Lysophosphatidylcholine Biomarkers of Lung Cancer Detected by Ultra-performance Liquid Chromatography Coupled with Quadrupole Time-of-flight Mass Spectrometry

Centrifugal ultrafiltration after methanol extraction of whole plasma was used as an optimal condition for the preparation of blood plasma before metabonomic studies. The plasma samples from 102 lung cancer patients and 34 healthy volunteers were prepared with this approach. With ultra-performance liquid chromatography（UPLC） coupled with quadrupole time-of-flight mass spectrometry（Q-TOF MS） analysis, the samples were investigated in order to find potential disease biomarkers. After data acquisition, orthogonal signal correction partial least squares models were built to differentiate the healthy volunteers from lung cancer patients and to identify metabolites that showed significantly different expression between the two groups. Several metabolite ions were identified as potential biomarkers according to the variable importance in the project（VIP） value in both ion modes. Five lysophosphatidylcholines were further identified as specifically lysoPC 16：0, isomer of lysoPC 16：0, lysoPC 18：0, lysoPC 18：1 and lysoPC 18：2. These results suggest that UPLC coupled with Q-TOF MS is an effective technique for the analysis of plasma metabolites in metabonomic studies.

UPLC-MS/MS法测定大鼠血浆中雷公藤内酯醇及其药代动力学

目的建立大鼠血浆中雷公藤内酯醇的超高效液相色谱质谱联用（ultra performance liquid chromatography-mass spectrometer/mass spectrometer,UPLC-MS/MS）法测定大鼠灌胃给药与静脉注射给药后的血浆药物浓度,并进行药代动力学参数评价。方法 Sprague Dawley大鼠分别经灌胃与尾静脉注射给药,经颈静脉取血,测定不同时间的大鼠血浆药物浓度,并计算其主要药代动力学参数。结果在0.1~200.0 ng/m L质量浓度范围内,血浆雷公藤内酯醇线性关系良好,血浆中雷公藤内酯醇的定量限为0.1 ng/m L,该药稳定性良好,回收率均在95%以上,日内与日间差异均小于15%。口服与静脉给药后,雷公藤内酯醇在大鼠体内的消除均较快,口服生物利用度为9.5%。结论本实验操作简便、稳定可靠、检测限低、效率高、重现性好,可以可靠地用于雷公藤内酯醇血药浓度测定及其药代动力学研究,本研究结果为雷公藤内酯醇的结构优化、剂型改进以及临床应用提供了实验依据。

Pharmacokinetic Comparison of Four Major Bio-Active Components of Naoxintong Capsule in Normal and Acute Blood Stasis Rats Using Ultra-Performance Liquid Chromatography Coupled with Triple-Quadrupole Mass Spectrometry

Objective: To compare the pharmacokinetic differences of the main components of Naoxintong capsule(NXTC) in normal and acute blood stasis rats. Materials and Methods: Rats were subcutaneously injected with adrenaline hydrochloride twice;during the two subcutaneous injections, the rats were placed in ice water for 4 min to reproduce the model rat of acute blood stasis. The normal and acute blood stasis rats were administrated a 5.04 g/kg dose of NXTC suspension. Then, blood samples were collected from the posterior retinal venous plexus at different time points. Plasma concentrations of four major bio-active components including caffeic acid, ferulic acid, formononetin, and tanshinone IIA in NXTC were measured using ultra-performance liquid chromatography coupled with triple-quadrupole mass spectrometry. Phoenix Win Nonlin v6.2 software was used to calculate the pharmacokinetic parameters. Results: Compared with the normal rats, the acute blood stasis rats showed a significant decrease in C_(max) of ferulic acid and formononetin, AUC_(all) of caffeic acid and ferulic acid, and AUC_(INF_obs) of ferulic acid. Conversely, an increase in the Vz_F_obs and MRT_(last) of ferulic acid and caffeic acid was observed. These findings demonstrate that the absorption of the four NXTC components was weakened in the acute blood stasis rats and that the elimination time was prolonged. Conclusions: The significant difference in some parameters of the four NXTC components between the normal and acute blood stasis rats might be caused by an increase in blood viscosity and the subsequent slowing down of blood flow in the acute blood stasis rats. The pharmacokinetic study conducted in pathological state can provide important information and scientific basis for further rational clinical application of NXTC.

Rapid Differentiation of Aconiti Kusnezoffii Radix from Different Geographic Origins Using Ultra-Performance Liquid Chromatography Coupled with Time-of-Flight Mass Spectrometry

Objective:There are different geographic origins of Aconiti Kusnezoffii Radixs(AKRs)sold in the market with different quality.This study aims to establish a rapid analysis method to distinguish the different geographic origins of AKRs and to realize the rapid evaluation of their quality.Methods:An ultra-performance liquid chromatography coupled with time-of-flight mass spectrometry(UPLC-Q-TOF MS)method was utilized to acquire the constituents'information of AKRs from different geographic origins.MSE data and Progenesis QI software were employed to identify the chemical constitutes.Principal component analysis(PCA)was applied to comparing MS data to find the chemical markers of AKRs from different geographic origins.Results:Twenty-three components were detected and 17 out of them were identified,including diester-diterpenoid alkaloids,monoester-diterpenoid alkaloids,and amine-diterpenoid alkaloids.Three pairs of isomers were detected and two of them were distinguished by the retention time of standard samples.Thirteen chemical markers were screened out through PCA and orthogonal partial least square discriminant analysis.Through detecting Napelline or isomer of Napelline(m/z 360.2530)and Aconifine(m/z 662.3170),AKRs from inner Mongolia autonomous could be screened.According to the existence of benzoylaconine(m/z 604.3108)and Indaconitine(m/z 630.3159),it could be confirmed that the AKRs are from Xinjiang Uygur autonomous.AKRs that cannot detect compounds above-mentioned could be from Liaoning or Shanxi Province.Conclusions:The chemical profile could be used not only to distinguish the AKRs from different geographic origins but also to identify the true and false of AKRs.This study lays a foundation for the study of efficacy and toxic of AKRs.

Simultaneous Determination of Six Compounds in Rat Plasma by Ultra-Performance Liquid Chromatography with Tandem Mass Spectrometry: Application in the Pharmacokinetic Study of Qing Gan-Shu Yu-Fang

A rapid and high selective ultra-performance liquid chromatography(UPLC)with tandem mass spectrometry method for simultaneous determination of six compounds including albiflorin,paeoniflorin,picroside I,picroside II,saikosaponin A,and saikosaponin D in rat plasma was developed and validated using butyl p-hydroxybenzoate as an internal standard.One-step direct protein precipitation with acetonitrile was used to extract the compounds from the rat plasma samples.Chromatographic separation was achieved using an ACQUITY UPLC BEH C18 column(100 mm×2.1 mm,1.7μm)at a flow rate of 0.4 m L/min,using gradient mode containing 0.1%formic acid in water and acetonitrile were used as the Mobile phase A and B.Electrospray ionization in negative ion mode and multiple reaction monitoring were used to identify and quantify active components.Calibration curves showed good linearity(R^2>0.9908)over a wide concentration range for all compounds.The intra-and interday precision(relative standard deviation)ranged 2.4%–7.0%and 2.6%–8.0%,respectively.The accuracy(relative error)was from-13.0%to 13.2%at all quality control levels.The recovery ranged from 81.1%to 92.5%.The validated method was successfully applied to pharmacokinetic study in rats after oral administration of Qing Gan-Shu Yu-Fang.The results show that one can draw a conclusion that these six active ingredients can be quickly absorbed and play a pharmacodynamic role rapidly in vivo.

UPLC-MS/MS法同时测定妇科养荣丸(浓缩丸)中10种有效成分的含量

目的建立一种UPLC-MS/MS方法同时测定妇科养荣丸(浓缩丸)中芍药苷、阿魏酸、黄芪甲苷、甘草酸、鲁斯可皂苷元、松脂醇二葡萄糖苷、橙皮苷、毛蕊花糖苷、毛蕊异黄酮苷和益母草碱的含量。方法采用Thermo Syncronis C色谱柱(100 mm×2.1 mm,1.7μm),以体积分数0.1%甲酸水溶液-乙腈为流动相进行梯度洗脱,流速为0.4 mL·min,柱温为30℃,离子源为ESI源,多反应离子检测模式。结果10种化合物在其质量浓度范围内线性关系良好,其线性范围分别为益母草碱0.05010~1.002 mg·L^(-1)、芍药苷2.502~50.05 mg·L^(-1)、松脂醇二葡萄糖苷0.07560~1.512 mg·L^(-1)、阿魏酸0.2511~5.022 mg·L^(-1)、毛蕊异黄酮葡萄糖苷0.02505~0.5010 mg·L^(-1)、毛蕊花糖苷0.05030~1.006 mg·L^(-1)、橙皮苷1.252~25.04 mg·L^(-1)、黄芪甲苷0.05070~1.014 mg·L^(-1)、甘草酸1.001~20.02 mg·L^(-1)、鲁斯可皂苷元0.0501~1.002 mg·L^(-1),其r值均大于0.996,平均加样回收率为97.3%~99.2%,相对应的RSD值为2.3%~3.1%。结论本方法适用于测定妇科养荣丸(浓缩丸)中各种有效成分的含量。

An Automatic Solid Phase Extraction and Ultra-Performance Liquid Chromatography Tandem Mass Spectrometry for Determination of Seven Microcystins at Ultra-Trace Levels in Surface Water

A method was developed for the detection of seven microcystins(microcystin-LR, RR, YR, LA, LY, LW and LF) in surface water using automatic solid-phase extraction(A-SPE) coupled with ultra-performance liquid chromatography tandem mass spectrometry(UPLC-MS/MS). The automated solid-phase extraction system was used to extract microcystins(MCs) from water samples. UPLC-MS/MS was used to determine MCs concentrations in just 5 min. Method detection limits were from 0.3 to 0.9 ng/L, microcystin recoveries ranged from 83.8% to 114%, and the relative standard deviation(RSD) varied from 5.6% to 12.5%. This analytical approach was found to be simple, highly sensitive, accurate, which required little manual operation. Additionally, to validate this analytical method, A-SPE+UPLC-MS/MS was applied to characterize the concentration of MCs in Taihu Lake, Wuxi, China.

Development of a Rapid and Simultaneous Detection Method for Buprenorphine, Norbuprenorphine and Naloxone in Human Plasma Using Ultra-high Performance Liquid Chromatography- tandem Mass Spectrometer with Solid-phase Extraction

A simultaneous method was successfully established and validated for the separation and determination of bu- prenorphine （BP）, its primary metabolite, nor-buprenorphine （NBP） and a proposed co-formulate, naloxone （NLX） in human plasma. The method used buprenorphine-d4 （BP-D4）, nor-buprenorphine-d3 （NBP-D3）, naltrexone （NTX） as internal standards （ISs）. 100 μL of plasma sample fortified with the ISs was cleaned up by solid-phase extraction （SPE）, and was then separated on a Waters AcquityTM BEH C18 column with gradient elution using methanol and water （containing 0.2% formic） at a flow rate of 0.25 mL·min^-1. The mass spectrometer was used for detection and was operated in the positive electrospray ionization with multiple reaction monitoring （MRM） mode. The three compounds were effectively separated in 5 min. The linear ranges of the compounds were 0.1--25, 0.25--25 and 0.05--25 ng·mL^-1 for BP, NBP and NLX, respectively, with r≥0.9935. The method had high sensitivity （the lim- its of detection were 0.02, 0.1 and 0.01 ng.mL-1 for BP, NBP and NLX, respectively） and high recoveries （≥97.6%）. The result was shown to be linear and satisfactorily met current acceptance criteria for validation of bio- analytical method： intra and inter assay precisions within the required limits of ≤25% RSD. The LOQs fulfilled the LOQ requirements： precision≤25% RSD, and was fully validated according to the State Food and Drug Administration （SFDA） regulations. The results demonstrated that ultra-high performance liquid chromatography- tandem mass spectrometer （UPLC-MS/MS） with SPE was a powerful detection tool and contributed to pharmaceutical analysis in biological matrices.

Perfluoropentane-based oxygen-loaded nanodroplets reduce microglial activation through metabolic reprogramming

Microglia,the primary immune cells within the brain,have gained recognition as a promising therapeutic target for managing neurodegenerative diseases within the central nervous system,including Parkinson’s disease.Nanoscale perfluorocarbon droplets have been reported to not only possess a high oxygen-carrying capacity,but also exhibit remarkable anti-inflammatory properties.However,the role of perfluoropentane in microglia-mediated central inflammatory reactions remains poorly understood.In this study,we developed perfluoropentane-based oxygen-loaded nanodroplets(PFP-OLNDs)and found that pretreatment with these droplets suppressed the lipopolysaccharide-induced activation of M1-type microglia in vitro and in vivo,and suppressed microglial activation in a mouse model of Parkinson’s disease.Microglial suppression led to a reduction in the inflammatory response,oxidative stress,and cell migration capacity in vitro.Consequently,the neurotoxic effects were mitigated,which alleviated neuronal degeneration.Additionally,ultrahigh-performance liquid chromatography–tandem mass spectrometry showed that the anti-inflammatory effects of PFP-OLNDs mainly resulted from the modulation of microglial metabolic reprogramming.We further showed that PFP-OLNDs regulated microglial metabolic reprogramming through the AKT-mTOR-HIF-1αpathway.Collectively,our findings suggest that the novel PFP-OLNDs constructed in this study alleviate microglia-mediated central inflammatory reactions through metabolic reprogramming.

基于液相色谱-三重四级杆质谱仪的栀子及水栀子中萜类物质鉴定及成分差异研究

目的建立栀子中萜类物质的高通量分析方法,并将其应用于栀子与水栀子的成分差异研究。方法结合液相色谱-三重四级杆质谱仪中的全扫描、子离子扫描、母离子监测、中性丢失离子扫描、多反应离子监测、动态多反应离子监测等多种技术提出栀子中萜类物质的定性、定量分析策略,进一步应用主成分分析和正交偏最小二乘判别分析等多元变量分析方法研究栀子与水栀子的成分差异。结果共鉴定出并相对定量81种萜类物质,发现栀子与水栀子间萜类物质含量差异显著,其中13种物质可作为鉴别栀子与水栀子的差异标志物。结论所建立的方法操作便捷,结果准确,为栀子药材鉴别及质量评价提供了新思路,并为栀子资源的合理开发利用提供了参考依据。

用UPLC-MS/MS法测定人血浆中雌二醇及雌酮的浓度

目的建立一种同时测定患者血浆中雌二醇(E2)及雌酮(E1)浓度的超高效液相色谱-质谱联用(UPLC-MS/MS)法。方法用液液萃取法对样品进行前处理,内标为17β-雌二醇-D3。色谱柱:ACQUITY UPLC BEH C18(2.1 mm×100.0 mm,1.7μm),流动相:100%水含0.2 mmol氟化铵(NH_(4)F)-100%甲醇含0.2 mmol NH4F,流速:0.30 mL·min^(-1),柱温:30℃,自动进样器温度:15℃,进样量:6μL。用电喷雾离子化源,负离子模式,多反应监测。考察该方法的专属性、标准曲线与定量下限、精密度与回收率、基质效应及稳定性。结果雌二醇和雌酮均在20~1000 pg·mL^(-1)内,线性关系良好,其标准曲线雌二醇为y=1.87×10^(-3)x+3.71×10^(-2)(R=0.99927);雌酮为y=8.48×10^(-3)x+6.46×10^(-2)(R=0.99890),定量下限均为20 pg·mL^(-1),雌二醇和雌酮批内和批间相对标准偏差均小于9.46%,回收率在97.64%~107.35%,基质效应在93.29%~109.87%。结论本方法专属性强、灵敏度高,适用于雌二醇和雌酮的治疗药物监测研究。

基于高分辨液质联用的双标记氨基酸同位素丰度测定方法研究

近年来,稳定同位素标记化合物,尤其是同位素标记氨基酸在合成生物学、蛋白质组学、代谢组学等前沿科学研究中发挥着越来越重要的作用。建立了一种基于高分辨液质联用的双标记氨基酸同位素丰度检测方法,以^(13)C_(2),^(15)N-甘氨酸为例,考察了样品浓度、质谱扫描张数等影响因素对同位素丰度计算结果的影响,结果表明该方法具有良好的准确性和稳定性,所需样品用量少,操作简单,有望成为一种简便、通用的同位素试剂质控方法;也将为代谢组学、海洋学等同位素示踪技术应用研究提供数据支持。

基于广泛靶向代谢组学的石金钱龟龟甲不同熬制时间的水提物成分差异分析

目的分析石金钱龟龟甲在不同熬制时间差异水提物的变化情况。方法本研究选取9个样品,分为3组进行代谢研究;首先通过非靶向代谢组技术检测混样,鉴定出样本中的水提物,然后将这些水提物与自建数据库结合,并基于广泛靶向代谢组学(TM)技术进行代谢分析,采用基于液相色谱与四级杆飞行时间质谱联用技术(LC/QTOF-MS/MS)检测平台、自建数据库以及重复相关性评估、主成分分析、正交偏最小二乘法判别分析等多元统计分析相结合的手段研究样品间的代谢组差异。结果共鉴定出1248个水提物,共鉴定出1248个水提物,共有8大类(氨基酸及其代谢物,杂环化合物,脂肪酰类,甘油磷脂类,醛、酮、酯类,辅酶和维生素,色胺、胆碱、色素、其他);熬制终期差异水提物相对含量最为丰富。结论该研究从TM广靶代谢组学角度初步解释了龟甲3个不同熬制时间的代谢产物的差异性及特征化合物,鉴于石金钱龟的代谢产物与人们的健康和营养有着密切的关系,可为金钱龟龟甲深加工产品开发与应用提供参考信息和理论依据。

超高效液相色谱-三重四极杆质谱联用仪测定葡萄中的两种甜味剂

建立一种使用超高效液相色谱-三重四极杆质谱联用仪对葡萄样品中的甜蜜素(环己基氨基磺酸钠)和纽甜等甜味剂进行定性分析和定量检测的方法。样品经提取后上机,采用多反应监测模式,负离子扫描。在1~50ng·mL^(-1)呈良好的线性关系,加标回收率为87.6%~96.8%,相对标准偏差≤5.8%。该方法具有前处理方便快捷、检测效率高、抗干扰能力强、准确度高、重复性好等特点,可作为新鲜葡萄样品中部分甜味剂的检测方法,也可为检测其他固体样品提供参考。

Banxia xiexin decoction prevents the development of gastric cancer

BACKGROUND In China banxia xiexin decoction(BXD)has been used in treating gastric cancer(GC)for thousands of years and BXD has a good role in reversing GC histopathology,but its chemical composition and action mechanism are still unknown.AIM To investigate the mechanism of action of BXD against GC based on transcriptomics,network pharmacology,in vivo and in vitro experiments.METHODS The transplanted tumor model was prepared,and the nude mouse were pathologically examined after administration,and hematoxylin-eosin staining was performed.The active ingredients of BXD were quality controlled and identified using ultra-performance liquid chromatography tandem quadrupole electrostatic field orbitrap mass spectrometry(UPLC-Q-Orbitrap MS/MS),and traditional Chinese medicines systems pharmacology platform,drug bank and the Swiss target prediction platform to predict the relevant targets,the differentially expressed genes(DEGs)of GC were screened by RNA-seq sequencing,and the overlapping targets were analyzed to obtain the key targets and pathways.Cell Counting Kit-8,apoptosis assay,cell migration and Realtime fluorescence quantitative polymerase chain reaction were used for in vitro experiments.RESULTS All dosing groups inhibited the growth of transplanted tumors in laboratory-bred strain nude,with the capecitabine group and the BXD medium-dose group being the best.A total of 29 compounds and 859 potential targets in BXD were identified by UPLC-Q-Orbitrap MS/MS and network pharmacology,RNA-seq sequencing found 4767 GC DEGs,which were combined with network pharmacology and analyzed 246 potential therapeutic targets were obtained and pathway results showed that BXD may against GC through the Phosphoinositide 3-kinase(PI3K)/protein kinase B(AKt)signaling pathway.In vitro cellular experiments confirmed that BXDcontaining serum and LY294002 could inhibit the proliferation of GC cells,promote apoptosis,and inhibit the migration of GC cells by decreasing the expression of EGFR,PIK3CA,IL6,BCL2 and AKT1 in the PI3K-Akt pathway in MGC-803 expression.CONCLUSION BXD has the effect of inhibiting tumor growth rate and delaying the development of GC.Its mechanism of action may be related to the regulation of PI3K-Akt signaling pathway.

饲料中乙烯雌酚含量的检测方法研究

本研究旨在开发一种高效、经济的方法,利用高效能液相层析仪(HPLC)配合光二极管侦测器(PDA),对饲料中乙烯雌酚含量进行分析。为了避免在低浓度情况下的误判,同时采用高效液相层析质谱仪(HPLC/MS)进行了再确认。在实验中,饲料样品以甲醇为萃取液,经过振荡、离心、浓缩上清液,残渣用70%甲醇溶解,通过Oasis■HLB卡匣去除干扰物质,最后用甲醇进行冲提,冲提液经高效液相层析仪检测。实验结果表明,对于饲料添加0.5、1、3、5 ppm的情况,回收率在80.8%到107.5%。仅采用光二极管侦测器检测时,最低检测限为0.03 ppm;而采用质谱侦测器检测时,最低检测限可达0.005 ppm。研究结果表明,所开发的高效液相层析仪饲料中乙烯雌酚检测方法具有便捷、经济、可行的特点,可作为饲料中乙烯雌酚检验的一种有效方法。

超高效液相色谱串联质谱法测定植物源性食品中杀螟丹的残留

利用三重四极杆液相色谱-质谱联用技术对不同植物源性食品中杀螟丹进行定性、定量分析,通过对杀螟丹提取和净化条件进行筛选与优化,建立了植物源性食品中杀螟丹快速、便捷、准确、高效的分析方法。色谱柱选择ZORBAX SB-C18(2.1 mm×100 mm,3.5μm),流动相为水(0.1%甲酸)和甲醇,梯度洗脱,流速为0.3 mL/min,采用多反应监测模式(MRM),电喷雾离子源(ESI)选择正离子模式,定量离子对为238.1/149.9。样品经2%乙酸乙腈提取,QuEChERS法净化,过膜后直接测定,外标法定量。结果表明,目标物在5~200 ng/mL范围内线性关系良好,决定系数(R2)=0.9992,样品加标回收率为72%~86%,精密度试验相对标准偏差为0.7%~6.3%。该方法适用性广、操作便捷、回收率好、精密度高,可用于植物源性食品中杀螟丹残留的测定。

醚菌酯在土壤中的残留分析方法验证试验

文章中的试验验证了醚菌酯在土壤中的残留检测分析方法,采集的空白小区土壤样品用25.0 mL乙腈提取,使用C18吸附剂对提取液除杂,方法的定性定量分析使用安捷伦1290Ultivo型超高效液相色谱质谱联用仪,出峰时间采取(3.250min),定性离子对采取(314.1/115.7),残留定量方法采取标准曲线法(314.1/221.5),试验结果表明文章中的分析方法适合土壤中醚菌酯残留的检测。

大黄通便片中非法成分土大黄苷的定性筛查和定量测定研究

目的:建立大黄通便片中土大黄苷的定性筛查和定量测定方法。方法:采用薄层色谱(TLC)法初筛,高效液相色谱(HPLC)法定量测定及超高效液相色谱-质谱联用(UHPLC-MS)法定性确证。结果:TLC法检测结果显示专属性强,耐用性好。HPLC法方法学考察结果显示:土大黄苷质量浓度在2.59~259.6μg·mL^(-1)范围内呈良好的线性关系(R^(2)=0.9999);精密度、重复性、稳定性试验RSD均小于2.0%;平均加样回收率为97.89%(RSD=0.61%)。UHPLC-MS/MS法确证结果与TLC、HPLC法结果一致,阳性检出样品与土大黄苷对照品呈现同一保留时间的提取离子对(421.1→258.9,421.1→199.0)。结论:该方法灵敏、准确,可作为大黄通便片中非法成分土大黄苷的检查方法。

优化液质法测定多种基质中五氯酚残留量

在GB 23200.92-2016《动物源性食品中五氯酚残留量的测定液相色谱-质谱法》的基础上,改进了前处理方法,使用1%氨水乙腈提取,QuEChERS法净化,氮吹后甲醇复溶,并在多种基质中进行了方法学验证。结果表明,方法平均回收率为72.22%~89.51%;精密度试验中,相对标准偏差(RSD)为4.23%~10.28%(n=6);在1~10μg/L范围内线性关系良好(相关系数r>0.995);加标量为1.0μg/kg的情况下,信噪比>10;定量限小于1.0μg/kg,满足检测需求。该方法能够节省前处理时间,同时降低成本。