column 71 with a time lag.
[0021] The eluate from the column 71 is sent to a mass spectrometer 8 as a detector via a switching valve 9, and is sprayed from a spray nozzle of an ESI ion source 81 into an atmosphere at approximately atmospheric pressure, and the component molecules contained in the eluate are ionized. The generated ions are focused by an ion lens 82, separated by a quadrupole mass filter 83 according to their mass-to-charge ratio, and reach an ion detector 84 where they are detected. The types of components contained in the eluate, that is, the types of components subjected to mass analysis, change with the passage of time. The quadrupole mass filter 83 is driven in an ion selection (SIM) mode so as to detect ions of one or more mass-to-charge ratios set in advance. Therefore, the detection signals obtained by the ion detector 84 reflect each component, and a data processing unit (not shown) creates a mass chromatogram corresponding to the target component, an oxyhalogen acid compound, based on the detection signal, and performs qualitative and quantitative analysis of the target component based on the peaks appearing in the chromatogram.
[0022] The ion source of the mass spectrometer 8 is not limited to that based on ESI, but may be that based on atmospheric pressure chemical ionization (APCI) or atmospheric pressure photoionization (APPI). The mass separator is not limited to a quadrupole mass filter, but may be a time-of-flight mass analyzer or the like. Furthermore, the mass spectrometer may be a mass spectrometer that performs MS / MS analysis or MS, such as a triple quadrupole mass spectrometer. n It may also be a mass spectrometer capable of performing the analysis. As the liquid chromatograph connected to the mass spectrometer, various liquid chromatographs can be used, such as a nanoflow liquid chromatograph, a microflow liquid chromatograph, a high-performance liquid chromatograph, an ultra-high-performance liquid chromatograph, etc. In short, it is advisable to use a suitable combination of a mass spectrometer and a liquid chromatograph according to the type and properties of the sample and steroid compound to be analyzed.
[0023] Next, the procedure for preparing a mixed sample solution prior to the analysis of steroid compounds in a biological sample will be described with reference to FIG. 3. The procedure shown below is an example of the procedure when estradiol, a steroid compound, is the subject of analysis. FIG. 2 shows the chemical structure of estradiol. Estradiol is a type of female hormone (estrogen) produced in the adrenal cortex and has two hydroxyl groups and one methyl group. Estradiol is converted into a derivative (NMP-E2) by introducing a 2-fluoro-1-methylpyridinium group into one of the two hydroxyl groups, and then analyzed by a mass spectrometer 8.
[0024] [Solid phase extraction] A solid-phase extraction column (or solid-phase extraction cartridge) is used to fractionate the steroid compounds contained in the sample into a plurality of liquid fractions. The solid-phase extraction column is made of a cartridge filled with a packing material as a solid phase. The packing material used is one that can separate the steroid compounds to be analyzed from the other steroid compounds contained in the sample solution, and for example, Water's mixed packing material of reversed phase and anion exchange systems (product name: Oasis MAX) can be used.
[0025] [Conditioning the solid phase extraction column: Step 101] Conditioning is performed to wet the packing material of the solid-phase extraction column and activate the functional groups of the packing material. Conditioning liquids used for conditioning include methanol, a mixed solution of 100 mmol / L ammonium bicarbonate / 25% ammonium water (50:2, v / v), 90% acetonitrile, a mixed solution of water / methanol / acetic acid (90:10:1, v / v / v), and the like. The operation of wetting the packing material with the conditioning liquid may be performed once or multiple times.
[0026] [Separation of steroid compounds from biological samples] Prior to separating the steroid compounds in the biological sample, a sample for separation is prepared (step 102). First, methanol and water / methanol / acetic acid in a volume ratio of 90:10:1 (v / v / v) are added to a container containing the biological sample, and the mixture is stirred with a vortex mixer. This results in a sample for separation. As the biological sample, tissue, blood, urine, bile, etc. collected from a subject can be used.
[0027] The sample to be separated is then loaded onto the packing material of the conditioned solid-phase extraction column (step 103). Next, an appropriate washing solution that matches the polarity of the steroid compounds and unnecessary components contained in the sample to be separated is passed through the packing material (wash) (step 104). This allows the unnecessary components adsorbed to the packing material to be discharged from the solid-phase extraction column. Examples of the washing solution that can be used include 25% methanol, 100 mmol ammonium bicarbonate / 25% aqueous ammonia (50:2 (v / v)), and water / 25% aqueous ammonia (95:5 (v / v)).
[0028] Next, an eluent is passed through the packing material of the solid-phase extraction column to elute some of the steroid compounds retained in the packing material (elution 1, step 105). In this case, an eluent is used that retains the target steroid compounds in the packing material or elutes the target steroid compounds from the packing material. Here, an eluent (hereinafter referred to as a first eluent) that retains the target steroid compounds including estradiol in the packing material is used. An example of such a first eluent is 90% acetonitrile. As a result, a fraction containing the target steroid compounds (target fraction) remains in the solid-phase extraction column, and a fraction not containing the target compounds (hereinafter referred to as a non-target fraction) is eluted from the solid-phase extraction column. Note that the target fraction may contain steroid compounds other than estradiol, but an appropriate first eluent is selected so that the non-target fraction does not contain estradiol.
[0029] Next, the eluted non-target fractions are collected (step 106) and evaporated to dryness at 40° C. using nitrogen gas (step 107). On the other hand, an eluent (second eluent) different from the first eluent is passed through the packing of the solid-phase extraction column in which the target fraction is held (elution 2, step 108), and the target fraction is eluted from the packing and collected (step 109). As the second eluent, for example, a solution prepared so that ACN:water:acetic acid=45:25:30 can be used. As the acetic acid, for example, 30% acetic acid can be used.
[0030] The collected target fraction is evaporated to dryness (step 110), and a derivatization reagent is added thereto (step 111). The derivatization reagent is a mixture of 2-fluoro-1-methylpyridinium p-toluenesulfonate (FMP-TS), triethylamine, and acetonitrile. The target fraction to which the derivatization reagent has been added is then heated at 40°C for 15 minutes (step 112). This results in the derivatization of estradiol. The derivatized target fraction is evaporated to dryness again (step 113), and the dried target fraction is mixed with the dried non-target fraction obtained in step 107, and 100 μL of water / acetonitrile (4:1 (v / v)) is added thereto to prepare a mixed sample solution (step 114).
[0031] The mixed sample solution obtained in step 114 is introduced into the above-mentioned HPLC / MS, and the steroid compounds in the mixed sample solution are analyzed simultaneously. Generally, the content of estradiol is low compared to other steroid compounds, and when estradiol is analyzed by mass spectrometry together with other steroid compounds, the sensitivity is low and the accuracy is poor. In contrast, in the above embodiment, estradiol is derivatized and then analyzed by mass spectrometry together with other steroid compounds, so that the content of estradiol can be measured with high sensitivity.
[0032] When the mixed sample solution is introduced into the HPLC / MS for analysis, during the time period when the unreacted derivatization reagent contained in the mixed sample solution is eluted from the column 71, the switching valve 9 may be switched so that the eluate is not sent to the mass spectrometer 8. If the unreacted derivatization reagent is introduced into the mass spectrometer 8 together with the steroid compound, the background increases, reducing the analytical sensitivity of the target component and causing the mass spectrometer 8 to become dirty, but such problems can be prevented by switching as described above.
[0033] [Various aspects] It will be appreciated by those skilled in the art that the above-described exemplary embodiments are illustrative of the following aspects:
[0034] (1) The method for analyzing steroid compounds according to the first aspect of the present invention comprises: A fractionation step of fractionating a sample containing a steroid compound into a plurality of liquid fractions; a processing step in which each of the plurality of liquid fractions is subjected to a different processing; a mixing step of mixing the plurality of liquid fractions after the treatment to prepare one mixed sample solution; an analysis step of analyzing the steroid compounds contained in the mixed sample solution by a liquid chromatograph mass spectrometer; It includes.
[0035] According to the steroid compound analysis method of paragraph 1, when a single sample contains multiple types of steroid compounds, the steroid compounds can be fractionated into multiple liquid fractions, and the steroid compounds contained in each liquid fraction can be subjected to different processing, and further, the liquid fractions containing the treated steroid compounds can be combined into one and subjected to liquid chromatography mass spectrometry, thereby shortening the time required for analysis.
[0036] (2) The steroid compound analysis method according to the second aspect of the present invention comprises: 2. The method for analyzing a steroid compound according to claim 1, In the processing step, the processing performed on one of the multiple liquid fractions may include a derivatization process for a steroid compound having a specific structure, and the processing performed on at least one of the remaining liquid fractions may not include the derivatization process.
[0037] The analytical sensitivity of a steroid compound having a specific structure can be improved according to the steroid compound analysis method of item 2. The derivatization treatment includes a treatment of replacing a specific structure with another structure, a treatment of replacing one or more elements contained in the specific structure with isotope elements, etc.
[0038] (Item 3) The method for analyzing a steroid compound according to item 3 is the method for analyzing a steroid compound according to item 1, In the fractionation step, the single sample is fractionated into two liquid fractions, In the processing step, the processing carried out on one of the two liquid fractions may include a derivatization treatment of estradiol, and the processing carried out on the other liquid fraction may not include the derivatization treatment.
[0039] According to the steroid compound analysis method of item 3, estradiol can be analyzed with high sensitivity even if the sample contains only a trace amount of estradiol.
[0040] (4) The method for analyzing steroid compounds according to the fourth aspect of the present invention comprises: The method for analyzing a steroid compound according to claim 1, wherein the fractionation step comprises fractionating the single sample into a plurality of liquid fractions by introducing the single sample into a solid-phase extraction column.
[0041] (Item 5) The method for analyzing steroid compounds according to item 5 is the method for analyzing steroid compounds according to item 4, wherein the solid-phase extraction column is an ion-exchange type solid-phase extraction column.
[0042] (Item 6) The method for analyzing steroid compounds according to Item 6 comprises: In the steroid compound analysis method according to claim 5, the solvent of the mixed sample solution contains an organic acid or an organic acid salt. [Explanation of symbols]
[0043] 1…First mobile phase container 2…First liquid delivery pump 3…Second mobile phase container 4…Second liquid delivery pump 5…Mixer 6…Injector 7. Column 8...Mass spectrometer 81...ESI ion source 82…Ion lens 83...Quadrupole mass filter 84…Ion detector
Claims
1. 1. A method for analyzing a steroid compound in a sample, comprising: A fractionation step of fractionating a sample containing a steroid compound into a plurality of liquid fractions; a processing step in which each of the plurality of liquid fractions is subjected to a different processing; a mixing step of mixing the plurality of liquid fractions after the treatment to prepare one mixed sample solution; an analysis step of analyzing the steroid compounds contained in the mixed sample solution by a liquid chromatograph mass spectrometer; A method for analyzing a steroid compound, comprising:
2. A steroid compound analysis method as described in claim 1, wherein the treatment performed on one of the multiple liquid fractions in the processing step includes a derivatization treatment of a steroid compound having a specific structure, and the treatment performed on at least one of the remaining liquid fractions does not include the derivatization treatment.
3. In the fractionation step, the single sample is fractionated into two liquid fractions, The steroid compound analysis method according to claim 1, wherein in the processing step, the processing performed on one of the two liquid fractions includes a derivatization treatment of estradiol, and the processing performed on the other liquid fraction does not include said derivatization treatment.
4. The method for analyzing a steroid compound according to claim 1, A steroid compound analysis method, wherein the fractionation step comprises fractionating the single sample into a plurality of liquid fractions by introducing the single sample into a solid-phase extraction column.
5. The method for analyzing a steroid compound according to claim 4, A method for analyzing a steroid compound, wherein the solid-phase extraction column is an ion-exchange type solid-phase extraction column.
6. The method for analyzing a steroid compound according to claim 5, A method for analyzing a steroid compound, wherein the solvent of the mixed sample solution contains an organic acid or an organic acid salt.