Study On The Constitutes And Distributing Of Selenium Speciation In Enriched Biomaterials By ICP-MS And HPLC-ICP-MS

The element Se is known to have enzymatic roles and imuninty.The essentiality of Se was established in the observation of deficiency syptoms which were reduced after Se supplementation.Many regions in the world,including our country are lack of Se.The Se products are greatly expected.The doses of Se is narrow,Besides the total content of selenium,the chemical form is also important due to the differences in bioavailabilty and toxity of the different forms.So the establishment of simple,effective qualative and quantitative analysis technique are of important.As Se is unstabile and trace in amount in biomaterials.The former complex extraction may cause the lose and exchange of Se specitaion.Although there are a few study on the determination of total Se,to the best of our knowledge,the Se sepeciation determination are still of challenge.Laminaria japonica is widely produced in China,by treated with Se enriched nutrients in the laboratory mode for 8 days,different Se species can be got,and the enriched role of Se were firstly studied by ICP-MS.As RP-HPLC-ICP-MS technich has the specility of high seperation,high sensitivity and low detection,we established three chromatagraph systems with ICP-MS detection,three Se specices standards are well seperated.We also compared four solutions(0.1mol/L HCl,0.1mol/L NaOH,20mmol/L NH₄COOH-CH₃OH(5%), Proteaseâ…©â…£)with ultrasonic extraction,and established a RP-HPLC-ICP-MS method to determine the Se speciation for the first time.with 0.1mol/L HCl,the linearity,precision and recovery are well,as a result,the method is easy and effective and acurate for Se species determination in Laminaria japonica.we continued to have garlic sprout enriched with Selinite for one months,and determined the tatol Se and Se species with the established method,and do further methodology validation to establish a theoretic fundation for the application of Se enriched Laminaria japonica.Besides,19 elements of 23 large economical algae in ShanDong Province were determined by ICP-MS with closed digestion,and then identified by clustering analysis.Furthermore,some Se enriched products at present time were also determind for scientific and accurate data with microwave-ICP-MS.The main results were summarized as follows:(1)Determination of 19 elements in 23 large economical algae by ICP-MS with closed digestion.in the result,the algea are rich of the body necessary elements,such as Na,Mg,Al,K,Ca,Zn,Fe,and so on.13 algea are all of K,Ca,P macroelements and Fe,Mn,Cu,Zn microelements.Laminaria has the high ability of enriching elements,itt was rich of K,Ca,Na,Mg,Zn,Mn,As etal,and selenium is about 0.281μg/g,As one of the economic and edible algea,it was prouduced widely in our country,so was choosed as the study object.The elements concentration in those samples were calculated and the algae sort were identified according to the elements components and concentration by the PCA and CA methods.such as,Scytosiphon lomentarius(Lyngb.)J.Ag and Dictyopteris divaricata(Okamura)Okamura,Laminaria japonica Aresch and Sarcodia montagneana(Hooker et Harvey)J.Agardh clustered as one group seperately,which all belongs to phaeophyta.The Ceramium kondoi Yendo,Gracilaria vermiculophylla, Grateloupia filicina(Lamx.)C.Ag,Porphyra yezoensis Ueda and Corallina sp.are all Rhodophyta.so the same phylum are of similar elements symptom the research provides new theory for the identification of algea.(2)We established three chromatographic system in conjunction with ICP-MS to separate Se species standards(Na₂SeO₃,MeSeCys,SeMet).The chromatographic systemsâ‘¢were:Alltima C18 column(250mm×4.6mm,5μm),with pre-column,mobile phase:20mmol/L ammonium acetic-methanol(95:5),flow 1.0ml/min,injection volumn 20μL.The systemâ‘ has a low pH,so it was of a higher requirement of the column.the standards' peaks were relatively sharp and seperated well in short time by systemâ‘¢,and the system was also suitable for further LC-MS,TOF-MS analysis,and so were choosed as the system for the next quantative analysis of Se.(3)A new RP-HPLC-ICPMS method to determin different Se species in Laminaria japonica with Comparing four solution of 0.1mol/L HCl,0.1mol/L NaOH, 20mmol/L ammonium acetic-methanol(5%),nonspecific Proteaseâ…©â…£,with ultrasonic extraction were studied,and the methodology validation were also done.With 0.1mol/L HCl,three species(Na₂Se₂O₃,MeSeCys,SeMet)and one unknown species were identified and determined,Na₂SeO₃ was 72.26%,and MeSeCys 5.73%of the sum.the linearity(y=2325.9x,r=0.9999) and the precision(RSD=0.5%, n=7) are good by methodology validation,the recovery reached 82.00%.As a result,the method is easy,usible and reliable.First,A laboratory method was established for Cultivating Laminaria japonica with the addition of selenite(10mg/L) and N(300μg/L),P(30μg/L) nutrient solution for 8 days,and the tatol Se and the enriched role were also studied by the new technich ICP-MS for the first time.the enriched rate was fastest in the first few hours and the Se concentration of the enriched Laminaria japonica reached 900μg/g,about 3000 times of the unriched(0.281μg/g).Second,with 0.1mol/L NaOH,20mmol/L ammonium acetic-methanol(5%) and nonspecific Proteaseâ…©â…£as the extract solution,the main Se species identified were Na₂Se₂O₃ and SeMet,the SeMeCys was not detected.and the recoverity was low relatively,so different extract solution result in different Se specices,more study are needed for the quantitive and qualitive analysis of HPLC-ICP-MS on bio-Se.(4)We continued to determine the Se species in Se enriched garlic sprouts by the established RP-HPLC-ICPMS methods for further methodological validation.Se concentration reached 250μg/g by cultivating in Se enriched solution for one monthes,about 2066 times of the unenriched(0.121μg/g).six species,including selenite,MeSeCys,SeMet and three unknown species were well separated and determined,selenite were greatly transformed to MeSeCys and SeMet,Se recovery was 102.5%by 0.1mol/L HCl extract solution with ultrasonic processing.with 20mmol/L ammonium acetic-methanol(5%) and proteaseâ…©â…£solution extraction,the recovery were 82.87%and 82.81%.the results provided further methodological validation for the established RP-HPLC-ICPMS technich for Se species determination in biomaterials.(5)The Se enriched eggs,onion,tea and rice sold in the market were studied by ICP-MS with microwave digestion.The Se concentration was all improved to some extend.such as,the Se of enriched egg white was 1.50*10⁴μg/g,3.69 times of the unenriched(4.07*10³Î¼g/g);the enriched egg yellow was 6.31*10³Î¼g/g,2.04 times of the unenriched(3.09*10³Î¼g/g);the enriched rice(113.5μg/g),tea(459.8μg/g),green chinese onion(214μg/g),onion(1.22*10⁴μg/g)were 241,72,64,20.1 times of that of the unenriched rice(54μg/g),tea(72μg/g),green chinese onion(12μg/g),onion(342.8μg/g). from the results,we can also find that the plants have the higher ability of enriching microelements.the Se species of the Se enriched product are also expected for further study.