毛细管反相液相色谱-串联质谱用于肽的鉴定和相对定量分析

建立了一种基于毛细管反相液相色谱-串联质谱联用技术和质谱峰强度数据处理的肽段鉴定和相对定量分析方法。该方法无需对样品中的肽进行化学标记,在对样品进行反相色谱分离和串联质谱分析后,将二级质谱扫描数据进行蛋白质数据库搜索,获得所鉴定肽段的序列、保留时间、质荷比、带电荷数等定性信息;再以此为定位依据,在全扫描质谱数据中提取该肽段对应的离子峰并以该离子峰的峰强度作为定量信息,从而实现对不同样品中的共有肽段进行差异比较分析。以标准蛋白酶解混合肽段为实验对象,以肽段相对强度的相对标准偏差为指标,考察了该方法用于肽段相对定量分析的重现性、检测动态范围以及浓度标准曲线等,为将该方法用于生物样品中内源性肽的差异分析奠定了基础。     

Determination of flunixin and ketoprofen in milk by liquid chromatography-tandem mass spectrometry

A relatively fast, simple and very selective liquid chromatography-tandem mass spectrometry (LC-MS-MS) method for the detection of flunixin, its 5-hydroxymetabolite and ketoprofen in raw milk has been developed and validated. After a simple extraction with acetonitrile and partial evaporation of the organic phase, the extract was filtered and directly injected into the LC-MS-MS system on a Symmetry C₁₈ column. The parent ions were selected for further fragmentation with argon. The method developed was partially validated according to Commission Decision 2002/657/EC [Commission Decision 2002/657/EC implementing Council Directive 96/23/EC concerning the performance of analytical methods and the interpretation of results]. The validation parameters were linearity, specificity, repeatability, recovery and decision limit (CCα). CCα varied from 0.5 μg kg⁻¹ for flunixin and 5-hydroxyflunixin to 1 μg kg⁻¹ for ketoprofen.Holstein-Friesian cows were given either Ketofen^® or Finadyne^® via an intravenous injection at the maximum dose as written in the instructions. Cows were milked twice a day and all samples were analysed by the method described. The highest concentrations found for ketoprofen, flunixin and 5-hydroxyflunixin were 2.5, 6.7 and 590 μg l⁻¹, respectively. The concentration of 5-hydroxyflunixin declined rapidly to concentrations below the MRL value of 40 μg l⁻¹. It can be concluded that the withdrawal time proposed by the pharmaceutical companies, 12 h after the last dosing, is acceptable for both compounds.     

Simultaneous quantification of fatty acids in serum with dual derivatization by liquid chromatography-tandem mass spectrometry

Fatty acids have multitudinous biological functions and play a crucial role in many biological processes, but due to poor ionization efficiency and lack of appropriate internal standards, the comprehensive quantification of fatty acids by liquid chromatography-tandem mass spectrometry is still challenging. In this study, a new, accurate, and reliable method for quantifying 30 fatty acids in serum using dual derivatization was proposed. Indole-3-acetic acid hydrazide derivants of fatty acids were used as the internal standard and indole-3-carboxylic acid hydrazide derivants of them were used to quantify. The derivatization conditions were systematically optimized and the method validation results showed good linearity with R² > 0.9942, low detection limit (0.03–0.6 nM), precision (1.6%–9.8% for intra-day and 4.6%–14.1% for inter-day), recovery (88.2%–107.2% with relative standard deviation < 10.5%), matrix effect (88.3%–105.2% with the relative standard deviation < 9.9%) and stability (3.4%–13.8% for fatty acids derivants in 24 h at 4°C and 4.2%–13.8% for three freeze-thaw cycles). Finally, this method was successfully applied to quantify fatty acids in serum samples of Alzheimer's patients. In contrast to the healthy control group, nine fatty acids showed a significant increase in the Alzheimer's disease group.     

高效液相色谱-串联质谱法检测婴幼儿血清中4种双酚类环境激素残留

目前,双酚类化合物是重要的工业原料,常用来制造塑料(奶)瓶、幼儿用吸口杯、食品和饮料(奶粉)罐内侧涂层,其具有类似雌激素的作用,摄入低剂量的双酚类物质便会引起机体尤其婴幼儿体内激素水平的调节。建立了一种同时测定婴幼儿血清中双酚A(BPA)、双酚B(BPB)、双酚F(BPF)、双酚S (BPS)4种双酚类环境激素的高效液相色谱-串联质谱法(HPLC-MS/MS)。以甲基叔丁基醚(MTBE)为提取溶剂,采用液液萃取的方法进行样品处理,同时对影响4种双酚类环境激素提取效率的提取溶剂、提取时间、提取溶剂体积等影响因素进行了优化。100μL血清样本在40℃下经400μL MTBE提取15 min后,采用Waters ACQUITY UPLC BEH C18柱(50 mm×2.1 mm, 1.7μm)进行分离,以超纯水和含0.5 mmol/L乙酸铵的甲醇溶液为流动相进行梯度洗脱,流速为0.2 mL/min,采用电喷雾电离、负离子模式扫描,在多反应监测(MRM)模式下测定。BPA、BPB、BPS在0.25～100μg/L范围内线性关系良好,BPF在1～00μg/L范围内线性关系良好,相关系数为0.9...     

液相色谱-串联质谱法测定猪肉中咪唑类药物及其代谢物

建立了高效液相色谱-串联质谱同时测定猪肉中29种咪唑类药物及其代谢物残留的检测方法。样品采用乙酸乙酯提取,浓缩复溶后加入乙腈饱和正己烷净化除脂,以0.3%(体积分数)甲酸水溶液和乙腈为流动相,反相C_(18)色谱柱梯度洗脱分离,采用电喷雾正离子(ESI+)源多反应监测(MRM)模式进行检测。29种化合物在0.05~20.0μg/L范围内线性关系良好,相关系数r~20.99。在1.0~5.0μg/kg添加范围内,平均回收率为65.4%~103%,相对标准偏差(RSD)为1.3%~6.8%。方法检出限为0.02~0.3μg/kg,定量限为0.1~1μg/kg。该方法简便、快速、灵敏,适用于猪肉中咪唑类及其代谢物残留的检测。     

Improving quantitative precision and throughput by reducing calibrator use in liquid chromatography-tandem mass spectrometry

To improve efficiency in our mass spectrometry laboratories we have made efforts to reduce the number of calibration standards utilized for quantitation over time. We often analyze three or more batches of 96 samples per day, on a single instrument, for a number of assays. With a conventional calibration scheme at six concentration levels this amounts to more than 5000 calibration points per year. Modern LC-tandem mass spectrometric instrumentation is extremely rugged however, and isotopically labelled internal standards are widely available. This made us consider whether alternative calibration strategies could be utilized to reduce the number of calibration standards analyzed while still retaining high precision and accurate quantitation.     

基于液相色谱-串联质谱的结肠癌血清氧固醇标志物筛选

结肠癌(CC)是全球常见恶性肿瘤之一,发病率呈逐年上升趋势,目前没有有效的标志物用于疾病早期诊断和干预跟踪。胆固醇及其氧化衍生物氧固醇在众多恶性肿瘤发生发展中发挥关键作用。该研究采用液相色谱-串联质谱(LC-MS/MS)技术,对CC临床血清样本中胆固醇及相关10种氧固醇代谢物进行了定性定量分析,并采用偏最小二乘判别分析(PLS-DA)和正交偏最小二乘判别分析(OPLS-DA)进行多元统计分析,发现上述目标代谢物能够较好地区分CC组与健康对照组。为防止数据过拟合,该研究在PLS-DA模型各代谢物变量投影重要性(VIP)基础上,结合最优组分数及K-均值聚类结果,筛选得到3种代谢标志物。通过受试者操作特征曲线(ROC)的曲线下面积(AUC)分析,发现筛选得到的3种潜在标志物联合预测CC达到0.998,说明模型性能优良。GO(基因本体论)富集分析显示3种潜在标志物主要分布在内质网和包被囊泡上,参与胆固醇代谢、运输、低密度脂蛋白重塑等生物进程,发挥胆固醇运输活性和低密度脂蛋白颗粒受体结合的分子功能。KEGG(京都基因与基因组百科全书)通路分析显示3种潜在标志物富集于类固醇生物合成、PPAR(过氧化物酶体增殖物激活受体)信号通路及ABC(ATP结合盒)转运等通路上。该研究为寻找CC标志物及进一步阐明胆固醇及氧固醇在CC发病过程中的作用奠定了一定的基础。     

液相色谱-串联质谱法测定卷烟主流烟气中的四种致癌物

N-亚硝基降烟碱（NNN）、4-（亚硝基甲氨基）-1-（3-吡啶基）-1-丁酮（NNK）、N-亚硝基新烟草碱 （NAT）和N-亚硝基假木贼碱（NAB）是4种广泛存在于烟草和烟气中的致癌物,准确测定其含量对评估其对人体健康的影响有着重要的作用。采用液相色谱-电喷雾串联质谱(LC-ESI MS/MS)技术建立了卷烟主流烟气中NNN、NNK、NAT和NAB的测定方法,并将其用于中国烤烟和混合型卷烟主流烟气的分析。卷烟主流烟气通过剑桥滤片捕集,捕集烟气后的滤片在加入100 μL氘代混合内标后用10 mL 100 mmol/L醋酸铵水溶液萃取,萃取液过水相滤膜后直接进行LC-ESI MS/MS检测。选用Agilent Zorbax Eclipse XDB-C18色谱柱,以流动相0.1%(v/v)乙酸水溶液和0.1%（v/v）乙酸甲醇溶液梯度洗脱,质谱检测采用正离子扫描,多反应监测模式。NNN、NNK、NAT和NAB的检出限分别为0.019、0.002、0.008和0.007 μg/L,回收率为84.9%～104.5%,相对标准偏差(n=8)为2.96%～6.65%。该方法的检出限低,特异性好,适用于卷烟主流烟气中NNN、NNK、NAT和NAB释放量的检测。     

液相色谱-串联质谱法研究涕灭威及其代谢物在白菜中的动态行为

采用液相色谱-串联质谱法(LC-MS/MS)研究了白菜中涕灭威及其代谢物的残留动态行为。按农民常用施药量(3000 g/hm²)将涕灭威沟施予田地后,定期取样检测,并建立其残留行为的数学模型。结果表明,用LC-MS/MS测定涕灭威及其代谢物,在0.005~0.2 mg/L范围内线性关系良好,平均回收率为78.9%~108.5%,相对标准偏差为2.03%~8.91%(n=8)。涕灭威在白菜中的浓度升高和降低的过程均符合一级动力学模型(c=0.020e^0.136t和c=0.65e^-0.059t),相关系数(r²)分别为0.888和0.979,半衰期为29.1天。代谢产物涕灭威砜和涕灭威亚砜在白菜中的浓度降低时同样符合一级动力学方程(c=23.4e^-0.044t和c=4.54e^-0.027t), r²分别为0.916和0.972。涕灭威、涕灭威砜和涕灭威亚砜在白菜中的含量要降到国内外限量要求0.01 mg/kg,分别需要70.7、226.6和176.3天。白菜生长周期为120天,收获时涕灭威砜和涕灭威亚砜含量仍超过国内外限量要求。因此,在本文施药量下,涕灭威不能使用。该数据为涕灭威的安全使用和农药残留动态行为研究提供了理论依据。     

高效液相色谱-串联质谱法测定蔬菜水果中双甲脒及其代谢产物

建立了高效液相色谱-串联质谱（HPLC-MS/MS）检测蔬菜水果中双甲脒（AMZ）及其降解或代谢产物单甲脒（DMPF）、2，4-二甲基苯基甲酰胺（DMF）和2，4-二甲基苯胺（DMA）的分析方法。样品经0.1 mol/L NaOH溶液稀释、正己烷-异丙醇（2：1，v/v）混合提取液提取，然后采用HPLC-MS/MS对目标物进行定性确证和定量分析。采用Phenomenex Kinetex C18色谱柱（100 mm×4.6 mm，2.6 μm）分离，以0.1%（v/v）甲酸水溶液和甲醇为流动相进行梯度洗脱，采用电喷雾电离（ESI）源、正离子监测模式进行检测。在1.0~200.0 μg/L范围内，4种目标化合物呈良好的线性关系，相关系数（r）大于0.99；方法的定量限为0.01~0.4 μg/kg。4种目标化合物在0.5、5.0和20.0 μg/kg 3个水平下的加标回收率为62.5%~105.0%，RSD为7.5%~17.6%。该法简便快捷，准确可靠，灵敏度高，定量限满足目前国内外残留限量要求。     

High-performance liquid chromatography-tandem mass spectrometry with a new quadrupole/linear ion trap instrument

The use of a new hybrid quadrupole/linear ion trap known as the Q TRAP offers unique benefits as a LC-MS-MS detector for both small and large molecule analyses. The instrument combines the capabilities of a triple quadrupole mass spectrometer and ion trap technology on a single platform. Product ion scans are conducted in a hybrid fashion with the fragmentation step accomplished via acceleration into the collision cell followed by trapping and mass analysis in the Q3 linear ion trap. This results in triple quadrupole fragmentation patterns with no inherent low molecular mass cutoff. In-trap fragmentation is also possible in order to provide triple MS (MS3) capabilities. There are also several scan modes that are not possible on conventional instruments that enable identification of analytes within complex biological matrixes for subsequent high sensitivity product ion scans. This report will describe the new hybrid instrument and the principles of operation, and also provide examples of the unique scan modes and capabilities of the Q TRAP for LC-MS-MS detection in metabolism identification.     

Identification of tanshinone IIA metabolites in rat liver microsomes by liquid chromatography-tandem mass spectrometry

Tanshinone IIA, the major component extracted from Radix salvia miltiorrhiza, has been observed to possess various kinds of pharmacological activities including antioxidant, prevention of angina pectoris and myocardial infarction and anticancer. Tanshinone IIA was incubated with rat liver microsomes and the resulting metabolites were identified by liquid chromatography/tandem mass spectrometry. The results showed the formation of three main hydroxyl metabolites. The three hydroxyl metabolites of tanshinone IIA were proved to be tanshinone IIB, hydroxytanshinone IIA and przewaquinone A by comparing the tandem mass spectra and the chromatographic retention time with that of the respective authentic compounds. Tanshinone IIB, hydroxytanshinone IIA and przewaquinone A are all the chemical components of total tanshinones. It was reasonable to presume that the three hydroxy metabolites of tanshinone IIA were pharmacologically active the same as tanshinone IIA and the total tanshinones.     

Identification and quantification of cucurbitacin in watermelon frost using molecular networking integrated with ultra-high-performance liquid chromatography-tandem mass spectrometry

Watermelon frost, a traditional Chinese medicine produced using watermelon and Glauber's salt, has been widely used for the therapy of oral and throat disorders. Watermelon contains various phytochemical compounds including cucurbitacins and their glycoside derivatives, which have attracted considerable attention because of their medicinal values. However, whether the composition of cucurbitacins existed in watermelon frost was rarely reported. In this study, three cucurbitacins including cucurbitacin B, isocucurbitacin B, and cucurbitacin E were found from watermelon frost extract assisted by ultra-high-performance liquid chromatography-tandem mass spectrometry and molecular networking guided strategy, and the compounds were verified using standard solutions. Furthermore, a quantification method for simultaneously targeted analysis of cucurbitacins was established using ultra-high-performance liquid chromatography-tandem mass spectrometry operating in the multiple reaction monitoring mode. Among them, cucurbitacin B and cucurbitacin E in watermelon frost samples were determined, and the concentrations were 3.78 ± 0.18 and 0.86 ± 0.19 ng/ml, respectively. While isocucurbitacin B was not detected due to the lower content possibly. In conclusion, ultra-high-performance liquid chromatography-tandem mass spectrometry combined with molecular networking is a very useful technique for the rapid identification of unknown cucurbitacin components in watermelon frost.     

液相色谱-串联质谱法检测血清中15种甾体激素的动态变化

建立了用液相色谱-串联质谱检测血清中15种甾体激素的方法。在血清样品中加入含内标的乙腈沉淀蛋白质后,用100 mmol/L盐酸羟胺衍生,经Agilent-C18柱(50 mm×3.0 mm,2.7μm)分离,内标法定量。质谱分析采用电喷雾电离(ESI)正离子扫描,多反应监测(MRM)模式检测。数据显示,13种激素在0.05~20 ng/mL范围内线性关系良好(相关系数(R2)不小于0.995 6),检出限不大于1 ng/mL;皮质醇及脱氢表雄酮硫酸酯在50~2 000ng/mL范围内线性关系良好(R2不小于0.997 9),检出限不大于0.5 ng/mL。将该方法应用于女性月经周期激素变化规律的研究,结果表明,15种甾体激素在一个完整的月经周期内有不同特征的动态变化,其中雄激素在滤泡期的后期达到最高值,孕(雌)激素在黄体期达到最高值,而皮质激素则波动不明显。该方法灵敏度高、重复性好,为临床诊疗提供了参考。     

Drugs of abuse and their metabolites in waste and surface waters by liquid chromatography-tandem mass spectrometry

A solid‐phase extraction and liquid chromatography‐tandem mass spectrometry (SPE/LC‐MS‐MS) method was developed and validated for the simultaneous determination of nicotine, five drugs of abuse (morphine, cocaine, codeine, methadone, and 2‐ethylidene‐1,5‐dimethyl‐3,3‐diphenylpyrrolidine) and four metabolites (dihydrocodeine, 6‐acetylmorphine, 11‐nor‐carboxy‐Δ⁹‐tetrahydrocannabinol, and benzoylecgonine) in water samples. A Fused‐Core? particle column was used as an alternative to sub‐2‐μm particles in chromatographic separations to work with low backpressures and high efficiencies in short analysis times. Drugs were extracted from waste and surface water with SPE using Oasis MCX cartridges. Electrospray (ESI) in positive and negative mode and tandem MS selected reaction monitoring mode were used for identification and quantification. Calibration by linear regression analysis with deuterated internal standards was used to compensate the matrix effects. Limits of detection were found as low as 0.5–1 ng/L (surface water) and 1–50 ng/L (wastewater). The method was applied to the analysis of different kinds of samples. Wastewater from a sewage treatment plant was collected from three sampling points (after primary, secondary, and tertiary treatments) for a week. The analysis of the samples revealed a significant presence of these drugs in samples from primary treatments, where maximum concentrations of nicotine (1105 ng/L) and benzoylecgonine (3336 ng/L) were found. Most of the compounds showed values between 相似文献   

液相色谱-串联质谱法测定黄瓜和苹果中氟啶虫酰胺及其代谢产物残留量

建立了黄瓜和苹果中氟啶虫酰胺及其3种代谢产物［N-(4-trifluoromethylnicotinoyl)glycine(TFNG)、4-tri-fluoromethylnicotinic acid(TFNA)和4-trifluoromethylnicotinamide(TFNA-AM)］同时测定的液相色谱-串联质谱分析方法。样品用磷酸盐缓冲液提取两次,调节pH值至1.5～2.0后,再用乙酸乙酯提取,液相色谱-串联质谱分析。采用Acquity BEH C18色谱柱分离,0.1%甲酸水-甲醇作为流动相进行梯度洗脱,电喷雾正离子(ESI+)模式电离,多反应监测(MRM)模式检测,外标法定量。氟啶虫酰胺、TFNG、TFNA和TFNA-AM的检出限分别为0.17、0.20、0.35和0.60 μg/kg。在黄瓜和苹果样品中添加5.0～2000 μg/kg水平的氟啶虫酰胺、TFNG、TFNA和TFNA-AM,其平均添加回收率在82.9%～104.1%范围内,批内分析相对标准偏差(RSD)在3.6%～6.9%之间。4种物质的峰面积与其浓度在0.50～200 μg/L范围内均呈良好的线性关系,线性回归系数均大于0.998。前处理步骤仅用有机溶剂6 mL。整个方法具有高灵敏度、准确、稳定的特点。     

固相萃取-液相色谱-串联质谱法测定蔬菜中4种有机磷农药及其代谢产物

在蔬菜种植中经常使用的有机磷农药马拉硫磷、甲基对硫磷、敌百虫及乙酰甲胺磷可能转化的主要代谢产物分别为O,O-二甲基二硫代磷酸酯、对硝基酚、敌敌畏及甲胺磷。根据蔬菜色素等基质的含量不同采用不同的净化方法,色素含量高的蔬菜采用活性炭和弗罗里硅土串联固相萃取小柱净化,其他蔬菜采用弗罗里硅土固相萃取小柱净化。色谱分离选择ACQUITY UPLC BEH HILIC色谱柱,以乙腈和5 mmol/L乙酸铵水溶液为流动相进行梯度洗脱,得到的分离效果较好;质谱采用电喷雾正或负离子电离、多反应监测模式检测。液相色谱-质谱检测的基质效应为15.3%~45.1%;4种有机磷农药及其代谢物的方法回收率为76.9%~102.8%,相对标准偏差为5.92%~10.19%;定量限范围为0.001~0.01 mg/L;在0.01~1.00 mg/L范围内线性相关系数为0.9982~0.9999。方法具有良好的回收率、相对标准偏差、定量限及线性关系,适合蔬菜中有机磷及代谢物的检测,应用该检测方法对农贸市场购买的白菜、辣椒、西红柿及洋葱进行了检测。     

Determination of digoxin and its metabolites in biological samples by solidphase supported liquid-liquid extraction-liquid chromatography-tandem mass spectrometryEI北大核心CSCD

采用固相支撑液液萃取-超高效液相色谱-串联质谱（SLE-UPLC-MS/MS）技术建立了生物样本血液、尿液和肝组织中地高辛（DG）及其3种代谢物的分析方法。生物样本经匀浆、蛋白沉淀后,通过含有硅藻土的固相支撑液液萃取（SLE）柱净化富集,经洗脱、定容后进行LC-MS/MS分析。结果表明,血液基质中,地高辛在0.1～100 ng/mL浓度范围内线性关系良好;肝脏和尿液基质中,地高辛在0.2～100 ng/mL浓度范围内线性关系良好,地高辛的3种代谢物在0.5～100 ng/mL浓度范围内线性关系良好,3个浓度水平（10, 50和100 ng/mL）的加标回收率为60.5%～95.6%,基质效应80.7%～113.6%,日内、日间相对标准偏差（RSD）均小于13%,检出限为0.1～0.5 ng/mL。所建立的方法可用于生物样本中地高辛及其代谢物的定性定量分析。     

Nonaqueous capillary electrophoresis method for the analysis of tamoxifen, imipramine and their main metabolites in urine

The viability of nonaqueous capillary electrophoresis (NACE) was investigated for the simultaneous determination of tamoxifen, imipramine and their main metabolites (4-hydroxytamoxifen and desipramine, respectively). Baseline separation of the studied solutes was obtained on a 57 cm × 75 μm capillary using a nonaqueous solution composed of 17 mM ammonium acetate and 1.25% acetic acid in 80:20 (v:v) methanol-acetonitrile, temperature and voltage 22 °C and 15 kV, respectively, and hydrodynamic injection. Paroxetine was used as internal standard. Different aspects including linearity, accuracy, ruggedness and precision was studied. Detection limits between 9.0 and 15.0 μg L⁻¹ were obtained for all the studied compounds. The developed method is simple, rapid and sensitive and has been used to determine tamoxifen, imipramine and their metabolites at clinically relevant levels in human urine. Before NACE determination, a solid phase extraction (SPE) procedure with a C₁₈ cartridge was necessary. Real determination of these analytes in three females urines were done.     

超高效液相色谱-串联质谱法测定畜禽毛发中利巴韦林及其代谢物

建立了一种测定畜禽毛发中利巴韦林及其代谢物1H-1,2,4-三氮唑-3-甲酰胺(TCONH2)残留的检测方法。1%十二烷基硫酸钠清洗毛发,2%甲酸-甲醇(2:98,V/V)溶液提取,13C-利巴韦林(13CRBV)内标法定量,乙二胺-N-丙基硅烷(PSA)和十八烷基硅烷(C18)基质分散净化,Agilent ZORBAX SB-Aq色谱柱分离,电喷雾串联质谱多反应监测模式测定。利巴韦林和TCONH2在毛发中线性关系良好(R^2>0.99)在2,10,100μg/kg3个添加水平下利巴韦林和代谢物TCONH2的回收率分别为101.5%~108.5%和98.5%~101.5%相对标准偏差均小于7%,检出限和定量限分别为0.2μg/kg和0.5μg/kg。该方法适合于畜禽毛发中违禁抗病毒药物利巴韦林及其代谢物TCONH2的测定。