Objective The aim of this study was to develop a method for the determination of nine vulcanizing agents and accelerators in pharmaceutical rubber stoppers by headspace gas chromatography with sulfur chemiluminescence detector(HS-GC-SCD).Methods A DB-1 capillary column(30 m×0.32 mm×0.25 μm)was used with He as the carrier gas.The injection port temperature was 220 ℃,the split ratio was 10∶1,and the carrier gas flow rate was 1.5 mL·min-1.The temperature program was as follows:the starting temperature was 40 ℃,and it was increased to 60 ℃ at a rate of 5 ℃ per minute,and then to 260 ℃ at a rate of 15℃per minute.SCD detector parameters:base temperature was 280℃,combustion chamber temperature was 800 ℃,air flow rate was 60 mL·min-1,and hydrogen flow rate was 40 mL·min-1.Results The sample pretreatment method was simple and convenient,and it effectively reduced the extraction loss.The methodological results showed that all sulfides showed good linear relationships within the concentration range(r2≥ 0.990).The RSD was less than 2％;and the average recovery rate was 86.4％-97.5％.From the analysis of the sample test results,free sulfur is commonly found in rubber stoppers.Among all the samples of the vulcanization system,the free sulfur detected in the sulfur vulcanization system is higher than the free sulfur produced by degradation of other vulcanization systems.Conclusions This method is simple to operate,compared with other existing detection methods.HS-GC-SCD has a smaller matrix effect,higher sensitivity and selectivity,and is appropriate for quantifying the concentrations of sulfide present in various rubber stoppers.

Objective The aim of this study was to develop a method for the determination of nine vulcanizing agents and accelerators in pharmaceutical rubber stoppers by headspace gas chromatography with sulfur chemiluminescence detector(HS-GC-SCD).Methods A DB-1 capillary column(30 m×0.32 mm×0.25 μm)was used with He as the carrier gas.The injection port temperature was 220 ℃,the split ratio was 10∶1,and the carrier gas flow rate was 1.5 mL·min-1.The temperature program was as follows:the starting temperature was 40 ℃,and it was increased to 60 ℃ at a rate of 5 ℃ per minute,and then to 260 ℃ at a rate of 15℃per minute.SCD detector parameters:base temperature was 280℃,combustion chamber temperature was 800 ℃,air flow rate was 60 mL·min-1,and hydrogen flow rate was 40 mL·min-1.Results The sample pretreatment method was simple and convenient,and it effectively reduced the extraction loss.The methodological results showed that all sulfides showed good linear relationships within the concentration range(r2≥ 0.990).The RSD was less than 2％;and the average recovery rate was 86.4％-97.5％.From the analysis of the sample test results,free sulfur is commonly found in rubber stoppers.Among all the samples of the vulcanization system,the free sulfur detected in the sulfur vulcanization system is higher than the free sulfur produced by degradation of other vulcanization systems.Conclusions This method is simple to operate,compared with other existing detection methods.HS-GC-SCD has a smaller matrix effect,higher sensitivity and selectivity,and is appropriate for quantifying the concentrations of sulfide present in various rubber stoppers.

Mutants of proteins are the basis for studying their structure and function, this work aimed to establish an efficient and rapid method for constructing multi-site mutants. When four or more adjacent amino acid residues need to be mutated, firstly, two long and two short primers (long primers Ⅰ/Ⅰ, short primersⅡ/Ⅱ) were designed: the long primers contain mutated sites, and the number of mutant bases is ≤20 bp, the short primers do not contain mutated sites; GC contents of the long and short primers are ≤80%, and the difference of annealing temperature is ≤40 °C. Then two sets of reverse PCR amplifications were performed using primer pairs (Ⅰ/Ⅱand Ⅰ/Ⅱ) and templates, respectively. After amplification, each system can obtain non-methylated linear plasmids which contain mutated sites, and the breakpoints of the two sets of linear plasmids amplified by primers Ⅰ/Ⅱ and Ⅲ/Ⅳ were distributed on both sides of the mutated sites. Followed by digested by DpnⅠ to remove the methylated templates, the recovered PCR products, which were mixed in an equimolar ratio, were performed another round of denaturation and annealing: the two sets of linear plasmids were denatured at 95 °C and then annealed with each other's single-stranded DNA as templates to form open-loop plasmids, and then the transformants containing the mutations will be obtained after transformed the open-loop plasmids into Escherichia coli competent cells. Results showed that, this method can mutate 4 to 11 consecutive amino acid residues (8-20 bp) simultaneously, which will greatly simplify the construction of multi-site mutants, Thereby improve the efficiency of protein structure and function research further.
DNA Primers ; genetics ; Escherichia coli ; Mutagenesis, Site-Directed ; methods ; Plasmids ; genetics ; Polymerase Chain Reaction

BACKGROUND: It has been explained in many big sample experiments that community comprehensive prevention from the risks of cardiocerebrovascular disease can reduce its morbidity and mortality. But, it is required more samples to verify the evaluations of the process and effect of intervention.OBJECTIVE: To understand the effect of community comprehensive prevention on cardiocerebrovascular disease and hygienic costs demanded and summarizes the suitable patterns of comprehensive prevention on cardiocerebrovascular disease in countryside.DESIGN: Healthy people in community were taken as the objects and community intervention was designed.SETTING: Department of Community Hygienic Service of People's Hospital in Guzheng Town, Zhongshan City, Guangdong Province and Neurological Institute of Guangzhou Medical College.PARTICIPANTS: Totally 32 000 inhabitants were collected from 8 villages in Guzheng Town, Zhongshan City, Guangdong Province from March 1992 to March 2002, aged varied from 20 to 74 years, of which, 14 600inhabitants were male and 17 400 inhabitants were female.METHODS: The intervention of community-based prevention was applied on cardiocerebrovascular disease, including managementof hypertension,smoking control, rational nutrient, nationwide body building and diabetic management.MAIN OUTCOME MEASURES: [1] Awareness, attitude and behavior related to cardiocerebrovascular disease. [2] Management rate and control rate of hypertension. [3] Morbidity and mortality of cerebral apoplexy.RESULTS: [1] By 10-year intervention, awareness rate of hypertension in whole group was 70.29%. [2] Management rate and control rate of hypertension were increased yearly, in which, the management rate was increased from 25.66% in 1997 to 80.50% in 2002, the total control rate was increased from 4.43% in 1997 to 45.80% in 2002, the control rate of blood pressure ＜160/95 mm HG (1 mm Hg=0.133 kPa) was 58.80%and that ＜140/90 mm Hg was 32.80%. [3] The systolic and diastolic pressures in intervention community were decreased averagely, in which the D-values are 7.23 mm Hg and 3.92 mm Hg respectively. [4] The awareness, attitude and behavior related to cardiocerebrovascular disease were enhanced remarkably. [5] Morbidity and mortality of cerebral apoplexy were in tendency of decreasing yearly, from 146.90/100 000 and 108.63/100 000 in 1997 to 105.83/100 000 and 69.90/100 000 in 2002 respectively, by 41.07/100 000 and 38.73/100 000 respectively in 6 years and the age of incidence was postponed. [6] It was indicated in economic analysis that the average ratio of profit to cost between 1997 and 2001 was 2.32.CONCLUSION: Community-based comprehensive prevention on cardiocerebrovascular disease improves the awareness, attitude and behavior in community group and reduces morbidity and mortality of cerebral apoplexy.