HPLC-ICP-MS (High Performance Liquid Chromatography-Inductively Coupled Plasma-Mass Spectrometry) isocratic rapid analysis method for forms and / or valence states of six arsenic

Abstract

The invention discloses an analysis method for isocratically and rapidly analyzing forms and / or valence states of six arsenic through HPLC-ICP-MS (High Performance Liquid Chromatography-Inductively Coupled Plasma-Mass Spectrometry). The method provided by the invention comprises the following steps: analyzing a sample to be detected by using HPLC-ICP-MS (High Performance Liquid Chromatography-Inductively Coupled Plasma-Mass Spectrometry), the elution mode of the HPLC-ICP-MS is isocratic elution, and the mobile phase of the HPLC-ICP-MS comprises an ammonium dihydrogen phosphate aqueous solution and methanol.

Description

Technical Field

[0001] The invention relates to the field of analysis and detection, and in particular to an analysis method for rapid isocratic analysis of six types of arsenic forms and / or valence states by HPLC-ICP-MS. Background Art

[0002] At present, the method of analyzing the form and valence of arsenic in samples by high performance liquid chromatography-inductively coupled plasma mass spectrometry (HPLC-ICP-MS) has been widely used in the fields of food and medicine. HPLC-ICP-MS analysis of arsenic form and valence uses HPLC as a separation method. After the arsenic form and valence in the sample are eluted and separated with a specific mobile phase on an anion exchange chromatography column, they enter the ICP-MS nebulizer for atomization, ionize in the plasma, and the ions are extracted by the lens and enter the quadrupole for detection.

[0003] In HPLC-ICP-MS analysis, the ICP-MS signal response of the ion to be measured is closely related to the co-flow matrix. Therefore, a co-flow matrix with a composition as constant as possible is conducive to signal stability. In addition, the long-term stability of ICP-MS analysis is worse than that of conventional UV detectors. Completing the injection in the shortest possible time is conducive to analytical stability and reduces cone loss.

[0004] In the existing national standards and literature reports on the HPLC-ICP-MS analysis methods for arsenic forms and valence states, when using anion exchange chromatography columns for analysis, diammonium phosphate, ammonium carbonate or other buffer salts of different concentrations and pH values ​​are often selected as mobile phases, and gradient elution can be used to achieve the separation of 5 to 6 arsenic forms and valence states, such as arsenocholine (AsC), arsenobetaine (AsB), trivalent arsenic (AsⅢ), dimethyl arsenic (DMA), monomethyl arsenic (MMA) and pentavalent arsenic (AsⅤ). However, gradient elution causes the co-flow matrix entering the ICP-MS to change over time, which can easily cause signal drift and unstable response. In addition, the chromatographic column needs to be re-balanced for each injection, which prolongs the analysis time and is not conducive to the long-term stability of large-scale analysis. There are also methods for analyzing arsenic forms and valence states using isocratic elution methods, but the currently reported isocratic elution methods can only achieve the separation of 4 arsenic forms and valence states, and cannot meet the requirements of separating 6 arsenic forms and valence states at the same time.

[0005] Therefore, there is an urgent need in the art to develop a method for isocratic elution analysis of arsenic in different forms and valence states. Summary of the invention

[0006] The object of the present invention is to provide a method for analyzing the form and / or valence state of arsenic.

[0007] In order to solve the above technical problems, the first aspect of the present invention provides a method for analyzing the form and / or valence state of arsenic, comprising the steps of:

[0008] The sample to be tested is analyzed by using HPLC-ICP-MS, wherein the elution mode of the HPLC-ICP-MS is isocratic elution, and the mobile phase of the HPLC-ICP-MS includes an aqueous solution of ammonium dihydrogen phosphate and methanol.

[0009] In some preferred embodiments, the concentration of the aqueous solution of ammonium dihydrogen phosphate is 20-30 mmol / L.

[0010] In some preferred embodiments, the pH value of the aqueous solution of ammonium dihydrogen phosphate is 5.5-6.5.

[0011] In some preferred embodiments, the mobile phase consists of an aqueous solution of ammonium dihydrogen phosphate and methanol.

[0012] In some preferred embodiments, the volume percentage of the methanol is 5-10%.

[0013] In some preferred schemes, the mobile phase of the HPLC-ICP-MS consists of an aqueous phase and an organic phase, the aqueous phase is an aqueous solution of ammonium dihydrogen phosphate, and the organic phase is methanol; wherein the molar concentration of the aqueous solution of ammonium dihydrogen phosphate is 20-30 mmol / L, and the pH of the aqueous solution of ammonium dihydrogen phosphate is 5.5-6.5; the volume ratio of the aqueous phase to the organic phase is (85-95):(5-10), preferably 90:10.

[0014] In some preferred embodiments, the liquid chromatography column of the HPLC-ICP-MS is an anion exchange chromatography column, the carrier material of the anion exchange chromatography column is a polystyrene-divinylbenzene copolymer, and an anion exchange functional group is bonded to the anion exchange chromatography column, and the anion exchange functional group is an organic ammonium, such as trimethylammonium or quaternary ammonium.

[0015] In some preferred embodiments, the chromatographic conditions of the HPLC-ICP-MS are as follows:

[0016] The mobile phase consists of an aqueous phase and an organic phase, the aqueous phase is an aqueous solution of ammonium dihydrogen phosphate, wherein the concentration of the substance of ammonium dihydrogen phosphate is 25 mmol / L, and the pH value of the aqueous phase is adjusted to 6.0 with ammonia water; the organic phase is methanol; the volume ratio of the aqueous phase to the organic phase is 90:10;

[0017] The column equilibration time is 15 to 30 minutes;

[0018] The column flow rate is 0.3-0.5 mL / min;

[0019] The elution mode of the chromatographic column is isocratic elution;

[0020] The column temperature is room temperature (about 24-27°C); and / or

[0021] The ICP-MS mass spectrometry conditions of the HPLC-ICP-MS are as follows:

[0022] The injection volume is 5 to 20 μl;

[0023] The flow rate of the mobile phase is 0.8~1.2ml / min.

[0024] In some preferred embodiments, the method is used to simultaneously and rapidly analyze the contents of six arsenic forms and valence states, namely, AsC, AsB, AsⅢ, MDA, MMA and AsⅤ, in a sample by isocratic elution.

[0025] In some preferred embodiments, the sample to be tested is food or medicine, more preferably a traditional Chinese medicine, such as earthworm, phoenix tail grass, and realgar.

[0026] Compared with the prior art, the present invention has at least the following advantages:

[0027] (1) The method of the present invention can realize the simultaneous determination of six arsenic forms and valence states in a complex matrix by isocratic elution by optimizing the composition ratio of the mobile phase and the type of chromatographic column, thereby compensating for the drift and response instability of the ICP-MS signal caused by the gradient elution method in the prior art, and the large error in the detection results. It can improve the analysis efficiency and analysis stability and reduce the loss of the cone, which is of great significance to improving the quantitative accuracy of arsenic forms and valence states.

[0028] (2) The method of the present invention shortens the analysis time relative to gradient elution, and can achieve isocratic elution and simultaneous analysis of six arsenic forms and valence states within 10 minutes.

[0029] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] One or more embodiments are exemplarily described by the pictures in the corresponding drawings, and these exemplary descriptions do not constitute limitations on the embodiments.

[0031] Figure 1 HPLC-ICP-MS chromatograms of the six arsenic forms and valence state reference solutions in Example 1;

[0032] Figure 2 The HPLC-ICP-MS chromatogram of arsenic forms and valence states in the earthworm sample in Example 1;

[0033] Figure 3 HPLC-ICP-MS chromatograms of 6 arsenic forms and valence state reference solutions in Comparative Example 1;

[0034] Figure 4 The HPLC-ICP-MS chromatogram of arsenic forms and valence states in the earthworm sample in Comparative Example 1;

[0035] Figure 5 HPLC-ICP-MS chromatograms of the six arsenic forms and valence state reference solutions in Example 2;

[0036] Figure 6 The HPLC-ICP-MS chromatogram of arsenic forms and valence states in the pteris viridis sample in Example 2;

[0037] Figure 7 HPLC-ICP-MS chromatograms of 6 arsenic forms and valence state reference solutions in Comparative Example 2;

[0038] Figure 8 The HPLC-ICP-MS chromatogram of arsenic forms and valence states in the pteris viridis sample in Comparative Example 2;

[0039] Fig. 9 HPLC-ICP-MS chromatograms of the six arsenic forms and valence state reference solutions in Example 3;

[0040] Fig.10 HPLC-ICP-MS chromatogram of arsenic forms and valence states in the realgar sample in Example 3;

[0041] Fig.11 HPLC-ICP-MS chromatograms of 6 arsenic forms and valence state reference solutions in Comparative Example 3;

[0042] Fig.12 This is the HPLC-ICP-MS chromatogram of the arsenic form and valence state in the realgar sample in Comparative Example 3.

[0043] Fig.13 This is the separation result diagram of the PRPX-100 chromatographic column (5um, 4.6*150mm) in Comparative Example 4;

[0044] Fig.14 This is the separation result diagram of the PRPX-100 chromatographic column (10um, 4.6*250mm) in Comparative Example 4. DETAILED DESCRIPTION

[0045] HPLC-ICP-MS analysis of arsenic forms and valence states has become a mature technology in the field of food and medicine. Existing standards and literature all use anion exchange columns and volatile salt solutions of a certain concentration and pH as mobile phases. Only by gradient elution can the six arsenic forms and valence states be well separated. In the gradient elution method, the composition of the mobile phase changes over time with each injection, causing the composition of the ICP-MS co-flow matrix to change, resulting in drift and unstable response of the ICP-MS signal; and each injection requires the chromatographic column to be rebalanced, which prolongs the analysis time and is not conducive to the stability of the analysis. The existing isocratic elution method cannot achieve the separation of only 4 or 5 arsenic forms and valence states, let alone meet the demand for simultaneous good separation of the six arsenic forms and valence states.

[0046] After extensive and in-depth research, the inventors found that the concentration of buffer salts and the composition of mobile phase in a specific range are conducive to the analysis of multiple arsenic species in complex matrix samples. The mobile phase is optimized and combined with the optimization of chromatography and mass spectrometry conditions to develop an isocratic elution HPLC-ICP-MS method for simultaneously analyzing 6 arsenic forms and valence states, including the steps of: using HPLC-ICP-MS to analyze the sample to be tested, wherein the elution mode of HPLC-ICP-MS is isocratic elution, and the mobile phase of the HPLC-ICP-MS includes an aqueous solution of ammonium dihydrogen phosphate and methanol. The method of the present invention makes up for the shortcomings of the existing gradient and isocratic elution methods, solves the problems of long analysis time and poor stability of the current 6 arsenic forms and valence states, and has strong pertinence.

[0047] In some preferred embodiments, the concentration of the aqueous solution of ammonium dihydrogen phosphate is 20-30 mmol / L, such as 25 mmol / L.

[0048] In some preferred embodiments, the pH value of the aqueous solution of ammonium dihydrogen phosphate is 5.5-6.5, for example 6.0.

[0049] In some preferred embodiments, the mobile phase consists of an aqueous solution of ammonium dihydrogen phosphate and methanol.

[0050] In some preferred embodiments, the volume percentage of methanol in the mobile phase is not less than 1%, more preferably not less than 2%, more preferably not less than 3%, more preferably not less than 4%, for example, 5-10%.

[0051] In some preferred embodiments, the volume percentage of methanol in the mobile phase is not higher than 15%, more preferably not higher than 12%, more preferably not higher than 10%, for example, 5-10%.

[0052] In some preferred embodiments, the volume percentage of methanol in the mobile phase is 5-10%.

[0053] In some preferred embodiments, the sample to be tested is food or medicine, more preferably a traditional Chinese medicine, such as earthworm, phoenix tail grass, and realgar.

[0054] In some preferred embodiments, the method is capable of detecting 2, preferably 3, preferably 4, preferably 5, and most preferably 6 different forms of arsenic in a sample.

[0055] In some preferred embodiments, the mobile phase of the HPLC-ICP-MS comprises: an aqueous phase and an organic phase, the aqueous phase comprises an aqueous solution of ammonium dihydrogen phosphate, and the organic phase comprises methanol; wherein the volume ratio of the aqueous phase to the organic phase is (85-95):(5-10), preferably 90:10.

[0056] In some preferred embodiments, the mobile phase of the HPLC-ICP-MS consists of an aqueous phase and an organic phase, the aqueous phase is an aqueous solution of ammonium dihydrogen phosphate, and the organic phase is methanol; wherein the molar concentration of the aqueous solution of ammonium dihydrogen phosphate is 20-30 mmol / L, and the volume ratio of the aqueous phase to the organic phase is (85-95):(5-10), preferably 90:10.

[0057] In some preferred embodiments, the mobile phase of the HPLC-ICP-MS consists of an aqueous phase and an organic phase, the aqueous phase is an aqueous solution of ammonium dihydrogen phosphate, and the organic phase is methanol; wherein the pH of the aqueous solution of ammonium dihydrogen phosphate is 5.5-6.5, and the volume ratio of the aqueous phase to the organic phase is (85-95):(5-10), preferably 90:10.

[0058] In some preferred embodiments, the mobile phase of the HPLC-ICP-MS consists of an aqueous phase and an organic phase, the aqueous phase is an aqueous solution of ammonium dihydrogen phosphate, and the organic phase is methanol; wherein the molar concentration of the aqueous solution of ammonium dihydrogen phosphate is 20-30 mmol / L, and the pH of the aqueous solution of ammonium dihydrogen phosphate is 5.5-6.5; the volume ratio of the aqueous phase to the organic phase is (85-95):(5-10), preferably 90:10.

[0059] In some preferred embodiments, the liquid chromatography column of the HPLC-ICP-MS is an anion exchange chromatography column. Preferably, the carrier material of the anion exchange chromatography column is a polystyrene-divinylbenzene copolymer, and an anion exchange functional group is bonded to the anion exchange chromatography column. More preferably, the anion exchange functional group is an organic ammonium, such as trimethylammonium or quaternary ammonium. In some preferred schemes, the anion exchange chromatography column is a polystyrene-divinylbenzene copolymer carrier bonded with a trimethylammonium anion exchange chromatography column.

[0060] In some preferred embodiments, the HPLC-ICP-MS chromatographic conditions are as follows:

[0061] The mobile phase consists of an aqueous phase and an organic phase, the aqueous phase is an aqueous solution of ammonium dihydrogen phosphate, wherein the concentration of the substance of ammonium dihydrogen phosphate is 25 mmol / L, and the pH value of the aqueous phase is adjusted to 6.0 with ammonia water; the organic phase is methanol; the volume ratio of the aqueous phase to the organic phase is 90:10;

[0062] The column equilibration time is 15 to 30 minutes;

[0063] The column flow rate is 0.3-0.5 mL / min;

[0064] The elution mode of the chromatographic column is isocratic elution;

[0065] The column temperature was room temperature (about 24-27°C).

[0066] In some preferred embodiments, the ICP-MS mass spectrometry conditions of the HPLC-ICP-MS are as follows:

[0067] The injection volume is 5 to 20 μl;

[0068] The flow rate of the mobile phase is 0.8~1.2ml / min.

[0069] In some preferred embodiments, before the injection, an ICP-MS aging step is also included. Preferably, the ICP-MS aging step includes: setting the instrument mobile phase flow rate to 0.8-1.2 ml / min, the column temperature to room temperature for aging, and the aging time is 30-60 min.

[0070] In some preferred embodiments, before detecting the sample, the step of preparing a standard curve is also included, and the step of preparing a standard curve includes preparing at least two (preferably at least 3, preferably at least 4, preferably at least 5) standard curve solutions of different concentrations for each form or valence state of arsenic, injecting the samples in sequence, and drawing a standard curve with the peak area as the ordinate and the concentration of the standard curve solution as the abscissa.

[0071] In some preferred embodiments, before detecting the sample, a step of preparing a standard curve is also included, and the step of preparing a standard curve includes:

[0072] Draw standard curves of 2 (preferably at least 3, preferably at least 4, preferably at least 5, and more preferably at least 6) different forms or valence states of arsenic. Preferably, draw standard curves of AsC, AsB, AsIII, MDA, MMA, and AsV.

[0073] In some preferred embodiments, standard curves of arsenic in different forms or valence states are plotted separately.

[0074] In some preferred embodiments, a mixed reference solution is prepared, and standard curves of arsenic in different forms or valence states are simultaneously plotted.

[0075] In some preferred embodiments, the preparation of a mixed reference solution comprises the steps of: mixing at least 2 (preferably at least 3, preferably at least 4, preferably at least 5, and more preferably at least 6) standard arsenic solutions of different forms or valences.

[0076] In some preferred embodiments, a mixed standard curve solution is diluted to obtain a mixed standard curve solution containing at least 2 (preferably at least 3, preferably at least 4, preferably at least 5, and more preferably at least 6) arsenic in different forms or valence states, and at least two (preferably at least 3, preferably at least 4, and preferably at least 5) mixed standard curve solutions of different concentrations are prepared, and then the mixed standard curve solution is used to simultaneously draw standard curves of arsenic in different forms or valence states.

[0077] In some preferred embodiments, after drawing the standard curve, a step of sample injection detection is further included, in which 5 to 20 μl of a sample solution diluted to a suitable concentration is drawn for detection under the same chromatographic and ICP-MS mass spectrometric conditions.

[0078] In some preferred embodiments, the method comprises the steps of:

[0079] Step S1, setting the chromatographic column conditions and balancing the chromatographic column; the mobile phase is an aqueous solution of ammonium dihydrogen phosphate, the organic phase is methanol, and the volume ratio of the aqueous phase to the organic phase is 90:10; the flow rate is 0.3-0.5 mL / min, the column temperature is room temperature, and the equilibration time is 15-30 min;

[0080] Step S2, connecting the outlet of the chromatographic column of the liquid chromatography to the ICP-MS nebulizer device;

[0081] Step S3, aging the ICP-MS cone; setting the mobile phase flow rate to 0.8-1.2 ml / min, the column temperature to room temperature for aging, and the aging time to 30-60 min;

[0082] Step S4, 5 to 20 μl of the prepared standard curve solution and the test solution are respectively taken and injected into the liquid chromatograph for determination. The standard curve is drawn with the peak area of ​​different forms or valence states of arsenic in the standard curve solution as the ordinate and the concentration as the abscissa, and the content of the corresponding arsenic form or valence state in the test solution is calculated.

[0083] In some preferred embodiments, the method is used to analyze various forms and valence states of arsenic in a sample by isocratic elution.

[0084] In some preferred embodiments, the method is used to analyze the content of AsC, AsB, AsIII, MDA, MMA and / or AsV in a sample by isocratic elution.

[0085] In some preferred embodiments, the water used in the method is selected from one or more of HPLC grade water, ultrapure water and deionized water.

[0086] In some preferred embodiments, the methanol used in the method is HPLC grade, chromatographic grade or mass spectrometry grade.

[0087] To make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the present invention is further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples that do not specify specific conditions are usually based on conventional conditions or the conditions recommended by the manufacturer. Unless otherwise specified, percentages and parts are weight percentages and weight parts. The experimental materials and reagents used in the following examples can be obtained from commercial channels unless otherwise specified.

[0088] Unless otherwise specified, the technical and scientific terms used herein have the same meaning as commonly understood by ordinary technicians in the technical field to which the application belongs. It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments of the present application.

[0089] Unless otherwise indicated, the term "or" means and is used interchangeably with the term "and / or".

[0090] As used herein, including the appended claims, singular forms of words such as "a," "an," and "the" include their corresponding plural referents unless the context clearly dictates otherwise.

[0091] The present invention will be further described below in conjunction with specific implementation modes, and the advantages and features of the present invention will become clearer as the description proceeds. However, the present invention is not limited to the following implementation modes.

[0092] In the following Examples 1-3, the main instruments used include:

[0093] Agilent 8800 inductively coupled plasma mass spectrometer (Agilent, USA);

[0094] 1260 high performance liquid chromatograph (Agilent, USA, with binary pump and online degasser);

[0095] Water bath (SW22, Julabo);

[0096] Balance (Sartorius, Germany);

[0097] Centrifuge (Eppendorf, Germany);

[0098] In the following Examples 1-3, the main materials and reagents used include:

[0099] Anion chromatography column I (Hamilton PRP-X100 (250 mm × 4.1 mm, 5 μm)

[0100] Anion chromatography column II (Hamilton PRP-X100 (250 mm × 4.1 mm, 10 μm)

[0101] Arsenite (AsⅢ, batch number: J14N53, AsfaAesar)

[0102] Arsenate (AsⅤ, batch number: L19P58, AsfaAesar)

[0103] Monomethylarsenic (MMA, batch number: 40617, Dr. Ehrenstorfer Company)

[0104] Dimethylarsenic (DMA, batch number: 41001, Dr. Ehrenstorfer Company);

[0105] Arsenic choline (As C, batch number: DSM7387 Wako Company)

[0106] Arsenobetaine (As B, batch number: AWM0904 Wako Company)

[0107] Ammonium dihydrogen phosphate (superior grade, Shanghai Sinopharm Group);

[0108] Pepsin (P7125, Sigma);

[0109] Nitric acid, hydrochloric acid and ammonia water are all of high-grade purity;

[0110] Water was deionized water treated with Milli-Q (18.2ΩM);

[0111] High-purity liquid argon (purity> 99.99%) and helium (purity 99.999%) were provided by Shanghai Chenggong Gas Industry Co., Ltd.

[0112] Example 1

[0113] In this embodiment, anion exchange chromatography column I was used, and an established analytical method was used to analyze the arsenic form and valence state of the Chinese medicinal material earthworm. The reference solution and the sample solution were sequentially injected and analyzed according to established conditions, and the methodology was verified.

[0114] 1.1 Solution configuration:

[0115] Preparation of mixed reference solution:

[0116] Accurately weigh appropriate amounts of As C, As B, MMA, DMA, As III and As V reference substances, add water to make 1 mg ml -1 Accurately pipette an appropriate amount of each reference substance stock solution and add water to make a 10 μg·mL -1 of the reference mixed solution.

[0117] Accurately pipette an appropriate amount of reference substance and mix, then add 0.02 mol·L-1 EDTA-2Na solution to make concentrations of 0, 1, 5, 10, 20, 50, and 100 μg·L -1 A series of standard curve solutions of (all in terms of arsenic).

[0118] Preparation of sample solution (prepared by ultrasonic extraction of artificial gastric juice using nitric acid):

[0119] Weigh 0.2g of earthworm powder (pass through No. 3 sieve) accurately, add 20ml of nitric acid artificial gastric juice accurately, shake in a 37℃ water bath for 22h, take out, let stand for 2h, centrifuge, take the supernatant, filter through a disposable 10+3μm filter membrane, take the filtrate, centrifuge at high speed (13000rpm, 2min), take the supernatant, and prepare the blank solution in the same way.

[0120] 1.2 Instrument working conditions:

[0121] HPLC conditions:

[0122] Chromatographic column: polystyrene-divinylbenzene copolymer carrier bonded with alkyl quaternary ammonium salt anion exchange material (Hamilton PRP-X100 (250 mm × 4.1 mm, 5 μm);

[0123] Mobile phase: 25mmol·L -1 Ammonium dihydrogen phosphate (adjust pH to 6.0 with aqueous ammonia) - methanol (90:10);

[0124] Flow rate: 1.0mL / min.

[0125] ICP-MS conditions:

[0126] Platinum skimmer and sampling cones, Micromist concentric nebulizer, and helium collision mode analysis were used.

[0127] RF power 1550W, RF voltage 1.80V, plasma gas flow rate 15.0L·min -1 , sampling depth 8.0mm, atomizing gas flow rate 0.9L·min -1 , auxiliary gas flow rate is 0.3L·min -1 , spray chamber temperature 2.0℃, helium flow rate 0.4ml·min -1 .

[0128] The isotopes selected for determination are 75 As, the acquisition mode selects the time resolution mode, the acquisition time is 0.5s, and the peristaltic pump flow rate is 0.4r / s.

[0129] 1.3 Methodological Validation:

[0130] 1.3.1 Linear relationship, limit of quantification and limit of detection:

[0131] Six arsenic forms were accurately drawn into a series of mixed standard solutions and injected into HPLC-ICP-MS for analysis. The peak area of ​​each analyte was used as the ordinate (y) and the concentration of each analyte was used as the abscissa (x). A standard curve was drawn to obtain the regression equation and correlation coefficient. -1 The linear relationship was good within the range, and the correlation coefficients were all greater than 0.999. The detection limit and quantification limit of each arsenic form were calculated using 3-fold and 10-fold signal-to-noise ratios. See Table 1.

[0132] Table 1-1. Linear equations, correlation coefficients, detection limits and quantification limits of six arsenic forms

[0133]

[0134] 1.3.2 Specificity test

[0135] Take earthworm medicinal materials, prepare according to the test solution preparation, and analyze according to the established method. The six arsenic forms of mixed reference substances are well separated ( Figure 1 ), AsB, AsⅢ and AsⅤ were detected in earthworm samples ( Figure 2 ), the analyte had no interference from other chromatographic peaks, and the method had good specificity.

[0136] 1.3.3 Precision

[0137] 1.3.3.1 Injection precision

[0138] Take six kinds of arsenic forms mixed standard solution (10μg·L -1 ), and the sample was injected 6 times continuously. The relative standard deviation (RSD) of the measured concentration of each arsenic form was less than 5%, indicating that the instrument has good injection precision, see Table 1-2.

[0139] Table 1-2. Precision test results (unit: μg·L -1 )

[0140] serial number As C As B AsⅢ DMA MMA AsⅤ 1 10.711 11.030 10.538 10.688 9.967 10.049 2 11.207 11.050 10.910 10.925 9.496 9.956 3 10.852 10.693 10.174 10.759 9.916 9.584 4 10.733 10.980 11.146 11.508 9.436 10.007 5 11.078 10.630 11.022 11.305 9.793 10.189 6 10.885 10.753 10.694 10.956 9.353 9.847 AVE 10.911 10.856 10.747 11.023 9.660 9.939 RSD 1.79％ 1.71％ 3.32％ 2.90％ 2.73％ 2.08％

[0141] 1.3.3.2 Repeatability

[0142] Take earthworm slices and prepare 6 copies according to the test solution. The content of AsC, DMA and MMA is very low. The repeatability is examined by adding samples. 6 copies are prepared according to the preparation method of the test solution. The results show that the RSD of the determination results of As B, AsⅢ and AsⅤ are all less than 5%, and the RSD of the sample recovery determination results of MMA, As C and DMA are all less than 5%, indicating that the method has good repeatability, see Table 1-3.

[0143] Table 1-3. Repeatability test results

[0144]

[0145] 1.3.3.3 Intermediate precision

[0146] At different times, different personnel analyzed the test solution and the spiked test solution according to the established method to examine the durability of the method. The results showed that the RSDs of the determination results of As B, AsⅢ and AsⅤ were all less than 10%, and the RSDs of the spiked recovery determination results of MMA, As C and DMA were all less than 3%, indicating that the method had good durability, as shown in Table 1-4.

[0147] Table 1-4. Intermediate precision test results

[0148]

[0149] 1.3.3 Stability

[0150] Take the first sample solution and the first sample solution under the repeatability item, and analyze them at 0, 2, 4, 8, 12, 16, and 24 hours. The RSDs of the determination results of As B, AsⅢ and AsⅤ in the solution within 24 hours were all less than 3%, and the RSDs of the sample recovery determination results of MMA, As C and DMA were all less than 3%, indicating that each arsenic form was basically stable within 24 hours, as shown in Table 1-5.

[0151] Table 1-5. Stability test results

[0152]

[0153]

[0154] 1.3.4 Accuracy

[0155] Weigh 0.1g of earthworm powder accurately, and add appropriate amount of 6 kinds of arsenic forms mixed standard solution into 12 parts, and conduct three levels of low, medium and high sample recovery experiments. The results show that the sample recovery rate of 6 kinds of arsenic forms is 90.63% to 109.85%, and the RSD is less than 5%, indicating that the method has good accuracy, see Table 1-6.

[0156] Table 1-6. Accuracy test results

[0157] serial number As C As B AsⅢ DMA MMA AsⅤ Low concentration 1 104.80％ 100.70％ 95.68％ 103.59％ 93.67％ 109.85％ Low concentration 2 102.79％ 101.98％ 100.97％ 100.29％ 100.07％ 103.88％ Low concentration 3 103.32％ 101.96％ 104.90％ 105.06％ 94.00％ 105.51％ Medium concentration 1 103.47％ 96.94％ 100.32％ 95.37％ 92.17％ 106.45％ Medium concentration 2 98.50％ 97.22％ 103.21％ 97.89％ 93.95％ 108.84％ Medium concentration 3 106.30％ 98.82％ 99.53％ 102.43％ 94.05％ 103.02％ Medium concentration 4 98.18％ 102.91％ 99.22％ 99.31％ 97.01％ 106.08％ Medium concentration 5 104.20％ 100.50％ 104.85％ 101.49％ 96.02％ 108.29％ Medium concentration 6 104.71％ 94.08％ 102.24％ 98.31％ 95.90％ 109.14％ High concentration 1 99.13％ 98.87％ 99.09％ 97.95％ 91.86％ 100.55％ High concentration 2 100.90％ 100.71％ 99.69％ 95.70％ 90.63％ 105.49％ High concentration 3 106.42％ 96.57％ 101.22％ 101.96％ 92.13％ 105.36％ average 102.73％ 99.27％ 100.91％ 99.95％ 94.29％ 106.04％ RSD 2.82％ 2.68％ 2.60％ 3.05％ 2.78％ 2.58％

[0158] Comparative Example 1

[0159] The arsenic form and valence state analysis method in Chinese Pharmacopoeia 2322 was used to analyze the arsenic form and valence state in earthworms, and compared with the analysis results of Example 1 of the present invention, the main steps are as follows:

[0160] HPLC conditions

[0161] Chromatographic column: polystyrene-divinylbenzene copolymer carrier bonded with alkyl quaternary ammonium salt anion exchange material (Hamilton PRP-X100 (250 mm × 4.1 mm, 5 μm);

[0162] Mobile phase: 25mmol·L -1 Ammonium dihydrogen phosphate (adjusted to pH 8.0 with ammonia water) was used as mobile phase A; water was used as mobile phase B, gradient elution, 0-15 min A: 0→100%, B: 100%→0%; 15-120 min A: 100→0%, B: 0%→100%; 0-15 min B: 100%;

[0163] Flow rate: 1.0mL / min.

[0164] IICP-MS conditions, preparation of mixed reference solution and preparation of sample solution were the same as in Example 1.

[0165] After the above operations are completed, take the mixed reference solution and the test solution, inject and analyze them, and record the chromatogram. Figures 3-4 shown.

[0166] contrast Figures 1-2 and Figures 3-4 It can be seen that the six arsenic forms and valence states are well separated in the two elution modes; however, the running time of each needle in the method of the present invention is about 10 minutes, and the baseline is stable, while the running time of each needle in Comparative Example 1 is close to 25 minutes, and after 10 minutes, the baseline begins to drift upward due to the change of salt entering the ICP-MS.

[0167] Example 2

[0168] In this example, the herbal medicine Pteris villosa was used as a research object to investigate the applicability of the method of the present invention.

[0169] 2.1 Solution preparation

[0170] Preparation of mixed reference solution is the same as 1.1.

[0171] Preparation of sample solution:

[0172] Weigh 0.2g of the powder of the herb (passed through a No. 3 sieve) accurately, add 20ml of nitric acid artificial gastric juice accurately, shake in a 37℃ water bath for 10h, take out, let stand for 2h, centrifuge, take the supernatant, filter through a disposable 0.45μm filter membrane, and take the filtrate. Prepare a blank solution in the same way.

[0173] 2.2 Determination conditions

[0174] The HPLC conditions and ICP-MS conditions were the same as those in Example 1.

[0175] 2.3 Methodological validation

[0176] 2.3.1 Linear relationship and limit of quantification and limit of detection

[0177] Six arsenic forms and valence states were accurately aspirated and injected into HPLC-ICP-MS for analysis. The standard curve was drawn with the peak area of ​​each analyte as the ordinate (y) and the concentration of each analyte as the abscissa (x). The regression equation and correlation coefficient were obtained. The six arsenic forms and valence states had a good linear relationship in the range of 0 to 200 μg·L-1, and the correlation coefficients were all greater than 0.999. The detection limit and quantification limit of each arsenic form and valence state were calculated using 3 times and 10 times the signal-to-noise ratio, as shown in Table 2-1.

[0178] Table 2-1 Linear equations, correlation coefficients, detection limits and quantification limits of six arsenic forms and valence states

[0179]

[0180]

[0181] 2.3.2 Specificity test

[0182] Take the herb of Pteris corylifolia and prepare it according to the preparation of the test solution. Inject and analyze it according to the established method. The six arsenic forms of the mixed reference substance are well separated ( Figure 5 ), AsⅤ and a small amount of AsⅢ and AsB were detected in the samples of Pteris fasciata. Figure 6 ), the analyte had no interference from other chromatographic peaks, and the method had good specificity.

[0183] 2.3.3 Precision test

[0184] 2.3.3.1 Injection precision test

[0185] Take six kinds of arsenic forms and valence state mixed standard solutions (10μg·L -1 ), and the sample was injected 6 times continuously. The relative standard deviation (RSD) of the measured concentrations of each arsenic form and valence state was less than 3%, indicating that the instrument had good injection precision, see Table 2-2.

[0186] Table 2-2 Precision test results (unit: μg·L -1 )

[0187] serial number As C As B AsⅢ DMA MMA AsⅤ 1 9.867 10.415 10.618 10.436 8.669 10.149 2 10.512 10.421 10.478 10.874 8.749 10.074 3 10.192 10.206 10.344 10.494 9.141 9.907 4 10.082 10.537 10.393 10.576 8.721 9.784 5 10.113 10.124 10.725 10.112 8.939 10.455 6 10.284 10.446 10.278 10.314 9.176 9.960 AVE 10.175 10.358 10.473 10.468 8.899 10.055 RSD 2.12％ 1.53％ 1.63％ 2.45％ 2.48％ 2.33％

[0188] 2.3.3.2 Repeatability test

[0189] Take the slices of phoenix tail grass and prepare 6 copies of the test solution. The repeatability of the undetected arsenic form and valence state was investigated by the addition method. The test solution was prepared in 6 copies according to "2.1". The results showed that the RSD of the determination results of As B, AsⅢ, DMA, MMA and AsⅤ detected in the sample was less than 10%, and the RSD of the repeatability result characterized by the addition recovery rate of As C that was not detected was less than 3%, indicating that the method has good repeatability, see Table 2-3.

[0190] Table 2-3 Repeatability test results

[0191]

[0192] 2.3.3.3 Intermediate precision test

[0193] At different times, different personnel analyzed the test solution and the spiked test solution according to the established method to examine the durability of the method. The results showed that the RSDs of the determination results of As B, AsⅢ, DMA, MMA and AsⅤ detected in the samples were all less than 5%, and the RSD of the spiked recovery determination results of As C that was not detected was less than 3%, indicating that the method had good durability, as shown in Table 2-4.

[0194] Table 2-4 Intermediate precision test results

[0195]

[0196]

[0197] 2.3.4 Stability test

[0198] Take the first sample solution and the first sample solution under the repeatability item, and analyze them at 0, 2, 4, 8, 12, 16, and 24 hours. The RSD of the determination results of As B, AsⅢ, DMA, MMA, and AsⅤ detected in the solution within 24 hours is less than 10%, and the RSD of the determination result of the sample recovery rate of As C that was not detected is less than 3%, indicating that each arsenic form and valence state are basically stable within 24 hours, see Table 2-5.

[0199] Table 2-5 Stability test results

[0200]

[0201] 2.3.5 Accuracy test

[0202] Weigh 0.1g of the herbal slices of Pteris fasciata accurately, and add appropriate amounts of 6 arsenic forms and valence mixed standard solutions in 12 copies, and perform three levels of low, medium and high sample recovery tests. The results show that the sample recovery rates of the 6 arsenic forms and valences are 91.78% to 109.65%, and the RSDs are all less than 10%, indicating that the method has good accuracy, see Table 2-6.

[0203] Table 2-6 Accuracy test results

[0204]

[0205]

[0206] 2.3.6 Sample determination

[0207] The established HPLC-ICP-MS method was used to analyze the arsenic form and valence state of the herb slices of Pteris viridis. The results showed that the extraction rate of arsenic form and valence state of the herb slices was basically above 85%. AsⅤ, AsⅢ, AsB, DMA and MMA were detected in all five batches of herb slices of Pteris viridis, which were mainly inorganic arsenic, accounting for 70% to 90% of the total arsenic content.

[0208] Comparative Example 2

[0209] The arsenic form and valence state analysis method in Chinese Pharmacopoeia 2322 was used to analyze the arsenic form and valence state in Pteris viridis, and compared with the analysis results of Example 1 of the present invention. The main steps are as follows:

[0210] HPLC conditions

[0211] Chromatographic column: polystyrene-divinylbenzene copolymer carrier bonded with alkyl quaternary ammonium salt anion exchange material (Hamilton PRP-X100 (250 mm × 4.1 mm, 5 μm);

[0212] Mobile phase: 25mmol·L-1 Ammonium dihydrogen phosphate (adjusted to pH 8.0 with ammonia water) was used as mobile phase A; water was used as mobile phase B, gradient elution, 0-15 min A: 0→100%, B: 100%→0%; 15-120 min A: 100→0%, B: 0%→100%; 0-15 min B: 100%;

[0213] Flow rate: 1.0mL / min.

[0214] IICP-MS conditions, preparation of mixed reference solution and preparation of sample solution were the same as in Example 1.

[0215] After the above operations are completed, take the mixed reference solution and the test solution, inject and analyze them, and record the chromatogram. Figures 7-8 shown.

[0216] contrast Figures 5-6 and Figures 7-8 It can be seen that the six arsenic forms and valence states are well separated in the two elution methods, and the arsenic forms detected in Pteris viridis under the two systems are consistent; however, the running time of each needle in the method of the present invention is about 10 minutes, and the baseline is stable, while the running time of each needle in Comparative Example 1 is close to 25 minutes, and after 10 minutes, the baseline begins to drift upward due to the change of salt entering the ICP-MS.

[0217] Example 3

[0218] In this embodiment, the mineral medicine realgar was used as a research object to investigate the applicability of the method of the present invention.

[0219] 3.1 Solution configuration:

[0220] 3.1.1 Preparation of reference stock solution

[0221] Take appropriate amount of trivalent arsenic (AsⅢ) and pentavalent arsenic (AsⅤ) standard solutions respectively, add water to make mixed solutions containing 2.0 μg (both as arsenic) of each solution per 1 ml.

[0222] 3.1.2 Preparation of standard curve solution

[0223] Accurately pipette an appropriate amount of the reference stock solution, add 0.02 mol / L disodium ethylenediaminetetraacetic acid solution to prepare a series of solutions containing 5, 20, 50, 100, 200, and 500 ng (all calculated as arsenic) of two valence states per 1 ml.

[0224] 3.1.3 Preparation of test solution

[0225] Take 30mg of the powder (pass through No. 5 sieve), weigh accurately, put it in a 250ml plastic volumetric bottle, add artificial intestinal fluid to the mark, shake well (shake thoroughly to dissolve the realgar powder), seal, weigh, put it in a 37℃ water bath for ultrasonic treatment (power 300W, frequency 45kHz) for 2 hours, let it cool, weigh it again, make up the lost weight with artificial intestinal fluid, shake well, take an appropriate amount to a 50ml plastic centrifuge tube, let it stand at 25℃ for 20-24 hours, blow off the upper surface solution gently with an ear-washing bulb, absorb about 15ml of the middle solution (avoid bringing in particles when absorbing), filter with a microporous filter membrane (10μm), discard the initial filtrate, accurately measure 5ml of the subsequent filtrate, put it in a 50ml plastic volumetric bottle, add 0.02mol / L disodium ethylenediaminetetraacetic acid solution to dilute to the mark, shake well, and get it. Prepare the reagent blank solution in the same way.

[0226] 3.2 Determination conditions

[0227] The HPLC conditions and ICP-MS conditions were the same as those in Example 1 and Example 2.

[0228] 3.3 Methodological validation

[0229] 3.3.1 Linear relationship and limit of quantification and limit of detection

[0230] AsⅢ and AsⅤ mixed series standard solutions were accurately aspirated and injected into HPLC-ICP-MS for analysis. The standard curve was drawn with the chromatographic peak area of ​​each analyte as the ordinate (y) and the concentration of each analyte series as the abscissa (x), and the regression equation and correlation coefficient were obtained. Trivalent arsenic and pentavalent arsenic have good linear relationships in the range of 0 to 500 μg·L-1, and the correlation coefficients are all greater than 0.999. The detection limit and quantification limit of each arsenic form and valence state were calculated at 3 times and 10 times the signal-to-noise ratio, see Table 3-1.

[0231] Table 3-1 Linear relationship results

[0232] Arsenic forms and valence states Standard curve equation Correlation coefficient Detection limit / (%) Limit of quantitation / (%) AsⅢ y=3156.80x+2391.63 1.0000 0.01 0.03 AsⅤ y=3194.684x-2164.88 1.0000 0.04 0.12

[0233] 3.3.2 Exclusivity

[0234] Take the medicinal material of Pteris fasciata and Realgar, prepare it according to the preparation of the test solution, and analyze it according to the established method. The six arsenic forms of the mixed reference substance are well separated ( Fig. 9 ), AsⅤ and AsⅢ were mainly detected in realgar samples ( Fig.10 ), the analyte had no interference from other chromatographic peaks, and the method had good specificity.

[0235] 3.3.3 Repeatability

[0236] Take about 30 mg of realgar powder (passed through a No. 5 sieve), weigh accurately, and perform six replicates. Perform the operation in accordance with the test solution preparation, calculate the average value and RSD value of the six replicates, and the results are shown in Table 3-2. The results show that the repeatability results are all less than 5%, and the method has good repeatability.

[0237] Table 3-2. Repeatability results (unit: %)

[0238] Valence Arsenic 1 2 3 4 5 6 AVE RSD AsⅢ 0.911 0.851 0.894 0.865 0.87 0.876 0.9 2.4％ AsⅤ 0.03 0.029 0.03 0.03 0.031 0.031 0.03 2.5％

[0239] 3.3.3 Sample recovery

[0240] Take about 15 mg of the powder (passed through a No. 5 sieve), weigh accurately, place in a 250 ml plastic volumetric bottle, add 297.9 μL of trivalent arsenic reference solution, 59.9 μL of pentavalent arsenic reference solution, add about 200 ml of artificial intestinal fluid, shake well, and operate as the test solution. The results show that the recovery rates of AsⅢ and AsⅤ are between 95% and 105%, and the relative deviation of duplicates is less than 5%, and the method has good accuracy.

[0241] Comparative Example 3

[0242] The arsenic form and valence state analysis method in Chinese Pharmacopoeia 2322 was used to analyze the arsenic form and valence state in realgar, and compared with the analysis results of Example 1 of the present invention, the main steps are as follows:

[0243] HPLC conditions

[0244] Chromatographic column: polystyrene-divinylbenzene copolymer carrier bonded with alkyl quaternary ammonium salt anion exchange material (Hamilton PRP-X100 (250 mm × 4.1 mm, 5 μm);

[0245] Mobile phase: 25mmol·L -1 Ammonium dihydrogen phosphate (adjusted to pH 8.0 with ammonia water) was used as mobile phase A; water was used as mobile phase B, gradient elution, 0-15 min A: 0→100%, B: 100%→0%; 15-120 min A: 100→0%, B: 0%→100%; 0-15 min B: 100%;

[0246] Flow rate: 1.0mL / min.

[0247] IICP-MS conditions, preparation of mixed reference solution and preparation of sample solution were the same as in Example 1.

[0248] After the above operations are completed, take the mixed reference solution and the test solution, inject and analyze them, and record the chromatogram. Figures 11-12 Show.

[0249] contrast Figures 9-10 and Figures 11-12 It can be seen that the six arsenic forms and valence states are well separated in the two elution modes, and the arsenic forms detected in realgar are consistent in the two systems; however, the running time of each injection in the method of the present invention is about 10 minutes, and the baseline is stable, while the running time of each injection in Comparative Example 3 is close to 25 minutes, and after 10 minutes, the baseline begins to drift upward due to the change of salt entering the ICP-MS.

[0250] Comparative Example 4

[0251] The method of reference (HPLCICP-MS coupling technology to study the effect of methanol on the results of arsenic form analysis. Spectroscopy and Analysis. Vol. 38, No. 6. June 2018) was used to detect 6 forms of arsenic, and 30mmM ammonium carbonate was used to separate the 6 forms and valence states of arsenic. The results showed that

[0252] 1) Using the first PRPX-100 column (5um, 4.6*150mm), the six arsenic forms could not be effectively separated, among which AsC and AsB could not be completely separated, AsⅢ and DMA completely overlapped, and only four arsenic forms could be separated, see Fig.13 .

[0253] 2) A second PRPX-100 column (10um, 4.6*250mm) was used. This column was consistent with the literature, but the six arsenic forms could not be effectively separated. AsC and AsB did not reach baseline separation, and AsⅢ and DMA were not completely separated. Only four arsenic forms could be separated. See Fig.14 .

[0254] Those skilled in the art will appreciate that the above-mentioned embodiments are specific examples for implementing the present invention, and in actual applications, various changes may be made thereto in form and detail without departing from the spirit and scope of the present invention.

Claims

1. A method for analyzing the form and / or valence of arsenic, It is characterized in that The method comprises the steps of: using HPLC-ICP-MS to analyze a sample to be tested, wherein the elution mode of the HPLC-ICP-MS is isocratic elution, and the mobile phase of the HPLC-ICP-MS comprises an aqueous solution of ammonium dihydrogen phosphate and methanol.

2. The method according to claim 1, It is characterized in that The concentration of the diammonium phosphate aqueous solution is 20-30 mmol / L.

3. The method according to claim 1 or 2, It is characterized in that The pH value of the diammonium phosphate aqueous solution is 5.5-6.

5.

4. The method according to claim 1 or 2, It is characterized in that The mobile phase consists of an aqueous solution of ammonium dihydrogen phosphate and methanol.

5. The method according to claim 4, It is characterized in that The volume percentage of the methanol is 5-10%.

6. The method according to claim 5, It is characterized in that The mobile phase of the HPLC-ICP-MS consists of an aqueous phase and an organic phase, the aqueous phase is an aqueous solution of ammonium dihydrogen phosphate, and the organic phase is methanol; wherein the molar concentration of the aqueous solution of ammonium dihydrogen phosphate is 20-30 mmol / L, and the pH of the aqueous solution of ammonium dihydrogen phosphate is 5.5-6.5; the volume ratio of the aqueous phase to the organic phase is (85-95):(5-10), preferably 90:

10.

7. The method according to claim 5, It is characterized in that The liquid chromatography column of the HPLC-ICP-MS is an anion exchange chromatography column, the carrier material of the anion exchange chromatography column is a polystyrene-divinylbenzene copolymer, and an anion exchange functional group is bonded to the anion exchange chromatography column, and the anion exchange functional group is an organic ammonium, such as trimethylammonium or quaternary ammonium.

8. The method according to claim 7, It is characterized in that The chromatographic conditions of the HPLC-ICP-MS are as follows: The mobile phase consists of an aqueous phase and an organic phase, the aqueous phase is an aqueous solution of ammonium dihydrogen phosphate, wherein the concentration of the substance of ammonium dihydrogen phosphate is 25 mmol / L, and the pH value of the aqueous phase is adjusted to 6.0 with ammonia water; the organic phase is methanol; the volume ratio of the aqueous phase to the organic phase is 90:10; The column equilibration time is 15 to 30 minutes; The column flow rate is 0.3-0.5 mL / min; The elution mode of the chromatographic column is isocratic elution; The column temperature is room temperature (about 24-27°C); and / or The ICP-MS mass spectrometry conditions of the HPLC-ICP-MS are as follows: The injection volume is 5 to 20 μl; The flow rate of the mobile phase is 0.8~1.2ml / min.

9. The method according to claim 8, It is characterized in that The method is used to analyze the contents of at least five (more preferably at least six) selected from AsC, AsB, AsⅢ, MDA, MMA and AsⅤ in a sample in an isocratic elution manner.

10. The method according to claim 4, It is characterized in that The sample to be tested is food or medicine, more preferably traditional Chinese medicine, such as earthworm, phoenix tail grass, and realgar.

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