Kit for liquid phase detection of amino acid, pretreatment method and liquid phase detection method

Abstract

The invention relates to the technical field of detection, in particular to a kit for liquid phase detection of amino acid, a pretreatment method and a liquid phase detection method. The kit comprises a protective agent, an activating solution, a leacheate, an eluent and a resolvent, the protective agent comprises a first ammonium acetate solution; the activating solution comprises methanol and a second ammonium acetate solution; the leacheate comprises a formic acid aqueous solution and methanol; the eluent comprises a methanol solution with the pH value of 9 to 13. The detection method using the kit can meet the requirements of related laws and regulations on linearity, repeatability, batch-to-batch difference, accuracy and the like of cystine, lysine, ornithine and arginine, and has important reference significance for evaluating whether cystinuria exists or not.

Description

Technical Field

[0001] The present invention relates to the field of detection technology, and in particular to a kit for liquid-phase detection of amino acids, a pretreatment method, and a liquid-phase detection method. Background Art

[0002] Cystinuria is a congenital metabolic disease caused by poor absorption and reabsorption of the amino acid cystine in the intestine and kidney. Cystinuria is classified into subtypes I, II, and III (types II and III are also called non-type I) according to the excretion amount of urinary cystine in heterozygous parental specimens. This leads to the accumulation of poorly soluble cystine in the urine and the formation of kidney stones (urolithiasis). Symptoms may include acute attacks of abdominal pain or low back pain, blood in the urine (hematuria), and repeated kidney stones may lead to frequent urinary tract infections and ultimately may lead to renal insufficiency.

[0003] Homozygotes or compound heterozygotes of cystinuria excrete a large amount of cystine in the urine, but the quantity varies significantly and the excretion amounts of other dibasic amino acids (arginine, lysine, and ornithine) in the urine usually also increase.

[0004] Homozygotes and heterozygous individuals of non-type I cystinuria can be distinguished by the urinary amino acid excretion pattern: homozygous individuals secrete a large amount of cystine and all three dibasic amino acids (arginine, lysine, and ornithine), while heterozygous individuals secrete more lysine and cystine than arginine and ornithine.

[0005] There are many detection articles on cystine, lysine, ornithine, and arginine, but few articles report the simultaneous determination of these four substances. Summary of the Invention

[0006] The present invention provides a kit and method for accurately and simultaneously detecting cystine, lysine, ornithine, and arginine in human urine. This method can meet the requirements of relevant regulations for the linearity, repeatability, batch-to-batch difference, accuracy, etc. of cystine, lysine, ornithine, and arginine, and has important reference significance for evaluating whether there is cystinuria.

[0007] Based on this, the present invention has the following technical solutions: In a first aspect, the present invention provides a kit for liquid-phase detection of amino acids, comprising: a protective agent, an activation solution, a washing solution, an elution solution, and a reconstitution solvent; The protective agent comprises a first ammonium acetate solution; The activation solution comprises methanol and a second ammonium acetate solution; The washing solution comprises a formic acid aqueous solution and methanol; The elution solution comprises a methanol solution with a pH value of 9-13.

[0008] In the present invention, a volatile alkaline solution can be used to adjust the pH value of methanol to 9 - 13, such as any value among 9, 10, 11, 12, and 13, or a value range with any two of the above values as endpoints.

[0009] The present invention discovers that using a methanol solution within the above pH value range as the eluent can improve the extraction efficiency and selectivity of four amino acids, and contribute to obtaining target compounds with high purity and high recovery rate.

[0010] Preferably, the eluent is prepared by adjusting the pH value of methanol to 9 - 13 with ammonia water having a concentration of 2wt% - 8wt%. The concentration of the ammonia water can be any value among 2wt%, 3wt%, 4wt%, 5wt%, 6wt%, 7wt%, 8wt%, or a value range with any two of the above values as endpoints.

[0011] Preferably, the concentrations of the first ammonium acetate solution and the second ammonium acetate solution are independently, identically, or differently 0.10 - 0.35 mol / L, such as 0.10 mol / L, 0.11 mol / L, 0.12 mol / L, 0.13 mol / L, 0.14 mol / L, 0.15 mol / L, 0.18 mol / L, 0.17 mol / L, 0.18 mol / L, 0.19 mol / L, 0.20 mol / L, 0.21 mol / L, 0.22 mol / L, 0.23 mol / L, 0.24 mol / L, 0.25 mol / L, 0.26 mol / L, 0.27 mol / L, 0.28 mol / L, 0.29 mol / L, 0.30 mol / L, 0.31 mol / L, 0.32 mol / L, 0.33 mol / L, 0.34 mol / L, 0.35 mol / L, or a value range with any two of the above values as endpoints.

[0012] Preferably, the content of formic acid in the aqueous formic acid solution is 1wt% - 3wt%, such as any value among 1wt%, 2wt%, 3wt%, or a value range with any two of the above values as endpoints.

[0013] Preferably, the complex solvent is an aqueous methanol solution containing 1 - 10 mmol / L perfluorooctanoic acid, wherein the volume ratio of methanol to water is 1:8 - 10.

[0014] Preferably, the kit further contains a mobile phase; the mobile phase includes mobile phase A and mobile phase B. Mobile phase A is an aqueous solution of 1 - 10 mmol / L perfluorooctanoic acid, and mobile phase B is a methanol solution of 1 - 10 mmol / L perfluorooctanoic acid or an acetonitrile solution of 1 - 10 mmol / L perfluorooctanoic acid.

[0015] Preferably, the kit further contains an amino acid quality control product and / or an amino acid standard; the amino acids include one or more of cystine, lysine, ornithine, and arginine.

[0016] Second, the present invention provides a pretreatment method for liquid-phase detection of amino acids, including: adjusting the pH value of the sample to be measured to 5-6, and then performing centrifugation; subjecting the supernatant obtained by centrifugation to solid-phase extraction; the solid-phase extraction includes: S1: Activating the solid-phase extraction column: sequentially adding methanol and a second ammonium acetate solution for activation; S2: Loading: adding the supernatant to the solid-phase extraction column; S3: Rinsing: sequentially adding a formic acid aqueous solution and methanol to the solid-phase extraction column; S4: Eluting: eluting with a methanol solution having a pH of 9-13, and then performing nitrogen blowing for reconstitution; The amino acids include one or more of cystine, lysine, ornithine, and arginine.

[0017] Preferably, in S4, the eluent is prepared by adjusting the pH value of methanol to 9-13 with ammonia water having a concentration of 2wt%-8wt%.

[0018] Preferably, the solid-phase extraction column is a cation exchange column; more preferably, the solid-phase extraction column has a silica gel matrix and a bonded benzenesulfonic acid group as the stationary phase; further preferably, the specification of the solid-phase extraction column is 200mg / 3mL.

[0019] Preferably, the pH value of the sample to be measured is adjusted to 5-6 with a first ammonium acetate solution, and then centrifugation is performed.

[0020] Preferably, the concentrations of the first ammonium acetate solution and the second ammonium acetate solution are 0.15-0.25mol / L.

[0021] Preferably, the content of formic acid in the formic acid aqueous solution is 1%-3%.

[0022] Preferably, the flow rate of loading is controlled to be 0.1mL / min-1mL / min. For example, it can be any value among 0.1mL / min, 0.2mL / min, 0.3mL / min, 0.4mL / min, 0.5mL / min, 0.6mL / min, 0.7mL / min, 0.8mL / min, 0.9mL / min, 1mL / min, or a value range with any two of the above values as endpoints.

[0023] Thirdly, the present invention provides a liquid-phase detection method for amino acids, comprising: treating a sample to be detected using the above-mentioned pretreatment method for liquid-phase detection of amino acids, and then performing liquid-phase detection; the chromatographic column for the liquid-phase detection is an amino column.

[0024] More preferably, the chromatographic column is ChromCore HILIC-Amide, and further preferably ChromCore HILIC-Amide 5μm, 2.1×100mm.

[0025] Preferably, the mobile phase for the liquid-phase detection comprises mobile phase A and mobile phase B, mobile phase A is an aqueous solution of perfluorooctanoic acid at 1~10 mmol / L, and mobile phase B is a methanol solution of perfluorooctanoic acid at 1~10 mmol / L or an acetonitrile solution of perfluorooctanoic acid at 1~10 mmol / L.

[0026] Preferably, the gradient elution degree for the liquid-phase detection comprises:

[0027] % represents volume percentage, and the sum of the volume percentages of mobile phase A and mobile phase B is 1.

[0028] Preferably, the gradient elution degree for the liquid-phase detection comprises:

[0029] % represents volume percentage, and the sum of the volume percentages of mobile phase A and mobile phase B is 1.

[0030] Preferably, the liquid-phase detection method for amino acids comprises: Adjusting the pH value of the sample to be detected to 5~6 using a first ammonium acetate solution, and then performing centrifugation; subjecting the supernatant obtained by centrifugation to the following solid-phase extraction treatment, and then performing liquid-phase detection: S1: Activating the solid-phase extraction column: sequentially adding methanol and a second ammonium acetate solution for activation; the solid-phase extraction column is a cation exchange column; S2: Loading: adding the supernatant to the solid-phase extraction column, and controlling the flow rate to be 0.1 mL / min~1 mL / min; S3: Rinsing: sequentially adding a formic acid aqueous solution and methanol to the solid-phase extraction column; S4: Eluting: eluting using an eluent, and then performing nitrogen blowing and reconstitution using a reconstitution solvent; The eluent is prepared by adjusting the pH value of methanol to 9~13 with ammonia water at a concentration of 2wt%~8wt%; the reconstitution solvent is an aqueous methanol solution containing 1~10 mmol / L perfluorooctanoic acid; in the reconstitution solvent, the volume ratio of methanol to water is 1:8~10; The chromatographic column for the liquid phase detection is an amino column; the mobile phase for the liquid phase detection includes mobile phase A and mobile phase B. Mobile phase A is an aqueous solution of perfluorooctanoic acid at 1 - 10 mmol / L, and mobile phase B is a methanol solution of perfluorooctanoic acid at 1 - 10 mmol / L; The gradient elution degree for the liquid phase detection includes:

[0031] % represents volume percentage, and the sum of the volume percentage of mobile phase A and the volume percentage of mobile phase B is 1; The sample to be tested includes one or more of quality control products, standard products, and urine; the amino acids include a mixture of one or more of cystine, lysine, ornithine, and arginine.

[0032] The kit, pretreatment method, and liquid phase detection method for amino acid liquid phase detection provided by the present invention can simultaneously detect four amino acids, namely cystine, lysine, ornithine, and arginine, and have the characteristics of high sensitivity, good repeatability, high accuracy, and good specificity. For this method, the linear range of cystine is 4 - 100 μg / mL, lysine is 5 - 125 μg / mL, ornithine is 2 - 50 μg / mL, and arginine is 2 - 50 μg / mL, and the correlation coefficient r ≥ 0.990; the coefficient of variation (CV) of the repeatability of the low-value quality control product ≤ 15%, and the coefficient of variation (CV) of the repeatability of the high-value quality control product ≤ 15%; the relative deviation (B) of the accuracy ≤ ±15%, and the spiked recovery rate is 85% - 115%. Brief Description of the Drawings

[0033] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0034] Figure 1 It is the liquid chromatogram of cystine, lysine, ornithine, and arginine in Example 1 provided by the present invention.

[0035] Figure 2 It is the liquid chromatogram of cystine, lysine, ornithine, and arginine in Comparative Example 1 provided by the present invention.

[0036] Figure 3 It is the liquid chromatogram of cystine, lysine, ornithine, and arginine in Comparative Example 2 provided by the present invention. Detailed Embodiments

[0037] To make the objectives, technical solutions, and advantages of the present invention clearer, the following will clearly and completely describe the technical solutions in the present invention in conjunction with the accompanying drawings in the present invention. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments in the present invention without making creative efforts fall within the scope of protection of the present invention.

[0038] Unless otherwise specified, all kinds of raw materials used in the examples and comparative examples are commercially available conventional raw materials, and the technical means used are conventional means well-known to those skilled in the art. Since it is difficult to obtain human samples without background, in the following examples, artificial urine is used as a substitute matrix for the human body to prepare each concentration point and quality control of the standard curve. The artificial urine used is purchased from the Addison brand, 500 mL / bottle. In the following examples, the solid-phase extraction plate uses Cleanert SCXPR5006 of Agela.

[0039] Example 1 This example first provides a kit for liquid-phase detection of cystine, lysine, ornithine, and arginine, mainly including the following reagents: Table 1 Main components of the product

[0040] 1. Preparation of the kit and the mobile phase: Preparation of 1 mol / L perfluorooctanoic acid solution: Weigh 3.64 g of perfluorooctanoic acid accurately with an electronic balance, add 10 mL of pure water, prepare 10 mL of 1 mol / L perfluorooctanoic acid, and place it in a refrigerator at 4°C for later use.

[0041] Preparation of 0.2 mol / L ammonium acetate solution: Weigh 1.54 g of ammonium acetate accurately with an electronic balance, add 100 mL of pure water, prepare 100 mL of 0.2 mol / L ammonium acetate, and place it in a refrigerator at 4°C for later use.

[0042] Preparation of mobile phase A solution: Accurately transfer 500 mL of water with a measuring cylinder, add 2.5 mL of 1 mol / L perfluorooctanoic acid solution, and prepare a 5 mmol / L perfluorooctanoic acid solution for later use.

[0043] Preparation of mobile phase B solution: Accurately transfer 500 mL of methanol with a measuring cylinder, add 2.5 mL of 1 mol / L perfluorooctanoic acid solution, and prepare a 5 mmol / L perfluorooctanoic acid solution for later use.

[0044] Preparation of the needle washing solution: Accurately transfer 500 mL of methanol and 500 mL of water, mix them evenly, filter, and perform ultrasonic degassing for later use.

[0045] 2. Preparation of stock solutions of cystine, lysine, ornithine and arginine: Preparation of arginine stock solution: Using an electronic balance with a precision of one in a hundred thousand, accurately weigh 50 mg of arginine, place it in a 10 mL volumetric flask, dissolve it with water and make up to the calibration mark to prepare a stock solution with a concentration of 5 mg / mL. Transfer it to a 15 mL HDPE plastic bottle, label it and store it at -20 °C.

[0046] Preparation of lysine stock solution: Using an electronic balance with a precision of one in a hundred thousand, accurately weigh 50 mg of lysine, place it in a 10 mL volumetric flask, dissolve it with water and make up to the calibration mark to prepare a stock solution with a concentration of 5 mg / mL. Transfer it to a 15 mL HDPE plastic bottle, label it and store it at -20 °C.

[0047] Preparation of ornithine stock solution: Using an electronic balance with a precision of one in a hundred thousand, accurately weigh 50 mg of ornithine, place it in a 10 mL volumetric flask, dissolve it with water and make up to the calibration mark to prepare a stock solution with a concentration of 5 mg / mL. Transfer it to a 15 mL HDPE plastic bottle, label it and store it at -20 °C.

[0048] Preparation of cystine stock solution: Using an electronic balance with a precision of one in a hundred thousand, accurately weigh 50 mg of cystine, place it in a 10 mL volumetric flask, dissolve it with 1 mol / L hydrochloric acid solution and make up to the calibration mark to prepare a stock solution with a concentration of 5 mg / mL. Transfer it to a 15 mL HDPE plastic bottle, label it and store it at -20 °C.

[0049] 3. Preparation of calibrators S1 to S6 The preparation of standards is carried out according to the high-low preparation mode, that is, after preparing S1 and S6, S2 to S5, LQC, MQC and HQC are prepared by proportional mixing.

[0050] Preparation of calibrator S6: Use a pipette with a suitable specification to respectively take 0.72 mL of cystine stock solution, 0.9 mL of lysine stock solution, 0.36 mL of ornithine stock solution, and 0.36 mL of arginine stock solution, place them in a suitable HDPE tube, add 33.66 mL of commercially available artificial urine solution to prepare a standard solution with cystine 100 μg / mL, lysine 125 μg / mL, ornithine 50 μg / mL, and arginine 50 μg / mL. After thorough mixing, label it and store it at -20 °C.

[0051] Preparation of calibrator S1: Accurately pipette 9.6 mL of artificial urine and 0.4 mL of S6 standard, mix them thoroughly to prepare a standard solution with cystine 4 μg / mL, lysine 5 μg / mL, ornithine 2 μg / mL, and arginine 2 μg / mL. After thorough mixing, label it and store it at -20 °C.

[0052] Preparation of calibrator S2: Accurately pipette 9.2 mL of artificial urine and 0.8 mL of S6 standard. Mix well to prepare a standard solution containing 8 μg / mL of cystine, 10 μg / mL of lysine, 4 μg / mL of ornithine, and 4 μg / mL of arginine. After thorough mixing, label it and store at -20°C.

[0053] Preparation of calibrator S3: Accurately pipette 8.4 mL of artificial urine and 1.6 mL of S6 standard. Mix well to prepare a standard solution containing 16 μg / mL of cystine, 20 μg / mL of lysine, 8 μg / mL of ornithine, and 8 μg / mL of arginine. After thorough mixing, label it and store at -20°C.

[0054] Preparation of calibrator S4: Accurately pipette 6.8 mL of artificial urine and 3.2 mL of S6 standard. Mix well to prepare a standard solution containing 32 μg / mL of cystine, 40 μg / mL of lysine, 16 μg / mL of ornithine, and 16 μg / mL of arginine. After thorough mixing, label it and store at -20°C.

[0055] Preparation of calibrator S5: Accurately pipette 4 mL of artificial urine and 6 mL of S6 standard. Mix well to prepare a standard solution containing 60 μg / mL of cystine, 75 μg / mL of lysine, 30 μg / mL of ornithine, and 30 μg / mL of arginine. After thorough mixing, label it and store at -20°C.

[0056] 4. Preparation of quality control samples Preparation of low-concentration quality control sample LQC: Accurately pipette 8.8 mL of artificial urine and 1.2 mL of S6 standard. Mix well to prepare a standard solution containing 12 μg / mL of cystine, 15 μg / mL of lysine, 6 μg / mL of ornithine, and 6 μg / mL of arginine. After thorough mixing, label it and store at -20°C.

[0057] Preparation of medium-concentration quality control sample MQC: Accurately pipette 4.8 mL of artificial urine and 5.2 mL of S6 standard. Mix well to prepare a standard solution containing 52 μg / mL of cystine, 65 μg / mL of lysine, 26 μg / mL of ornithine, and 26 μg / mL of arginine. After thorough mixing, label it and store at -20°C.

[0058] Preparation of high-concentration quality control sample HQC: Accurately pipette 2.8 mL of artificial urine and 7.2 mL of S6 standard. Mix well to prepare a standard solution containing 72 μg / mL of cystine, 90 μg / mL of lysine, 36 μg / mL of ornithine, and 36 μg / mL of arginine. After thorough mixing, label it and store at -20°C.

[0059] This embodiment further provides a method for detecting cystine, lysine, ornithine, and arginine using the above kit, including: Before the test, restore the product and the samples to room temperature.

[0060] Urine dilution: Take 0.2 mL of urine and add 0.2 mL of ammonium acetate solution. After mixing, adjust the pH value of the urine to 5.4. Urine centrifugation: Centrifuge the urine using a high-speed centrifuge at 10000 rmp for 2 minutes. Purification: Use a pipette to accurately transfer 2 mL of methanol into the solid-phase extraction column. Use a positive pressure device to slowly flow the methanol through the extraction column. Then add 2 mL of ammonium acetate solution to the solid-phase extraction column. After the same removal, add 0.2 mL of the diluted sample to be tested (urine, calibrator, quality control sample). Control the flow rate, remove the filtrate, wash the solid-phase extraction column with 1 mL of 2% formic acid and 1 mL of methanol solution respectively, remove the filtrate. Finally, use a mixed solution of 1 mL of 5% ammonia water and 200 μL of methanol solution as the eluent (the pH value of the eluent is 10) to wash down the compound and collect it. After drying with nitrogen blowing, re-dissolve it with 200 μL of the reconstitution solvent and inject it into the instrument. The liquid chromatogram of quality control sample 3 of cystine, lysine, ornithine and arginine is shown in Figure 1 .

[0061] The liquid phase detection conditions include the following method settings: Liquid phase conditions: Chromatographic column: ChromCore HILIC-Amide 5μm, 2.1×100mm or equivalent; Column temperature: 40°C; Injection volume: 10 μL (can be adjusted according to the instrument sensitivity); Sample manager temperature: 10°C; Table 2 Test conditions

[0062] % represents volume percentage.

[0063] Test examples 1. Linear range test data 1.1. Verification method: Process the calibrator solutions S1 - S6 of the product to be tested according to the sample treatment method described for the determination of cystine, lysine, ornithine and arginine in human urine by high performance liquid chromatography-tandem mass spectrometry. Each concentration is tested 3 times. The correlation coefficient r of linear regression can be calculated by referring to the formula, and the correlation coefficient r should be ≥0.990.

[0064] r: Correlation coefficient of linear regression x i : Concentrations of S1 - S6 y i: Mean of the peak area ratio of the calibrator to its internal standard in the corresponding concentration solution 1.2 Acceptance criteria: The correlation coefficient r for the regression of cystine, lysine, ornithine, and arginine is ≥ 0.990.

[0065] 1.3 Experimental results Linear range: The linear range of cystine is 4 - 100 μg / mL, lysine is 5 - 125 μg / mL, ornithine is 2 - 50 μg / mL, and arginine is 2 - 50 μg / mL.

[0066] Table 3 Cystine linear data

[0067] Table 4 Lysine linear data

[0068] Table 5 Ornithine linear

[0069] Table 6 Arginine linear

[0070] 1.4 Conclusion: The correlation coefficient r for cystine, lysine, ornithine, and arginine is ≥ 0.990, meeting the acceptance criteria.

[0071] 2. Repeatability test data 2.1 Verification method: Process the calibrator solutions S1 - S6 and quality control samples of the product to be tested according to the sample processing method described for the determination of cystine, lysine, ornithine, and arginine in human urine by high performance liquid chromatography - tandem mass spectrometry. Use the actual human sample test background as the low value and the sample added with high - concentration standard as the high value. Each sample is measured 5 times and tested in three batches. The coefficient of variation (CV) of repeatability can be calculated using the reference formula, and the CV of the quality control sample ≤ 15%.

[0072] CV = S / × 100% CV: Coefficient of variation of repeatability : Mean of the 5 measurement results S: Standard deviation of the 5 measurement results 2.2 Acceptance criteria: The coefficient of variation CV of the low - value quality control sample ≤ 15%, and the coefficient of variation CV of the high - value quality control sample ≤ 15%.

[0073] Table 7 Intra - batch and inter - batch precision of cystine

[0074] Table 8 Intra - batch and inter - batch precision of lysine

[0075] Table 9 Intra - batch and inter - batch precision of ornithine

[0076] Table 10 Intra - batch and inter - batch precision of arginine

[0077] 2.3. Experimental results 2.4. Conclusion: The coefficient of variation CV of the low - value quality control product ≤ 15%, and the coefficient of variation CV of the high - value quality control product ≤ 15%, meeting the acceptance criteria.

[0078] 3. Accuracy 3.1. Test procedure The standard addition method was adopted. 200 μL of low - and high - concentration accuracy working solution samples (concentrations were: background value + LQC, background value + HQC) were taken for pretreatment respectively; 3 samples were parallel at each concentration level.

[0079] 3.2. Acceptance criteria Recovery rate: 85% - 115%.

[0080] Table 11 Accuracy of cystine

[0081] Table 12 Accuracy of lysine

[0082] Table 13 Accuracy of ornithine

[0083] Table 14 Accuracy of arginine

[0084] 3.3. Summary of the experiment The accuracy meets the requirement within ± 15% of the labeled value.

[0085] Example 2 This example provides a method for liquid - phase detection of cystine, lysine, ornithine and arginine. The difference from Example 1 is only that the eluent is replaced with methanol with a pH value of 13.

[0086] The experimental results show that the detection effects of this example and Example 1 are equivalent.

[0087] Example 3 This embodiment provides a method for liquid-phase detection of cystine, lysine, ornithine and arginine. The difference from Example 1 is only that when diluting urine, ammonium acetate is replaced by borate buffer solution, and the pH value of urine is adjusted to 5-6 with borate buffer solution.

[0088] The results show that the accuracy detection effect of lysine in amino acids is slightly poor, as shown in Table 15: Table 15

[0089] Example 4 This embodiment provides a method for liquid-phase detection of cystine, lysine, ornithine and arginine. The difference from Example 1 is only that methanol in mobile phase B is replaced by acetonitrile.

[0090] The results show that the accuracy detection effect of arginine in amino acids is slightly poor, and the recovery rate of arginine at low point exceeds the standard, as shown in Table 16: Table 16

[0091] Comparative Example 1 This comparative example provides a method for liquid-phase detection of cystine, lysine, ornithine and arginine. The difference from Example 1 is only that the chromatographic column is replaced by a C18 column. Specifically, arginine and ornithine cannot be separated, and only three peaks appear, as Figure 2 shown.

[0092] Comparative Example 2 This comparative example provides a method for liquid-phase detection of cystine, lysine, ornithine and arginine. The difference from Example 1 is only that the eluent is replaced by methanol and the pH value is about 7. The results are as Figure 3 shown. Compared with Figure 1 the response values of arginine, ornithine and cystine decrease to varying degrees.

[0093] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements on some of the technical features. These modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A kit for liquid phase detection of amino acids, characterized in that: include: Protective agent, activating solution, eluent, eluting solution and resolvent; The protective agent includes a first ammonium acetate solution; The activation solution includes methanol and a second ammonium acetate solution; The eluent includes aqueous formic acid solution and methanol; The eluent includes a methanol solution with a pH value of 9 to 13.

2. The kit for liquid phase detection of amino acids according to claim 1, characterized in that: The eluent is prepared by adjusting the pH value of methanol to 9-13 by using aqueous ammonia with a concentration of 2wt%-8wt%; And / or, the concentrations of the first ammonium acetate solution and the second ammonium acetate solution are independently, identically or differently, 0.10 to 0.35 mol / L; And / or, the content of formic acid in the formic acid aqueous solution is 1wt%~3wt%.

3. The kit for liquid phase detection of amino acids according to claim 1 or 2, characterized in that: The complex solvent is a methanol aqueous solution containing 1-10 mmol / L perfluoroheptanoic acid, wherein the volume ratio of methanol to water is 1:8-10; Preferably, the kit further contains a mobile phase; the mobile phase includes a mobile phase A and a mobile phase B, the mobile phase A is a 1-10 mmol / L aqueous solution of perfluoroheptanoic acid, and the mobile phase B is a 1-10 mmol / L methanol solution of perfluoroheptanoic acid or a 1-10 mmol / L acetonitrile solution of perfluoroheptanoic acid; Preferably, the kit further contains amino acid quality control products and / or amino acid standards; the amino acids include one or more of cystine, lysine, ornithine and arginine; Preferably, the kit further comprises a solid phase extraction column, and the solid phase extraction column is a cation exchange column; more preferably, the solid phase extraction column uses silica gel as a matrix and bonded benzenesulfonic acid groups as a stationary phase.

4. A pretreatment method for liquid phase detection of amino acids, characterized in that: include: The pH value of the sample to be tested is adjusted to 5-6, and then centrifuged; The supernatant obtained by centrifugation is subjected to solid phase extraction; The solid phase extraction process comprises: S1: Activation of solid phase extraction column: adding methanol and the second ammonium acetate solution in sequence for activation; S2: loading: adding the supernatant into a solid phase extraction column; S3: Elution: adding formic acid aqueous solution and methanol to the solid phase extraction column in sequence; S4: Elution: Use a methanol solution with a pH of 9 to 13 for elution, and then perform nitrogen blowing for re-dissolution; The amino acids include one or more of cystine, lysine, ornithine and arginine.

5. The pretreatment method for liquid phase detection of amino acids according to claim 4, characterized in that: In S4, the pH value of methanol is adjusted to 9-13 by using aqueous ammonia with a concentration of 2wt%-8wt% to obtain the eluent.

6. The pretreatment method for liquid phase detection of amino acids according to claim 4 or 5, characterized in that: The solid phase extraction column is a cation exchange column; preferably, the solid phase extraction column uses silica gel as a matrix and bonded benzenesulfonic acid groups as a stationary phase.

7. The pretreatment method for liquid phase detection of amino acids according to any one of claims 4 to 6, characterized in that: Using a first ammonium acetate solution, the pH value of the sample to be tested is adjusted to 5-6, and then centrifuged; Preferably, the concentrations of the first ammonium acetate solution and the second ammonium acetate solution are independently, identically or differently, 0.10 to 0.35 mol / L; Preferably, the concentration of the formic acid aqueous solution is 1wt%~3wt%.

8. The pretreatment method for liquid phase detection of amino acids according to any one of claims 4 to 7, characterized in that: In S2, the loading flow rate is controlled to be 0.1 mL / min~1 mL / min.

9. A liquid phase detection method for amino acids, characterized in that: include: The sample to be tested is treated by the pretreatment method for liquid phase detection of amino acids according to any one of claims 4 to 8, and then liquid phase detection is performed; the chromatographic column for liquid phase detection is an amino column; Preferably, the mobile phase of the liquid phase detection comprises a mobile phase A and a mobile phase B, wherein the mobile phase A is a 1-10 mmol / L aqueous solution of perfluoroheptanoic acid, and the mobile phase B is a 1-10 mmol / L methanol solution of perfluoroheptanoic acid or a 1-10 mmol / L acetonitrile solution of perfluoroheptanoic acid; Preferably, the gradient elution degree of the liquid phase detection includes: % represents volume percentage, and the sum of the volume percentage of mobile phase A and the volume percentage of mobile phase B is 1.

10. The liquid phase detection method of amino acids according to claim 9, characterized in that: include: Using a first ammonium acetate solution, the pH value of the sample to be tested is adjusted to 5-6, and then centrifuged; The supernatant obtained by centrifugation was subjected to the following solid phase extraction treatment and then to liquid phase detection: S1: Activate the solid phase extraction column: add methanol and the second ammonium acetate solution in sequence for activation; the solid phase extraction column is a cation exchange column; S2: Loading: Add the supernatant to the solid phase extraction column and control the flow rate to 0.1 mL / min~1 mL / min; S3: Elution: adding formic acid aqueous solution and methanol to the solid phase extraction column in sequence; S4: Elution: use an eluent for elution, and then use a resolvent for nitrogen blowing for re-dissolution; The eluent is prepared by adjusting the pH value of methanol to 9-13 with 2wt%-8wt% ammonia water; the resolvent is a methanol aqueous solution containing 1-10mmol / L perfluoroheptanoic acid; in the resolvent, the volume ratio of methanol to water is 1:8-10; The chromatographic column for liquid phase detection is an amino column; the mobile phase for liquid phase detection includes mobile phase A and mobile phase B, the mobile phase A is a 1-10 mmol / L aqueous solution of perfluoroheptanoic acid, and the mobile phase B is a 1-10 mmol / L methanol solution of perfluoroheptanoic acid; The gradient elution degree of the liquid phase detection includes: % represents volume percentage, and the sum of the volume percentage of mobile phase A and the volume percentage of mobile phase B is 1; The sample to be tested includes one or more of a quality control product, a standard product and urine; the amino acid includes one or a mixture of cystine, lysine, ornithine and arginine.

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