目的：探索前处理方法和外源性物质对药物中 β-硫化汞（β-HgS）转化为甲基汞带来的影 响。方法：在前处理溶液体积为 5 mL 的条件下,考察超纯水、酸性前处理溶剂、碱性前处理溶剂、酸浓度、物理浸萃前处理手段和外源性物质对 β-HgS 转化为甲基汞的影响,采用高效液相色谱与冷蒸气-原子荧光光谱法（HPLC-CV-AFS）在线耦合测定甲基汞。色谱柱为 Agilent Eclipse Plus C18 柱（4.6 mm× 150 mm, 5 μm）,以甲醇 -0.06 mol·L-1 乙酸铵溶液（含 0.1% L-半胱氨酸）（5∶95）为流动相, 流速 1 mL·L-1,进样体积 100 μL,检测波长 254 nm。结果：15 mg β-HgS 经超纯水前处理后,测得甲基汞浓度低于检测下限;经 5 mol·L-1 盐酸或 25% 氢氧化钾-甲醇溶液分别前处理后,发现有 β-HgS 转化为甲基汞,甲基汞质量浓度为（0.63±0.01）μg·L-1 和（2.32±0.07）μg·L-1,占可溶性汞的比值分别为 0.067% 和 1.162%;β-HgS 经加热法前处理后,转化甲基汞质量浓度为（0.71±0.03）μg·L-1,显著高于振荡法[（0.52±0.02）μg·L-1]和超声法[（0.61±0.01）μg·L-1],3 种物理浸萃方法的甲基汞 占可溶出汞比值无显著性差异,分别为 0.059%、0.061% 和 0.062%;盐酸浓度为 0.5~5.0 mol·L-1 时, 测得甲基汞质量浓度为（0.52±0.02）~（0.63±0.04）μg·L-1（占可溶性汞的比值为 0.454%~0.069%）, 8 mol·L-1 盐酸浓度下甲基汞的浓度显著升高为（1.50±0.08）μg·L-1（占可溶性汞的比值为 0.018%）;与 β-HgS 对照组[甲基汞浓度（0.64±0.02）μg·L-1,占可溶性汞的比值为 0.069%]相比,加入纤维素和植物混合组分后甲基汞浓度为（0.76±0.03）μg·L-1 和（1.44±0.07）μg·L-1,占可溶性汞的比值分别为 0.070% 和 0.114%。结论：本研究表明,当样品中存在较高含量 β-HgS（≥ 15 mg）时,在 5 mol·L-1 盐酸或 25% 氢氧化钾-甲醇溶液做前处理溶剂的条件下,β-HgS 经前处理后会转化为甲基汞,转化的甲基汞浓度与酸浓 度呈正相关,加热能够促进甲基化的发生,某些外源性物质如纤维素等可促进高含量 β-HgS样品转化为甲基汞。
Objective：To explore the effects of pretreatment methods and exogenous substances on theconversion of β-HgS to methylmercury in drugs. Methods：With 5 mL pretreatment solution, β-HgS convert to methylmercury caused by ultrapure water, acidic pretreatment solvent, alkaline pretreatment solvent, acid concentration, physical leaching pretreatment methods and exogenous substances were investigated. Speciation analysis of methylmercury was done by on-line coupling of high performance liquid chromatography with cold vapor generation atomic fluorescence spectrometry（HPLC-CV-AFS）. Agilent Eclipse Plus C18 was uesd and the mobile phase consisted of 5% methanol,0.06 mol·L-1 ammonium acetate solution contained 0.1% L-cysteine（5∶95）at a flow rate of 1 mL·L-1。The injection value was 100 μL and the detection wavelength was 254 nm. Results：After 15 mg β-HgS was pretreated with ultrapure water,the measured methylmercury concentration did not reach the lowest detection limit of this LC-AFS. After pretreating with 5 mol·L-1 hydrochloric acid and 25% potassium hydroxide-methanol solutions,the mercury element derived from β-HgS was methyl mercury concentrations in both kinds of solutions were（0.63±0.01）μg·L-1 and（2.32±0.07）μg·L-1,accounting for 0.067% and 1.162% in dissolved mercury respectively. β-HgS was converted into methylmercury after pre-treatment by heating method,and the concentration was（0.71±0.03）μg·L-1,accounting for 0.059% of the dissolved mercury,which was significantly higher than the shaking method（0.52±0.02）μg·L-1 and ultrasonic method（0.61±0.01）μg·L-1. There was no significant difference in the ratio of methylmercury to dissolved mercury in the three physical extraction methods,which were 0.059%,0.061% and 0.062% respectively. When the concentration of hydrochloric acid was 0.5-5.0 mol·L-1,the measured methylmercury concentration was（0.52±0.02）-（0.63±0.04）μg·L-1（accounting for 0.454%-0.069% of the concentration of dissolved mercury）,and the methylmercury concentration increases significantly to（1.50±0.08）μg·L-1 at 8 mol·L-1 acid concentration（accounting for 0.018% of the concentration rate of dissolved mercury）. compared with the β-HgS control group（0.64±0.02）μg·L-1,the methylmercury concentrations after adding cellulose and plant mixture were（0.76±0.03）μg·L-1 and（1.44±0.07）μg·L-1,accounting for 0.070% and 0.114% of dissolved mercury, respectively. Conclusion：We demonstrate that when there is a high content of β-HgS（≥ 15 mg）in the sample, with 5 mol·L-1 hydrochloric acid or 25% potassium hydroxide-methanol solution as the pretreatment solvent,β-HgS can be converted into methylmercury after pretreatment. The converted methylmercury concentration is positively correlated with the acid concentration. Heating can promote methylation of β-HgS. Some exogenous substances such as cellulose can promote the conversion of samples with high content of β-HgS into methylmercury.
[1] LI C,XU W,CHU S,et al.The chemical speciation,spatial distribution and toxicity of mercury from Tibetan medicine Zuotaibeta-HgS and HgCl2 in mouse kidney[J].J Trace Elem Med Biol,2018,45:104
[2] 陈朝军,武世奎,李刚,等.民族医药中汞的形态分析研究进展[J].药物分析杂志,2013,33(1):165
CHEN CJ,WU SK,LI G,et al.Research progress of mercury speciation analysis in minority medicine[J].Chin J Pharm Anal,2013,33(1):165
[3] CHEN Y,YIN Y,SHI J,et al.Analytical methods,formation,and dissolution of cinnabar and its impact on environmental cycle of mercury[J].Crit Rev Environ Sci Technol,2018,47(24):2415
[4] ZHANG BB,LI WK,HOU WY,et al.Zuotai and HgS differ from HgCl2 and methyl mercury in Hg accumulation and toxicity in weanling and aged rats[J].Toxicol Appl Pharmacol,2017,331(15):76
[5] SYVERSEN T,KAUR P.The toxicology of mercury and its compounds[J].J Trace Elem Med Biol,2012,26(4):215
[6] HINTELMANN H,FALTER R,ILGEN G,et al.Determination of artifactual formation of monomethylmercury (CH3Hg+) in environmental samples using stable Hg2+ isotopes with ICP-MS detection:calculation of contents applying species specific isotope addition[J].Fresenius J Anal Chem,1997,358(3):363
[7] CARRASCOL,VASSILEVA E.Determination of methylmercury in marine sediment samples:Method validation and occurrence data[J].Anal Chim Acta,2015,853:167
[8] YUN Z,HE B,WANG Z,et al.Evaluation of different extraction procedures for determination of organic mercury species in petroleum by high performance liquid chromatography coupled with cold vapor atomic fluorescence spectrometry[J].Talanta,2013,106:60
[9] PIETILA H,PERAMAKI P,PIISPANEN J,et al.Determination of low methylmercury concentrations in peat soil samples by isotope dilution GC-ICP-MS using distillation and solvent extraction methods[J].Chemosphere,2015,124:47
[10] JAGTAP R,MAHER W.Measurement of mercury species in sediments and soils by HPLC-ICPMS[J].Microchem J,2015, 121:65
[11] HJ 977-2018 水质烷基汞的测定吹扫捕集/气相色谱-冷原子荧光光谱法[S].2018
HJ 977-2018 Water Quality-determination of Alkyl Mercury-purge and Trap/Gas Chromatography Cold Atomic Fluorescence Spectrometry[S].2018
[12] HAMMERSCHMIDT CR,FITZAGERALD WF.Formation of artifact methylmercury during extraction from a sediment reference material[J].Anal Chem,2001,73(24):5930
[13] LIU M,HE Y,BAUMANN Z,et al.Traditional tibetan medicine induced high methylmercury exposure level and environmental mercury burden in Tibet,China[J].Environ Sci Technol,2018,52(15):8838
[14] RODRÍGUEZ MARTÍN-DOIMEADIOS RC,MONPERRUS M,KRUPP E,et al.Using speciated isotope dilution with GC-inductively coupled plasma MS to determine and unravel the artificial formation of monomethylmercury in certified reference sediments[J].Anal Chem,2003,75(13):3202
[15] DELGADO A,PRIETO A,ZULOAGA O,et al.Production of artifact methylmercury during the analysis of certified reference sediments:use of ionic exchange in the sample treatment step to minimise the problem[J].Anal Chim Acta,2007,582(1):109
[16] GB 5009.17-2014 食品安全国家标准食品中总汞及有机汞的测定[S].2015
GB 5009.17-2014 National Food Safety Standard,Determination of Total Mercury And Organic Mercury in Foods[S].2015
[17] 中华人民共和国药典 2015 年版.四部华人民共和国药典 2015 年版.四部[S].2015:330 ChP 2015.Vol Ⅳ[S].2015:330
[18] 李岑,占堆,楞本才让,等.藏药佐太的化学成分_ 汞配位结构及微观形貌分析[J].光谱学与光谱分析,2015,35(4):210
LI C,ZHAN D,LENG-BEN-CAI-RANG,et al.Analysis of the chemical composition,mercury coordination structure and micro-morphology of Tibetan medicine Zuotai[J].Spectrosc Spect Anal, 2015,35(4):210
[19] 杨红霞,于明杰,斗周才让,等.当佐与安置精华散对人体肝肾功能的影响[J].华西药学杂志,2016,31(5):548
YANG HX,YU MJ,DOU-ZHOU-CAI-RANG.et al.Effects of Dangzuo and Anzhi Jinghua powder on the function of liver and kidney[J].West China Pharm Sci,2016,31(5):548
[20] NAP.Dietary Reference Intakes for Energy,Carbohydrate,Fiber, Fat,Fatty Acids,Cholesterol,Protein,and Amino Acids[M]. Washington,DC:The National Academies,2005:107
[21] BLOOM NS,COLMAN JA,BARBER L.Artifact formation of methyl mercury during aqueous distillation and alternative techniques for the extraction of methyl mercury from environmental samples[J]. Fresenius J Anal Chem,1997,358(3):371
[22] HORVAT M,BLOOM NS,LIANG L.Comparison of distillation with other current isolation methods for the determination of methyl mercury compounds in low level environmental samples:Part 1. sediments[J].Anal Chim Acta,1993,281(1):135
[23] TSENG CM,DIEGO AD,WASSERMAN JC,et al.Potential interferences generated during mercury species determination using acid leaching,aqueous ethylation,cryogenic gas chromatography and atomic spectrometry detection techniques[J].Chemosphere, 1999,39(7):1119
[24] QIN C,CHEN M,YAN H,et al.Compound specific stable isotope determination of methylmercury in contaminated soil[J].Sci Total Environ,2018,644:406
[25] MIRANDA-ANDRADES JR,KHAN S,PEDROZO-PENAFIEL MJ,et al.Combination of ultrasonic extraction in a surfactant-rich medium and distillation for mercury speciation in offshore petroleum produced waters by gas chromatography cold vapor atomic fluorescence spectrometry[J].Spectrochim Acta B,2019,105641. doi:10.16/j.sab.2019.105641
[26] GB 2762-2017 食品中污染物限量食品安全国家标准[S].2017 GB 2762-2017 Maximum Levels of Contaminants in Foods,National Food Safety Standard[S].2017
