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An offline silver-impregnated silica gel solid-phase extraction (Ag-SPE) approach combined with a programmed temperature vaporization-gas chromatography-flame ionization detector (PTV-GC-FID) was proposed for routine analysis of mineral oil saturated hydrocarbons (MOSH) in chocolates. The MOSH in chocolates were extracted by n-hexane and 1 mL of extract was purified by offline Ag-SPE column. The SPE columns packed with 0.3％ Ag-activated silica gel were used to separate MOSH from triglycerides and olefins in chocolates. The eluent of MOSH fraction was only 5 mL and then concentrated to 0.2 mL through nitrogen blowing with little evaporation loss. The PTV parameters were as follows: the initial temperature was set at 45℃ and held for 1 min (split ratio was 200∶1), then warmed up to 360℃ at linear gradient of 250℃ minSymbolm_1 and held for 27 min (split valve was closed for 2 min followed by split ratio of 100∶1). The GC injection volume was 40 μL. The GC column was heated from 35℃ (3 min) to 350℃ at 25℃/min, and then raised to 370℃ (10 min) at 5℃/min. The flow rate of the carrier gas was 1.3 mL/min (and pressure was 60 kPa), FID temperature was set at 380℃. The limit of quantification (LOQ) and the recoveries of the method were 0.5 mg/kg and 84.9％-108.6％, respectively, with the relative standard deviations (RSD) of 0.2％-1.5％. Twenty-five commercial chocolate samples were analyzed with the proposed method, and it was found that the MOSH in three samples were not detected, and the concentrations of MOSH in other 22 samples were 1.09-8.15 mg/kg (the concentrations of MOSH with C16-C35 component were 0.56-4.43 mg/kg). The results suggested that it was necessary to routinely detect mineral oil contamination in chocolates for food safety.

8.Mutant prevention concentration of moxifloxacin combined with cefoperazone/sulbactam against carbapenem-resistant acinetobacter baumannii

Xinyue ZHANG ; Chengchun SUN ; Yanwen GONG ; Chuanwei YANG ; Ying LIU

Chinese Pharmacological Bulletin 2014;(6):825-828

9.Mutation of HPV 18 E6 inhibits the growth of Hela cells and induces apoptosis

Lijun ZUO ; Baojie WU ; Fei LIU ; Yanwen WU ; Liqiang FAN

Chinese Journal of Biochemical Pharmaceutics 2014;(1):51-55

Objective To construct pcDNA 3.1(+)/HPV 18 E 6 fusion gene and a single-codon mutation pcDNA 3.1(+)/E6 F49R or pcDNA 3.1(+)/E6 F127R fusion gene in eukaryotic expression vector and study the effects on proliferation and apoptosis of cervical carcinoma cell line Hela. Method HPV 18 E6 gene sequence and the single-point mutation HPV 18 E6 F49R or HPV 18 E6 F127R were amplified from total RNA of Hela cell line by reverse transcription- polymerase chain reaction ( RT- PCR), then the gene sequences were respectively inserted into pcDNA 3.1(+) vector to reconstruct recombinant plasmids which were transfected transiently into Hela cells. MTT and RT-PCR were used to test the expression levels of HPV 18 E6 and the growth of HeLa cells after transfected about 48 h. The proliferation and apoptosis of Hela cells were detected respectively by cell counting and AO/EB fluorescent vital staining. Results The pcDNA 3.1(+)/HPV 18 E6, pcDNA 3.1(+)/E6 F49R and pcDNA 3.1(+)/E6 F127R eukaryotic expression vectors were successfully constructed. The gene of HPV 18 E6 was discriminably detected in the HeLa cells which were transfected with the recombinant plasmids. After several days, the proliferation of Hela cells transfected with pcDNA 3.1(+)/E6 F49R or pcDNA 3.1(+)/E6 F127R plasmid were obviously inhibited and the apoptotic rates were significantly increased, then the proliferation of cells transfected with pcDNA(+)/HPV 18 E6 was rather increased slightly, and we could observe the phenomena of early apoptosis and the formation of thekaryopyknosis by fluorescent microscope in the cells transfected with pcDNA 3.1(+)/E6 F49R or pcDNA 3.1(+)/E6 F127R. Conclusion The eukaryotic expressing vectors encoding HPV 18 E6 F49R and HPV 18 E6 F127R provide fundamental basis for the further study on HPV 18 E6 mechanism as well as prevention and treatment of uterine cancer.