Study On The In Vitro Metabolism Of Organophosphate Diesters And Their Detection Characteristics In Biological Samples

Organophosphate diesters(di-OPEs)are often regarded as the metabolism or degradation products of organophosphate triesters(tri-OPEs),which are widely used as flame retardants.However,according to investigations,some di-OPEs have industrial production sources and uses including flame retardants and plasticizers.In recent years,di-OPEs have been found in a variety of environments or biological media in many regions of the world,and the concentration levels of some di-OPEs are comparable to their parent tri-OPEs.In this study,the instrument method of 14 kinds of target di-OPEs were optimized,and then the in vitro metabolism of target di-OPEs in liver microsomal metabolic system was explored.Finally,the detection characteristics of target di-OPEs and its potential phase I and phase II metabolites in human serum and wild fish samples were analyzed.This study found that the use of PFP column and mobile phase system with a certain concentration of formic acid is conducive to the detection of di-OPEs.Among 14 kinds of target di-OPEs,bis(2-ethylhexyl)phosphate(DEHP),isopropyl diphenyl phosphate(ip-DPHP),di-ortho-cresyl phosphate(Do CP),di-meta-cresyl phosphate(Dm CP)and di-para-cresyl phosphate(Dp CP),which have higher Log Kow values,could be significantly degraded in vitro when incubated with rat or human liver microsomes for 2 hours.In metabolic kinetics experiments,the fitted metabolic kinetic parameters Vmax and Km values of ip-DPHP are 0.493± 0.164 nmol/min/mg and 3.69 ± 2.42 μM,respectively.Do CP,Dm CP and Dp CP are not significantly metabolized within 5 min.DEHP is still in the first-order reaction stage under the initial concentration range of 0.1 ~ 10 μM.The presumed main metabolic pathway of the above five metabolizable di-OPEs is hydroxylation.In addition,DEHP may undergo the metabolic process of dealkylation and hydroxyl further oxidation to aldehyde or carbonyl group,and ip-DPHP may also have the metabolic process of dealkylation and oxidative ring-opening.There are 8 di-OPEs in human serum(n = 120),but the overall concentration level is low.The concentration range of ∑8di-OPEs is ND ~ 1.64 ng/m L.Bis(1,3-dichloro-2-propyl)phosphate(BDCIPP)is the main di-OPEs in human serum samples in this study.There are 6di-OPEs in wild fish(28 muscle samples and 7 liver samples).The concentration range of∑6di-OPEs in muscle samples is 0.48 ~ 5.92 ng/g ww,and the concentration range of∑5di-OPEs in liver samples(33.2 ~ 262 ng/g ww)is much higher than that in muscle samples.Di-n-butyl phosphate(Dn BP),ip-DPHP and DEHP are the main di-OPEs in Taihu wild fish.The potential phase I and phase II metabolites of di-OPEs in human serum(n = 30)and all wild fish samples were screened(matching parent ion and isotope peaks).It was found that there were suspicious metabolites of di-OPEs in both biological samples.Suspicious di-OPEs that may be isomer of(OH)-DEHP were screened in human serum samples,and a variety of potential phase I and phase II metabolites of DEHP were also screened in wild fish samples,which further confirmed the possibility of DEHP metabolism in vivo.However,the suspicious di-OPEs screened in this paper require more accurate structural confirmation.