A method for determining cinnamoyl glycine in a biological sample based on LC-MS

Abstract

The application belongs to the technical field of analysis and detection, and particularly relates to a method for determining cinnamoyl glycine in a biological sample based on LC-MS, which comprises the following steps: mixing the biological sample with an extraction solution, obtaining supernatant after extraction treatment; evaporating and concentrating, and then re-dissolving in an acetonitrile aqueous solution to obtain a solution to be tested; diluting a cinnamoyl glycine standard stock solution with a PBS solution to obtain a standard solution with a concentration gradient; mixing the standard solution with the extraction solution, and obtaining a mixed standard solution after extraction treatment; testing the mixed standard solution by LC-MS to obtain a standard curve; and testing the solution to be tested, and obtaining the content of cinnamoyl glycine in the solution to be tested according to the standard curve. The method can accurately qualitatively and quantitatively analyze cinnamoyl glycine in a sample, has the advantages of simple processing, high throughput and reliable results, and is easy to popularize and popularize clinically.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of analytical detection technology, 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 C. difficile colonization, 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:

[0007] The application provides a method for determining cinnamoyl glycine in a biological sample based on LC-MS, comprising the following steps:

[0008] 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;

[0009] The extraction solution comprises an internal standard solution and a solvent, and 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;

[0010] The cinnamoyl glycine standard solution stock solution is diluted with a PBS solution to obtain a standard solution with a concentration gradient;

[0011] 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.

[0012] 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.

[0013] In some other embodiments, the extraction solution comprises an internal standard solution and a solvent, and 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.

[0014] [ 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 H7) label 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 traditional labels such as 13 C / 15 N labeling due to natural isotopic peaks, and the mass spectrometry of the 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 quantification can be significantly improved, especially 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, the results show that when the solvent is acetonitrile, the extraction efficiency of cinnamoyl glycine is the highest.

[0015] In some other embodiments, the biological sample is one of plasma, serum, blood, urine, feces and saliva.

[0016] 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.

[0017] 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.

[0018] 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.

[0019] 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%.

[0020] 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.

[0021] 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;

[0022] 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.

[0023] 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.

[0024] 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.

[0025] Due to the complexity of the components in the plasma and the influence of the 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 will reduce the response of CMG, formic acid and acetic acid will 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 the interferents.

[0026] 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 rates, the response values and peak shapes of CMG under different flow rates are compared, and 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.

[0027] 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.

[0028] 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.

[0029] The beneficial effects of the present application are:

[0030] (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 and rapid, the analysis time is only 8 minutes, has the advantages of high throughput and low cost, and has guiding significance for early diagnosis of gestational diabetes mellitus, and is easy to popularize and popularize clinically.

[0031] (2) The extraction method used in the present application can remove more impurities, reduce the matrix effect during detection, reduce ion interference, and is simple and fast in operation, and can sensitively detect the low content of cinnamoyl glycine in the 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.

[0032] In summary, the present 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 in operation, 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 the human body is effectively monitored, the early diagnosis of gestational diabetes mellitus is facilitated, has guiding significance, is easy to popularize and popularize in clinic. BRIEF DESCRIPTION OF DRAWINGS

[0033] The drawings accompanying the specification of the present application serve to provide a further understanding of the present application, and the illustrative embodiments of the present application and the description thereof serve to explain the present application, and do not constitute an improper limitation on the present application.

[0034] Figure 1 The mass spectrum of the cinnamoyl glycine standard in Example 1 of the present application is shown in the figure;

[0035] Figure 2 The mass spectrum of the internal standard of the cinnamoyl glycine standard in Example 1 of the present application is shown in the figure; 2 The mass spectrum of the internal standard of the cinnamoyl glycine standard in Example 1 of the present application is shown in the figure;

[0036] Figure 3 The standard curve established in Example 1 of the present application is shown in the figure. DETAILED DESCRIPTION

[0037] Those skilled in the art will understand that the following examples are only used to illustrate the present application, and should not be regarded as limiting the scope of the present 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 all conventional products that can be obtained by marketing.

[0038] 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, and small flux. The amino acid analyzer determination method has the defects 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. The concentration content of cinnamoyl glycine in blood 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 condition are changed, so that the cinnamoyl glycine in the biological sample is qualitatively and quantitatively analyzed.

[0039] The application provides a method for determining cinnamoyl glycine in a biological sample based on LC-MS, which comprises the following steps.

[0040] The biological sample is mixed with an extraction liquid to obtain supernatant after extraction treatment; the supernatant is evaporated and concentrated, re-dissolved in an acetonitrile aqueous solution, filtered to obtain a to-be-tested solution;

[0041] 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;

[0042] The cinnamoyl glycine standard solution is diluted with a PBS solution to obtain a standard solution with a concentration gradient;

[0043] 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.

[0044] The scheme of the application is further described in combination with specific embodiments as follows:

[0045] Embodiment 1

[0046] A method for determining cinnamoyl glycine in a biological sample based on LC-MS, which comprises the following steps.

[0047] (1) Biological sample pretreatment: 200 μL of a biological sample (for example, blood 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, and supernatant is prepared. 1 mL of the supernatant is concentrated and dried, 50 μL of 40 % acetonitrile aqueous solution is added for re-dissolution, and the to-be-tested biological sample is obtained after filtration with a 0.2 μm filter membrane.

[0048] The extraction liquid is prepared by using 2 H7]-N-cinnamoyl glycine (product number: IR-72407) as an isotopic internal standard (mass spectrum as shown in Figure 2Isotope internal standard was dissolved in methanol / water solution (1:1) to prepare internal standard solution with a concentration of 1 μg / mL. The internal standard solution was diluted with acetonitrile to prepare acetonitrile containing internal standard solution with a concentration of 2 ng / mL as the extraction solution.

[0049] (2) Preparation of standard working solution:

[0050] The biological sample actually detected, such as the composition of plasma, has 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. Substitution matrix can reduce the interference on the detection of target analyte. Substitution matrix has specific chemical or biological properties, which can specifically interact with target analyte or can specifically reflect the presence of target analyte.

[0051] During the research, 5 %-20 % bovine serum albumin (BSA), 0.9 % sodium chloride, phosphate buffered saline (PBS), 5 % bovine serum albumin-phosphate buffered saline (BSA-PBS) were used as substitution matrix, respectively. It was found that BSA and 0.9 % sodium chloride had matrix effect, and the addition of PBS could reduce the matrix effect. Therefore, PBS was finally selected as the substitution matrix.

[0052] Specifically, the preparation of the standard working solution is as follows: cinnamoyl glycine standard (mass spectrum as shown in Figure 1 The figure) was added to water to prepare a 10 ng / mL cinnamoyl glycine standard stock solution. Then, the 10 ng / mL cinnamoyl glycine standard stock solution was gradiently diluted with PBS solution (as a substitute for plasma) 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, respectively.

[0053] (3) Liquid chromatography tandem mass spectrometry (LC-MS) detection:

[0054] Liquid chromatography parameters: the chromatographic column is selected as APCELL PAK ADME HR (S-3), 2.1 μm*100 mm, the column temperature is 25 °C; the flow rate is 0.35 mL / min, the injection amount 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. A 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 used for balancing, which is convenient for multiple continuous injections.

[0055] Table 1 Gradient elution conditions

[0056]

[0057] Mass spectrometry parameters: negative ion electrospray ionization in multiple ion reaction monitoring mode, mass spectrometry parameter settings include 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.

[0058] Table 2 MRM mass spectrometry parameters

[0059]

[0060] MRM: multiple ion reaction monitoring mode. It refers to the mass spectrometry method in which ions are generated and detected.

[0061] 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".

[0062] 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.

[0063] CXP: collision cell exit voltage. CXP focuses, accelerates and transports ions from Q2 to Q3.

[0064] 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 large noise and high baseline, which affects the detection of low content samples. The 103 / 109 ion pair has low baseline, but 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.

[0065] (4) Preparation of standard curve:

[0066] To the 10 concentrations (0.02, 0.04, 0.08, 0.16, 0.31, 0.625, 1.25, 2.5, 5, 10 ng / mL) of standard solution prepared in step (2), 1 mL of acetonitrile containing internal standard solution was added as the extraction liquid, and other preparation methods were the same as in Example 1 to prepare 10 concentrations of mixed standard working solution.

[0067] The peak area of each mixed standard working solution was measured 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 .

[0068] Table 3 Linear range and equation of cinnamoyl glycine

[0069]

[0070] As shown in 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 %.

[0071] The peak area of the test biological sample was substituted into the standard curve equation to calculate the concentration of cinnamoyl glycine in the biological sample.

[0072] (5) Reproducibility

[0073] The 10 ng / mL cinnamoyl glycine standard stock solution in step (2) was diluted with PBS to 6 portions of 0.5 ng / mL standard solution, 1 mL of acetonitrile containing internal standard solution was added as the extraction liquid, and other preparation methods were the same as in Example 1, and the sample was injected. The RSD was 1.6 %, and the RSD was less than 15.0 %.

[0074] (6) Spiked recovery rate

[0075] The 10 ng / mL cinnamoyl glycine standard solution was gradiently diluted with a PBS solution 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, and a standard curve was obtained by injection. A lower concentration clinical sample was selected as a basic sample, the standard solution was added to prepare low, medium and high concentration test samples (0.05, 0.5 and 8 ng / mL), each concentration sample was divided into 5 parts, the standard addition recovery was calculated according to formula (1), and the standard addition recovery should meet the requirement of 85%-115%, and the analysis results are shown in Table 4.

[0076] Formula (1)

[0077] R - recovery rate, C - the average measured concentration of the low concentration clinical sample after standard addition, ng / mL; C 0 - the average measured concentration of the low concentration clinical sample, ng / mL; C s - the standard addition amount, ng / mL.

[0078] Table 4 Recovery rates of cinnamoyl glycine at different concentrations

[0079]

[0080] As shown in Table 4, the recovery rates at different concentrations are all within the range of 85%-115%, and the relative standard deviations of 9 recovery rate data are less than 10.0%. The recovery rate meets the requirement.

[0081] (7) Clinical sample detection

[0082] 20 normal human plasma samples (labeled as samples 1-20) were selected, the test biological samples were prepared according to the pretreatment steps in step (1), the test samples were detected by the detection method in step (3), and the concentration of cinnamoyl glycine in each test sample was measured by the standard curve equation in step (4), and the detection results are shown in Table 5.

[0083] Table 5 Clinical sample detection results

[0084]

[0085] As shown in Table 5, the cinnamoyl glycine in the plasma is detected by the high performance liquid chromatography-mass spectrometry method, which has high specificity, high sensitivity, high throughput, objective results and is easy to analyze, and is especially suitable for clinical popularization and application.

[0086] Comparative Example 1

[0087] A method for determining cinnamoyl glycine in a biological sample based on LC-MS, different from Example 1, is that in step (1), during the pretreatment of the biological sample, 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 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 in 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.

[0088] Table 6 CMG response values obtained by different extraction methods

[0089]

[0090] As can be seen from Table 6, when the extraction solvent is serum and the volume ratio of acetonitrile is 1:5, the extraction efficiency of cinnamoyl glycine is the highest.

[0091] Example 2

[0092] A method for determining cinnamoyl glycine in a biological sample, 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 other conditions are the same as those in Example 1. The separation results are shown in Table 7.

[0093] Table 7 Results of separating cinnamoyl glycine by different types of chromatographic columns

[0094]

[0095] In Table 7, “√” represents good, and “×” represents poor.

[0096] Cinnamoyl glycine has a very low content in blood and a low response in mass spectrometry, so it is not easy to detect. By comparing the retention time, peak shape, and response value when separating by different types of chromatographic columns from Table 7, it is found that C18 columns can obtain good retention time and peak shape. Since 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, APCELL PAK ADME HR column is finally determined.

[0097] To sum up, the application first realizes the purpose of detecting cinnamoyl glycine in biological samples by using a liquid chromatography-mass spectrometry method, reduces the influence of interfering substances, and has the advantages of simple operation, rapidness (analysis time is only 8 min), high throughput, low cost and the like. Meanwhile, the application quantifies by using an internal standard method, ensures the accuracy of the detection result, can effectively monitor the cinnamoyl glycine level in the human body, has guiding significance for early diagnosis of gestational diabetes, and is easy to popularize and popularize clinically.

[0098] The above merely describes preferred embodiments of the present application but is not intended to limit the present application, and various modifications and changes can be made by those skilled in the art based on the spirit and principles of the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. A method for determining cinnamyl glycine in biological samples based on LC-MS, characterized in that, Includes the following steps: Biological samples were mixed with extraction solution and extracted to obtain supernatant; the supernatant was evaporated and concentrated, redissolved in acetonitrile aqueous solution, filtered, and the solution to be tested was obtained. The extract includes an internal standard solution and a solvent, the internal standard solution comprising [ 2 [H7]-N-cinnamylglycine and methanol / water solution, wherein the solvent is acetonitrile; The cinnamyl glycine standard stock solution was diluted with phosphate buffer solution to obtain a concentration gradient of standard solutions. The content of cinnamyl glycine in the test solution was obtained by LC-MS according to the standard curve. In the liquid chromatography analysis, an APCELL PAK ADME HR column was used. Mobile phases A and B were employed: mobile phase A was acetonitrile containing 0.03-0.06% formic acid, and mobile phase B was water containing 0.05-0.15% formic acid. A gradient elution program was 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. The column temperature was 25-30℃; the flow rate was 0.3-0.4 mL / min; and the injection volume was 8-12 μL. In the mass spectrometry analysis, the ion pairs used were 103 / 137 ion pairs, and the multi-ion reaction monitoring mode of negative ion electrospray ionization was adopted. The curtain gas pressure was 30-35 kPa, the spray voltage was 3700-3900 V, the desolventization temperature was 550-650 ℃, the nebulizer gas GS1 was 25-35 kPa, and the auxiliary gas GS2 was 70-80 kPa. The volume ratio of the biological sample to the extract is 1:(3-6). The extract contains [ 2 The concentration of [H7]-N-cinnamylglycine was 1-3 ng / mL.

2. The method for determining cinnamyl glycine in biological samples 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 cinnamyl glycine in biological samples based on LC-MS according to claim 1, characterized in that, The volume ratio of the biological sample to the extract is 1:5; The extract contains [ 2 The concentration of [H7]-N-cinnamylglycine was 2 ng / mL.

4. The method for determining cinnamyl glycine in biological samples based on LC-MS according to claim 1, characterized in that, The extraction process involves sequentially performing vortex oscillation and centrifugation. 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%.

5. The method for determining cinnamyl glycine in biological samples based on LC-MS according to claim 4, 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%.

6. The method for determining cinnamyl glycine in biological samples based on LC-MS according to claim 1, characterized in that, The column temperature was 25℃; the flow rate was 0.35 mL / min; the injection volume was 10 μL; mobile phase A was acetonitrile containing 0.05% formic acid; and mobile phase B was water containing 0.1% formic acid.

7. The method for determining cinnamyl glycine in biological samples based on LC-MS according to claim 1, characterized in that, In the mass spectrometry analysis, the multi-ion reaction monitoring mode of negative ion electrospray ionization was adopted, with curtain gas: 30 kPa, spray voltage: 3800 V, desolventization temperature: 600℃, nebulizer gas GS1: 30 kPa, and auxiliary gas GS2: 77 kPa.

Images