The standard for determination of mercury and arsenic element forms and valence states of Chinese medicinal materials in the 2020 edition of the Chinese Pharmacopoeia is planned to be upgraded

On December 18, 2019, centering closely on the overall goal of the 13th five year plan for national drug safety, taking the establishment of "the most rigorous standard" as the criterion and benchmarking against the international advanced standards, the State Pharmacopoeia Commission issued 2322 "determination of mercury, arsenic element forms and their valence states" (the third draft for comments) on its website, with a publicity period of one month, which is of far-reaching significance to the traditional Chinese medicine industry Due to the success of traditional Chinese medicine Because of its complexity, the current standard is difficult to evaluate the quality of drugs comprehensively and find potential quality problems This paper reviews the knowledge of mercury and arsenic speciation and their valence determination 1、 The speciation of mercury and arsenic in traditional Chinese medicine and the status quo of their valence state determination In terms of the control of heavy metals and harmful elements in traditional Chinese medicine, most national pharmacopoeia have mainly formulated residue standards such as mercury and arsenic The 2015 edition of China Pharmacopoeia has technically realized the simultaneous detection of many heavy metals and harmful elements It is also the first time to explore and formulate the forms and prices of mercury and arsenic elements according to the characteristics of different pharmacological and toxic effects of heavy metals in different valence states It can be said that the state-of-the-art international experience and scientific concepts have been used for reference in the detection level of heavy metals and harmful elements, and more attention has been paid to the unified coordination with the International Pharmaceutical General technical standards 2、 A comparative study of three editions of the method for the determination of the forms and valence states of mercury and arsenic According to the work schedule of China Pharmacopoeia 2020 edition, the first, second and third drafts of 2322 mercury and arsenic speciation and valence determination method have been solicited on the official website of the National Pharmacopoeia Committee in November 2018, may 2019 and December 2019 respectively for public opinions from all walks of life According to the characteristics of different pharmacological and toxic effects of heavy metals in different valence states, they have different pharmacological and toxic effects , gradually revised and improved the determination method of mercury and arsenic forms and their valence states It can be said that in the detection level of heavy metals and harmful elements, after revision and improvement, China's pharmacopoeia 2020 edition can reach the international advanced level The revised contents of the first draft are marked in red The light orange shading is the revised content of the second draft The light blue shading is the revised content of the third draft 2322 determination of the speciation and valence state of mercury and arsenic in the test sample by high performance liquid chromatography inductively coupled plasma mass spectrometry As the pretreatment method of element form and valence analysis is closely related to the sample, the preparation method of test solution should be specified separately under the item of variety if there are special requirements 1、 The speciation and valence determination of mercury are determined by high performance liquid chromatography inductively coupled plasma mass spectrometry (general rule 0412) Chromatographic and mass spectrometric conditions and system suitability tests: octadecylsilane bonded silica gel was used as filler (150 mm × 4.6 mm, 5 μ m); methanol-0.01 mol / L ammonium acetate solution (containing 0.12% L-cysteine, pH value adjusted by ammonia to 7.5) (8:92) was used as mobile phase; flow rate was 1.0 ml / min Inductively coupled plasma mass spectrometry (ICP-MS) with a coaxial atomizer and a collision pool was used for detection The isotope was 202hg, and the normal mode or the collision pool reaction mode was selected according to the interference The resolution of three different forms of mercury and different valence mercury should be greater than 1.5 (Figure 1) Fig 1 Diagram for determination of mercury form and valence 1 Mercuric chloride (divalent mercury); 2 Methylmercury; 3 Preparation of ethyl mercury reference stock solution Take appropriate amount of mercuric chloride, methylmercury and ethyl mercury reference respectively, accurately weigh them, and then accurately absorb appropriate amount of mercury standard solution (1mg / ml, medium type is nitric acid) Add 8% methanol is made into a solution containing 100ng (all in mercury) per 1ml Preparation of standard curve solution: accurately suck a proper amount of reference stock solution, add 8% methanol to prepare a series of solutions containing 0.5ng, 1ng, 5ng, 10NG and 20ng (all calculated by mercury) per 1ml respectively Preparation of test solution (1) mineral medicine and its preparation: unless otherwise specified, take an appropriate amount of test sample, take a test sample powder (passing No.4 sieve) with mercury content of 20-30mg, weigh accurately, add an appropriate amount of artificial gastric juice or artificial intestinal juice precisely, place it in a 37 ℃ water bath for ultrasonic treatment for an appropriate time, shake it well, take an appropriate amount, and leave it standing for about 24 20-36 Take a proper amount of middle layer solution, filter it with microporous filter membrane (10 μ m), accurately measure a proper amount of continuous filtrate, dilute it with 0.125mol/l hydrochloric acid solution to a certain volume, and shake it well Prepare blank solution by the same method (2) Animal and plant traditional Chinese medicine (except class A and hair): unless otherwise specified, take 0.2-0.5g of test sample powder (passing No.3 sieve), weigh accurately, add 200-600 μ l of 0.1mol/l silver nitrate solution, add proper amount of nitric acid artificial gastric juice precisely, heat it in a 37-45 ℃ water bath for about 20-24 hours, take it out, shake it well, and keep it at room temperature Put for 2 hours, take the supernatant, filter it with disposable double-layer filter membrane (10 μ m + 3 μ m), take the continuous filtrate, and then obtain Prepare blank solution by the same method A series of standard curve solutions and test sample solutions were respectively aspirated 20-100 μ L by the determination method, and then injected into the liquid chromatograph for determination Take the peak area of different forms of mercury or different valence mercury in the series of standard curve solutions as the ordinate and the concentration as the abscissa, draw the standard curve and calculate the content of different forms or different valence mercury in the test solution 2、 The speciation and valence determination of arsenic are determined by high performance liquid chromatography inductively coupled plasma mass spectrometry (general rule 0412) Chromatographic and mass spectrometric conditions and system suitability test: Polystyrene divinylbenzene copolymer carrier bonded trimethylammonium anion exchange material or equivalent material was used as filler (250 × 4.1mm; 10 μ m); 0.025mol/l ammonium dihydrogen phosphate solution (adjust pH value of ammonia to 8.0) was used as mobile phase a, water as mobile phase B, and gradient elution was carried out according to the following table; flow rate was 1.0ml/min Inductively coupled plasma mass spectrometry (ICP-MS) with coaxial atomizer and collisional pool was used for detection The isotope of 75as was selected for determination, and the reaction mode of collisional pool was selected or the appropriate correction equation was selected according to the requirements of different instruments for correction The resolution of six different forms of arsenic shall meet the requirements, among which the resolution of arsenicone, betaine and arsenite shall not be less than 1.0 (Figure 2) Fig 2 Determination Atlas of arsenic form and valence state 1 Arsenicone; 2 Arsenical betaine; 3 Arsenite (trivalent arsenic) 4 Dimethylarsenic; 5 Dimethylarsenic; 6 Preparation of arsenic acid (pentavalent arsenic) reference stock solution take arsenious acid, arsenic acid, dimethylarsenic, dimethylarsenic, arsenicone and arsenical betaine reference respectively, weigh accurately, and add water to make each reference 1ml of reference solution containing 2.0 μ g (all calculated as arsenic) is obtained Preparation of standard curve solution: accurately suck a proper amount of reference stock solution, add 0.02mol/l ethylenediamine tetraacetic acid disodium solution to make a solution containing 1ng, 5ng, 20ng, 50NG, 100ng, 200ng and 500ng (all calculated as arsenic) series concentrations every 1ml, and shake it up Preparation of test solution (1) mineral medicine and its preparation: unless otherwise specified, take an appropriate amount of test sample, take 20-30mg of test sample powder equivalent to arsenic content (passing No.4 sieve), weigh precisely, add an appropriate amount of artificial intestinal fluid precisely, place it in a 37 ℃ water bath for ultrasonic treatment for an appropriate time, shake it well, take an appropriate amount, and leave it standing for about 24 20-36 Take a proper amount of middle layer solution, filter it with microporous filter membrane (10 μ m), accurately measure a proper amount of continuous filtrate, dilute it with 0.02mol/l disodium ethylenediamine tetraacetate solution to a certain volume, and shake it well Prepare blank solution by the same method (2) Animal and plant traditional Chinese medicine (except class A and hair): unless otherwise specified, take 0.2-0.5g of test sample powder (passing No.3 sieve), weigh accurately, add a proper amount of nitric acid artificial gastric juice precisely, heat it in a water bath at 37-45 ℃ for about 20-24 hours, take it out, shake it well, put it for 2 hours, take the supernatant, filter it with a disposable double-layer filter membrane (10 μ m + 3 μ m), take the continuous filtrate, then get 。 Prepare blank solution by the same method A series of standard curve solutions and test sample solutions were respectively aspirated 20-100 μ L by the determination method, and then injected into the liquid chromatograph for determination Take the peak area of different forms of arsenic or different valence arsenic in the series of standard curve solutions as the ordinate and the concentration as the abscissa, draw the standard curve and calculate the content of different forms or different valence arsenic in the test solution [note] ① before use, all glass instruments shall be soaked in 20% nitric acid solution (V / V) for 24 hours or other appropriate methods to avoid interference ② Under the condition of meeting the applicability of the system, it is not necessary to prepare three kinds of mercury or six kinds of arsenic forms and their series of standard curve solutions for each determination According to the actual situation, only the series of standard curve solutions for mercury or arsenic forms and their valence states need to be analyzed can be prepared ③ In the analysis of mercury speciation and its valence state, the column efficiency of Hg2 + was lost quickly because of the influence of the incompletely capped silicon hydroxyl in the column It is suggested to use the chromatographic column with high end coverage, and if necessary, at a certain injection interval, to use the valve switching technology to wash the chromatographic column with high proportion of organic phase, and then continue the analysis ④ Nitric acid artificial gastric juice: take 32.8ml of dilute nitric acid, add 800ml of water and 10g of artificial pepsin, shake well, add water and dilute to 1000ml ⑤ Because the components of traditional Chinese medicine are complex and the contents of arsenic and mercury are quite different, the sample size in this method is only for reference The sampling quantity of mineral medicine and its preparation should be converted to 20-30mg of arsenic or mercury content; the sampling quantity of animal and plant traditional Chinese medicine (except class A and hair) should be determined according to the content of arsenic or mercury in the sample, generally 0.2-0.5g ⑥ The preparation method of test solution specified in this method is a general recommendation method In practice, the parameters can be adjusted appropriately according to the different sample matrix, and specific provisions can be made under each variety At the same time, necessary method verification can be carried out ⑦ The limit of mercury or arsenic form or valence state in the test article shall conform to the provisions of each variety 3、 Prospect after the implementation of Chinese Pharmacopoeia 2020 According to the regulatory needs and the international advanced standards, the State Pharmacopoeia Commission released 2322 "determination method of mercury and arsenic forms and their valence states" (the third draft for comments), which included the test methods with strong specificity, good discrimination and high maturity into the standard, and reasonably upgraded the determination standard of mercury and arsenic forms and their valence states The upgrading of the standard promotes the international development of Chinese herbal medicine Standardization development will play a better leading role in promoting the international influence and competitiveness of traditional Chinese medicine products and promoting the international trade of traditional Chinese medicine products The State Food and drug administration has made it clear that good drugs are all over the world, and inferior drugs are hard to move forward It has once again sounded an alarm for the quality supervision of the traditional Chinese medicine industry The preparation and revision of the 2020 edition of the Chinese Pharmacopoeia are more grounded, closely linked with the actual supervision, and meet the regulatory needs in a timely manner, fully reflecting the main function of the pharmacopoeia standard, that is, encouraging good drugs, eliminating inferior drugs, identifying the true and the false, judging the good and better In order to serve the supervision, relevant enterprises need to understand the innovation and change of mercury and arsenic speciation and its valence determination standard in advance, so as to make clear the connotation of each standard and understand the content of the standard