Method for determining cinnamyl glycine in biological sample based on LC-MS (Liquid Chromatography-Mass Spectrometer)

Abstract

The invention belongs to the technical field of analysis and detection, and particularly relates to a method for determining cinnamyl glycine in a biological sample based on LC-MS. The method comprises the following steps: mixing the biological sample with an extracting solution, and performing extraction treatment to obtain supernate; evaporating and concentrating, and re-dissolving in an acetonitrile water solution to obtain a solution to be detected; diluting the cinnamyl glycine standard stock solution with a PBS (Phosphate Buffer Solution) to obtain a standard solution with a concentration gradient; mixing the standard substance solution with the extracting solution, performing extraction treatment to obtain a mixed standard solution, and testing the mixed standard solution through LC-MS to obtain a standard curve; and testing the solution to be tested, and obtaining the content of cinnamyl glycine in the solution to be tested according to the standard curve. The method can be used for accurately qualitatively and quantitatively analyzing the cinnamyl glycine in the sample, has the advantages of simplicity in treatment, high flux and reliable result, and is easy to clinically popularize and popularize.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of analytical detection, and particularly relates to a method for determining cinnamoyl glycine in a biological sample based on LC-MS. BACKGROUND

[0002] Metabolomics is an important part of systems biology, aiming to comprehensively analyze the dynamic changes of small molecule metabolites (molecular weight <1500 Da) in the body, and reveal the metabolic response of the organism under physiological, pathological or external stimulation. Metabolomics technology includes chromatographic technology, mass spectrometry technology and magnetic resonance technology, etc. It has the advantages of fast detection speed, high degree of instrument automation, high throughput, controllable detection quality and good repeatability, and is often used for the detection of small molecule metabolites in metabolomics.

[0003] In recent years, with the development of metabolomics technology, metabolomics has gradually become a recognized effective research method for studying metabolic diseases (including type 2 diabetes and gestational diabetes mellitus (GDM)). Cinnamoyl glycine (CMG) is a glycine conjugate of cinnamic acid, produced by intestinal microorganisms, and is abundant in the serum of conventional mice, but the concentration in the serum of germ-free mice is extremely low. The urinary clearance rate of cinnamoyl glycine is much higher than that of creatinine, so it will accumulate in the plasma under the condition of reduced kidney function. After adjusting creatinine, the association between cinnamoyl glycine and diversity and clinical characteristics is not affected. Although its functional effect in humans is unknown, the urinary excretion level of cinnamoyl glycine has been proposed as a marker of resistance to colonization by Clostridium difficile, i.e. as a marker of a healthy gut microbiome that can inhibit the growth of pathogenic microorganisms.

[0004] Liquid chromatography-mass spectrometry is widely used in biological sample detection due to its high sensitivity, high precision and high throughput. Due to the complexity of biological samples, many interference factors, and the fact that cinnamoyl glycine is an endogenous metabolite, its content in biological samples is extremely low and difficult to detect, limiting the application of cinnamoyl glycine as a marker in early diagnosis of diabetes. SUMMARY

[0005] The purpose of the present application is to provide a method for determining cinnamoyl glycine in a biological sample based on LC-MS, in order to overcome the shortcomings of the prior art and establish an analysis method suitable for endogenous metabolites in the human body. This method is a high-efficiency, rapid and accurate detection method that can meet the detection needs of a large number of clinical samples, and can provide important support for the application of metabolomics technology in clinical disease screening or diagnosis.

[0006] In order to achieve the above-mentioned purpose, the technical scheme of the present application is as follows: The application provides a method for determining cinnamoyl glycine in a biological sample based on LC-MS, comprising the following steps: The biological sample is mixed with an extraction solution, and after extraction treatment, a supernatant is obtained; the supernatant is evaporated and concentrated, redissolved in an acetonitrile aqueous solution, and filtered to obtain a to-be-tested solution; The extraction solution comprises an internal standard solution and a solvent, the internal standard solution comprises 2 H7]-N-cinnamyl glycine and a methanol / water solution, and the solvent is one or more of methanol, acetonitrile, methanol-acetonitrile, 1% formic acid-methanol-water and 0.1% formic acid-acetonitrile; The cinnamoyl glycine standard solution stock solution is diluted with a PBS solution to obtain a standard solution with a concentration gradient; The to-be-tested solution is tested by LC-MS, and the content of cinnamoyl glycine in the to-be-tested solution is obtained according to a standard curve.

[0007] The application uses liquid chromatography-tandem mass spectrometry (LC-MS) to quantitatively detect the content of cinnamoyl glycine in a biological sample, establishes an analysis method suitable for human endogenous metabolites, and provides important support for the screening or diagnosis of clinical diseases by the metabolomics technology.

[0008] In some other embodiments, the extraction solution comprises an internal standard solution and a solvent, the internal standard solution comprises 2 H7]-N-cinnamyl glycine and a methanol / water solution, and the solvent is one or more of methanol, acetonitrile, methanol-acetonitrile, 1% formic acid-methanol-water, 0.1% formic acid-acetonitrile. More specifically, methanol-acetonitrile (1:1), methanol-acetonitrile (1:9), 1% formic acid-methanol:water (9:1), 0.1% formic acid-acetonitrile. Further, the solvent is acetonitrile.

[0009] [ 2 H7]-N-cinnamyl glycine is used as an isotopic internal standard of cinnamoyl glycine, and the two have high consistency in structure and physicochemical properties. 2 The deuterium labeling only replaces hydrogen atoms, does not change the molecular structure, polarity or chromatographic behavior, ensures complete consistency with the target (cinnamoyl glycine) in retention time, ionization efficiency and matrix effect, and avoids signal interference caused by natural isotopic peaks in traditional labeling such as 13 C / 15 N labeling. The mass spectrometry of deuterium labeling has a larger shift, reduces the influence of background noise, and avoids the recovery deviation caused by structural differences. 2H7]-N-cinnamyl glycine and methanol / water solution consisting of internal standard solution, with solubility and stability. By optimizing the concentration and storage conditions, the accuracy of LC-MS quantitative can be significantly improved, especially suitable for the detection of cinnamoyl glycine in complex biological samples (plasma, urine). Through research, it is found that using methanol, acetonitrile, methanol:acetonitrile 1:1, methanol:acetonitrile 1:9, 1% formic acid-methanol:water 9:1, 0.1% formic acid-acetonitrile for extraction, the results show that when the solvent is acetonitrile, the extraction efficiency of cinnamoyl glycine is the highest.

[0010] In some other embodiments, the biological sample is one of plasma, serum, blood, urine, feces and saliva.

[0011] The method of the present application has high flux and low cost, effectively monitors the level of cinnamoyl glycine in the body, and is easy to popularize and popularize clinically.

[0012] In some other embodiments, the volume ratio of the biological sample to the extraction solution is 1:(3-6); the concentration of the internal standard in the extraction solution is 1-3 ng / mL. The extraction rate of cinnamoyl glycine in the biological sample in this range is higher.

[0013] In some other embodiments, the volume ratio of the biological sample to the extraction solution is 1:5; the concentration of the internal standard in the extraction solution is 2 ng / mL. The extraction rate of cinnamoyl glycine in the biological sample under this ratio is the highest.

[0014] In some other embodiments, the extraction process is carried out by vortex oscillation and centrifugal treatment in sequence; the volume ratio of the supernatant to the acetonitrile aqueous solution is (3-5):1; the volume concentration of the acetonitrile aqueous solution is 35-45%.

[0015] Exemplarily, the volume ratio of the supernatant to the acetonitrile aqueous solution is 3:1, 4:1, 5:1; the volume concentration of the acetonitrile aqueous solution is 35%, 40%, 45%. Further, the volume ratio of the supernatant to the acetonitrile aqueous solution is 4:1; the volume concentration of the acetonitrile aqueous solution is 40%. The volume ratio of the supernatant to the acetonitrile aqueous solution in this numerical range has the best protein precipitation efficiency, target recovery rate and matrix effect.

[0016] In some other embodiments, the chromatographic column used in liquid chromatography analysis is one or more of C18 column, ADME column and HILIC column, the column temperature is 25-30℃; the flow rate is 0.3-0.4 mL / min, the injection amount is 8-12 μL; The mobile phase is divided into mobile phase A and mobile phase B, the mobile phase A is acetonitrile containing 0.03-0.06% formic acid, the mobile phase B is water containing 0.05-0.15% formic acid, and gradient elution separation is adopted.

[0017] More specifically, the C18 column used in the liquid chromatography analysis is Agilent RRHD EclipsePlus C18 column or Titank C18 column, the ADME column is APCELL PAK ADME HR column, and the HILIC column is PC HILIC column; preferably, the column is APCELL PAK ADME HR (S-3), 2.1 μm x 100 mm, the column temperature is 25°C, 30°C; the flow rate is 0.3, 0.35, 0.4 mL / min, the injection volume is 8, 10, 12 μL; the mobile phase A is acetonitrile containing 0.03%, 0.04%, 0.05%, 0.06% formic acid, and the mobile phase B is water containing 0.05%, 0.1%, 0.15% formic acid.

[0018] In some other embodiments, the column temperature used in the liquid chromatography analysis is 25°C; the flow rate is 0.35 mL / min, the injection volume is 10 μL; the mobile phase A is acetonitrile containing 0.05% formic acid, the mobile phase B is water containing 0.1% formic acid, and the gradient elution program is used for separation: 0-1 min, 30% A, 1-2 min, 30%-50% A, 2-2.5 min, 50%-90% A, 2.5-5 min, 90% A, 5.1-8 min, 30% A.

[0019] Due to the complexity of components in the plasma and the influence of interferents, by comparing the advantages and disadvantages of different types of chromatographic columns in separation, such as Titank C18, C18-AQ, PC HILIC, etc., the APCELL PAK ADME HR chromatographic column is finally determined. Since CMG is a negative ion, it is difficult to scan positively and negatively, so different proportions of ammonium fluoride, formic acid and acetic acid are added to the mobile phase to improve the signal response of CMG. Analysis shows that ammonium fluoride reduces the response of CMG, formic acid and acetic acid improve the response of CMG, and adding formic acid to the mobile phase is beneficial to improve the separation degree, so that cinnamoyl glycine and interferents can be better separated. Further exploration of adding different proportions of formic acid in AB two phases shows that the response is the highest when the mobile phase A is acetonitrile containing 0.05% formic acid and the mobile phase B is water containing 0.1% formic acid, and it is not affected by interferents.

[0020] Gradient elution condition and flow rate: The flow rate has an effect on separation time and peak shape. By trying 0.3 mL / min, 0.35 mL / min, 0.4 mL / min, etc. flow rate, comparing the response value and peak shape of CMG under different flow rates, it is found that when the flow rate is 0.35 mL / min, the response value is high and the peak shape is narrow. In addition, the gradient elution condition is explored, and the initial elution proportion of the mobile phase is changed, and finally the best elution condition is obtained. The analysis time under this condition is short, only 5 min, and finally balanced for 3 min, which is convenient for multiple continuous sample injection.

[0021] In some other embodiments, in the mass spectrometric analysis, a multiple ion reaction monitoring mode of negative ion electrospray ionization is adopted, the curtain gas is 30-35 kPa, the spray voltage is 3700-3900 V, the desolvation temperature is 550-650 DEG C, GS1 is 25-35 kPa, and GS2 is 70-80 kPa; one of the ion pairs 160 / 167, 130 / 137 and 103 / 109 is adopted. For example, the curtain gas is 30, 35 kPa, the spray voltage is 3700, 3800, 3900 V, the desolvation temperature is 550, 600, 650 DEG C, GS1 is 25, 30, 35 kPa, and GS2 is 70, 75, 80 kPa. The setting of the parameters in this range is better for the sensitivity, resolution, accuracy and reproducibility of CMG detection.

[0022] In some other embodiments, in the mass spectrometric analysis, a multiple ion reaction monitoring mode of negative ion electrospray ionization is adopted, the curtain gas is 30 kPa, the spray voltage is 3800 V, the desolvation temperature is 600 DEG C, GS1 is 30 kPa, and GS2 is 75 kPa; the ion pair 103 / 137 is adopted. The setting of the parameters in this range is the best for the sensitivity, resolution, accuracy and reproducibility of CMG detection.

[0023] Advantages of the present application: (1) The application discloses a method for detecting cinnamoyl glycine in a blood sample. The biological sample is pretreated by a simple liquid-liquid extraction method, and then subjected to chromatographic separation and mass spectrometric detection. A pair of qualitative ions and quantitative ions are selected, the relative retention time and the qualitative ion pair of cinnamoyl glycine are used as the qualitative basis, and the standard curve is prepared by using a standard product for quantification. Meanwhile, three levels of quality control products are used to investigate the accuracy and effectiveness of the method, so that the detection result is not distorted. The method is simple in operation, has a rapid analysis time of only 8 minutes, has advantages of high throughput and low cost, has guiding significance for early diagnosis of gestational diabetes, and is easy to popularize and popularize clinically.

[0024] (2) The extraction method used in the application can remove impurities, reduce matrix effect during detection, reduce ion interference, and is simple and fast, and can sensitively detect low content of cinnamoyl glycine in human body. The curve correlation of cinnamoyl glycine is good (R>0.99), the linear range is 0.02-10 ng / mL, the detection limit is 0.02 ng / mL, and the deviation is within ±15 %. The detection cost is significantly reduced, the time is saved, and the blood volume of the detected person is reduced.

[0025] In summary, the application first realizes the pretreatment of the plasma sample, and detects cinnamoyl glycine in the plasma sample by using the LC-MS technology, uses two pairs of ions for quantification and qualitative guarantee of the specificity of the detected substance, reduces the influence of interfering substances, and the method is simple and fast, and the analysis time is only 8 min, the flux is high, the cost is low, the performance of the method is verified, the stability is good, the cinnamoyl glycine level in human body is effectively monitored, the early diagnosis of gestational diabetes mellitus is facilitated, the guidance significance is provided, and the method is easy to popularize and popularize in clinic. BRIEF DESCRIPTION OF DRAWINGS

[0026] The drawings accompanying the specification of the application form part of the application and serve to provide further understanding of the application. The illustrative embodiments of the application and their description serve to explain the application without forming an improper limitation of the application.

[0027] Figure 1 The figure is the mass spectrum of the cinnamoyl glycine standard in example 1 of the application; Figure 2 The figure is the mass spectrum of the internal standard 2 H7]-N-cinnamyl glycine standard in example 1 of the application; Figure 3 The figure is the standard curve established in example 1 of the application. DETAILED DESCRIPTION

[0028] Those skilled in the art will understand that the following examples are only used to illustrate the application, and should not be regarded as limiting the scope of the application. The specific conditions are not indicated in the examples, and the conventional conditions or the conditions recommended by the manufacturer are used. The components used are not indicated by the manufacturer, and are conventional products that can be obtained by market.

[0029] Among the common methods for determining the content of amino acids in blood, the high performance liquid chromatography method and the capillary electrophoresis method have large result difference, poor linearity, complex derivatization operation, small throughput and other problems. The amino acid analyzer determination method has the disadvantages of large sample dosage and long analysis time. The derivatization step of the gas chromatography-mass spectrometry method is complex, and the derivatization reaction is disturbed by many factors. The concentration content of cinnamoyl glycine in plasma is extremely low and is not easy to be detected, the protein precipitation method is used to extract cinnamoyl glycine in the application, and the composition of the mobile phase, the chromatographic column and the mass spectrometry conditions are changed, so that the cinnamoyl glycine in the biological sample is qualitatively and quantitatively analyzed.

[0030] The application provides a method for determining cinnamoyl glycine in a biological sample based on LC-MS, which comprises the following steps: The biological sample is mixed with an extraction liquid to obtain supernatant after extraction treatment; the supernatant is evaporated and concentrated, redissolved in an acetonitrile aqueous solution, filtered to obtain a to-be-tested solution; The extraction liquid comprises an internal standard solution and a solvent, the internal standard solution comprises 2 H7]-N-cinnamoyl glycine and a methanol / water solution, and the solvent is methanol, acetonitrile, methanol-acetonitrile, 1 % formic acid-methanol-water and 0.1 % formic acid-acetonitrile; The cinnamoyl glycine standard solution is diluted with a PBS solution to obtain a standard solution with a concentration gradient; The to-be-tested solution is tested by LC-MS, and the content of cinnamoyl glycine in the to-be-tested solution is obtained according to a standard curve.

[0031] The scheme of the application will be further described in combination with specific examples: Example 1 A method for determining cinnamoyl glycine in a biological sample based on LC-MS, which comprises the following steps: (1) Biological sample pretreatment: 200 μL of a biological sample (for example, plasma) is taken, 1 mL of acetonitrile containing an internal standard solution is added as an extraction liquid, vortexed for 5 min, centrifuged at 14000 rpm for 5 min to obtain supernatant. 1 mL of the supernatant is concentrated and dried, 50 μL of 40 % acetonitrile aqueous solution is added for redissolution, and the to-be-tested biological sample is obtained after filtration with a 0.2 μm filter membrane.

[0032] The extraction liquid is prepared as follows: 2 H7]-N-cinnamoyl glycine (product number: IR-72407) is used as an isotopic internal standard (mass spectrum as shown in Figure 2 The isotopic internal standard is dissolved in a methanol / water solution (1:1) to prepare an internal standard solution with a concentration of 1 μg / mL. The internal standard solution is diluted with acetonitrile to prepare acetonitrile containing an internal standard solution with a concentration of 2 ng / mL as an extraction liquid.

[0033] (2) Preparation of standard working solution: The biological sample actually detected, such as plasma, has complex composition and many interference factors. Since cinnamoyl glycine is an endogenous metabolite, if plasma is used as the matrix, a complex pretreatment step is needed to remove interfering substances before detection. The surrogate matrix can reduce the interference of the target analyte detection. The surrogate matrix has specific chemical or biological properties, which can specifically interact with the target analyte or specifically reflect the presence of the target analyte.

[0034] During the research, 5%-20% bovine serum albumin (BSA), 0.9% sodium chloride, phosphate buffered saline (PBS), and 5% bovine serum albumin-phosphate buffered saline (BSA-PBS) were used as surrogate matrices, respectively. It was found that BSA and 0.9% sodium chloride had matrix effects, and the addition of PBS could reduce the matrix effect. Therefore, PBS was finally selected as the surrogate matrix.

[0035] Specifically, the preparation of the standard working solution is as follows: cinnamoyl glycine standard (mass spectrum as shown in Figure 1 ) is added to water to prepare a 10 ng / mL cinnamoyl glycine standard stock solution. Then, the 10 ng / mL cinnamoyl glycine standard stock solution is gradiently diluted with PBS solution (as a surrogate matrix for plasma), respectively, to prepare 10 standard solutions with concentrations of 0.02, 0.04, 0.08, 0.16, 0.31, 0.625, 1.25, 2.5, 5, and 10 ng / mL.

[0036] (3) Liquid chromatography tandem mass spectrometry (LC-MS) detection: The liquid chromatography parameters are set as follows: the chromatographic column is APCELL PAK ADME HR (S-3), 2.1 μm*100 mm, the column temperature is 25℃, the flow rate is 0.35 mL / min, and the injection volume is 10 μL. The mobile phase is divided into mobile phase A and mobile phase B, wherein the mobile phase A is acetonitrile containing 0.05% formic acid, and the mobile phase B is water containing 0.1% formic acid. Gradient elution program is used for separation, and the specific gradient elution program is shown in Table 1: 0-1 min, 30% A, 1-2 min, 30%-50% A, 2-2.5 min, 50%-90% A, 2.5-5 min, 90% A, 5.1 min, 30% A. The analysis time under this condition is short, only 5 min, and the last 3 min is for balancing, which is convenient for multiple continuous injections.

[0037] Table 1 Gradient elution conditions

[0038] Mass spectrometry parameter settings: negative ion electrospray ionization, multiple ion reaction monitoring mode, ion source parameter settings and MRM parameter settings (as shown in Table 2). Among them, the ion source parameters include: curtain gas: 30 kPa, spray voltage: 3800 V, desolvation temperature: 600 ℃, atomization gas (GS1) pressure: 30 kPa, auxiliary gas (GS2) pressure: 75 kPa.

[0039] Table 2 MRM mass spectrometry parameters

[0040] MRM: multiple ion reaction monitoring mode. It refers to the mass spectrometry method in which ions are generated and detected.

[0041] m / z: refers to the dimensionless quantity formed by dividing the mass number of an ion by its charge number. For a long time, it has been called "mass-to-charge ratio".

[0042] CE: collision energy. The precursor ions receive energy and accelerate into the collision cell, where they collide with gas molecules (CAD gas) and form fragment ions. The higher the collision energy, the more fragments are caused.

[0043] CXP: collision cell exit voltage. CXP focuses, accelerates and transmits ions from Q2 to Q3.

[0044] From Table 2, by optimizing the mass spectrometry conditions, a total of three ion pairs can be selected, specifically: 160 / 167, 130 / 137 and 103 / 109 ion pairs. Among them, the 160 / 167 ion pair has high noise and high baseline, which affects the detection of low content samples. Although the 103 / 109 ion pair has a low baseline, there is an interferent that affects the detection of the target compound. Finally, the 103 / 137 ion pair, i.e. the cinnamoyl glycine, is selected for detection.

[0045] (4) Preparation of standard curve: To the 10 concentration (0.02, 0.04, 0.08, 0.16, 0.31, 0.625, 1.25, 2.5, 5, 10 ng / mL) standard solution prepared in step (2), 1 mL of acetonitrile containing internal standard solution was added as extraction liquid, and other preparation methods were the same as in Example 1 to prepare 10 concentrations of mixed standard working solution.

[0046] The peak area of each mixed standard working solution was determined by LC-MS, and the concentration of each mixed standard working solution was taken as the x-axis and the peak area of each mixed standard working solution was taken as the y-axis. The linear range, standard curve equation, linear correlation coefficient and detection limit were obtained by linear fitting, as shown in Table 3, and the standard curve graph is shown in Figure 3 .

[0047] Table 3 Linear range and equation of cinnamoyl glycine

[0048] As can be seen from Table 3, the curve correlation of cinnamoyl glycine is good (R > 0.99), the linear range is 0.02-10 ng / mL, the detection limit is 0.02 ng / mL, and the deviation is within ± 15 %.

[0049] The peak area of the biological sample to be tested is substituted into the standard curve equation to inversely calculate the concentration of cinnamoyl glycine in the biological sample.

[0050] (5) repeatability The 10 ng / mL cinnamoyl glycine standard stock solution in step (2) is diluted with PBS to 6 portions of 0.5 ng / mL standard solution, 1 mL of acetonitrile containing internal standard solution is added as the extraction liquid, and other preparation methods are the same as in Example 1, and the sample is injected. The RSD is 1.6 %, and the RSD is less than 15.0 %. The repeatability test meets the requirements.

[0051] (6) recovery rate The 10 ng / mL cinnamoyl glycine standard stock solution is gradient diluted with PBS solution, and 10 standard solutions with concentrations of 0.02, 0.04, 0.08, 0.16, 0.31, 0.625, 1.25, 2.5, 5, and 10 ng / mL are prepared. The sample is injected to obtain the standard curve. The lower concentration clinical sample is selected as the basic sample, the standard is added, and the low, medium and high concentration test samples (0.05, 0.5, and 8 ng / mL) are prepared. Each concentration sample is divided into 5 parts, the recovery rate is calculated according to formula (1), which should meet the requirements of 85%-115 %, and the analysis results are shown in Table 4.

[0052] Formula (1) R - recovery rate, C - the average measured concentration of the low concentration clinical sample after adding the standard, ng / mL; C 0 - the average measured concentration of the low concentration clinical sample, ng / mL; C s - the amount of standard added, ng / mL.

[0053] Table 4 Recovery rate of cinnamoyl glycine at different concentrations

[0054] As can be seen from Table 4, the recovery rate at each concentration is within the range of 85 % - 115 %, and the relative standard deviation of the 9 recovery rate data is less than 10.0 %. The recovery rate meets the requirements.

[0055] (7) Clinical sample detection Select 20 normal human plasma samples (sample numbers 1-20), prepare the test biological sample according to the pretreatment steps of the biological sample in step (1), detect the test sample by the detection method in step (3), and measure the concentration of cinnamoyl glycine in each test sample by the standard curve equation in step (4). The detection results are shown in Table 5.

[0056] Table 5 Clinical sample detection results

[0057] As can be seen from Table 5, the present application uses high performance liquid chromatography-mass spectrometry to detect cinnamoyl glycine in plasma, which has strong specificity, high sensitivity, high throughput, objective results and is easy to analyze, and is especially suitable for clinical popularization and application.

[0058] Comparative Example 1 A method for determining cinnamoyl glycine in a biological sample based on LC-MS, which is different from Example 1, is that in the biological sample pretreatment process of step (1), methanol, acetonitrile, methanol: acetonitrile (volume ratio 1:1), methanol: acetonitrile (volume ratio 1:9), 1% formic acid-methanol: water (volume ratio 9:1), or 0.1% formic acid-acetonitrile containing an internal standard solution are used as extraction solvents to extract the biological sample (such as plasma), and other preparation methods are the same as those of Example 1. The extraction agent, the volume ratio of the extraction agent to the biological sample, and the CMG response value are shown in Table 6.

[0059] Table 6 CMG response values prepared by different extraction methods

[0060] As can be seen from Table 6, when the volume ratio of the extraction liquid serum to acetonitrile is 1:5, the extraction efficiency of cinnamoyl glycine is the highest.

[0061] Example 2 A method for determining cinnamoyl glycine in a biological sample, which is different from Example 1, is that in step (3), Agilent RRHD Eclipse Plus C18, APCELL PAK ADME HR, PC HILIC, and Titank C18 are selected as the chromatographic column, respectively, and the other conditions are the same as those of Example 1. The separation results are shown in Table 7.

[0062] Table 7 Results of separating cinnamoyl glycine by different types of chromatographic columns

[0063] In Table 7, "√" represents good, and "×" represents poor.

[0064] Cinnamoyl glycine is extremely low in blood, and its response in mass spectrometry is low, so it is not easy to detect. By comparing the retention time, peak shape and response value of different types of chromatographic columns from Table 7, it is found that the C18 column can obtain better retention time and peak shape. Since the APCELL PAK ADME HR is resistant to pure water, the mobile phase can be eluted from 0% pure water, and the response value is high, so the APCELL PAK ADME HR column is finally determined.

[0065] In summary, the application first realizes the purpose of detecting cinnamoyl glycine in biological samples by liquid chromatography-mass spectrometry, reduces the influence of interferents, and has the advantages of simple operation, rapidity (analysis time is only 8 min), high throughput, low cost and the like. At the same time, the application uses an internal standard method for quantification, ensures the accuracy of the detection results, can effectively monitor the level of cinnamoyl glycine in the human body, has guiding significance for early diagnosis of gestational diabetes, and is easy to popularize and popularize in the clinic.

[0066] The above is only a preferred embodiment of the application and is not used to limit the application. For those skilled in the art, the application can have various changes and variations. Any modification, equivalent replacement, improvement, etc. within the spirit and principles of the application shall be included in the protection scope of the application.

Claims

1. A method for determining cinnamoylglycine in a biological sample based on LC-MS, characterized in that: The following steps are involved: The biological sample is mixed with the extraction solution, and a supernatant is obtained after extraction treatment; the supernatant is evaporated and concentrated, redissolved in an acetonitrile aqueous solution, and filtered to obtain a test solution; The extract comprises an internal standard solution and a solvent, wherein the internal standard solution comprises [ 2 H7] -N-cinnamylglycine and a methanol / water solution, wherein the solvent is one or more of methanol, acetonitrile, methanol-acetonitrile, 1% formic acid-methanol-water, and 0.1% formic acid-acetonitrile; Dilute the cinnamoylglycine standard stock solution with PBS solution to obtain a concentration gradient of standard solution; The solution to be tested is tested by LC-MS, and the content of cinnamoylglycine in the solution to be tested is obtained according to the standard curve; The chromatographic columns used in the liquid chromatography analysis are one or more of a C18 column, an ADME column, and a HILIC column. The mobile phases are mobile phase A and mobile phase B. Mobile phase A is acetonitrile containing 0.03-0.06% formic acid, and mobile phase B is water containing 0.05-0.15% formic acid. The separation is performed using a gradient elution program: 0-1 min, 30% A, 1-2 min, 30%-50% A, 2-2.5 min, 50%-90% A, 2.5-5 min, 90% A, 5.1-8 min, 30% A. In mass spectrometry analysis, the ion pairs used are one or more of 160 / 167, 130 / 137 and 103 / 109 ion pairs.

2. The method for determining cinnamoylglycine in a biological sample based on LC-MS according to claim 1, characterized in that: The biological sample is one of plasma, serum, blood, urine, feces and saliva.

3. The method for determining cinnamoylglycine in a biological sample based on LC-MS according to claim 1, characterized in that: The mixing volume ratio of the biological sample to the extract is 1:(3-6); The extract [ 2 H7]-N-cinnamylglycine at a concentration of 1-3 ng / mL.

4. The method for determining cinnamoylglycine in a biological sample based on LC-MS according to claim 3, characterized in that: The mixing volume ratio of the biological sample to the extract is 1:5; The extract [ 2 H7]-N-cinnamylglycine was present at a concentration of 2 ng / mL.

5. The method for determining cinnamoylglycine in a biological sample based on LC-MS according to claim 1, characterized in that: The extraction process is to perform vortex oscillation and centrifugation in sequence; The volume ratio of the supernatant to the acetonitrile aqueous solution is (3-5):1; The volume concentration of the acetonitrile aqueous solution is 35-45%.

6. The method for determining cinnamoylglycine in a biological sample based on LC-MS according to claim 5, characterized in that: The volume ratio of the supernatant to the acetonitrile aqueous solution is 4:1; The volume concentration of the acetonitrile aqueous solution is 40%.

7. The method for determining cinnamoylglycine in a biological sample based on LC-MS according to claim 1, characterized in that: The chromatographic columns used in the liquid chromatography analysis were an Agilent RRHD Eclipse Plus C18 column or a Titank C18 column, an APCELL PAK ADME HR column, and a PC HILIC column. The column temperature was 25-30°C; the flow rate was 0.3-0.4 mL / min, and the injection volume was 8-12 μL.

8. The method for determining cinnamoylglycine in a biological sample based on LC-MS according to claim 7, characterized in that: The chromatographic column used in the liquid chromatography analysis was an APCELL PAK ADME HR column with a column temperature of 25°C; the flow rate was 0.35 mL / min, and the injection volume was 10 μL; the mobile phase A was acetonitrile containing 0.05% formic acid, and the mobile phase B was water containing 0.1% formic acid.

9. The method for determining cinnamoylglycine in a biological sample based on LC-MS according to claim 1, characterized in that: In the mass spectrometry analysis, negative ion electrospray ionization in multiple ion reaction monitoring mode was used, with curtain gas: 30-35 kPa, spray voltage: 3700-3900 V, desolvation temperature: 550-650 °C, GS1: 25-35 kPa, and GS2: 70-80 kPa.

10. The method for determining cinnamoylglycine in a biological sample based on LC-MS according to claim 9, characterized in that: In the mass spectrometry analysis, negative ion electrospray ionization was used in the multiple ion reaction monitoring mode, with a curtain gas of 30 kPa, a spray voltage of 3800 V, a desolvation temperature of 600°C, GS1 of 30 kPa, and GS2 of 77 kPa; the ion pair used was 103 / 137.

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