OBJECTIVE To establish HPLC methods with different separation principles to analyze the relevant impurities in the APIs of bivalirudin from seven enterprises, to provide a basis for the comprehensive control of related substances of bivalirudin. METHODS Reversed-phase high performance liquid chromatography(RP-HPLC) was used to separate and analyze 11 kinds of impurities. Hydrophilic chromatography(HILIC)-HPLC was used to control four process impurities. Polymers were determined by size exclusion chromatography(SEC)-HPLC. RESULTS The established RP-HPLC could effectively separate the principal component and 11 impurities, the correction factors of 11 impurities were between 0.8−1.2, the detection concentration of bivalirudin was 0.1 μg·mL−1, and the detection limit was 0.004%. The established HILIC-HPLC could effectively separate the principal components and four process impurities, and the detection concentration of bivalirudin was 0.3 μg·mL−1, and the detection limit was 0.01%. Under SEC-HPLC conditions, the polymer and bivalirudin peaked sequentially, the resolution of the two was 2.9, the detection concentration of bivalirudin was 6 ng·mL−1, and the detection limit was 0.000 6%. Fifteen kinds of known impurities and polymers in 15 batches of samples from 7 enterprises were calculated by the self-control method of principal components, and the impurity contents from different enterprises had a certain correlation with their production processes. CONCLUSION The three different principles of the method have good specificity, high sensitivity, good durability, and reliable results, and can be used for quality control of substances related to bivalirudin.

Objective:To determine the relative correction factor of pre-vitamin D and simplify the calculation method of vitamin D assay.Methods:By studying the calculation method of vitamin D content in drug standards of various countries,HPLC was used to determine the relative correction factor of pre-vitamin D,and the influencing factors of determination were investigated.Results:The relative correction factors of pre-vitamin D at 254 nm and 265nm wavelength were determined by statistical analysis of 7 laboratories in China.Conclusion:Using the pre-vi-tamin D relative correction factor method to calculate the total amount of vitamin D simplified the experimental steps can be simplified by the pre-vitamin D relative correction factor method to calculate the total amount of vitamin D and the random operating errors can be avoided.The method is rapid and accurate,and lay a solid foundation for further improving the standard of vitamin D preparations.

Host cell residue DNA is one of the most common impurity which can affect the safety of biological products,therefore,domestic and international regulatory agencies have required the limit for host cell residue DNA in different biological products,either at the final product qualification or the appropriate intermediate control stage.The removal effect is verified by monitoring the residue DNA of products in different production stages,which is beneficial for assuring the scientificity and stability of the production process.In order to strengthen the understanding of control strategy about host cell residual DNA,the paper reviews progress in host cell residual DNA in biological products by authors'work experience and other's research,which provides reference for future work.

Humans ; HIV Infections/drug therapy* ; HIV-1/genetics* ; Tenofovir/therapeutic use* ; Anti-HIV Agents/therapeutic use* ; RNA ; Drug Combinations

Abstract OBJECTIVE To establish a detection method for fingerprints and content determination of free sugar and hydrolyzed monosaccharide of intracellular glycopeptides in Coriolus versicolor. METHODS PMP derivatization after ultrasonic extraction with water as solvent was determined for free sugars, and PMP derivatization after acid hydrolysis was determined for hydrolyzed monosaccharides. The free sugars and hydrolyzed monosaccharides of raw materials and intermediates of intracellular glycopeptides from different manufacturers were analyzed by HPLC qualitatively and quantitatively. The fingerprints were analyzed by chemometrics analysis, and the differences between intracellular glycopeptides produced by different manufacturers were discussed. RESULTS Both two methods were validated by methodology. The samples from different manufacturers were clustered separately, and three free sugars and two hydrolyzed monosaccharides contributed greatly to the quality difference of intracellular glycopeptides of Coriolus versicolor. CONCLUSION The established analytical method can be effectively used for the determination of free sugars and hydrolyzed monosaccharides in the intracellular glycopeptide of Coriolus versicolor and the study of fingerprints. The adopted chemometrics research method provides guidance for the quality control of the intracellular glycopeptide of Coriolus versicolor.

Objective:To investigate the c-Jun N-terminal kinase (JNK), CCAAT/enhancer-binding protein homologous protein (CHOP) pathway apoptosis and the changes of cytokine levels in immune-related organs and tissues of sepsis mice at different time points.Methods:Twenty-seven male BALB/c mice were divided into normal group, sepsis 6 hours group and sepsis 12 hours group by the block randomization method, with 9 mice in each group. The sepsis model was made by cecal ligation and puncture (CLP). Blood sample was collected from each group at the corresponding time point, and the serum levels of tumor necrosis factor-α (TNF-α), interleukin (IL-1β, IL-10) were measured by enzyme linked immunosorbent assay (ELISA). The spleen, thymus and appendix tissues were taken from the mice to detect the expressions of phosphorylation-JNK (p-JNK), JNK1, CHOP and cleaved caspase-3 protein by Western Blot.Results:The level of cytokines, p-JNK/JNK1 ratio, CHOP and caspase-3 in spleen tissues, and the CHOP, caspase-3 in thymus and appendix tissue in the sepsis 6 hours group were significantly higher than those in the normal group [serum TNF-α (ng/L): 24.29±3.09 vs. 2.93±2.09, serum IL-1β (ng/L): 5.00±3.19 vs. 3.54±1.53, serum IL-10 (ng/L): 1 963.93±270.20 vs. 275.09±45.21, spleen p-JNK/JNK1 ratio: 0.257±0.126 vs. 0.154±0.068, spleen CHOP/β-actin: 0.201±0.131 vs. 0.142±0.068, spleen caspase-3/β-actin: 0.215±0.126 vs. 0.098±0.088, thymus CHOP/β-actin: 0.122±0.071 vs. 0.089±0.067, thymus caspase-3/β-actin: 0.258±0.145 vs. 0.108±0.045, appendix CHOP/β-actin: 0.361±0.134 vs. 0.215±0.112, appendix caspase-3/β-actin: 0.439±0.211 vs. 0.321±0.145, all P < 0.05]. However, there were no significant difference in the p-JNK/JNK1 ratio in thymus and appendix (thymus p-JNK/JNK1 ratio: 1.221±0.776 vs. 1.168±0.475, appendix p-JNK/JNK1 ratio: 2.014±1.227 vs. 1.828±0.915, both P > 0.05). Cytokine levels and the p-JNK/JNK1 ratio, CHOP, caspase-3 in spleen, thymus, and appendix in the sepsis 12 hours group were further increased when compared with those in the sepsis 6 hours group, except for a significant decrease in IL-10 level [serum IL-10 (ng/L): 1 698.98±210.52 vs. 1 963.93±270.20, serum TNF-α (ng/L): 41.66±6.57 vs. 24.29±3.09, serum IL-1β (ng/L): 10.37±4.14 vs. 5.00±3.19, spleen p-JNK/JNK1 ratio: 0.399±0.135 vs. 0.257±0.126, spleen CHOP/β-actin: 0.298±0.145 vs. 0.201±0.131, spleen caspase-3/β-actin: 0.353±0.145 vs. 0.215±0.126, thymus p-JNK/JNK1 ratio: 1.667±0.891 vs. 1.221±0.776, thymus CHOP/β-actin: 0.207±0.133 vs. 0.122±0.071, thymus caspase-3/β-actin: 0.416±0.179 vs. 0.258±0.145, appendix p-JNK/JNK1 ratio: 2.425±1.361 vs. 2.014±1.227, appendix CHOP/β-actin: 0.456±0.189 vs. 0.361±0.134, appendix caspase-3/β-actin: 0.635±0.289 vs. 0.439±0.211, all P < 0.05]. Conclusions:The endoplasmic reticulum pathway JNK and CHOP pathways are involved in immune-related cell apoptosis and cytokine expression in mice with sepsis. Apoptosis is more obvious at 12 hours than at 6 hours, and the inflammatory response is stronger.

Objective To explore the etiology and treatment of acute intestinal obstruction.Methods Clinical data of patients who underwent operation for acute intestinal obstruction in Zhongshan Hospital from May 2012 to May 2017 were collected and retrospectively analyzed.Results 721 patients were included and the ratio of males to females was 1.55 ∶ 1.There were 48.8％ in old-aged group and 51.1％ in young-middle-aged group.The most common causes of ileus included tumor in 376 cases (51.5％),adhesion in 168 cases (23.3％),hernia in 70 cases (9.7％),intraluminal obstruction in 42 cases (5.8％) and others in 79 cases.There was a significant difference between incarcerated hernia in elderly group and middle-young-aged group (4.6％ vs.15.1％,x2 =22.4,P ＜ 0.01).The length of hospitalized days in patients with tumor and incarcerated hernia in elderly group were significantly longer than young-middle-aged patients [(15.3±8.6)d vs.(13.4±6.3)d,t =-2.5,P＜0.05;(10.1 ± 6.7) d vs.(6.4 ± 2.9) d,t =-2.2,P ＜ 0.05].The length of hospitalized days by limited operation in patients with tumor were significantly shorter than those by emergency operation [(16 ± 12)d vs.(18 ± 24) d,t =-0.3,P ＞ 0.05].Conclusion Tumor and adhesion are the main causes of acute intestinal obstruction.Neoplastic bowel obstruction from small intestine or proximal colon requires emergency surgery.

OBJECTIVE To explore the effect of aqueous extract of Zheng Chaihu Yin(ZCH)on paracetamol(acetaminophen,APAP)-induced hepatotoxicity. METHODS Male ICR mice were divided into three scenarios randomly:the single treatment dose of ZCH,multiple treatment or pretreatment dose of ZCH. Each scenario had a up control group and an APAP model group,while single treatment dose of ZCH group had a ZCH group at the same time. The dose of APAP and ZCH was 500 mg·kg-1 and 36 g · kg- 1,respectively. 24 h after the last administration,plasma and liver samples were prepared. Ultra- performance liquid chromatography/quadrupole- time- of- flight mass spectrometry (UPLC-Q-TOF-MS)based metabolomics profiling was used to examine changes in plasma after expo?sure to ZCH,APAP or co-exposure to ZCH and APAP. Glutamic pyruvic transaminase (GPT) and glutamic oxaloacetic transaminas (GOT) values were determined by a biochemical auto analyzer in plasma. Histopathologic changes in the liver were observed and the area was calculated after HE staining. The data were analyzed with SPSS16.0 statistical software and the results were compared with the test between the two groups to find biomarkers. Also,SIMCA software was used for partial least squares-discriminant analysis (PLS-DA) pattern recognition. RESULTS Compared to control group, APAP dosing alone caused an increase in plasma transaminases and alterations in multiple metabolic pathways. Compared to APAP group,decrease in plasma transaminases was noted when ZCH was administered after or prior to APAP. Histopathologic results showed that in the single treatment group, multiple treatment group and pretreatment group,ZCH could alleviate the liver damage induced by APAP from (32.3 ± 12.0)% to (14.2 ± 9.9)%,(8.6 ± 7.9)% to (5.2 ± 1.7)% and (32.5 ± 10.0)% to (5.2 ± 6.4)%(P<0.05). Similarly,the PLS-DA of the LC-MS data showed that the groups dosed with APAP alone were the most distinct from controls,while animals dosed with ZCH prior to or after APAP treatment were located near control group. Metabolic spectrum results showed that ZCH could restore the changes in endogenous substances including lipid metabolism,amino acid metabolism,sugar metabolism and energy metabolism induced by APAP to normal. CONCLUSION ZCH water-extraction plays major roles in the regulation of metabolism on APAP-induced liver injury. These studies demonstrate that UPLC-Q-TOF-MS-based metabolomic analysis can be sensitively and accurately predict the initiation and progres?sion of liver injury and greatly contribute to a better understanding of the hepatoprotective effects of ZCH in a clinical environment.

Objective To evaluate the sensitizations of Hydroxyethyl Starch 40 Sodium Chloride Injection . Methods Active systemic anaphylaxis ( ASA) test, internal sensitive index ' s determination and screening and Passive Cutaneous Anaphylaxis ( PCA) test are carried out .Observe the allergy safety of the samples .Results We use guinea pigs to carry on ASA test and PCA test with Hydroxyethyl Starch 40 Sodium Chloride Injection from 45 batches of 3 companies and no changes have occurred .Measuring and comparing the sensitive index of HIS ,IgG, IgM and IgE in plasma, IgM and IgE are not obvious variation;HIS and IgG positive group and negative group and sample groups are different .Conclusions It is suggestion that Hydroxyethyl Starch 40 Sodium Chloride Injection safety evaluation should include allergic reactions , HIS and IgG sensitive index monitoring in clinical application of hydroxyethyl starch .