Durable and stable mercury element form and valence state analysis method

Abstract

The invention discloses a durable and stable mercury element form and valence state analysis method. In the application, the mobile phase with specific composition can provide stable and durable HPLC-ICP-MS analysis of three mercury forms and valence states such as divalent mercury (Hg < 2 + >), methyl mercury and ethyl mercury, and the defects that the Hg < 2 + > chromatographic peak column efficiency is reduced too fast and tailing is easy to generate in the existing analysis method are overcome; meanwhile, the invention further develops a pretreatment method and a mercury form and valence analysis strategy aiming at the snake sample, and the pretreatment method and the analysis strategy are used for efficiently and stably extracting and accurately quantifying three mercury forms and valence states including divalent mercury (Hg < 2 + >), methyl mercury and ethyl in the snake sample, especially long-noded pit viper.

Description

Technical Field

[0001] The invention relates to the technical field of drug analysis, and in particular to a durable and stable mercury element morphology and valence state analysis method. Background Art

[0002] High performance liquid chromatography-inductively coupled plasma mass spectrometry (HPLC-ICP-MS) has been widely used to analyze the form and valence of mercury in food and medicine. However, the existing literature and standard methods have been repeated and found that due to the influence of extraction solvent and sample matrix, divalent mercury (Hg 2+ ) The chromatographic peak column efficiency decreases rapidly with the extension of analysis time, affecting the long-term stability of the analysis method and high-throughput analysis of large batches of samples; when the existing mercury form and valence state extraction methods for drugs or foods are used to extract Agkistrodon acutus samples, there are problems such as low extraction efficiency, large interference and organic mercury degradation, which makes it impossible to effectively study the distribution and content of mercury forms and valence states in Agkistrodon acutus.

[0003] When HPLC is used to separate mercury forms and valence states, an octadecyl bonded silica column (C 18 ), by adding thiol-containing reagents (APDC, mercaptoethanol, cysteine) to the mobile phase, different mercury compounds form complexes with it, which are separated by chromatographic columns and then analyzed by ICP-MS. Hg 2+ With C 18 The -OH residues in the chromatographic column have strong binding, which requires shielding the silanol group under acidic conditions and selecting a chromatographic column with a high end-capping rate to improve the analysis. However, as the analysis time increases, the mobile phase and matrix components may cause the end-capping group to be lost, and Hg 2+ It is easy to have severe peak tailing, and even affect the separation of methylmercury (MeHg), resulting in poor analysis accuracy. Therefore, when HPLC-ICP-MS is used for long-term stable analysis, Hg 2+ Improvements in the robustness of chromatographic behavior are necessary.

[0004] The extraction method of mercury forms and valence states, common pretreatment methods include artificial gastric juice or intestinal fluid simulation extraction, hydrochloric acid extraction or L-cysteine ​​extraction, etc. The extraction efficiency of different extraction methods varies greatly and should be optimized according to the sample matrix. For the Agkistrodon acutus sample, the general chapter 2322 of the 2020 edition of the Chinese Pharmacopoeia on the forms and valence states of mercury in animal and plant drugs was used. The extraction rate of mercury forms in Agkistrodon acutus was only about 10%; the pretreatment method of GB 5009.17-2021 for the analysis of methylmercury in food was used, and the methylmercury extraction amount increased significantly, but Hg 2+ There is a large interference and it is impossible to determine simultaneously. In addition, under this condition, ethyl mercury (EtHg) is demethylated. L-cysteine ​​hydrochloride (monohydrate) is used in the literature and extracted by heating in a water bath at 60 degrees for 2 hours. Methyl mercury and Hg 2+The extraction rate is increased, and the morphological extraction efficiency is greater than 80%, but EtHg degradation still exists, which affects the comprehensive analysis of the mercury morphology and valence distribution in Agkistrodon acutus. Therefore, the field urgently needs to develop a stable analytical method for the morphology and valence of mercury in traditional Chinese medicine and Agkistrodon acutus. Summary of the invention

[0005] The object of the present invention is to provide a durable and stable method for analyzing the form and valence state of mercury element.

[0006] Another object of the present invention is to provide a snake sample pretreatment method.

[0007] Another object of the present invention is to provide a method for analyzing the form and valence state of mercury in snake samples.

[0008] To solve the above technical problems, the first aspect of the present invention provides a durable and stable method for analyzing the form and valence state of mercury, comprising the steps of: using HPLC-ICP-MS to analyze a sample to be tested, wherein the chromatographic conditions of the HPLC-ICP-MS include using a mobile phase containing gold to elute and separate different forms of mercury in the sample to be tested.

[0009] In some embodiments, the form and valence state of the mercury element include Hg 2+ , MeHg and EtHg.

[0010] In some embodiments, the sample to be tested is a traditional Chinese medicine, a health product or a food, preferably a snake, and more preferably Agkistrodon acutus.

[0011] In some embodiments, the elution is isocratic elution.

[0012] In some embodiments, the mobile phase includes: a cysteine ​​system in which gold element is dissolved and methanol.

[0013] In some embodiments, the cysteine ​​system containing dissolved gold element contains 0.1-0.2 mg·L -1 A mixed solution of 0.1% L-cysteine ​​hydrochloride and 0.1% L-cysteine ​​with gold element.

[0014] In some embodiments, the cysteine ​​system containing dissolved gold element contains 0.1 mg·L -1 The cysteine ​​system of gold element is a mixed solution of 0.1% L-cysteine ​​hydrochloride and 0.1% L-cysteine.

[0015] In some embodiments, the mass concentration percentage of methanol in the mobile phase is 0-5%, preferably 5%.

[0016] In some embodiments, the preparation of the mobile phase comprises the steps of accurately weighing 1.00 g of L-cysteine ​​hydrochloride, adding 1.00 g of L-cysteine ​​to 200 ml of ultrapure water to dissolve it, and then accurately adding a gold element standard solution (1 g·L -1 )100μL, add ultrapure water to make up to 1000mL, shake well, and you have it.

[0017] In some embodiments, the HPLC-ICP-MS chromatographic conditions further include: using C 18 The chromatographic column analyzes the sample to be tested, and the stationary phase of the chromatographic column is bonded with silanol groups.

[0018] In some embodiments, the chromatographic conditions of the HPLC-ICP-MS are:

[0019] Chromatographic column: octadecylsilane bonded silica gel chromatographic column;

[0020] Mobile phase: methanol-0.1% L-cysteine ​​and 0.1% L-cysteine ​​hydrochloride solution (containing 0.1 mg / L gold element) (5:95);

[0021] Mobile phase flow rate: 1.0 mL / min.

[0022] In some embodiments, the mass spectrometry conditions of the HPLC-ICP-MS include: selecting isotopes as 202 Hg was analyzed.

[0023] In some embodiments, the mass spectrometry conditions of the HPLC-ICP-MS include: isotopes: 202 Hg;

[0024] Acquisition mode: time-resolved mode;

[0025] The acquisition time is 1.0s;

[0026] The peristaltic pump flow rate is 0.4r / s.

[0027] Another aspect of the present invention provides a method for pre-processing snake samples, the method comprising two independent sub-methods:

[0028] The first sub-method includes the steps of extracting EtHg: extracting the snake sample powder using artificial intestinal fluid and diluting to a suitable volume;

[0029] The second sub-method involves the extraction of Hg 2+ , MeHg and EtHg steps: Use 1% L-cysteine ​​hydrochloride solution to dissolve the snake sample powder and dilute to an appropriate volume.

[0030] In some embodiments, the snake is Agkistrodon acutus, Black-necked Snake, Golden-money Agkistrodon, Red-banded Snake, Rape Snake (Elaphe kingus), Python; preferably Agkistrodon acutus.

[0031] In some embodiments, the first sub-method comprises the steps of: accurately weighing Agkistrodon acutus sample powder (passing through a No. 3 sieve);

[0032] 0.1g, put it in a polypropylene centrifuge tube, add 8ml artificial intestinal fluid, shake in a 50℃ water bath for 24h, add 8ml 5% hydrochloric acid, make up to 20ml with artificial intestinal fluid, and shake well.

[0033] In some embodiments, the second sub-method comprises the steps of: accurately weighing Agkistrodon acutus sample powder (passed through a No. 3 sieve);

[0034] 0.1g, put it in a polypropylene centrifuge tube, accurately add 20ml of 1% L-cysteine ​​hydrochloride solution, weigh it, shake it well, heat it, take it out, cool it to room temperature, weigh it again, make up the lost weight with 1% L-cysteine ​​hydrochloride, and shake it well.

[0035] In some embodiments, the heating is performed by water bath heating or graphite heating.

[0036] In some embodiments, the heating is performed by water bath heating or graphite heating. The heating is performed at 60° C. for 15-30 min.

[0037] In some embodiments, the heating is carried out in a water bath at 60° C. for 30 min.

[0038] In some embodiments, the 1% L-cysteine ​​hydrochloride solution can convert EtHg in snake samples into Hg 2+ .

[0039] In some embodiments, the preparation method of the artificial intestinal fluid is: take 6.8g of potassium dihydrogen phosphate, add 500ml of water to dissolve it, and adjust the pH value to 6.8 with 0.1mol / L sodium hydroxide solution; take 10g of pancreatic enzyme, add appropriate amount of water to dissolve it, mix the two liquids, and dilute it to 1000ml with water.

[0040] Another aspect of the present invention provides a method for analyzing the form and valence state of mercury in snake samples, comprising the steps of:

[0041] S1, sample pretreatment, the sample pretreatment comprises the steps of: taking two equal amounts of samples to be tested and treating them using different pretreatment methods, wherein the first sample to be tested is treated with a first sub-method to extract EtHg to obtain a first extract, and the second sample to be tested is treated with a second sub-method, wherein the second sub-method converts part of the EtHg in the sample into Hg 2+, while extracting Hg 2+ , MeHg and unconverted EtHg to obtain a second extract;

[0042] S2, sample analysis, the sample analysis comprises the steps of: using HPLC-ICP-MS to analyze the first extract to obtain the EtHg content; using the same method to analyze the second extract to obtain the Hg 2+ , MeHg and unconverted EtHg content;

[0043] S3, data analysis, the data analysis includes the steps of: EtHg content value and MeHg content value are respectively measured content values, Hg 2+ The content value of Hg in the second extract is 2+ The EtHg content value measured in the first extract is subtracted from the sum of the EtHg content values.

[0044] In some embodiments, the first sub-method includes the steps of extracting EtHg: extracting snake sample powder using artificial intestinal fluid and diluting to an appropriate volume; the second sub-method includes extracting Hg 2+ , MeHg and EtHg steps: Snake sample powder was extracted with 1% L-cysteine ​​hydrochloride solution and diluted to an appropriate volume.

[0045] In some embodiments, the 1% L-cysteine ​​hydrochloride solution can convert part of the EtHg in the snake sample into Hg 2+ .

[0046] In some embodiments, the 1% L-cysteine ​​hydrochloride solution is capable of converting about 80% of the EtHg in the snake sample into Hg 2+ .

[0047] In some embodiments, the snake is Agkistrodon acutus, Black-necked Snake, Golden-money Agkistrodon, Red-banded Snake, Rape Snake (Elaphe kingus), Python; preferably Agkistrodon acutus.

[0048] In some embodiments, the first sub-method comprises the steps of accurately weighing 0.1 g of Agkistrodon acutus sample powder (passed through No. 3 sieve), placing it in a polypropylene centrifuge tube, adding 8 ml of artificial intestinal fluid, shaking in a 50°C water bath for 24 hours, adding 8 ml of 5% hydrochloric acid, making the volume up to 20 ml with artificial intestinal fluid, and shaking well.

[0049] In some embodiments, the second sub-method comprises the steps of: accurately weighing 0.1 g of Agkistrodon acutus sample powder (passed through No. 3 sieve), placing it in a polypropylene centrifuge tube, accurately adding 20 ml of 1% L-cysteine ​​hydrochloride solution, weighing the weight, shaking well, heating, taking out, cooling to room temperature, weighing the weight again, supplementing the lost weight with 1% L-cysteine ​​hydrochloride, and shaking well.

[0050] In some embodiments, the heating is performed by water bath heating or graphite heating.

[0051] In some embodiments, the heating is performed by water bath heating or graphite heating.

[0052] In some embodiments, the heating is performed at 60° C. for 15-30 min.

[0053] In some embodiments, the heating is carried out in a water bath at 60° C. for 30 min.

[0054] In some embodiments, the preparation method of the artificial intestinal fluid is: take 6.8g of potassium dihydrogen phosphate, add 500ml of water to dissolve it, and adjust the pH value to 6.8 with 0.1mol / L sodium hydroxide solution; take 10g of pancreatic enzyme, add appropriate amount of water to dissolve it, mix the two liquids, and dilute it to 1000ml with water.

[0055] In some embodiments, the sample analysis includes the step of using HPLC-ICP-MS to analyze the sample to be tested, and the chromatographic conditions of the HPLC-ICP-MS include using a mobile phase containing gold element to elute the sample to be tested.

[0056] In some embodiments, the form and valence state of the mercury element include Hg 2+ , MeHg and EtHg.

[0057] In some embodiments, the elution is isocratic elution.

[0058] In some embodiments, the mobile phase includes: a cysteine ​​system in which gold element is dissolved and methanol.

[0059] In some embodiments, the cysteine ​​system containing dissolved gold element contains 0.1-0.2 mg·L -1 A mixed solution of 0.1% L-cysteine ​​hydrochloride and 0.1% L-cysteine ​​with gold element.

[0060] In some embodiments, the cysteine ​​system containing dissolved gold element contains 0.1 mg·L -1 The cysteine ​​system of gold element is a mixed solution of 0.1% L-cysteine ​​hydrochloride and 0.1% L-cysteine.

[0061] In some embodiments, the mass concentration percentage of methanol in the mobile phase is 0-5%, preferably 5%.

[0062] In some embodiments, the preparation of the mobile phase comprises the steps of accurately weighing 1.00 g of L-cysteine ​​hydrochloride, adding 1.00 g of L-cysteine ​​to 200 ml of ultrapure water to dissolve it, and then accurately adding a gold element standard solution (1 g·L -1 )100μL, add ultrapure water to make up to 1000mL, shake well, and you have it.

[0063] In some embodiments, the HPLC-ICP-MS chromatographic conditions further include: using C 18 The chromatographic column analyzes the sample to be tested, and the stationary phase of the chromatographic column is bonded with silanol groups.

[0064] In some embodiments, the chromatographic conditions of the HPLC-ICP-MS are:

[0065] Chromatographic column: octadecylsilane bonded silica gel chromatographic column;

[0066] Mobile phase: methanol-0.1% L-cysteine ​​and 0.1% L-cysteine ​​hydrochloride solution (containing 0.1 mg / L gold element) (5:95);

[0067] Mobile phase flow rate: 1.0 mL / min.

[0068] In some embodiments, the mass spectrometry conditions of the HPLC-ICP-MS include: selecting isotopes as 202 Hg was analyzed.

[0069] In some embodiments, the mass spectrometry conditions of the HPLC-ICP-MS include: isotopes: 202 Hg;

[0070] Acquisition mode: time-resolved mode;

[0071] The acquisition time is 1.0s;

[0072] The peristaltic pump flow rate is 0.4r / s.

[0073] In some embodiments, the method for analyzing the form and valence state of mercury in snake samples comprises the steps of:

[0074] Analysis and extraction steps of Sa and EtHg: accurately weigh 0.1 g of Agkistrodon acutus sample powder (passed through No. 3 sieve), place in a polypropylene centrifuge tube, add 8 ml of artificial intestinal fluid, shake in a 50°C water bath for 24 h, add 8 ml of 5% hydrochloric acid, make up to 20 ml with artificial intestinal fluid, shake well, take an appropriate amount, centrifuge at high speed (15000 rpm), take the supernatant, and prepare a blank solution in the same way;

[0075] Sb,Hg 2+, MeHg, and unconverted EtHg analysis and extraction steps; accurately weigh 0.1 g of Agkistrodon acutus sample powder (passed through No. 3 sieve), put it in a polypropylene centrifuge tube, accurately add 20 ml of 1% L-cysteine ​​hydrochloride solution, weigh it, shake it well, heat it, take it out, cool it to room temperature, weigh it again, make up the lost weight with 1% L-cysteine ​​hydrochloride, shake it well, take an appropriate amount, centrifuge it at high speed (15000 rpm), take the supernatant, and prepare a blank solution in the same way;

[0076] Sc, respectively take the test solution and the corresponding blank solution in the above Sa and Sb, adopt the mobile phase of the first aspect of the present invention, and analyze by HPLC-ICP-MS;

[0077] If no EtHg chromatographic peak appears in the above Sa test solution, the Hg in Agkistrodon acutus samples will be further analyzed with Sb test solution. 2+ and MeHg, and calculate the content (in Hg);

[0078] Se, if EtHg chromatographic peak appears in the above Sa test solution, calculate the EtHg1 content in the test solution, and determine the Hg in the Sb test solution 2+ The sum of the contents of EtHg and Hg2 (both in Hg), Hg in the test sample 2+ The content is the difference between the contents of ΣHg2 and EtHg1.

[0079] Compared with the prior art, the present invention has the following advantages:

[0080] (1) The embodiment of the present invention uses HPLC-ICP-MS to analyze the form and valence of mercury, innovatively adding an appropriate amount of gold to the mobile phase to reduce the silanol group of the chromatographic column to Hg 2+ The adsorption effect of the mobile phase increases the relative Hg 2+ Elution efficiency, effectively improve Hg 2+ The chromatographic peak shape and analytical durability effectively solve the problem of unstable column efficiency and rapid decline of divalent mercury in the existing method for determining the form and valence of mercury. In addition, the -SH of cysteine ​​can complex with Hg, and chloride ions can react with Hg 2+ It also has a good complexing effect, further reducing Hg 2+ The residual silanols adsorbed the mercury in various forms and valence states, making Hg 2+ The tailing of the chromatographic peak was significantly improved; it was applied to more than 1000 injections of different Chinese medicine matrix samples, and the Hg 2+ There is still no obvious tailing of the chromatographic peak or decrease in column efficiency, which solves the problem of Hg appearing after about 100 injections. 2+ It can solve the problems of decreased chromatographic peak column efficiency and affected separation, and has the advantages of wide application range, good stability and durability.

[0081] (2) The embodiments of the present invention provide a method for pre-treatment of snake samples. In view of the poor stability of EtHg in organic mercury and the easy demethylation during the extraction process, a method for stably and effectively extracting organic mercury (EtHg and MeHg) is invented for confirming the distribution of organic mercury forms in samples; furthermore, a stable and efficient method for extracting Hg 2+ The MeHg extraction method can simultaneously extract and analyze unconverted EtHg, effectively solving the problem of low extraction efficiency in the analysis of mercury forms and valence states in animal-based traditional Chinese medicines.

[0082] (3) The embodiment of the present invention is also based on the aforementioned double extraction snake sample pretreatment method, and provides a comprehensive analysis and content calculation method of three mercury forms and valence states for the presence of EtHg in the sample. This method can accurately study the distribution of mercury forms and valence states in Agkistrodon acutus, and can also provide a reference for the analysis and determination of mercury forms and valence states in other traditional Chinese medicines or foods; it has the advantages of simple operation, good specificity, and high accuracy.

[0083] 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

[0084] 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.

[0085] Figure 1 The chromatograms of three mercury forms under different pH values ​​of the mobile phase in the embodiment of the present invention are shown;

[0086] Figure 2 The chromatograms of three mercury forms with different concentrations of ammonium acetate added to the mobile phase in the embodiment of the present invention;

[0087] Figure 3 The chromatograms of three mercury forms with different concentrations of gold added to the mobile phase in the embodiment of the present invention;

[0088] Figure 4 The chromatograms of three mercury forms after multiple matrix injection analysis under two different mobile phase conditions in the embodiment of the present invention;

[0089] Figure 5 The chromatograms of three mercury forms in the mobile phase containing different methanol ratios in the embodiment of the present invention are shown;

[0090] Figure 6 The chromatograms of three mercury forms obtained by multiple injections on eight different chromatographic columns in the embodiment of the present invention are as follows;

[0091] Figure 7 The chromatogram of the mercury form and valence state test sample solution in Agkistrodon acutus in the embodiment of the present invention is shown;

[0092] Figure 8 It is the chromatogram of the general method of the four parts of the 2020 edition of the Chinese Pharmacopoeia in the embodiment of the present invention (mobile phase ①);

[0093] Figure 9 Chromatogram of mobile phase analysis of methylmercury analysis in the United States Pharmacopoeia 2024 edition in the embodiment of the present invention (mobile phase ②);

[0094] Figure 10 Analytical chromatogram of the mobile phase in the embodiment of the present invention (mobile phase ③);

[0095] Figure 11 The uncapped chromatographic column (SB-C 18 ) in different mobile phases. DETAILED DESCRIPTION

[0096] After extensive and in-depth research, the inventors have developed a mobile phase with a specific composition that can provide a stable and durable HPLC-ICP-MS analysis of the three mercury forms and valence states in Agkistrodon acutus, which makes up for the existing analytical methods of Hg 2+ The column efficiency of the chromatographic peak decreases too quickly, which is prone to tailing. At the same time, the present invention also develops a specific pretreatment procedure for efficient and stable extraction and accurate quantification of mercury forms and valence states in Agkistrodon acutus. The present invention is of great significance for improving the stability and durability of the existing HPLC-ICP-MS analysis method for mercury forms and valence states in traditional Chinese medicine, as well as the specificity and accuracy of the determination of mercury forms and valence states in Agkistrodon acutus.

[0097] 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.

[0098] 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.

[0099] Unless otherwise indicated, the term "or" means and is used interchangeably with the term "and / or".

[0100] 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.

[0101] In the following Examples 1-3, the main instruments used include:

[0102] Agilent 7700 inductively coupled plasma mass spectrometer (Agilent, USA);

[0103] 1260 high performance liquid chromatograph (Agilent, USA, with binary pump and online degasser);

[0104] Water bath (SW22, Julabo);

[0105] Balance (Sartorius, Germany);

[0106] Centrifuge (Eppendorf, Germany);

[0107] Mettler Toledo XSE205DU 1 / 100,000 balance, Mettler Toledo XS204 1 / 10,000 balance (Mettler Toledo, Germany);

[0108] Water purifier (Milli-Q, USA).

[0109] In the following examples, the main materials and reagents used include:

[0110] Synergi C 18 Chromatographic column (lot 1, Hydro-RP 80A 150×4.6 mm; 4 μm, Phenomenex)

[0111] Synergi C 18 Chromatographic column (lot 2, Hydro-RP 80A 150×4.6 mm; 4 μm, Phenomenex)

[0112] X select HSS T3 C 18 Chromatographic column (4.6×150 mm, 5 μm, Waters)

[0113] X select HSS T3 C 18 Chromatographic column (4.6×150 mm, 3.5 μm, Waters)

[0114] ZORBAX SB C 18Chromatographic column (4.6×150 mm, 5 μm, Agilent)

[0115] Eclipse Plus C 18 Chromatographic column (4.6×150 mm, 5 μm, Agilent)

[0116] ODS-100V C 18 Chromatographic column (4.6×150mm, 5μm, TSKgel)

[0117] Inert Sustain AQ-C 18 Chromatographic column (4.6×150mm, 5μm, GL)

[0118] Total mercury (divalent mercury) standard solution (1000 μg / ml, batch number: GBW08617); methylmercury solution standard substance (c(MeHg) = 76.3 ± 2.9 μg / g, batch number: GBW08675); ethylmercury solution standard substance (c(CH3CH2Hg) = 74.2 ± 2.7 μg / g, batch number: GBW(E)081524) were all purchased from the National Institute of Metrology, China.

[0119] L-cysteine ​​hydrochloride, L-cysteine, ammonium acetate, nitric acid, hydrochloric acid, ammonia water, and silver nitrate were all of high purity; pepsin (enzyme activity of 1:27000) and pancreatin (casein conversion capacity ≥25.0) were all biochemical reagents; deionized water (resistivity of 18.2 MΩ·cm).

[0120] High-purity liquid argon (purity> 99.99%) and helium (purity 99.999%) were provided by Shanghai Chenggong Gas Industry Co., Ltd.

[0121] Embodiment 1

[0122] In this embodiment, an octadecylsilane bonded silica gel chromatographic column is used to specifically solve the problem of Hg 2+ The problems of rapid column efficiency decline and chromatographic peak tailing were solved, and the durability and stability of the invented mobile phase were verified by using different types of chromatographic columns and matrices.

[0123] 1. Solution configuration:

[0124] Accurately pipette an appropriate amount of mercury standard solution (1000μg / ml), add 5% hydrochloric acid to make a 1μg / ml divalent solution, and set aside; take an appropriate amount of methylmercury solution standard substance and ethylmercury solution standard substance, accurately weigh, and accurately pipette an appropriate amount of 1μg / ml divalent solution, add 5% methanol to make solutions containing 100ng (all in terms of mercury) per 1ml, to obtain a reference substance mixed stock solution.

[0125] Accurately pipette an appropriate amount of the mixed stock solution of the above-mentioned reference substances, and use 8% methanol to prepare mixed standard solutions containing 1 ng and 10 ng of mercury per 1 ml respectively.

[0126] 2 Instrument working conditions:

[0127] HPLC conditions:

[0128] Chromatographic column: octadecylsilane bonded silica gel chromatographic column;

[0129] Mobile phase: methanol-0.1% L-cysteine ​​and 0.1% L-cysteine ​​hydrochloride solution (containing 0.1 mg / L gold element) (5:95)

[0130] Flow rate: 1.0mL / min.

[0131] ICP-MS conditions:

[0132] Platinum skimmer and sampling cones, Micromist concentric nebulizer, and helium collision mode analysis were used.

[0133] 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 .

[0134] The isotopes selected for determination are 202 Hg, the acquisition mode is selected as time-resolved mode, the acquisition time is 1.0s, and the peristaltic pump flow rate is 0.4r / s.

[0135] 3. Investigation of mobile phase:

[0136] According to domestic and foreign standards and literature, adding L-cysteine ​​to the mobile phase is more common, as it has the advantages of high cost performance, easy availability, and harmlessness. The study found that different mobile phases were used in the literature. 2+ Susceptible to C 18 The influence of residual silanol in the column, the mobile phase reported in the literature, when the sample was injected less, the separation effect of the three mercury forms in the reference solution and the test solution was good; but after more injections, Hg 2+ The chromatographic peak broadens, the column efficiency deteriorates, and the separation degree from methylmercury gradually deteriorates until the last peak disappears, forming a dead adsorption (see Comparative Example 1 for details). The main influencing factors of mobile phase separation were investigated.

[0137] 3.1 Effect of mobile phase pH on the separation of three mercury forms and valence states:

[0138] Literature reports that the mobile phase under acidic conditions can inhibit the ionization of silanol groups, and the Cl - Can be used with Hg 2+ The formation of water-soluble complexes, different pH values ​​may lead to differences in the effect of silanol groups. Take a mixed reference solution of three mercury forms (concentration of 10 ng / mL), and inject 20 μl of each solution under different mobile phases. Investigate the chromatographic peak shapes and peak response values ​​of the three mercury forms in mobile phases with different pH values ​​(see Figure 1 and Table 1), the results show that under mobile phase ① conditions, Hg 2+ The peak shape is the best and the response value is the highest. As the pH value of the mobile phase increases, the tailing of the Hg2+ peak becomes more serious.

[0139] Table 1. Response values ​​of chromatographic peaks of three mercury species in mobile phases with different pH values

[0140]

[0141]

[0142] 3.2 Effect of adding different concentrations of ammonium acetate in the mobile phase on the separation of three mercury forms and valence states

[0143] The addition of ammonium acetate additive to the mobile phase for methyl mercury determination in food national standard GB5009.17-2021 is related to the improvement of peak shape. The study took a mixed reference solution of three mercury forms (all with a concentration of 10 μg / l) and examined the chromatographic peak shape and peak response values ​​of the three mercury forms in the mobile phase with different concentrations of ammonium acetate added (see Figure 2 and Table 2).

[0144] The results show that the mobile phase ①, Hg 2+ The peak shape is good and the response value is high. As the concentration of ammonium acetate in the mobile phase increases, Hg 2+ The peak tailing was more serious, and the peak shape was not significantly improved compared with that without adding ammonium acetate. 2+ The response was slightly reduced, so the addition of ammonium acetate to the mobile phase was not considered.

[0145] Table 2. Response values ​​of chromatographic peaks of three mercury species in mobile phases with different concentrations of ammonium acetate

[0146]

[0147] 3.3 Effect of adding gold to the mobile phase on mercury speciation and valence analysis

[0148] Trace gold element for Hg 2+It has a stabilizing effect. A mixed reference solution of three mercury forms (all with a concentration of 10 μg / l) was taken to investigate the chromatographic peak shapes, peak response values ​​and chromatographic peak conditions of the three mercury forms when different concentrations of gold were added to the mobile phase (see Figure 3 and Table 3). The results show that when gold is added to the mobile phase, Hg 2+ The peak shape is sharper, with different concentrations of gold, Hg 2+ There were no significant differences in peak shape and response.

[0149] Table 3. Response values ​​of chromatographic peaks of three mercury species in mobile phases with different concentrations of gold added

[0150]

[0151] 3.4 Durability of mobile phase

[0152] Chromatographic columns ①Phenomenex SynergiHydro-RP 80A (150×4.6mm, 4μm) and ②TSK gelODS-100V (4.6×150mm, 5μm) were selected, and different matrix samples were multi-injected under the mobile phases ①0.1% L-cysteine ​​and 0.1% L-cysteine ​​hydrochloride mixed solution-methanol (95:5) and ②0.1% L-cysteine ​​and 0.1% L-cysteine ​​hydrochloride mixed solution (containing 0.1μg / ml gold)-methanol (95:5) to investigate the durability of the chromatographic columns. Results Chromatographic columns ① and ② were injected with 430 injections of mobile phase ①, Hg 2+ The response gradually decreased, which may be related to column adsorption; chromatographic columns ① and ② were injected with 1100 needles in mobile phase ②, Hg 2+ The response remains basically unchanged, indicating that the system with trace gold added to the mobile phase has better durability (see Figure 4 ), which is beneficial to eliminate Hg 2+ The binding effect with silanol was finally selected to add 0.1 μg / ml gold in the mobile phase.

[0153] 3.5 Investigation of different organic phase ratios in mobile phase

[0154] Take the mixed reference solution of three mercury forms (concentration of each is 10 μg / l) and examine the chromatographic peak shape and peak response value of the three mercury forms with different methanol ratios in the mobile phase (see Figure 5 and Table 4).

[0155] The results showed that there was no significant difference in the chromatographic peak shape and response of the three mercury forms in the mobile phase with different methanol ratios. The greater the methanol ratio in the mobile phase, the shorter the retention time of the chromatographic peaks of the three mercury forms; considering that a small amount of methanol can shorten the analysis time and is suitable for most C18 chromatographic columns, a mixed solution of 0.1% L-cysteine ​​and 0.1% L-cysteine ​​hydrochloride (containing 0.1μg / ml gold)-methanol (95:5) was finally selected.

[0156] Table 4. Response values ​​of chromatographic peaks of three mercury species in mobile phases with different methanol ratios

[0157]

[0158] 3.6 Durability study of chromatographic columns of different brands and different batches of the same brand

[0159] Previous studies have found that the durability of chromatographic columns of different brands and of the same brand but different batches varies greatly when used for mercury speciation and valence state analysis. In order to investigate the feasibility and versatility of the invented mobile phase, (1) Phenomenex Synergi Hydro-RP 80A (150×4.6mm, 4μm, batch 1); (2) Phenomenex Synergi Hydro-RP 80A (150×4.6mm, 4μm, batch 2); (3) Waters Xselect HSS T3 (4.6×150mm, 5μm); (4) Waters Xselect HSS T3 (4.6×150mm, 3.5μm); (5) Agilent ZORBAX SB-C 18 (4.6×150mm, 5μm); (6) Agilent Eclipse Plus C18 (4.6×150mm, 5μm); (7) TSKgel ODS-100V (4.6×150mm, 5μm); (8) GL InertSustain AQ-C 18 8 different chromatographic columns (4.6×150mm, 5μm) were used to inject different matrix samples (cinnabar, earthworm, Agkistrodon acutus, leech, honeysuckle, etc.). The results showed that the three mercury forms showed good separation and durability on each chromatographic column. The column efficiency and separation of the chromatographic peaks of each mercury form and valence state were maintained well. (See Figure 6 ). The mobile phase of the invention has good analytical durability on different chromatographic columns.

[0160] Embodiment 2

[0161] In this example, a pretreatment method and an analytical determination strategy were established to efficiently and stably extract the mercury forms and valence states in Agkistrodon acutus.

[0162] 1. Solution configuration:

[0163] 1.1 Preparation of standard curve solution

[0164] Accurately measure an appropriate amount of divalent mercury standard solution (1000μg / ml), add 5% hydrochloric acid to make a 1μg / ml divalent mercury solution, and set aside; take an appropriate amount of methylmercury solution standard substance and ethylmercury solution standard substance, accurately weigh, then accurately pipette an appropriate amount of 1μg / ml divalent solution, add 5% methanol to make a solution containing 100ng (all in terms of mercury) per 1ml, and obtain the reference substance mixed stock solution.

[0165] Accurately pipette an appropriate amount of the mixed stock solution of the above-mentioned reference substances, and use 5% methanol to prepare a series of standard solutions containing 0.5 ng, 1 ng, 5 ng, 10 ng and 20 ng of mercury per 1 ml.

[0166] 1.2 Preparation of test solution

[0167] Step (1) Ethyl mercury: Take 0.1 g of Agkistrodon acutus sample powder (all passed through No. 3 sieve), weigh accurately, place in a polypropylene centrifuge tube, add 8 ml of artificial intestinal fluid, shake in a 50°C water bath for 24 hours, take out and cool to room temperature, add 8 ml of 5% hydrochloric acid, and make up to 20 ml with artificial intestinal fluid. Shake well, take an appropriate amount and centrifuge at high speed (15000 rpm), take the supernatant, and prepare a blank solution in the same way.

[0168] Step (2) divalent mercury and methyl mercury: Take 0.1 g of Agkistrodon acutus sample powder (all passed through No. 3 sieve), weigh accurately, place in a polypropylene centrifuge tube, accurately add 20 ml of 1% L-cysteine ​​hydrochloride, weigh, shake, heat in a 60°C water bath for 30 min, take out, cool to room temperature, weigh again, make up the lost weight with 1% L-cysteine ​​hydrochloride, shake, take an appropriate amount, centrifuge at high speed (15000 rpm), and take the supernatant. Prepare a blank solution in the same way.

[0169] 2 Instrument working conditions: Same as implementation case 1

[0170] 3. Sample pretreatment investigation

[0171] The key to elemental morphological extraction is that the morphology does not change during extraction. The main extraction methods include enzymatic hydrolysis, ultrasonic extraction, microwave-assisted extraction, etc. The extraction solvents commonly used are relatively low concentration acids, enzymes, methanol-water systems, etc.

[0172] Literature research shows that under extraction conditions such as hydrochloric acid, EtHg and MeHg may be demethylated during the extraction process and then degraded to Hg 2+Therefore, the extraction process should focus on the transformation of organic mercury. The extraction with sample addition and the determination of total mercury in the extract can clarify the extraction stability and efficiency of mercury forms and valence states, and clarify whether there are mercury forms other than the three mercury forms and valence states in the extract.

[0173] 3.1 Determination of the analytical strategy for mercury speciation and valence in Agkistrodon acutus

[0174] The results of the references and related published standard methods show that the stability of different mercury forms and valence states in Agkistrodon acutus varies greatly during the extraction process (see Comparative Example 2 for details). 2+ The requirements for the stability of the extraction solvents are different, and it is impossible to extract them stably and completely at the same time. Therefore, in order to clarify the distribution and content of the three mercury forms and valence states in Agkistrodon acutus samples, it is necessary to combine the two extraction methods to determine the analytical strategy for the forms and valence states of mercury in Agkistrodon acutus.

[0175] Generally speaking, ethyl mercury is unstable under natural conditions and is prone to demethylation, which is relatively rare in organisms. The present invention determines the distribution and content of organic mercury in Agkistrodon acutus by a two-step extraction strategy combined with a relevant calculation method: 1) firstly effectively extracting organic mercury (methyl mercury and ethyl mercury) in Agkistrodon acutus by using step (1) of 1.2, and performing determination and analysis to clarify whether the sample contains ethyl mercury and its content; 2) then extracting divalent mercury and methyl mercury in Agkistrodon acutus by using step (2) of 1.2, and performing determination and analysis; 3) comprehensive calculation: comprehensively analyzing and calculating the measured results according to step (1) and step (2): if the sample does not contain ethyl mercury, the determination result of step (2) is the mercury form and valence state result in Agkistrodon acutus; if ethyl mercury is contained, the divalent mercury (in terms of Hg) measured in step (2) needs to be deducted from the result after the ethyl mercury conversion part (the difference between the ethyl mercury obtained by the two extraction methods).

[0176] 3.2 Specificity Experiment

[0177] According to 1.2, the test solution and blank sample solution were prepared respectively. The results showed that in the pretreatment step (1), only MeHg peak was detected in the Agkistrodon acutus sample, and Hg 2+ , MeHg and EtHg were well separated, and the blank sample had no interference; the pretreatment step (2) showed that Hg was detected in the Agkistrodon acutus sample 2+ and MeHg, the blank sample had no interference, and the method had good specificity (see Figure 7 ).

[0178] 3.2 Investigation of different time and heating methods for the extraction steps of divalent mercury and methylmercury

[0179] The extraction results of heating extraction for 15min, 30min, 60min and graphite extraction for 30min were investigated respectively. The results showed (see Table 5) that after heating extraction for 30min, the extraction of divalent mercury and methylmercury was basically stable, so heating for 30min was selected. The results of graphite heating and water bath heating for 30min were basically the same, and both heating methods were acceptable.

[0180] Table 5. Investigation of different extraction times and heating methods (mg / kg)

[0181]

[0182] 3.3 Investigation of different material-liquid ratios in the extraction steps of divalent mercury and methylmercury

[0183] The extraction effects of sampling amounts of 0.1g, 0.2g and 0.5g were investigated respectively. The results showed (see Table 6) that the extraction efficiency of different sampling amounts was basically the same. Increasing the sampling amount was conducive to the detection of trace components, but it might cause components with larger content to exceed the standard curve. Considering the general reference limit of mercury is 0.2mg / kg, sampling 0.1g can meet the requirement, so the sampling amount was determined to be 0.1g.

[0184] Table 6. Investigation of different sampling amounts (mg / kg)

[0185]

[0186] 3.4 Investigation of the extraction stability of mercury forms and valence states in different extraction steps

[0187] Weigh 0.1 g of Agkistrodon acutus sample powder (all passed through No. 3 sieve) and add Hg 2+ , MeHg and EtHg reference solution, prepare the test solution according to the test solution preparation steps (1) and (2) in 1.2, and examine the extraction and recovery of the three mercury forms and valence states in different extraction steps to characterize whether the mercury form is stable during the extraction process. At the same time, accurately weigh 0.1g of Agkistrodon acutus powder and add Hg 2+ , MeHg and EtHg mixed reference solution, and the sample recovery was carried out according to the above steps to investigate the mutual transformation of different mercury forms during the extraction process.

[0188] The results show that in step (1), Hg 2+ , MeHg and EtHg were added to the reference substance for extraction, and the recoveries of were 47.8%, 103.5% and 92.8% respectively; the recoveries of and were added to the mixed reference substance solution for extraction were 46.4%, 101.9% and 95.6% respectively, indicating that the organic mercury extraction in this step is stable and Hg 2+ Extraction is incomplete; Hg in step (2) 2+The recoveries of MeHg, EtHg and EtHg added to the reference substance were 114.8%, 98.1% and 19.6% respectively, and the recoveries of MeHg and EtHg added to the mixed reference substance solution were 181.7%, 101.9% and 20.6% respectively; this indicates that about 80% of EtHg was converted to Hg by alkylation during the extraction process. 2+ The MeHg extraction stability in both extraction steps was good, and the measured MeHg contents were basically consistent (relative average deviation was less than 2%).

[0189] In summary, the two-step extraction method is adopted. Step (1) can accurately determine the ethyl mercury content, which is the content of Hg in step (2). 2+ Provides basic data, such as if EtHg is detected in step (1), it will be partially or completely converted into Hg in step (2) 2+ , according to the EtHg content in step (1), the Hg measured in step (2) 2+ The sum of the Hg and EtHg (if any) minus the EtHg content measured in step (1) is the Hg content in the sample. 2+ content.

[0190] 4 Methodological validation

[0191] 4.1 Specificity test

[0192] According to the established chromatographic conditions, Hg 2+ The three forms and valence states of mercury, MeHg, and EtHg, were separated well. The sample solution preparation step (2) was used to detect Hg in the sample solution of Agkistrodon acutus. 2+ and MeHg, and the blank sample had no interference (see Figure 8 ); using the test solution preparation step (1), Agkistrodon acutus organic mercury extraction test solution, blank solvent in Hg 2+ There is interference in the retention time, but no interference in the retention time of MeHg and EtHg chromatographic peaks. The chromatographic peaks of MeHg and EtHg in the sample solution are well separated, and the method has good specificity.

[0193] 4.2 Linearity and range

[0194] The series of standard curve solutions were accurately drawn and sampled for analysis, and the standard curve was drawn with the peak area of ​​each analyte as the ordinate and the concentration of each analyte as the abscissa. The results are shown in Table 7. Within the range of 0.5-20 μg / L, the limits of the three mercury forms and valence states were well related.

[0195] Table 7. Standard curve equations and correlation coefficients for three mercury forms

[0196]

[0197] 4.2 Precision test

[0198] 4.2.1 Injection precision test

[0199] A mixed standard solution of three mercury forms (10 μg / L) was taken and sampled 6 times continuously. The RSDs of the measured concentrations of the three mercury forms and valence states were all less than 5%, indicating that the instrument had good injection precision. The results are shown in Table 8.

[0200] Table 8. Injection precision test

[0201]

[0202] 4.2.2 Repeatability test

[0203] Take Agkistrodon acutus sample powder (all passed through No. 3 sieve), weigh 6 copies, and prepare the test solution according to the test solution preparation step (2) to examine the repeatability of the method for divalent mercury and methyl mercury.

[0204] Since ethyl mercury was not detected in the sample, the repeatability of the ethyl mercury method was investigated by adding sample recovery: the fine powder of Agkistrodon acutus sample (all passed through No. 3 sieve) was taken, weighed into 6 copies, and 0.06 ml of 0.001 mg / ml ethyl mercury standard solution was accurately added. The test solution was prepared according to the test solution preparation step (1) and the determination was carried out according to the law.

[0205] The results showed that Hg 2+ The RSDs of the determination results of Hg and MeHg were both less than 5%, and the recovery of EtHg in 6 replicates was less than 5%, indicating that the method had good repeatability (see Table 9).

[0206] Table 9. Repeatability test results

[0207]

[0208] 4.2.3 Intermediate precision test

[0209] The samples were analyzed by different personnel at different times according to this method to investigate the intermediate precision of the method. 2+ The RSDs of the determination results of Mg and MeHg were all less than 3%, indicating that the method had good robustness (see Table 10).

[0210] Table 10. Intermediate precision test results

[0211]

[0212] 4.3 Accuracy test

[0213] Accurately weigh 0.1 g of the test sample powder and add 0.06 ml, 0.08 ml and 0.10 ml of the mixed reference solution (containing Hg 2+0.001mg / ml, MeHg 0.002mg / ml (both in terms of Hg)) were prepared according to the test solution preparation step 2); 0.1g of the test sample powder was accurately weighed, and 12 copies were added, and 0.06ml, 0.08ml and 0.10ml of EtHg reference solution (concentration of 0.001mg / ml in terms of Hg) were accurately added, respectively, according to the test solution preparation step 2); three levels of spike recovery experiments were performed, low, medium and high. The results showed that Hg 2+ The recoveries of MeHg and EtHg were 116.9% and 104.8% respectively, and the recoveries of EtHg were all RSD less than 5% (see Table 11), indicating that the determination of Hg in step 2) 2+ The MeHg method has good accuracy.

[0214] Table 11. Accuracy test results

[0215]

[0216] 4.4 Solution stability test (durability test)

[0217] Take the first solution of different preparation methods under the repeatability item and analyze it at 0, 4, 8, 12, and 24 hours. 2+ The measured concentration RSDs of MeHg and EtHg were all less than 5%, indicating that the test solution was basically stable within 24 h, see Table 12.

[0218] Table 12. Stability test results (unit: μg / L)

[0219]

[0220]

[0221] 4.5 Limit of detection and limit of quantification

[0222] Take blank solvent and accurately add Hg 2+ and MeHg reference substance, 4 ng each, prepare the test solution according to the test sample preparation method step (2), and determine the Hg in the chromatogram 2+ and MeHg chromatographic peak signal-to-noise ratio, and converted them according to signal-to-noise ratios of 10 and 3, respectively, and brought into the method sampling volume to calculate the method quantitative limit and then the detection limit. The results showed that Hg 2+ The quantification limit of the method for MeHg is 0.03 mg / kg (sampling size 0.1 g), and the detection limit of the method is 0.01 mg / kg.

[0223] Take 0.1 g of Agkistrodon acutus sample powder, accurately add 4 ng of ethyl mercury reference substance, prepare ethyl mercury sample solution according to the test solution preparation method step (1), measure the signal-to-noise ratio of EtHg chromatographic peak, convert it according to the signal-to-noise ratio of 10 and 3 respectively, and bring it into the method sampling amount to calculate the method quantitative limit and then the detection limit. The results show that the method quantitative limit of EtHg is 0.04 mg / kg (sampling amount 0.1 g), and the method detection limit is 0.02 mg / kg.

[0224] According to the established method analysis, the quantitative limits and detection limits of the three mercury forms and valence states can meet the safety risk assessment requirements of mercury forms and valence states in Agkistrodon acutus samples.

[0225] 5. Sample determination

[0226] According to the established method, 13 batches of Agkistrodon acutus samples were collected and tested. The results showed that the total mercury content in Agkistrodon acutus was high, and no ethyl mercury was detected. The mercury forms and valence states were mainly divalent mercury and methyl mercury. The methyl mercury content accounted for a relatively high proportion, ranging from 0.29 to 2.0 mg / kg, and the divalent mercury content ranged from 0.036 to 0.15 mg / kg.

[0227] Comparative Example 1

[0228] According to domestic and foreign standards and literature, different chromatographic analysis methods were compared and Synergi C 18 The chromatographic column (batch number 1, Hydro-RP 80A 150×4.6mm; 4μm, Phenomenex) was used with the mobile phase for mercury form and valence state analysis in 2322 of the General Rules of Part IV of the 2020 Edition of the Chinese Pharmacopoeia: ① methanol-0.01mol / L ammonium acetate solution (containing 0.12% L-cysteine, and the pH value was adjusted to 7.5 with ammonia water) (8:92); the mobile phase for methylmercury analysis in 2232 of the United States Pharmacopoeia (USP 2024 Edition) ② a mixed solution of 0.1% L-cysteine ​​and 0.1% L-cysteine ​​hydrochloride was used, and the mobile phase of the patent of the present invention was used for comparative study, and Chinese medicine matrices such as Acuta acutus and Cinnabar were used for applicability and durability investigations.

[0229] All three mobile phases can achieve good separation of the three mercury forms and valence states, but Hg 2+ The chromatographic peak response gradually decreased. After about 20 injections of the sample matrix, Hg 2+ The response decreases and the chromatographic peak column efficiency decreases ( Figure 8 ); After about 400 injections of Agkistrodon acutus and Cinnabaris samples, Hg 2+ The response decreases and the chromatographic peak column efficiency decreases ( Figure 9) ; Mobile phase ③ was injected about 1000 times, Hg 2+ The chromatographic peak response remains essentially unchanged ( Figure 10) .

[0230] Using uncapped C 18 Chromatographic column (Agilent ZOBAX SB-C 18 ), under the conditions of mobile phase ① and mobile phase ②, Hg 2+ No peaks were observed, but Hg 2+ The chromatographic peak shape is good, and the three mercury forms and valence states are well separated ( Figure 11) . In summary, the mobile phase of this patent invention has better durability and is applicable to a wider range of chromatographic columns.

[0231] Comparative Example 2

[0232] The pretreatment methods of the test solution reported in the existing national standards or literature and the method of this patent invention were used to conduct a comparative study on the extraction and analysis of mercury forms and valence states in Agkistrodon acutus. The extraction efficiency of Hg forms and valence states was determined by the ratio of the sum of Hg forms and valence states extracted by different analytical methods to the total mercury content of the sample (1.99 mg / kg); the recovery rates of the three mercury forms and valence states added to the sample matrix for extraction were used to confirm whether the conversion of different mercury forms occurred during the extraction process.

[0233] (1) Refer to the analysis of mercury speciation in animal and plant medicines in Appendix 2322 of the 2020 edition of the Chinese Pharmacopoeia.

[0234] Preparation method of sample solution: accurately weigh Agkistrodon acutus powder (all passed through No. 3 sieve), accurately add 20 ml nitric acid artificial gastric juice and 600 μl 0.1 mol / L silver nitrate solution, heat and extract in a 37°C water bath for 24 hours, take out, shake well, leave at room temperature for 2 hours, take the supernatant, filter with a disposable double-layer filter membrane (10 μm + 3 μm), and take the filtrate. Prepare blank solution in the same way.

[0235] (2) Refer to the preparation method of the test solution for methylmercury analysis in 2232 of the United States Pharmacopoeia 2024:

[0236] Weigh Agkistrodon acutus powder (all passed through No. 3 sieve), accurately add 20 ml of 1% L-cysteine ​​hydrochloride monohydrate, heat and extract at 60±4℃ for 120 min, take out, shake well, cool to room temperature, take the supernatant and filter through 0.45μm filter membrane, take the filtrate, and prepare blank solution in the same way.

[0237] (3) Refer to GB 5009.17-2021 Pretreatment method for methylmercury analysis in food: Accurately weigh Agkistrodon acutus powder

[0238] 0.1g of the powder (all passed through No. 3 sieve) is placed in a 15mL polypropylene centrifuge tube, and 10mL of hydrochloric acid solution (5mol / L) is added. Ultrasonic water bath extraction is performed at room temperature for 60min, and shaking is performed several times during the process. Centrifuge at 8000r / min for 15min at 4℃. Accurately transfer 2.0mL of supernatant to a 5mL volumetric flask or graduated test tube, and add ammonia solution (1+1) dropwise until the sample solution has a pH of 3-7. Add 0.1mL of L-cysteine ​​solution (10g / L), dilute with water and make up to the scale, shake well, filter through a 0.45μm filter membrane, and take the filtrate. Prepare a blank solution in the same way.

[0239] (4) Reference: Weigh 0.1 g of Agkistrodon acutus powder (all passed through No. 3 sieve) accurately, add 5.0 ml of 25% tetramethylammonium hydroxide solution, and treat with ultrasound at 60°C for 60 minutes; take out and cool, add 40% HNO 3 Adjust the pH to about 4-7; add 0.5 ml of methanol and dilute to 10 ml with water. Prepare a blank solution in the same way.

[0240] (5) Reference: Weigh 0.1 g of Agkistrodon acutus powder (all passed through No. 3 sieve), add 8 ml of 10% hydrochloric acid, treat with ultrasound at 60°C for 1 hour, add ammonia water to adjust the pH to about 4-7, and add water to make up to 10 ml. Prepare blank solution in the same way.

[0241] (6) Weigh 0.1 g of Agkistrodon acutus powder (all passed through No. 3 sieve) accurately, add 20 ml of nitric acid to the artificial stomach

[0242] Liquid, heat in 37℃ water bath for 24h; take out, shake well, leave at room temperature for 2h, take supernatant, filter with disposable double-layer filter membrane (10μm+3μm), take filtrate, and prepare blank solution in the same way.

[0243] (7) Accurately weigh 0.1 g of Agkistrodon acutus powder (all passed through No. 3 sieve), accurately add 20 ml of artificial gastric juice, and extract in a 37°C water bath for 24 h; remove, shake well, and leave at room temperature for 2 h. Take the supernatant, filter with a disposable double-layer filter membrane (10 μm + 3 μm), and take the filtrate. Prepare a blank solution in the same way.

[0244] (8) Accurately weigh 0.1 g of Agkistrodon acutus powder (all passed through No. 3 sieve), accurately add 20 ml of artificial intestinal fluid, heat and extract in a 37°C water bath for 24 h; remove, shake well, and leave at room temperature for 2 h. Take the supernatant, filter with a disposable double-layer filter membrane (10 μm + 3 μm), and take the filtrate. Prepare a blank solution in the same way.

[0245] (9) Method for extracting ethyl mercury using the patented method of the present invention (extraction step (1)): see Example 2 1.2 for details

[0246] Preparation steps of test solution (1).

[0247] (10) The method for extracting divalent mercury and methylmercury according to the present invention (extraction step (2)): See Example 2 1.2 Test solution preparation step (2) for details.

[0248] The results show (see Table 5) that all 10 extraction methods showed that the mercury form in Agkistrodon acutus was mainly methylmercury, containing trace amounts of divalent mercury, and no ethylmercury was detected. The extraction efficiency of methylmercury using methods (2), (9), and (10) was basically the same, and was higher than that of other extraction solvents; the corresponding blank solvents of extraction methods (3) and (6) had a high concentration of Hg 2+ There is a large interference at the chromatographic peak position, resulting in high determination results and sample recovery, which is introduced by the high concentration hydrochloric acid sample, indicating that this extraction method is not suitable for the determination of divalent mercury; the extraction efficiency of artificial gastric juice or artificial intestinal juice of other reference methods is lower than that of this patent method; except for methods (8) and (9), the sample recovery tests of other extraction methods show that there is different degrees of conversion of ethyl mercury, and the conversion is more obvious under hydrochloric acid or cysteine ​​extraction conditions. The extraction efficiency of method (9) for methyl mercury and ethyl mercury is better than that of method (8).

[0249] In summary, it is impossible to effectively extract the three mercury forms and valence states at the same time. Based on the extraction efficiency of different methods, the method (9) of the present invention is determined to be effective for the extraction of organic mercury (methylmercury and ethylmercury) from Agkistrodon acutus, and the method (10) of the present invention is used for the extraction of divalent mercury and methylmercury from Agkistrodon acutus.

[0250] Table 5. Comparative study results of the preparation of test solutions in existing standards or literature and the preparation of test solutions in this patent

[0251]

[0252] The above description shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited by the above embodiments. The above embodiments are only preferred embodiments of the present invention and cannot be used to limit the scope of the present invention. That is, equivalent changes and modifications made according to the patent scope of the present invention and the content of the specification should still be within the scope of the present invention. The scope of protection required by the present invention is defined by the attached claims and their equivalents.

[0253] 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 mercury element form and valence state, characterized in that: The method comprises the steps of: using HPLC-ICP-MS to analyze a sample to be tested, wherein the chromatographic conditions of the HPLC-ICP-MS include using a mobile phase containing gold elements to elute and separate different forms of mercury in the sample.

2. The method according to claim 1, characterized in that The form and valence of the mercury element include Hg 2+ , MeHg and EtHg.

3. The method according to claim 1, characterized in that The mobile phase comprises: a cysteine ​​system in which gold is dissolved and methanol; preferably, the cysteine ​​system in which gold is dissolved contains 0.1 to 0.2 mg·L -1 A mixed solution of 0.1% L-cysteine ​​hydrochloride and 0.1% L-cysteine ​​containing gold element; preferably, the mass concentration percentage of methanol in the mobile phase is 0-5%, preferably 5%.

4. The method according to claim 1, characterized in that The chromatographic conditions of the HPLC-ICP-MS also include: using C 18 The chromatographic column analyzes the sample to be tested, and the stationary phase of the chromatographic column is bonded with silanol groups.

5. The method according to any one of claims 1 to 4, characterized in that: The sample to be tested is a traditional Chinese medicine, a health product or a food, preferably a snake, and more preferably Agkistrodon acutus.

6. A method for pre-treatment of snake samples, characterized in that: The method comprises two independent sub-methods: The first sub-method includes the steps of extracting EtHg: using artificial intestinal fluid, only snake sample powder can be extracted and fixed to a suitable volume; The second sub-method involves the extraction of Hg 2+ , MeHg and EtHg steps: Use 1% L-cysteine ​​hydrochloride solution to dissolve the snake sample powder and dilute to an appropriate volume.

7. A method for analyzing the form and valence of mercury in snake samples, characterized in that: The method comprises the steps of: S1, sample pretreatment, the sample pretreatment comprises the steps of: taking two equal amounts of samples to be tested and treating them using different pretreatment methods, wherein the first sample to be tested is treated with a first sub-method to extract EtHg to obtain a first extract, and the second sample to be tested is treated with a second sub-method, wherein the second sub-method converts at least part of the EtHg in the sample into Hg 2+ , while extracting Hg 2+ , MeHg and unconverted EtHg to obtain a second extract; S2, sample analysis, the sample analysis comprises the steps of: using HPLC-ICP-MS to analyze the first extract to obtain the EtHg content; using the same method to analyze the second extract to obtain the Hg 2+ , MeHg and unconverted EtHg content; S3, data analysis, the data analysis includes the steps of: EtHg content value and MeHg content value are respectively measured content values, Hg 2+ The content value of Hg in the second extract is 2+ and the sum of the EtHg content values ​​- the EtHg content value measured in the first extract.

8. The method according to claim 7, characterized in that The first sub-method comprises the steps of extracting EtHg: extracting the snake sample powder using artificial intestinal fluid and fixing the volume to a suitable volume; The second sub-method includes extracting Hg 2+ , MeHg and EtHg steps: Snake sample powder was extracted with 1% L-cysteine ​​hydrochloride solution and diluted to an appropriate volume.

9. The method according to claim 8, characterized in that The first sub-method comprises the steps of: accurately weighing 0.1 g of snake sample powder (passed through a No. 3 sieve), placing it in a polypropylene centrifuge tube, adding 8 ml of artificial intestinal fluid, shaking in a 50° C. water bath for 24 hours, adding 8 ml of 5% hydrochloric acid, making the volume up to 20 ml with artificial intestinal fluid, and shaking well; and / or The second sub-method comprises the steps of: accurately weighing 0.1 g of snake sample powder (passed through No. 3 sieve), placing it in a polypropylene centrifuge tube, accurately adding 20 ml of 1% L-cysteine ​​hydrochloride solution, weighing the weight, shaking well, heating, taking out, cooling to room temperature, weighing the weight again, supplementing the lost weight with 1% L-cysteine ​​hydrochloride, and shaking well.

10. The method according to any one of claims 6 to 9, characterized in that: The snake is a white-flowered snake, a black-banded snake, a golden-money white-flowered snake, a red-banded snake, a rat snake (Elaphe kingus), and a python; preferably, Agkistrodon acutus.

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