虎纹捕鸟蛛毒素-Ⅴ去折叠过程的色谱分析

将纯化的天然虎纹捕鸟蛛毒素 Ⅴ经盐酸胍变性 30min后 ,在pH 3 0、反应温度为 37℃的条件下与三羧甲基磷酸 (TCEP)反应 12min ,用反相高效液相色谱分离得到其全部去折叠中间体 ,通过基体辅助激光解吸电离飞行时间质谱 (MALDI TOFMS)进行鉴定 ,并利用烷基化反应对这些去折叠中间体予以进一步确证。根据其保留时间 ,分析虎纹毒素 Ⅴ各去折叠中间体的色谱行为 ,初步探讨了多肽或蛋白质构象异构体反相色谱行为的多样性。     

部分还原和分步序列测定法定位虎纹捕鸟蛛毒素-Ⅳ二硫键

结合部分还原和分步序列测定法确定了虎纹捕鸟蛛毒素-的二硫键配对方式.在pH=3和40℃的条件下与还原剂三羧甲基磷酸(TCEP)反应10min,利用RP-HPLC分离并分别收集含有一对和两对二硫键被还原的中间体,分别与0.5mol/L碘乙酰胺溶液(pH=8.3)反应1min,使游离巯基烷基化后,测定各中间体的氨基酸序列,从而确定虎纹捕鸟蛛毒素-的3对二硫键分别为Cys2-Cys17,Cys9-Cys24和Cys-Cys31(1-4,2-5和3-6).     

(S)-与(R)-五味子丙素的HPLC手性分离及质谱研究

建立了一种反相高效液相色谱法分析(S)-和(R)-五味子丙素对映异构体的方法.选用Chiralcel OD-RH手性色谱柱,以甲醇和水(体积比78 : 22、97 : 3)为流动相,流速0.5 mL/min,254 nm波长下检测,五味子丙素合成中间体噁唑啉取代联苯对映异构体2和2'的保留时间分别为22.2 min和19.9 min,分离度为2.5,五味子丙素对映异构体3'和3的保留时间分别为7.5、9.0 min,与相邻杂质峰分离度分别为3.2和2.7.经液相色谱-质谱实现了分离与鉴定.方法灵敏度高、重复性好,可用于五味子丙素对映异构体的立体选择性研究及质量控制.     

用高效疏水色谱法对多种脲变α-淀粉酶折叠中间体的研究

用高效疏水色谱法对用脲变性的α-淀粉酶的体外折叠中间体进行了分离，发现脲变α-淀粉酶折叠至少有19个中间体，而且，这些中间体在色谱流出液中可稳定一周．这一结论已由电泳、离子交换色谱和体积排阻色谱法证实．此外，还用紫外吸收光谱和荧光发射光谱研究了这些折叠中间体与天然α-淀粉酶构象之间的差异．     

液相色谱-串联质谱法测定双壳类水产品中原多甲藻酸类贝类毒素

建立了双壳类水产品中原多甲藻酸贝类毒素Azaspiracid 1(AZA1)、Azaspiracid 2(AZA2)和Azaspir-acid 3(AZA3)的液相色谱-电喷雾串联质谱检测方法。选用具有基质代表性的扇贝、牡蛎和杂色蛤为研究对象,样品经80%甲醇-水混合溶液提取,正己烷脱脂、氯仿反提萃取并旋转蒸发浓缩后,MAX混合型阴离子交换反相吸附固相萃取柱净化,Luna C18色谱柱(150 mm×2.0 mm,5μm,Phenomenex公司)反相液相色谱法分离,乙腈和0.1%甲酸溶液组成的流动相梯度洗脱,正离子扫描,多反应监测(MRM)模式下电喷雾串联质谱法测定和确证,外标法定量。结果表明,3种原多甲藻酸类贝类毒素均在4 min内出峰,检出限均为1.5μg/kg,在0.3～30μg/L范围内线性良好,平均回收率大于80%,相对标准偏差(RSD)小于12%(n=6)。     

反相高效液相色谱法监测蔗糖-6-乙酸酯合成

4,1′,6′-三氯-4,1′,6′-三脱氧半乳蔗糖(4,1′,6′-Trichloro-4,6′-trideoxygalactosucrose,简称TGS,又称sucralose即蔗糖素)甜度高、低热量、无毒、抗龋。蔗糖-6-乙酸酯(A)是合成三氯蔗糖的重要中间体,其合成路线见图1,本文采用反相高效液相色谱法(RP-HPLC)分析。在选定的色谱条件下,分离效果满意,对其合成工艺有良好的指示作用。     

反相高效液相色谱法快速分析咖啡因合成过程中的两个中间体

1,3-二甲基-5-亚硝基-6-氨基脲嘧啶（ANDMU）是咖啡因合成中的一个非常重要的中间体,其加氢还原生成1,3-二甲基-5,6-二氨基脲嘧啶（DADMU）的反应是咖啡因合成过程中的关键一环。本文采用反相高效液相色谱（HPLC）对ANDMU及DADMU进行分析,操作简便快速,结果准确,重现性好,适合于实验室研究和工业检测的要求。     

化学合成虎纹捕鸟蛛毒素-Ⅰ的色谱行为及分离纯化

 利用反相高效液相色谱 (RP HPLC)和离子交换色谱 (IEC)比较研究了固相化学合成的多肽类神经毒素虎纹捕鸟蛛毒素 Ⅰ (HWTX Ⅰ )与其对应的天然产物的色谱行为差异及分离纯化方法。发现在完全相同的实验条件下 ,复性处理后的合成HWTX Ⅰ的谱峰有别于还原变性后又复性的天然HWTX Ⅰ的谱峰 ,主要表现为扩散度较大 ,对称性较差 ,说明合成产物存在更大分子构象的不均一性。推测除了在合成HWTX Ⅰ复性过程中有少数分子发生二硫键错配外 ,还有部分分子在固相合成中发生了消旋作用 ,需要交替采取多种色谱法才能完全分离纯化复性后的合成多肽产物。     

基于串联反应的吡虫啉新合成工艺研究

以2-氯-5-氯甲基吡啶(CCMP)、无水乙二胺和硝基胍为原料,运用串联反应方法合成了吡虫啉[化学名:1-(6-氯-3-吡啶甲基)-N-硝基-2-咪唑啉亚胺]。该方法反应中间体无需纯化处理,操作简单,同时解决了活性中间体进一步进行副反应的难题。通过单因素实验,探讨了pH、反应溶剂、温度以及时间等因素对产物收率的影响,得到的优化工艺条件为:以乙腈为溶剂,nCCMP∶n无水乙二胺∶n硝基胍=1∶5∶1,于30℃反应120min,产率可达96.35%。其结构经1H NMR、13C NMR、IR表征。     

基于串联反应的吡虫啉新合成工艺研究

以2-氯-5-氯甲基吡啶(CCMP)、无水乙二胺和硝基胍为原料,运用串联反应方法合成了吡虫啉[化学名:1-(6-氯-3-吡啶甲基)-N-硝基-2-咪唑啉亚胺]。该方法反应中间体无需纯化处理,操作简单,同时解决了活性中间体进一步进行副反应的难题。通过单因素实验,探讨了pH、反应溶剂、温度以及时间等因素对产物收率的影响,得到的优化工艺条件为:以乙腈为溶剂,nCCMP∶n无水乙二胺∶n硝基胍=1∶5∶1,于30℃反应120min,产率可达96.35%。其结构经1H NMR、13C NMR、IR表征。     

高效液相色谱-串联质谱法测定葡萄中的吡效隆和赤霉素

采用高效液相色谱-串联质谱法（HPLC-MS/MS）建立了葡萄果实中吡效隆（CPPU）和赤霉素（GA3）两种植物生长调节剂残留量的定性定量分析方法。样品用0.5%甲酸水溶液和乙腈（体积比4:1）混合溶剂提取,经Strata-X固相萃取净化,使用Agilent SB-C18 色谱柱（50mm×2.1mm,1.8μm）,以0.5%甲酸水溶液-乙腈为流动相,流量0.3mL/min,采用电喷雾离子化源（ESI）多反应监测（MRM）模式下HPLC-MS/MS检测。在最佳检测条件下,得到吡效隆和赤霉素在2.0～100.0μg/L检测范围内线性关系良好,相关系数大于0.999;检出限(LOD)分别为0.3μg/L、0.5μg/L,定量限(LOQ)分别为1.0μg/L、2.0μg/L,回收率和精密度均能达到要求,表明该法准确,可用于葡萄中吡效隆和赤霉素残留量测定。     

高效液相色谱-串联质谱法同时测定贝类中22种农药残留

建立了高效液相色谱-三重四极杆质谱（HPLC-MS/MS）同时测定贝类中22种农药残留的分析方法。样品经含0.1%（v/v）甲酸的乙腈提取，N-丙基乙二胺（PSA）和石墨化碳黑（GCB）净化，然后采用ACE UltraCore 2.5 SuperC18柱（100 mm×2.1 mm，2.5 μm）分离，以甲醇-0.1%（v/v）甲酸水溶液为流动相梯度洗脱，流速为0.4 mL/min，柱温为35 ℃，然后以电喷雾电离（ESI）源，在多反应监测（MRM）、正离子模式下，采用三重四极杆质谱检测。22种农药在各自的线性范围内线性关系良好，相关系数均大于0.997，检出限为0.1~0.3 μg/kg，定量限为0.3~1.0 μg/kg。在3个添加水平下，22种农药的平均回收率为65.2%~109.4%，相对标准偏差为1.3%~15.2%（n=6）。该方法操作简单，快速，准确度高，灵敏度高，可用于贝类中22种农药残留的同时检测。     

Direct-injection high performance liquid chromatography ion trap mass spectrometry for the quantitative determination of olanzapine,clozapine and N-desmethylclozapine in human plasma

A specific and sensitive direct-injection high performance liquid chromatography electrospray ionization tandem mass spectrometry (HPLC/ESI-MS/MS) method has been developed for the rapid identification and quantitative determination of olanzapine, clozapine, and N-desmethylclozapine in human plasma. After the addition of the internal standard dibenzepin and dilution with 0.1% formic acid, plasma samples were injected into the LC/MS/MS system. Proteins and other large biomolecules were removed during an online sample cleanup using an extraction column (1 x 50 mm i.d., 30 microm) with a 100% aqueous mobile phase at a flow rate of 4 mL/min. The extraction column was subsequently brought inline with the analytical column by automatic valve switching. Analytes were separated on a 5 microm Symmetry C18 (Waters) analytical column (3.0 x 150 mm) with a mobile phase of acetonitrile/0.1% formic acid (20:80, v/v) at a flow rate of 0.5 mL/min. The total analysis time was 6 min per sample. The inter- and intra-assay coefficients of variation for all compounds were <11%. By eliminating the need for extensive sample preparation, the proposed method offers very large savings in total analysis time.     

液相色谱-四极杆-飞行时间质谱法快速筛查与确证紫甘蓝中415种农药残留

应用液相色谱-四极杆-飞行时间质谱(LC-QTOF/MS)建立了一次进样可同时对紫甘蓝中415种农药残留进行快速筛查和准确确证的分析方法。实验采用1%(v/v)醋酸乙腈溶液提取,无水硫酸镁和氯化钠进行盐析,ZORBAX SB-C18色谱柱(100 mm×2.1 mm, 3.5 μm)分离,以0.1%(v/v)甲酸水溶液(含5 mmol/L乙酸铵)-乙腈为二元流动相进行梯度洗脱,应用LC-QTOF/MS在电喷雾电离、全离子MS/MS(All Ions MS/MS)扫描正模式下进行检测,基质匹配外标法定量分析。通过优化全自动MS/MS采集模式(Auto MS/MS)和全离子MS/MS采集模式下的不同参数,得到每种采集模式下的最佳条件。然后在2种不同采集模式的最佳条件下对比,最终选取All Ions MS/MS采集模式。实验结果表明,采用所建立的分析方法可以准确定性和定量筛查紫甘蓝中415种农药残留,所有415种农药在各自的范围内线性相关系数(r²)均大于0.990,其中411种农药的筛查限(SDL)≤5 μg/kg, 413种农药的定量限(LOQ)≤10 μg/kg。在1倍、2倍和10倍LOQ添加水平下,农药的回收率分别为65.7%~118.4%、72.0%~118.8%和70.2%~111.2%,相对标准偏差分别为0.9%~19.7%、0.2%~19.9%和0.6%~19.9%。将该方法应用于2019年欧盟能力验证项目的紫甘蓝样品中未知农药残留筛查方法和定量方法考核样的检测,所有添加农药均被准确定性筛查和定量检测,没有假阳性和假阴性。结果表明,该方法快速、准确、可靠,适用于对紫甘蓝中多种农药残留的高通量定性筛查和准确定量,可以扩展到其他果蔬基质中多农残的高通量筛查。     

凝胶渗透色谱净化-高效液相色谱法测定鱼肉中的5种激素类药物残留

建立了凝胶渗透色谱净化-高效液相色谱法测定鱼肉中己烯雌酚、雌二醇、炔雌醇、炔诺酮、炔诺孕酮5种激素类药物残留的方法。样品用乙酸乙酯-甲醇(8:2, v/v)溶液提取,提取液经Pharmadex LH-20凝胶柱(450 mm×15 mm)净化,并用甲醇-乙酸乙酯-乙酸(800:200:2, v/v/v)溶液洗脱。采用Agilent TC-C18柱(250 mm×4.6 mm, 5 μm)分离净化后的样品,用乙腈-水(45:55, v/v)溶液洗脱,流速为1.2 mL/min,双检测波长为245 nm和222 nm。5种激素类药物在0.05～2.5 mg/L范围内有良好的线性关系(r ＞0.999),检出限为10～24 μg/kg,平均加标回收率为60.1%～89.0%,相对标准偏差为2.0%～7.4%。该方法快速、简单,可应用于鱼肉中激素类药物残留量的检测。     

Simultaneous quantitation of acetylsalicylic acid and clopidogrel along with their metabolites in human plasma using liquid chromatography tandem mass spectrometry

The interest in therapeutic drug monitoring has increased over the last few years. Inter‐ and intra‐patient variability in pharmacokinetics, plasma concentration related toxicity and success of therapy have stressed the need of frequent therapeutic drug monitoring of the drugs. A sensitive, selective and rapid liquid chromatography coupled with tandem mass spectrometry (LC‐MS/MS) method was developed for the simultaneous quantification of acetylsalicylic acid (aspirin), salicylic acid, clopidogrel and carboxylic acid metabolite of clopidogrel in human plasma. The chromatographic separations were achieved on Waters Symmetry Shield^TM C₁₈ column (150 × 4.6 mm, 5 µm) using 3.5 mm ammonium acetate (pH 3.5)–acetonitrile (10:90, v/v) as mobile phase at a flow rate of 0.75 mL/min. The present method was successfully applied for therapeutic drug monitoring of aspirin and clopidogrel in 67 patients with coronary artery disease. Copyright © 2015 John Wiley & Sons, Ltd.     

QuEChERS-超高效液相色谱-串联质谱法测定牛奶中6种头孢菌素类抗生素残留

建立了QuEChERS-超高效液相色谱-串联质谱（UPLC-MS/MS）同时测定牛奶中6种头孢菌素类抗生素的分析方法。牛奶样品加入含1%（v/v）醋酸的乙腈溶液提取,于45℃氮吹浓缩,经100 mg C18吸附剂净化后,采用HSS T3柱（100 mm×2.1 mm,1.8 μm）分离,以0.1%（v/v）甲酸水溶液和乙腈为流动相进行梯度洗脱,在电喷雾离子源、正离子模式（ESI⁺）下电离,多反应监测（MRM）模式下检测,外标法定量。结果表明,6种头孢菌素类抗生素在2~200 μg/L质量浓度范围内均呈良好的线性关系,相关系数（r）均大于0.999,方法的检出限为0.2~0.6 μg/kg、定量限为0.8~2.0 μg/kg。在8、16和80 μg/kg的加标水平下,6种头孢菌素类抗生素的回收率为75.1%~94.4%,相对标准偏差为0.63%~8.3%。该方法简单快捷,准确可靠,适用于牛奶中头孢菌素类抗生素残留的同时测定。     

Simultaneous determination of isochamaejasmin,neochamaejasmin A and aphnoretinin rat plasma by UPLC‐MS/MS and its application to a pharmacokinetic study of Stellera chamaejasme L. extract

Isochamaejasmin, neochamaejasmin A and daphnoretin derived from Stellera chamaejasme L. are important because of their reported anticancer properties. In this study, a sensitive UPLC‐MS/MS method for the determination of isochamaejasmin, neochamaejasmin A and daphnoretin in rat plasma was developed. The analyte and IS were separated on an Acquity UPLC HSS T3 column (100 × 2.1 mm, 1.8 μm) using gradient elution with the mobile phase of aqueous solution (methanol–water, 1:99, v/v, containing 1 mm formic acid) and organic solution (methanol–water, 99:1, v/v, containing 1 mm formic acid) at a flow rate of 0.3 mL/min. Multiple reaction monitoring mode with negative electrospray ionization interface was carried out to detect the components. The method was validated in terms of specificity, linearity, accuracy, precision, stability, etc. Excellent linear behavior was observed over the certain concentration ranges with the correlation coefficient values >0.99. Intra‐ and inter‐day precisions (RSD) were <6.7% and accuracy (RE) ranged from −7.0 to 12.0%. The validated method was successfully applied to investigate the pharmacokinetics of three chemical ingredients after oral administration of S. chamaejasme L. extract to rats.     

Validation of liquid chromatography and liquid chromatography/tandem mass spectrometry methods for the determination of etoricoxib in pharmaceutical formulations

Reversed-phase liquid chromatography (LC) and LC/tandem mass spectrometry (LC/MS/MS) methods were developed and validated for the determination of etoricoxib in pharmaceutical dosage forms. The LC method was performed by reversed-phase chromatography on a Synergi fusion C18 column (150 x 4.6 mm id) maintained at ambient temperature. The mobile phase consisted of 0.01 M phosphoric acid, pH 3.0-acetonitrile (62 + 38, v/v) at a flow rate of 1.0 mL/min, and photodiode array detection at 234 nm was used. The chromatographic separation was obtained within 7.0 min, and calibration curves were linear in the concentration range of 0.02-150 microg/mL. The LC/MS/MS method was performed on a Luna C18 column (50 x 3.0 mm id). The mobile phase consisted of acetonitrile-water (95 + 5)-0.1% acetic acid (90 + 10, v/v). Detection was performed by positive electrospray ionization in the multiple reaction monitoring mode, monitoring the transitions 359.3 > 280.0 and 332.0 > 95.0 for etoricoxib and piroxicam (internal standard), respectively. The chromatographic separation was obtained within 2.0 min, and calibration curves were linear in the concentration range of 1-5000 ng/mL. Validation parameters, such as specificity, linearity, precision, accuracy, and robustness, were evaluated, which gave results within the acceptable range for both methods. Moreover, the proposed methods were successfully applied for routine quality control analysis of pharmaceutical products and showed significant correlation (r = 0.9999) of the results.     

超高效液相色谱-串联质谱法测定中药材中三唑类杀菌剂及三嗪类除草剂的残留量

建立了超高效液相色谱-串联质谱(UPLC-MS/MS)同时测定甘草、西洋参、三七、人参、丹参5种中药材中10种三唑类杀菌剂、18种三嗪类除草剂(包括有毒代谢物)残留量的分析方法。采用QuEChERS前处理方法,样品经1%(v/v)醋酸乙腈提取,乙二胺-N-丙基硅烷(PSA)净化处理,以Shim-pack XR-ODSII(75 mm×2.0 mm)为色谱柱,0.05%(v/v)甲酸溶液-乙腈为流动相梯度洗脱,在多反应监测(MRM)模式下测定。25种农药及其3种有毒代谢物的定量限(S/N≥10)为0.20～5.52 μg/kg;检出限(S/N≥3)为0.10～2.57 μg/kg;在各自的考察浓度范围内线性关系良好(r≥0.999);在0.20～55.2 μg/kg添加水平内,平均加标回收率为70.6%～125.7%,RSD为0.7%～14.2%。该方法样品前处理简单、快速、灵敏,可用于中药材中三唑类杀菌剂和三嗪类除草剂农药残留量的快速筛查。