Speciation Analysis Of Arsenic In Blood And Urine And Its Application

Arsenic species are widely distributed in nature,its abundance ranks 20 th.Most of them exist in sedimentary rocks and siliceous rocks.Every year,the arsenic compounds produced are mainly used for arsenic metal,special alloys,pesticide,wood preservative,catalyst,pigment and glass industry and other fields.Because of such wide range of use,artificial or accidental arsenic poisoning or exposure accidents occurred frequently.Although people's consciousness of environmental protection has been greatly enhanced and the use and production of arsenic compounds has been dropped significantly,there are still arsenic compounds in air,soil,water and sediment,in the form of arsenic trioxide(As2O3),arsenite(As?),arsenate(As?),monomethylarsonic acid(MMA),dimethylarsonic acid(DMA),arsenobetaine(As B),arsenocholine(As C).Long exposure to arsenic in the environment,can lead to acute and chronic arsenic poisoning;severe cases can even lead to human gene mutation,cell cancer and fetal malformations.Therefore,the International Agency for Research on Cancer(LARC),the US Centers for Disease Control(CDC)has identified arsenic as a first class of carcinogens,and arsenic compounds are also one of the 30 key pollutants monitored globally.There were many death cases of arsenic compounds in Chinese and foreign history,for example the ‘Morinaga milk arsenic poisoning' incident.It can be seen that arsenic and arsenic compounds have been the focus of forensic toxicology since ancient times.At present,the application of arsenic poisoning in the forensic practice is mainly based on the test results of the total arsenic content.The arsenic with different forms has different physical and chemical properties and biological toxicity.Therefore,when evaluating arsenic poisoning degree,the implementation of only the total arsenic content is not rigorous.For example,the toxicity of inorganic arsenic compounds in As? is 60 times that of As?,and 70 times that of the methyl arsenic compounds(such as MMA and DMA).The toxicity order has been widely studied and arsenic compounds in the order of toxic sequence are: arsine(Arsine,As H3),AsIII,AsV,MMA,DMA,Trimetlylamine oxide(TMAO),As C,As B,the latter two are generally considered non-toxic.In summary,Due to the differences of the biological toxicity of arsenic compounds,arsenic poisoning forensic evaluation should be based on speciation analysis of arsenic compounds as the basis for the evaluation of the content.The aim of this study was to establish the arsenic compounds as the target,and to establish the detection technology of arsenic compounds to meet the needs of forensic evaluation of arsenic poisoning and the requirements of evidence reliability.High-performance liquid chromatography(HPLC)is a good technology for compound separation,while inductively coupled plasma mass spectrometry(ICP-MS)is widely used in metallic element analysis for its high sensitivity and reliability.The combination of the two techniques suitable for metal element analysis,the study will use the joint technology to establish a method for arsenic speciation analysis.This study focuses on the characters of different arsenic species,taking blood and urine as biological specimens to explore a scientific,reliable and specific determination methods of arsenic compounds by HPLC-ICP-MS.The validation of the methods proves them effective.The established methods were applied to the practice of forensic investigation and are suitable to make up for the gaps the judicial practice of arsenic poisoning cases only with total arsenic content determination.The first part of this study was the HPLC-ICP-MS method for 6 different kinds of arsenic speciation in blood.500?L blood sample was used in the method.Triton was first applied to damage cells and EDTA disodium salt to complex arsenic compounds in cells.Acetonitrile was added for protein deposition after sonication.The separation was carried out with the anion-exchange column Hamilton PRP-X100(4.1×250mm)as the solid phase.And the supernatant was injected for analysis by HPLC-ICP-MS.The mobile phase was a gradient composition consisting of 100 m M ammonium carbonate with the p H adjusted to 9.5 and Milli-Q water.The analysis took 20 minutes.The limits of detection of As C,As B,As?,DMA,MMA,AsV were all 1.66-10ng/m L,while the lower limits of quantifation were 5-30ng/m L for As C,As B,As?,DMA,MMA,AsV.The linearity ranges of six normal arsenic compounds as As C,As B,As?,DMA,MMA,AsV were 5-250ng/m L,5-250ng/m L,15-500ng/m L,5-250ng/m L,15-500ng/m L,30-1000ng/m L respectively.The linearity correlation coefficients were 0.999 for all the analytes.The RSD for inter-and intra-day were less than 6.1%.The matrix effect and extraction recovery were 86.4%-118.2%,90.2%-104.7%,respectively.The second part of this present study was to establish a HPLC-ICP-MS method measuring 6 different kinds of arsenic speciation in urine.With references of related analysis methods of metallic element,the study was researched the arsenic speciation analysis methods based on Milli-Q water.The urine sample was mixed with 0.1% Triton with 50 m M EDTA disodium salt.The mixture was vortexed briefly,sonicated for 40 minutes and then centrifuged for 10 minutes.The supernatant was then mixed with acetonitrile(1:1)and centrifuged for 5 minutes,after which the supernatant was transferred into a new vial for analysis.The limits of detection of As C,As B,As?,DMA,MMA,AsV were 0.5,0.6,5,1,3,10ng/m L,respectively.The lower limits of quantifation in urine is 5,5,15,5,15,30ng/m L for each analyte.The linearity range of six normal arsenic compounds is 5-250ng/m L,5-250ng/m L,15-500ng/m L,5-250ng/m L,15-500ng/m L,30-1000ng/m L,respectively.The linearity correlation coefficients were greater than 0.995 for all the analytes.The results of room temperature stability and repeated freeze-thaw stability were good.The established method was applied to an actual arsenic poisoning case for forensic investigation.Combined with the total arsenic content test results,the results of the speciation analysis of human blood arsenic compounds were used to exclude the controversial focus of the case that edible seafood and injection of vitamin C would generate arsenic(AsIII)as the conclusion of poisoning.The results of this study can provide technical support and key basis for arsenic poisoning cases and related cases in forensic examination,so that the results of forensic evaluation of arsenic poisoning cases are more scientific,rigorous and reliable.