Liquid quality method for rapidly detecting phytic acid and degradation products thereof in underground water

Abstract

The invention discloses a liquid chromatography-mass spectrometry method for rapidly detecting phytic acid and degradation products thereof in underground water, which specifically comprises the following steps: preparing standard substance solutions of phytic acid and degradation products thereof with different concentrations, obtaining mass spectrum parameters in a mass spectrum direct sample injection mode, combining optimized chromatographic conditions, and performing sample injection detection by using a liquid chromatography-mass spectrometer. Obtaining chromatograms of phytic acids with different concentrations and degradation products thereof, and drawing a standard curve according to the chromatograms; the method comprises the following steps: pre-treating a to-be-detected sample by using a silver-hydrogen composite column, passing the treated sample through a selected filter membrane, and carrying out sample injection detection by using a liquid chromatography-mass spectrometer; substituting the detection result into the standard curve of the phytic acid and the degradation product of the phytic acid, and calculating to obtain the concentration of the sample to be detected. The method can meet the detection of phytic acid and degradation products thereof in related fields.

Description

Technical Field

[0001] The invention relates to the field of liquid quality methods for phytic acid and its degradation products, and in particular to a liquid quality method for rapidly detecting phytic acid and its degradation products in groundwater. Background Art

[0002] Phytic acid is the main organophosphorus, also known as inositol hexaphosphate or hexaphosphate of cyclohexanehexol (InsP6). Its structure is an inositol derivative formed by the phosphate esterification reaction of the six hydroxyl groups of inositol, containing six phosphate groups and 12 dissociable hydrogen atoms. Under certain conditions, phytic acid can be degraded into low-grade inositol phosphates or inositol. Studies have shown that most degradation products play a certain role in the cell signal transduction process and metabolic activities in the body, and some have been found to have anti-cancer and heart regulation functions in clinical medicine.

[0003] Phytic acid has strong chelating properties and can combine with some mineral elements and proteins to form water-insoluble phytates. Phytates in soil account for about 20% to 50% of soil organic phosphorus, and the accumulation of phytates in soil depends largely on the climate environment and soil type. In the groundwater environment, phytic acid can interact with iron and aluminum (hydroxides), clay minerals, etc. The strength and degree of its combination with minerals also affect the release of orthophosphate. When the degradation products of phytic acid under certain conditions are discharged into the environment, they will become an important source of phosphorus release. Different inositol phosphates produce different numbers of phosphate groups, causing different degrees of soil pollution and water eutrophication.

[0004] At present, the detection of phytic acid and its degradation products is mainly divided into pretreatment, detection and analytical detection: acid leaching, organic solvents, ultrasound, microwave and solid phase extraction are commonly used methods in pretreatment; qualitative and quantitative detection is mainly based on spectrophotometry, ion chromatography and liquid mass spectrometry. Spectrophotometry involves adsorption and desorption of anion exchange resins. The phytic acid in the eluent reacts with the ferric chloride-sulfosalicylic acid mixture to discolor. The detection process is complicated, costly and has low sensitivity. In addition, as the final degradation product of phytic acid, the simultaneous detection of inositol with phytic acid and its degradation products has not been reported in the literature. In addition, in order to remove the influence of ion interference when studying the interaction between phytic acid and iron minerals, the solid phase extraction column used in the existing literature needs to go through the steps of equilibration, elution and elution when processing samples, which is a cumbersome process.

[0005] Therefore, it is necessary to optimize or develop new detection methods to meet the detection of phytic acid and its degradation products in related fields. Summary of the invention

[0006] In view of the above-mentioned technical deficiencies, the object of the present invention is to provide a liquid quality method for rapidly detecting phytic acid and its degradation products in groundwater.

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

[0008] The present invention provides a liquid quality method for rapidly detecting phytic acid and its degradation products in groundwater, comprising the following steps:

[0009] S1. Prepare standard solutions of phytic acid and its degradation products of different concentrations, obtain mass spectrometry parameters by direct mass spectrometry injection to obtain mass spectrometry conditions, combine the optimized chromatographic conditions, use liquid chromatography-mass spectrometer injection detection to obtain chromatograms of phytic acid and its degradation products of different concentrations, and draw a standard curve according to the chromatograms;

[0010] S2. Pre-treating the sample to be tested by using a silver-hydrogen composite column, and then testing the treated sample by using a liquid chromatography-mass spectrometer after passing through a selected filter membrane; substituting the test result into a standard curve of phytic acid and its degradation products to calculate and obtain the concentration in the sample to be tested;

[0011] Preferably, in step S1, the concentration of the phytic acid standard solution is 5 μg / mL, 10 μg / mL, 20 μg / mL, 40 μg / mL, 50 μg / mL; the concentration of the standard solution of six degradation products of phytic acid (inositol pentaphosphate, inositol tetraphosphate, inositol triphosphate, inositol diphosphate, inositol monophosphate and inositol) is 1 μg / mL, 2 μg / mL, 5 μg / mL, 10 μg / mL, 20 μg / mL.

[0012] Preferably, the chromatographic conditions in step S1 are: using a Waters ACQUITY high performance liquid chromatograph; using an ACQUITY UPLC BEH C18 column to separate phytic acid and its degradation products; mobile phase A is 50 mM ammonium acetate solution, and mobile phase B is acetonitrile; gradient elution conditions are: 0-2 min, 95% A; 2-3 min, 80% A; 3-4 min, 80% A; 4-4.5 min, 95% A; 4.5-5.5 min, 95% A; flow rate is 0.2 mL / min; column temperature is 40° C.; injection volume is 10 μL.

[0013] Preferably, the mass spectrometry conditions are: using a Waters Xevo TQ-S triple quadrupole mass spectrometer; electrospray ion source negative ion mode; multiple reaction monitoring mode; ion source temperature of 150°C; capillary voltage of 2.5KV; cone voltage of 23V; desolvation gas temperature of 550°C; and desolvation gas flow rate of 1000L / h.

[0014] Preferably, in step S2, the treated sample is filtered through a 0.22 μm filter membrane and then tested using a liquid chromatography-mass spectrometer.

[0015] Preferably, when acetonitrile is used as the organic phase, 50 mmol / L ammonium acetate aqueous solution and acetonitrile are selected as the mobile phase.

[0016] Preferably, when the phytic acid concentration is 10 μg / mL, the sample injection volume is selected to be 10 μL.

[0017] Preferably, ultrapure water and groundwater are used to prepare the phytic acid solution.

[0018] The beneficial effects of the present invention are as follows: the advantages of the present method over the prior art are that the six degradation products including inositol can be detected simultaneously; and the pretreatment method uses an Ag-H solid phase extraction column, which does not require the tedious steps of balancing, leaching and eluting, and only requires activation with ultrapure water before loading, which can improve the detection efficiency and accuracy, while reducing the detection cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0020] Figure 1 This is a multiple reaction monitoring chromatogram of phytic acid and its degradation products by the high performance liquid chromatography-tandem mass spectrometry method provided in Example 1 of the present invention. DETAILED DESCRIPTION

[0021] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0022] Example 1, a liquid quality method for rapid detection of phytic acid and its degradation products in groundwater, comprising the following steps:

[0023] S1. Prepare standard solutions of phytic acid and its degradation products of different concentrations, obtain mass spectrometry parameters by direct mass spectrometry injection to obtain mass spectrometry conditions, combine the optimized chromatographic conditions, use liquid chromatography-mass spectrometer injection detection to obtain chromatograms of phytic acid and its degradation products of different concentrations, and draw a standard curve according to the chromatograms;

[0024] S2. Pre-treating the sample to be tested by using a silver-hydrogen composite column, and then testing the treated sample by using a liquid chromatography-mass spectrometer after passing through a selected filter membrane; substituting the test result into a standard curve of phytic acid and its degradation products to calculate and obtain the concentration in the sample to be tested;

[0025] In step S1, the concentrations of the phytic acid standard solution are 5 μg / mL, 10 μg / mL, 20 μg / mL, 40 μg / mL, and 50 μg / mL; the concentrations of the standard solutions of six degradation products of phytic acid (inositol pentaphosphate, inositol tetraphosphate, inositol triphosphate, inositol diphosphate, inositol monophosphate and inositol) are 1 μg / mL, 2 μg / mL, 5 μg / mL, 10 μg / mL, and 20 μg / mL.

[0026] The chromatographic conditions in step S1 are: using a Waters ACQUITY high performance liquid chromatograph; using an ACQUITYUPLC BEH C18 column to separate phytic acid and its degradation products; mobile phase A is 50 mM ammonium acetate solution, and mobile phase B is acetonitrile; gradient elution conditions are: 0-2 min, 95% A; 2-3 min, 80% A; 3-4 min, 80% A; 4-4.5 min, 95% A; 4.5-5.5 min, 95% A; flow rate is 0.2 mL / min; column temperature is 40° C.; injection volume is 10 μL.

[0027] Mass spectrometry conditions: Waters Xevo TQ-S triple quadrupole mass spectrometer; electrospray ion source negative ion mode; multiple reaction monitoring mode; ion source temperature 150°C; capillary voltage 2.5KV; cone voltage 23V; desolvation gas temperature 550°C; desolvation gas flow rate 1000L / h. The mass spectrometry conditions of phytic acid and its degradation products are shown in Table 2:

[0028] Table 2

[0029]

[0030]

[0031] Note: * is the quantitative ion

[0032] (1) Accuracy and precision testing

[0033] The precision and accuracy of the method were evaluated by repeating the measurement three times with 10 μg / mL phytic acid standard solution and degradation product standard solution (see Table 3).

[0034] Table 3

[0035] Compound Name Inositol IP1 IP2 IP3 IP4 IP5 IP6 Recovery rate 95.32％ 113.24％ 102.51％ 98.72％ 108.11％ 116.15％ 104.25％ RSD 0.54％ 0.47％ 0.26％ 0.10％ 0.97％ 1.02％ 0.43％

[0036] (2) Results Analysis

[0037] With the concentration of phytic acid and its degradation product standards as the abscissa and the peak area as the ordinate, a standard curve for the determination of phytic acid and its degradation product contents was established. The results are shown in Table 4:

[0038] Table 4

[0039] Compound Name Regression equation Correlation coefficient Detection limit (μg / mL) Limit of quantification (μg / mL) Phytic Acid y=2324.9x+367.62 0.9937 0.19 0.58 Inositol Pentaphosphate y=50.59x+140.71 0.9796 0.48 1.43 Inositol Tetraphosphate y=481.13x+1341.7 0.9863 0.38 1.15 Inositol triphosphate y=4484.7x+4089.1 0.9978 0.24 0.72 Inositol diphosphate y=2961.4x+2017.7 0.9983 0.10 0.31 Inositol monophosphate y=8321.2x+19117 0.9781 0.27 0.82 Inositol y=21.5x+7.7 0.9989 0.07 0.18

[0040] Example 2: Mobile phase selection experiment

[0041] In this embodiment, when acetonitrile is used as the organic phase, the effects of 10, 30, and 50 mmol / L ammonium acetate as the aqueous phase on the separation of phytic acid and its degradation products are compared. It was found that when the aqueous phase was 50 mmol / L ammonium acetate aqueous solution, phytic acid and its degradation products were better separated, and the peak shape was better. Therefore, 50 mmol / L ammonium acetate aqueous solution and acetonitrile were finally selected as mobile phases.

[0042] Example 3: Experiment on determining the injection volume

[0043] In this example, when the phytic acid concentration was 10 μg / mL, the effects of injection volumes of 5 μL and 10 μL on the detected object were compared. When the injection volume was 5 μL, two peaks appeared with poor peak shape and poor symmetry. Therefore, the sample injection volume of 10 μL was selected and the response value was higher.

[0044] Example 4: Groundwater matrix influence experiment

[0045] In this example, ultrapure water and groundwater were used to prepare 10 μg / mL phytic acid solutions, respectively. The concentrations were detected and the recovery of phytic acid was calculated according to the aforementioned quantitative method. Three groups of samples were set in parallel. The influence of the matrix effect on phytic acid in actual groundwater was determined based on the test results of each group. The results are shown in Table 5.

[0046] Table 5

[0047] Ultrapure water groundwater RSD 0.43％ 7.72％ Average recovery rate 104.25％ 26.25％

[0048] Example 4: Solid Phase Extraction Column Selection Experiment

[0049] In groundwater, phytic acid combines with iron (hydroxide). Under the action of microorganisms or organic acids, iron minerals undergo reduction dissolution to release phytic acid, and phytic acid is degraded into low-grade inositol phosphate and phosphate under the action of microorganisms or phytase. In the process of exploring the interaction between phytic acid and iron minerals caused by reduction dissolution of iron minerals under the action of microorganisms, the detection of phytic acid and its degradation products is particularly important. In order to reduce the influence of groundwater matrix and iron ions on the experiment, the sample needs to be pretreated. Solid phase extraction technology is an ideal pretreatment method. The sample treatment effects of Ag-H column and HLB column are compared. The results are shown in Table 6. When the HLB column is used, the recovery rate is poor, only 13.4-56%, and the solid phase extraction column needs to go through the steps of equilibration, elution, and elution, and the process is relatively cumbersome. When the Ag-H column is used, the recovery rate of phytic acid and its degradation products is good, ranging from 85.24% to 115.24%.

[0050] Table 6

[0051]

[0052] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A liquid quality method for rapid detection of phytic acid and its degradation products in groundwater, characterized in that: The following steps are involved: S1. Prepare standard solutions of phytic acid and its degradation products of different concentrations, obtain mass spectrometry parameters by direct mass spectrometry injection, combine the optimized chromatographic conditions, use liquid chromatography-mass spectrometer injection detection to obtain chromatograms of phytic acid and its degradation products of different concentrations, and draw a standard curve according to the chromatograms; S2. Pre-treat the sample to be tested by using a silver-hydrogen composite column. After the treated sample passes through a selected filter membrane, it is sampled and tested by liquid chromatography-mass spectrometry; the test result is substituted into the standard curve of phytic acid and its degradation products to calculate and obtain the concentration in the sample to be tested.

2. A liquid quality method for rapid detection of phytic acid and its degradation products in groundwater according to claim 1, characterized in that: In step S1, the concentrations of the phytic acid standard solution are 5 μg / mL, 10 μg / mL, 20 μg / mL, 40 μg / mL, and 50 μg / mL; the concentrations of the six degradation product standard solutions of phytic acid are 1 μg / mL, 2 μg / mL, 5 μg / mL, 10 μg / mL, and 20 μg / mL; the six degradation products are inositol pentaphosphate, inositol tetraphosphate, inositol triphosphate, inositol diphosphate, inositol monophosphate, and inositol.

3. A liquid quality method for rapid detection of phytic acid and its degradation products in groundwater as claimed in claim 2, characterized in that: The chromatographic conditions in step S1 are: using a Waters ACQUITY high performance liquid chromatograph; using an ACQUITY UPLCBEH C18 column to separate phytic acid and its degradation products; mobile phase A is 50 mM ammonium acetate solution, and mobile phase B is acetonitrile; gradient elution conditions are: 0-2 min, 95% A; 2-3 min, 80% A; 3-4 min, 80% A; 4-4.5 min, 95% A; 4.5-5.5 min, 95% A; flow rate is 0.2 mL / min; column temperature is 40° C.; injection volume is 10 μL.

4. A liquid quality method for rapid detection of phytic acid and its degradation products in groundwater as claimed in claim 3, characterized in that: The mass spectrometry conditions and parameters were as follows: using a Waters Xevo TQ-S triple quadrupole mass spectrometer; electrospray ion source negative ion mode; multiple reaction monitoring mode; ion source temperature of 150°C; capillary voltage of 2.5KV; cone voltage of 23V; desolvation gas temperature of 550°C; and desolvation gas flow rate of 1000L / h.

5. A liquid quality method for rapid detection of phytic acid and its degradation products in groundwater according to claim 1, characterized in that: In step S2, the treated sample is filtered through a 0.22 μm filter membrane and then tested using a liquid chromatography-mass spectrometer.

6. A liquid quality method for rapid detection of phytic acid and its degradation products in groundwater as claimed in claim 2, characterized in that: When acetonitrile is used as the organic phase, 50 mmol / L ammonium acetate aqueous solution and acetonitrile are selected as the mobile phase.

7. A liquid quality method for rapid detection of phytic acid and its degradation products in groundwater as claimed in claim 2, characterized in that: When the phytic acid concentration is 10 μg / mL, the sample injection volume is selected to be 10 μL.

8. A liquid quality method for rapid detection of phytic acid and its degradation products in groundwater according to claim 1, characterized in that: Ultrapure water and groundwater were used to prepare phytic acid solution.

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