CN113917008A - Application of product for detecting metabolite level in urine by mass spectrometry in preparation of product for early evaluation of intestinal polyp and colorectal cancer - Google Patents
Application of product for detecting metabolite level in urine by mass spectrometry in preparation of product for early evaluation of intestinal polyp and colorectal cancer Download PDFInfo
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Abstract
The invention discloses an application of a product for detecting metabolite levels in urine by mass spectrometry in preparation of products for early evaluation of intestinal polyps and colorectal cancer, wherein by using the product, a urine sample is subjected to impurity removal and then is directly introduced into a liquid chromatography tandem mass spectrometer for determination, so that the contents of N1, N12-diacetylspermine, dog urea and succinic acid in the urine can be rapidly and accurately detected. According to the invention, the corresponding standard substances of N1, N12-diacetyl spermine, dog urea and succinic acid are used as internal standard quantitative analytes, so that the error in the sample pretreatment process can be reduced; the selectivity and the accuracy of the method are improved by utilizing the liquid chromatography-tandem mass spectrometry; the selection of chromatographic column and elution condition shortens the analysis time. Meanwhile, a high-efficiency and feasible technical method is provided for the research and development of products for early diagnosis of adenomatous polyps and/or colorectal cancer.
Description
Technical Field
The invention relates to the technical field of clinical examination and diagnosis, in particular to application of a product for detecting the level of metabolites in urine by a mass spectrum in preparation of products for early evaluation of intestinal polyps and colorectal cancer.
Background
The world health organization proposed: one third of cancers can be prevented; one third of cancers can be cured by early screening findings; one third of the cancers can prolong life, relieve pain and improve life quality by using the existing medical measures. Currently, cancer prevention and control can be performed by three levels of prevention: the primary prevention is the disease cause prevention and reduces the damage of external adverse factors; the secondary prevention is early screening discovery, early diagnosis and early treatment; the third-level prevention is to improve the quality of life and prolong the survival time. According to the report of 'the national cancer center annual work report' in 2020, about 1930 ten thousand of newly added cancer cases are added in the world, and about 23% of China. In 2020, the number of cancer deaths worldwide is about 1000 million, and China accounts for about 30%. The incidence rate of colorectal cancer is the third (23.9 ten thousand/10 ten thousand people) and the death rate is the fifth (12.0 ten thousand/10 ten thousand people). The literature reports that more than 90% of colon cancers (colon cancer) develop from adenomatous polyps (adenomatous polyps). The number of patients who have been in the middle and late stages at the initial diagnosis of colorectal cancer in China is 80%, and the 5-year survival rate of colon cancer is greatly reduced as the cancer worsens. According to statistics, after conventional screening of cancers is carried out in the U.S. from 2001 to 2010, the death rate of intestinal cancer is reduced by 17.1%, only 3 of 10 intestinal cancer patients are subjected to conventional intestinal cancer screening in China due to death of the intestinal cancer, and 6 of 10 intestinal cancer patients are subjected to death of the intestinal cancer due to the disease. Thus, early screening, regular screening, and removal of polyps can greatly reduce the incidence of colorectal cancer.
In the process of tumor development and development, there are some specific biological metabolic processes. The metabonomics technology is utilized to monitor the change or fluctuation of metabonomics in the processes, so that the tumor progress can be diagnosed and predicted early. The previous methods for detecting metabolites of the human body are all radioactive labeling methods. However, radiolabelling has many limitations or problems, including (1) problems with radiation and contamination; (2) the scale of detection is limited, and high-throughput screening cannot be carried out. Liquid chromatography tandem mass spectrometry (LC-MS/MS) is widely applied to detection of proteins, amino acids or metabolites in biological matrixes such as blood, urine and the like due to high sensitivity and selectivity. By analyzing medium-complex metabolite samples with high mixing degree by LC-MS/MS, dozens to hundreds of compounds can be identified at one time, and high coverage rate is obtained. And directionally monitoring the metabolite groups in the urine sample to be detected through the constructed mass spectrum result information of the metabolites, so as to realize the quantitative detection of the contents of the metabolites in the urine.
There is currently no detection technique for simultaneous detection of liquid chromatography tandem mass spectrometry of N1, N12-diacetylspermine (change of the huxin ornithine metabolic pathway in colon cancer and application of multivariate data modeling in its diagnosis [ D ]. university of medical science, brocade, 2021), canine urea (zhangjianhexing, luxuemei, jamaici, chenbo, zhuanglan. high performance liquid chromatography-ultraviolet, fluorescent dual detector simultaneously measuring kynurenine and kynurenic acid [ J ]. test medicine in human urine 2009,24(09): 659-. The invention adopts a liquid chromatography-tandem mass spectrometry method to simultaneously detect the contents of N1, N12-diacetylspermine, dog urea and succinic acid in urine, and provides a high-efficiency and feasible technical method for the research and development of products for early diagnosis of adenomatous polyps and/or colorectal cancer.
Disclosure of Invention
The invention aims to overcome the defects in the prior art, and provides an application of a product for detecting the level of metabolites in urine by a mass spectrum in the preparation of products for early evaluation of intestinal polyps and colorectal cancer, wherein the urine sample is subjected to impurity removal and then directly introduced into a liquid chromatography tandem mass spectrometer for determination, so that the contents of N1, N12-diacetylspermine, dog urea and succinic acid in the urine can be quickly and accurately detected. According to the invention, the corresponding standard substances of N1, N12-diacetyl spermine, dog urea and succinic acid are used as internal standard quantitative analytes, so that the error in the sample pretreatment process can be reduced; the selectivity and the accuracy of the method are further improved by utilizing a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method; by selecting the chromatographic column and the elution condition, the time of chromatographic analysis is shortened. Meanwhile, an efficient and feasible technical method is provided for the research and development of products for early diagnosis of adenomatous polyps and/or colorectal cancer.
It is a first object of the present invention to provide a mobile phase for liquid chromatography.
The second purpose of the invention is to provide a product for simultaneously detecting one or more of N1, N12-diacetyl spermine, dog urea and/or succinic acid in urine.
It is a third object of the present invention to provide a product for early diagnosis of adenomatous polyps and/or colorectal cancer.
A fourth object of the invention is to provide the use of said mobile phase.
In order to achieve the purpose, the invention is realized by the following scheme:
a mobile phase for liquid chromatography comprising a reagent A and a reagent B, wherein the reagent A is a 10mM ammonium formate aqueous solution, and the reagent B is a 90% acetonitrile solution by volume concentration containing 10mM ammonium formate.
Preferably, the volume concentration of the reagent A is 95% and the volume concentration of the reagent B is 5%.
A product for simultaneously detecting one or more of N1, N12-diacetylspermine, canine urea and/or succinic acid in urine comprises the mobile phase.
The application of the mobile phase in preparing a product for simultaneously detecting one or more of N1, N12-diacetyl spermine, dog urea and/or succinic acid in urine.
The application of the mobile phase in preparing products for early diagnosis of adenomatous polyps and/or colorectal cancer.
A product for early diagnosis of adenomatous polyps and/or colorectal cancer contains the mobile phase.
Preferably, the product also contains an internal reference mixed solution, and the internal reference mixed solution contains standard products of N1, N12-diacetyl spermine, dog urea and succinic acid.
Preferably, the product further comprises a chromatographic column, wherein the chromatographic column is an Agilent Zorbax Eclipse XDB-C18 chromatographic column with the specification of 100mm multiplied by 2.1mm and 3.5 mu m.
Preferably, the method of use of the product comprises the steps of:
s1, removing impurities from a urine sample, and mixing the urine sample with an internal reference mixed solution, wherein the internal reference mixed solution contains standard substances of N1, N12-diacetyl spermine, dog urea and succinic acid;
s2, detecting a product in the step S1 by using a liquid chromatography-tandem mass spectrometry:
the chromatographic analysis conditions were: the chromatographic column is an Agilent Zorbax Eclipse XDB-C18 chromatographic column with the specification of 100mm x 2.1mm, 3.5 μm, the column temperature of 35 ℃, the autosampler temperature of 10 ℃, the mobile phase system of 95 percent of the reagent A described in claim 1 and 5 percent of the reagent B described in claim 1, the elution conditions are that 60 percent of the reagent A described in claim 1 and 40 percent of the reagent B described in claim 1 are eluted isocratically, the flow rate of the mobile phase is 0.55mL/min, and the analysis time is 4 min.
Preferably, the mass spectrometer detector conditions are: adopting an electrospray ion source and a multi-reaction monitoring positive ion scanning mode; the electrospray voltage was: a positive ion of 5500V and a negative ion of-4500V; the atomization gas is 40psi, the gas curtain gas amount is 30psi, the flow rate of the auxiliary heating gas is 40ps, the ion source temperature is 600 ℃, and the residence time is as follows: 100 ms.
Preferably, in step S1, the step of removing impurities from the urine sample and mixing with the internal reference mixture solution comprises:
collecting urine sample, centrifuging, filtering with membrane, and adding mixed solution of standard N1, N12-diacetyl spermine, dog urea and succinic acid.
More preferably, the concentrations of the standard N1, N12-diacetylspermine, canine urea and succinic acid in the product of step S1 are 10ng/mL, 200ng/mL and 400ng/mL, respectively.
Preferably, in step S2, the linear regression equations of N1, N12-diacetylspermine, canine urea and succinic acid are respectively: y 1.66x +0.0457, y 0.117x +0.0179, and y 0.0671x +0.082, wherein y represents the ratio of the analyte to the peak area of the internal standard, x represents the concentration of the target analyte in the urine, and the correlation coefficients are: r-0.9921, r-0.9941 and r-0.9913.
The product is used, and a urine sample is subjected to impurity removal and then is directly introduced into a liquid chromatography tandem mass spectrometer for determination, so that the contents of N1, N12-diacetylspermine, dog urea and succinic acid in the urine can be rapidly and accurately detected. According to the invention, the corresponding standard substances of N1, N12-diacetyl spermine, dog urea and succinic acid are used as internal standard quantitative analytes, so that the error in the sample pretreatment process can be reduced; the liquid chromatography-tandem mass spectrometry (LC-MS/MS) method is simple to operate, convenient to apply, accurate in measurement result and less in measurement interference, and the selectivity and the accuracy of the method are further improved; by selecting the chromatographic column and the elution condition, the time of chromatographic analysis is shortened. Meanwhile, an efficient and feasible technical method is provided for the research and development of products for early diagnosis of adenomatous polyps and/or colorectal cancer.
Compared with the prior art, the invention has the following beneficial effects:
1. LC-MS/MS does not need a specific antibody reagent, has the advantages of high selectivity, high specificity, high sensitivity and the like, and is a post-transcriptional modification and mutation form of protein;
2. because the mass-to-charge ratio of the measured object is directly measured by the target mass spectrum, the International System of Units (SI) traceability is easily realized by using an isotope or other marker dilution calibration System (including internal standardization and external calibration). The immune method has the problems of non-specific antibody combination, autoantibody interference and the like, so that the traceability of the immune method has greater challenge;
3. the operation is simple, and the result is stable;
4. the early screening kit is suitable for early diagnosis of colorectal cancer, can be used for early screening of various diseases by one tube of urine specimen, and has strong universality. Adenomatous polyps, tumors and the like of the left colon and the right colon can be simultaneously found;
5. the application range is wide, the urine sampling is convenient, the device is completely noninvasive, and the device can sample at home. Contamination of the urine sample with DNA, hemoglobin, albumin, lipids, etc. will not affect the results.
Drawings
FIG. 1 is a liquid chromatography tandem mass spectrometry (LC-MS/MS) chromatogram of N1, N12-diacetylspermine, wherein A is an LC-MS/MS chromatogram of an aqueous solution of N1, N12-diacetylspermine standard; b is LC-MS/MS chromatogram of N1, N12-diacetyl spermine in urine sample.
FIG. 2 is an LC-MS/MS chromatogram of canine urea, wherein A is an LC-MS/MS chromatogram of an aqueous solution of a canine urea standard; and B is an LC-MS/MS chromatogram of the canine urea in the urine sample.
FIG. 3 is an LC-MS/MS chromatogram of succinic acid, wherein A is an LC-MS/MS chromatogram of an aqueous solution of a succinic acid standard; and B is an LC-MS/MS chromatogram of succinic acid in the urine sample.
FIG. 4 shows the urine contents of N1, N12-diacetylspermine (A), canine urea (B) and succinic acid (C) in healthy subjects, adenomatous polyps and stage I-II colorectal cancer subjects, respectively.
FIG. 5 is an observer operating characteristic curve (ROC) of three metabolites, N1, N12-diacetylspermine, canine urea and succinic acid, as markers of adenomatous polyposis and colorectal cancer.
Detailed Description
The present invention will be described in further detail with reference to the drawings and specific examples, which are provided for illustration only and are not intended to limit the scope of the present invention. The test methods used in the following examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are, unless otherwise specified, commercially available reagents and materials.
Example 1 preparation of a Standard working solution of N1, N12-diacetylspermine, Canine Urea and succinic acid
1. Sources of standards
The standard N1, N12-diacetylspermine with the specification of 2.5mg and the purity of more than 98 percent;
the standard dog urea product has the specification of 100mg and the purity of more than 98 percent;
the specification of the succinic acid standard substance is 250mg, and the purity is more than 98%.
The above standards were purchased from Sigma.
2. Preparation of N1, N12-diacetyl spermine, dog urea and succinic acid standard working solution
Each of the above standards was diluted with ultrapure water to 10 gradients, respectively.
The concentrations of N1 and N12-diacetylspermine after dilution are respectively 0, 0.05, 0.4, 2, 4, 8, 16, 24, 32 and 40 ng/mL;
the concentration of diluted dog urea is 0, 0.5, 4, 20, 40, 80, 160, 240, 320 and 400ng/mL respectively;
the concentration of the diluted succinic acid was 0, 1, 8, 40, 80, 160, 320, 480, 640 and 800ng/mL, respectively.
EXAMPLE 2 determination of the content of N1, N12-diacetylspermine, dog Urea and succinic acid in an aqueous solution of standards
10 μ L of the calibration working solution prepared in example 1 at different concentrations was pipetted and detected by LC-MS/MS under the following conditions.
(1) Chromatographic conditions are as follows:
a chromatographic column: an Agilent Zorbax Eclipse XDB-C18 column (100 mm. times.2.1 mm, 3.5 μm);
mobile phase reagent a: 10mM aqueous ammonium formate solution (pH 3), mobile phase reagent B: a 90% strength by volume acetonitrile solution of 10mM ammonium formate (pH 3);
the column temperature is 35 ℃;
the sample volume is 10 mu L;
the mobile phase system selects a reagent A with 95% volume concentration and a reagent B with 5% volume concentration, the elution conditions select the reagent A with 60% volume concentration and the reagent B with 40% volume concentration to elute isocratically, the sample is eluted with isocratic gradient at the flow rate of 0.55mL/min, and the analysis time is 4 min.
(2) Mass spectrum conditions:
electrospray ion source, multi-reaction monitoring positive ion scanning mode: IS: positive ion 5500V, negative ion-4500V, Gas 1: 50psi, CUR: 25psi, Gas 2: 50psi, TEM: 600 ℃, CXP: -15V, residence time: 100 ms.
Selecting m/z287.2/170.09, 287.2/99.7 as quantitative ion pairs, and selecting m/z 290.3/103.2, 290.3/174.1 as corresponding standard N1, N12-diacetylspermine (D6); selecting 209.2/94.2 and 209.2/191.7 as quantitative ion pairs, wherein m/z 215.37/98.1 and 215.37/198.1 are corresponding standard dog urea (ring-D4, 3, 4-D2); the m/z 117.9/73.8 and 117.9/98.9 are selected as quantitative ion pairs, and the m/z121.39/76.9 and 121.39/101.1 are corresponding standard products of succinic acid (D4). See table 1 for details.
TABLE 1 LC-MS/MS analysis Mass Spectrometry Condition parameter Table
Linear regression equations for N1, N12-diacetylspermine, canine urea and succinic acid were 1.66x +0.0457, 0.117x +0.0179, 0.0671x +0.082, respectively, with correlation coefficients r 0.9921, r 0.9941, and r 0.9913, respectively, where y represents the ratio of the peak area of the analyte to the internal standard and x represents the concentration of the target analyte in the sample.
Example 3 detection of the content of N1, N12-diacetylspermine, dog Urea and succinic acid in urine
First, sample pretreatment
1mL of urine sample is centrifuged at 12000r/min for 10min, the centrifuged mixture is filtered through a 0.22 μm filter membrane, and 200 μ L of a mixture of standard N1, N12-diacetylspermine (D6), dog urea (cyclo-D4, 3, 4-D2) and succinic acid (D4) are added to a 10mL brown centrifuge tube for later use at concentrations of 10ng/mL, 200ng/mL and 400ng/mL, respectively.
Second, liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis
The analysis was performed using the LC-MS/MS conditions of example 2. The related chromatograms of N1, N12-diacetylspermine, canine urea and succinic acid are shown in figures 1-3. The result shows that chromatographic peaks of N1, N12-diacetyl spermine, dog urea and succinic acid can be identified through chromatograms of N1, N12-diacetyl spermine, dog urea and succinic acid quasi-aqueous solution and urine, and by adopting the liquid chromatography tandem mass spectrometry detection method, the chromatographic peak shape, retention time and separation effect are good, and the contents of N1, N12-diacetyl spermine, dog urea and succinic acid in the urine can be rapidly and accurately detected.
Example 4 clinical validation
First, experiment method
The clinical verification of 151 study subjects showed no statistical significance of differences in the onset of gender, age, blood pressure, smoking, drug use, etc., among groups, and were comparable. Urine samples of 66 healthy subjects (Control), 39 Adenomatous Polyps (AP), 53 colorectal cancers at stage I (CC-I), and 59 colorectal cancers at stage II (CC-II) were collected and tested for the contents of N1, N12-diacetylspermine, dog urea, and succinic acid in the urine samples of each group using the method of example 2.
Second, experimental results
As shown in FIG. 4, the average concentrations of N1 and N12-diacetylspermine in the urine of the above groups are 0.9366 + -0.8050 ng/mL, 11.2800 + -6.9070 ng/mL, 16.7300 + -7.1630 ng/mL and 22.0100 + -9.7280 ng/mL respectively;
the average concentration of the dog urea in the urine of each group is 9.1530 + -6.0630 ng/mL, 31.9800 + -9.9970 ng/mL, 34.2300 + -11.0900 ng/m and 38.5200 + -12.4500 ng/mL;
the average concentration of succinic acid in the urine of the above groups was 20.9200 + -14.9100 ng/mL, 89.7800 + -22.4000 ng/mL, 103.8000 + -27.1000 ng/mL and 109.9000 + -33.3600 ng/mL.
Compared with the healthy group, the urine specimens of patients with adenomatous polyposis and stage I-II colorectal carcinoma have obviously higher contents of N1, N12-diacetylspermine, canine urea and succinic acid, and the difference has statistical significance.
Using observer operating characteristic curve (ROC) analysis, as shown in fig. 5, in the grouped specimens of N1, N12-diacetyl spermine detection, the area under the ROC curve (AUC) was 0.9701 (95% CI,0.9492to 0.9910), and the difference had statistical significance (p < 0.0001);
in canine urea assay cohorts, the area under the ROC curve (AUC) was 0.9742 (95% CI,0.9579to 0.9904), and the difference was statistically significant (p < 0.0001);
in the succinic acid assay grouped specimens, the area size under the ROC curve (AUC) was 0.9863 (95% CI,0.9746to 0.9979), and the difference was statistically significant (p < 0.0001).
And, the cutoff value (cutoff value) of N1, N12-diacetylspermine can be analytically calculated to be 3.1980ng/mL (above which value is for adenomatous polyps or colorectal cancer patients, below which value is for healthy people), the Sensitivity (Sensitivity) is 92.05%, and the Specificity (Specificity) is 98.48%;
the cutoff value (cutoff value) of the dog urea can be analytically calculated to be 18.41ng/mL (above which is the adenomatous polyp or colorectal cancer patient, below which is the healthy person), the Sensitivity (Sensitivity) is 89.40%, and the Specificity (Specificity) is 95.45%;
the analysis calculated a succinic acid cut-off value of 55.69ng/mL, (above this value for patients with adenomatous polyps or colorectal cancer, below this value for healthy persons), a Sensitivity (Sensitivity) of 93.38% and a Specificity (Specificity) of 98.48%.
Therefore, one or more measured values of N1, N12-diacetylspermine, canine urea and succinic acid in the urine sample are higher than corresponding cut-off values, and are patients with adenomatous polyposis or colorectal cancer, and are healthy people when the measured values are lower than the corresponding cut-off values.
It should be finally noted that the above examples are only intended to illustrate the technical solutions of the present invention, and not to limit the scope of the present invention, and that other variations and modifications based on the above description and thought may be made by those skilled in the art, and that all embodiments need not be exhaustive. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included in the protection scope of the claims of the present invention.
Claims (10)
1. A mobile phase for liquid chromatography comprising reagent a and reagent B, said reagent a being a 10mM aqueous ammonium formate solution and reagent B being a 90% strength by volume acetonitrile solution containing 10mM ammonium formate.
2. The mobile phase according to claim 1, wherein the volume concentration of reagent a is 95% and the volume concentration of reagent B is 5%.
3. A product for simultaneously detecting one or more of N1, N12-diacetylspermine, canine urea and/or succinic acid in urine, comprising the mobile phase of claim 1.
4. Use of the mobile phase of claim 1 in the preparation of a product for simultaneous detection of one or more of N1, N12-diacetylspermine, canine urea and/or succinic acid in urine.
5. Use of the mobile phase according to claim 1 for the preparation of a product for the early diagnosis of adenomatous polyps and/or colorectal cancer.
6. A product for the early diagnosis of adenomatous polyps and/or colorectal cancer comprising the mobile phase of claim 1.
7. The product of any one of claims 3 or 6, further comprising an internal reference mixture comprising standard N1, N12-diacetylspermine, canine urea and succinic acid.
8. The product according to any one of claims 3 or 6, further comprising a chromatography column, wherein the chromatography column is an Agilent Zorbax Eclipse XDB-C18 chromatography column with a specification of 100mm x 2.1mm, 3.5 μm.
9. The product according to claim 6, characterized in that the method of use of the product comprises the following steps:
s1, removing impurities from a urine sample, and mixing the urine sample with an internal reference mixed solution, wherein the internal reference mixed solution contains standard substances of N1, N12-diacetyl spermine, dog urea and succinic acid;
s2, detecting a product in the step S1 by using a liquid chromatography-tandem mass spectrometry:
the chromatographic analysis conditions were: the chromatographic column is an Agilent Zorbax Eclipse XDB-C18 chromatographic column with the specification of 100mm x 2.1mm, 3.5 μm, the column temperature of 35 ℃, the autosampler temperature of 10 ℃, the mobile phase system of 95 percent of the reagent A described in claim 1 and 5 percent of the reagent B described in claim 1, the elution conditions are that 60 percent of the reagent A described in claim 1 and 40 percent of the reagent B described in claim 1 are eluted isocratically, the flow rate of the mobile phase is 0.55mL/min, and the analysis time is 4 min.
10. The product of claim 9, wherein the mass spectrometer detector conditions in step S2 are: adopting an electrospray ion source and a multi-reaction monitoring positive ion scanning mode; the electrospray voltage was: a positive ion of 5500V and a negative ion of-4500V; the atomization gas is 40psi, the gas curtain gas amount is 30psi, the flow rate of the auxiliary heating gas is 40ps, the ion source temperature is 600 ℃, and the residence time is as follows: 100 ms.
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| CN114755422A (en) * | 2022-06-10 | 2022-07-15 | 杭州凯莱谱精准医疗检测技术有限公司 | Biomarker for colorectal cancer detection and application thereof |
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