CN120801576A

CN120801576A - Method for determining cinnamoyl glycine in biological sample based on LC-MS

Info

Publication number: CN120801576A
Application number: CN202511299380.8A
Authority: CN
Inventors: 高媛; 步思情; 邹洋; 高选; 陈子江; 马金龙
Original assignee: Shandong University
Current assignee: Shandong University
Priority date: 2025-09-12
Filing date: 2025-09-12
Publication date: 2025-10-17
Anticipated expiration: 2045-09-12
Also published as: CN120801576B

Abstract

The present invention belongs to the field of analysis and detection technology, and specifically relates to a method for determining cinnamoylglycine in a biological sample based on LC-MS, comprising the following steps: mixing the biological sample with an extract, obtaining a supernatant after extraction treatment; concentrating by evaporation and redissolving in an acetonitrile aqueous solution to obtain a solution to be tested; diluting a cinnamoylglycine standard stock solution with a PBS solution to obtain a standard solution with a concentration gradient; mixing the standard solution with the extract, obtaining a mixed standard solution after extraction treatment, testing the mixed standard solution by LC-MS to obtain a standard curve; testing the solution to be tested, and determining the content of cinnamoylglycine in the solution to be tested according to the standard curve. This method can perform accurate qualitative and quantitative analysis of cinnamoylglycine in a sample, has the advantages of simple processing, high throughput, and reliable results, and is easy to promote and popularize in clinical practice.

Description

Method for determining cinnamoyl glycine in biological sample based on LC-MS

Technical Field

The invention belongs to the technical field of analysis and detection, and particularly relates to a method for measuring cinnamoyl glycine in a biological sample based on LC-MS.

Background

Metabolomics (Metabolomics) is an important component of systematic biology, aimed at comprehensively analyzing the dynamic changes of small molecule metabolites (molecular weight <1500 Da) in organisms, revealing the metabolic response of the organisms under physiological, pathological or external stimuli. The metabonomics technology comprises a plurality of technical means such as chromatography technology, mass spectrometry technology, magnetic resonance technology and the like, has the advantages of high detection speed, high instrument automation degree, high flux, controllable detection quality, good repeatability and the like, and is commonly used for detecting small molecular metabolites in metabonomics.

In recent years, with the development of metabonomics technology, metabonomics has gradually become a well-established research method for studying metabolic diseases, including type 2 diabetes and Gestational Diabetes (GDM). Cinnamoyl glycine (CMG) is a glycine conjugate of cinnamic acid produced by intestinal microorganisms and is abundant in serum of conventional mice, but has a very low concentration in serum of sterile mice. The urine clearance of cinnamoyl glycine is much higher than creatinine and therefore accumulates in plasma under conditions of reduced renal function. After creatinine adjustment, the association between cinnamoyl glycine and diversity and clinical features 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 clostridium difficile colonization resistance, i.e. as a marker of healthy intestinal microbiome that can inhibit the growth of pathogenic microorganisms.

The liquid chromatography-mass spectrometry method has the advantages of high sensitivity, high precision and high flux, and is widely applied to biological sample detection. Because the biological sample has complex components and multiple interference factors, and the cinnamoyl glycine is an endogenous metabolite, the content of the cinnamoyl glycine in the biological sample is extremely low, and the detection is difficult, so that the application of the cinnamoyl glycine serving as a marker in early diagnosis of diabetes is limited.

Disclosure of Invention

The invention aims to provide a method for measuring cinnamoyl glycine in a biological sample based on LC-MS (liquid chromatography-mass spectrometry), which aims to overcome the defects of the prior art and establish an analysis method suitable for endogenous metabolites of a human body.

In order to achieve the above purpose, the technical scheme of the invention is as follows:

the invention provides a method for determining cinnamoyl glycine in a biological sample based on LC-MS, which comprises the following steps:

mixing the biological sample with the extracting solution, extracting to obtain a supernatant, evaporating and concentrating the supernatant, redissolving the supernatant in an acetonitrile aqueous solution, and filtering to obtain a solution to be detected;

the extracting solution comprises an internal standard solution and a solvent, wherein the internal standard solution comprises [ ²H₇ ] -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;

diluting the cinnamoyl glycine standard stock solution with PBS solution to obtain a standard solution with a concentration gradient;

And testing the solution to be tested through LC-MS, and obtaining the content of cinnamoyl glycine in the solution to be tested according to a standard curve.

The invention uses liquid chromatography-tandem mass spectrometry (LC-MS) to quantitatively detect the content of cinnamoyl glycine in a biological sample, and establishes an analysis method suitable for endogenous metabolites of human bodies, which is a high-efficiency, rapid and accurate detection method capable of meeting the detection requirements of mass samples in clinic, and provides important support for metabonomics technical means in screening or diagnosis of clinical diseases.

In some other embodiments, the extract comprises an internal standard solution comprising [ ²H₇ ] -N-cinnamylglycine and a methanol/water solution and a solvent that is 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.

The [ ²H₇ ] -N-cinnamyl glycine is used as an isotope internal standard substance of cinnamoyl glycine, the structure and physicochemical properties of the two are highly consistent, the deuterated (²H₇) mark only replaces hydrogen atoms, the molecular structure, polarity or chromatographic behavior is not changed, the complete consistency with a target substance (cinnamoyl glycine) in retention time, ionization efficiency and matrix effect is ensured, the signal interference caused by natural isotope peaks of the traditional mark such as ¹³C/¹⁵ N mark is avoided, the mass spectrum deviation of the deuterated mark is larger, the influence of background noise is reduced, and the recovery rate deviation caused by structural difference is avoided. [ ²H₇ ] -N-cinnamyl glycine and methanol/water solution form an internal standard solution, and the internal standard solution has solubility and stability. By optimizing the concentration and the storage condition, the accuracy of LC-MS quantification can be remarkably improved, and the method is particularly suitable for detecting cinnamoyl glycine in complex biological samples (blood plasma and urine). It was found by study that extraction was performed using methanol, acetonitrile, methanol: acetonitrile 1:1, methanol: acetonitrile 1:9, 1% formic acid-methanol: water 9:1, 0.1% formic acid-acetonitrile, etc., and the results showed that the extraction efficiency of cinnamoyl glycine was highest when the solvent was acetonitrile.

In some other embodiments, the biological sample is one of plasma, serum, blood, urine, stool, and saliva.

The method has high flux and low cost, effectively monitors the level of the cinnamoyl glycine in the organism, and is easy to clinically popularize.

In some other embodiments, the mixing volume ratio of the biological sample to the extract is 1 (3-6), and the concentration of the internal standard in the extract is 1-3 ng/mL. The extraction rate of cinnamoyl glycine in the biological sample in the range is higher.

In some other embodiments, the mixing volume ratio of the biological sample to the extract is 1:5 and the concentration of the internal standard in the extract is 2 ng/mL. The extraction rate of cinnamoyl glycine in the biological sample is highest at the ratio.

In some other embodiments, the extraction process is sequentially performed by vortex oscillation and centrifugation, the volume ratio of the supernatant to the acetonitrile aqueous solution is (3-5): 1, and the volume concentration of the acetonitrile aqueous solution is 35-45%.

Illustratively, the volume ratio of the supernatant to the acetonitrile aqueous solution is 3:1, 4:1, 5:1, and 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, and the volume concentration of the acetonitrile aqueous solution is 40%. The volume ratio of the supernatant to the acetonitrile aqueous solution in the numerical range can be used for preparing the best protein precipitation efficiency, target recovery rate and matrix effect.

In some other embodiments, the chromatographic column used in the liquid chromatographic analysis is one or more of C18 column, ADME column and HILIC column, the column temperature is 25-30deg.C, the flow rate is 0.3-0.4 mL/min, and the sample injection amount is 8-12 μl;

the mobile phase is divided into a mobile phase A and a mobile phase B, wherein the mobile phase A is acetonitrile containing 0.03-0.06% of formic acid, and the mobile phase B is water containing 0.05-0.15% of formic acid, and gradient elution separation is adopted.

More specifically, the C18 column in the chromatographic column used in the liquid chromatography is AGILENT RRHD ECLIPSE Plus C18 column or Titank C column, the ADME column is APCELL PAK ADME HR column, the HILIC column is PC HILIC column, the chromatographic column is APCELL PAK ADME HR (S-3), 2.1 μm×100mm, the column temperature is 25 ℃ and 30 ℃, the flow rate is 0.3, 0.35 and 0.4 mL/min, the sample injection amount is 8, 10 and 12 μl, the mobile phase A is acetonitrile containing 0.03%, 0.04%, 0.05% and 0.06% formic acid, and the mobile phase B is water containing 0.05%, 0.1% and 0.15% formic acid.

In some other embodiments, the column temperature used in the liquid chromatography is 25 ℃, the flow rate is 0.35 mL/min, the sample loading is 10 μl, mobile phase A is acetonitrile containing 0.05% formic acid, mobile phase B is water containing 0.1% formic acid, and the separation is performed using a gradient elution procedure of 0-1min,30% A,1-2min,30% -50% A,2-2.5min,50% -90% A,2.5-5min,90% A,5.1-8 min,30% A.

The plasma has complex components and has the influence of interferents, and the separation of different types of chromatographic columns is compared with the advantages and disadvantages, such as Titank C, C18-AQ, PC HILIC and the like, and finally the chromatographic column is APCELL PAK ADME HR. Because CMG is negative ion, the positive and negative simultaneous scanning is difficult, so that the signal response of CMG is improved by trying to add ammonium fluoride, formic acid and acetic acid in different proportions into the mobile phase. Analysis shows that ammonium fluoride can reduce the response of CMG, formic acid and acetic acid can improve the response of CMG, and the addition of formic acid in the mobile phase is beneficial to improving the separation degree, so that cinnamoyl glycine and an interfering substance can be well separated, and further, the addition of formic acid with different proportions in the AB two phases is further searched, and the mobile phase A is acetonitrile containing 0.05% of formic acid, the mobile phase B is highest in water response containing 0.1% of formic acid and is not influenced by the interfering substance.

Gradient elution conditions and flow rate has an effect on separation time and peak shape. By trying the flow rates of 0.3 mL/min, 0.35 mL/min, 0.4 mL/min, and the like, the response value and peak shape of the CMG at different flow rates were compared, and as a result, the response value was found to be high and the peak shape was found to be narrow when the flow rate was 0.35 mL/min. In addition, gradient elution conditions are searched, the initial elution proportion of the mobile phase is changed, and finally the optimal elution conditions are obtained. The condition analysis time is short, only 5min is needed, and the final balance is 3 min, so that continuous sample injection for multiple times is convenient.

In some other embodiments, in mass spectrometry, a multi-ion reaction monitoring mode of anion electrospray ionization is used, a gas curtain gas is 30-35 kPa, a spray voltage is 3700-3900V, a desolvation temperature is 550-650 ℃, GS1 is 25-35 kPa, GS2 is 70-80 and kPa, and the ion pair used is one of 160/167,130/137,103/109 ion pairs. Exemplary, air curtains 30, 35 kPa, spray voltages 3700, 3800, 3900, V, desolvation temperatures 550, 600, 650 ℃, GS1:25, 30, 35 kpa, gs2:70, 75, 80, kPa. The parameter setting in the range has better sensitivity, resolution, accuracy and reproducibility for CMG detection.

In some other embodiments, in mass spectrometry, a multi-ion reaction monitoring mode of anion electrospray ionization is used, gas curtain gas is 30 kPa, spray voltage is 3800V, desolvation temperature is 600 ℃, GS1 is 30 kPa,GS2:75 kPa, and the ion pairs used are 103/137 ion pairs. The setting of parameters in this range is the best for sensitivity, resolution, accuracy and reproducibility of the CMG detection.

The invention has the beneficial effects that:

(1) The invention discloses a method for detecting cinnamoyl glycine in a blood sample, which comprises the steps of preprocessing a biological sample by a simple liquid-liquid extraction method, then performing chromatographic separation and mass spectrum detection, selecting a pair of qualitative ions and quantitative ions, taking the relative retention time and the qualitative ion pair of the cinnamoyl glycine as qualitative basis, and preparing a standard curve for quantification by a standard substance. Meanwhile, the accuracy and the effectiveness of the quality control product inspection method at three levels are applied, and the distortion of the detection result is avoided. The method is simple and convenient to operate, has the advantages of quick analysis time of only 8 minutes, high throughput, low cost and the like, has guiding significance for early diagnosis of gestational diabetes mellitus, and is easy to clinically popularize.

(2) The extraction method used in the invention can remove more impurities, reduce matrix effect during detection, reduce ion interference, and has simple and rapid operation, and can sensitively detect the cinnamoyl glycine with lower content in human body. The curve correlation of the 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 obviously reduced, the time is saved, and the blood volume of the detected person is reduced.

In summary, the invention realizes the aim of preprocessing the plasma sample for the first time, uses the LC-MS technology to detect the cinnamoyl glycine in the plasma sample, uses two pairs of ions to quantitatively and qualitatively ensure the specificity of the detected object, reduces the influence of interfering substances, has simple and rapid analysis time of only 8 min, has high flux and low cost, performs performance verification on the method, has good stability, effectively monitors the cinnamoyl glycine level in a human body, is convenient for early diagnosis of gestational diabetes, has guiding significance, and is easy to clinically popularize.

Drawings

The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention.

FIG. 1 is a mass spectrum of a cinnamoyl glycine standard in example 1 of the present invention;

FIG. 2 is a mass spectrum of an internal standard [ ²H₇ ] -N-cinnamyl glycine standard in example 1 of the present invention;

Fig. 3 is a standard graph established in example 1 of the present invention.

Detailed Description

It will be appreciated by those skilled in the art that the following examples are illustrative of the present invention and should not be construed as limiting the scope of the invention. Specific conditions are not noted in the examples, and are carried out according to conventional conditions or conditions recommended by the manufacturer. The components used are not specific to the manufacturer and are all commercially available conventional products.

Aiming at the problems of large result difference, poor linearity, complex derivatization operation, small flux and the like in the method for measuring the common amino acid content in blood by a high performance liquid chromatography method and a capillary electrophoresis method. The amino acid analyzer assay has the disadvantages of large sample usage and long analysis time. The derivatization step of the gas chromatography-mass spectrometry is complex, and the derivatization reaction is much interfered. The concentration content of the cinnamoyl glycine in the blood plasma is extremely low and is not easy to detect, the method adopts a protein precipitation method to extract the cinnamoyl glycine, and the composition of a mobile phase, a chromatographic column and mass spectrum conditions are changed, so that qualitative and quantitative analysis is carried out on the cinnamoyl glycine in a biological sample.

The extracting solution comprises an internal standard solution and a solvent, wherein the internal standard solution comprises [ ²H₇ ] -N-cinnamyl 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 following is a further description of the present invention with reference to specific examples:

Example 1

A method for LC-MS based determination of cinnamoyl glycine in a biological sample, comprising the steps of:

(1) Pretreatment of biological sample by taking 200. Mu.L of biological sample (for example, plasma), adding acetonitrile containing 1mL of internal standard solution as an extracting solution, swirling for 5min, centrifuging at 14000 rpm for 5min, and obtaining supernatant. Concentrating and drying 1mL supernatant, adding 50 mu L of 40% acetonitrile water solution for redissolving, and filtering by adopting a 0.2 mu m filter membrane to obtain a biological sample to be detected.

Wherein the extract is prepared by adopting [ ²H₇ ] -N-cinnamyl glycine (product number: IR-72407) as isotope internal standard (mass spectrum is shown in figure 2). The isotope internal standard is dissolved in methanol/water solution (1:1) to prepare an internal standard solution with the concentration of 1 mug/mL. The internal standard solution was diluted with acetonitrile to prepare acetonitrile containing the internal standard solution at a concentration of 2 ng/mL as an extract.

(2) Preparation of a standard working solution:

The composition of a biological sample such as plasma to be actually detected is complex and the interference factors are many. Since cinnamoyl glycine is an endogenous metabolite, if plasma is used as a substrate, a complex pretreatment step is required to remove interfering substances before detection. The surrogate matrix reduces interference with detection of the target analyte. The surrogate matrix has specific chemical or biological properties, is capable of specifically interacting with or reflecting the presence of the target analyte.

In the research process, 5% -20% Bovine Serum Albumin (BSA), 0.9% sodium chloride, phosphate Buffer Solution (PBS) and 5% bovine serum albumin-phosphate buffer solution (BSA-PBS) are respectively adopted as the alternative matrixes. It was found that BSA and 0.9% sodium chloride both had a matrix effect, whereas the addition of PBS reduced the matrix effect. Thus, PBS was ultimately selected as an alternative matrix.

Specifically, the standard working solution is prepared by adding a cinnamoyl glycine standard substance (mass spectrum is shown in figure 1) into water to prepare 10 ng/mL of cinnamoyl glycine standard substance stock solution. 10 ng/mL of the cinnamoyl glycine standard stock solutions were then each serially diluted with PBS solution (as a plasma replacement matrix) to give 10 total concentrations (0.02, 0.04, 0.08, 0.16, 0.31, 0.625, 1.25, 2.5, 5, 10 ng/mL) of standard solutions.

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

Liquid chromatography parameters were set up by selecting APCELL PAK ADME HR (S-3) columns, 2.1 μm x 100mm column temperature 25 ℃, flow rate 0.35 mL/min and sample injection volume 10. Mu.L. The mobile phase is divided into a mobile phase A and a mobile phase B, wherein the mobile phase A is acetonitrile containing 0.05% of formic acid, and the mobile phase B is water containing 0.1% of formic acid. The separation is carried out by adopting a gradient elution program, wherein the specific gradient elution program is shown in the table 1, namely 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 and 30% A. The condition analysis time is short, only 5min is needed, and the final balance is 3 min, so that continuous sample injection for multiple times is convenient.

TABLE 1 gradient elution conditions

Mass spectrometry parameter settings multi-ion reaction monitoring mode using negative ion electrospray ionization, mass spectrometry parameter settings included ion source parameter settings and MRM parameter settings (as shown in table 2). The ion source parameters include gas curtain gas 30 kPa, spray voltage 3800V, desolvation temperature 600 ℃, atomizing gas (GS 1) pressure 30 kPa, auxiliary gas (GS 2) pressure 75 kPa.

Table 2 MRM mass spectral parameters

MRM Multi-ion reaction monitoring mode. Refers to mass spectrometry methods in which ions are generated and detected.

M/z refers to a dimensionless number formed by dividing the mass number of an ion by its charge number. It has long been known as the "mass to charge ratio".

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.

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

As can be seen from Table 2, by optimizing the mass spectrometry conditions, a total of three ion pairs, specifically 160/167, 130/137 and 103/109, can be selected. Wherein 160/167 ion pairs are noisy, have a high baseline, and affect low content sample detection. The 103/109 ion pair, although baseline is low, the presence of interferents affects the detection of the target compound, and the final selection, cinnamoyl glycine selection 103, and the internal standard selection 137, are detected by the 103/137 ion pair.

(4) Drawing a standard curve:

to 10 standard solutions of the concentrations (0.02, 0.04, 0.08, 0.16, 0.31, 0.625, 1.25, 2.5, 5, 10 ng/mL) obtained in the step (2), acetonitrile containing an internal standard solution of 1:1 mL was added as an extraction solution, and 10 mixed standard working solutions of the concentrations were obtained in the same manner as in example 1.

The peak area of each mixed standard working solution is measured by using LC-MS, the concentration of each mixed standard working solution is taken as an x axis, the peak area of each mixed standard working solution is taken as a y axis, a linear range, a standard curve equation, a linear correlation coefficient and a detection limit are obtained after linear fitting, the linear range, the standard curve equation, the linear correlation coefficient and the detection limit are respectively shown in a table 3, and a standard curve graph is shown in fig. 3.

TABLE 3 linear range and equation of cinnamoyl glycine

As is clear from Table 3, the curve correlation of cinnamoyl glycine was good (R > 0.99), the linear range was 0.02-10 ng/mL, the detection limit was 0.02 ng/mL, and the deviation was within.+ -. 15%.

Substituting the peak area of the biological sample to be detected into a standard curve equation, and reversely calculating the concentration of cinnamoyl glycine in the biological sample.

(5) Repeatability of

10 Ng/mL of cinnamoyl glycine standard stock solution in the step (2) is diluted into 6 parts of 0.5 ng/mL of standard solution by PBS in parallel, acetonitrile containing an internal standard solution of 1 mL is added as an extracting solution respectively, and other preparation methods are the same as in the example 1, and sample injection is carried out. RSD of 1.6% and RSD less than 15.0% repeatability test is satisfactory.

(6) Recovery rate of adding mark

10 Ng/mL of cinnamoyl glycine standard stock solution is subjected to gradient dilution by using PBS solution to prepare 10 standard substance solutions with concentrations (0.02, 0.04, 0.08, 0.16, 0.31, 0.625, 1.25, 2.5, 5 and 10 ng/mL) respectively, and standard curves are obtained by sample injection. And (3) selecting a lower concentration clinical sample as a basic sample, adding a standard substance, preparing to-be-detected samples (0.05, 0.5 and 8 ng/mL) with low, medium and high concentrations, processing each concentration sample in 5 parts, calculating a standard adding recovery rate according to a formula (1), and meeting the requirement of 85-115%, wherein the analysis result is shown in a table 4.

Formula (1)

R-recovery rate, average measured concentration of C-low concentration clinical samples after adding the standard, ng/mL, average measured concentration of C ₀ -low concentration clinical samples, ng/mL, C _s -adding the scalar, ng/mL.

TABLE 4 recovery of cinnamoyl glycine at various concentrations

As can be seen from Table 4, the recovery at each concentration was in the range of 85% -115%, and the relative standard deviation of the 9 recovery data was less than 10.0%. The recovery rate meets the requirement.

(7) Clinical sample detection

And (3) selecting 20 plasma samples (respectively numbered samples 1-20) of normal people, preparing a biological sample to be detected according to the pretreatment step of the biological sample in the step (1), detecting the sample to be detected by adopting the detection method in the step (3), and measuring the concentration of cinnamoyl glycine in each sample to be detected by using the standard curve equation in the step (4), wherein the detection result is shown in table 5.

TABLE 5 clinical sample test results

As can be seen from Table 5, the method for detecting cinnamoyl glycine in plasma by using high performance liquid chromatography-mass spectrometry has the advantages of strong specificity, high sensitivity, high flux, objective and easy analysis of results, and is particularly suitable for clinical popularization and application.

Comparative example 1

Unlike example 1, in the pretreatment of the biological sample in 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 an extraction solution to extract the biological sample (e.g., plasma), and other preparation methods are the same as in example 1. The extractant, the volumetric method of extractant and biological sample, and the CMG response values are shown in table 6.

TABLE 6 CMG response values from different extraction methods

As can be seen from Table 6, the extraction efficiency of cinnamoyl glycine was highest when the volume ratio of serum to acetonitrile in the extract was 1:5.

Example 2

A method for measuring cinnamoyl glycine in a biological sample is different from example 1 in that AGILENT RRHD ECLIPSE Plus C18, APCELL PAK ADME HR, PC HILIC, titank C and the like are selected as columns in step (3), respectively, and the other conditions are the same as in example 1. The separation results are shown in Table 7.

TABLE 7 results of separation of cinnamoyl glycine by different types of chromatographic columns

In table 7, "v" indicates good and "×" indicates poor.

Since the content of cinnamoyl glycine in blood is extremely low, the response in mass spectrum is low, and detection is difficult. As a result of comparing the retention time, peak shape and response value of the different types of chromatographic columns in Table 7, it was found that the C18 column could obtain a better retention time and peak shape, and the mobile phase could be eluted from 0% pure water due to APCELL PAK ADME HR resistance to pure water, and the response value was high, and finally determined as APCELL PAK ADME HR column.

In conclusion, the method for detecting the cinnamoyl glycine in the biological sample by using the liquid chromatography-mass spectrometry method for the first time achieves the aim of detecting the cinnamoyl glycine in the biological sample, reduces the influence of interfering substances, and has the advantages of simplicity, convenience, rapidness (the analysis time is only 8 min), high flux, low cost and the like. Meanwhile, the invention adopts an internal standard method for quantification, ensures the accuracy of a detection result, can effectively monitor the level of cinnamoyl glycine in a human body, has guiding significance for early diagnosis of gestational diabetes mellitus, and is easy to clinically popularize.

The above is only a preferred embodiment of the present invention, and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for determining cinnamoylglycine in a biological sample based on LC-MS, comprising the following steps:

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 H ₇ ]-N-cinnamylglycine 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;

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, wherein 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, wherein the mixing volume ratio of the biological sample to the extract is 1:(3-6);

The concentration of [ ² H ₇ ]-N-cinnamylglycine in the extract is 1-3 ng/mL.

4. The method for determining cinnamoylglycine in a biological sample based on LC-MS according to claim 3, wherein the mixing volume ratio of the biological sample to the extract is 1:5;

The concentration of [ ² H ₇ ]-N-cinnamylglycine in the extract is 2 ng/mL.

5. The method for determining cinnamoylglycine in a biological sample based on LC-MS according to claim 1, wherein the extraction process comprises 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%.

6. The method for determining cinnamoylglycine in a biological sample based on LC-MS according to claim 5, wherein 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 C18 column in the chromatographic column used in the liquid chromatography analysis is an Agilent RRHD Eclipse Plus C18 column or a Titank C18 column, the ADME column is an APCELL PAK ADME HR column, and the HILIC column is a PC HILIC column; the column temperature is 25-30°C; the flow rate is 0.3-0.4 mL/min, and the injection volume is 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 is an APCELL PAK ADME HR column, the column temperature is 25°C; the flow rate is 0.35 mL/min, and the injection volume is 10 μL; mobile phase A is acetonitrile containing 0.05% formic acid, and mobile phase B is 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 is used, curtain gas: 30-35 kPa, spray voltage: 3700-3900 V, desolvation temperature: 550-650°C, GS1: 25-35 kPa, 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 in multiple ion reaction monitoring mode is used, 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; and the ion pair used is a 103/137 ion pair.