衍生化GC法测定生物转化产品L-山梨糖及其中残余D-山梨醇的含量

设计了生物转化产品L-山梨糖及其中残余D-山梨醇的衍生化GC测定方法。以乙酸酐—吡啶(1∶1)为衍生化试剂对样品中残余D-山梨醇进行乙酰化GC测定,再以四氢硼钠为还原剂、乙酸酐为乙酰化试剂对样品中L-山梨糖进行还原乙酰化GC测定。结果表明,D-山梨醇及L-山梨糖分别在0.01999～2.999μg及4.00～24.00μg范围内呈线性。本法精密度及回收率均较好,可用于监测L-山梨糖生物转化终点及其成品中L-山梨糖及残余D-山梨醇的含量。     

HPLC测定D-对羟基苯甘氨酸甲酯中的手性杂质

目的 建立HPLC测定D-对羟基苯甘氨酸甲酯中潜在的手性杂质[杂质1：L-对羟基苯甘氨酸,杂质2：L-对羟基苯甘氨酸甲酯,杂质3：D-对羟基苯甘氨酸]的含量和限度。方法 采用Crownpak CR（-）手性色谱柱（150 mm×4.0 mm,5 μm）;流动相：高氯酸溶液（pH 1.7）-甲醇（90：10）;检测波长为226 nm;流速：0.7 mL·min^-1;柱温：25℃。结果 杂质1、杂质2和杂质3均在其定量限浓度~2.400 μg·mL^-1内线性良好（r分别为1.000 0,0.999 9,0.999 9）,平均回收率分别为99.97%,100.30%和103.18%,RSD分别为0.30%,0.64%和0.62%（n=9）。结论 该方法专属性强,准确、方便,可以作为D-对羟基苯甘氨酸甲酯中手性杂质1、杂质2和杂质3的液相分析方法。     

D-柠檬烯和L-柠檬烯对盐酸川芎嗪透皮吸收的影响

目的研究手性对映体D-柠檬烯和L-柠檬烯对盐酸川芎嗪透皮吸收的影响及其作用机制。方法采用Franz-扩散池测定盐酸川芎嗪通过离体猪皮的透过量；红外光谱法测定角质层脂质中C-H对称和不对称伸缩振动吸收峰峰位及其峰面积；采用扫描电镜观察皮肤表面形态学变化，并新引入量化的概念——表观密度衡量角质鳞片脱落的程度。结果D-柠檬烯和L-柠檬烯对盐酸川芎嗪的促透量均高于对照组和氮酮组，这两种对映体促透剂产生的促透量无统计学差异(P>0.05)， L-柠檬烯的时滞是D-柠檬烯的2.55倍。红外光谱结果显示： 角质层脂质中的C-H伸缩振动吸收峰位移和峰面积的变化取决于所用的促透剂；两种对映体促透剂均引起对称振动和不对称振动吸收峰位移增大和峰面积减少。扫描电镜结果表明，不同的促透剂抽提角质层中的脂质，引起角质鳞片不同程度的脱落。结论D-柠檬烯和L-柠檬烯的促透量均高于对照组和氮酮组，并且以D-柠檬烯的时滞最短。两种对映体促透剂对盐酸川芎嗪的促透机制是多种原因共同作用的结果。     

重组L-天门冬酰胺酶II的液相色谱/电喷雾离子化质谱法分析

目的　对基因重组L-天门冬酰胺酶II产品进行一级结构分析。方法　采用流动注射方式,电喷雾离子化质谱法测定分子量;三氯乙酸变性,胰蛋白酶水解后,HPLC测定肽图谱;结合质谱源内碰撞诱导解离技术,解析酶解肽段的氨基酸顺序。结果　分子量测定值与理论值不符,质谱解析表明样品中存在3个氨基酸变异现象,由此计算的分子量与测定值吻合。结论　液相色谱/电喷雾质谱法为基因重组蛋白质药物的一级结构分析和质量控制提供了新途径。     

L-盐酸赖氨酸对局部脑缺血大鼠的治疗作用

研究L-盐酸赖氨酸对大鼠局限性脑缺血的治疗作用。方法：采用电凝阻断大鼠一侧大脑中动脉造成局限性脑缺血模型。结果：一侧大脑中动脉阻断后15minipL-盐酸赖氨酸621.5及310.8mg.kg^-1,能显著降低局限性脑缺血大鼠的脑梗塞面积,减少相关脑区变性、坏死神经元的数量,而且L-盐酸赖氨酸621.5mg.kg^-1还能明显改善大鼠的神经功能缺失,降低行为评分。 结论：L-盐酸赖氨酸对脑缺血有治疗作用。     

丹参酰-L-丙氨酰-L-脯氨酸冰片酯单晶的制备及其结构表征

目的 制备丹参酰-L-丙氨酰-L-脯氨酸冰片酯的单晶，并对其进行结构表征。方法 通过溶剂挥发法制备单晶，采用X射线单晶衍射对其晶体空间结构进行了解析，采用X射线粉末衍射进行晶型分析，通过热重分析（TGA）法–差示扫描量热法（DSC）分析热稳定性。结果 晶体呈现无色透明针状，属于单斜晶系，空间群为P2₁。1个晶胞由4个分子组成，氢键主要存在于分子间，分子中存在6个手性碳原子，R、S型各有3个碳原子。粉末X射线衍射实测粉末衍射图特征峰位置与理论衍射峰大致相同。DSC结果与TGA图结果一致。结论 确证了丹参酰-L-丙氨酰-L-脯氨酸冰片酯的立体构型和热稳定性。     

衍生化法分离（2R，4R）-4-甲基-2-哌啶甲酸乙酯酒石酸盐手性异构体

目的 建立衍生化法分离（2R,4R）-4-甲基-2-哌啶甲酸乙酯酒石酸盐（MPFET）3种手性异构体。方法 以2,3,4,6-四-O-乙酰基-β-D-吡喃葡萄糖异硫氰酸酯（GITC）为柱前衍生化试剂,对MPFET手性异构体进行分离,并对衍生化条件进行优化。采用Venusil AQ C₁₈（250 mm×4.6 mm,5 μm）色谱柱作为固定相进行分离、监测和定量。以0.01 mol/L磷酸二氢钾溶液（用磷酸调pH至3.6）-乙腈（60∶40）为流动相,体积流量1.5 mL/min进行等度洗脱,采用紫外检测器,检测波长266 nm;柱温30℃;进样量20 μL。结果 MPFET与2S,4S-异构体分离度为1.76。2S,4R-异构体的线性范围为1.500～8.999 μg/mL,2R,4S-异构体的线性范围为0.255 2～1.531 0 μg/mL,2S,4S-异构体的线性范围为0.250 1～75.000 0 μg/mL,MPFET的线性范围为0.250 1～600.100 0 μg/mL,回收率均在90%～108%内,RSD均不大于3.0%。结论 柱前衍生化法分离MPFET中3种手性异构体,专属性强、准确度高、灵敏度高、重复性好,可用于MPFET手性异构体的分离和质量控制。     

一氧化氮合酶抑制剂(L-NAME)的药理作用与慢性血管效应

目的：探讨N^ω-硝基L-精氨酸甲酯(L-NAME)对不同动脉的慢性血管效应及其与抑制内皮依赖性一氧化氮(EDNO)合成的关系。方法：采用大鼠离体动脉环的张力测定,一氧化氮合酶活性测定及组织学、生化学等方法。结果：给药8周后大鼠尾动脉压明显升高、体重及胸主动脉的内皮依赖性舒张功能明显下降并释放内皮依赖性收缩因子,肠系膜微动脉的内皮依赖性舒张功能无改变。给药8周后出现了动脉中膜肥厚及其周围纤维化。N^ω-硝基D-精氨酸甲酯(D-NAME)的慢性给药亦可引起与L-NAME同样的动脉周围纤维化。结论：L-NAME对大鼠胸主动脉及肠系膜微动脉的作用不同,其慢性血管效应与抑制EDNO合成以外的其它机制相关。     

L-和DL-正丁基东莨菪碱对大鼠子宫作用的比较

用放射配基结合法测定L-正丁基东莨菪碱，DL-正丁基东莨菪碱与阿托品对大鼠子宫M 胆碱受体的亲和力。结果表明，L-与DL-正丁基东莨菪碱对大鼠子宫M胆碱受体的亲和力十分相近，但都比阿托品弱。比较L-正丁基东莨菪碱、DL-正丁基东莨菪碱和阿托品对离体大鼠子宫的影响及对抗乙酰胆碱所致的子宫收缩作用，表明两种正丁基东莨菪碱对离体大鼠子宫无影响，而能对抗乙酰胆碱所致的子宫收缩，且作用相近，但都比阿托品弱。说明生物效应与其受体亲和力的大小相关。     

N-硬脂酰酪氨酸的理化性质研究

目的 表征N-硬脂酰酪氨酸及其盐的理化常数和血浆蛋白结合率。方法 采用摇瓶法及HPLC-UV法测定N-硬脂酰酪氨酸及其盐在不同介质和pH值下的溶解度、表观脂水分配系数,采用平衡透析法测定血浆蛋白结合率。结果 N-硬脂酰酪氨酸盐的溶解度和血浆蛋白结合率明显优于其原型药物;N-硬脂酰酪氨酸的溶解度为（27.62±0.43）mg/L,其二钾盐的溶解度为（51.70±0.29）mg/L;N-硬脂酰酪氨酸及其二钾盐的血浆蛋白结合率分别为20.96%～23.11%、35.41%～40.38%。结论 N-硬脂酰酪氨酸成盐后其理化性质得到明显改善。     

首批L-犬尿氨酸国家药品对照品的研制

目的：建立首批L-犬尿氨酸国家对照品。方法：采用紫外吸收光谱、红外吸收光谱、核磁共振谱及高分辨质谱对犬尿氨酸进行结构确证，测定了原料的水分含量、炽灼残渣量及元素含量，并考察了其引湿性、均匀性和稳定性，最后采用氮分析法、液相色谱法及定量核磁共振法对其含量进行测定，最后采用质量平衡法对其含量进行赋值。结果：确证了犬尿氨酸的结构，以质量平衡法计算，将本批次犬尿氨酸的含量定值为97.4%。结论：经过结构确证和含量测定，本批次犬尿氨酸能够满足相关药品检查使用，首批L-犬尿氨酸国家对照品研制成功并通过审定，可用于相关氨基酸药物的检查，有利于提高相关药品质量，保证用药安全。     

Chiral purity assay for Flindokalner using tandem mass spectrometry: method development, validation, and benchmarking

The present work demonstrates the application and validation of a mass spectrometry method for quantitative chiral purity determination. The particular compound analyzed is Flindokalner, a Bristol-Myers Squibb drug candidate for post-stroke neuroprotection. Chiral quantification of Flindokalner was achieved using tandem mass spectrometry (MS/MS) and the kinetic method, a gas phase method used for thermochemical and chiral determinations. The MS/MS method was validated and benchmarked against two separate chromatographic techniques, chiral high performance liquid chromatography with ultra-violet detection (LC/UV) and achiral high performance liquid chromatography with circular dichroism detection (LC/CD). The chiral purity determination of Flindokalner using MS/MS proved to be rapid (3 min run time for each sample) and to have accuracy and precision comparable to the chiral LC/UV and achiral LC/CD methods. This method represents an alternative to commonly used chromatographic techniques as a means of chiral purity determination and is particularly useful in rapid screening experiments.     

Metabolite fingerprinting of Camptotheca acuminata and the HPLC–ESI-MS/MS analysis of camptothecin and related alkaloids

The major phytochemical constituents, namely, alkaloids, flavonoids and ellagic acid derivatives, of leaves of Camptotheca acuminata were identified using high performance liquid chromatography (HPLC) coupled with electrospray mass spectrometry (ESI-MS) in extracts of plants cultivated in Italy and collected at different growth stages. Alkaloids related to camptothecin were identified and quantified by HPLC coupled with ESI-tandem mass spectrometry (MS/MS) employing, respectively, an ion trap and a triple quadrupole mass analyser. The fragmentation patterns of alkaloids related to camptothecin were analysed and a specific Multiple Reaction Monitoring HPLC–MS/MS method was developed for the quantitative determination of these constituents. The described method provides high sensitivity and specificity for the characterisation and quantitative determination of the alkaloids in C. acuminata.     

化学—酶法立体控制合成光学纯L-羟基苯丙氦酸的新方法

Optically pure L-3(2-hydroxyphenyl) alanine(L-o-tyrosine ,Ⅲa,),L-3-(3-hydroxyphenyl) alanine(L-m-tyrosine,Ⅲb )and L-3-(4-hydroxyphenyl )alanine(L-p-tyrosine,Ⅲc )were synthesized by the stereocontrolled amination of corresponding hydroxycinnamic acld(Ⅱ)catalyzed by L-phenylalanine ammonia-lyase(PAL,EC4.3.1.5 )contained in Rhodoterula rubramycelium. The amination of compound Ⅱ was completed in aqueous ammonia solution( 6.4mol·L^-1,pH10.5, 30℃) with the conversion of 74.9%(Ⅱa),21.1%(Ⅱb)and 20.6%(Ⅱc)respectively.The absolute configuration of the products Ⅲa～c were confirmed by circular dichroism(CD),and chiral high-performance ligand exchange chromatography(HPLEC)showed that productsⅢ were optically pure L-isomers.     

左旋门冬酰胺酶化疗方案致不良反应分析

目的 探讨左旋门冬酰胺酶化疗方案致不良反应发生的规律和特点，为临床合理用药提供参考。方法 检索1989年1月—2018年11月中国学术期刊（网络版）、万方数字化期刊全文库、中文科技期刊全文数据库（维普）、Pubmed中关于左旋门冬酰胺酶不良反应的不良反应个案报道，并进行回顾性分析。结果 共检索到56篇文献，82例病例。年龄集中在6～10岁。给药方式主要为静脉滴注。原患疾病主要以急性淋巴细胞白血病为主。不良反应大多发生于用药48 h内，构成比为45.1%。左旋门冬酰胺酶引发的不良反应主要涉及消化系统损害、皮肤及附件损害、血液系统损害和中枢神经系统损害，消化系统损害例数最多，临床表现主要为恶心呕吐、血浆淀粉酶升高、肝功能异常等。结论 临床应重视左旋门冬酰胺酶不良反应的危害性，加强药学监护，尽量避免或减少其所致不良反应的发生，确保患者用药安全。     

The use of mass spectrometry in the study of chemically-reactive drug metabolises. Application of MS/MS and LC/MS to the analysis of glutathione- and related S-linked conjugates of N-methylformamide

The S-(N-methylcarbamoyl) derivatives of glutathione, cysteine and N-acetylcysteine, the S-linked conjugates derived from a reactive metabolite of N-methylformamide (NMF), were studied in mice dosed with an equimolar mixture of NMF and deuterium-labelled NMF. Following preparation of N-benzyloxycarbonyl derivatives in aqueous media, the title conjugates were isolated, purified as their methyl esters and subjected to analysis by fast atom bombardment mass spectrometry (FAB/MS), fast atom bombardment tandem mass spectrometry (FAB/MS/MS) or thermospray liquid chromatography/mass spectrometry (TSP LC/MS). Characteristic isotope clusters in the FAB or TSP mass spectra facilitated recognition of drug metabolites, while constant neutral loss (89 u) and daughter ion scanning tandem mass spectrometry (MS/MS) experiments provided unique structural information on the conjugates of interest. It is concluded that the combined use of stable isotopes, aqueousphase derivatization and contemporary mass spectrometric techniques represents a powerful approach for the analysis of glutathione adducts and related S-linked conjugates of chemically-reactive drug metabolites.     

Isolation and characterization of surfactin produced by <Emphasis Type="Italic">Bacillus polyfermenticus</Emphasis> KJS-2

Bacillus polyfermenticus KJS-2 (BP-KJS-2) was used to produce a lipopeptide-type surfactin. To accomplish this, a surfactin-producing BP-KJS-2 was fermented by soybeans. The surfactin was then purified by a procedure including ethanol treatment and preparative chromatography. Next, the biochemical structure of the purified surfactin was analyzed by electrospray ionization mass spectrometry (ESI-MS) and high-resolution ESI Q-Tof mass spectrometry (Q-Tof MS). In addition, the masses of the four peaks were determined to be 1007, 1021, 1035, and 1049 m/z revealing that the compound was mixture with quasi-molecular ions. Taken together, these findings indicated that the lipopeptide had a cyclic structure and amino acid composition of Gln-Leu-Leu-Leu-Val-Asp-Leu-Leu, and that the major lipopeptide product of BP-KJS-2 is the surfactin isoform. In addition, this lipopeptide showed strong antimicrobial activity against bacteria at the level of 0.05 mg/mL.     

左卡尼汀通过增加UCP1和p-AKT的表达活化高脂血症大鼠棕色脂肪细胞的研究

目的探讨左卡尼汀对高脂喂养大鼠棕色脂肪的影响及其相关机制。方法将30只SD大鼠随机分为对照组、模型组和左卡尼汀低、中、高剂量(100、200、300mg/kg)组,每组各6只。每日ig给药,治疗12周后称重、处死大鼠,收集血清和棕色脂肪组织,检测血清相关生化指标,测定棕色脂肪组织中解偶联蛋白1(UCP1)和磷酸化AKT(p-AKT)的表达水平。结果与模型组相比,左卡尼汀100、200、300 mg/kg组的总胆固醇(TC)、三酰甘油(TG)明显降低,高密度脂蛋白胆固醇(HDL-C)明显升高(P0.05);左卡尼汀100、200 mg/kg组的体质量明显降低,棕色脂肪组织(BAT)质量、BAT质量/体质量明显升高(P0.05)。与模型组相比,左卡尼汀100、200mg/kg组BAT中UCP1RNA明显增加,差异有统计学意义(P0.05)。与模型组相比,左卡尼汀组100、200、300mg/kg组BAT中UCP1和p-AKT蛋白表达明显增加,差异有统计学意义(P0.05、0.01),且呈剂量相关性。结论左卡尼汀具有降脂和促进棕色脂肪活化的作用,这可能与其增加棕色脂肪组织中UCP1和p-AKT的表达有关。     

Characterization of Recombinant Cytokine Fragments Using Isotachophoresis-Capillary Zone Electrophoresis,Reversed-Phase High Performance Liquid Chromatography,and Mass Spectrometry

Purpose. Capillary zone electrophoresis with isotachophoretic sample preconcentration (ITP-CZE) and reversed-phase high performance liquid chromatography (RP-HPLC) with UV detection and on-line coupling to electrospray-ionization mass spectrometry were investigated for their potential to separate and identify fragments of recombinant human interleukin-6 formed during acidic stress of the parent protein. Results. Based on the orthogonal separation principles governing ITP-CZE and RP-HPLC, different peak patterns were observed using both methods. The selectivity of ESI-MS allowed identification of several co-migrating compounds. Data obtained by on-line ESI-MS were compared to results from off-line investigations by MALDI-TOF-MS performed with single fractions collected from the RP-HPLC system. Cleavage of the protein backbone occurred preferably at acid-labile Asp-sites. The total amount of rhIL-6 needed for ITP-CZE-ESI-MS identification of all fragments was only in the upper femtomole range, while RP-HPLC required amounts of protein three orders of magnitude higher. On the other hand, the low CE sample volume opposes the collection of fractions to perform off-line analysis. Conclusions. Growing acceptance of CE with on-line MS detection for pharmaceutical quality control of proteins is expected.