HPLC/ESI-Q-TOF MS鉴定淋巴癌患者尿液中的修饰核苷

尿液修饰核苷反映了机体RNA的代谢速率及细胞增殖状况, 可以作为非常有发展潜力的肿瘤标志物进行研究. 尿液中的修饰核苷采用Oasis®HLB固相萃取柱进行纯化, 利用高效液相色谱与电喷雾质谱联用(HPLC-ESI-MS)、高分辨质谱(HRMS)及串联质谱(MS/MS)技术进行分离鉴定. 对淋巴癌患者尿中修饰核苷研究发现, 9种尿液核苷与标样的信息完全一致, 17种无标样的尿液修饰核苷也被鉴定, 其中包括3-甲基腺苷、7-甲基腺嘌呤、5′-脱氢-2′-脱氧次黄苷、3-甲基鸟嘌呤、O6-甲基鸟苷和7-甲基-1-乙基鸟苷6种未见报道的新尿液修饰核苷. 此方法能在无对照品的情况下快速、准确地提纯、分离和鉴定复杂的生物样品.     

GC/MS和ESI/MS/MS同位素内标法检测甲基丙二酸血症

以甲基丙二酸血症为对象,分别用GC/MS和ESI/MS/MS方法对该疾病进行了定性和定量检测.通过对样品前处理和分离条件的改善,对疾病的标识化合物之一甲基丙二酸进行了定量测定,其稳定性、精密度和回收率结果很好.同时比较了GC/MS和ESI/MS/MS两种方法的特点,发现两种方法的结合不仅可满足新生儿代谢疾病筛查的要求,同时还可对高危人群进行诊断.     

林蛙油中农药残留的GC-MS/MS检测方法

首次建立了林蛙油中15种农药（p，p′DDE，O，p′-DDD，o p′-DDT，p，p′-DDT，BHC（α、β、γ、δ），异丙威，甲拌磷，六氯苯，百菌清，皮蝇磷，倍硫磷，甲氰菊酯）的GC—MS／MS残留检测方法。样品用乙酸乙酯提取、Florisil层析柱净化后，经GC—MS／MS检测，用外标法定量。15种农药在0．050～10．0mg／L范围内，线性关系良好，相关系数r≥0．996。当添加量在0．050—2．00mg／kg间，平均加标回收率为81％～117％，RSD为2．4％-18．2％，方法的检出限为0．050mg／kg。该方法准确、灵敏，适用于农药残留的分析。     

基于HPLC-TOF/MS与HPLC-MS/MS的养心氏片主要成分研究

采用高效液相色谱-高分辨飞行时间质谱联用技术(HPLC-TOF/MS),建立了快速鉴定养心氏片化学成分的定性方法,并利用高效液相色谱-串联三重四极杆质谱(HPLC-MS/MS)对养心氏片的主要成分进行定量分析。HPLC-TOF/MS法以ZORBAX Eclipse Plus C18(100 mm×3.0 mm,1.8μm)为色谱柱,乙腈-0.1%甲酸为流动相,梯度洗脱,流速为0.2 mL·min~(-1),柱温30℃,质谱采用正、负离子扫描,可推断鉴定养心氏片中的30种成分。HPLC-MS/MS法采用Waters XBridgeBEH C18(150 mm×4.6 mm,2.5μm)为色谱柱,乙腈-0.1%甲酸为流动相,梯度洗脱,流速0.4 mL·min~(-1),柱温30℃,Scheduled MRM正负离子切换监测模式定量分析。对养心氏片中16种主要成分进行HPLC-MS/MS分析,16种成分的质量浓度与峰面积呈良好线性,相关系数r≥0.997,检出限为0.039 1~78.1μg/L,定量下限为0.156~312μg/L,平均加标回收率为96.8%~103.8%,相对标准偏差(RSD)为1.0%~3.5%。结果表明,该方法操作简便、结果准确、重复性好,可用于养心氏片中多组分的同时定性和定量分析,为养心氏产品的质量控制、药理研究和应用奠定了基础。     

SPE-LC/MS/MS快速测定卷烟丝中的烟草特有亚硝胺类化合物

建立了卷烟丝中烟草特有亚硝胺类化合物(TSNAs)的SPE-LC/MS/MS分析方法,可一次性对卷烟烟丝中4种TSNAs进行定量分析.该方法弥补了传统的烟丝中TSNAs分析方法样品处理步骤多,检出限高,适应范围窄等缺点.4种TSNA的回收率的范围在95.7%～99.2%之间;相对标准偏差均小于8%;方法检出限均低于1.0 ng/g.可应用于国内外各类型卷烟的分析.     

粮谷中灭草松残留量检测的GC及GC-MS/MS研究

试验证明GC-ECD及GC-MS／MS两种方法均能用于粮谷中残留除草剂灭草松的测定。GC-MS／MS法可同时进行定性和定量分析,测定结果的可靠性较高。GC-ECD法具有较高的灵敏度,用于定量分析的效果较好。提出一种可与上述两种方法接用的试样预处理方法。10 g试样用甲醇提取其中的测定组分,再用正己烷萃取除去脂肪。用稀盐酸调节水相的酸度至pH≤3, 用二氯甲烷将灭草松萃取至有机相。在45℃减压干燥除去溶剂,所得浓缩的残渣溶于0．2 mL甲醇中并进行甲烷化和纯化(用固相萃取法),所得残留灭草松溶于1 mL正己烷中供GC-ECD或 GC-MS／MS测定。用两种方法进行粮谷试样的分析,测定结果的RSD值依次为2．3％-4．7％和 3．5％-6．7％。用标准加入法测得两方法的回收率依次为77％-98％和80％-102％,方法的检出限为0．01 mg·kg-1。     

硒芳香杂环化合物的GC/MS分析

本文对合成的7种含硒芳香杂环化合物进行了GC／MS分析研究。结果表明：BS、MB、BBS和DBBS等4个化合物在色谱柱内的保留时间与它们的相对分子质量呈线性关系。所有化合物均可获得特征质谱,表现出含单个硒原子的分子离子或碎片离子特征峰簇,硒的两种主要同位素在峰簇中表现为主要峰M与（M-2）的相对丰度比约为2：1,可为鉴定含硒分子离子或碎片离子提供重要信息。新化合物1,2,5-硒二唑并[3,4-d]嘧啶-5,7-（4H,6H）二酮（SPDO）在色谱柱内出现11．83min和7．96min两个具有相同的质谱的色谱峰,被认为是互变异构体的峰。     

采用配备GPC纯化装置的GC/MS进行农产品中农药残留的快速分析

本文采用GPC-GC／MS在线连接系统和固相萃取的方法快速测定农产品中的农药残留。该方法首先用液-液分配法从均化的食物样品中提取农药,然后用PSA结合相进行固相萃取,最后进行GPC-GC／MS分析。GPC-GC／MS系统包括一套GPC净化装置,可以简化样品前处理步骤。把浓度为0．1 μg／g的97种农药添加到马铃薯、甘蓝和胡萝卜中,得到了比较好的回收率。整个分析过程大约需要50 min。     

LC/MS法分析头孢替坦二钠原料中的杂质

本文应用LC/MS技术对头孢替坦二钠原料中的4种杂质进行了快速鉴定.根据头孢菌素的降解反应机制设计加速实验,确定头孢替坦的2个主要杂质为其碱水解产物;以1%冰醋酸溶液-乙腈-甲醇为流动相,经C18柱分离,通过电喷雾串联质谱负离子检测,获得各杂质的相对分子质量信息和碎片信息,并辅助UV特征对杂质结构进行了鉴定.在所建立的LC/MS条件下,头孢替坦及其杂质得到有效的分离,4个杂质经分析分别为5-巯基-1-甲基-四氮唑、头孢替坦内酯、头孢替坦脱羧物和头孢替坦异构体.本研究表明,利用LC/MS技术可推测头孢菌素类抗生素中杂质的结构,且本方法快速、灵敏、专属性高.     

GC/MS测定马面鲀鱼油中脂肪酸

本实验利用GC/MS电子轰击电离和化学电离两种方法对马面鲀鱼油中饱和、不饱和脂肪酸(经甲酯化后)进行了测定,用色谱法作了定量,共鉴定出16种脂肪酸。     

UPLC‐MS/MS based diagnostics for epithelial ovarian cancer using fully sialylated C4‐binding protein

Serum levels of fully sialylated C4‐binding protein (FS‐C4BP) are remarkably elevated in patients with epithelial ovarian cancer (EOC) and can be used as a marker to distinguish ovarian clear cell carcinoma from endometrioma. This study aimed to develop a stable, robust and reliable liquid chromatography–hybrid mass spectrometry (UPLC‐MS/MS) based diagnostic method that would generalize FS‐C4BP as a clinical EOC biomarker. Glycopeptides derived from 20 μL of trypsin‐digested serum glycoprotein were analyzed via UPLC equipped with an electrospray ionization time‐of‐flight mass spectrometer. This UPLC‐MS/MS‐based diagnostic method was optimized for FS‐C4BP and validated using sera from 119 patients with EOC and 127 women without cancer. A1958 (C4BP peptide with two fully sialylated biantennary glycans) was selected as a marker of FS‐C4BP because its level in serum was highest among FS‐C4BP family members. Preparation and UPLC‐MS/MS were optimized for A1958, and performance and robustness were significantly improved relative to our previous method. An area under the curve analysis of the FS‐C4BP index receiver operating characteristic curve revealed that the ratio between A1958 and A1813 (C4BP peptide with two partially sialylated biantennary glycans) reached 85%. A combination of the FS‐C4BP index and carbohydrate antigen‐125 levels further enhanced the sensitivity and specificity.     

毛细管电泳-小波神经网络法辅佐诊断乳腺癌的研究

采用毛细管电泳法测定了46个健康人和26个乳腺癌病人尿样中的13种正常核苷和修饰核苷,以小波神经网络作为模式识别工具对健康人和乳腺癌病人的分类作了研究,随机选取的训练集的识别率达到100％,相应的预测集判别率正确性在96％以上,与经典的前向多层神经网络相比,小波神经网络具有更强的信息提取和逼近能力.研究结果还表明,小波神经网络的预测能力强于主成分分析和线性判别分析,毛细管电泳法与小波神经网络的结合有望成为乳腺癌的辅助诊断手段.     

Comprehensive analysis of proteins of pH fractionated samples using monolithic LC/MS/MS, intact MW measurement and MALDI-QIT-TOF MS

A comprehensive platform that integrates information from the protein and peptide levels by combining various MS techniques has been employed for the analysis of proteins in fully malignant human breast cancer cells. The cell lysates were subjected to chromatofocusing fractionation, followed by tryptic digestion of pH fractions for on-line monolithic RP-HPLC interfaced with linear ion trap MS analysis for rapid protein identification. This unique approach of direct analysis of pH fractions resulted in the identification of large numbers of proteins from several selected pH fractions, in which approximately 1.5 microg of each of the pH fraction digests was consumed for an analysis time of ca 50 min. In order to combine valuable information retained at the protein level with the protein identifications obtained from the peptide level information, the same pH fraction was analyzed using nonporous (NPS)-RP-HPLC/ESI-TOF MS to obtain intact protein MW measurements. In order to further validate the protein identification procedures from the fraction digest analysis, NPS-RP-HPLC separation was performed for off-line protein collection to closely examine each protein using MALDI-TOF MS and MALDI-quadrupole ion trap (QIT)-TOF MS, and excellent agreement of protein identifications was consistently observed. It was also observed that the comparison to intact MW and other MS information was particularly useful for analyzing proteins whose identifications were suggested by one sequenced peptide from fraction digest analysis.     

Detection of flavonoids from Spinacia oleracea leaves using HPLC-ESI-QTOF-MS/MS and UPLC-QqQLIT-MS/MS techniques

Spinacia oleracea L. (Spinach) is a leafy vegetable which is considered to have a high nutritional value. Flavonoids in spinach were reported to act as antimutagenic property. Rapid detection of these flavonoids in Spinach was achieved by using HPLC-ESI-QTOF-MS/MS. Thirty six compounds were tentatively identified based on their retention times, accurate mass and MS/MS spectra. The fragmentation patterns of known compounds were applied to elucidate the structure of their corresponding derivatives having the same basic skeleton. Out of thirty six peaks, three peaks were assigned as patuletin and six peaks were assigned as spinacetin derivatives. Twelve compounds were first time identified following the fragmentation pattern of known compounds. Five of the identified compounds i.e., spinacetin, 5,3′,4′-trihydroxy-3-methoxy-6,7-methylenedioxyflavone, protocatechuic acid, ferulic acid and coumaric acid were simultaneously quantified in spinach leaves by a validated UPLC-ESI-MS/MS method under MRM mode.     

Derivatization of chiral carboxylic acids with (S)‐anabasine for increasing detectability and enantiomeric separation in LC/ESI‐MS/MS

A simple and practical derivatization procedure for increasing the detectability and enantiomeric separation of chiral carboxylic acids in LC/ESI‐MS/MS has been developed. (S)‐Anabasine (ANA) was used as the derivatization reagent and rapidly reacted with carboxylic acids [3‐hydroxypalmitic acid (3‐OH‐PA), 2‐(β‐carboxyethyl)‐6‐hydroxy‐2,7,8‐trimethylchroman (γ‐CEHC), and etodolac] in the presence of 4‐(4,6‐dimethoxy‐1,3,5‐triazin‐2‐yl)‐4‐methylmorpholium chloride. The resulting ANA‐derivatives were highly responsive in ESI‐MS operating in the positive‐ion mode and gave characteristic product ions during MS/MS, which enabled sensitive detection using selected reaction monitoring; the detection responses of the ANA‐derivatives were increased by 20–160‐fold over those of the intact carboxylic acids and the limits of detection were in the low femtomole range (1.8–11 fmol on the column). The ANA‐derivatization was also effective for the enatiomeric separation of the chiral carboxylic acids; the resolution was 1.92, 1.75, and 2.03 for 3‐OH‐PA, γ‐CHEC, and etodolac, respectively. The derivatization procedure was successfully applied to a biological sample analysis; the derivatization followed by LC/ESI‐MS/MS enabled the separation and detection of trace amounts of 3‐OH‐PA in neonatal dried blood spot and γ‐CEHC in human saliva with a simple pretreatment and small sample volume.     

Screening of exon methylation biomarkers for colorectal cancer via LC‐MS/MS strategy

The identification of biomarkers would be of benefit for the diagnosis and treatment of colorectal cancer. DNA methylation in specific genomic regions, which had shown strongly association with disease genotypes, was an effective indicator to reveal the occurrence and development of cancers. To screen out methylation biomarkers for colorectal cancer (CRC), genomic DNA was isolated from colorectal cancerous and corresponding cancer‐adjacent tissues collected from 30 CRC patients and then bisulfite‐converted. The exon regions of 5 targeted genes (CNRIP1 , HIC1 , RUNX3 , p15 , and SFRP2 ) were amplified by using nested polymerase chain reaction with specific primers, and the amplicon was purified and hydrolyzed. The methylation levels of these specific regions were detected by liquid chromatography tandem mass spectrometry (LC‐MS/MS). The results showed that 5 targeted exon regions were successfully amplified and confirmed by sequencing. The methodological validations indicated that LC‐MS/MS was highly sensitive and accurate. The methylation levels of CNRIP1 and RUNX3 were remarkably high in CRC tissues with statistical difference when compared with corresponding cancer‐adjacent individuals, while that of HIC1 , p15 , and SFRP2 had no difference between 2 subjects. These findings supported CNRIP1 and RUNX3 as potential DNA methylation biomarkers for CRC diagnosis and treatment, and our LC‐MS/MS approach exhibited great advantages in the identification of regional DNA methylation biomarkers.     

Detection and Sequence Identification of Dinucleotides Produced from N‐Phosphoryl Alanine and Four Nucleosides by HPLC‐ESI‐MS/MS

本文利用液相色谱质谱联用技术研究了N－磷酰化丙氨酸和四种核苷（腺苷,尿苷,胞苷和鸟苷）的模板反应产物。结果表明生成了不同类型的单核苷酸和二核苷酸,并且生成的二核苷酸序列也得到了确证。研究结果揭示,二核苷酸骨架裂解形成的c离子可以作为确证二核苷酸序列的诊断离子。本文首次证明不论是在正离子模式还是在负离子模式下,c离子都可以用来确定此反应体系中生成的二核苷酸产物的序列。