Method for detecting contents of VMA, HVA and 5-HIAA in urine by using liquid chromatography-tandem mass spectrometry

Abstract

The invention discloses a method for detecting the contents of VMA, HVA and 5-HIAA in urine by using liquid chromatography-tandem mass spectrometry. The method comprises the following steps: S1, removing VMA, HVA and 5-HIAA in urine to obtain a blank urine matrix; s2, weighing VMA, HVA and 5-HIAA solid powder, fixing the volume with methanol, mixing the dissolved liquids, diluting with methanol-water to obtain a VMA, HVA and 5-HIAA working solution, and finally diluting step by step with a blank urine matrix to obtain a calibration product; s3, respectively taking a urine sample to be detected, the calibration substance and the quality control substance, adding the mixed internal standard working solution of VMA, HVA and 5-HIAA isotopes, uniformly mixing, centrifuging, and taking supernate; s4, separating the supernate by using liquid chromatography; s5, the separated supernate enters a mass spectrum to be detected, and the content of VMA, HVA and 5-HIAA in the urine sample to be detected is obtained. The detection method disclosed by the invention is simple and rapid in pretreatment, high in specificity, low in reagent consumption cost, free of environmental pollution, small in required sample size, easy in high-throughput operation and suitable for being developed into a kit for conventional clinical detection.

Description

Technical Field

[0001] The invention belongs to the technical field of biological sample detection, and in particular relates to a method for detecting the contents of VMA, HVA and 5-HIAA in urine by using liquid chromatography-tandem mass spectrometry. Background Art

[0002] Vanillylmandelic acid (VMA) and homovanillic acid (HVA) are auxiliary diagnostic indicators for the screening of pheochromocytoma, paraganglioma and neuroblastoma, while 5-hydroxyindoleacetic acid (5-HIAA) is the recommended first-line indicator for carcinoid tumor screening. Therefore, the detection of these three indicators is of great significance for the diagnosis of neuroendocrine tumors.

[0003] Traditional detection methods for VMA, HVA and 5-HIAA include microcolumn colorimetry, enzyme-linked immunosorbent assay, liquid chromatography-electrochemical detection and gas chromatography-mass spectrometry. These methods all have the disadvantages of complex pretreatment processes and cumbersome steps, as well as limited detection sensitivity. In recent years, liquid chromatography-tandem mass spectrometry (LC-MS / MS) has been increasingly used for the detection of VMA, HVA and 5-HIAA. This method has the advantages of high sensitivity, good specificity, high throughput and short time consumption. In order to remove the interference of sample proteins, samples usually need to be pretreated before detection. Most of the pretreatment methods reported in the published literature are solid phase extraction (SPE). These pretreatment methods have the problems of operating steps, large sample consumption, long time consumption, high reagent and consumable costs and environmental pollution.

[0004] Patent CN202310280452.9 discloses a method for detecting vanillylmandelic acid and homovanillic acid in urine and its application. Sample pretreatment includes extracting the sample to be tested with an extract containing an organic phase, wherein the organic phase includes n-hexane, dichloromethane or ethyl acetate. Before extraction, the sample to be tested is first diluted with a diluent, and then extracted with the extract. After extraction, the separated organic phase is blown dry with nitrogen, and then re-dissolved and detected by high performance liquid chromatography. However, the invention requires liquid-liquid extraction and drying of urine, and the operation steps are complicated and time-consuming. The use of unconventional organic solvents will cause environmental pollution, which is not conducive to rapid analysis of clinical tests. In addition, the invention does not mention the required sample size. Patent 201911363651.6 discloses a method for determining the concentration of endogenous homovanillic acid and vanillylmandelic acid in human urine by LC-MS / MS. Sample pretreatment includes adding 50.0 μL of internal standard working solution to each of the above-mentioned standard curve samples and control matrix samples, and then adding 500 μL of 0.2% formic acid in acetonitrile solution for protein precipitation. After centrifugation, 50.0 μL of supernatant is taken, and 50.0 μL of acetonitrile-water solution containing 0.2% formic acid (1:1, v / v) and 300 μL of 0.1% formic acid aqueous solution are added. After shaking and centrifugation, it is provided for LC-MS / MS determination. However, the pretreatment of this invention requires dilution after protein precipitation, which has many steps and is not suitable for clinical testing. Patent 201911170671 discloses a method and kit for rapid detection of homovanillic acid, vanillylmandelic acid, 5-hydroxyindoleacetic acid and creatinine in human urine. The sample pretreatment includes taking a urine sample under light-proof conditions and adding EDTA solution, Na2S2O3 solution and acetic acid thereto for acidification, and mixing them evenly to obtain a urine sample to be tested; then taking 10 μL each of the urine sample to be tested, calibration material, and urine quality control material, adding them to their respective corresponding 96 deep-well plates, and then adding 190 μL of sample dilution working solution to each well, sealing the film, vortexing, centrifuging, and taking the supernatant for detection by liquid chromatography-mass spectrometry; however, the invention requires the addition of EDTA solution, Na2S2O3 solution and acetic acid to acidify the urine before sample processing, involving the use of many types of solvents, and Na2S2O3 has a sulfur dioxide smell, which is not suitable for routine applications in clinical testing.

[0005] Therefore, it is necessary to develop a simple, rapid, stable, reliable and quantitative detection and analysis method with small sample volume to accurately determine the contents of VMA, HVA and 5-HIAA in urine. Summary of the invention

[0006] The object of the present invention is to provide a method for detecting the contents of VMA, HVA and 5-HIAA in urine by liquid chromatography-tandem mass spectrometry, which is simple, rapid, stable and reliable and requires a small amount of sample.

[0007] In order to achieve the above purpose, this application adopts the following technical solutions:

[0008] The present invention provides a method for detecting the contents of VMA, HVA and 5-HIAA in urine by liquid chromatography-tandem mass spectrometry, comprising the following steps:

[0009] S1, preparation of blank urine matrix: removing free VMA, HVA and 5-HIAA in urine by anion exchange resin to obtain the blank urine matrix;

[0010] S2, preparation of calibration products: weigh a certain amount of VMA, HVA and 5-HIAA solid powder respectively, make up the volume with methanol (for example: 1-2 mg / ml), and use the dissolved liquid as the mother liquor of VMA, HVA and 5-HIAA. The three are mixed in a certain ratio (for example: 0.5:1:1 or 1:1:1) and then diluted with methanol-water in a certain volume ratio (for example: 1:3, 1:1 or 3:1) to obtain VMA, HVA and 5-HIAA working solution, and finally dilute stepwise with the blank urine matrix to obtain the calibration products of different concentrations;

[0011] S3, sample pretreatment: respectively taking the urine sample to be tested, the calibration product and the quality control product, adding the mixed internal standard working solution of VMA, HVA and 5-HIAA isotopes, vortexing and mixing, centrifuging, taking the supernatant, and obtaining the urine sample supernatant to be tested, the calibration product supernatant and the quality control product supernatant;

[0012] S4, liquid chromatography separation: using liquid chromatography and corresponding mobile phases to separate the above-mentioned supernatants respectively;

[0013] S5, tandem mass spectrometry detection: the supernatant separated by liquid chromatography directly enters the mass spectrometer for detection, and a standard curve is drawn to obtain the contents of VMA, HVA and 5-HIAA in the urine sample to be tested.

[0014] In the above technical solution, in S2, the concentration of the calibration substance is controlled between 1.0-500 μmol / L.

[0015] In the above technical solution, in S3, the solvent of the mixed internal standard working solution is a methanol-water mixture, and the volume ratio of methanol to water is 1:3, 1:1 or 3:1.

[0016] In the above technical solution, in S3, the volume ratio of the urine sample to be tested / calibrator / quality control product to the mixed internal standard working solution is 1:(3-5).

[0017] In the above technical solution, in S4, the conditions of liquid chromatography are:

[0018] Chromatographic column: C18 column;

[0019] Column temperature: 35±5℃;

[0020] Injection volume: 2-10 μL;

[0021] Flow rate: 0.1-0.4 mL / min;

[0022] Mobile phase A is 0-0.1 wt % formic acid aqueous solution, mobile phase B is 0-0.1 wt % formic acid methanol solution, and isocratic elution is adopted.

[0023] In the above technical solution, in S4, the liquid phase gradient is:

[0024] 0-1.80min, the volume percentage of mobile phase A is 50%-70%, and the volume percentage of mobile phase B is 30%-50%;

[0025] 1.81 min, the volume percentage of mobile phase A is 0-10%, and the volume percentage of mobile phase B is 90%-100%;

[0026] 1.81-3.00min, the volume percentage of mobile phase A is 0-10%, and the volume percentage of mobile phase B is 90%-100%;

[0027] 3.01min, the volume percentage of mobile phase A is 50%-70%, and the volume percentage of mobile phase B is 30%-50%;

[0028] 3.01-4.00min, the volume percentage of mobile phase A is 50%-70%, and the volume percentage of mobile phase B is 30%-50%.

[0029] In the above technical solution, in S5, the mass spectrometry conditions are: using electrospray ionization, negative ion mode, and multiple reaction monitoring mode;

[0030] The ion source parameters include: curtain gas: 30±2psi, ion source temperature: 550±10℃, ion source voltage: -4500±10V, nebulizer gas 40±5psi, auxiliary gas: 50±5psi.

[0031] In the above technical solution, the mass spectrometry parameters are:

[0032] VMA ion pair: 197.0>137.0;

[0033] VMA-d3 ion pair: 200.0>140.0;

[0034] HVA ion pair: 181.0>122.0;

[0035] HVA-d3 ion pair: 184.0>122.0;

[0036] 5-HIAA ion pair: 190.0>143.9;

[0037] 5-HIAA-d4 ion pair: 195.0>147.9.

[0038] In the above technical solution, in S3, the centrifugal condition is room temperature, the centrifugal speed is above 12000 rpm, and the time is 2-10 min.

[0039] The beneficial effects of the present invention are: the detection method of the present invention has simple and rapid pretreatment, strong specificity, low reagent consumption cost, no environmental pollution, small sample size required, easy high-throughput operation, and is suitable for development into a kit for routine clinical testing. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 is a flow chart of liquid chromatography-tandem mass spectrometry detection of the present invention;

[0041] Figure 2 is a chromatogram of a urine sample to be tested provided by an embodiment of the present invention;

[0042] Figure 3 It is a matrix effect diagram provided by an embodiment of the present invention. DETAILED DESCRIPTION

[0043] In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments. The present invention can be implemented in many different forms and should not be construed as being limited to the embodiments set forth herein. On the contrary, these embodiments are provided so that this disclosure will be thorough and complete, and the concept of the present invention will be fully conveyed to those skilled in the art, and the present invention will only be limited by the claims.

[0044] The present invention provides a method for detecting the contents of VMA, HVA and 5-HIAA in urine by liquid chromatography-tandem mass spectrometry, comprising the following steps:

[0045] S1, preparation of blank urine matrix: anion exchange resin was used to remove free VMA, HVA and 5-HIAA in urine to obtain blank urine matrix for preparation of calibrants;

[0046] S2, preparation of calibrants: Weigh a certain amount of VMA, HVA and 5-HIAA solid powder respectively, make up to volume with methanol, and use the dissolved liquid as the mother liquor of VMA, HVA and 5-HIAA. The three are mixed in a certain proportion and then diluted with a certain volume ratio of methanol-water to obtain VMA, HVA and 5-HIAA working solutions. Finally, they are diluted step by step with blank urine matrix to obtain calibrants of different concentrations.

[0047] Among them, the concentration of the calibration material is controlled between 1.0-500umol / L.

[0048] S3, sample pretreatment: respectively take urine samples to be tested, calibrators and quality control products, add mixed internal standard working solutions of VMA, HVA and 5-HIAA isotopes, vortex mix and centrifuge, take the supernatant, and obtain the supernatant of the sample to be tested, the supernatant of the calibrator and the supernatant of the quality control product.

[0049] The sampling volume of the urine sample to be tested is controlled at 10-50 μL. Within the range of the sampling volume, the sample can be controlled to ensure that the sample for testing is sufficient, thereby ensuring the accuracy of the test.

[0050] The solvent of the mixed internal standard working solution is a methanol-water mixture with a volume ratio of 1:3, 1:1 or 3:1, preferably 1:1.

[0051] The volume ratio of the urine sample to be tested / calibrator / quality control product to the mixed internal standard working solution is 1:(3-5), preferably 1:4.

[0052] The centrifugation conditions are room temperature, a centrifugal speed of 12000 rpm or more, and a time of 2-10 min, preferably 2 min. Controlling the specific parameters of the centrifugation can ensure the removal of impurities in the urine sample to be tested, thereby improving the detection accuracy of the subsequent liquid chromatography-tandem mass spectrometry.

[0053] S4, liquid chromatography separation: use liquid chromatography and corresponding mobile phase to separate the supernatant of the sample to be tested, the supernatant of the calibration sample and the supernatant of the quality control sample.

[0054] The conditions of liquid chromatography are:

[0055] Chromatographic column: C18 column;

[0056] Column temperature: 35±5℃;

[0057] Injection volume: 2-10 μL, preferably 5 μL;

[0058] Flow rate: 0.1-0.4 mL / min;

[0059] Mobile phase A is 0-0.1 wt % formic acid aqueous solution, mobile phase B is 0-0.1 wt % formic acid methanol solution, and isocratic elution is adopted.

[0060] By controlling the specific parameters of liquid chromatography, the specific column temperature, injection volume and flow rate can be coordinated with the reverse phase chromatography column, and the mobile phase A and mobile phase B of the gradient elution can be further limited, so as to ensure that the substances in the urine test sample are separated after liquid chromatography, thereby ensuring the accuracy of subsequent mass spectrometry detection.

[0061] The liquid phase gradient is:

[0062] 0-1.80min, the volume percentage of mobile phase A is 50%-70%, and the volume percentage of mobile phase B is 30%-50%;

[0063] 1.81min, the volume percentage of mobile phase A is 0-10%, and the volume percentage of mobile phase B is 90%-100%;

[0064] 1.81-3.00min, the volume percentage of mobile phase A is 0-10%, and the volume percentage of mobile phase B is 90%-100%;

[0065] 3.01min, the volume percentage of mobile phase A is 50%-70%, and the volume percentage of mobile phase B is 30%-50%;

[0066] 3.01-4.00min, the volume percentage of mobile phase A is 50%-70%, and the volume percentage of mobile phase B is 30%-50%.

[0067] Preferably:

[0068] 0-1.80min, the volume percentage of mobile phase A is 60±5%, and the volume percentage of mobile phase B is 40±5%;

[0069] 1.81 min, the volume percentage of mobile phase A was 0±5%, and the volume percentage of mobile phase B was 95±5%;

[0070] 1.81-3.00min, the volume percentage of mobile phase A is 0±5%, and the volume percentage of mobile phase B is 95±5%;

[0071] 3.01 min, the volume percentage of mobile phase A is 60±5%, and the volume percentage of mobile phase B is 40±5%;

[0072] 3.01-4.00 min, the volume percentage of mobile phase A is 60±5%, and the volume percentage of mobile phase B is 40±5%.

[0073] Controlling the ratio of mobile phase A and mobile phase B in the specific gradient elution can ensure the complete separation of VMA, HVA and 5-HIAA from other interferences.

[0074] S5, tandem mass spectrometry detection: the supernatant of the sample to be tested, the supernatant of the calibrator and the supernatant of the quality control separated by liquid chromatography enter the mass spectrometer for detection, and the multiple reaction monitoring mode in the triple quadrupole is used to specifically detect the contents of VMA, HVA and 5-HIAA. A standard curve is drawn based on the known concentration of the calibrator and the internal standard to obtain a quantitative correction equation, and the contents of VMA, HVA and 5-HIAA are calculated based on the signal response of the analyte in the sample.

[0075] Among them, the mass spectrometry conditions are: using electrospray ionization, negative ion mode, and multiple reaction monitoring mode;

[0076] The ion source parameters include: curtain gas: (30±2)psi, ion source temperature: (550±10)oC, ion source voltage: (-4500±10)V, nebulizer gas (40±5)psi, auxiliary gas: (50±5)psi.

[0077] The mass spectrometry parameters are:

[0078] VMA ion pair: 197.0>137.0;

[0079] VMA-d3 ion pair: 200.0>140.0;

[0080] HVA ion pair: 181.0>122.0;

[0081] HVA-d3 ion pair: 184.0>122.0;

[0082] 5-HIAA ion pair: 190.0>143.9;

[0083] 5-HIAA-d4 ion pair: 195.0>147.9.

[0084] By controlling the specific ion pairs of VMA, HVA and 5-HIAA in the mass spectrometer and coordinating with the solutes of the mixed internal standard working solution of VMA, HVA and 5-HIAA isotopes, the contents of VMA, HVA and 5-HIAA in the urine sample to be tested can be accurately determined, thereby realizing the large-scale determination of VMA, HVA and 5-HIAA in urine.

[0085] Example 1

[0086] This embodiment provides a method for detecting the content of VMA, HVA and 5-HIAA in urine using liquid chromatography-tandem mass spectrometry, comprising the following steps:

[0087] 1. Preparation of blank urine matrix: Mix at least 100 mL of human urine sample, adsorb VMA, HVA and 5-HIAA in urine with anion exchange resin, and collect the effluent during loading as blank urine matrix; judge whether VMA, HVA and 5-HIAA in urine matrix are completely removed by the peak at retention time of liquid chromatography-tandem mass spectrometry. If they can still be detected, repeat the above operation with new anion exchange resin until VMA, HVA and 5-HIAA are no longer present in urine matrix. The prepared blank urine matrix is ​​used for the preparation of calibration products.

[0088] 2. Preparation of calibration products: Weigh 0.0120g of VMA, HVA and 5-HIAA solid powder respectively, dilute to 10mL in a volumetric flask with 50% methanol-water, dissolve to obtain a mother solution with a concentration of 1.2mg / mL, dilute with methanol-water (1:1, v / v) to obtain a working solution with a concentration of 12μg / mL, and finally dilute with the prepared blank urine matrix step by step to obtain the calibration product. The concentration range of VMA is 1-250μM, the concentration range of HVA is 2-500μM, and the concentration range of 5-HIAA is 2-500μM.

[0089] 3. Preparation of isotope internal standard working solution: dilute the internal standard stock solution with methanol-water (1:1 v / v) to prepare internal standard mixed working solutions with concentrations of 1.62 μM, 1.45 μM and 15.3 μM for VMA, HVA and 5-HIAA, respectively.

[0090] 4. Sample pretreatment: Take 50 μL of urine sample / calibrator / quality control product, mix with 200 μL of stable isotope internal standard working solution (1:9, v / v), vortex for 2 min to mix, centrifuge for 5 min (room temperature, 14800 rpm), take 200 μL of supernatant into the injection vial, and test on the machine (such as Figure 1 shown).

[0091] 5. Instrument conditions: Chromatographic conditions are as follows:

[0092] Chromatographic column: Synergl RP C18 chromatographic column (100×2.0mm, Luna, 3μm Synergl RP C18, pore size, Phenomenex, Torrance, CA, USA).

[0093] Column temperature: 40°C

[0094] Injection volume: 5 μL

[0095] Flow rate: 0.2 mL / min;

[0096] Mobile phase A is an aqueous solution containing 0.05% formic acid, and mobile phase B is a methanol solution, and isocratic elution is used. In some embodiments, the liquid phase gradient of the high performance liquid chromatography-tandem mass spectrometer is:

[0097] 0-1.80min, the volume percentage of mobile phase A is 60%, and the volume percentage of mobile phase B is 40%;

[0098] 1.81 min, the volume percentage of mobile phase A is 0%, and the volume percentage of mobile phase B is 100%;

[0099] 1.81-3.00min, the volume percentage of mobile phase A is 0%, and the volume percentage of mobile phase B is 100%;

[0100] 3.01 min, the volume percentage of mobile phase A is 60%, and the volume percentage of mobile phase B is 40%;

[0101] 3.01-4.00 min, the volume percentage of mobile phase A is 60%, and the volume percentage of mobile phase B is 40%.

[0102] Under this gradient elution condition, the retention times of VMA, HVA and 5-HIAA were approximately 1.92 min, 3.01 min and 2.33 min (e.g. Figure 2 The present invention achieves short detection time, fast peak time and high detection throughput.

[0103] 6. Instrument conditions: Mass spectrometry conditions are as follows:

[0104] An AB Sciex 4500 triple quadrupole mass spectrometer was used; atmospheric pressure chemical dissociation (ESI), negative ion mode, multiple reaction monitoring mode (MRM), and MRM scan mode were used. The specific mass spectrometry parameters are shown in Table 1; the ion source parameters included: spray voltage: -4500 V, ion source temperature: 550°C, curtain gas: 30 psi, auxiliary gas 40 psi, and heating gas 50 psi.

[0105] Table 1. Mass spectrometry parameters

[0106] Analyte name MRM ion pairs DP(V) EP(V) CE(V) CXP(V) VMA 197.0>137.0 -60 -10 -30 -7 VMA-d3 200.0>140.0 -50 -10 -30 -6 HVA 181.0>122.0 -45 -10 -25 -4 HVA-d3 184.0>122.0 -45 -10 -22 -7 5-HIAA 190.0>143.9 -75 -10 -28 -8 5-HIAA-d5 195.0>147.9 -76 -10 -30 -7

[0107] 7. The effect of Example 1 is confirmed by verifying the method. The specific verification parameters and verification results are as follows:

[0108] (1) Linear range

[0109] Each concentration curve point was tested 6 times, and the average value, CV and recovery rate of each concentration sample were calculated;

[0110] Table 2. Linear range experimental data

[0111]

[0112]

[0113] From Table 2 and Figure 3It can be seen that the linear ranges of urinary VMA, HVA and 5-HIAA are 1-250μM, 1-500μM and 1-500μM, respectively, and the linear correlation coefficients of the goodness of fit of the six curves are 0.9998, 0.9970 and 0.9980, respectively, indicating that the linearity of the curve fits the measured value very well and meets the needs of clinical sample testing.

[0114] (2) Precision

[0115] Precision evaluation includes intra-batch precision and inter-batch precision evaluation. Purchased urine quality control samples were used. Each sample was measured 6 times to obtain the intra-batch precision, and the inter-batch precision was obtained by continuous measurement for 6 days.

[0116] Table 3. Intra-batch and inter-batch precision experimental data

[0117]

[0118] The results of precision investigation showed that the intra-batch precision of VMA, HVA and 5-HIAA was ≤4.94%, and the inter-batch precision was ≤5.96%, which had very good precision and met the requirements of quantitative analysis.

[0119] (3) Accuracy

[0120] The recovery experiment was used to evaluate the accuracy: four different concentrations of the analyte were added to one patient sample at a ratio of 9:1 (v / v), and the final concentration after the addition was required to cover the entire medical decision level; an equal volume of solvent was added to another patient sample, and the two samples were measured 6 times to calculate the recovery rate. The recovery rate was calculated as follows: Recovery rate % = (measured concentration - basic concentration) / spiked theoretical concentration * 100%.

[0121] Table 4. Recovery rate experimental data

[0122]

[0123] The recovery rate investigation results showed that the spiked recoveries of VMA, HVA and 5-HIAA were all between 88% and 109%, meeting the recovery rate requirement of 88.4% to 119%, and this result met the requirements of quantitative analysis.

[0124] (4) Analytical sensitivity

[0125] The limit of detection (LOD) is defined as the minimum detectable concentration (S / N>3); analytical sensitivity, also known as the limit of quantification (LOQ), is defined as the lowest concentration of the analyte that can be reliably quantified, requiring S / N>10, coefficient of variation CV<20%, and the bias between the theoretical value and the measured value not exceeding 15%.

[0126] Table 5. Experimental data of limit of quantitation and limit of detection

[0127] Analytes Limit of quantification LOQ (μM) Limit of detection LOD(μM) VMA 0.50 0.25 HVA 0.50 0.25 5-HIAA 1.00 0.50

[0128] The above quantitative limits meet the needs of clinical testing.

[0129] (5) Matrix effect

[0130] The post-column infusion method was used: the analyte and internal standard solution (10 μg / mL) were injected from the mass spectrometer at a flow rate of 10 μL / min, and the pure matrix was prepared and injected from the liquid phase under running conditions to observe whether there was a matrix effect.

[0131] The results are as follows Figure 3 As shown, the retention times of VMA, HVA and 5-HIAA were approximately 1.92 min, 3.01 min and 2.33 min, and there was no matrix enhancement or reduction effect at the retention times.

[0132] Comparative Example

[0133] Compared with the pre-treatment methods of liquid-liquid extraction or solid-phase extraction reported in existing literature or patents, the present invention adopts a direct dilution method, by directly adding an internal standard solution to the sample, mixing and centrifuging, and then testing on the machine. From sampling to completing the entire sample processing process, only 4 operations are required, and the sample volume is only 50μL; on the contrary, if the liquid-liquid extraction method is used to process the sample, the whole process requires 7 steps, while the protein precipitation method requires 5 steps. In contrast, liquid-liquid extraction and solid-phase extraction are not only complicated to operate, time-consuming, environmentally polluting, and costly to process samples, but also require the use of high-cost disposable consumables extraction columns. As shown in Table 6, taking the processing of 1000 samples as an example, the differences between liquid-liquid extraction and solid-phase extraction and the present invention in terms of pre-processing time, reagents and consumables costs are analyzed.

[0134] Table 6. Comparison of sample pretreatment methods among liquid-liquid extraction, protein precipitation, acidification dilution and direct dilution

[0135]

[0136] As can be seen from Table 6, compared with liquid-liquid extraction, solid phase extraction, protein precipitation and acidification dilution methods, the direct mixing and dilution method not only has fewer total operation steps and shorter pretreatment time, but also uses fewer steps of organic solvents in the entire process, with less environmental pollution, and has significant advantages in promoting its application in clinical laboratory testing.

[0137] The present invention provides a method for detecting the content of VMA, HVA and 5-HIAA in urine by liquid chromatography-tandem mass spectrometry within 12 minutes (including sampling + pre-treatment + on-machine analysis time) on the premise that the methodology verification meets the requirements of clinical application. Compared with the prior art, the method of the present invention has fewer sample processing steps, shorter time, lower reagent and consumables costs and less environmental pollution. In addition, the standard curve drawn by preparing the blank urine matrix and preparing the standard product can better reflect the actual concentration of the urine sample. These advantages are conducive to the promotion and application of this method in the high-throughput and accurate detection of urine VMA, HVA and 5-HIAA in clinical laboratories.

[0138] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. A method for detecting the content of VMA, HVA and 5-HIAA in urine using liquid chromatography-tandem mass spectrometry, characterized in that: The following steps are involved: S1, preparation of blank urine matrix: removing free VMA, HVA and 5-HIAA in urine by anion exchange resin to obtain the blank urine matrix; S2, preparation of calibration products: weigh a certain amount of VMA, HVA and 5-HIAA solid powder respectively, make up to volume with methanol, and use the dissolved liquid as the mother liquor of VMA, HVA and 5-HIAA. The three are mixed in a certain proportion and then diluted with a certain volume ratio of methanol-water to obtain VMA, HVA and 5-HIAA working solution, and finally diluted step by step with the blank urine matrix to obtain the calibration products of different concentrations; S3, sample pretreatment: respectively taking the urine sample to be tested, the calibration product and the quality control product, adding the mixed internal standard working solution of VMA, HVA and 5-HIAA isotopes, vortexing and mixing, centrifuging, taking the supernatant, and obtaining the urine sample supernatant to be tested, the calibration product supernatant and the quality control product supernatant; S4, liquid chromatography separation: using liquid chromatography and corresponding mobile phases to separate the above-mentioned supernatants respectively; S5, tandem mass spectrometry detection: the supernatant separated by liquid chromatography directly enters the mass spectrometer for detection, and a standard curve is drawn to obtain the contents of VMA, HVA and 5-HIAA in the urine sample to be tested.

2. The method according to claim 1, characterized in that: In S2, the concentration of the calibration substance is controlled between 1.0-500 μmol / L.

3. The method according to claim 1, characterized in that: In S3, the solvent of the mixed internal standard working solution is a methanol-water mixture, and the volume ratio of methanol to water is 1:3, 1:1 or 3:

1.

4. The method according to claim 1, characterized in that: In S3, the volume ratio of the urine sample to be tested / calibrator / quality control product to the mixed internal standard working solution is 1:(3-5).

5. The method according to claim 1, characterized in that: In S4, the conditions of liquid chromatography are: Chromatographic column: C18 column; Column temperature: 35±5℃; Injection volume: 2-10 μL; Flow rate: 0.1-0.4 mL / min; Mobile phase A is 0-0.1 wt % formic acid aqueous solution, mobile phase B is 0-0.1 wt % formic acid methanol solution, and isocratic elution is adopted.

6. The method according to claim 1, characterized in that: In S4, the liquid phase gradient is: 0-1.80min, the volume percentage of mobile phase A is 50%-70%, and the volume percentage of mobile phase B is 30%-50%; 1.81min, the volume percentage of mobile phase A is 0-10%, and the volume percentage of mobile phase B is 90%-100%; 1.81-3.00min, the volume percentage of mobile phase A is 0-10%, and the volume percentage of mobile phase B is 90%-100%; 3.01min, the volume percentage of mobile phase A is 50%-70%, and the volume percentage of mobile phase B is 30%-50%; 3.01-4.00min, the volume percentage of mobile phase A is 50%-70%, and the volume percentage of mobile phase B is 30%-50%.

7. The method according to claim 1, characterized in that: In S5, the mass spectrometry conditions were: electrospray ionization, negative ion mode, multiple reaction monitoring mode; The ion source parameters include: curtain gas: 30±2psi, ion source temperature: 550±10℃, ion source voltage: -4500±10V, nebulizer gas 40±5psi, auxiliary gas: 50±5psi.

8. The method according to claim 1, characterized in that: In S5, the mass spectrometry parameters are: VMA ion pair: 197.0>137.0; VMA-d3 ion pair: 200.0>140.0; HVA ion pair: 181.0>122.0; HVA-d3 ion pair: 184.0>122.0; 5-HIAA ion pair: 190.0>143.9; 5-HIAA-d4 ion pair: 195.0>147.

9.

9. The method according to claim 1, characterized in that: In S3, the centrifugation condition is room temperature, the centrifugation speed is above 12000 rpm, and the time is 2-10 min.

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