CN114924016B - Method for simultaneously detecting sulfated and non-sulfated gastrin G17 and detection kit thereof - Google Patents
Method for simultaneously detecting sulfated and non-sulfated gastrin G17 and detection kit thereof Download PDFInfo
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- 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
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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
- 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
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- 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
- G01N30/86—Signal analysis
- G01N30/8624—Detection of slopes or peaks; baseline correction
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Abstract
The invention relates to the technical field of analysis and detection, in particular to a method for simultaneously detecting sulfated and non-sulfated gastrin G17 and a detection kit thereof. The method comprises the steps of preprocessing a blood sample to be detected, extracting the sample by adopting a weak anion solid-phase extraction mode, detecting the sample on a machine, separating two components by liquid phase, respectively entering a mass spectrometer, generating specific mass-to-charge ratios by electrospray ionization, and quantitatively detecting the Unsulfated-G17 and the Sulfated-G17 in the blood sample by adopting an isotope internal standard method. The method has the advantages of less sample amount, high detection speed, higher accuracy and precision, capability of obviously improving the accuracy and the effectiveness of the detection of the gastrin, namely, the Unsulfated-G17 and the Sulfated-G17, and good clinical application prospect.
Description
Technical Field
The invention relates to the technical field of analysis and detection, in particular to a method for simultaneously detecting sulfated and non-sulfated gastrin G17 and a detection kit thereof.
Background
Gastrin is a polypeptide gastrointestinal hormone secreted by G cells of antrum, and the main active ingredients comprise two subtypes G34 and G17, namely sulfonated and non-sulfonated forms. Wherein G17 has the functions of stimulating gastric acid secretion, promoting gastric mucosa cell proliferation and differentiation and the like, is one of sensitive indexes reflecting gastric sinus endocrine function, and can prompt gastric sinus mucosa atrophy or whether abnormal increase exists. When serum G17 levels rise, suggesting a risk of gastric cancer, it is therefore newly added as a project for early gastric cancer screening guidelines. Studies have indicated that there is a difference in the ratio of non-sulfonated gastrin to sulfonated gastrin in different disease states, suggesting that the ratio has the potential to assess gastric cancer risk and to make differential diagnosis with other diseases. The sequences of Unsulfat ed-G17 and Sulfated-G17 are shown in FIG. 1.
Most of the existing methods for detecting gastrin in blood samples are based on immunological methods (radioimmunoassay, chemiluminescence, immunological methods and the like), and the immunological methods most used in clinic at present are convenient, quick and high in flux, but can only detect total gastrin and cannot distinguish various subtypes. The tandem mass spectrometry method based on liquid chromatography has the advantages of strong specificity and high sensitivity, and can distinguish and detect a plurality of structural analogues simultaneously, thereby playing an increasingly important role in clinical detection. For example, yu S, et al analytical and Clinical Performance of a Liquid Chromatography-Tandem Mass Spectrometry Method for Measuring Gastrin Subtypes G and G17 in serum Clin chem.2021Sep 1;67 1220-1229.Doi:10.1093/clinchem/hvab097.PMID 34383899. Although this method does not allow simultaneous determination of Unsulfated-G17 and Sulfated-G17. In view of the fact that quantitative ratios of Unsulfated-G17 and Sulfated-G17 in the same test sample have practical clinical significance in different disease states, and particularly have potential capability of assisting diagnosis in the aspects of risk assessment of gastric cancer and differential diagnosis of other diseases, it is necessary to develop a method capable of simultaneously and quantitatively detecting Unsulfated-G17 and Sulfated-G17 in a sample to be tested, and convert the quantitative ratios into a kit for clinical detection.
Disclosure of Invention
The invention aims to provide a method for simultaneously detecting gastrin, namely, unsulfated-G17 and Sulfated-G17 and a detection kit thereof.
The detection method is established based on a liquid chromatography tandem mass spectrometry detection technology, acid compounds are judged according to pKa values of Unsulfated-G17 and Sulfated-G17, a weak anion solid phase extraction mode is adopted to extract samples, then the samples are effectively separated on a liquid chromatography instrument, and the samples enter a mass spectrometer respectively to generate specific mass/charge ratios (mass to charge ratios) through electrospray ionization, and the quantification of Unsulfated-G17 and Sulfated-G17 is realized by adopting an isotope internal standard method. Determination of Unsulfated-G17 and Sulfated-G17 calibration curves were prepared with the respective mass concentrations of the standard, the mass concentration of the standard solution was taken as the X axis, the ratio of the peak areas of the standard and the internal standard was taken as the Y axis, and linear regression analysis was performed to obtain the regression equation by the "1/X" weights. Substituting the area ratio of the components to be detected in the sample to the internal standard peak into a standard curve equation, and calculating the concentration of each component to be detected in the serum sample. The ratio of the sample detection signal intensity to the isotope internal standard is in direct proportion to the concentration of the component to be detected in the sample.
Specifically, the invention provides the following technical scheme:
in a first aspect, the present invention provides a method for simultaneous detection of gastrin, unqualified-G17 and Sulfated-G17, comprising the steps of:
(1) Adding a sample preparation liquid 1 into a sample to be detected, centrifuging, taking supernatant, and adding a sample preparation liquid 2; the sample preparation liquid 1 is acetonitrile; the sample preparation liquid 2 is water;
(2) Carrying out solid phase extraction and positive pressure elution on the solution prepared in the step (1), sequentially adding water and the solid phase extract 1 for cleaning, then adding the solid phase extract 2 for cleaning, carrying out positive pressure elution, and collecting the eluent; or (b)
Enriching the solution prepared in the step (1) by an immunomagnetic bead method;
(3) And (3) drying the eluent, redissolving the eluent by using a sample redissolution, and detecting by using a liquid chromatography-tandem mass spectrometry method.
In the method, the solid phase extract 1 is 10% -80% of methanol, and the solid phase extract 2 is 1% -10% of ammonia water methanol solution.
The sample complex solution is 0.1% -1% ammonia water 5% -50% acetonitrile water solution.
The sample to be tested in the step (1) of the method is whole blood, blood plasma or blood serum; the sample injection amount for detection in the step (3) by adopting a liquid chromatography tandem mass spectrometry is 1-10 mu L; the chromatographic separation uses ACQUITY UPLC Peptide BEH C18 chromatographic column.
The present invention discovers that when the chromatographic separation is carried out by combining the method of the present invention, the conventional chromatographic columns such as HSS T3, HSS PFP, BEH C18, BEH C8, BEH Phenyl, etc. can not realize effective separation, and only ACQUITY UPLC Peptide BEH C chromatographic column has the best separation effect by combining other steps, reagents and parameters in the method of the present invention.
Further, in the step (1) of the method, the volume ratio of the sample to be detected to the sample preparation liquid 1 is 1:1-5; the volume ratio of the sample preparation liquid 1 to the sample preparation liquid 2 is 1:2-10;
preferably, in the step (1), the volume ratio of the sample to be tested to the sample preparation liquid 1 is 1:2; the volume ratio of the sample preparation liquid 1 to the sample preparation liquid 2 is 1:4.25;
in the step (2), the added water is 1-5 times of the volume of the sample to be detected in the step (1), the added amount of the solid phase extract 1 is equal to the added amount of the water, and the volume ratio of the solid phase extract 1 to the solid phase extract 2 is 1:0.1-0.8;
preferably, in the step (2), the amount of water added is 2 times the volume of the sample to be detected in the step (1), the addition amount of the solid phase extract 1 is equal to the amount of the water, and the volume ratio of the solid phase extract 1 to the solid phase extract 2 is 1:0.4;
in the step (3), the amount of the sample complex solution is equivalent to the volume of the sample to be measured in the step (1).
In the liquid chromatography tandem mass spectrometry, a mobile phase of liquid chromatography comprises a mobile phase A and a mobile phase B, wherein the mobile phase A is 0.3% ammonia water solution, and the mobile phase B is pure acetonitrile.
In the method of the invention, the mobile phase of the liquid chromatograph is subjected to gradient elution according to the following gradient: 0-1.0min, mobile phase A95%, 1.0-3.5min, mobile phase A95-50%, 3.5-4.0min, mobile phase A50%, 4.0-4.01min, mobile phase A50-2%, 4.01-4.5min, mobile phase A2%, 4.5-6.0min, mobile phase A95%;
the invention discovers that the optimal separation degree and the optimal peak shape can be obtained by adopting the mobile phase system and the elution program, and the accuracy and the precision of the detection of the Unsulfated-G17 and the Sulfated-G17 are improved.
Preferably, the flow rate of the mobile phase is 0.3mL/min.
In the method of the invention, mass spectrum detection conditions are as follows: multi-ion monitoring reaction mode, anion mode, electrospray ionization, ion source temperature 400-600 ℃, monitoring ion pairs of Unsulfated-G17 and Sulfated-G17 and internal standards thereof: unsulfated-G17-quantification: m/z523.5→ 610.9; un-programmed-G17-qualitative 418.6 → 457.9; sulfated-G17-quantification: m/z 434.6 →477.9; sulfated-G17-qualitative m/z 543.3 →477.9; an internal standard of the Unsulfated-G17 isotope: m/z 525.5→ 610.8.
The detection method of the invention can be used for qualitative detection or quantitative detection of the Unsulfated-G17 and the Sulfated-G17 in blood samples. When quantitative detection is carried out, standard curves are manufactured by using standard substances with different concentrations, the ratio of the standard substance concentration to the internal standard peak area is taken as an X axis, linear regression analysis is carried out, and a regression equation is obtained through 1/X weight. Substituting the area ratio of the to-be-detected component in the sample and the internal standard peak thereof into a standard curve equation, and calculating the concentration of the Unsulfated-G17 and the Sulfated-G17 in the sample. The ratio of the sample detection signal intensity to the isotope internal standard is in direct proportion to the concentration of the component to be detected in the sample. The invention discovers that the detection method provided by the invention is adopted for detection, and the correlation coefficient of the standard curve is more than 0.995, so that the requirement of quantitative detection can be met.
In a second aspect, the present invention provides a kit for detecting gastrin, ensulted-G17 and sulFated-G17, comprising:
reagent I: pure acetonitrile is used for sample preparation;
reagent II: pure water for sample preparation;
reagent III:10% -80% of methanol used for solid phase extraction;
reagent IV:1% -10% ammonia water methanol solution for solid phase extraction;
reagent V:0.1% -1% ammonia water 10% acetonitrile aqueous solution for sample re-dissolution;
reagent VI:0.3% aqueous ammonia solution as mobile phase a separated by liquid chromatography;
reagent VII: pure acetonitrile as mobile phase B separated by liquid chromatography;
preferably, the kit further comprises:
reagent VIII: g17 isotope internal standard, the concentration is 10-50ng/mL;
reagent IX: a Unsulfated-G17 standard and/or a Sulfated-G17 standard.
In a third aspect, the invention provides the use of the above-described kit in the qualitative or quantitative detection of gastrin, unsulfated-G17 and Sulfated-G17.
The invention has the beneficial effects that: the invention establishes a method for simultaneously measuring the Unsulfated-G17 and the Sulfated-G17 by using the LC-MS/MS without being interfered by non-specific cross-talk based on the liquid chromatography tandem mass spectrometry technology, and the method has the advantages of less requirement on the dosage of a sample to be detected, high detection speed, higher accuracy and precision, and can accurately, efficiently and sensitively realize quantitative detection of the Unsulfated-G17 and the Sulfated-G17, thereby having good application prospects in the aspects of risk assessment of gastric cancer and differential diagnosis of other diseases.
Drawings
FIG. 1 shows the amino acid sequences of the Unsulfated-G17 (A) and Sulfated-G17 (B) according to the invention.
FIG. 2 shows the MRM chromatograms of the detection of Unsulfated-G17 and Sulfated-G17 in serum samples according to example 1 of the present invention.
FIG. 3 shows the linear ranges of the Unsulfated-G17 and Sulfated-G17 determined by the LC-MS/MS method.
FIG. 4 is a graph showing the results of detection of different sample preparations 2.
FIG. 5 is a graph showing the comparison of the detection results of different sample solutions.
FIG. 6 is a graph showing the results of liquid chromatography on different columns.
Fig. 7A is a graph comparing the gradient of different mobile phases to the detection results.
Fig. 7B is a graph comparing the gradient of different mobile phases to the detection results.
Detailed Description
The following examples are illustrative of the invention and are not intended to limit the scope of the invention. If not illustrated, reagents, consumables, and instruments used in the examples of the present invention are commercially available.
Sample preparation liquid 1: acetonitrile;
sample preparation liquid 2: water;
solid phase extract 1:50% aqueous methanol;
solid phase extract 2:5% ammonia methanol solution;
sample complex solution: 1% ammonia water 10% acetonitrile aqueous solution;
mobile phase a: contains 0.3% ammonia solution. Mobile phase B acetonitrile (pure acetonitrile).
Calibration solution and internal standard solution: accurately weighing the Unsulfated-G17 and the Sulfated-G17 and the respective internal standards thereof to a constant volume bottle, preparing a mother solution by using 1% ammonia water and 10% acetonitrile water solution, and then preparing working calibration solutions with the concentration of 5, 10, 20, 50, 100, 200, 500, 1000, 2000, 5000 and 10000pg/mL by using 1% ammonia water and 10% acetonitrile water solution for serial dilution; accurately weighing isotope internal standard of Unsulfated-G17 to a constant volume bottle, preparing mother liquor by using 1% ammonia water and 10% acetonitrile water solution, and diluting the mother liquor into internal standard working solution (10 ng/mL) by using 1% ammonia water and 10% acetonitrile water solution.
Example 1
The embodiment provides a method capable of simultaneously detecting gastrin, namely, the gastrulated-G17 and the Sulfated-G17, wherein the method adopts liquid chromatography tandem mass spectrometry for detection, and the method specifically comprises the following steps:
1. sample pretreatment
Taking 100 mu L of blood sample or calibration solution, respectively adding 1.5ml of plastic tube, respectively adding 200 mu L of sample preparation liquid 1, mixing, vibrating, centrifuging, taking supernatant into a new tube, respectively adding 850 mu L of sample preparation liquid 2; then adding the sample into the activated WAX solid phase extraction plate, performing positive pressure elution, sequentially adding 200 mu L of water and 200 mu L of solid phase extraction liquid 1 for cleaning, finally adding 80 mu L of solid phase extraction liquid 2, performing positive pressure elution, collecting the eluent in the step, drying by nitrogen, re-dissolving by using 80 mu L of sample re-solution, waiting for the test of a machine, and performing sample injection volume: 10 mu L.
2. Liquid chromatography detection
The chromatographic separation was carried out using a ACQUITY UPLC Peptide BEH C column, and the liquid chromatography mobile phase a and mobile phase B were set up according to the gradient of table 1 below:
TABLE 1
| Time/min | % mobile phase A | % mobile phase B | Flow rate mL/min |
| 0 | 95 | 5 | 0.3 |
| 1.0 | 95 | 5 | 0.3 |
| 3.5 | 50 | 50 | 0.3 |
| 4 | 50 | 50 | 0.3 |
| 4.01 | 2 | 98 | 0.3 |
| 4.5 | 2 | 98 | 0.3 |
| 6.0 | 95 | 5 | 0.3 |
3. Mass spectrometry detection
The mass spectrum selects a multi-ion monitoring reaction mode, a negative ion mode and electrospray ionization, wherein the temperature of an ion source is 400-600 ℃, and simultaneously ion pairs of a Unsulfated-G17 and a Sulfated-G17 and internal standards thereof are monitored: unsulfated-G17 (quantitative): 523.5→ 610.9; un-programmed-G17 (qualitative) 418.6 → 457.9; sulfated-G17 (quantitative): 434.6 →477.9; sulfated-G17 (qualitative) 543.3 →477.9; an internal standard of the Unsulfated-G17 isotope: 525.5- & gt 610.8
Representative chromatograms of the detection of ensulfated-G17 and sulfliated-G17 in serum samples using the methods described above are shown in fig. 2.
4. Linear correlation analysis
The correlation of the Unsulfated-G17 linearity was >0.995 in the range of 10-10000pg/mL, and the correlation of the Sulfatted-G17 linearity was >0.99 in the range of 20-10000pg/mL, see FIG. 3.
5. Precision evaluation
Three horizontal serum samples were assayed in three batches, 5 replicates each, to give in-batch and total imprecision as in table 2:
TABLE 2 LC determination of Unsulfamed-G17 and Sulfamed-G17 imprecision by MS/MS method
6. Recovery rate and accuracy evaluation:
the results of the additive recovery test using the Unsulfated-G17 and Sulfated-G17 standards are shown in Table 3.
TABLE 3 LC-MS/MS method for determining recovery of Unsulfamed-G17 and Sulfamed-G17
Comparative example 1
Under the same conditions as in example 1, only sample preparation 2 was different, and 850. Mu.L of 2% Formic Acid (FA) solution was used in place of sample preparation 2 in example 1 in this comparative example, and the results are shown in FIG. 4.
Compared with the results of example 1, the replacement of sample preparation liquid 2 of example 1 with 850. Mu.L of 2% Formic Acid (FA) solution resulted in a decrease in the extraction efficiency of the rensfated-G17, indicating that the extraction effect with sample preparation liquid 2 of example 1 was superior to that with 850. Mu.L of 2% Formic Acid (FA) solution.
Comparative example 2
Under the same conditions as in example 1, the comparative example uses a 1% aqueous ammonia solution and 20% acetonitrile aqueous solution instead of the sample complex solution in example 1, and the results are shown in fig. 5.
As compared with the results of example 1, the use of 1% aqueous ammonia and 20% acetonitrile in water has a solvent effect, which causes cleavage of the chromatographic peak of Unsulfated-G17. The sample complex solution in example 1 is described as having an optimal ratio to avoid solvent effects.
Comparative example 3
In this comparative example, based on example 1, three different chromatographic columns were selected for liquid chromatography detection, and other conditions and method parameters were the same as in example, and the three chromatographic columns were respectively:
A.HSS C18,2.1mm*100mm,1.8μm,
B.BEH C18,2.1mm*100mm,1.7μm,
C.Peptide BEH C18,2.1*100mm,1.7μm,
wherein C is the chromatographic column used in example 1. The final results are shown in FIG. 6, wherein panels A, B and C correspond to the three columns described above. The results demonstrate that the chromatographic column C employed in example 1 of the invention has the best retention and separation.
Comparative example 4
In this comparative example, the influence of different mobile phase gradients (tables 4 and 5) on the detection results when liquid chromatography detection is performed was examined, and other method step parameters are all described in example 1. A. The results of the detection of the two gradients are shown in fig. 7A and 7B. The results demonstrate that the optimized gradient in example 1 ensures optimal chromatographic separations for both the ensulfated-G17 and the sulfocured-G17.
Table 4A gradient:
table 5B gradient:
| time/min | % mobile phase A | % mobile phase B | Flow rate mL/min |
| 0 | 95 | 5 | 0.3 |
| 1.0 | 95 | 5 | 0.3 |
| 3.5 | 50 | 50 | 0.3 |
| 4 | 50 | 50 | 0.3 |
| 4.01 | 2 | 98 | 0.3 |
| 4.5 | 2 | 98 | 0.3 |
| 6.0 | 95 | 5 | 0.3 |
While the invention has been described in detail in the foregoing general description and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.
Claims (5)
1.A method for simultaneously detecting gastrin, unqualified-G17 and Sulfated-G17, comprising the steps of:
(1) Adding a sample preparation liquid 1 into a sample to be detected, centrifuging, taking supernatant, and adding a sample preparation liquid 2; the sample preparation liquid 1 is acetonitrile; the sample preparation liquid 2 is water;
the sample to be tested in the step (1) is whole blood, blood plasma or blood serum;
(2) Carrying out solid phase extraction and positive pressure elution on the solution prepared in the step (1), sequentially adding water and the solid phase extract 1 for cleaning, then adding the solid phase extract 2, carrying out positive pressure elution, and collecting the eluent;
the solid phase extract 1 is 10% -80% of methanol aqueous solution, and the solid phase extract 2 is 1% -10% of ammonia water methanol solution;
(3) Drying the eluent, redissolving the eluent by using a sample redissolution, and detecting by using a liquid chromatography tandem mass spectrometry;
the sample complex solution is 0.1% -1% ammonia water 10% acetonitrile water solution;
the chromatographic separation adopts a ACQUITY UPLC Peptide BEH C18 chromatographic column;
in the liquid chromatography tandem mass spectrometry, a mobile phase of liquid chromatography comprises a mobile phase A and a mobile phase B, wherein the mobile phase A is 0.3% ammonia water solution, and the mobile phase B is acetonitrile;
the mobile phase was gradient eluted according to the following gradient: 0-1.0min, mobile phase A95%, 1.0-3.5min, mobile phase A95-50%, 3.5-4.0min, mobile phase A50%, 4.0-4.01min, mobile phase A50-2%, 4.01-4.5min, mobile phase A2%, 4.5-6.0min, mobile phase A95%.
2. The method of claim 1, wherein the sample size for detection in step (3) is 1-10 μl by liquid chromatography tandem mass spectrometry.
3. The method of claim 1, wherein the step of determining the position of the substrate comprises,
in the step (1), the volume ratio of the sample to be detected to the sample preparation liquid 1 is 1:1-5; the volume ratio of the sample preparation liquid 1 to the sample preparation liquid 2 is 1:2-10;
in the step (2), the added water is 1-10 times of the volume of the sample to be detected in the step (1), the added amount of the solid phase extract 1 is equal to the added amount of the water, and the volume ratio of the solid phase extract 1 to the solid phase extract 2 is 1:0.1-0.5;
in the step (3), the amount of the sample complex solution is equivalent to the volume of the sample to be measured in the step (1).
4. The method of claim 1, wherein the mobile phase has a flow rate of 0.3-0.5 mL/min.
5. The method of any one of claims 1-4, wherein mass spectrometry detection conditions are as follows: multi-ion monitoring reaction mode, anion mode, electrospray ionization, ion source temperature of 400-600 ℃, and monitoring ion pairs of Unsulfated-G17 and Sulfated-G17 and internal standards thereof: unsulfated-G17-quantification: m/z523.5→ 610.9; un-programmed-G17-qualitative 418.6 → 457.9; sulfated-G17-quantification: m/z 434.6 →477.9; sulfated-G17-qualitative m/z 543.3 →477.9; an internal standard of the Unsulfated-G17 isotope: m/z 525.5→ 610.8.
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| WO2008150488A1 (en) * | 2007-06-01 | 2008-12-11 | Laboratory Corporation Of America Holdings | Methods and systems for quantification of peptides and other analytes |
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| WO2019196233A1 (en) * | 2018-04-09 | 2019-10-17 | 深圳市宇驰检测技术股份有限公司 | Method for measuring contents of various endocrine disruptors in estuary sediment |
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