Analysis Of Arsenic Speciation And Arsenic Metabolism Of Realgar In The Intestinal Flora

The clinical use of Realgar in China can date back to thousands of years ago.It was firstly recorded "The Shennong Ben Cao Jing",with insecticide detoxification,dampness and phlegm,antimalaria efficacy,the clinical use in our country has been for thousands of years.Since the 1950s,some domestic doctors achieved remarkable therapeutic effects by using realgar compound preparation or single prescription to treat hematological diseases,malignant lymphoid diseases and even solid tumors.Due to its toxicity,the safe use of Realgar and its realgar-containing formulations is of utmost importance.The toxicity of realgar is not only related to the total amount of arsenic,but also to the chemical speciation of arsenic and its metabolites.With the different valence states and chemical forms,the toxicity of arsenic compounds performed huge difference.The intestinal tract is an important site for oral drug metabolism in the body,and a large number of bacteria in the intestinal tract have played a vital role in the biotransformation of drugs.Objective:Firstly,to determine the total arsenic in Realgar and optimize the extraction conditions of soluble arsenic in it.Secondly,through the detection of soluble arsenic speciation in Realgar,we preliminarily studied the arsenic species and arsenic metabolism of soluble arsenic in As single-element and realgar under the action of gut microbiota.Moreover,the amount of glutathione(GSH)in gut microbiota was determined to reveal the mechanism.Methods:1.The total arsenic in Realgar was determined by microwave digestion hydride generation atomic absorption spectrometry and microwave digestion inductively coupled plasma mass spectrometry(ICP-MS).2.The extraction technology of soluble arsenic from Realgar were optimized by Plackett-Burman and Box-Behnken design.3.A method was developed for simultaneous determination of six As species in AsB,AsC,As(Ⅲ),As(Ⅴ),MMA,and DMA using high performance liquid chromatography(HPLC)coupled with inductively coupled plasma mass spectrometry(ICP-MS).The established method was used to detect arsenic species of As single element and Realgar in the role of gut microbiota.4.DTNB method was established to determine the reduced Glutathione(GSH)in gut microbiota.Results:1.Determination of arsenic in Realgar with microwave digestion-hydride atomic absorption spectrometer and microwave digestion-inductively coupled plasma-mass Spectrometry(ICP-MS)was 96.20%and 97.07%,respectively,based on As2S2.2.Optimization of extraction process of soluble arsenic in realgar hrough Plackett-Burman and Box-Behnken design and the parameter of optimum extraction method were as follows:water as extraction solvent,Realgar 15 mg,water 10 mL,extraction temperature 80 ℃,ultrasonic extraction 3 times,60 minutes each time,Finally,soluble arsenic in Realgar accounted for 4.16%of total arsenic.3.The method of HPLC(HPLC)-inductively coupled plasma mass spectrometry(icp-ms)was used to separate AsB,AsC,As(III),As(V),MMA and DMA.A Dionex IonPacTM AS7 RFICTM anion exchange column(4x250 mm)was used as the analytical column.The mobile phase was Phase A 3.75 mM ammonium carbonate,mobile Phase B:100 mM ammonium carbonate.Gradient elution,0.00～3.40 min,100%A;3.50 min 20%A,80%B;4.50-6.00 min 100%B;6.10-8.00 min 100%A.The flow rate is 1.2 mL/min,and the sample volume is 20 μL.The experimental results show that the linear range of six arsenic species is 1～100 ng/mL,the R2 is greater than 0.9990,the detection limit is 0.1～0.2 ng/mL,and the precision RSD is 0.93%～1.83%,all less than 2%.The recovery of standard addition is 97.28%～105,24%,and RSD is less than 6%.The optimized extraction process was used to extract soluble arsenic from Realgar.The results showed that:arsenic in soluble arsenic of Realgar is As(Ⅲ),As(Ⅴ),and As(Ⅲ)content is higher than As(Ⅴ).As single element contains only As(V).The gut microbiota samples were extracted by ultrasonic water bath.The experimental results show that:the As(Ⅲ)and As(Ⅴ)arsenic species detected by HPLC-ICP-MS under the action of intestinal micro flora by soluble arsenic of realgar and As single element.4.The glutathione(GSH)in the gut microbiota of 0～48h was measured by DTNB method,GSH was not detected in the first 0～2 h,but the amount of it peaked at 4 h,was 50.36 μg/mL,and stabilized after 12～48 h.At 48 h,it reduced to 13.30 μg/mL.Conclusions:1.The arsenic content in Realgar is greater than 90%in the Chinese pharmacopoeia of 2015,which is in accordance with the requirements of pharmacopoeia.2.Plackett-Burman and Box-Behnken design and optimize the extraction process of soluble arsenic in Realgar is stable and reliable,and the experimental accuracy is high,which is basically consistent with the predicted value of the model.3.Simultaneous determination of six arsenic species was established by HPLC-ICP-MS.This method used As-7 anion column to separate six arsenic species compounds.The separation effect was good.Each arsenic species compound was completely separated and the detection results were more accurate and reliable.It can be applied to arsenic in soluble arsenic in realgars and analysis of arsenic species in intestinal flora.4.GSH in intestinal flora was determined by DTNB method for the first time,which indicated that GSH in gut microbiota was reduced to glutathione,which may play an important role in the methylation and metabolism of inorganic arsenic.The methylation and metabolism of inorganic arsenic in intestinal flora need to be further studied.