免疫亲和质谱法研究／β2-微球蛋白抗原表位

采用免疫亲和分离与质谱分析相结合的方法,对β2-微球蛋白抗原表位进行了系统研究.完整的抗原分子和已固定在载体(CNBr-activated Sepharose beads)上的单克隆抗体发生免疫亲和反应后,用Endoproteinase Glu-C,Trypsin,AminopeptidaseM和carboxypeptidase Y四种不同的蛋白酶依次酶解抗原分了,并采用基质辅助激光解析电离飞行时间质谱(MALDI-TOF-MS)技术对与抗体连接受保护而未发生水解的肽段进行了研究.结果表明:β2-微球蛋白抗原表位位于整个蛋白分了氨基酸序列的61～67位,即为SFYLLYY.通过合成肽段的分析,证明了SFYLLYY即为抗原表位,与亲和质谱方法分析结果一致.     

免疫亲和质谱法研究β2-微球蛋白抗原表位

采用免疫亲和分离与质谱分析相结合的方法, 对β2-微球蛋白抗原表位进行了系统研究. 完整的抗原分子和已固定在载体(CNBr-activated Sepharose beads)上的单克隆抗体发生免疫亲和反应后, 用Endoproteinase Glu-C, Trypsin, Aminopeptidase M和carboxypeptidase Y四种不同的蛋白酶依次酶解抗原分子, 并采用基质辅助激光解析电离飞行时间质谱(MALDI-TOF-MS)技术对与抗体连接受保护而未发生水解的肽段进行了研究. 结果表明: β2-微球蛋白抗原表位位于整个蛋白分子氨基酸序列的61～67位, 即为SFYLLYY. 通过合成肽段的分析, 证明了SFYLLYY即为抗原表位, 与亲和质谱方法分析结果一致.     

溶剂诱导萃取/UPLC-MS/MS法检测农田土壤中20种除草剂

采用优化的溶剂诱导萃取前处理方法联合超高效液相色谱-串联质谱（UPLC-MS/MS）,建立了同时快速测定土壤中20种除草剂的方法。采用1.5 mL二氯甲烷为萃取剂,5 mL乙腈为提取剂,涡旋时间8 min进行提取,以ZORBAX Eclipse Plus C18色谱柱（100 mm×2.1 mm,1.8μm）进行分离,0.1%甲酸水和乙腈为流动相,电喷雾离子源（ESI）正离子方式扫描。结果显示,20种除草剂在对应质量浓度范围内线性良好,相关系数均不小于0.999 0,方法检出限为0.1～1.0μg/kg,平均回收率为77.6%～115%,相对标准偏差为0.40%～3.8%。该方法简便快捷、灵敏度高、有机溶剂用量少,适用于实际样品中20种除草剂的高通量检测。     

溶剂诱导萃取/UPLC-MS/MS法高通量检测环境水体中12种除草剂

以二氯甲烷为萃取剂,建立了测定环境水样中12种除草剂的溶剂诱导萃取/超高效液相色谱-串联质谱（UPLC-MS/MS）法。通过单因素实验确定提取剂乙腈的体积、萃取剂的种类和体积、离心时间,并结合响应面分析,得到最优的前处理方法;在仪器方法建立过程中对质谱参数、色谱柱、流动相进行了优化。在最优条件下,12种除草剂的线性关系良好,相关系数（r）均大于0.999 5,方法检出限为0.05～0.5μg/L,定量下限为0.2～5.0μg/L,平均回收率为83.0%～110%,相对标准偏差为0.30%～5.0%。该方法简便快捷、灵敏度高、有机溶剂用量少,适用于环境水体中除草剂的高通量检验。     

虾过敏原Pen a1抗原表位的关键氨基酸分析

建立了一种利用表位抗体筛选虾过敏原Pen a1抗原表位中关键氨基酸的方法。利用生物信息学软件MEGA5计算Pen a1中表位的氨基酸组成和出现频率,并采用DNAMAN软件对SDAP数据库中致敏食物原肌球蛋白的氨基酸保守性分析,综合两种方法筛选出各表位可能的关键氨基酸,Epitope 1:K,E,N;Epitope2:K,L,E;Epitope 3:E,R,D,L,Q;Epitope 5:K,L,Q。用丙氨酸取代并合成突变多肽。利用表位抗体,通过竞争性免疫斑点法及间接ELISA方法检测突变多肽的抗体结合能力,筛选出各表位的关键氨基酸。分别为:Epitope1:谷氨酰胺;Epitope2:亮氨酸和谷氨酸;Epitope3:亮氨酸和天冬氨酸;Epitope5:亮氨酸。实验结果证明了此种筛选关键氨基酸的方法的可行性,为Pen a1致敏机理的研究及利用基因修饰脱敏提供了一定的理论依据。     

溶剂诱导相变萃取法用于高效液相色谱-质谱分析的血浆样品前处理

在匀相的乙腈-水体系中添加一种疏水但与乙腈互溶的有机溶剂(如氯仿), 能诱导乙腈-水体系分相, 基于此现象建立了一种血浆样品处理方法溶剂诱导相变萃取(SIPTE). 与传统的液-液萃取法相比, 该法最大的优势为新形成的有机相为乙腈(仅含少量氯仿), 与常用的反相C₁₈柱兼容, 能直接进行高效液相色谱-质谱分析. 此外, 通过减少乙腈及氯仿的用量还可实现样品的自动浓缩. 本文通过测定血浆中尼群地平, 验证了该方法的有效性. 使用反相C₁₈柱, 以V(乙腈)∶V(水)=70∶30(含20 mmol/L甲酸铵)为流动相, 尼莫地平为内标, ESI正离子模式下选择离子监测(SIM)测定了血浆中尼群地平的浓度. 实验所得的线性、精密度和准确度结果良好, 表明所建立的方法灵敏、准确、简单、快速, 可用于药物代谢动力学研究.     

溶剂诱导固相合成MAGE-2表位多肽异构的LC-MS研究

近年来随着黑色素瘤特异性免疫治疗研究的不断深入,其肿瘤特异性抗原的发现及其细胞毒性T细胞(CTL)表位的预测及鉴定成为特异性免疫治疗及其多肽疫苗研制的重要前提.预测黑色素瘤抗原基因-2(MAGE-2)的HLA-A2限制性CTL表位的方法也日显成熟,预测出的表位其组成通常为10个氨基酸左右的多肽,随后进行固相合成.因不同氨基酸活化方式及偶联效率的差异,通常对合成的表位多肽进行梯度反相高压液相色谱(RP-HPLC-UV)分析制备,以确保下一步细胞功能性研究即细胞杀伤验证的特异性;MAGE-2众多表位多肽都表现较强的疏水性,虽然分析制备前可供疏水肽选择的有机溶剂系统较多,乙醇及甲醇溶剂系统可能最具代表性.当用乙醇或甲醇溶解众多MAGE-2疏水性表位多肽样品后进行色谱分析时发现,合成的MAGE-2表位多肽的RP-HPLC分析时,214 nm峰面积积分百分比都偏小,显示其合成时氨基酸偶联率较低,由此色谱分析结果推出的合成的低偶联率与理论上单个氨基酸99.5%的平均偶联率差距太大;随后的表位氨基酸组成分析也未发现有诸如半胱氨酸等可能导致折叠异构的氨基酸存在;如何消除这一困惑?正基于此,本实验随机选用了固相合成MAGE-2表位(161-169),通过不同溶剂处理后,在LC-MS技术平台上对其反相行为作了初步的研究.     

脑啡肽的固相合成与LC-MS/MS分离鉴定

多肽在生命过程中扮演着重要的角色,对其生理生化功能的研究与应用,离不开对单一多肽物质的需求,而化学合成法是获取目标多肽的最有效方法之一。对合成产物的分离与鉴定,是优化合成条件,以得到高产率的重要保证。以两种内源性神经肽亮氨酸脑啡肽和甲硫氨酸脑啡肽为模型,利用Fmoc固相多肽合成策略对其进行合成,并建立了HPLC-ESI-MS/MS新方法用于所制备的亮氨酸脑啡肽和甲硫氨酸脑啡肽的分离与结构鉴定。研究结果显示,主要合成产物均为目标多肽,副产物主要包括C端丢失1个氨基酸所形成的四肽,以及由于甲硫氨酸残基氧化而形成的含甲硫氨酸亚砜的多肽。该研究为高效合成含敏感氨基酸的生理活性多肽提供了新信息。     

HPLC-ELSD与GC-MS法测定牛乳甘油三酯sn-2位脂肪酸组成

研究建立了快速\\准确测定牛乳脂甘油三酯sn-2位脂肪酸组成的方法,利用胰酶专一水解甘油三酯sn-1和sn-3位置上的脂肪酸得到sn-2单甘油酯和游离脂肪酸,再通过蒸发光散射高效液相色谱分离出sn-2位单甘油酯,然后对其进行衍生,用气相色谱质谱联用仪对sn-2脂肪酸组成进行分析,结果显示用蒸发光散射高效液相色谱法分离sn-2位单甘油酯时方法的回收率达到83.3%~85.1%,该法省去了传统测定中费时费力的薄层色谱分离步骤.用气相色谱质谱联用法对产物进行分析,精密度高,结果可靠,分析结果表明,牛乳脂肪sn-2位脂肪酸由2.57%月桂酸、7.68%豆蔻酸、34.74%棕榈酸、11.56%亚油酸、22.53%油酸和15.21%硬酯酸组成.     

液相色谱-质谱/质谱法测定猪尿中β_2-受体激动剂残留量

本文应用固相萃取净化-液相色谱.质谱/质谱法同时测定尿液中8种β2-受体激动荆(妥布特罗、马布特罗、马贲特罗、特布他林、克伦特罗、塞布特罗、沙丁胺醇、莱克多巴胺)的残留量.对样品前处理中的提取液、SPE净化柱等参数进行了讨论优化,最低检测浓度(LOD):沙丁胺醇0.1μg/L,其他0.08μg/L,在阴性猪尿样品中添加回收率为68.2%～110.8%,8种β2-受体激动剂线性范围为0.1～4.0μg/L,RSD 3.2%～13.5%.     

Pharmacokinetics and bioavailability of ipatasertib in dog plasma using LC/MS/MS

A rapid, sensitive, and reliable liquid chromatography-tandem mass spectrometric method was developed to quantify ipatasertib in dog plasma. The dog plasma sample was deproteinated by using acetonitrile with ulixertinib as an internal standard followed by separation on a Spursil C₁₈-EP column with a gradient mobile phase comprising 2 mM ammonium acetate containing 0.1% formic acid and acetonitrile. Positive ion electrospray was used, and multiple reaction monitoring transitions were m/z 458.2 > 387.2 for ipatasertib and m/z 433.1 > 262.1 for the internal standard. The developed method was validated with a linear range of 0.3–1500 ng/mL, and with correlation coefficient greater than 0.9989. The lower limit of quantification was 0.3 ng/mL. The intra- and inter-day precision ranged from 3.58 to 14.32%, whereas the intra- and inter-day accuracy was in the range of −2.50–13.25%. No carry-over and matrix effects were observed under the current conditions. The extraction recovery was demonstrated to be greater than 85.43%. Ipatasertib was stable during the storage, processing, and determination. The validated assay was further successfully applied to a pharmacokinetic study of ipatasertib in dogs after oral and intravenous administrations. The bioavailability of ipatasertib was determined to be 19.3%.     

Simultaneous determination of anti‐diabetes/anti‐obesity drugs by LC/PDA,and targeted analysis of sibutramine analog in dietary supplements by LC/MS/MS

The safety of dietary supplements is questionable as there have been occasional reports of products contaminated with illegal adulterants. The present study was carried out to develop trustworthy methodologies to screen for six anti‐diabetic drugs (phenformin, rosiglitazone, glipizide, glimepiride, glybenclamide and gliclazide) and six anti‐obesity drugs (ephedrine, fenfluramine, T3, T4, fluoxetine and sibutramine) in dietary supplements. A simultaneous determination method of the 12 drugs by liquid chromatography coupled with a photodiode array (LC/PDA) was established and was validated for linearity (r² > 0.99), precision (RSD <13.3%), recoveries (88.8–115.9%) and reproducibility. Sibutramine and its analogs, N‐desmethylsibutramine, were subject to further investigation by LC/MS/MS because they were one of the major illegal adulterants. Our proposed method to monitor illegal drug adulterations in dietary supplements using LC/PDA is a simple and reliable, and therefore applicable to routine drug‐adulteration screening. Copyright © 2009 John Wiley & Sons, Ltd.     

LC–MS/MS assay for the quantification of foretinib in rat plasma and its application to preclinical pharmacokinetic study

A simple and sensitive ultra-high-performance liquid chromatography tandem mass spectrometric method was developed and validated for the determination of foretinib in rat plasma. The analyte and internal standard were extracted from the bio-samples with acetonitrile and then separated on an Acquity UPLC BEH C₁₈ column (50 × 2.1 mm, 1.7 μm) using 0.1% formic acid aqueous and acetonitrile as mobile phase, at a flow rate of 0.4 ml/min. The mass detection was performed in positive selected reaction monitoring mode with precursor-to-product transitions at m/z 317.1 > 128.1 for foretinib and m/z 502.2 > 323.1 for internal standard. The assay was demonstrated to be linear in the concentration range of 0.5–1000 ng/ml, with correlation coefficient >0.999. The mean extraction recovery of foretinib from rat plasma was within the range of 84.55–88.09%, while the matrix effect was in the range of 88.56–99.21%. The intra- and inter-day precisions were <12.95% and the accuracy ranged from −7.55 to 8.57%. Foretinib was stable in rat plasma under the tested storage conditions. The validated assay was successfully applied to the pharmacokinetic study of foretinib in the rats. The results revealed that foretinib showed moderate elimination half-life, low clearance and dose-independent pharmacokinetic profiles inrats.     

Analysis of turkish lignite tar by coupled LC/GC,GC/MS,and capillary SFC

This work describes the analysis of a pyrolysis product of a lignite sample obtained from the Turkish Goynuk reserve. The aliphatic, aromatic and polar compounds present in the tar are separated and identified by various chromatographic techniques: Capillary gas chromatography/mass spectrometry (GC/MS), on-line high performance microbore liquid chromatography/capillary gas chromatography (LC/GC) and capillary supercritical fluid chromatography (SFC). The suitability of each technique for this particular application is discussed, and semi-quantitative results are presented for the major components detected.     

A validated LC–MS/MS method for the quantification of capivasertib in dog plasma: Application to its pharmacokinetics study

In this study, a simple and reliable liquid chromatography tandem mass spectrometric method was first developed for the determination of capivasertib in dog plasma with ipatasertib as internal standard. The plasma samples were deproteinated using acetonitrile. An Acquity BEH C₁₈ column (1.7 μm, 2.1 × 50 mm) maintained at 40°C was used for chromatographical separation, with water containing 0.1% formic acid and acetonitrile as mobile phase. Multiple reaction monitoring transitions were m/z 429.2 > 135.1 for capivasertib and m/z 458.2 > 387.2 for ipatasertib, respectively. Excellent linearity was achieved in the concentration range of 1–1,000 ng/ml with a correlation coefficient of >0.9981. The lower limit of quantification was 1 ng/ml. The extraction recovery of capivasertib from dog plasma was >85.81% and no significant matrix effect was found. The intra- and inter-day precision was <9.58% and the accuracy ranged from −10.60% to 12.50%. The validated method was further applied to the pharmacokinetic study of capivasertib in dog plasma after single oral (5 mg/kg) and intravenous (1 mg/kg) administrations. The results revealed that capivasertib was rapidly absorbed into plasma with good bioavailability (47.04%) and low clearance.     

LC–MS/MS method for the determination of erianin in rat plasma: Application to a pharmacokinetic study

Erianin is one of the bibenzyl ingredients isolated from Dctidrobium chrysotoxum Lindl. In recent years, erianin has attracted attention owing to its antitumor activity. In this study, an LC–MS/MS method was established to measure erianin in rat plasma. Gigantol was used as the internal standard. A Waters Acquity UPLC BEH C₁₈ column was employed for chromatographic separation. The mobile phase consisted of water containing 0.1% formic acid and acetonitrile with a gradient elution at the flow rate of 0.4 ml/min. Selective reaction monitoring mode was used for quantitative analysis of erianin in positive electrospray ionization. In the concentration range of 0.1–1200 ng/ml, erianin in rat plasma was linear with correlation coefficient >0.999. The lowest limit of quantification was 0.1 ng/ml. The intra- and inter-day RSDs were <9.69%, while the RE was in the range of −8.59–11.24%. The mean recovery was >85.37%. Erianin was stable in rat plasma after storage under certain conditions. The validated method was demonstrated to be selective, sensitive and reliable, and has been successfully applied to pharmacokinetic study of erianin in rat plasma. Erianin was rapidly eliminated from rat plasma with a short half-life (〜1.5 h) and low oral bioavailability (8.7%).     

A review of direct liquid introduction interfacing for LC/MS Part II: Mass spectrometry and applications

Summary The coupling liquid chromatography and mass spectrometry is still growing in popularity. Direct liquid introduction has become one of the most important interfaces in LC/MS coupling. The various aspects of the interface have been investigated by several groups. This paper is the second part of a review of the developments in direct liquid introduction. Instrumental aspects of direct liquid introduction interfacing were discussed in the first part. This part will deal with the mass spectrometric aspects, i.e. chemical ionization and the influence of the various experimental conditions on the spectra. The applications of direct liquid introduction will also be reviewed briefly.     

溶剂诱导相变萃取-UPC~2快速测定中成药及原料药中的香豆素

使用溶剂诱导相变萃取对中成药及原料药进行预处理,建立了中成药中香豆素的超高效合相色谱(UPC2)检测方法。样品经粉碎(60目)、乙腈-水(7∶3,体积比)提取,二氯甲烷诱导分相后,使用UPC2对样品中的香豆素进行检测。以3倍信噪比(S/N)确定香豆素的检出限为0.1 mg/L;线性范围为0.3~10.0mg/L;加标回收率为89.8%~102.3%;相对标准偏差为0.31%~1.0%。同时与UPLC进行比较,结果表明该方法的选择性高,检测成本低,且分析时间仅为UPLC的1/4。该研究为高通量检测香豆素提供了一种全新的前处理及检测方法。     

SPE/RIA vs LC/MS for measurement of low levels of budesonide in plasma

A radioimmunoassay is described that measures budesonide in plasma after solid-phase extraction (SPE/RIA) of the analyte. The performance of the assay was compared with that of a selective LC/MS method. The limit of quantitation of budesonide determined for the LC/MS and SPE/RIA assay was 50 pg/mL and 120 pg/mL, respectively. Based on quality control samples, a higher variability was observed for the SPE/RIA (CV between 4.5 and 23.0%) than for the LC/MS method (CV between 7.5 and 12.5%). Plasma samples obtained from healthy volunteers after administration of budesonide rectal foam were assayed by both methods. In a subset of samples, these results were compared with those measured by direct RIA to evaluate the selectivity of two assays. About two times higher budesonide levels were measured with the direct RIA (lacking the extraction step), presumably because of cross-reactivity with budesonide metabolites, indicating that the extraction step in SPE/RIA is necessary for selectivity. Both SPE/RIA and LC/MS methods were found to be selective, sensitive and suitable for pharmacokinetic studies. Results obtained from the two methods were compared with a number of statistical methods. Ratios of results obtained for the clinical samples were close to 1 (ratio LC-MS/ SPE/RIA = 0.98 +/- 0.27). Linear regression indicated a slope of 1.17 +/- 0.0378. The concordance correlation (r = 0.91) indicated that the agreement between both methods was fair while the Bland-Altman plot indicated that the agreement was less pronounced at higher concentrations (1-3 ng/mL). In summary, the results confirm that the SPE/RIA is an alternative to HPLC/MS and that among the statistical methods tested the concordance correlation analysis was judged to be the most informative test to assess the comparability of two methods.