Detection method of vanillylmandelic acid and homovanillic acid in urine and its application

Abstract

The present invention provides a method for detecting vanillylmandelic acid and homovanillic acid in urine and its application, relating to the field of metabolite detection technology. The method comprises subjecting a pretreated sample to be tested to high-performance liquid chromatography (HPLC), substituting the test value into a standard curve, and calculating the content of vanillylmandelic acid and homovanillic acid in the urine sample; the pretreatment comprises extracting the sample to be tested using an extract containing an organic phase; and the extracted sample to be tested using HPLC. This method alleviates the technical problems of high cost and complex detection methods in the prior art for detecting vanillylmandelic acid and homovanillic acid in urine.

Description

Technical Field

[0001] The present invention relates to the technical field of metabolite detection, and in particular to a method for detecting vanillylmandelic acid and homovanillic acid in urine and an application thereof. Background Art

[0002] Vanillylmandelic acid (VMA) is the primary end metabolite of epinephrine and norepinephrine. Pheochromocytoma patients secrete large amounts of epinephrine and norepinephrine, approximately 60% of which is ultimately converted to VMA and excreted in the urine. Elevated urinary VMA levels are an important indicator for the clinical diagnosis of pheochromocytoma.

[0003] Homovanillic acid (HVA), structurally similar to VMA, is the terminal metabolite of the monoamine neurotransmitter dopamine. Neuroblastoma patients can simultaneously secrete excessive amounts of epinephrine, norepinephrine, and dopamine. Their metabolites, VMA and HVA, are excreted through the kidneys and can be elevated early in the disease. Simultaneous urinary testing for VMA and HVA not only indirectly reflects catecholamine secretion but also plays a crucial role in the early diagnosis and differentiation of central nervous system diseases such as pheochromocytoma and neuroblastoma.

[0004] Methods for detecting vanillic acid primarily include electrochemical analysis, high-performance liquid chromatography (HPLC), gas chromatography, homogeneous enzyme immunoassay (HEIA), latex-enhanced immunoturbidimetry (LTTI), and liquid chromatography-tandem mass spectrometry (LC-MS / MS). Liquid chromatography-mass spectrometry (LC-MS / MS) offers excellent results for vanillic acid, meeting regulatory requirements for sensitivity, specificity, and accuracy. However, the complex equipment and operation of LC-MS / MS place higher demands on operators, and the expensive equipment and maintenance costs require significant capital investment. Therefore, reducing the cost of vanillic acid detection, simplifying the detection process, and maintaining the sensitivity, specificity, and accuracy of the test results remain unresolved.

[0005] In view of this, the present invention is proposed. Summary of the Invention

[0006] The purpose of the present invention is to provide a method for detecting vanillylmandelic acid and homovanillic acid in urine, which alleviates the technical problems of high detection cost and complex detection method of vanillylmandelic acid and homovanillic acid in urine in the prior art.

[0007] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0008] According to one aspect of the present invention, a method for detecting vanillylmandelic acid and homovanillic acid in urine is provided, comprising: subjecting a pre-treated sample to be tested to high performance liquid chromatography, inserting the test value into a standard curve, and calculating the content of vanillylmandelic acid and homovanillic acid in the urine sample;

[0009] The pretreatment includes extracting the sample to be tested using an extraction liquid containing an organic phase, and detecting the extracted sample to be tested using high performance liquid chromatography.

[0010] Preferably, the organic phase comprises n-hexane, dichloromethane or ethyl acetate;

[0011] Preferably, the organic phase comprises ethyl acetate;

[0012] Preferably, the sample to be tested is diluted with a diluent before extraction, and then extracted with an extraction solution. After extraction, the separated organic phase is blown dry with nitrogen, and then re-dissolved and detected by high performance liquid chromatography;

[0013] Preferably, the extract is mixed with the diluted sample to be tested, vortexed for 3 to 10 minutes, and then allowed to stand until the layers are separated;

[0014] Preferably, the diluent comprises any one or more of a hydrochloric acid aqueous solution, an acetic acid aqueous solution and a boric acid aqueous solution; preferably comprises a hydrochloric acid aqueous solution;

[0015] Preferably, the diluent is a 10-50 mmol / L hydrochloric acid aqueous solution;

[0016] Preferably, the nitrogen blowing temperature is 30 to 70°C, preferably 60°C;

[0017] Preferably, ultrapure water is used to reconstitute the sample to be tested that has been dried by nitrogen blowing;

[0018] Preferably, the volume ratio of the sample to be tested to the diluent is 1:(1-5), preferably 1:3;

[0019] Preferably, the volume ratio of the sample to be tested to the extract is 1:(5-12), preferably 1:10;

[0020] Preferably, 100 μL to 300 μL of the sample to be tested is taken for pretreatment, preferably 100 μL.

[0021] Preferably, the mobile phase for the HPLC detection comprises a mobile phase A and a mobile phase B; the mobile phase A is an aqueous phase, and the mobile phase B is an organic phase; the mobile phase A contains potassium dihydrogen phosphate, disodium ethylenediaminetetraacetic acid, and concentrated phosphoric acid; the concentrated phosphoric acid is HPLC grade phosphoric acid with a concentration of ≥85%;

[0022] Preferably, the mobile phase A contains 15-25 mM potassium dihydrogen phosphate, 0.15-0.25 mM disodium ethylenediaminetetraacetic acid, and 0.001-0.02% v / v concentrated phosphoric acid;

[0023] Preferably, the mobile phase A contains 20 mM potassium dihydrogen phosphate, 0.2 mM disodium ethylenediaminetetraacetic acid and 0.005% v / v concentrated phosphoric acid;

[0024] Preferably, the mobile phase B is methanol or acetonitrile, preferably methanol.

[0025] Preferably, the high performance liquid chromatography detection uses a PFP chromatographic column or a C18 chromatographic column;

[0026] Preferably, the high performance liquid chromatography detection uses a PFP chromatographic column;

[0027] Preferably, the PFP chromatographic column is selected from Agilent Pursuit PFP chromatographic column;

[0028] Preferably, the column temperature is 35-45°C;

[0029] Preferably, the column temperature is 40°C;

[0030] Preferably, the elution procedure for the high performance liquid chromatography detection is:

[0031] 0.0 min, mobile phase A 90%, mobile phase B 10%;

[0032] 3.5 min, mobile phase A 90%, mobile phase B 10%;

[0033] 5.5 min, mobile phase A 95%, mobile phase B 5%;

[0034] 15.0 min, mobile phase A 80%, mobile phase B 20%;

[0035] 15.1 min, mobile phase A 40%, mobile phase B 60%;

[0036] 17.0 min, mobile phase A 40%, mobile phase B 60%;

[0037] 17.1 min, mobile phase A 90%, mobile phase B 10%;

[0038] 20.0 min, mobile phase A 90%, mobile phase B 10%.

[0039] Preferably, the standard curve is the correspondence between vanillylmandelic acid or homovanillic acid in the calibrator and the detection value of the calibrator;

[0040] Preferably, a linear regression is performed with the chromatographic peak area of ​​the calibrator as the dependent variable and the concentration of vanillylmandelic acid or homovanillic acid in the calibrator as the independent variable to obtain a standard curve of vanillylmandelic acid or homovanillic acid.

[0041] Preferably, the calibrator consists of several calibrators with a series of concentrations, wherein the concentration of vanillylmandelic acid in the several calibrators with a series of concentrations is distributed between 0.4 and 80 μg / mL, and the concentration of homovanillic acid is distributed between 0.6 and 120 μg / mL;

[0042] Preferably, the detection method further comprises detecting a quality control product;

[0043] According to another aspect of the present invention, the present invention also provides the use of the above detection method in preparing a product for detecting vanillylmandelic acid and homovanillic acid in urine.

[0044] Compared with the prior art, the present invention has the following beneficial effects:

[0045] The detection method of vanillylmandelic acid and homovanillic acid in urine provided by the present invention adopts liquid-liquid extraction to pre-treat the sample to be tested, with high accuracy and low pre-treatment cost, and the pre-treatment effect is more accurate than the more expensive solid phase extraction method. The present invention adopts high performance liquid chromatography for sample analysis, which is lower in cost and simpler to operate than liquid chromatography-mass spectrometry (LC-MS / MS) for determining vanillylmandelic acid and homovanillic acid, and has lower requirements for equipment and operators, significantly simplifies the operating procedure, reduces the detection cost, and can complete the test of vanillylmandelic acid and homovanillic acid within 20 minutes, which is convenient, fast, and can achieve high-throughput detection. At the same time, the detection method provided by the present invention can still meet the requirements of relevant laws and regulations for linearity, repeatability, batch difference, accuracy, stability, etc. for the detection of vanillylmandelic acid and homovanillic acid. The linear range of this method is 0.4-80 μg / mL for vanillylmandelic acid, and 0.6-120 μg / mL for homovanillic acid, with correlation coefficients r≥0.999; the coefficient of variation (CV) of the repeatability of the low-value quality control product is ≤10%, the coefficient of variation (CV) of the repeatability of the mid-value quality control product is ≤10%, and the coefficient of variation (CV) of the repeatability of the high-value quality control product is ≤10%; the relative deviation (B) of the accuracy is ≤±15%, and the spiked recovery rate is 85% to 115%. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the specific embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0047] Figure 1 This is a peak diagram of vanillylmandelic acid and homovanillic acid under the test conditions of the detection method of Example 1 of the present invention;

[0048] Figure 2 Comparison of the peak patterns of vanillylmandelic acid and homovanillic acid under the test conditions of the detection methods of Example 1 and Example 5 of the present invention;

[0049] Figure 3 1 is a comparison of the peak patterns of vanillylmandelic acid and homovanillic acid under the test conditions of the detection methods of Example 1 and Example 6 of the present invention. DETAILED DESCRIPTION

[0050] The following will clearly and completely describe the technical solutions of the present invention in conjunction with the embodiments. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0051] According to one aspect of the present invention, the present invention provides a method for detecting vanillylmandelic acid and homovanillic acid in urine, which comprises detecting a pre-treated sample using high performance liquid chromatography, substituting the detection value into a standard curve, and calculating the content of vanillylmandelic acid and homovanillic acid in the urine sample.

[0052] The present invention uses liquid-liquid extraction to extract vanillylmandelic acid and homovanillic acid from the sample to be tested. The inventors verified a variety of pretreatment methods, such as filtration, solid phase extraction, and liquid-liquid extraction, and found that filtration cannot effectively remove impurities in urine samples. When the sample is pretreated by filtration, the impurities cannot be effectively separated from the target compound after high-performance liquid chromatography detection; the processing cost of solid phase extraction is higher than that of liquid-liquid extraction, and the accuracy of solid phase extraction is significantly lower than that of liquid-liquid extraction. Therefore, the present invention uses liquid-liquid extraction to pretreat the sample. The liquid-liquid extraction method uses an extraction solution containing an organic phase to extract the sample, which can effectively separate the impurities in the sample to be tested from vanillylmandelic acid and homovanillic acid. The organic phase in the extraction solution preferably includes n-hexane, dichloromethane or ethyl acetate, more preferably ethyl acetate.

[0053] Other conditions for pretreatment are preferably as follows:

[0054] In some preferred embodiments, the sample to be tested is diluted with a diluent before extraction. The diluent preferably includes any one or more of aqueous hydrochloric acid, aqueous acetic acid, and aqueous boric acid, more preferably aqueous hydrochloric acid. The preferred concentration of the aqueous hydrochloric acid is 10 to 50 mmol / L, for example, but not limited to 10, 20, 30, 40, or 50 mmol / L, preferably 30 mmol / L. The volume ratio of the sample to be tested to the diluent can optionally be 1:(1 to 5), for example, but not limited to 1:1, 1:2, 1:3, 1:4, or 1:5, preferably 1:3. Optionally, 100 to 300 μL of the sample to be tested is taken for pretreatment, for example, but not limited to 100, 150, 200, 250, or 300 μL. Specific embodiments include: taking 100 μL of the sample to be tested and optionally mixing it with 100, 200, 300, 400, or 500 μL of the diluent for dilution.

[0055] The extraction solution is then added to the diluted sample to be tested, where the volume ratio of the sample to the extraction solution can be 1:(5-12), for example, but not limited to, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, or 1:12, preferably 1:10. For example, 100 μL of the diluted sample can be optionally mixed with 500, 600, 700, 800, 900, 1000, 1100, or 1200 μL of the extraction solution for liquid-liquid extraction.

[0056] A preferred extraction method is to mix the extract with the diluted sample, then vortex mix for 3 to 10 minutes, for example, but not limited to, 3, 4, 5, 6, 7, 8, 9, or 10 minutes, and then allow the mixture to separate into layers, thereby separating the upper organic phase. A more preferred extraction method is to mix the extract with the diluted sample, then vortex mix for 5 minutes, and then allow the mixture to stand for 1 minute.

[0057] The organic phase separated after extraction is dried with nitrogen blown to dryness, and the nitrogen blowing temperature is preferably 30-70°C, for example, but not limited to 30, 40, 50, 55, 60, 65 or 70°C, preferably dried at 60°C.

[0058] The dried sample is then reconstituted, optionally with ultrapure water, in an amount of 100 to 600 μL, for example, but not limited to, 100, 200, 300, 400, 500, or 600 μL, preferably 300 μL. High-performance liquid chromatography is performed after reconstitution.

[0059] In some optional embodiments, the method for detecting vanillylmandelic acid and homovanillic acid in urine provided by the present invention further optimizes chromatographic conditions.

[0060] In some optional embodiments, the mobile phase for HPLC detection is optimized. The mobile phase comprises mobile phase A and mobile phase B, wherein mobile phase A is aqueous and mobile phase B is organic. Mobile phase A preferably also contains potassium dihydrogen phosphate, disodium ethylenediaminetetraacetic acid, and concentrated phosphoric acid (HPLC-grade phosphoric acid with a concentration of ≥85%). The addition of these components can effectively alleviate the severe tailing defect of the homovanillic acid peak, enabling efficient separation of the target substance from impurities. The preferred concentration of potassium dihydrogen phosphate is 15-25 mM, for example, but not limited to 15, 20, or 25 mM, preferably 20 mM; the preferred concentration of ethylenediaminetetraacetic acid disodium salt is 0.15-0.25 mM, for example, but not limited to 0.15, 0.2, or 0.25 mM, preferably 0.2 mM; the preferred concentration of concentrated phosphoric acid is 0.001-0.02% v / v, for example, but not limited to 0.001, 0.005, 0.01, 0.015, or 0.02% v / v. The mobile phase B can be methanol or acetonitrile, preferably methanol.

[0061] In some optional embodiments, the high performance liquid chromatography detection uses a PFP column or a C18 column, preferably a PFP column; more preferably, the high performance liquid chromatography detection uses an Agilent Pursuit PFP column, which has significantly improved accuracy compared to similar columns from other manufacturers, such as ChromCore PFP columns.

[0062] Preferably, the column temperature is 35-45°C, preferably 40°C.

[0063] In some preferred embodiments, the elution procedure for high performance liquid chromatography detection is:

[0064] 0.0 min, mobile phase A 90%, mobile phase B 10%;

[0065] 3.5 min, mobile phase A 90%, mobile phase B 10%;

[0066] 5.5 min, mobile phase A 95%, mobile phase B 5%;

[0067] 15.0 min, mobile phase A 80%, mobile phase B 20%;

[0068] 15.1 min, mobile phase A 40%, mobile phase B 60%;

[0069] 17.0 min, mobile phase A 40%, mobile phase B 60%;

[0070] 17.1 min, mobile phase A 90%, mobile phase B 10%;

[0071] 20.0 min, mobile phase A 90%, mobile phase B 10%.

[0072] In some optional embodiments, the standard curve is the correspondence between vanillylmandelic acid or homovanillic acid in the calibrator and the detection value of the calibrator. The detection value can be, for example, but not limited to, peak height or peak area. Preferably, linear regression is performed with the chromatographic peak area of ​​the calibrator as the dependent variable and the concentration of vanillylmandelic acid or homovanillic acid in the calibrator as the independent variable to obtain a standard curve of vanillylmandelic acid or homovanillic acid.

[0073] In some preferred embodiments, the calibrator comprises a plurality of calibrators of a concentration series, wherein the concentration of vanillylmandelic acid in the plurality of calibrators is distributed between 0.4 and 80 μg / mL, and the concentration of homovanillic acid is distributed between 0.6 and 120 μg / mL; a plurality of calibrators of a concentration series refers to a calibrator comprising a plurality of calibrators of different concentrations, wherein the concentration of vanillylmandelic acid in each calibrator is distributed between 0.4 and 80 μg / mL and includes the endpoint values ​​(0.4 μg / mL and 80 μg / mL), and the concentration of homovanillic acid is distributed between 0.6 and 120 μg / mL and includes the endpoint values ​​(0.6 μg / mL and 120 μg / mL). Due to the difficulty in obtaining human samples, the matrix for preparing the calibrator can optionally be normal saline, artificial urine, or PBS buffer.

[0074] In some preferred embodiments, the concentration points of vanillylmandelic acid in the calibrators in the concentration series are set to 0.4 μg / mL, 1 μg / mL, 4 μg / mL, 20 μg / mL, 40 μg / mL and 80 μg / mL.

[0075] In some preferred embodiments, the concentration points of homovanillic acid in the several calibrators in the concentration series are set to 0.6 μg / mL, 1.5 μg / mL, 6 μg / mL, 30 μg / mL, 60 μg / mL and 120 μg / mL.

[0076] In some optional embodiments, the detection method further includes testing a quality control product to verify whether the test results are credible. The quality control products preferably include at least two of a low-value quality control product, a median quality control product, and a high-value quality control product. The concentration of vanillylmandelic acid in the low-value quality control product is 2 μg / mL, and the concentration of homovanillic acid is 3 μg / mL; the concentration of vanillylmandelic acid in the median quality control product is 40 μg / mL, and the concentration of homovanillic acid is 60 μg / mL; the concentration of vanillylmandelic acid in the high-value quality control product is 60 μg / mL, and the concentration of homovanillic acid is 90 μg / mL. If human samples are difficult to obtain, the matrix for preparing the quality control product can optionally be normal saline, artificial urine, or PBS buffer.

[0077] It should be noted that the method for detecting vanillylmandelic acid and homovanillic acid in urine provided by the present invention is not intended for diagnostic or therapeutic purposes. Although the levels of vanillylmandelic acid and homovanillic acid in urine are associated with central nervous system diseases such as pheochromocytoma and neuroblastoma, this does not mean that the levels of vanillylmandelic acid and homovanillic acid in urine can directly indicate whether the source of the test sample has a disease.

[0078] The present invention does not limit the source of the urine of the detection subject. The sources of urine include but are not limited to mice, rats, guinea pigs, hamsters, rabbits, ferrets, cats, dogs, goats, sheep, cows, pigs, horses, monkeys or humans. It is preferred to detect urine from humans, or in animal experiments, urine from, for example but not limited to, mice, rats or rabbits is detected.

[0079] The detection method provided by the present invention can simultaneously determine the contents of vanillylmandelic acid and homovanillic acid in a single test, and has the advantages of high sensitivity, good repeatability, high accuracy, and good specificity. The linear range of this method is 0.4-80 μg / mL for vanillylmandelic acid and 0.6-120 μg / mL for homovanillic acid, with correlation coefficients r ≥ 0.999. The coefficient of variation (CV) of the repeatability of the low-value quality control sample is ≤ 10%, the coefficient of variation (CV) of the repeatability of the mid-value quality control sample is ≤ 10%, and the coefficient of variation (CV) of the repeatability of the high-value quality control sample is ≤ 10%. The relative deviation (B) of the accuracy is ≤ ±15%, and the spiked recovery rate is 85% to 115%.

[0080] According to another aspect of the present invention, the present invention also provides the use of the above-mentioned detection method in the preparation of a product for detecting vanillylmandelic acid and homovanillic acid in urine. The above-mentioned detection method can be used as an evaluation standard to evaluate the detection effect of other products for detecting vanillylmandelic acid and homovanillic acid in urine; or the above-mentioned detection method can be pre-installed in the detection product as an operating module, and the reagents used in the above-mentioned detection method can be used as supporting reagents in the detection product.

[0081] The technical solutions and effects of the present invention are further described below in conjunction with preferred embodiments.

[0082] The reagents used in the following examples are shown in Table 1.

[0083] Table 1. Calibrators and reagents

[0084]

[0085] The equipment and instruments used in the following examples are shown in Table 2.

[0086] Table 2. Instruments and Equipment

[0087] Serial number Device Name Model / Specifications 1 Liquid chromatograph LC2300 2 Fluorescence detector RF-20AXS 3 96-well plate shaker MB100-4A 4 Nitrogen blowdown apparatus MV-96-GS 5 2ml centrifuge tube 2mL 6 100 μL pipette 10-100 μL 7 1000 μL pipette 100-1000μL 8 96-well plate 1mL 9 Balance One in 100,000

[0088] Example 1

[0089] (1) Reagent preparation:

[0090] (1) Preparation of vanillylmandelic acid and homovanillic acid stock solutions:

[0091] Preparation of vanillylmandelic acid stock solution: Use a 1 / 100,000 electronic balance to accurately weigh 20 mg of vanillylmandelic acid, place it in a 10 mL volumetric flask, dissolve it in methanol and dilute to the mark to prepare a 2 mg / mL stock solution. Transfer it to a 15 mL brown glass bottle, label it, and store it at -80°C.

[0092] Preparation of homovanillic acid stock solution: Using a 1 / 100,000 electronic balance, accurately weigh 20 mg of homovanillic acid and place it in a 10 mL volumetric flask. Dissolve it in methanol and dilute to the mark to prepare a 2 mg / mL stock solution. Transfer it to a 15 mL brown glass bottle, label it, and store it at -80°C.

[0093] (2) Preparation of secondary stock solutions of vanillylmandelic acid and homovanillic acid:

[0094] Preparation of secondary stock solutions of vanillylmandelic acid and homovanillic acid: Use a pipette to transfer 4 mL of vanillylmandelic acid stock solution and 6 mL of homovanillic acid, respectively, and mix them in a ratio of 2:3 to prepare secondary stock solutions with a concentration of (vanillylmandelic acid: 0.8 mg / mL, homovanillic acid: 1.2 mg / mL). Transfer to a 15 mL brown glass bottle, label it, and store at -80°C.

[0095] (3) Preparation of calibrators S1 to S6:

[0096] The calibrators were prepared according to the high-low preparation mode, that is, after H0 and S6 were prepared, they were mixed in proportion to prepare S1 to S5 and LQC, MQC and HQC.

[0097] Preparation of Calibrator S6: Use a pipette of appropriate size to transfer 1 mL of the secondary stock solutions of vanillylmandelic acid and homovanillic acid into a suitable HDPE tube. Add 9 mL of normal saline to prepare a standard solution containing 80 μg / mL vanillylmandelic acid and 120 μg / mL homovanillic acid. Mix thoroughly, label, and store at -20°C.

[0098] Prepare calibrator S1: Use an appropriate pipette to transfer 12 μL of S6 calibrator into a suitable HDPE tube. Add 2.41 mL of normal saline to prepare a standard solution containing 0.40 μg / mL vanillylmandelic acid and 0.60 μg / mL homovanillic acid. Mix thoroughly, label, and store at -20°C.

[0099] Preparation of calibrator S2: Accurately pipette 30 μL of S6 calibrator, add 2.39 mL of normal saline and mix thoroughly to prepare 1 μg / mL vanillylmandelic acid and 1.5 μg / mL homovanillic acid standard solutions. After thorough mixing, label and store at -20°C.

[0100] Preparation of calibrator S3: Accurately pipette 121 μL of S6 calibrator, add 2.30 mL of normal saline and mix thoroughly to prepare 4 μg / mL vanillylmandelic acid and 6 μg / mL homovanillic acid standard solutions. After thorough mixing, label and store at -20°C.

[0101] Preparation of calibrator S4: Accurately pipette 605 μL of S6 calibrator, add 1.82 mL of normal saline, mix thoroughly to prepare 20 μg / mL vanillylmandelic acid and 30 μg / mL homovanillic acid standard solutions. After thorough mixing, label and store at -20°C.

[0102] Preparation of calibrator S5: Accurately pipette 1.21 mL of S6 calibrator, add 1.21 mL of normal saline, mix thoroughly to prepare 40 μg / mL vanillylmandelic acid and 60 μg / mL homovanillic acid standard solutions. After thorough mixing, label and store at -20°C.

[0103] (4) Preparation of quality control products:

[0104] Preparation of low-concentration quality control (LQC): Accurately pipette 61 μL of S6 calibrator, add 2.36 mL of normal saline, mix thoroughly to prepare 2 μg / mL vanillylmandelic acid and 3 μg / mL homovanillic acid standard solutions. After thorough mixing, label and store at -20°C.

[0105] Preparation of medium-concentration quality control (MQC): Accurately pipette 1.21 mL of S6 calibrator, add 1.21 mL of normal saline, mix thoroughly to prepare 40 μg / mL vanillylmandelic acid and 60 μg / mL homovanillic acid standard solutions. After thorough mixing, label and store at -20°C.

[0106] Preparation of high-concentration quality control (HQC): Accurately pipette 1.815 mL of S6 calibrator, add 0.61 mL of normal saline, mix thoroughly to prepare 60 μg / mL vanillylmandelic acid and 90 μg / mL homovanillic acid standard solutions. After thorough mixing, label and store at -20°C.

[0107] (5) Preparation of sample diluent:

[0108] Preparation of sample diluent: Pipette 0.276 μL of concentrated hydrochloric acid into a 100 mL solvent bottle, accurately add 99.724 mL of ultrapure water, mix thoroughly and set aside to obtain a 30 mmol / L hydrochloric acid aqueous solution.

[0109] (6) Preparation of mobile phase:

[0110] Preparation of mobile phase A: Use an electronic balance to accurately weigh 2.72 g of potassium dihydrogen phosphate and 74.5 mg of ethylenediaminetetraacetic acid disodium salt, add 1000 mL of pure water to prepare a solution, then add 50 μL of concentrated phosphoric acid, mix, filter and ultrasonicate for use.

[0111] Preparation of fluidity liquid B: methanol solution, filter and degas by ultrasonication, and set aside.

[0112] (2) Pre-treatment steps:

[0113] (1) Add sample: Pipette 100 μL of sample (test sample / calibrator solution / quality control solution) and add them to new 2 ml centrifuge tubes respectively;

[0114] (2) Add diluent: Pipette 300 μL of sample diluent into the above centrifuge tubes and mix thoroughly;

[0115] (3) Extraction: Pipette 1 ml of ethyl acetate into a well-mixed centrifuge tube, vortex and extract for 5 min. After standing for 1 min, take 600 μL of the upper organic phase and transfer it to a new 96-well plate;

[0116] (4) Nitrogen blowing: blow with nitrogen at 60°C until dry;

[0117] (5) Redissolution: Add 300 μL of ultrapure water to a 96-well plate, seal the plate, and mix for 3 min;

[0118] (6) Detection: Place the 96-well plate in the instrument and use liquid phase LC 2300 for detection.

[0119] (III) Chromatographic conditions:

[0120] (1) Chromatographic column: Agilent Pursuit PFP, (3 μm, 150 × 4.6 mm);

[0121] (2) Mobile phase: Phase A: water (containing 20 mM potassium dihydrogen phosphate, 0.2 mM disodium ethylenediaminetetraacetic acid, and 0.005% concentrated phosphoric acid); Phase B: methanol.

[0122] (3) Gradient elution procedure:

[0123] Table 3. Gradient conditions

[0124] T(min) ％A ％B 0.0 90 10 3.5 90 10 5.5 95 5 15.0 80 20 15.1 40 60 17.0 40 60 17.1 90 10 20.0 90 10

[0125] (4) Flow rate: 1.0 mL / min;

[0126] (5) Column temperature: 40°C;

[0127] (6) Automatic sampler temperature: 10°C;

[0128] (7) Needle wash solution: methanol: water (3:2 v / v);

[0129] (8) Injection volume: 5 μL.

[0130] The peaks of vanillylmandelic acid and homovanillic acid under the test conditions of the detection method of Example 1 are as follows: Figure 1 shown.

[0131] Example 2

[0132] The only difference between Example 2 and Example 1 is that the pretreatment steps are different. In Example 2, PAX solid phase extraction is used to purify the sample, and the sample is directly loaded for detection after nitrogen drying and redissolution.

[0133] Example 3

[0134] The only difference between Example 3 and Example 1 is that the chromatographic column is different. This example uses ChromCore PFP (3 μm, 150×4.6 mm) as the analytical chromatographic column.

[0135] Example 4

[0136] The only difference between Example 4 and Example 1 is that the mobile phase A is different. Water is used as the mobile phase A in this example.

[0137] Example 5

[0138] The only difference between Example 5 and Example 1 is the pre-treatment steps. In Example 5, the sample is purified by filtration before being directly tested on the machine.

[0139] Example 6

[0140] The only difference between Example 6 and Example 1 is that the elution procedure in the liquid phase method is different. Example 6 uses the elution procedure in Table 4 for detection.

[0141] Table 4. Gradient conditions

[0142] T(min) ％A ％B 0.0 90 10 3.5 90 10 4.5 80 20 13.0 80 20 13.1 60 40 15.1 60 40 15.2 90 10 18.0 90 10

[0143] Effect Example 1

[0144] Comparison of the test results of Example 1 and Example 2: A certain amount of calibrator was added to blank human urine to prepare high concentration and low concentration samples. The samples were tested using the detection methods of Example 1 and Example 2, respectively, and the external standard method was used for calculation. The results are shown in Tables 5 and 6.

[0145] Table 5. Accuracy results of Example 2

[0146]

[0147] Table 6. Accuracy results of Example 1

[0148]

[0149] By comparing Table 5 and Table 6, the results show that the recovery rate of vanillylmandelic acid and homovanillic acid is significantly improved by using ethyl acetate liquid-liquid extraction pretreatment, and the detection method provided in Example 1 is better.

[0150] Effect Example 2

[0151] Comparison of the test results of Example 1 and Example 3: A certain amount of calibrant was added to blank human urine to prepare high-concentration and low-concentration samples. The samples were tested using Example 1 and Example 3, respectively, and the external standard method was used for calculation. The results are shown in Table 7. A comparison of the recovery rates of the two chromatographic columns shows that the Agilent Pursuit PFP column achieves superior accuracy after testing to the ChromCore PFP column, demonstrating that the detection method provided in Example 1 is superior.

[0152] Table 7. Comparison of test results of Example 1 and Example 3

[0153]

[0154] Effect Example 3

[0155] Samples were tested using the detection methods of Examples 1 and 4, respectively. The detection method of Example 4 exhibited severe tailing of the homovanillic acid peak, shifting the retention time later and resulting in poor separation from impurities. However, the addition of potassium dihydrogen phosphate, EDTA-2Na, and 0.005% concentrated phosphoric acid to Example 1 significantly improved the peak shape and clear separation from impurities. The results are shown in Table 8.

[0156] Table 8. Effect of different flow rates on the results

[0157]

[0158] Effect Example 4

[0159] The samples were tested using the pretreatment methods of Example 1 and Example 5, respectively. After the filtration pretreatment of Example 5, homovanillic acid HVA was inseparable from the impurities, while after the liquid-liquid extraction method of Example 1, homovanillic acid was clearly separated from the impurities. The results are shown in Figure 5. Figure 2 .

[0160] Effect Example 5

[0161] The samples were tested using the liquid phase elution procedures of Example 1 and Example 6, respectively. The retention time of homovanillic acid HVA was advanced by the elution procedure of Example 6, and a miscellaneous peak appeared near the elution peak. The results are shown in FIG. Figure 3.

[0162] Effect Examples 6 to 11 all used the detection method of Example 1 to test the samples.

[0163] Effect Example 6

[0164] Linear range test data:

[0165] (1) Verification Method: Treat the test product and calibrator solutions S1 to S6 according to the test method of Example 1 and repeat the test for three days. Calculate the correlation coefficient r of the linear regression using the following formula; the correlation coefficient r should be ≥ 0.990.

[0166]

[0167] r: linear regression correlation coefficient;

[0168] xi: concentrations of S1–S6;

[0169] yi: mean peak area of ​​calibrator in corresponding concentration solution.

[0170] (2) Acceptance criteria: The linear regression correlation coefficients r of vanillylmandelic acid and homovanillic acid were both ≥ 0.990.

[0171] (3) Experimental results:

[0172] Linear range: Vanillylmandelic acid had a linear range of 0.4-80 μg / mL, and homovanillic acid had a linear range of 0.6-120 μg / mL. Under the test conditions, the correlation coefficient r was greater than 0.999, meeting the relevant requirements. The results are shown in Tables 9 and 10.

[0173] Table 9. Vanillylmandelic acid linearity data

[0174]

[0175]

[0176] Table 10. Homovanillic acid linearity data

[0177]

[0178] Effect Example 7

[0179] Precision test plan and data:

[0180] (1) Validation method: For intra-batch precision, at least one analytical batch of three concentrations is required, including low, medium, and high concentration levels, with at least five samples for each concentration. For inter-batch precision, at least three analytical batches (at least three days) are required, including low, medium, and high concentration levels, with at least five samples for each concentration. The coefficient of variation is used to evaluate inter-batch precision.

[0181]

[0182] CV: coefficient of variation of repeatability;

[0183] The average value of 5 measurements;

[0184] S: standard deviation of 5 measurements.

[0185] (2) Acceptance criteria: The intra-batch coefficient of variation shall generally not exceed 15%; the inter-batch coefficient of variation shall generally not exceed 15%.

[0186] (3) Experimental results:

[0187] Intra-batch precision of vanillylmandelic acid: After three days of testing, the highest coefficients of variation of different concentrations of vanillylmandelic acid were: 1.94% at the low concentration level, 2.18% at the medium concentration level, and 1.59% at the high concentration level, which met the requirements.

[0188] Intra-batch precision of homovanillic acid: After three days of testing, the highest coefficients of variation of different concentrations of homovanillic acid were 3.34% at the low concentration level, 2.67% at the medium concentration level, and 2.33% at the high concentration level, which met the requirements.

[0189] The inter-batch precision of vanillylmandelic acid: the coefficient of variation at the low concentration level was 4.44%, the coefficient of variation at the medium concentration level was 4.14%, and the coefficient of variation at the high concentration level was 4.03%, which met the requirements.

[0190] The inter-batch precision of homovanillic acid: the coefficient of variation at the low concentration level was 2.88%, the coefficient of variation at the medium concentration level was 2.20%, and the coefficient of variation at the high concentration level was 1.56%, which met the requirements.

[0191] Table 11. Intra-assay and inter-assay precision data of vanillylmandelic acid

[0192]

[0193]

[0194] Table 12 Intra-batch precision and inter-batch precision data of homovanillic acid

[0195]

[0196]

[0197] Effect Example 8

[0198] Residue studies:

[0199] (1) Validation method: Carryover may affect the accuracy of sample determination, so it should be investigated and minimized during method development. Evaluate whether the carryover meets the performance evaluation requirements. Use one analytical batch for validation, with high and low concentration samples injected alternately and continuously, repeated six times.

[0200] (2) Acceptance criteria: Two samples of high and low concentrations are injected alternately and continuously. The test results of the lower concentration sample should meet the accuracy requirements. The deviation (% Dev) of the accuracy of the low concentration sample (i.e., the LQC) from the theoretical value should be less than 15.0%, and the coefficient of variation (CV) of the six LQC test concentrations in each batch should be less than 10.0%.

[0201] (3) Experimental results:

[0202] The high and low concentration samples were cross-injected continuously and repeated 6 times. The test results of low concentration samples of vanillylmandelic acid and homovanillic acid met their accuracy requirements and satisfied the residue acceptance criteria.

[0203] Table 13. Vanillylmandelic acid residue evaluation

[0204]

[0205] Table 14. Homovanillic acid residue evaluation

[0206]

[0207] Effect Example 9

[0208] Accuracy:

[0209] (1) Verification method: This protocol evaluates accuracy by detecting vanillylmandelic acid and high vanillic acid accuracy samples and calculating their recovery rates. Evaluate whether the accuracy meets the requirements of performance evaluation. Add a certain amount of the working solution of the analyte to the blank human matrix to prepare three concentrations of recovered samples LQC, MQC, and HQC (i.e., quality control samples). The concentration selection includes low concentration level, medium concentration level, and high concentration level (75%-80% concentration of the upper limit of the standard curve). Take three analysis batches, repeat the measurement of 5 samples at each concentration, and test continuously for three days.

[0210] (2) Acceptance criteria: Recovery rate: 85%-115%.

[0211] (3) Experimental results:

[0212] Accuracy of vanillylmandelic acid: After three days of testing, the accuracy of vanillylmandelic acid at different concentrations was 100.3%-111.5% at the low concentration level, 100.9%-109.4% at the medium concentration level, and 98.7%-107.6% at the high concentration level, meeting the requirements.

[0213] Accuracy of homovanillic acid: After three days of testing, the accuracy of homovanillic acid at different concentrations was 101.1%-110.5% at the low concentration level, 99.7%-106.9% at the medium concentration level, and 102.1%-105.8% at the high concentration level, meeting the requirements.

[0214] Table 15. Vanillylmandelic acid accuracy evaluation table

[0215]

[0216] Table 16. Homovanillic acid accuracy evaluation table

[0217]

[0218] Effect Example 10

[0219] Interference evaluation:

[0220] (1) Verification method: Prepare interfering substance storage solution. According to the interfering substance concentration requirements of the experimental design, add a certain amount of interfering substance storage solution to the quality control product as the test sample. At the same time, add the interfering substance storage solution solvent to the same quality control product as the control sample. The addition amount in this experimental protocol is uniformly 5% of the addition amount of the basic sample. Pre-process the test sample and the control sample. Test the test sample and the control sample separately in the alternating order, i.e., C1T1C2T2C3T3C4T4C5T5.

[0221] (2) Acceptance criteria: If Dev% = (Xtest-Xcontrol) / Xcontrol≤15%, the potential interfering substance has no effect on the determination of the analyte; otherwise, the potential interference can be judged to be established.

[0222] (3) Experimental results: Dev% of the test specimens and control specimens of low and high quality control samples of vanillylmandelic acid and homovanillic acid was ≤15%, indicating that the potential interference had no effect on the determination of the analyte.

[0223] Table 17. Evaluation table of interfering substances of vanillylmandelic acid

[0224]

[0225] Table 18. Homovanillic acid interfering substance evaluation table

[0226]

[0227]

[0228] Effect Example 11

[0229] Experimental process:

[0230] (1) Validation method: High and low concentration quality control samples were placed at 2-8°C and stability was investigated at 0, 3, and 7 days. At each time point, a calibration curve was obtained using freshly prepared calibration standards. Stability quality control samples were analyzed based on the calibration curve. Each sample was tested twice.

[0231] (2) Acceptance criteria: The deviations of low and high quality control samples within 3 days and 7 days compared with day 0 were both within ±15%, and were generally stable, indicating that vanillylmandelic acid and homovanillic acid samples were stable in the alternative matrix.

[0232] (3) Experimental results:

[0233] The deviations of the low and high quality control samples of vanillylmandelic acid and homovanillic acid within 3 days and 7 days compared with day 0 were all within the range of ±10%, meeting the requirements.

[0234] Table 19. Vanillylmandelic acid stability results

[0235]

[0236]

[0237] Table 20. Homovanillic acid stability results

[0238]

[0239] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for detecting vanillylmandelic acid and homovanillic acid in urine, characterized in that: The method includes detecting the pre-treated sample using high performance liquid chromatography and a fluorescence detector, inserting the detection value into a standard curve, and calculating the content of vanillylmandelic acid and homovanillic acid in the urine sample; The pretreatment includes extracting the sample to be tested with an extraction liquid containing an organic phase, diluting the sample to be tested with a diluent before extraction, then adding the extraction liquid for extraction, blowing the separated organic phase to dryness with nitrogen, and then re-dissolving and detecting with high performance liquid chromatography; the organic phase is ethyl acetate; the diluent includes any one or more of a hydrochloric acid aqueous solution, an acetic acid aqueous solution, and a boric acid aqueous solution; and re-dissolving the nitrogen-dried sample to be tested with ultrapure water; The HPLC detection uses a PFP column; the mobile phase for the HPLC detection includes mobile phase A and mobile phase B; the mobile phase A is an aqueous phase containing 15-25 mM potassium dihydrogen phosphate, 0.15-0.25 mM disodium ethylenediaminetetraacetic acid, and 0.001-0.02% v / v concentrated phosphoric acid; the mobile phase B is methanol or acetonitrile; The elution procedure of the high performance liquid chromatography detection is: 0.0 min, mobile phase A 90%, mobile phase B 10%; 3.5 min, mobile phase A 90%, mobile phase B 10%; 5.5 min, mobile phase A 95%, mobile phase B 5%; 15.0 min, mobile phase A 80%, mobile phase B 20%; 15.1 min, mobile phase A 40%, mobile phase B 60%; 17.0 min, mobile phase A 40%, mobile phase B 60%; 17.1 min, mobile phase A 90%, mobile phase B 10%; 20.0 min, mobile phase A 90%, mobile phase B 10%.

2. The detection method according to claim 1, wherein Mix the extract with the diluted sample, vortex mix for 3-10 minutes, and then let it stand until the layers separate.

3. The detection method according to claim 2, characterized in that The extract was mixed with the diluted sample, vortexed for 5 minutes, and then allowed to stand for 1 minute.

4. The detection method according to claim 1, wherein The diluent includes aqueous hydrochloric acid solution.

5. The detection method according to claim 4, characterized in that The diluent includes a 10-50 mmol / L hydrochloric acid aqueous solution.

6. The detection method according to claim 5, characterized in that The diluent includes a 30 mmol / L hydrochloric acid aqueous solution.

7. The detection method according to claim 1, characterized in that The nitrogen blowing temperature is 30~70℃.

8. The detection method according to claim 7, characterized in that The nitrogen blowing temperature was 60°C.

9. The detection method according to claim 1, wherein The amount of ultrapure water added is 100~600μL.

10. The detection method according to claim 9, characterized in that: The amount of ultrapure water added was 300 μL.

11. The detection method according to claim 1, characterized in that The volume ratio of the sample to be tested to the diluent is 1:1~5.

12. The detection method according to claim 11, characterized in that The volume ratio of the sample to be tested to the diluent is 1:

3.

13. The detection method according to claim 1, characterized in that The volume ratio of the sample to be tested to the extract is 1:5~12.

14. The detection method according to claim 13, characterized in that The volume ratio of the sample to be tested to the extract was 1:

10.

15. The detection method according to claim 1, characterized in that Take 100μL~300μL of the sample to be tested for pretreatment.

16. The detection method according to claim 15, characterized in that: Take 100 μL of the sample to be tested for pretreatment.

17. The detection method according to claim 1, wherein The mobile phase A contained 20 mM potassium dihydrogen phosphate, 0.2 mM disodium ethylenediaminetetraacetic acid, and 0.005% v / v concentrated phosphoric acid.

18. The detection method according to claim 1, characterized in that The mobile phase B is methanol.

19. The detection method according to claim 1, wherein The PFP chromatographic column is selected from Agilent Pursuit PFP chromatographic column.

20. The detection method according to claim 1, characterized in that The column temperature is 35~45℃.

21. The detection method according to claim 20, characterized in that The column temperature was 40°C.

22. The detection method according to claim 1, characterized in that The sources of urine include mice, rats, guinea pigs, hamsters, rabbits, ferrets, cats, dogs, goats, sheep, cows, pigs, horses, monkeys or humans.

23. The detection method according to any one of claims 1 to 22, characterized in that The standard curve is the corresponding relationship between the vanillylmandelic acid or homovanillic acid in the calibrator and the detection value of the calibrator.

24. The detection method according to claim 23, characterized in that Linear regression was performed with the chromatographic peak area of ​​the calibrator as the dependent variable and the concentration of vanillylmandelic acid or homovanillic acid in the calibrator as the independent variable to obtain the standard curve of vanillylmandelic acid or homovanillic acid.

25. The detection method according to claim 23, characterized in that The calibrators are composed of several calibrators with a series of concentrations. The concentrations of vanillylmandelic acid in the several calibrators with a series of concentrations are distributed between 0.4 and 80 μg / mL, and the concentrations of homovanillic acid are distributed between 0.6 and 120 μg / mL.

26. The detection method according to claim 25, characterized in that The concentration points of vanillylmandelic acid in the several calibrators of the concentration series were set to 0.4 μg / mL, 1 μg / mL, 4 μg / mL, 20 μg / mL, 40 μg / mL and 80 μg / mL.

27. The detection method according to claim 25, characterized in that The concentration points of homovanillic acid in the several calibrators of the concentration series were set to 0.6 μg / mL, 1.5 μg / mL, 6 μg / mL, 30 μg / mL, 60 μg / mL and 120 μg / mL.

28. The detection method according to claim 23, characterized in that The matrix of the calibrator is physiological saline, artificial urine or PBS buffer.

29. The detection method according to any one of claims 1 to 22, characterized in that The detection method also includes detecting quality control products.

30. The detection method according to claim 29, characterized in that The quality control products include at least two of low-value quality control products, medium-value quality control products and high-value quality control products; The concentration of vanillylmandelic acid in the low-value quality control product is 2 μg / mL, and the concentration of homovanillic acid is 3 μg / mL; the concentration of vanillylmandelic acid in the median quality control product is 40 μg / mL, and the concentration of homovanillic acid is 60 μg / mL; the concentration of vanillylmandelic acid in the high-value quality control product is 60 μg / mL, and the concentration of homovanillic acid is 90 μg / mL.

31. The detection method according to claim 29, characterized in that The matrix of the quality control product is physiological saline, artificial urine or PBS buffer.

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