直接竞争ELISA法检测动物源食品中利巴韦林残留

本研究针对动物源食品中利巴韦林残留的问题,建立了直接竞争酶联免疫分析方法,快速高效地检测利巴韦林残留。采用活泼酯法将利巴韦林(RBV)与钥孔血蓝蛋白(KLH)偶联制备利巴韦林完全抗原(KLH-RBV),通过免疫新西兰大耳白兔得到多抗血清,经Protein A-Sepharose 4B凝胶柱纯化后得到纯化抗体,在此基础上建立直接竞争ELISA检测方法。该方法的IC₅₀为10.84 ng/mL,最低检测限达0.002 ng/mL,对鸡肉、鸡肝、猪肉、猪肝等实际样品进行检测,加标回收率为80.23%~90.07%。采用高效液相色谱法进行验证,两种方法测定结果呈现良好的线性关系(R²≥0.99)。本文建立的方法灵敏度高,简便快速,适合现场大批量快速检测动物源食品中利巴韦林残留。     

Precision Medicine: Determination of Ribavirin Urinary Metabolites in Relation to Drug Adverse Effects in HCV Patients

The most commonly used antiviral treatment against hepatitis C virus is a combination of direct-acting antivirals (DAAs) and ribavirin (RBV), which leads to a shortened duration of therapy and a sustained virologic response until 98%. Nonetheless, several dose-related side effects of RBV could limit its applications. This study aims to measure the urinary concentration of RBV and its main metabolites in order to evaluate the drug metabolism ability of HCV patients and to evaluate the adverse effects, such as anemia, with respect to RBV metabolite levels. RBV and its proactive and inactive metabolites were identified and quantified in the urine of 17 HCV males with severe liver fibrosis using proton nuclear magnetic resonance (¹H-NMR) at the fourth week (TW4) and at the twelfth week of treatment (EOT). Four prodrug urinary metabolites, including RBV, were identified and three of them were quantified. At both the TW4 and EOT stages, six HCV patients were found to maintain high concentrations of RBV, while another six patients maintained a high level of RBV proactive metabolites, likely due to nucleosidase activity. Furthermore, a negative correlation between the reduction in hemoglobin (Hb) and proactive forms was observed, according to RBV-triphosphate accumulation causing the hemolysis. These findings represent a proof of concept regarding tailoring the drug dose in relation to the specific metabolic ability of the individual, as expected by the precision medicine approach.     

二维高效液相色谱-三重四极杆/复合线性离子阱质谱联用法快速测定鸡肉和鸡蛋中利巴韦林总残留量

建立了二维超高效液相色谱-三重四极杆/复合线性离子阱质谱联用方法快速测定鸡肉、鸡蛋中利巴韦林总残留量。样品在0.10 mol/L乙酸铵缓冲液(pH 4.8)中,于37 ℃下经酸性磷酸酯酶酶解和提取,提取液经超滤后直接进样分析。样品先经Zorbax SB-Aq色谱柱(3.0 mm×150 mm×1.8 μm)分离,将分离出含利巴韦林的流分切割至捕集柱中捕集,然后再将捕集柱切换至第2维色谱流路中,于Hypercarb PGC色谱柱(2.1 mm×150 mm×3 μm)分离,利用电喷雾正离子模式多离子监测触发的增强子离子扫描方式（MRM-IDA-EPI）检测,稳定同位素内标法定量。结果表明：鸡肉、鸡蛋中利巴韦林的平均加标回收率为87.5%~97.7%,相对标准偏差在2.8%~8.3%之间 (n=6),方法的检出限（S/N=3）为0.2 μg/kg。本法操作简单、灵敏度高、选择性好,已成功应用于食品安全风险检测。     

同位素稀释-超高效液相色谱-串联质谱法测定禽类食品中利巴韦林和金刚烷胺类化合物残留量

目的建立同位素稀释-超高效液相色谱-串联质谱(UPLC-MS/MS)法检测禽类食品中利巴韦林和金刚烷胺类化合物(金刚烷胺、金刚烷甲胺、金刚烷乙胺、3,5-二甲基金刚胺)的残留量。方法样品经酶解,三氯乙酸沉淀蛋白,低温高速离心,上清液调节pH值后经PBA/PCX复合固相萃取柱净化,Agilent ZORBAX SB-Aq柱(3.0 mm×100 mm,1.8μm)分析利巴韦林,Waters BEH C_(18)色谱柱(2.1 mm×100 mm,1.7μm)分析金刚烷胺类化合物,串联质谱测定,同位素内标法定量。结果利巴韦林在1.0～100 ng/mL、金刚烷胺类化合物在0.2～20 ng/mL范围内呈良好的线性关系,相关系数(r)均为0.999。利巴韦林的检出限和定量限分别为0.5和1.5μg/kg;金刚烷胺类化合物的检出限和定量限分别为0.1和0.3μg/kg。利巴韦林(1.5～15μg/kg)和金刚烷胺类化合物(0.3～3.0μg/kg)添加3个浓度的检测结果显示,利巴韦林的回收率为91.4%～103.7%,金刚烷胺类化合物的回收率为94.3%～108.2%,相对标准偏差(RSD)均小于10%。结论本方法具有简便快捷、灵敏度高、定性准确等特点。     

三氮唑核苷的合成

肌苷与醋酐〔n(肌苷 )∶n(醋酐 ) =1 0 0∶2 .18〕在催化剂对甲基苯磺酸的催化作用下制得 1,2 ,3,5 O 四乙酰 β D 呋喃核糖 (Ⅰ) ,收率为 84%。1,2 ,4 三氮唑 3 羧酸甲酯与N ,O 二 (三甲基硅烷基 )乙酰胺在乙腈中硅烷化反应后 ,直接与Ⅰ在催化剂CF3SO2 OSi(CH3) 3的作用下缩合〔n(1,2 ,4 三氮唑 3 羧酸甲酯 )∶n(Ⅰ)∶n(催化剂 ) =1∶1∶2〕制得 1 (2 ,3,5 三 O 乙酰基 β D 呋喃核糖基 ) 1,2 ,4 三氮唑 3 羧酸甲酯 (Ⅱ) ,收率为 83 4% ,Ⅱ经氨解制得三氮唑核苷。总收率为 5 5 6 %。     

不同给药方式在大鼠内对利巴韦林的药代动力学和组织分布的影响

目的：利用HPLC-QqQ-MS多反应监测（multiple reaction monitoring, MRM）技术检测方法探究口服和呼吸道给药方式对利巴韦林药代动力学及组织分布情况的影响。方法：实验建立了检测利巴韦林的高灵敏度液质联用分析方法,经过了线性、专属性、回收率、准确度、精密度的考察。用建立的方法对口服和呼吸道给药两种方式的药代动力学和组织分布情况进行考察。结果：呼吸道给药0～72 h血药浓度较高,药物吸收较快,呼吸道给药Cmax（46.1±10.6） μg/L和AUC(0-t)（276±68） μg·L-1·h显著性高于口服给药Cmax（8.9±3.8）μg/L和AUC(0-t)（142±63） μg·L-1·h。组织分布结果表明,呼吸道给药肺组织利巴韦林含量明显高于口服给药,同时呼吸道给药肝脏组织含量较低。 结论：与口服给药相比,呼吸道给药具有较高的血药浓度,及在肺组织含量较高,揭示了吸入给药的组织靶向作用。HPLC-QqQ-MS检测方法简单有效、准确度高、重复性好、可用于大鼠血浆,组织利巴韦林的含量测定。     

混合菌发酵法生产三氮唑核苷的工艺条件优化

以枯草芽孢杆菌（Bacillus substilis）TM903和谷氨酸棒杆菌（Corynebacterium glutamicum）TL1105为供试菌株,进行混合菌发酵法生产三氮唑核苷的研究。通过对TL1105接种量、KH2PO4、Mn2+以及前体物1H-1,2,4-三氮唑-3-甲酰胺（简称TCA）添加量的研究初步确定三氮唑核苷的发酵工艺条件。采用MINITAB软件对葡萄糖、KH2PO4、Mn2+及TCA的添加方式进行析因实验设计,得出最佳发酵条件,即一次性投糖,KH2PO4、Mn2+及TCA则进行分批流加。在最佳发酵条件下进行三氮唑核苷混合菌发酵,经过发酵60h,发酵液中可积累4.69g•L-1的三氮唑核苷。     

A Protease Inhibitor with Induction Therapy with Natural Interferon-β in Patients with HCV Genotype 1b Infection

The restoration of innate immune responses has potential as a novel therapeutic strategy for chronic hepatitis C (CHC). We compared the efficacy and safety of induction therapy (IT) with natural interferon-β (n-IFN-β) followed by pegylated-IFN-α/ribavirin (PR) alone (group A, n = 30) and IT with a protease inhibitor (PI) (simeprevir or vaniprevir)/PR (group B, n = 13) in CHC patients with genotype 1b and high viral loads. During IT with nIFN-β, virologic response rates in group A and group B were 10% and 8% (p = 0.6792) at week 4, 30% and 16% (p = 0.6989) at week 12 and 47% and 20% (p = 0.0887) at week 24 respectively. During and after the treatment with PR alone or PI/PR, virologic response rates in groups A and B were 50% and 82% (p = 0.01535) at week 4, 53% and 91% (p = 0.006745) at week 8, 57% and 91% (p = 0.001126) at week 12, 57% and 100% (p < 0.001845) at the end of the treatment and 57% and 80% (p < 0.005166) after treatment cessation. IT with PI/PR linked to the restoration of innate immune response was tolerated well, overcame virological breakthrough, enhanced early virologic responses, and resulted in a sustained virologic response in difficult-to-treat CHC patients. IT with PI/PR is beneficial for treating difficult-to-treat CHC patients.     

非食品性动物专用利巴韦林可溶性粉的制备

介绍了利巴韦林可溶性粉的处方和制备方法。以利巴韦林原粉为主药,辅以磷霉素钠、香料、可溶性矫味剂和可溶性填料,投料比(4~25)∶(1~15)∶(0.1~1)∶(1~20)∶余量,经由烘干、粉碎、称量、混匀、检验、计量、包装等常规操作混合配制而成。结果表明,可根据需要制备出不同含量的利巴韦林可溶性粉剂,产品水溶性好,适合非食品性动物使用。