Method for detecting content of amantadine in water body

Abstract

The invention provides a method for detecting the content of amantadine in a water body. The method is a liquid chromatography-tandem mass spectrometry method for detecting the content of amantadine in a water body (including seawater and fresh water), and aims at providing a rapid, sensitive and accurate detection means to meet the requirement for safety monitoring of a water body environment.

Description

Technical Field

[0001] The invention belongs to the technical field of water body detection, and particularly relates to a method for detecting the content of amantadine in water. Background Art

[0002] Amantadine is the earliest antiviral drug used to inhibit influenza virus. It is contained in many cold medicines. It is also an anti-tremor paralysis drug and has a good effect on Parkinson's disease, but it also has great side effects on the human body. It may cause adverse reactions such as nausea, vomiting, and dizziness, which can easily increase the burden on the patient's kidneys and affect the normal metabolism of the kidneys. Recent food safety testing and environmental monitoring have found that aquatic shellfish and environmental water quality (including seawater and freshwater) are contaminated with residual amantadine.

[0003] Judging from the existing detection method standards, the detection method standard for adamantane in water bodies is "DB37 / T4299-2020 Determination of adamantane in seawater by liquid chromatography tandem mass spectrometry", but it was found during the experiment that this detection method standard has defects such as too large sample volume, insufficient capacity for direct solid phase extraction column, and long column time.

[0004] At present, there is no national standard for the determination of amantadine residues in environmental water bodies (including seawater and fresh water) in my country. In order to investigate the pollution degree of amantadine in environmental water quality and accurately monitor the content of amantadine in environmental water bodies, it is of great significance to study the pollution of amantadine residues in environmental water bodies (including seawater and fresh water). Summary of the invention

[0005] In order to solve the problems and shortcomings in the prior art, the present invention provides a method for detecting the content of amantadine in water. The method is a liquid chromatography tandem mass spectrometry method for detecting the content of amantadine in water (including seawater and fresh water), aiming to provide a rapid, sensitive and accurate detection means to meet the needs of water environment safety monitoring.

[0006] The invention provides a method for detecting the content of amantadine in water, comprising the following steps: S1. water sample collection and filtration: collecting a water sample to be tested, filtering to remove suspended matter and impurities in the water sample to be tested, and obtaining a clear water sample to be tested; S2. liquid-liquid extraction: adding a first amantadine internal standard working liquid to the clear water sample to be tested and mixing evenly, then adding an organic solvent thereto to perform a liquid-liquid extraction operation, obtaining two completely separated phases, wherein the upper layer is a water phase and the lower layer is an organic phase, and collecting the organic phase; the organic solvent comprises at least one of dichloromethane and chloroform; in the first amantadine internal standard working liquid, the internal standard comprises D 15-adamantanamine; S3. continue to add organic solvent to the remaining aqueous phase, perform liquid-liquid extraction operation 1 to 2 times, combine the organic phases to obtain an organic extract; S4. remove the solvent in the organic extract to obtain a residue, add a complex solution to the residue to re-dissolve it, filter it, and then inject it into a liquid chromatography-tandem mass spectrometer for detection, and substitute the detection result into the standard curve to determine the content of adamantane in the water sample to be tested.

[0007] At present, recent food safety testing and environmental monitoring have found that aquatic products, shellfish and environmental water (including seawater and freshwater) are contaminated with residual amantadine. Therefore, how to further accurately detect the content of amantadine in environmental water is of great significance for investigating amantadine contamination in environmental water and improving water quality.

[0008] Environmental water bodies (including seawater and fresh water) generally have complex components. Therefore, if the conventional external standard method is used for testing, the test results may be inaccurate, and the accuracy of the external standard method depends on the accuracy and repeatability of the standard curve, which is greatly affected by the systematic error. The difference between the internal standard method and the external standard method is that the internal standard method: a known amount of internal standard substance is added to the sample to be tested, and the content of the component to be tested is calculated by comparing the peak area or peak height of the component to be tested with the internal standard substance, combined with the known concentration of the internal standard substance and the correction factor. The internal standard method is a relative quantitative method. By adding an internal standard substance, errors caused by injection volume, operating conditions, etc. can be corrected. External standard method: using a standard substance of known concentration, by drawing a standard curve (the relationship between peak area or peak height and concentration), according to the peak area or peak height of the sample to be tested, the content of the component to be tested is directly read from the standard curve. The external standard method is an absolute quantitative method, and its accuracy depends on the accuracy and repeatability of the standard curve.

[0009] Therefore, the present invention uses the internal standard method to detect the content of amantadine in water, which can effectively avoid the influence of other complex components in the environmental water on the detection of the content of amantadine, reduce the system error or the error caused by the injection amount, operating conditions, etc., and effectively improve the detection accuracy and sensitivity of the content of amantadine in water. Specifically, the internal standard substance used in the internal standard method provided by the present invention is also amantadine (D 15 -adamantanamine), so it can have the same solubility as the residual adamantane in the water body and can stably exist in water or other solvents. In addition, the original water body to be tested generally does not contain D 15 -Adamantane, which has a similar retention time to adamantane (analyte) commonly found in water, is a high-purity standard substance with a certain chemical stability under given chromatographic conditions. 15-Adamantane amine, as an internal standard substance, can effectively eliminate the influence of sample volume, mobile phase, detector, etc., greatly improving the accuracy and sensitivity of detecting adamantane amine in water.

[0010] Secondly, the present invention also uses a liquid-liquid extraction method to extract adamantane in the water body to be tested. The organic solvent used includes dichloromethane or chloroform. Dichloromethane and chloroform can more fully extract adamantane in the water body, improve the detection accuracy and sensitivity, and at the same time avoid introducing too many impurities in the water body to affect the detection results.

[0011] Therefore, in the method for detecting the content of adamantane in water provided by the present invention, by adopting a more suitable internal standard substance and extraction solvent, the accuracy and sensitivity of detecting the content of adamantane in water are effectively improved, that is, the method has a lower detection limit and quantitative limit, and has a higher recovery rate and better precision.

[0012] Preferably, in S2, the organic solvent includes dichloromethane. Compared with chloroform, the boiling point of dichloromethane is 39.8°C (the boiling point of chloroform is 61.3°C), the rotary evaporation time is fast, and the experimental efficiency is high. And chloroform is too toxic, which is not conducive to the personal safety of the test personnel during the experiment. Taking all factors into consideration, it is more ideal for this method to use dichloromethane as the extraction solution of adamantane in water.

[0013] Preferably, in S2, the volume ratio of the clear water sample to be tested to the first adamantane amine internal standard solution is 900-1100:1.

[0014] Preferably, in S2, the volume ratio of the clear water sample to be tested to the first adamantane amine internal standard solution is 1000:1.

[0015] Preferably, in S2, the concentration of the first amantadine internal standard solution is 90 to 110 ng / mL.

[0016] Preferably, in S2, the concentration of the first amantadine internal standard solution is 100 ng / mL.

[0017] Preferably, in S2, the volume ratio of the clear water sample to be tested to the organic solvent is 1.5-2.5:1.

[0018] Preferably, in S2, the volume ratio of the clear water sample to be tested to the organic solvent is 2:1.

[0019] Preferably, in S2, during the liquid-liquid extraction, after the extraction container is sealed, it is vigorously shaken for 4 to 8 minutes and then allowed to stand for 25 to 35 minutes.

[0020] Preferably, in S4, the volume of the reconstituted solution is 0.5 to 3 mL.

[0021] Preferably, in S4, the volume of the reconstituted solution is 1 mL.

[0022] Preferably, in S4, the re-solution includes acetonitrile-water solution or acetonitrile-methanol-water solution. The use of acetonitrile-water solution or acetonitrile-methanol-water solution can well re-dissolve the extracted amantadine, and can reduce the influence of the solvent on the detection result during the liquid chromatography tandem mass spectrometry analysis, improve the chromatographic response intensity of amantadine detection, and thus improve the detection accuracy and sensitivity.

[0023] Preferably, in S4, when the reconstituted solution is an acetonitrile-water solution, the volume ratio of acetonitrile to water is 4.5-5.5:4.5-5.5; when the reconstituted solution is an acetonitrile-methanol-water solution, the volume ratio of acetonitrile, methanol and water is 6-8:0.5-1.5:1-3. When the reconstituted solution is a different combination of solvents and is within the above volume ratio range, the chromatographic response of adamantane is higher, which is more conducive to improving the detection accuracy and sensitivity.

[0024] Preferably, in S4, the reconstituted solution is an acetonitrile-methanol-water solution, and the volume ratio of acetonitrile, methanol and water is 7:1:2.

[0025] Preferably, in S4, in the liquid chromatography analysis test, the eluent includes mobile phase A and mobile phase B, mobile phase A is 0.08-0.12% formic acid aqueous solution, and mobile phase B is acetonitrile; the flow rate of the eluent is 0.25-0.35 mL / min; and the elution process adopts gradient elution. The combination of the above mobile phase A and mobile phase B and the gradient elution method can improve the separation degree, improve the peak shape, and improve the detection sensitivity.

[0026] Preferably, in the liquid chromatography analysis test, the mobile phase is A: 0.1% formic acid aqueous solution, B: acetonitrile; the gradient elution conditions are as follows:

[0027]

[0028] Preferably, in the liquid chromatography analysis test, the column temperature is 24-26° C.; and the injection volume is 4.5-5.5 μL.

[0029] Preferably, the chromatographic column model is Zorbax SB-C18; the chromatographic column specification is 2.1 mm×50 mm.

[0030] Preferably, in S4, the conditions of mass spectrometry are as follows:

[0031] Ion source: AJS ESI ionization mode, positive ion scanning; detection mode: multiple reaction ion monitoring; capillary voltage positive ion 4450~4550V; drying gas temperature: 340~360℃; drying gas flow rate: 4.5~5.5L / min; nebulizing gas pressure: 44~46psi; sheath gas temperature: 290~310℃; sheath gas flow rate: 10.5~11.5L / min; nozzle voltage 495~505V.

[0032] Preferably, in S4, the standard curve is Y=a×C+b, and the correlation coefficient is not less than 0.99; wherein a is a constant, and its value range is 5.9 to 6.1; b is a constant, and its value range is -0.21 to -0.18; in the process of drawing the standard curve, the measured target peak area and the internal standard peak area ratio are used as the ordinate Y, and the corresponding standard working solution concentration is used as the abscissa X.

[0033] Preferably, the concentration of the standard working solution of the standard curve is 0.5 ng / mL, 1.0 ng / mL, 2.0 ng / mL, 5.0 ng / mL, 10 ng / mL, and 20 ng / mL.

[0034] Preferably, the process of preparing the standard curve comprises the following steps: step 1, mixing the amantadine standard working solution, the second amantadine internal standard working solution and the second reconstitution solution to prepare standard working solutions in which the amantadine standard concentrations are 0.5 ng / mL, 1.0 ng / mL, 2.0 ng / mL, 5.0 ng / mL, 10 ng / mL and 20 ng / mL respectively, and the amantadine internal standard concentration is 10 ng / mL; step 2, drawing the standard curve with the measured target peak area and the internal standard peak area ratio as the ordinate Y and the corresponding standard working solution concentration as the abscissa C; the second reconstitution solution is consistent with the first reconstitution solution.

[0035] Preferably, the content of amantadine in the water sample to be tested is calculated according to the following formula:

[0036]

[0037] Where:

[0038] X——the content of the measured component amantadine in the sample (corrected content), in ng / L;

[0039] C——Concentration of the analyte in the sample solution, in ng / mL;

[0040] C i ——Concentration of internal standard in the sample solution, in ng / mL;

[0041] C si ——Concentration of internal standard in standard solution, in ng / mL;

[0042] A——peak area of ​​the analyte in the sample solution;

[0043] A i ——Peak area of ​​internal standard in sample solution;

[0044] A s ——Peak area of ​​the analyte in the standard solution;

[0045] A si ——Peak area of ​​internal standard in standard solution;

[0046] V1——the fixed volume of the sample solution, in mL;

[0047] V2——The volume of the test sample, in L.

[0048] Above The formula is a calibration curve, that is, the final test result of the adamantane content of the water sample to be tested is further calibrated. Due to the internal standard method adopted in the present invention, after the curve calibration, the obtained test result is more accurate, that is, the accuracy and sensitivity of the detection are improved.

[0049] In summary, the method for detecting the content of amantadine in water provided by the present invention can improve the accuracy and sensitivity of detecting amantadine in water, and the detection method is fast and easy to operate. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 This is the LC-MS / MS total ion chromatogram of the amantadine standard solution.

[0051] Figure 2 This is the standard curve of amantadine.

[0052] Figure 3 This is the mass spectrum of adamantane compound.

[0053] Figure 4 This is a diagram showing the effect of liquid-liquid extraction in Example 1.

[0054] Figure 5 This is the chromatogram of the petroleum ether extraction result in Example 2.

[0055] Figure 6 The chromatogram is the dichloromethane extraction result in Example 2.

[0056] Figure 7 The chromatogram is the chloroform extraction result in Example 2.

[0057] Figure 8 The chromatographic response of the parallel standard samples with acetonitrile-water solution (volume ratio of 5:5) as the reconstitution solution.

[0058] Fig. 9 It is the chromatographic response of parallel standard samples with acetonitrile-methanol-water solution (volume ratio of 5:1:4) as the re-solution.

[0059] Fig.10 It is the chromatographic response of parallel standard samples with acetonitrile-methanol-water solution (volume ratio of 7:1:2) as the re-solution. DETAILED DESCRIPTION

[0060] In order to enable those skilled in the art to better understand the solutions of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only embodiments of a part of the present invention, rather than all embodiments.

[0061] The reagents and instruments used in the method of the present invention are specifically as follows:

[0062] 1. Amantadine standards were purchased from Beijing Zhenxiang Co., Ltd. with the following specifications: amantadine 100 μg / mL, 1.0 mL; D15-amantadine 1000 μg / mL, 1.0 mL.

[0063] 2. Acetonitrile (chromatographically pure, Wokai, Sinopharm Chemical Reagent Co., Ltd.); dichloromethane (chromatographically pure, Wokai, Sinopharm Chemical Reagent Co., Ltd.); methanol (chromatographically pure, Wokai, Sinopharm Chemical Reagent Co., Ltd.).

[0064] 3. Two-dimensional ultra-high performance liquid chromatography / triple tandem quadrupole mass spectrometer (Agilent 2D-LC 6470, Agilent, USA);

[0065] 4. Rotary evaporator (Hei-VAP Precision model, heidolph, Germany).

[0066] 5. Vortex shaker (MS3 basic, IKA, Germany).

[0067] 6. Filtration mechanical pump (Tianjin Jinteng).

[0068] Example 1

[0069] Follow the steps below to test the amantadine content in seawater:

[0070] S1. The water sample to be tested is filtered through a 0.45 μm water phase filter membrane to remove suspended matter and impurities therein to obtain a clear water sample to be tested;

[0071] S2. Accurately measure 100.0 mL (accurate to 0.1 mL) of the clear water sample after filtration and place it in a 250 mL separatory funnel; pay attention to anti-pollution measures during the operation; add 100 μL of adamantane internal standard solution (internal standard is D 15 -Amantadine, 100 ng / mL), ensure sufficient mixing for subsequent quantitative calibration;

[0072] Add 50 mL of dichloromethane to the separatory funnel, seal it and shake it vigorously for 5 minutes to allow the two phases to fully contact (liquid-liquid extraction method); let it stand for 30 minutes until the two phases are completely separated (reference Figure 4 , which is a liquid-liquid extraction effect diagram), carefully transfer the lower dichloromethane layer to a 250mL chicken heart bottle;

[0073] S3. Repeat the above extraction step once, that is, add 50 mL of dichloromethane to the original separatory funnel again, shake, let stand and collect the lower dichloromethane layer;

[0074] S4. Place the combined dichloromethane extracts on a rotary evaporator and evaporate them at 45°C until they are nearly dry; add 1.0 mL of a reconstituted solution (acetonitrile-methanol-water solution, volume ratio of 7:1:2) to the residue and vortex mix to completely dissolve the residue; filter the reconstituted solution through a 0.22 μm microporous filter membrane (organic phase) for liquid chromatography-tandem mass spectrometry analysis; substitute the test results into the standard curve to calculate the content of adamantane in the water sample to be tested.

[0075] (1) The conditions of the above-mentioned liquid chromatography tandem mass spectrometry analysis test are as follows:

[0076] Liquid chromatography conditions: Column model: Zorbax SB-C18 Column specification: 2.1mm×50mm1.8μm; Column temperature: 25℃; Injection volume: 5μL; Eluent flow rate: 0.3mL / min; Mobile phase A: 0.1% formic acid aqueous solution, Mobile phase B: acetonitrile; Gradient elution. The specific conditions of gradient elution are as follows:

[0077]

[0078] Mass spectrometry reference conditions: ion source: AJS ESI ionization mode, positive ion scanning; detection mode: multiple reaction ion monitoring; capillary voltage positive ion 4500V; drying gas temperature: 350℃; drying gas flow rate: 5L / min; nebulizer gas pressure: 45psi; sheath gas temperature: 300℃; sheath gas flow rate: 11L / min; nozzle voltage 500V. The qualitative and quantitative ions are shown in Table 1. The retention time of the monitoring ions determined with adamantane is shown in Table 1.

[0079] Table 1 Retention time, detection limit, monitoring ions and collision energy of the analytes

[0080]

[0081] *Quantitative ion

[0082] And you can refer to Figure 3 , Figure 3 This is the mass spectrum of adamantane compound.

[0083] (2) The preparation of the standard curve is as follows:

[0084] Step 1: Accurately measure an appropriate amount of amantadine standard working solution (1.0 μg / mL) and amantadine internal standard working solution (1.0 μg / mL) in a 10 mL volumetric flask, and dilute to volume with a reconstitution solution (acetonitrile: methanol: water = 7:1:2, volume ratio) to prepare standard working solutions with concentrations of 0.5 ng / mL, 1.0 ng / mL, 2.0 ng / mL, 5.0 ng / mL, 10 ng / mL, and 20 ng / mL, with an amantadine internal standard concentration of 10 ng / mL; Reference Figure 1 , which is a LC-MS / MS total ion current chromatogram of the amantadine standard solution;

[0085] Step 2: Draw a standard curve with the ratio of the target peak area to the internal standard peak area as the ordinate Y and the corresponding standard working solution concentration as the abscissa X. The obtained standard curve regression equation is Y = 6.007307X-0.19482, with a correlation coefficient of r = 0.9995. Figure 2 This indicates that this method has a good linear relationship in the linear range of 5.0 ng / L to 200.0 ng / L.

[0086] Regarding the calculation formula and data processing, this method uses the internal standard method to calibrate multiple points to quantitatively calculate the content of adamantane in the sample (ng / L). The blank value must be deducted from the calculation result, and the measurement result is expressed as the arithmetic mean of two parallel measurements, retaining three significant figures. The calculation formula is:

[0087]

[0088] Where:

[0089] X——the content of the measured component amantadine in the sample (corrected content), in ng / L;

[0090] C——Concentration of the analyte in the sample solution, in ng / mL;

[0091] C i ——Concentration of internal standard in the sample solution, in ng / mL;

[0092] C si——Concentration of internal standard in standard solution, in ng / mL;

[0093] A——peak area of ​​the analyte in the sample solution;

[0094] A i ——Peak area of ​​internal standard in sample solution;

[0095] A s ——Peak area of ​​the analyte in the standard solution;

[0096] A si ——Peak area of ​​internal standard in standard solution;

[0097] V1——the fixed volume of the sample solution, in mL;

[0098] V2——The volume of the test sample, in L.

[0099] (3) Regarding the method for detecting the content of amantadine in water provided in this embodiment, the sensitivity, accuracy and precision thereof were also tested. The test results are as follows:

[0100] ①Sensitivity:

[0101] This method pre-processed the samples and put them on the instrument. The detection limit and quantification limit of the method were determined by 3 times and 10 times the signal-to-noise ratio of the target response. The detection limit was 2.5ng / L and the quantification limit was 5.0ng / L. It is better than DB37 / T 4299-2020.

[0102] ②Accuracy:

[0103] When the concentration of the adamantane amine spiked solution in water is 5.0 ng / L to 200.0 ng / L, the recovery rate is 90.1% to 105%, and both the linear range and the recovery rate are better than those of DB37 / T 4299-2020.

[0104] Further, this embodiment uses 5 types of water to verify the actual application of the method, and the recovery rate is used as a verification index. Take 5 different types of water, and determine the background content of amantadine in the above 5 products according to the established method. At the same time, 3 groups of parallel spiked tests are performed on the 5 products, wherein the concentration of amantadine in the spiked solution is 20 ng / L. The experimental results are shown in Table 2. The data in Table 2 show that the average recovery rate is between 95.2% and 99.2%, so the method established by the present invention has good accuracy, and the method can also well meet the requirements for determining the content of amantadine in water.

[0105] Table 2 Spiked test data of 5 different types of water

[0106]

[0107]

[0108] ③Precision:

[0109] Furthermore, the precision experiment of this method was carried out at three levels: method quantification limit, twice the method quantification limit and ten times the method quantification limit. A water sample with a background content of 0.000 ng / L was selected, and spiked experiments were carried out at three levels: 1 times the quantification limit (5.00 ng / L), 5 times the quantification limit (25.00 ng / L) and 10 times the quantification limit (50.00 ng / L). Each level was tested in parallel 6 times to calculate the recovery rate and relative standard deviation. The experimental results are shown in Table 3.

[0110] Table 3 Recovery and relative standard deviation of amantadine (n=6)

[0111]

[0112]

[0113] As shown in Table 3, when the amount of amantadine added is 1 times the quantitative limit, the recovery rate is 95.4% to 99.9%, and the relative standard deviation is 2.0%; when the amount of amantadine added is 5 times the quantitative limit, the recovery rate is 94.4% to 99.9%, and the relative standard deviation is 2.3%; when the amount of amantadine added is 10 times the quantitative limit, the recovery rate is 91.1% to 98.7%, and the relative standard deviation is 2.7%. It can be seen that when the amount of amantadine added is 1, 5, and 10 times, there are good recovery rates and relative standard deviations. Therefore, the method for determining the content of amantadine in water provided by the present invention has high accuracy.

[0114] It should be noted here that when determining and analyzing the adamantane content in the water sample to be tested, qualitative analysis is generally performed first and then quantitative analysis.

[0115] Qualitative analysis: Determine the sample and establish a standard working curve according to the above-mentioned liquid chromatography and mass spectrometry (liquid chromatography tandem mass spectrometry) conditions. If the retention time of the mass chromatographic peak of the compound in the sample is within the allowable range of ±2.5% compared with the standard solution; the relative abundance of the qualitative ion pair of the compound to be tested is compared with the standard solution of equivalent concentration, and the relative abundance deviation does not exceed the provisions of Table 1, then it can be determined that the corresponding target compound exists in the sample.

[0116] Table 4 The allowable deviation of relative ion abundance in qualitative confirmation is in percentage.

[0117] Relative ion abundance >50 >20～≤50 >10～≤20 ≤10 Permissible relative deviation ±20 ±25 ±30 ±50

[0118] Quantitative analysis: Quantitative calculations were performed according to the methods provided above.

[0119] Example 2: Selection of extraction solution

[0120] When investigating the extraction strategy of adamantane in water, the extraction efficiency of three different solvents, petroleum ether, dichloromethane and chloroform, was systematically evaluated. Figures 5 to 7 , which are the liquid chromatography-tandem mass spectrometry test results under different solvent extractions of petroleum ether, dichloromethane and chloroform, respectively. The liquid chromatography-mass spectrometry conditions refer to Example 1.

[0121] The experimental results show that: petroleum ether as an extractant has an unstable chromatographic peak baseline, large noise, low response and poor qualitative analysis (see Figure 5 ), which is not suitable for the determination of adamantane in water. Using dichloromethane as the extractant, a good chromatographic response can be obtained, and the quantitative and qualitative results are accurate (see Figure 6 ), with a high recovery rate. In experimental operation, the boiling point of dichloromethane is 39.8°C, the rotary evaporation time is fast, and the experimental efficiency is high. When chloroform is used as the extraction solvent, a good chromatographic response can also be obtained for the target adamantane amine in water (see Figure 7 ). However, due to its high toxicity, it is not conducive to the personal safety of the testers during the experiment; secondly, the boiling point of chloroform is 61.3℃, and the rotary evaporation time in sample pretreatment is longer than that of dichloromethane, which increases the working time of the experiment. Comprehensive consideration, it is more ideal to use dichloromethane as the extraction solution of adamantane in water in this method.

[0122] Example 3: Optimization of reconstitution solution

[0123] In order to optimize the solvent ratio of the reconstitution solution, the present invention compares the experimental effects of three reconstitution solutions of 1. acetonitrile-water solution (volume ratio of 5:5), 2. acetonitrile-methanol-water solution (volume ratio of 5:1:4), and 3. acetonitrile-methanol-water solution (volume ratio of 7:1:2) V / V / V:7 / 1 / 2 in actual sample seawater spiked with 10 ng / mL (here refers to the concentration of the standard solution of amantadine). For details, see Figures 8 to 10 .

[0124] The results showed that when acetonitrile-methanol-water solution (volume ratio 7:1:2) was used as the resolubilization solution, the average response peak area of ​​the target was 271723.5, which was 340.4% and 36.3% higher than that of acetonitrile-methanol-water solution (volume ratio 5:1:4) and acetonitrile-water solution (volume ratio 5:5) respectively; secondly, when acetonitrile-methanol-water solution (volume ratio 7:1:2) was used as the resolubilization solution, there was no matrix interference peak near the target, and the relative deviation of parallel sample measurement at this spiked concentration was 5.5%, which met the experimental requirements. Therefore, acetonitrile-methanol-water solution (volume ratio 7:1:2) was selected as the optimal resolubilization solution.

[0125] Comparative Example 1: Determination of Amantadine by DB37 / T 4299-2020

[0126] First, according to DB37 / T 4299-2020 "Determination of adamantane in seawater by liquid chromatography-tandem mass spectrometry", 500 mL of seawater was accurately measured, 100 μL of internal standard solution and 10 mL of 0.2 mol / L hydrochloric acid were added, and an activated MCX column (5 mL of methanol and 5 mL of water for activation) was used, rinsed with 5 mL of water, and eluted with 10 mL of 5% ammonia methanol. The eluate was collected and blown to near dryness at 40°C with a nitrogen blower, and the volume was made up to 1 mL with a constant volume solution for liquid chromatography-tandem mass spectrometry analysis.

[0127] The results showed that adamantane was determined using the DB37 / T 4299-2020 method and enriched and purified using a solid phase extraction column (MCX column 60 mg / 3 mL). However, due to the large volume of seawater samples passing through the column, enrichment and purification took a long time, and the filler of the solid phase extraction column was limited. If the prepared water sample matrix was complex, the enrichment and purification effect was not good, and the extraction recovery rate of the analyte was poor.

[0128] The above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the protection scope of the present invention. Although the present invention is described in detail with reference to the above embodiments, ordinary technicians in the relevant field should understand that the technical solutions of the present invention can be modified or equivalently replaced, but these modifications or replacements are all within the protection scope of the present invention.

Claims

1. A method for detecting the content of amantadine in water, characterized in that: The steps include: S1. Water sample collection and filtration: Collect the water sample to be tested, filter it to remove suspended matter and impurities in the water sample to be tested, and obtain a clear water sample to be tested; S2. Liquid-liquid extraction: adding the first adamantane internal standard solution to the clear water sample to be tested and mixing evenly, then adding an organic solvent thereto to perform a liquid-liquid extraction operation to obtain two completely separated phases, the upper layer being an aqueous phase and the lower layer being an organic phase, and collecting the organic phase; the organic solvent comprises at least one of dichloromethane and chloroform; the internal standard substance in the first adamantane internal standard solution comprises D 15 -Amantadine; S3. continuing to add the organic solvent to the remaining aqueous phase, and then performing liquid-liquid extraction operation 1 to 2 times, combining the organic phases to obtain an organic extract; S4. removing the solvent from the organic extract to obtain a residue, adding a re-solution to the residue to redissolve it, filtering it, and then injecting it into a liquid chromatography-tandem mass spectrometer for detection, and substituting the detection result into a standard curve to determine the content of adamantane in the water sample to be tested.

2. The method for detecting the content of amantadine in water according to claim 1, characterized in that: In S4, the complex solution includes acetonitrile-water solution and acetonitrile-methanol-water solution.

3. The method for detecting the content of amantadine in water according to claim 2, characterized in that: In S4, when the reconstituted solution is the acetonitrile-water solution, the volume ratio of acetonitrile to water is 4.5-5.5:4.5-5.5; when the reconstituted solution is the acetonitrile-methanol-water solution, the volume ratio of acetonitrile, methanol and water is 6-8: 0.5～1.5：1～3。 4. The method for detecting the content of amantadine in water according to claim 3, characterized in that: In the S4, the reconstituted solution is the acetonitrile-methanol-water solution, and the volume ratio of acetonitrile, methanol and water is 7:1:

2.

5. The method for detecting the content of amantadine in water according to claim 1, characterized in that: In S4, in the liquid chromatography analysis test, the eluent includes mobile phase A and mobile phase B, mobile phase A is 0.08-0.12% formic acid aqueous solution, and mobile phase B is acetonitrile; the flow rate of the eluent is 0.25-0.35 mL / min; and the elution process adopts gradient elution.

6. The method for detecting the content of amantadine in water according to claim 5, characterized in that: In the liquid chromatography analysis test, the mobile phase is A: 0.1% formic acid aqueous solution, B: acetonitrile; the gradient elution conditions are as follows:

7. The method for detecting the content of amantadine in water according to claim 1, characterized in that: In said S4, the conditions of mass spectrometry are as follows: Ion source: AJS ESI ionization mode, positive ion scanning; Detection mode: multiple reaction ion monitoring; Capillary voltage positive ion 4450~4550V; Drying gas temperature: 340~360℃; Drying gas flow rate: 4.5~5.5L / min; Nebulizer gas pressure: 44~46psi; Sheath gas temperature: 290~310℃; Sheath gas flow rate: 10.5~11.5L / min; nozzle voltage 495~505V.

8. The method for detecting the content of amantadine in water according to claim 1, characterized in that: In S4, the standard curve is Y=a×C+b, and the correlation coefficient is not less than 0.99; wherein a is a constant, and its value range is 5.9 to 6.1; b is a constant, and its value range is -0.21 to -0.18; In the process of drawing the standard curve, the ratio of the measured target peak area to the internal standard peak area is used as the ordinate Y, and the corresponding standard working solution concentration is used as the abscissa C.

9. The method for detecting the content of amantadine in water according to claim 8, characterized in that: The process of making the standard curve comprises the following steps: Step 1: Mix the amantadine standard working solution, the second amantadine internal standard working solution and the second reconstituted solution to prepare standard working solutions with amantadine standard concentrations of 0.5 ng / mL, 1.0 ng / mL, 2.0 ng / mL, 5.0 ng / mL, 10 ng / mL and 20 ng / mL, respectively, and with the amantadine internal standard concentration of 10 ng / mL; Step 2, plotting the standard curve with the ratio of the measured target peak area to the internal standard peak area as the ordinate Y and the corresponding standard working solution concentration as the abscissa C; The second reconstitution solution is consistent with the first reconstitution solution.

10. The method for detecting the content of amantadine in water according to claim 9, characterized in that: The content of amantadine in the water sample to be tested is calculated according to the following formula: Where: X——the content of the tested component amantadine in the sample, in ng / L; C——Concentration of the analyte in the sample solution, in ng / mL; C i ——Concentration of internal standard in the sample solution, in ng / mL; C si ——Concentration of internal standard in standard solution, in ng / mL; A——peak area of ​​the analyte in the sample solution; A i ——Peak area of ​​internal standard in sample solution; A s ——Peak area of ​​the analyte in the standard solution; A si ——Peak area of ​​internal standard in standard solution; V1——the fixed volume of the sample solution, in mL; V2——The volume of the test sample, in L.

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