Method for simultaneously determining PAGln and PAGly in plasma based on ultra-high performance liquid chromatography triple quadrupole tandem mass spectrometer

Abstract

The invention discloses a method for simultaneously determining PAGln and PAGly in plasma based on an ultra-high performance liquid chromatography triple quadrupole tandem mass spectrometer, and belongs to the technical field of clinical biological sample detection. The method comprises the following steps: 1) uniformly mixing a quality control sample and a plasma sample with a precipitation reagent respectively, fully extracting, centrifuging, and taking supernate, namely a sample to be detected; (2) detecting the sample to be detected by using an ultra-high performance liquid chromatography triple quadrupole tandem mass spectrometer, wherein the chromatographic conditions are as follows: a chromatographic column is a C18 chromatographic column; carrying out gradient elution by adopting the mobile phase A and the mobile phase B; and 3) measuring the peak areas of PAGln and PAGly in the sample to be detected, and respectively substituting the peak areas into the standard curve for calculation to obtain the contents of PAGln and PAGly in the sample to be detected, the two metabolites PAGln and PAGly can be detected at the same time, and the method has the advantages of being high in accuracy, sensitivity and specificity, small in needed sample size, simple in sample pretreatment and the like.

Description

Technical Field

[0001] The present invention belongs to the technical field of clinical biological sample detection, and in particular relates to a method for simultaneously determining PAGln and PAGly in plasma based on an ultra-high performance liquid chromatography triple quadrupole tandem mass spectrometer. Background Art

[0002] Phenylacetylglutamine (PAGln) and phenylacetylglycine (PAGly) are two metabolites of the intestinal microbiota. Phenylacetylglutamine (PAGln) is a metabolite produced by the metabolism of phenylalanine (Phe) by intestinal microorganisms. Specifically, it is formed by the combination of phenylacetic acid (PAA) and glutamine (Glutamine). In humans, PAGln is mainly formed through glutamine conjugation reactions in the liver and kidneys. Phenylacetylglycine (PAGly) is mainly formed in rodents and less common in humans. PAGly is similar to PAGln and is also produced through microbial metabolic pathways, but it is formed by the combination of phenylacetic acid and glycine.

[0003] Modern studies have shown that increased levels of PAGln and PAGly are associated with an increased risk of cardiovascular disease. PAGln can enhance platelet activation mediated by G protein-coupled receptors (GPCRs) receptors α2A, α2B, and β2ADRs, affect platelet function, enhance platelet adhesion to collagen matrix, and promote platelet stimulation-dependent Ca + PAGly can also affect the function of platelets. Increased PAGly levels can promote platelet reactivity and thrombosis, increase the risk of inflammation, etc. In addition, PAGly can also affect cardiac function by activating β2-adrenergic receptors.

[0004] Although some progress has been made in the detection methods of the two metabolites of phenylacetylglutamine (PAGln) and phenylacetylglycine (PAGly) in the existing technology, there are still limitations and defects. First, due to the low concentrations of PAGln and PAGly in the human body, the sample processing process may encounter problems of insufficient sensitivity, and these metabolites may be degraded or transformed during processing and storage, resulting in inaccurate data; secondly, for some existing analytical methods, the detection limit and quantification limit may not be low enough to accurately capture low concentrations of PAGln or PAGly, especially in healthy individuals; in addition, for large-scale population screening, efficiency and cost-effectiveness need to be considered.

[0005] Liquid chromatography triple quadrupole tandem mass spectrometer (LC-MS / MS with triple quadrupole massspectrometer) is an analytical tool for liquid chromatography-mass spectrometry technology. It uses liquid chromatography as the separation system and mass spectrometry as the detection system. It combines the separation capability of liquid chromatography (LC) and the detection capability of mass spectrometry (MS), realizing the complementary advantages of chromatography and mass spectrometry. It has the advantages of high separation and high selectivity, and can be used for qualitative and quantitative analysis of specific compounds in complex mixtures. It is particularly suitable for application fields that require high specificity and sensitivity, such as drug analysis, clinical diagnosis, environmental monitoring, and food safety inspection.

[0006] Currently, there are technical solutions for determining PAGln in plasma using liquid chromatography-mass spectrometry methods. For example, a Chinese patent with publication number CN112834656A and application date of January 27, 2021 discloses a UPLC-MS / MS-based targeted metabolomics quantification method for plasma cardiovascular disease-related biomarkers. By optimizing mass spectrometry parameters and liquid chromatography methods, 20 biomarkers of metabolism including aromatic amino acid catabolism, trimethylamine oxide biosynthesis and histidine metabolism are detected with high throughput, high sensitivity and high separation. However, the 20 biomarkers of this method do not include PAGly, and the simultaneous detection of PAGln and PAGly cannot be achieved.

[0007] Japanese patent with publication number JP4555779B2 and application date of December 24, 2004 provides a method for predicting dyslipidemia, comprising detecting (a) phenylacetylglycine and / or phenylacetylglutamine, or a metabolic intermediate optionally selected in the metabolic pathway from phenylalanine to phenylacetylglycine or phenylacetylglutamine, and (b) any metabolic intermediate in the metabolic pathway from hippuric acid or phenylalanine to hippuric acid, and using the quantitative ratio of the two as an indicator for diagnosis. Although this method can achieve simultaneous detection of phenylacetylglycine and / or phenylacetylglutamine, it is necessary to additionally obtain the content of any metabolic intermediate in the metabolic pathway from hippuric acid or phenylalanine to hippuric acid during the determination, and use the quantitative ratio of the two as an indicator for compounding to determine the content, which not only makes the operation steps complicated, but also the detection results may be interfered by other detection factors.

[0008] In the disclosed prior art, the detection method of phenylacetylglycine (PAGly) usually includes the technical feature of "using hippuric acid-d5 as an internal standard", while the prior art scheme for detecting phenylacetylglutamine (PAGln) often requires mixing the sample to be tested with the isotope internal standard. Due to the different internal standard substances and pretreatment methods, when using liquid chromatography-mass spectrometry technology to detect PAGln and PAGly, it is necessary to collect and prepare the samples to be tested separately, and to separate the injection and set the detection and analysis conditions required for each. However, PAGln and PAGly are metabolites produced by the metabolism of intestinal microbiota, and are related to a variety of health conditions, especially cardiovascular disease and inflammatory bowel disease. They will face more and more application scenarios that require simultaneous detection of the two. Therefore, a method that uses liquid chromatography-mass spectrometry technology to simultaneously detect PAGln and PAGly, and is highly sensitive, fast and efficient, is developed. It is not only of great significance and reference value for experimental and clinical research on cardiovascular diseases, but also may provide new strategies for the prevention and treatment of cardiovascular diseases. Summary of the invention

[0009] To solve the problems existing in the prior art, the present invention provides a method for simultaneously determining PAGln and PAGly in plasma based on ultra-high performance liquid chromatography triple quadrupole tandem mass spectrometry, which has the advantages of high accuracy, high sensitivity, high specificity, small sample volume required, and simple sample pretreatment.

[0010] The technical solution of the present invention is as follows:

[0011] The object of the present invention is to provide a method for simultaneously determining PAGln and PAGly in plasma based on ultra-high performance liquid chromatography triple quadrupole tandem mass spectrometry, the determination method comprising the following steps:

[0012] S1. Sample processing: The quality control sample and plasma sample are mixed evenly with the precipitation reagent respectively, fully extracted and centrifuged to obtain the supernatant, which is the sample to be tested;

[0013] S2, using an ultra-high performance liquid chromatography triple quadrupole tandem mass spectrometer to detect the sample to be tested, sequentially undergoing liquid phase separation and mass spectrometry detection;

[0014] The liquid phase separation uses a C18 analytical column and a gradient elution method for separation. During the gradient elution process, mobile phase A accounts for 10 to 95% of the entire mobile phase.

[0015] The mass spectrometry detection adopts the multiple reaction detection SRM mode, the ion source is an electrospray ion source, the spray voltage is 3200V for positive ions, 3000V for negative ions, the atomization temperature is 350°C, the sheath gas pressure is 40Arb, and the auxiliary gas pressure is 15Arb;

[0016] S3. Measure the peak areas of PAGln and PAGly in the sample to be tested, and respectively bring them into the standard curve for quantification by linear equation to obtain the contents of PAGln and PAGly in the sample to be tested.

[0017] Furthermore, the quality control sample in S1 is prepared as follows:

[0018] Accurately weigh PAGln and PAGly and place them in a volumetric flask to prepare a mixed standard stock solution. Use methanol to gradually dilute the standard stock solution to prepare a series of mixed reference solutions.

[0019] Then, take 50 μL of each blank plasma sample and place it in a clean centrifuge tube, add low, medium and high concentrations of a series of mixed reference working solutions and mix thoroughly to obtain quality control samples.

[0020] Furthermore, the concentration range of PAGln in the series of mixed reference working solutions is 0.01-1000 ng / mL, and the concentration range of PAGly is 0.1-5000 ng / mL.

[0021] Furthermore, the series of mixed reference substance working solutions are set as follows:

[0022] Compound (concentration μg / L) STD1 STD2 STD3 STD4 STD5 STD6 STD7 STD8 PAGln 0.001 0.01 0.1 0.5 1 10 50 100 PAGly 0.01 0.1 1 5 10 100 500 1000

[0023] Furthermore, in the S1, a plasma sample and a quality control sample are taken respectively, a precipitating reagent is added and vortexed for 1 to 5 minutes for sufficient precipitation, and the supernatant is taken after centrifugation at 13000 rpm for 15 minutes at 4°C, and placed in a liquid phase vial as the sample to be tested; the precipitating reagent is methanol, and the volume ratio of the added precipitating agent to the plasma sample and the quality control sample is 3:1.

[0024] Furthermore, the analytical chromatographic column in S2 has a length of 100 mm, an inner diameter of 2.1 mm, a filler particle size of 1.7 μm, and a column temperature of 40°C.

[0025] Furthermore, the mobile phase in the gradient elution method in S2 consists of mobile phase A and mobile phase B, and the gradient elution procedure is as follows:

[0026]

[0027] The mobile phase A is a formic acid aqueous solution, an acetic acid aqueous solution or an ammonium formate solution, and the mobile phase B is acetonitrile or methanol.

[0028] Furthermore, the detection of PAGln in S2 adopts positive ion mode, detecting parent ion: 265.128m / z, daughter ions: 84.113, 91.054, 129.97m / z.

[0029] Furthermore, the detection of PAGly in S2 adopts negative ion mode, detecting parent ion: 191.930 m / z, daughter ions: 74.054, 90.97, 117.03 m / z.

[0030] Furthermore, the detection and analysis time in S2 is 8 minutes.

[0031] Further, the specific mass spectrum parameters are: PAGln: m / Z=129.40-130.40F:+CESSRMms2265.12884.112-84.114,91.053-91.055129.969-129.971];

[0032] PAGly:m / z=73.55-74.55F:-C ESSRM ms2 191.93074.053-74.055.90.969-90.971117.035-117.0371.

[0033] Furthermore, the linear equation in S3 is obtained as follows:

[0034] A1. Pipette 2 μL of the series of mixed reference working solutions into 1.5 mL clean centrifuge tubes, add 48 μL of blank group rat plasma thawed at room temperature, and mix to obtain a series of plasma samples containing different concentrations of standard substances;

[0035] A2. Add 150 μL of methanol to each series of plasma samples and vortex for 1 min. Centrifuge at 13,000 rpm for 15 min at 4°C and take the supernatant for ultra-high performance liquid chromatography tandem mass spectrometry detection. Measure the peak areas A of PAGln and PAGly of the series of plasma samples containing different concentrations of standard substances. n ;

[0036] A3. Take another 2 μL of blank methanol and place it in a centrifuge tube. Add 48 μL of blank group rat plasma thawed at room temperature and mix well to obtain a blank plasma sample.

[0037] A4. Add 150 μL of methanol to the blank plasma sample and vortex for 1 min. Centrifuge at 13,000 rpm for 15 min at 4°C and take the supernatant for ultra-high performance liquid chromatography tandem mass spectrometry to measure the peak areas A0 of PAGln and PAGly of the blank plasma sample.

[0038] A5. Perform regression calculation on the peak area ratio Y and the compound concentration X in plasma to obtain a linear equation.

[0039] Compared with the prior art, the beneficial effects of the present invention are:

[0040] 1. The present invention proposes for the first time a technical solution for simultaneously determining PAGln and PAGly in plasma using an ultra-high performance liquid chromatography triple quadrupole tandem mass spectrometer, combining the high separation ability of liquid chromatography (LC) with the high selectivity and sensitivity of mass spectrometry (MS), so that various complex samples to be tested can be separated in liquid chromatography first, and then accurately qualitatively and quantitatively analyzed by mass spectrometry, overcoming the defect in the prior art that PAGln and PAGly are difficult to be detected due to their low content in plasma. At the same time, the present invention does not need to set different internal standard substances and gradient elution methods when detecting phenylacetylglycine (PAGly) and phenylacetylglutamine (PAGln), and can achieve simultaneous detection of PAGln and PAGly in the same sample to be tested by only adjusting the positive and negative ion modes of the liquid chromatography triple quadrupole tandem mass spectrometer.

[0041] 2. The innovative determination method of the present invention adopts the multiple reaction monitoring (MRM) mode to improve the accuracy of quantitative analysis, can effectively identify the target compound, complete the analysis of the sample to be tested in a relatively short time, and realize the simultaneous determination of PAGln and PAGly in the plasma sample, which has important practical value and reference significance for the diagnosis of cardiovascular diseases. In addition, the determination method of the present invention has a simple pretreatment method, the sample to be tested is directly injected after being treated with methanol precipitation, and the liquid phase method does not need to use a hydrophilic filler analytical chromatographic column, which is suitable for conventional determination, and has the advantages of reducing detection costs, reducing sample usage, being convenient and fast, and improving work efficiency.

[0042] 3. The method for simultaneously determining PAGln and PAGly in plasma based on ultra-high performance liquid chromatography triple quadrupole tandem mass spectrometer of the present invention has strong anti-matrix interference ability, can detect a wide range of sample types, including biological samples, environmental samples and food samples, etc., and can detect the content of PAGln and PAGly in a variety of application scenarios. Under the method of the present invention, the retention time of PAGln is 5.27min, the retention time of PAGly is 5.67min, there is no impurity peak interference determination, and the linear correlation coefficients of PAGln and PAGly are both above 0.995, and the detection limit (LOD) and quantification limit (LOQ) have reached a good level, ensuring the reliability of the determination results. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 UPLC-MS / MS graph of the mixed reference substance of PAGln and PAGly in the determination method of the present invention;

[0044] Figure 2 UPLC-MS / MS diagram of PAGln and PAGly in rat plasma in the determination method of the present invention;

[0045] Figure 3 It is a standard curve diagram of the linear equation of PAGln and PAGly in rat plasma in the determination method of the present invention. DETAILED DESCRIPTION

[0046] The present invention is further described below in conjunction with preferred embodiments. The endpoints of the ranges disclosed in the present invention and any values ​​are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be deemed to be specifically disclosed herein.

[0047] The experimental methods in the following examples, unless otherwise specified, are conventional methods and are performed according to the techniques or conditions described in the literature in the art or according to the product instructions.

[0048] The PAGln used in the following examples is a standard product from selleck chemicals or of the same or higher grade;

[0049] The PAGly used is from MCE or a standard product of the same or higher grade;

[0050] The methanol and acetonitrile used were from Fisher Chemical or the same or higher grade reagents;

[0051] The analytical equipment used was a Thermo scientific TSQ Quantis liquid spectrometer. The same LC / MS / MS system can also be used.

[0052] Example 1

[0053] This embodiment provides a method for simultaneously determining PAGln and PAGly in plasma based on ultra-high performance liquid chromatography triple quadrupole tandem mass spectrometry, comprising the following steps:

[0054] (1) Accurately weigh 0.5 mg PAGln and 5 mg PAGly on an analytical balance and place them in a 5 ml volumetric flask. Dissolve them in methanol to make up to the volume and dilute them step by step to prepare a series of mixed reference working solutions.

[0055] (2) Take 50 μL of each of the six groups of blank plasma samples and place them in a 1.5 mL clean centrifuge tube. Add low, medium, and high concentrations of the series of mixed reference working solutions and mix thoroughly to obtain quality control samples.

[0056] (3) Use a pipette to transfer 50 μL of plasma sample and quality control sample into a centrifuge tube, add 150 μL of methanol and vortex for 4 min to fully precipitate, centrifuge at 13,000 rpm for 15 min at 4°C, take the supernatant, and place it in a liquid phase vial as the sample to be tested;

[0057] (4) The sample to be tested is detected using an ultra-high performance liquid chromatography triple quadrupole tandem mass spectrometer, and the detection and analysis time is 8 minutes. The conditions of the high performance liquid chromatography tandem triple quadrupole mass spectrometer are as follows:

[0058] Chromatographic conditions: The analytical column is Syncronis C18, with a column length of 100 mm, an inner diameter of 2.1 mm, a filler particle size of 1.7 μm, and a column temperature of 40°C; gradient elution is used for separation, and the mobile phase consists of 0.1% acetic acid aqueous solution and acetonitrile, with 0.1% acetic acid aqueous solution accounting for 10-95% of the entire mobile phase;

[0059] Mass spectrometry conditions: multiple reaction detection SRM mode was used, the ion source was an electrospray ion source, the spray voltage was 3200 V for positive ions and 3000 V for negative ions, the nebulization temperature was 350 °C, the sheath gas pressure was 40 Arb, and the auxiliary gas pressure was 15 Arb;

[0060] The positive ion mode was used for the detection of PAGln, and the parent ion was 265.128 m / z, and the daughter ions were 84.113, 91.054, and 129.97 m / z;

[0061] PAGly was detected in negative ion mode, with parent ion: 191.930 m / z, daughter ions: 74.054, 90.97, 117.03 m / z.

[0062] (5) The peak areas of PAGln and PAGly in the sample to be tested are measured, and the peak areas are respectively substituted into linear equations for quantification to obtain the contents of PAGln and PAGly in the sample to be tested. The linear equation of PAGln is as follows: y=2273.7x+3228; the linear equation of PAGly is as follows: y=376.49x+30589.

[0063] Example 2

[0064] This embodiment provides a method for simultaneously determining PAGln and PAGly in plasma based on ultra-high performance liquid chromatography triple quadrupole tandem mass spectrometry, comprising the following steps:

[0065] (1) Accurately weigh 0.5 mg PAGln and 5 mg PAGly on an analytical balance and place them in a 5 ml volumetric flask. Dissolve them in methanol to make up to the volume and dilute them step by step to prepare a series of mixed reference working solutions.

[0066] (2) Take 50 μL of each of the six groups of blank plasma samples and place them in a 1.5 mL clean centrifuge tube. Add low, medium, and high concentrations of the series of mixed reference working solutions and mix thoroughly to obtain quality control samples.

[0067] (3) Use a pipette to transfer 50 μL of plasma sample and quality control sample into a centrifuge tube, add 150 μL of methanol and vortex for 1 min to fully precipitate, centrifuge at 13,000 rpm for 15 min at 4°C, take the supernatant, and place it in a liquid phase vial as the sample to be tested;

[0068] (4) The sample to be tested is detected using an ultra-high performance liquid chromatography triple quadrupole tandem mass spectrometer, and the detection and analysis time is 8 minutes. The conditions of the high performance liquid chromatography tandem triple quadrupole mass spectrometer are as follows:

[0069] Chromatographic conditions: The analytical column is Syncronis C18, with a column length of 100 mm, an inner diameter of 2.1 mm, a filler particle size of 1.7 μm, and a column temperature of 40°C; gradient elution is used for separation, and the mobile phase consists of 0.1% formic acid aqueous solution and methanol, with 0.1% formic acid aqueous solution accounting for 10-95% of the entire mobile phase;

[0070] Mass spectrometry conditions: multiple reaction detection SRM mode was used, the ion source was an electrospray ion source, the spray voltage was 3200 V for positive ions and 3000 V for negative ions, the nebulization temperature was 350 °C, the sheath gas pressure was 40 Arb, and the auxiliary gas pressure was 15 Arb;

[0071] The positive ion mode was used for the detection of PAGln, and the parent ion was 265.128 m / z, and the daughter ions were 84.113, 91.054, and 129.97 m / z;

[0072] PAGly was detected in negative ion mode, with parent ion: 191.930 m / z, daughter ions: 74.054, 90.97, 117.03 m / z.

[0073] (5) The peak areas of PAGln and PAGly in the sample to be tested are measured, and the peak areas are respectively substituted into linear equations for quantification to obtain the contents of PAGln and PAGly in the sample to be tested. The linear equation of PAGln is as follows: y=2273.7x+3228; the linear equation of PAGly is as follows: y=376.49x+30589.

[0074] Example 3

[0075] This embodiment provides a method for simultaneously determining PAGln and PAGly in plasma based on ultra-high performance liquid chromatography triple quadrupole tandem mass spectrometry, comprising the following steps:

[0076] (1) Accurately weigh 0.5 mg PAGln and 5 mg PAGly on an analytical balance and place them in a 5 ml volumetric flask. Dissolve them in methanol to make up to the volume and dilute them step by step to prepare a series of mixed reference working solutions.

[0077] (2) Take 50 μL of each of the six groups of blank plasma samples and place them in a 1.5 mL clean centrifuge tube. Add low, medium, and high concentrations of the series of mixed reference working solutions and mix thoroughly to obtain quality control samples.

[0078] (3) Use a pipette to transfer 50 μL of plasma sample and quality control sample into a centrifuge tube, add 150 μL of methanol and vortex for 5 min to fully precipitate, centrifuge at 13,000 rpm for 15 min at 4°C, take the supernatant, and place it in a liquid phase vial as the sample to be tested;

[0079] (4) The sample to be tested is detected using an ultra-high performance liquid chromatography triple quadrupole tandem mass spectrometer, and the detection and analysis time is 8 minutes. The conditions of the high performance liquid chromatography tandem triple quadrupole mass spectrometer are as follows:

[0080] Chromatographic conditions: The analytical column is Syncronis C18, with a column length of 100 mm, an inner diameter of 2.1 mm, a filler particle size of 1.7 μm, and a column temperature of 40°C. Gradient elution is used for separation, and the mobile phase consists of 0.1% ammonium formate aqueous solution and acetonitrile, with 0.1% ammonium formate aqueous solution accounting for 10-95% of the entire mobile phase.

[0081] Mass spectrometry conditions: multiple reaction detection SRM mode was used, the ion source was an electrospray ion source, the spray voltage was 3200 V for positive ions and 3000 V for negative ions, the nebulization temperature was 350 °C, the sheath gas pressure was 40 Arb, and the auxiliary gas pressure was 15 Arb;

[0082] The positive ion mode was used for the detection of PAGln, and the parent ion was 265.128 m / z, and the daughter ions were 84.113, 91.054, and 129.97 m / z;

[0083] PAGly was detected in negative ion mode, with parent ion: 191.930 m / z, daughter ions: 74.054, 90.97, 117.03 m / z.

[0084] (5) The peak areas of PAGln and PAGly in the sample to be tested are measured, and the peak areas are respectively substituted into linear equations for quantification to obtain the contents of PAGln and PAGly in the sample to be tested. The linear equation of PAGln is as follows: y=2273.7x+3228; the linear equation of PAGly is as follows: y=376.49x+30589.

[0085] Performance Testing

[0086] 1. Linear range, limit of detection (LOD), limit of quantification (LOQ) detection

[0087] According to the determination conditions of Example 1, after pretreatment and injection, the concentration is measured from low to high, and the quantitative chromatographic peak area-concentration is plotted to obtain a standard curve of the linear equation as shown in the figure. Figure 3 The linear range, detection limit (LOD) and quantification limit (LOQ) are shown in the following table:

[0088] Analytes Linear equations Correlation coefficient Linear range (ng / mL) LOD(ng / mL) LOQ (ng / mL) PAGln y=2273.7x+3228 0.9999 0.01～1000 0.0002 0.0007 PAGly y=376.49x+30589 0.9971 0.1～5000 0.0372 0.1118

[0089] The results showed that the linear correlation coefficients for the detection of PAGln and PAGly were above 0.995. Within the concentration range of PAGln 0.01-1000 ng / mL and PAGly 0.1-5000 ng / mL, the detection limit (LOD) and quantification limit (LOQ) met the minimum level. The precision and accuracy of the method were verified, ensuring the reliability of the measurement results.

[0090] 2. Recovery and precision testing

[0091] Plasma samples containing low, medium and high concentrations were prepared respectively, and 6 copies of each concentration were prepared in parallel. The concentrations of PAGln and PAGly in each plasma sample were continuously measured and calculated. At the same time, standard working solutions of high, medium and low concentrations were prepared for precision experiments. The determination was carried out according to the determination method of Example 1, and 3 batches were repeatedly analyzed and determined. The precision test results are shown in the following table:

[0092] Low middle high PAGln Mean ± standard deviation (ug / mL) 24.62±0.18 18.50±0.77 124.28±1.27 Intra-assay CV (%) 0.74 2.71 1.02 Inter-assay CV (%) 4.87 0.37 1.98 PAGly Mean ± standard deviation (ug / mL) 103.80±5.11 172.18±2.17 1212.72±6.67 Intra-assay CV (%) 4.92 1.26 0.55 Inter-assay CV (%) 3.02 1.47 0.70

[0093] The results showed that the inter-group and intra-group CVs of PAGln and PAGly concentrations were less than 5%, indicating that this method had high precision and reliability.

[0094] 3. Recovery rate detection

[0095] 48 μL of rat plasma was aspirated, and 2 μL of PAG standard solutions of low, medium and high concentrations were added respectively. 150 μL of methanol was added for precipitation. Three replicates of three concentrations were prepared in parallel. The peak area was obtained after injection. The recovery rate was calculated as follows: recovery rate P = (C3-C1) / C2×100%, where C1 is the plasma background concentration, C2 is the spiked concentration, and C3 is the spiked sample concentration.

[0096] Analytes Low level Medium High level PAGln 100.58±4.64 101.20±2.79 100.54±4.73 PAGly 101.12±4.23 105.37±6.65 112.7796±2.50

[0097] The results showed that the recoveries of the two analytes at all levels were in the range of 95% to 115%, indicating that this analytical method is relatively reliable and can be used for the quantitative analysis of PAGln and PAGly in biological samples.

[0098] It should be noted that, in this article, relational terms such as "first" and "second" are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. The terms "include", "comprises" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of further restrictions, the elements defined by the sentence "comprises a ..." do not exclude the presence of other identical elements in the process, method, article or device including the elements.

[0099] The above are only preferred embodiments of the present application and do not constitute any form of limitation to the present application. Any technician familiar with the profession can make some changes or modify the technical contents disclosed above into equivalent embodiments with equivalent changes without departing from the scope of the technical solution of the present application. However, any simple modification, equivalent change and modification made to the above embodiments based on the technical essence of the present application without departing from the content of the technical solution of the present application still fall within the scope of the technical solution of the present application.

Claims

1. A method for simultaneously determining PAGln and PAGly in plasma based on ultra-high performance liquid chromatography triple quadrupole tandem mass spectrometry, characterized in that: The determination method comprises the following steps: S1. Sample processing: The quality control sample and plasma sample are mixed evenly with the precipitation reagent respectively, fully extracted and centrifuged to obtain the supernatant, which is the sample to be tested; S2, using an ultra-high performance liquid chromatography triple quadrupole tandem mass spectrometer to detect the sample to be tested, sequentially undergoing liquid phase separation and mass spectrometry detection; The liquid phase separation uses a C18 analytical column and a gradient elution method for separation. During the gradient elution process, mobile phase A accounts for 10 to 95% of the entire mobile phase. The mass spectrometry detection adopts the multiple reaction detection SRM mode, the ion source is an electrospray ion source, the spray voltage is 3200V for positive ions, 3000V for negative ions, the atomization temperature is 350°C, the sheath gas pressure is 40Arb, and the auxiliary gas pressure is 15Arb; S3. Measure the peak areas of PAGln and PAGly in the sample to be tested, and respectively bring them into the standard curve for quantification by linear equation to obtain the contents of PAGln and PAGly in the sample to be tested.

2. The method for simultaneously determining PAGln and PAGly in plasma based on ultra-high performance liquid chromatography triple quadrupole tandem mass spectrometry according to claim 1, characterized in that: The quality control sample in S1 is prepared as follows: Accurately weigh PAGln and PAGly and place them in a volumetric flask to prepare a mixed standard stock solution. Use methanol to gradually dilute the standard stock solution to prepare a series of mixed reference solutions. Take 50 μL of different blank plasma samples and place them in clean centrifuge tubes. Add low, medium and high concentrations of a series of mixed reference working solutions and mix thoroughly to obtain quality control samples.

3. The method for simultaneously determining PAGln and PAGly in plasma based on ultra-high performance liquid chromatography triple quadrupole tandem mass spectrometry according to claim 2, characterized in that: The concentration range of PAGln in the series of mixed reference solutions is 0.01-1000 ng / mL, and the concentration range of PAGly is 0.1-5000 ng / mL.

4. The method for simultaneously determining PAGln and PAGly in plasma based on ultra-high performance liquid chromatography triple quadrupole tandem mass spectrometry according to claim 1, characterized in that: In the S1, plasma samples and quality control samples are taken respectively, precipitating reagents are added and vortexed for 1 to 5 minutes to fully precipitate, and the supernatant is taken after centrifugation at 13000 rpm for 15 minutes at 4°C, and placed in a liquid phase vial as the sample to be tested; the precipitating reagent is methanol, and the volume ratio of the added precipitating agent to the plasma sample and the quality control sample is 3:

1.

5. The method for simultaneously determining PAGln and PAGly in plasma based on ultra-high performance liquid chromatography triple quadrupole tandem mass spectrometry according to claim 1, characterized in that: The analytical chromatographic column in S2 has a length of 100 mm, an inner diameter of 2.1 mm, a filler particle size of 1.7 μm, and a column temperature of 40° C.

6. The method for simultaneously determining PAGln and PAGly in plasma based on ultra-high performance liquid chromatography triple quadrupole tandem mass spectrometry according to claim 1, characterized in that: In the gradient elution method of S2, the mobile phase consists of mobile phase A and mobile phase B, and the gradient elution procedure is as follows: The mobile phase A is a formic acid aqueous solution, an acetic acid aqueous solution or an ammonium formate solution, and the mobile phase B is acetonitrile or methanol.

7. The method for simultaneously determining PAGln and PAGly in plasma based on ultra-high performance liquid chromatography triple quadrupole tandem mass spectrometry according to claim 1, characterized in that: The detection of PAGln in the S2 adopts the positive ion mode, detecting the parent ion: 265.128m / z, and the daughter ions: 84.113, 91.054, 129.97m / z.

8. The method for simultaneously determining PAGln and PAGly in plasma based on ultra-high performance liquid chromatography triple quadrupole tandem mass spectrometry according to claim 1, characterized in that: The detection of PAGly in the S2 adopts the negative ion mode, detecting the parent ion: 191.930 m / z, and the daughter ions: 74.054, 90.97, 117.03 m / z.

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