Determination of p-Phenylenediamine Antioxidants in Zebrafish Embryos by Gas Chromatography-Tandem Mass Spectrometry with Ultrasonic Extraction

p-Phenylenediamine antioxidants (PPDs) are rubber antioxidants that can suppress oxidation, heat, or light radiation to delay the aging of polymer components and extend the service life of rubber products. In recent years, PPDs have been found in the environment, and their ecological risks remain largely unknown, which cause widespread concern. In this work, a method of gas chromatography-tandem mass spectrometry (GC-MS/MS) with ultrasonic extraction was developed for the determination of PPDs in zebrafish embryos. In order to have good performance, extraction solvents and sorbents were optimized. Under optimal conditions, embryonic samples were combined with 5 mL of dichloromethane and ultrasonically extracted for 15 min. The extraction procedure was repeated twice, after that the resulting extracts were purified by 0.1 g primary secondary amine (PSA). Based on internal standard method, the analytes were detected using GC-MS/MS in selective reaction monitoring (SRM) mode with optimal parameters. The results showed that the chromatographic peaks of analyte were completely separated, and the baseline was smooth. The correlation coefficient (R2) of each analyte exceeded 0.99 in the concentration range of 0.1-200 μg/L. Method detection limits (MDLs) and method quantification limits (MQLs) were in the range of 0.38-0.68 ng/g and 1.28-2.28 ng/g, respectively. Except for the average recovery of N,N′-bis(methylphenyl)-1,4-benzenediamine (DTPD), which was higher than 120%, the average recoveries of N-(1,3-dimethylbutyl)-N′-phenyl-p-phenylenediamine (6PPD), N-phenyl-N′-cyclohexyl-p-phenylenediamine (CPPD) and N-isopropyl-N′-phenyl-1,4-phenylenediamine (IPPD) were 73.1%-109%, and the intra-day and inter-day relative standard deviations (RSDs) were 2.97%-15.0% and 4.76%-18.8%, respectively. In order to demonstrate the feasibility of this method, it was applied to detect PPDs in zebrafish embryos exposed to PPDs. The results indicated that PPD concentrations were 12.5-16.4 ng/g. After calculation, the measured bioconcentration factors of each analyte within 48 h were 1.70-33.6 L/kg. These values were much lower than the corresponding predicted bioconcentration factors, because PPD concentrations in vivo had not reached equilibrium during the exposure time of 48 h. This method is simple, sensitive, and suitable for the detection of PPDs in zebrafish embryos, which facilitates future bioaccumulation and toxicity mechanism studies for a comprehensive assessment of the ecological risks of PPDs

Effect of enzymolytic product on the curing characteristics and physico-mechanical properties of natural rubber

The natural latexes with different protein contents were prepared by centrifugation and then treated by alkaline protease to hydrolyze protein on the rubber particle surface, and the effects of protein hydrolysates on curing characteristics and dynamic behavior of natural rubber were studied. The results showed that with the increment of centrifugation times nitrogen mass fraction decreased from 0.27% to 0.13%, complex viscosity of raw rubber and curing rate of compound decreased, cross-linking density and mechanical properties of natural rubber vulcanizates declined, and storage modulus reduced under strain and frequency sweep. However, complex viscosity, curing rate, mechanical properties and storage modulus of natural rubber treated by multi-centrifugation and enzyme increased compared with those of natural rubber treated by only multi-centrifugation, due to the role of protein hydrolysates, and especially for the natural rubber treated by twice centrifugation and enzyme, curing rate could reach 17.8 min-1 and tensile strength could reach 23.73 MPa, which were significantly higher than curing rate of 9.59 min-1 and tensile strength of 20.52 MPa of natural rubber treated by only twice centrifugation, respectively