Valuation and Uncertainty Evaluation of the Purity Determination of Aminopyrine by Different Methods / 中国药师

Objective:To study the valuation and uncertainty evaluation of the purity determination of aminopyrine by two different principle methods. Methods:According to Technical Norm of Primary Reference Material and Technical Norm of Measurement,HPLC and acid-base titration were selected for studying the valuation of the purity determination of aminopyrine, and the uncertainty evaluation of the two different principle methods was systematically evaluated. Results:By using the two different principle methods,the standard value and uncertainty of aminopyrine content was 99.66% ± 0.08%(k = 2,P = 0.95). Conclusion:The valuation and uncertainty evaluation of the purity determination of aminopyrine by using HPLC and acid-base titration are accurate and reliable,which can avoid the defects by using single analysis method,and is helpful to improve the level of quality evaluation and control of aminopyrine. The study provides scientific basis for the development of aminopyrine purity reference materials.

Prevalence analysis of noninvasive follicular thyroid neoplasm with papillary-like nuclear features in a Third Class A Level Hospital in Guiyang / 中华内分泌代谢杂志

Objective To analyze of the prevalence of encapsulated follicular variant of papillary thyroid carcinoma (EFVPTC) and noninvasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTP) by 2017 World Health Organization ( WHO) classification of tumors of endocrine organs in Guiyang. Methods A retrospectively analysis of patients who had a thyroid surgery and confirmed thyroid cancer by pathological diagnosis in the Affiliated Hospital of Guizhou Medical University from 2009 to 2016. PTC and FVPTC by 2014 WHO classification of tumors of endocrine organs , and according to the 2017 WHO classification of tumors of endocrine organs ,the thyroid papillary carcinomas were reviewed and some had been confirmed as EFVPTC and NIFTP, with analysis of the prevalence and prognosis of NIFTP. Results Of the 1207 cases of thyroid carcinoma, 1150 cases were papillary carcinoma, the constituent ratio of thyroid carcinoma was 95.28％, the FVPTC was 72 cases, and the proportion of thyroid carcinoma was 5. 97％; the proportion of FVPTC in thyroid carcinoma decreased from 2009 to 2016 ( P <0.05). After pathological sections being reviewed, 10 cases had been confirmed as non-encapsulated infiltrative FVPTC, the ratio of thyroid cancer to thyroid cancer was 0.83％; EFVPTC was found in 62 cases, accounting for 5.14％ of thyroid carcinoma, these included 2 cases of NIFTP confirmed by the 2017 WHO classification of tumors of endocrine organs and 60 cases encapsulated invasive FVPTC, the percentage of thyroid carcinoma was 0.17％ and 4.97％. 62 cases of EFVPTC with the exception of 11 cases without further visit, while the remaining 51 cases of EFVPTC were followed up. Death, local or distant metastases were defined as adverse events. An adverse event was seen in 11 of 49 of the cases of invasive EFVPTC, including 2 died of disease; The NIFTP were alive with no evidence of disease. Conclusion The diagnosis of NIFTP according to new WHO classification of endocrine organ tumors in 2017 has little expected impact in Guiyang.

Constituent ratio and subtype changes of thyroid cancer in Guiyang during the 8 years before and after adjustment of iodized salt content / 中华内分泌代谢杂志

Objective To investigate the composition and pathological subtypes of thyroid cancer in patients with thyroid nodule surgery in Guiyang in recent 8 years, and to analyze their influencing factors. Methods A retrospective pathological analysis of 4 262 thyroid surgery cases in Affiliated Hospital of Guizhou Medical University during the 2009-2016 to investigate the proportion of thyroid cancer and pathological subtype. The iodine content of salt was reduced at the end of 2012 in Guiyang. 2009-2012 as the pre down-regulation group(n＝1 572), 2013-2016 as the after down-regulation group ( n＝2 690), then comparative analysis before and after the adjustment of the iodine content of salt composition of thyroid cancer and changes of pathological subtype was performed. Results From 2009 to 2016, the proportion of thyroid cancer were 17.08%, 17.52%,15.65%, 18.58%, 19.80%, 29.35%, 35.34%, and 48.33%, increased year by year (P＜0.05). Thyroid microcarcinoma were 2.14%, 4.74%, 3.40%, 3.65%, 3.80%, 7.03%, 9.10%, and 25.95%(P＜0.05). The constituent ratio of thyroid cancer after adjustment of salt iodine content was higher than before. Papillary thyroid carcinoma is the main pathological subtype before and after adjustment of salt iodine content. The proportion of female patients was higher than that of males. The age of patients with thyroid cancer after adjustmen was higher than before ( P＜0. 05). Conclusions In the past 8 years, the constituent ratio of thyroid cancer and thyroid microcarcinoma in Guiyang increased year by year. The reason may be related to the increase of radiation in the environment, the improvement of medical level and the higher detection rate of thyroid microcarcinoma. The relationship between iodine nutrition and thyroid cancer needs to be further studied.[Key words] Thyroid cancer; Thyroid microcarcinoma; Thyroid papillary carcinoma; Pathological type;Iodized salt; Iodine nutrition

Simultaneous Determination of Kanamycin and Kanamycin B in Kanamycin Sulfate Injection by HPLC-CAD / 中国药师

Objective: To establish an HPLC with charge aerosol detection (HPLC-CAD) method for the determination of kanamy-cin and kanamycin B in kanamycin sulfate injection. Methods: The samples were injected into a Boston Green ODS C18(250 mm× 4. 6 mm, 5 μm) column. The mobile phase was composed of 0. 2 mol·L-1trifluoroacetic acid aqueous solution-methanol (95: 5) , the flow rate was 1. 0 ml·min-1and the column temperature was maintained at 30 ℃. The nebulization temperature for the CAD was maintained at 55℃ and the gas pressure was 56. 4 psi. Results: The linear range of kanamycin was 0. 385-38. 500 μg·ml-1( r=0. 999 9), and the limit of detection was 0. 075μg·ml-1, the limit of quantitation was 0. 154 μg·ml-1, and the average recovery of the method was 100. 97% (n=9). The linear range of kanamycin B was 0. 374-37. 400 μg·ml-1(r=1. 000 0), and the limit of de-tection was 0. 075μg·ml-1, the limit of quantitation was 0. 150 μg·ml-1, and the average recovery of the method was 100. 44% (n=9). Conclusion: The method for the determination of kanamycin and kanamycin B by HPLC-CAD is simple and accurate, the limit of detection is lower than HPLC-ELSD, and the quality of kanamycin sulfate injection can be effectively controlled.

IgG antibodies against Dengue virus non-structure protein 1 mediate passive sys-temic anaphylaxis in mice / 中国免疫学杂志

Objective:To ascertain whether the immune complexes (ICs) formed by Dengue virus 1 non-structure protein 1 (DENV1 NS1)and its IgG antibodies could mediate passive systemic anaphylaxis (PSA) and to explain the pathogenesis of Dengue hemorrhagic fever or Dengue shock syndrome (DHF/DSS).Methods:The monoclonal antibodies (mAbs) or mAb cocktails from 20 IgG mAbs of DENV1 NS1 prepared in this lab were screened to initiate PSA and passive cutaneous anaphylaxis (PCA) in mice.Meanwhile, the effects of GdCl3 and platelet activating factor ( PAF) antagonist CV-3988 on PSA induced by the NS1-IgG ICs were observed.Results:Two groups of monoclonal antibody cocktails with purified NS 1 were proved to be capable of provoking PCA and PSA in mice,whereas the other mAbs or mAb cocktails could be not .The murine PSA initiated by NS1-IgG(5D25+3B1) ICs could be sig-nificantly inhibited by in vivo treatment with GdCl3 or PAF antagonist CV-3988.Conclusion: The NS1-IgG ICs formed with DENV1 NS1 and IgG mAb cocktails can mediate PSA and PCA ,but not all of ICs formed by DENV 1 NS1 mAbs or mAb cocktails with DENV 1 NS1 can induce PSA ,indicating that it may be related to the special epitopes of DENV 1 NS1.The monocyte/macrophages and PAF may be as major effector cells and the major mediator for PSA induced by NS 1-IgG ICs,respectively.

Determination of Content and Content Uniformity of Memantine Hydrochloride Dispersible Tablets by an Internal Standard Method of GC / 中国药师

Objective: To establish a GC method for the determination of content and content uniformity of memantine hydrochloride dispersible tablets.Methods: The sample was dissolved in water, alkalified by sodium hydroxide solution and extracted by methylene chloride.An HP-5 gas chromatography column (50 m×0.32 mm, 1.05 μm) was used.The column temperature was programming increased, and the initial temperature maintained at 120 ℃ for 3 min, and then raised to 220 ℃ at a rate of 10℃·min-1 and maintained for 7 min.A hydrogen flame ionization detector (FID) was used and the split ratio was 1∶1.The inlet temperature was 230 ℃ and the detector temperature was 260 ℃.The injection volume was 1 μl and the carrier gas was nitrogen with high purity at a flow rate of 3.0 ml·min-1.Adamantane was used as the internal standard, and the internal standard method was used for the calculation.Results: The calibration curve was linear over the range of 0.05-1.0 mg·ml -1 (r=0.999 7).The detection limit and the limit of quantification was 1.1 ng and 3.3 ng, respectively.The average recovery was 100.2% (RSD =0.73%, n=9).Conclusion: The method has the advantages of simple operation, small extraction process toxicity, little environmental pollution, high accuracy and high specificity, and can be used for the determination of content and content uniformity of memantine hydrochloride dispersible tablets.

Similarity Evaluation of Dissolution Curves for Generic Preparation and Reference Preparation of Bisaco-dyl Enteric-coated Tablets / 中国药房

OBJECTIVE:To investigate the similarity of dissolution curves between generic preparations and reference prepara-tions of Bisacodyl enteric-coated tablets in various dissolution mediums,and to provide reference for improving production technolo-gy and internal quality of generic preparations. METHODS:Paddle method was adopted with rotation speed of 75 r/min. The disso-lution test was performed using 1000 mL pH 6.0 phosphate buffer solution,pH 6.8 phosphate buffer solution,water containing 2% sodium lauryl sulfate. HPLC method was used to determine average accumulative dissolution of main components from 3 kinds of generic preparations and reference preparations at different time points to draw out dissolution curves. Similarity factor(f2)meth-od was used to the similarity of dissolution curves. RESULTS:Dissolution curves of reference preparations were basically the same in 3 kinds of dissolution mediums. But the dissolution curve f2 of one generic preparation among 3 manufactures to dissolution curve of reference preparation were ≥50,namely the similarity. CONCLUSIONS:The quality of generic Bisacodyl enteric-coat-ed tablets produced by different manufacturers is obviously different;the generic preparations needs to be further improved in the production technology and internal quality. For domestic generic preparation,it is necessary to strengthen the real-time monitoring of its dissolution curve,to ensure the drug quality.

Uncertainty Evaluation in the Determination of Sodium Valproate Tablets by GC with an Internal Standard Method / 中国药师

Objective:To evaluate the measurement uncertainty in the determination of sodium valproate tablets by GC with an in -ternal standard method , and determine the main sources of uncertainty .Methods:A GC internal standard method was selected to sys-tematically analyze the uncertainty in the determination of sodium valproate tablets , including the sample quantity , dilution ratio, purity and area repeatability of chromatographic peaks .Results: The expanded uncertainty of sodium valproate tablets was 2.7%, and the determination range of sodium valproate tablets was (96.3 ±2.7)%(k=2).Conclusion:The established GC internal standard meth-od for the uncertainty evaluation is reliable , which is helpful to improving the quality evaluation and control of sodium valproate tablets .

Simultaneous Residual Determination of Two Organic Solvents in Bisacodyl Raw Material by Head-space GC / 中国药房

OBJECTIVE:To establish a method for simultaneous residual determination of dichloromethane and ethyl acetate in bisacodyl raw material. METHODS:Head-space GC was performed on the capillary column of 6% cyanopropyl phenyl-94% di-methyl polysiloxane(DB-624)by temperature programming,the temperature of injector was 220 ℃,detector was flame ionization detector with temperature of 250 ℃,carrier gas was high purity nitrogen with the flow rate of 3.0 ml/min,split ration was 1∶10, headspace heating temperature was 70 ℃,equilibration time was 30 min,volume of headspace vial was 5 ml,and the injection volume was 1 ml. RESULTS:The linear range was 6-120μg/ml for dichloromethane(r=0.999 9)and 50-1 000μg/ml for ethyl ac-etate(r=0.999 9);the limit of quantitation was 0.2,1.7 μg,limit of detection was 0.06,0.5 μg;RSDs of precision,stability and reproducibility tests were no higher than 3%;recoveries were 100.30%-102.00%(RSD=0.63%,n=9) and 100.10 %-101.30%(RSD=0.44%,n=9). CONCLUSIONS:The method is simple and accurate,and can be used for the simultaneous residual deter-mination of dichloromethane and ethyl acetate in bisacodyl raw material.

Determination of Content and Content Uniformity of Bisacodyl Enteric-coated Tablet by HPLC / 中国药房

OBJECTIVE:To establish a method for the determination of content and content uniformity in Bisacodyl enter-ic-coated tablet. METHODS:HPLC method was performed on the column of Agilent ZORBAX C18 with mobile phase of acetoni-trile-20 mmol/L ammonium acetate(adjusted pH to 5.0 with acetic acid)(55∶45,V/V),the detection wavelength was 265 nm,col-umn temperature was 30℃,flow rate was 1.0 ml/min,and the volume injection was 20 μl. RESULTS:The linear range of bisaco-dyl was 50-1 000 μg/ml(r=0.999 9);RSDs of precision,stability and reproducibility tests were lower than 1%;recovery was 99.50%-101.17%(RSD=0.5%,n=9). CONCLUSIONS:The method is reproducible with high accuracy,and suitable for the quali-ty control of Bisacodyl enteric-coated tablet.

Study on the Determination Method for Related Substances in Bisacodyl Raw Material and Enteric-coated Tablet / 中国药房

OBJECTIVE:To establish a method for the determination of related substances in bisacodyl raw material and enteric-coated tablet. METHODS:HPLC was performed on the column of Hibar C18 with mobile phase of acetonitrile-20 mmol/L ammonium acetate (acetic acid adjust pH to 5.0)(55∶45,V/V),detection wavelength was 265 nm,flow rate was 1.0 ml/min, column temperature was 30℃,and the injection volume was 20 μl. RESULTS:The linear range of bisacodyl was 0.25-5.0 mg/ml (r=0.999 9);the limits of detection and quantification were 19-25 ng and 61-68 ng for bisacodyl and impurity A,B,C,D and E;RSDs of precision,stability and reproducibility tests were lower than 2%;recovery was 99.50%-101.00%(RSD=0.5%,n=9). CONCLUSIONS:The method is specific, sensitive and reproducible, and can be used for the determination of related substance in bisacodyl raw material and enteric-coated tablet.

Expression and identification of the functional domains of dengue virus type 1 envelope protein in 293T cells / 中华微生物学和免疫学杂志

Objective To construct a recombinant expression vector for expression of the function-al domains of dengue virus serotype 1 ( DENV1 ) envelope ( E ) protein in native soluble form. Methods The genes encoding the functional domains of DENV1-E protein (1-394 aa) were amplified with PCR and then cloned into the Psectag2B-Fc eukaryotic expression vector.The 293T cells were transfected with the recombinant vector by cationic lipid-based delivery.The cell clones expressing the fusion DENV1-E-Fc protein were screened out with 2 mg/ml of Zeocin.Immunofluorescence assay ( IFA) was performed to analyze the antigenicity and integrity of the fusion protein.The fusion proteins were purified from cell lysate with Protein-G and further identified by Western blot assay.Results The soluble form of fusion protein with a molecular weight of about 90×103 was obtained at a yield of about 25 μg per 1×107 cells.The results of IFA indicated that the fusion protein kept its integrity with right conformational epitopes.The fusion protein was successfully expressed with the advantage of good specificity as indicated by IFA and Western blot assay. Conclusion The recombinant fusion protein in soluble form was successfully expressed in eukaryotic ex-pression system, which paved the way for further investigation on the function of DENV1 E protein and its protective epitopes.

Determination of Purity and Uncertainty Evaluation of Bisacodyl by Differential Scanning Calorimetry / 中国药房

OBJECTIVE:To establish the purity determination of bisacodyl by differential scanning calorimetry(DSC)and the valuation of uncertainty. METHODS:DSC was conducted to detect the purity of bisacodyl and determine the optimal testing condi-tions. According to related standards,indium enthalpy change values,measurement repeatability,weighing process,instrument tem-perature deviation and system software deviation were systematically analyzed. The results were verified by HPLC. RESULTS:When the fiducial probability P was 0.95,the standard value and uncertainty of content of bisacodyl was (99.88 ± 0.06)% mea-sured by DSC. Weighing process,instrument temperature deviation and system software deviation had great effects on the total un-certainty. The result of HPLC and DSC were the same. CONCLUSIONS:The established DSC can quickly and accurately determine the chemical purity of bisacodyl. The uncertainty evaluation is reliable. Regularly calibrated and verificated equipment and strict con-trol of the weighing process will help to improve the accuracy measured by DSC;and it provides a new analysis method for the de-termination of purity of bisacodyl.

Comparing the multiplex RT-PCR method and liquichip technology in the detection of diarrhea-related virus / 中华检验医学杂志

Objective To compare the detection efficiency between multiplex RT-PCR method and liquichip technology for screening the viral etiological agents of diarrhea.Methods The development of the multiplex RT-PCR method.A total of 107 feces samples from patients who suffered from diarrhea and attended to Zhujiang Hospital of Southern University from September 2013 to February 2014 were collected and tested in parallel by both multiplex RT-PCR and xTAG Gastrointestinal Pathogen Panel ( xTAG GPP) for Adenovirus, Norovirus genogroupⅠandⅡ, as well as by both multiplex RT-PCR and monoplex RT-PCR for Astrovirus and Sapovirus.To evaluate the sensitivity and specificity of multiplex RT-PCR, xTAG GPP and monoplex RT-PCR were used as reference.Kappa coefficient test was used to evaluate the consistency among the methods.The detection limit and accuracy of multiplex RT-PCR were evaluated by detection of serial dilution of positive plasmids and products sequencing for the five viral agents.Results The multiplex RT-PCR showed high consistency with xTAG GPP and monoplex RT-PCR, in which Kappa value was 0.885 and 1.000 respectively( P=0.000 ).Compared to xTAG GPP, the sensitivity and specificity of the multiplex RT-PCR were at average of 80.8%( 21/26 ) and 100%( 295/295 ) respectively.The detection limit and accuracy of multiplex RT-PCR were 104 copies /μl-106 copies/μl.Conclusion The high consistency indicated that both the multiplex RT-PCR and xTAG GPP are useful as a special,sensitive, high throughput and rapid diagnostic tools for the detection of the major viral pathogens related to diarrhea in clinical laboratory.

Mapping of the B Cell Neutralizing Epitopes on ED III of Envelope Protein from Dengue Virus / 病毒学报

Dengue virus (DENV) envelope [E] protein is the major surface protein of the virions that indued neutralizing antibodies. The domain III of envelope protein (EDIII) is an immunogenic region that holds potential for the development of vaccines; however, the epitopes of DENV EDIII, especially neutralizing B-cell linear epitopes, have not been comprehensively mapped. We mapped neutralizing B-cell linear epitopes on DENV-1 EDIII using 27 monoclonal antibodies against DENV-1 EDIII proteins from mice immunized with the DENV-1 EDIII. Epitope recognition analysis was performed using two set of sequential overlapping peptides (16m and 12m) that spanned the entire EDIII protein from DENV-1, respectively. This strategy identified a DENV-1 type- specific and a group-specific neutralizing epitope, which were highly conserved among isolates of DENV-1 and the four DENV serotypes and located at two regions from DENV-1 E, namely amino acid residues 309-320 and 381-392(aa 309-320 and 381-392), respectively. aa310 -319(310KEVAETQHGT319)was similar among the four DENV serotypes and contact residues on aa 309 -320 from E protein were defined and found that substitution of residues E309 , V312, A313 and V320 in DENV-2, -3, -4 isolates were antigenically silent. We also identified a DENV-1 type-specific strain-restricted neutralizing epitope, which was located at the region from DENV-1 E, namely amino acid residues 329-348 . These novel type- and group-specific B-cell epitopes of DENV EDIII may aid help us elucidate the dengue pathogenesis and accelerate vaccine design.