Analysis method for simultaneously determining total phosphorus and 15 phosphorus compounds in lake sediment based on LC-ICP-MS (Liquid Chromatography-Inductively Coupled Plasma-Mass Spectrometry)

Abstract

The invention relates to the technical field of chemical analysis, in particular to an analysis method for simultaneously determining total phosphorus and 15 phosphorus compounds in lake sediment based on LC-ICP-MS. Preparing a phosphorus compound standard mixed solution and a total phosphorus standard solution with different concentration gradients, carrying out LC-ICP-MS determination, drawing to obtain a standard curve of phosphorus-containing compounds and a standard curve of total phosphorus, and obtaining a chromatogram of the standard mixed solution; the lake sediment is pretreated to obtain a sample to be detected, LC-ICP-MS determination is carried out, and all phosphorus compounds and total phosphorus are qualitatively and quantitatively analyzed. The method provided by the invention realizes one-time direct determination of total phosphorus and 15 types of phosphorus in the sediment extracting solution, greatly simplifies the operation process of obtaining the information of the phosphorus element in the sediment, and greatly simplifies the operation of morphological analysis of the phosphorus element in the sediment.

Description

Technical Field

[0001] The invention relates to the technical field of chemical analysis, and in particular to an analysis method for simultaneously determining total phosphorus and 15 phosphorus compounds in lake sediments based on LC-ICP-MS. Background Art

[0002] Phosphorus in lakes exists mainly in two forms: organic phosphorus (OP) and inorganic phosphorus (IP). Among them, inorganic phosphorus includes orthophosphate (Orth-P), pyrophosphate (Pyro-P) and triphosphate (Trip-P). Orthophosphate is considered to be the main form of phosphorus that can be directly used by aquatic primary producers and the main form of phosphorus in lakes. The structure of organic phosphorus compounds is more complex, including nucleotides, phospholipids, phosphonates, phosphosugars, inositol phosphates and organic complex phosphates. These organic phosphorus compounds can be mineralized into orthophosphates through processes such as biological hydrolysis or photolysis, and some organic phosphorus compounds have also been shown to directly promote the growth of algae. Sediments are the gathering place of phosphorus-containing substances in lakes, and the release of phosphorus-containing substances in sediments is one of the important sources of endogenous phosphorus pollution in lakes. Although there are many technologies that can be used to identify phosphorus-containing substances in sediments, due to the complexity of the composition of phosphorus-containing substances and the interference of complex matrices in lake sediments, these methods can often only provide relatively rough information, which seriously limits our research on the migration and transformation of phosphorus-containing substances in sediments.

[0003] The commonly used phosphorus morphological analysis method in sediments is the phosphorus nuclear magnetic resonance spectrum analysis method. For example, the Chinese patent with application number CN201710538876.5 discloses a method for analyzing phosphorus-containing substances in sediments by combining a chemical extraction method with a phosphorus nuclear magnetic resonance spectrum analysis method. However, this method has problems such as complicated operation steps, serious signal overlap of different phosphorus-containing substances, and excessive background noise. The liquid chromatography-electrospray ionization mass spectrometry (HPLC-ESI-MS / MS) technology commonly used for quantitative analysis of organic matter also has problems such as matrix effect and influence of ionization characteristics of the analyzed object, making it difficult to achieve morphological analysis of phosphorus in sediments. At present, there is an urgent need for accurate qualitative and quantitative analysis methods for a variety of complex phosphorus-containing molecules with simple operation procedures and strong anti-interference ability. Summary of the invention

[0004] The purpose of the present invention is to address the deficiencies of the above-mentioned prior art and to propose an analytical method for simultaneously determining total phosphorus and 15 phosphorus compounds in lake sediments based on LC-ICP-MS.

[0005] Firstly, a standard mixed solution of phosphorus-containing compounds and a standard solution of total phosphorus with different concentration gradients are prepared, and after analyzing the standard solution by LC-ICP-MS, a standard curve of phosphorus-containing compounds and a standard curve of total phosphorus are drawn with the concentration of various phosphorus-containing compounds and total phosphorus as the abscissa and the peak area as the ordinate, and a chromatogram of the standard mixed solution is obtained;

[0006] After pre-treatment of removing impurities, freeze-drying and grinding the lake sediment, a sample to be tested is obtained, an extractant is added to the sample to be tested, oscillation extraction is performed, the pH value is adjusted, and then the extract to be tested is obtained by filtration, the extract is measured by LC-ICP-MS to obtain a corresponding chromatogram, and the peak areas of each phosphorus-containing compound and total phosphorus measured in the sample are respectively brought into a standard curve of the phosphorus-containing compound and a standard curve of the total phosphorus, and the concentration of each phosphorus-containing compound and the total phosphorus concentration are quantitatively analyzed;

[0007] 15 phosphorus compounds including orthophosphate, pyrophosphate, tripolyphosphate, glucose-6-phosphate, β-glycerophosphate, phytic acid, thymidylic acid, 2'-deoxycytidine-5'-monophosphate, guanylic acid, adenosine-5'-phosphate, adenosine 5-diphosphate, adenosine-5'-triphosphate, β-nicotinamide adenine dinucleotide phosphate disodium salt, 2-aminoethylphosphonic acid, and ethanolamine phosphate;

[0008] The chromatographic column of the liquid chromatography is filled with a trimethylammonium anion exchange material bonded to a polystyrene-divinylbenzene copolymer carrier;

[0009] The mobile phases in the liquid chromatography include mobile phase A, mobile phase B and mobile phase C; the mobile phase A is 600 mM ammonium nitrate; the mobile phase B is 300 mM ammonium bicarbonate; the mobile phase C is pure water; the pH values ​​of mobile phases A and B are adjusted to 9.25±0.05 with ammonia water respectively;

[0010] Liquid chromatography was eluted by gradient elution; the gradient elution conditions of mobile phase A, mobile phase B and mobile phase C are shown in the following table:

[0011]

[0012]

[0013] Furthermore, the liquid chromatography flow rate was set to 1 mL / min.

[0014] Furthermore, the column temperature was maintained at 30 °C.

[0015] Furthermore, the extractant is a mixed solution containing 250 mM NaOH and 25 mM EDTA-Na2.

[0016] Furthermore, liquid chromatography can achieve simultaneous determination of total phosphorus and 15 types of phosphorus by multiple injections. The injection procedure is shown in the following table:

[0017]

[0018] Furthermore, an orthophosphoric acid solution with a concentration of 5 ppm in terms of phosphorus was prepared, and the solution was diluted to prepare orthophosphoric acid standard solutions with concentrations of 0.1, 0.2, 0.5, 1, and 2 ppm for a standard curve test of total phosphorus.

[0019] Furthermore, 15 mixed standard solutions with concentrations of phosphorus compounds of 0.1, 0.2, 0.5, 1, and 2 ppm were prepared for standard curve testing of phosphorus-containing compounds.

[0020] Furthermore, the mass spectrometer model is Agilent 7900ICP-MS.

[0021] Further, the mass spectrometry parameters are shown in the following table:

[0022]

[0023] The present invention adopts chemical extraction and liquid chromatography-inductively coupled plasma mass spectrometry (LC-ICP-MS) coupling method to analyze phosphorus components in lake sediments for the first time. Through flow path design, injection procedure and gradient elution design, the simultaneous determination of total phosphorus and 15 types of phosphorus in sediments is realized for the first time. Due to the strong anti-interference ability of inductively coupled plasma mass spectrometry, this method does not require freeze-drying and enrichment of the extract, and can be directly analyzed on the machine after adjusting the pH. It is also unnecessary to test the sediment extract multiple times to obtain data on total phosphorus and various forms of phosphorus respectively. For the first time, the one-time direct determination of total phosphorus and 15 types of phosphorus in the sediment extract is realized, which greatly simplifies the operation process of obtaining sediment phosphorus element information and the operation of sediment phosphorus element morphological analysis. And by comparing the sample after the extract is spiked with the standard sample, it is determined that there is almost no signal offset in this method.

[0024] The technology of the present invention has strong anti-interference ability, high sensitivity and precision, good calibration curve linearity, and most phosphorus-containing substances show a high recovery rate. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Schematic diagram of the connection between the liquid chromatography injector and the ICP-MS in Example 1;

[0026] Figure 2 LC-ICP-MS analysis results of the standard solution of Example 1;

[0027] Figure 3The LC-ICP-MS analysis results of the sample spiked with 500 ppb NaOH-EDTA extract of the 0-5 cm sediment sample in Example 1;

[0028] Figure 4 The LC-ICP-MS analysis results of the NaOH-EDTA extract of the 0-5 cm sediment sample in Example 1;

[0029] Figure 5 The LC-ICP-MS analysis results of the standard solution in Comparative Example 1;

[0030] Figure 6 This is the LC-ICP-MS analysis result of the standard solution in Comparative Example 2. DETAILED DESCRIPTION

[0031] The following are specific implementations of the present invention, and the technical solutions of the present invention are further described in conjunction with the accompanying drawings. These embodiments are only for the purpose of explanation and do not limit the scope and essence of the present invention.

[0032] The present invention provides an analytical method for simultaneously determining total phosphorus and 15 phosphorus compounds in lake sediments based on chemical extraction and LC-ICP-MS. The 15 phosphorus compounds include orthophosphate (Orth-P), pyrophosphate (Pyro-P), tripolyphosphate (Trip-P), glucose-6-phosphate (Glu-6), β-glycerophosphate (β-gly), phytic acid (IHP6), thymidylic acid (TMA), 2'-deoxycytidine-5'-monophosphate (dCMP), guanylic acid (GMP), adenosine-5'-phosphate (AMP), adenosine 5-diphosphate (ADP), adenosine-5'-triphosphate (ADP), β-nicotinamide adenine dinucleotide phosphate disodium salt (β-NADP), 2-aminoethylphosphonic acid (2-AEP) and ethanolamine phosphate (O-PEA).

[0033] This method greatly simplifies the analysis steps of phosphorus compounds in sediments and can simultaneously obtain quantitative data of total phosphorus and 15 phosphorus compounds through only one analysis.

[0034] An analytical method for simultaneous determination of total phosphorus and 15 phosphorus compounds in lake sediments based on chemical extraction and LC-ICP-MS comprises the following steps:

[0035] (1) After removing gravel and plant debris, the sediment was freeze-dried, ground and passed through a 200-mesh sieve.

[0036] (2) Add 20 mL of extractant to 2 g of the ground sediment and shake for 24 hours.

[0037] (3) The extract was then centrifuged at 10,000 r / min for 15 minutes. The upper layer solution was taken out and neutralized with acetic acid, and then the pH value of the solution was adjusted to 9 with ammonia water. Finally, the sample to be tested was filtered through a 0.22 μm polyethersulfone filter membrane.

[0038] (4) The extract was finally analyzed by liquid chromatography-inductively coupled plasma mass spectrometry (LC-ICP-MS).

[0039] In step (2), the extractant is a mixed solution containing 250 mM NaOH and 25 mM EDTA-Na2. The shaking temperature is 25°C and the shaking speed is 250 rpm.

[0040] In the present invention, the liquid chromatography injector and ICP-MS are connected as follows Figure 1 shown.

[0041] In step (4), the mobile phase in the liquid chromatography includes mobile phase A, mobile phase B and mobile phase C; the mobile phase A is 600 mM ammonium nitrate; the mobile phase B is 300 mM ammonium bicarbonate; and the mobile phase C is pure water. The pH values ​​of mobile phases A and B are adjusted to 9.25 (±0.05) with ammonia water, respectively.

[0042] In step (4), the liquid chromatography is eluted by gradient elution; the gradient elution conditions of the mobile phase A, mobile phase B and mobile phase C are shown in the following table:

[0043]

[0044] In step (4), liquid chromatography is used to simultaneously determine total phosphorus and 15 types of phosphorus by multiple injections. The injection procedure is shown in the following table:

[0045]

[0046] In the step (4), the liquid chromatography flow rate is set to 1 mL / min and the column temperature is maintained at 30°C.

[0047] In the step (4), the chromatographic column of the liquid chromatography is filled with a polystyrene-divinylbenzene copolymer carrier bonded with trimethylammonium anion exchange material (Hamilton PRP-X100; 250×4.1 mm; 10 μm).

[0048] In the present invention, the operation and detection conditions of the mass spectrometer may be conventional in the art.

[0049] Wherein, the instrument model of the mass spectrometer is Agilent 7900ICP-MS.

[0050] The instrument parameters are shown in the following table:

[0051]

[0052]

[0053] In the present invention, the detection results of the liquid chromatography-inductively coupled plasma mass spectrometry analysis method are compared with the standard curve of phosphorus-containing substances to obtain the concentrations of various phosphorus-containing compounds in the sediment extract;

[0054] The phosphorus-containing substance standard curve is obtained by fitting according to the test results after phosphorus-containing substance standards corresponding to different concentrations are tested by liquid chromatography-inductively coupled plasma mass spectrometry (LC-ICP-MS).

[0055] The standard curve of total phosphorus is obtained by fitting based on the test results of sodium hydrogen phosphate standards with different concentrations tested by liquid chromatography-inductively coupled plasma mass spectrometry.

[0056] Example 1

[0057] An analytical method for simultaneous determination of total phosphorus and 15 phosphorus compounds in lake sediments based on chemical extraction and LC-ICP-MS, the experimental steps are as follows:

[0058] (1) Sediment samples (0-40 cm) were collected from Nanhu Lake in Wuhan, my country, and the sediments were divided into different packages according to different depths (0-5 cm, 5-10 cm, 10-15 cm, 15-20 cm, 20-25 cm, 25-30 cm, 30-35 cm, and 35-40 cm). All sediments were freeze-dried, ground and sieved through a 200-mesh sieve, placed in sealed bags, and stored at -20°C.

[0059] (2) Weigh 2 g of the ground precipitate into a 50 mL centrifuge tube, add 20 mL of the extractant and shake for 24 hours.

[0060] (3) The extract was then centrifuged at 10,000 r / min for 15 minutes. The upper layer solution was taken out and neutralized with acetic acid, and then the pH value of the solution was adjusted to 9 with ammonia water. The sample to be tested was obtained after filtering through a 0.22 μm polyethersulfone filter membrane.

[0061] (4) Prepare a mixed solution of phosphorus standard compounds, including orthophosphate (Orth-P), pyrophosphate (Pyro-P), tripolyphosphate (Trip-P), glucose-6-phosphate (Glu-6), β-glycerophosphate (β-gly), phytic acid (IHP6), thymidylic acid (TMA), 2'-deoxycytidine-5'-monophosphate (dCMP), guanylic acid (GMP), adenosine-5'-phosphate (AMP), adenosine 5-diphosphate (ADP), adenosine-5'-triphosphate (ADP), β-nicotinamide adenine dinucleotide phosphate disodium salt (β-NADP), 2-aminoethylphosphonic acid (2-AEP) and ethanolamine phosphate (O-PEA) at a concentration of 5 ppm (in terms of phosphorus). Dilute the mixed standard solution with pure water to prepare mixed standard solutions with 15 phosphorus compound concentrations of 0.1, 0.2, 0.5, 1 and 2 ppm for the standard curve test of 15 phosphorus compounds.

[0062] An orthophosphoric acid solution with a concentration of 5 ppm (in terms of phosphorus) was prepared, and the solution was diluted to prepare orthophosphoric acid standard solutions with concentrations of 0.1, 0.2, 0.5, 1, and 2 ppm for the standard curve test of total phosphorus.

[0063] 0.5 ppm of mixed standard was added to the extract obtained in step (3) to verify whether there was a signal drift problem in the chromatographic peak.

[0064] The standard solution, extract and extract spiked samples were analyzed by liquid chromatography tandem inductively coupled plasma mass spectrometry (LC-ICP-MS).

[0065] In step (2), the extractant is a mixed solution containing 250 mM NaOH and 25 mM EDTA-Na2. The shaking temperature is 25°C and the shaking speed is 250 rpm.

[0066] In step (4), the liquid chromatography flow rate was set to 1 mL / min and the column temperature was maintained at 30°C.

[0067] In step (4), the chromatographic column of the liquid chromatography is filled with a polystyrene-divinylbenzene copolymer carrier bonded with trimethylammonium anion exchange material (Hamilton PRP-X100; 250×4.1 mm; 10 μm).

[0068] The chromatogram of the obtained standard solution is as follows Figure 2 The chromatogram of the spiked sample is shown in Figure 3 The chromatogram of the extract is shown in Figure 4 The data of phosphorus-containing substances in sediment samples at different depths are shown in Table 1. Figure 2It can be seen that this method successfully achieved the simultaneous determination of total phosphorus and 15 types of phosphorus, and the chromatographic peaks of the 15 phosphorus-containing substances had a high degree of separation, which could achieve the accurate identification of each phosphorus-containing substance. Figure 2 and Figure 3 By comparison, it can be determined that the peak time of each phosphorus-containing substance in the sediment extract sample is consistent with that in the standard solution, and this method has almost no signal drift problem. Figure 4 The results of the actual sediment sample tests confirmed the effectiveness of this method. The test targets of this method successfully covered several major phosphorus-containing substances in actual samples and it is a powerful tool that can be used for sediment phosphorus analysis.

[0069] Table 1 Data obtained from LC-ICP-MS analysis of NaOH-EDTA extract of sediment samples in Example 1

[0070]

[0071] Note: “-” indicates that the component was not detected;

[0072] Example 2

[0073] The detection method of Example 1 was verified with respect to standard curve (linearity, range), intra-day / inter-day precision, detection limit, quantitation limit, extraction recovery and matrix effect.

[0074] Preparation of standard samples of phosphorus compounds with different concentrations:

[0075] (1) Dissolving 15 kinds of phosphorus-containing compound standards in pure water to prepare a mixed standard stock solution of appropriate concentration, and diluting with pure water to obtain a plurality of mixed standard solutions with concentration gradients. The concentration gradients of the mixed standard solutions are 0.02 mg P / L, 0.05 mg P / L, 0.1 mg P / L, 0.2 mg P / L, 0.5 mg P / L, 1 mg P / L and 2 mg P / L;

[0076] (2) Sodium hydrogen phosphate is dissolved in pure water to prepare a standard stock solution of suitable concentration, and then diluted with pure water to obtain a plurality of standard working solutions with a plurality of concentration gradients. The concentration gradients of the standard working solutions are 0.5 mg P / L, 1 mg P / L, 2 mg P / L, 5 mg P / L, 10 mg P / L, 20 mg P / L and 50 mg P / L.

[0077] Sample preparation for recovery testing:

[0078] (1) 2 g of sediment was mixed with 0.4 mL of a mixed standard solution with a concentration of 5 mg P / L and allowed to stand overnight. Then, 20 mL of the same extractant as in Example 1 was added and shaken in a shaker at 250 rpm at 25°C for 24 hours. The extracted solution was then centrifuged at 10,000 r / min for 15 minutes, and the pH value of the supernatant was adjusted to 9 with acetic acid and ammonia water. Finally, the spiked samples of 15 kinds of phosphorus were obtained by filtration through a 0.22 μm polyethersulfone filter membrane.

[0079] (2) 2 g of the precipitate was mixed with 1 mL of a 100 mg P / L sodium hydrogen phosphate standard solution and allowed to stand overnight. Then, 20 mL of the same extractant as in Example 1 was added and shaken at 25° C. in a shaker at 250 rpm for 24 hours. The extracted solution was then centrifuged at 10,000 r / min for 15 minutes, and the pH value of the supernatant was adjusted to 9 with acetic acid and ammonia water. Finally, the total phosphorus and orthophosphoric acid spiked samples were obtained by filtration through a 0.22 μm polyethersulfone filter membrane.

[0080] (3) The sample obtained after treatment in steps (2) and (3) in Example 1 is the recovery reference sample.

[0081] Sample preparation for matrix effect testing:

[0082] (1) Dissolve 15 phosphorus-containing compound standards in pure water to prepare a mixed standard stock solution of appropriate concentration.

[0083] (2) After dilution with pure water, a mixed standard solution with multiple concentration gradients is obtained for testing the slope of the calibration curve in pure water. The concentration gradients of the mixed standard solution are 0.02 mg P / L, 0.05 mg P / L, 0.1 mg P / L, 0.2 mg P / L, 0.5 mg P / L, 1 mg P / L and 2 mg P / L;

[0084] (2) The standard solution was diluted with the sediment extract after being treated in steps (2) and (3) in Example 1 to obtain mixed standard solutions with multiple concentration gradients, and the mixed standard solutions were used to test the slope of the calibration curve in the matrix. The concentration gradients of the mixed standard solutions were 0.02 mg P / L, 0.05 mg P / L, 0.1 mg P / L, 0.2 mg P / L, 0.5 mg P / L, 1 mg P / L and 2 mg P / L.

[0085] Method Validation:

[0086] (1) Linear evaluation.

[0087] The prepared 15 phosphorus compound standard solutions with a concentration range of 0.02-2 mg P / L and the prepared sodium hydrogen phosphate standard solution with a concentration range of 0.5-50 mg P / L were detected by LC-ICP-MS, and the calibration curve was constructed using least squares linear regression and the regression coefficient (R 2 )

[0088] (2) Limit of detection and limit of quantification

[0089] The limits of detection (LOD) and quantification (LOQ) of the target compounds were determined based on 3- and 10-fold signal-to-noise ratios, respectively.

[0090] (3) Intra-day precision and inter-day precision

[0091] Sediment spiked samples of 15 phosphorus species and sediment spiked samples of total phosphorus were analyzed five times on the same day (intra-day precision) and five days (inter-day precision). Intra-day precision and inter-day precision were obtained based on the data of the five repeated analyses.

[0092] (4) Recovery rate

[0093] The recoveries of the analytical method were calculated based on five replicate sediment spiked samples, with the spiking level of 15 phosphorus compounds in the sediment samples being 1 mg P / kg. The data obtained from the analysis of the sediment spiked samples by LC-ICP-MS were subtracted from the data obtained from the recovery reference sample, divided by 1 mg P / kg, and then multiplied by 100 to obtain the recoveries of each phosphorus compound. Due to the high concentrations of TP and Orth-P in the sediment, the recoveries of these two test targets were analyzed and calculated at a spiking level of 50 mg P / kg, and the rest of the operations and data calculations were consistent with the 15 phosphorus compounds.

[0094] (5) Matrix effect

[0095] The prepared solution for matrix effect analysis was analyzed by LC-ICP-MS, and the obtained data was used to construct a calibration curve using least squares linear regression and obtain the slope of the calibration curve in pure water and the slope of the calibration curve in the matrix. The matrix effect (ME) is determined by the difference between the slope of the calibration curve at the calibration point in pure water and the slope of the calibration point in the matrix. The specific calculation formula is as follows:

[0096]

[0097] Table 2 is the method verification data of total phosphorus and 15 kinds of phosphorus in Example 2

[0098]

[0099] From the data in Table 2, it can be seen that the calibration curve shows an excellent linear relationship in the concentration range of 0.02 to 2 mg P / L, among which the regression coefficients (R 2 ) were all above 0.992. For total phosphorus, the regression coefficient reached 1.000 in a wider concentration range of 0.5 to 50 mg P / L. In addition, the limits of detection (LOD) in this method ranged from 30.4 μg P / kg to 246.0 μg P / kg, and the limits of quantification (LOQ) were between 101.4 μg P / kg and 820.0 μg P / kg, which indicated that this method was more reliable than other methods. 31 P NMR technology has better sensitivity. In addition, the intra-day precision and inter-day precision of all target compounds were less than 15%, showing good stability. The recoveries of 10 of the 16 target compounds in the sediment were within the acceptable range of 57.72%-128.73%. However, due to the insufficient extraction capacity of the NaOH-EDTA extractant and the degradation of some target compounds under these conditions, the recoveries of the remaining six targets were less than 50%. In addition, all target compounds showed a low matrix effect (|ME|<20%), reflecting the strong anti-interference ability of this method.

[0100] Comparative Example 1

[0101] An analytical method for simultaneously determining 15 phosphorus compounds in lake sediments based on LC-ICP-MS, compared with Example 1, is different in that:

[0102] The mobile phase in the liquid chromatography was changed, and the changed mobile phase included mobile phase A and mobile phase B; the mobile phase A was 300 mM ammonium nitrate (pH=9.25); the mobile phase B was pure water; the gradient elution conditions of the mobile phase A and the mobile phase B were shown in the following table:

[0103]

[0104]

[0105] The test results of 15 kinds of phosphorus compounds are as follows: Figure 4 As shown, compared with Example 1 Figure 2 The test results show that although the use of ammonium nitrate and pure water as the mobile phase can greatly reduce the peak time of each phosphorus-containing compound, it also causes serious peak overlap of several phosphorus-containing compounds with earlier peak times.

[0106] Comparative Example 2

[0107] An analytical method for simultaneously determining 15 phosphorus compounds in lake sediments based on LC-ICP-MS, compared with Example 1, is different in that:

[0108] The mobile phase in the liquid chromatography was changed, and the changed mobile phase included mobile phase A and mobile phase B; the mobile phase A was 300 mM ammonium bicarbonate (pH=9.25); the mobile phase B was pure water; the gradient elution conditions of the mobile phase A and the mobile phase B were shown in the following table:

[0109]

[0110] The test results of 15 kinds of phosphorus compounds are as follows: Figure 5 As shown, compared with Example 1 Figure 2 According to the test results, although the use of ammonium bicarbonate and pure water as mobile phases can achieve complete separation of several phosphorus-containing compounds with earlier peak elution time, due to the insufficient elution capacity of the ammonium bicarbonate mobile phase, even after 18 minutes, 300mM ammonium bicarbonate was continuously used to elute to 50 minutes, there were still two phosphorus-containing substances that failed to successfully elute. Combining the test results of Comparative Examples 1 and 2, it can be found that a single eluent is difficult to achieve peak separation of 15 target compounds. In order to achieve the simultaneous determination of 15 phosphorus-containing compounds, it is necessary to integrate different types of mobile phases and alternately perform gradient elution to achieve successful separation of the target compounds.

[0111] For matters not mentioned above, the prior art applies.

[0112] Although some specific embodiments of the present invention have been described in detail through examples, those skilled in the art should understand that the above examples are for illustration only and are not intended to limit the scope of the present invention. Those skilled in the art to which the present invention belongs may make various modifications or supplements to the specific embodiments described or replace them in a similar manner, but they will not deviate from the direction of the present invention or exceed the scope defined by the attached claims. Those skilled in the art should understand that any modifications, equivalent substitutions, improvements, etc. made to the above embodiments based on the technical essence of the present invention should be included in the protection scope of the present invention.

Claims

1. An analytical method for simultaneous determination of total phosphorus and 15 phosphorus compounds in lake sediments based on LC-ICP-MS, characterized in that: Firstly, a standard mixed solution of phosphorus-containing compounds and a standard solution of total phosphorus with different concentration gradients are prepared, and after analyzing the standard solution by LC-ICP-MS, a standard curve of phosphorus-containing compounds and a standard curve of total phosphorus are drawn with the concentration of various phosphorus-containing compounds and total phosphorus as the abscissa and the peak area as the ordinate, and a chromatogram of the standard mixed solution is obtained; After pre-treatment of removing impurities, freeze-drying and grinding the lake sediment, a sample to be tested is obtained, an extractant is added to the sample to be tested, oscillation extraction is performed, the pH value is adjusted, and then the extract to be tested is obtained by filtration, the extract is measured by LC-ICP-MS to obtain a corresponding chromatogram, and the peak areas of each phosphorus-containing compound and total phosphorus measured in the sample are respectively brought into a standard curve of the phosphorus-containing compound and a standard curve of the total phosphorus, and the concentration of each phosphorus-containing compound and the total phosphorus concentration are quantitatively analyzed; 15 phosphorus-containing compounds including orthophosphate, pyrophosphate, tripolyphosphate, glucose-6-phosphate, β-glycerophosphate, phytic acid, thymidylic acid, 2'-deoxycytidine-5'-monophosphate, guanylic acid, adenosine-5'-phosphate, adenosine 5-diphosphate, adenosine-5'-triphosphate, β-nicotinamide adenine dinucleotide phosphate disodium salt, 2-aminoethylphosphonic acid, and ethanolamine phosphate; The chromatographic column of the liquid chromatography is filled with a trimethylammonium anion exchange material bonded to a polystyrene-divinylbenzene copolymer carrier; The mobile phases in the liquid chromatography include mobile phase A, mobile phase B and mobile phase C; the mobile phase A is 600 mM ammonium nitrate; the mobile phase B is 300 mM ammonium bicarbonate; the mobile phase C is pure water; the pH values ​​of the mobile phases A and B are adjusted to 9.25±0.05 with ammonia water respectively; Liquid chromatography was eluted by gradient elution; the gradient elution conditions of mobile phase A, mobile phase B and mobile phase C are shown in the following table:

2. The analytical method for simultaneously determining total phosphorus and 15 phosphorus compounds in lake sediments based on LC-ICP-MS as claimed in claim 1, characterized in that: The HPLC flow rate was set at 1 mL / min.

3. The analytical method for simultaneously determining total phosphorus and 15 phosphorus compounds in lake sediments based on LC-ICP-MS as claimed in claim 1, characterized in that: The column temperature was maintained at 30 °C.

4. The analytical method for simultaneously determining total phosphorus and 15 phosphorus compounds in lake sediments based on LC-ICP-MS as claimed in claim 1, characterized in that: The extractant was a mixed solution containing 250 mM NaOH and 25 mM EDTA-Na2.

5. The analytical method for simultaneously determining total phosphorus and 15 phosphorus compounds in lake sediments based on LC-ICP-MS as claimed in claim 1, characterized in that: Liquid chromatography achieves simultaneous determination of total phosphorus and 15 types of phosphorus by multiple injections. The injection procedure is shown in the following table:

6. The analytical method for simultaneously determining total phosphorus and 15 phosphorus compounds in lake sediments based on LC-ICP-MS as claimed in claim 1, characterized in that: An orthophosphoric acid solution with a concentration of 5 ppm in terms of phosphorus was prepared, and the solution was diluted to prepare orthophosphoric acid standard solutions with concentrations of 0.1, 0.2, 0.5, 1, and 2 ppm for the standard curve test of total phosphorus.

7. The analytical method for simultaneously determining total phosphorus and 15 phosphorus compounds in lake sediments based on LC-ICP-MS as claimed in claim 1, characterized in that: Fifteen mixed standard solutions of phosphorus compounds with concentrations of 0.1, 0.2, 0.5, 1, and 2 ppm were prepared for the standard curve of phosphorus-containing compounds.

8. The analytical method for simultaneously determining total phosphorus and 15 phosphorus compounds in lake sediments based on LC-ICP-MS as claimed in claim 1, characterized in that: The mass spectrometer model is Agilent 7900ICP-MS.

9. The analytical method for simultaneously determining total phosphorus and 15 phosphorus compounds in lake sediments based on LC-ICP-MS as claimed in claim 1, characterized in that: The mass spectrometry parameters are shown in the following table:

Images