Determination of Ten Kinds of Alpha-2 Agonists Residues in Animal Derived Food by UHPLC-Triple Quadrupole/Composite Linear Ion Trap Mass Spectrometry

[Objectives]The paper was to establish an ultra high performance liquid chromatography-quadrupole/linear ion trap complex mass spectrometry for the determination of 10 kinds ofα2-receptor agonists in animal derived food.[Methods]The samples were extracted with sodium carbonate buffer solution and ethyl acetate,and analyzed by mass spectrometry after solid phase extraction and high performance liquid chromatography separation.[Results]Ten kinds ofα2-receptor agonists showed a good linear relationship in the range of 1-100μg/mL,with the average recovery of over 69%and the relative standard deviation less than 8.32%.The detection limit of 10 kinds of α_(2)-receptor agonists was up to 1μg/kg.[Conclusions]The method has good selectivity and strong anti-interference ability,and can meet the requirements of 10 kinds ofα2-receptor agonists residues in animal derived food.

Simultaneous identification and quantification of tetrodotoxin in fresh pufferfish and pufferfish-based products using immunoaffinity columns and liquid chromatography/quadrupole-linear ion trap mass spectrometry

In this study, we established a comprehensive method for simultaneous identification and quantification of tetrodotoxin （TTX） in fresh pufferfish tissues and pufferfish-based products using liquid chromatography/quadrupole-linear ion trap mass spectrometry （LC-QqLIT-MS）. TTX was extracted by 1% acetic acid-methanol, and most of the lipids were then removed by freezing lipid precipitation, followed by purification and concentration using immunoaffinity columns （IACs）. Matrix effects were substantially reduced due to the high specificity of the IACs, and thus, background interference was avoided. Quantitation analysis was therefore performed using an external calibration curve with standards prepared in mobile phase. The method was evaluated by fortifying samples at 1, 10, and 100 ng/g, respectively, and the recoveries ranged from 75.8%--107%, with a relative standard deviation of less than 15%. The TTX calibration curves were linear over the range of 1-1 000 ~tg/L, with a detection limit of 0.3 ng/g and a quantification limit of 1 ng/g. Using this method, samples can be further analyzed using an information- dependent acquisition （IDA） experiment, in the positive mode, from a single liquid chromatography-tandem mass spectrometry injection, which can provide an extra level of confirmation by matching the full product ion spectra acquired for a standard sample with those from an enhanced product ion （EPI） library. The scheduled multiple reaction monitoring method enabled TTX to be screened for, and TTX was positively identified using the IDA and EPI spectra. This method was successfully applied to analyze a total of 206 samples of fresh pufferfish tissues and pufferfish-based products. The results from this study show that the proposed method can be used to quantify and identify TTX in a single run with excellent sensitivity and reproducibility, and is suitable for the analysis of complex matrix pufferfish samples.

Trace-Level Analysis of Hexavalent Chromium in Lake Sediment Samples Using Ion Chromatography Tandem Mass Spectrometry

The analysis of hexavalent chromium, Cr(VI), in soil and sediment samples has been predominantly carried out in materials containing elevated levels. Reliable analysis of trace-level of Cr(VI) in sediment samples remains challenging. Cr(VI) analyses with multipoint calibration and speciated isotope dilution (SID) adapted from U.S. EPA method 6800 were used to measure lower-level Cr(VI) on an ion chromatograph coupled with a tandem mass spectrometer (IC-MS/MS). Lake sediment samples were collected from various locations in Northern Ontario and Cr(VI) was extracted using both alkaline digestion and ethylene diaminetetraacetic acid (EDTA) extraction. Certified reference materials were extracted and analyzed by IC-MS/MS and UV-VIS detection. The SID-MS approach allowed for the quantification of Cr(VI) in samples with concentration levels below 0.5 μg.g-1 wet weight.

Studies on Fragmentation Pathways of N-Ethoxy(phenyl) phosphoryl Amino Acids by Electrospray Ionization Tandem Mass Spectrometry

The positive and negative ESI-MS/MS spectra of N-ethoxy（phenyl） phosphoryl amino acids（EPP-AA） were investigated by electrospray ionization（ESI） ion trap mass spectrometry. The fragmentation pathways of [ M ＋ Na]^＋ and [ M - H]^- ions are proposed and rationalized. The observation may have some potential applications in the interpretation of the MS/MS spectra of novel N-phosphoryl compounds. The complexity of MS/MS spectra of EPP-AA [ M ＋ Na]^＋ ions is decreased compared with that of N-dialkyloxyphosphoryl amino acid. Therefore, the new phosphonamidate method may be considered one of the superior methods that can be used in sequencing peptides and proteins extensively.

Studies on Fragmentation of Peptide by Nano-electrospray Ionization Fourier Transform Ion Cyclotron Resonance Multi-stage Tandem Mass Spectrometry

The sequence analysis of peptides was performed by nano-electrospray ionization Fourier transform ion cyclotron resonance tandem mass spectrometry（Nano-ESI-FT-ICR-MSn） and several peptides were chosen as examples. With the aid of the collision induced dissociation（CID）, FT-ICR provides not only precise mass/charge ratio, but also structure information of the selected peptides. The fragment ions were identified according to the observed molecular weights and peptide sequence was determined successfully. So Nano-ESI-FT-ICR-MSn is a useful tool for identification of the amino acid sequence of peptides with high confidence. Besides, a pathway for the dehydration of y ions without amino acids containing carboxylic acid under sustained off-resonance irradiation collision-induced dissociation（SORI-CID） condition was proposed.

基于UFLC-QTrap-MS/MS结合多元统计分析的不同产地、不同部位乌药中核苷和氨基酸类成分分析

本研究建立了一种超快速液相色谱-三重四极杆/线性离子阱-串联质谱(UFLC-QTrap-MS/MS)同时测定乌药中11种核苷类和14种氨基酸类成分的分析方法,并结合多元统计分析比较不同产地、不同部位乌药的质量。采用Waters XBridge Amide色谱柱(2.1 mm×100 mm×3.5μm),以0.2%甲酸水溶液(A)-0.2%甲酸乙腈溶液(B)为流动相进行梯度洗脱;在电喷雾正离子模式下,以多反应监测(MRM)进行质谱检测;基于各成分的质量浓度,用方差分析(ANOVA)、主成分分析(PCA)、正交偏最小二乘法判别分析(OPLS-DA)、层次聚类分析(HCA)、逼近理想解排序法(TOPSIS)以及灰色关联度分析(GRA),对不同产地、不同部位乌药样品进行综合评价。结果表明,所测成分在一定浓度范围内具有良好的线性关系,相关系数均大于0.9924;精密度、重复性、稳定性良好,平均加样回收率为94.86%~106.47%,相对标准偏差(RSD)均小于4.55%。不同产地、不同部位乌药样品间存在一定的差异,HCA和PCA 2种方法均将样品分为3类。OPLS-DA模型分析通过VIP值共筛选得到6个差异性化合物,分别为尿苷、丙氨酸、胞嘧啶、黄嘌呤、腺嘌呤、鸟嘌呤。该方法简便、灵敏度高、重现性好,可用于乌药中核苷和氨基酸类成分的同时测定,为药材质量评价和综合利用提供方法参考。

基于UPLC-Q-Orbitrap-MS对铁皮石斛水提物不同极性溶剂萃取物化学成分分析

目的建立超高效液相色谱串联—四极杆—静电场轨道阱质谱技术(ultra-performance liquid chromatography-linear ion trap quadrupole-Orbitrap-mass spectrometry,UPLC-Q-Orbitrap-MS)对铁皮石斛(Dendrobium officinale Kimura&Migo)水提物石油醚、乙酸乙酯、正丁醇3种萃取组分进行定性表征的方法。方法提取铁皮石斛水提物并依次用石油醚、乙酸乙酯、正丁醇萃取,旋蒸浓缩后,得到石油醚、乙酸乙酯、正丁醇萃取组分。样品经UPLC-Q-Orbitrap-MS分析,根据元素组成、分子量、质荷比、离子碎片等确定化合物信息并推导裂解规律,通过与文献、MzCloud、MzVault、PudChem、本地数据库比对确定鉴定结果。结果在正负离子模式下共鉴定出化合物62种,包括黄酮类成分20种,联苄类成分6种,羧酸及其衍生物6种,脂肪酸及其衍生物6种,酚类成分4种,氨基酸及其衍生物3种,糖类成分2种,生物碱类、苯丙素、蒽醌、萜类化合物各1种,其他类成分3种,还检测到8种含量较高、质谱信号较强的未知成分。其中水提石油醚萃取物中鉴定出化合物7种,乙酸乙酯萃取物中鉴定出化合物36种,正丁醇萃取物中鉴定出化合物48种。通过与文献比对,62种化合物中有7种化合物为首次从铁皮石斛中提取得到。结论该方法可快速有效分析铁皮石斛中多种化学成分,可为铁皮石斛质量控制及药效物质基础研究提供参考。

Screening and Identifying of Nephrotoxic Compounds in Lithospermum erythrorhizon Using Live-cell Fluorescence Imaging and Liquid Chromatography Coupled with Tandem Mass Spectrometry

In order to identify the potential nephrotoxic compounds in traditional Chinese medicine Lithospermum erythrorhizon,it was separated into serial fractions according to their polarities.An in vitro method was utilized to determine the nephrotoxicity of these fractions with the help of fluorescence image analysis.As a result,the primary fraction A05 and its secondary fractions C06 ＂C09 and C12 ＂C14 were found to have significant toxicity to LLC-PK1 cell line,as determined by the survive rate less than 20% after they were treated with these fractions.These potential nephrotoxic fractions were further analyzed by multistage and high resolution mass spectrometry.The main compounds in these fractions were tentatively identified to be acetylshikonin,isobutyrylshikonin,β,β＇-dimethyla-cryloylshikonin,and isovalerylshikonin,which may bring nephrotoxicity.

QTRAP LC-MS/MS法检测血、尿中莫达非尼

本文采用超高效液相色谱-四级杆/线性离子阱质谱技术,建立血液、尿液中莫达非尼的检测方法。选取卡马西平为内标,血液、尿液经甲醇/乙腈(体积比1︰1)混合提取剂提取,高速离心,取上清液过滤膜,以0.1%(体积分数)甲酸/水溶液和0.1%(体积分数)甲酸/乙腈作为流动相,经Phenomenex Kinetex~?C18(2.1mm×100 mm×2.6μm)色谱柱分离,以电喷雾正离子多反应监测-信息依赖性采集-增强离子扫描(MRMIDA-EPI),结合二级谱库检索进行分析。结果显示,莫达非尼在1~80 ng/mL范围内线性关系良好,线性相关系数大于0.997;血液、尿液方法检出限(S/N≥3)均为0.5 ng/mL,定量限(S/N≥10)均为0.8 ng/mL;2、20、80 ng/mL低、中、高添加浓度水平血液、尿液回收率分别为80.1%~110.3%、87.9%~109.1%,相对标准偏差均小于10%。此方法可满足实际办案中对莫达非尼滥用者血液、尿液的定性定量分析。

MALDI coupled with laser-postionization and trapped ion mobility spectrometry contribute to the enhanced detection of lipids in cancer cell spheroids

Cancer cell spheroids(CCS) are a valuable three-dimensional cell model in cancer studies because they could replicate numerous characteristics of solid tumors. Increasing researches have used matrix-assisted laser desorption/ionization mass spectrometry imaging(MALDI-MSI) to investigate the spatial distribution of endogenous compounds(e.g., lipids) in CCS. However, only limited lipid species can be detected owing to a low ion yield by using MALDI. Besides, it is still challenging to fully characterize the structural diversity of lipids due to the existence of isomeric/isobaric species. Here, we carried out the initial application of MALDI coupled with laser-postionization(MALDI-2) and trapped ion mobility spectrometry(TIMS) imaging in HCT116 colon CCS to address these challenges. We demonstrated that MALDI-2 is capable of detecting more number and classes of lipids in HCT116 colon CCS with higher signal intensities than MALDI. TIMS could successfully separate numerous isobaric/isomeric species of lipids in CCS. Interestingly, we found that some isomeric/isobaric species have totally different spatial distributions in colon CCS. Further MS/MS imaging analysis was employed to determine the compositions of fatty acid chains for isomeric species by examining disparities in signal intensities and spatial distributions of product ions. This work stresses the robust ability of TIMS and MALDI-2 imaging in analyzing endogenous lipids in CCS, which could potentially become powerful tools for future cancer studies.

基于UHPLC-QTRAP-MS/MS的淫羊藿中多元活性成分动态累积的分析与评价

建立了超高效液相色谱-三重四极杆线性离子阱串联质谱(UHPLC-QTRAP-MS/MS)同时测定淫羊藿中黄酮类、生物碱类、酚酸类、氨基酸类及核苷类共50种活性成分的分析方法,并对其多元活性成分的动态累积进行分析与评价。采用Agilent ZORBAX Extend-C18(2.1 mm×100 mm,1.8μm)色谱柱,以水(含0.2%甲酸)-乙腈为流动相梯度分离,流速0.3 mL/min,柱温40℃。质谱配备电喷雾离子源,在正、负离子模式下采用多反应监测模式(MRM)进行测定;采用单因素实验及响应面法优化样品制备条件,灰色关联度分析(GRA)对不同采收期淫羊藿样品进行分析与评价。结果显示,50种目标成分在一定范围内线性关系良好(r2≥0.9990),精密度、重复性和稳定性良好,平均加标回收率为94.5%~107%,相对标准偏差(RSD)均小于5.0%。淫羊藿中多元活性成分累积量在7月中旬出现峰值,不同类别成分累积规律具有一定差异性。6月下旬至7月下旬所采收的淫羊藿药材综合质量较好,与当地传统采收期基本吻合。该研究所构建的方法准确、可靠,可为探究淫羊藿中多元活性成分动态累积规律及确定适宜采收期提供依据,同时也箭叶淫羊藿药材质量的综合评价和整体控制提供方法参考。

Fitting Nonlinear Calibration Curves: No Models Perfect

The study of the calibration of a series of compounds of environmental concern (six perfluoroalkyl compounds (perfluorooctane sulfonic acid and five perfluoroalkyl carboxylic acids), three preservatives (methyl-, ethyl- and propylparabens) and the brominated flame retardant hexabromocyclododecane) by LC-MS/MS has been carries out, with a view to their simultaneous determination in samples of environmental interest. In some cases nonlinear calibration curves are obtained, but restricting the concentration range a linear model may be used to fit the data. Residual analysis has been performed in order to verify which models fit the data better, opting for a compromise decision given the apparent complexity of residuals plots. As Box states there are no perfect models (but models that work better than others).

Classification of B and Y Ions in Peptide MS/MS Spectra Based on Machine Learning

In proteomics, b and y ions serve as the backbone ions for peptide sequencing in tandem mass spectrometry. Leveraging the existing ion recognition and separation methods, this article proposes a novel ion classification approach that combines machine learning with graph theory. By incorporating graph features, the method achieves higher accuracy and efficiency in ion type recognition, with the graph features playing a critical role in the classification process. Specifically, the method achieves a recall rate of nearly 90% for b and y ions, demonstrating its effectiveness in pre-processing de novo sequencing and improving its accuracy. The proposed method represents advancement in ion classification and has the potential to improve the accuracy and efficiency of de novo sequencing.

固相萃取-超高效液相色谱-三重四极杆/线性离子阱质谱法检测水中大环内酯类抗生素

采用固相萃取-超高效液相色谱-三重四极杆线性离子阱质谱技术(SPE-UPLC-QTRAP MS)建立了环境水样中阿奇霉素、林可霉素、克林霉素、红霉素及替米考星5种大环内酯类抗生素的检测方法。调节水样pH为10后经HLB固相萃取柱净化、富集,然后用Kinetex F5色谱柱进行分离,用0.1%甲酸水溶液和0.1%甲酸乙腈溶液进行洗脱,采用电喷雾正离子源(ESI+),多反应监测(MRM)-信息依赖采集(IDA)-增强子离子(EPI)扫描模式对样品进行检测,用多反应监测(MRM)进行定量,增强子离子(EPI)谱图库辅助定性。结果表明:5种大环内酯类抗生素线性关系良好,相关系数均大于0.9992;检出限为0.01~0.30μg·L^(-1),定量限为0.10~0.50μg·L^(-1);3种不同浓度的水样加标回收率为71.60%~111.05%,相对标准偏差均在10%以内,EPI谱库比对纯度值均大于90%。该方法将传统的MRM扫描模式结合EPI谱图库检索,实现了同时定性和定量分析,对未知物准确定性定量检测提供了重要参考。

Fragmentation study of two brassinolides by ion trap tandem mass spectrometry

The fragmentation pathways of two isomers of brassinosteroids,28-homobrassinolide(28-h-BL)and 28-epihomobrassinolide(28-eh-BL),have been investigated by tandem mass spectrometry(MSn,n=1,2,3,4)with the electrospray ionization(ESI) source.The ESI mass spectrometric fragmentation pathways of protonated 28-h-BL and 28-eh-BL were comprehensively elucidated,and the principles revealed in this investigation could potentially be used to identify and distinguish brassinosteroids and their isomers.

自主研制四极杆-线形离子阱质谱仪测定血清中甲氨蝶呤

本研究基于自主研制的QLIT-6610MD液相色谱-四极杆-线形离子阱-质谱联用仪,建立了准确测量血清中甲氨蝶呤的同位素稀释质谱法。采用甲醇沉淀血清样品的蛋白后测定上清液。通过基质匹配标准曲线定量,在10~5000μg/L浓度范围内,甲氨蝶呤的线性系数R^(2)>0.9990,定量限为10μg/L,质控品精密度优于6.94%,血清样本添加回收率为86.39%~97.84%,变异系数(CV)≤8.04%。应用QLIT-6610MD与AB QTRAP 6500+液相色谱-质谱联用仪同时测定70例临床血清样本,并采用Pearson系数与BLand-ALtman法进行比较。结果表明,2台仪器所测结果一致,QLIT-6610MD可达到三重四极杆质谱仪定量测定血清中甲氨蝶呤的水平,能够满足临床诊断需求,为临床质谱分析提供了一种新的国产仪器选择。此外,本研究还初步探讨了多级碎裂技术(MSn)测定血清中甲氨蝶呤的可能性,为后续研究奠定基础。

液相色谱-串联质谱法与离子色谱-串联质谱法测定食品中氯酸盐和高氯酸盐方法比较研究

目的考察离子色谱-串联质谱法(ion chromatography-tandem mass spectrometry,IC-MS/MS)和液相色谱-串联质谱法(liquid chromatography-tandem mass spectrometry,LC-MS/MS)在测定不同食品中氯酸盐和高氯酸盐的一致性。方法利用乙腈/水(3:2,V:V)提取鸡蛋、奶粉和菠菜中的目标物,经石墨化炭黑(graphitized carbon black,GCB)小柱净化,以IonPac^(TM)AS20离子色谱柱和CSHTMFluoro-Phenyl液相色谱柱为分析柱,三重四极杆质谱仪检测,内标法定量。对两种方法测得的数据进行正态性检验和差异性分析。比较两种方法的保留时间漂移,考察色谱稳定性。对两种方法测得的浓度进行线性回归和Bland-Altman(B-A)图可视化考察方法一致性。结果两种方法测得氯酸盐(高氯酸盐)的质量浓度在1~500(0.1~50.0)μg/L范围内线性良好(r^(2)>0.999)。IC-MS/MS与LC-MS/MS测定高氯酸盐的定量限均为0.5μg/kg,氯酸盐的定量限分别为5.7μg/kg和4.2μg/kg。回收率范围分别为82.3%~107.4%和94.8%~109.4%,相对标准偏差分别为1.5%~13.6%和1.2%~9.6%。IC-MS/MS(LC-MS/MS)测得鸡蛋、奶粉、菠菜中氯酸盐和高氯酸盐的基质效应分别为12.1%(22.1%)、9.0%(27.4%)、-24.0%(-11.8%)和-16.8%(-18.5%)、-18.6%(-28.4%)、-20.2%(-21.1%)。线性回归法显示两种方法测定氯酸盐和高氯酸盐浓度的回归曲线斜率分别为1.0144和1.0908。B-A图显示绝大多数数据点位于平均值±1.96标准偏差范围。结论两种方法的准确度和精密度均符合化学检验方法验证通则的要求,两种方法测得的氯酸盐、高氯酸盐浓度结果基本一致,但氯酸盐在LC-MS/MS系统中的保留时间稳定性更好,IC-MS/MS在测定食品中氯酸盐、高氯酸盐时的抗基质干扰能力更强。

LTQ-Orbitrap组合式高分辨质谱法快速筛查毛发中7种毒品及代谢物

采用超高压液相色谱-二维线性离子阱结合静电场轨道阱组合式高分辨质谱联用技术(Accela U HPLC/LTQ Orbitrap XL),建立了人毛发中吗啡、O^6-单乙酰吗啡、可待因、乙酰可待因、氯胺酮、去甲氯胺酮和美沙酮毒品及代谢物快速筛查方法。取毛发样品经表层清洗后冷冻研磨粉碎,置于硼酸盐缓冲液(pH 9.2)中超声90 min,离心取上清液,用Oasis HLB柱固相萃取制备。通过静电场轨道阱全扫描得到毒品及其代谢物的精确相对分子质量,同时进行7种毒品及其代谢物的快速筛查。高分辨率质谱可有效去除毛发基质干扰,毒品及其代谢物筛查检出限在0.001～0.02 ng/mg,在0.05～50 ng/mg范围内存在良好线性关系(r>0.9975);本方法平均加标回收率为76.1%～109.6%;日内及日间精密度RSD≤14.9%。本方法灵敏度高,样品制备简便,适用于常见毒品的快速筛查。

UPLC-LTQ/Orbitrap MS快速筛查确证化妆品中89种禁用物质

采用超高效液相色谱-线性离子阱/静电场轨道阱结合高分辨质谱法（UPLC-LTQ/Orbitrap MS）同时测定化妆品中抗生素、激素、邻苯二甲酸酯、农药以及孔雀石绿和结晶紫等5大类共89种化合物。不同剂型的化妆品基质用甲醇超声提取,以Waters Acquity UPLC BEH C18色谱柱（2.1mm×100mm×1.7μm）分离;正离子模式下,以含0.1%甲酸的5mmol/L乙酸铵溶液和乙腈为流动相;负离子模式下,以5mmol/L乙酸铵溶液和乙腈为流动相,进行梯度洗脱。以保留时间和Orbitrap获得的一级母离子精确质量数以及同位素丰度比,实现对化妆品中多种类物质的快速筛查;以Orbitrap碰撞诱导解离（CID）获得的碎片离子精确质量数进行确证。结果表明：化合物的线性关系良好,线性相关系数R2〉0.99;除邻苯二甲酸二苯酯外,其他化合物的最低检出限（LOQ）均在5~10μg/kg之间;在添加浓度为1倍、2倍、10倍LOQ 3个水平下,绝大多数化合物的平均回收率在60%~117%之间,相对标准偏差小于13%。该方法简便、快速、可靠性高,适用于化妆品中多种类别禁用物质的检测。

基质溶剂校正结合高温离子源GC-MS/MS同时分析烟气中16种多环芳烃

本研究建立了高温离子源气相色谱-三重四极杆串联质谱(GC-MS/MS)法结合基质溶剂校正,使用样品萃取溶液直接进样,同时测定卷烟主流烟气中16种多环芳烃(PAHs)。考察了常温和高温离子源对目标物响应情况的影响,探究基质溶剂制备方法及基质效应对目标物定量结果的影响,分析市售不同圆周卷烟烟气的释放分布情况。结果表明,16种PAHs的定量工作曲线在各自线性范围内的相关系数R2为0.996 4~0.999 8,检出限为0.29~1.48 ng/支,定量限为0.95~4.94 ng/支,在低、中、高3个不同添加水平下的平均回收率为87.71%~123.26%,精密度为1.58%~9.45%。通过分析参比卷烟3R4F,该方法的检测结果与文献报道结果无明显差异。采用该方法检测市售58个品牌卷烟样品中的PAHs,总释放量为742.76~1 505.99 ng/支,并随卷烟焦油量减少而下降。该方法具有前处理简便,准确及重复性好的优点,适用于卷烟主流烟气中痕量PAHs的定量分析。