LC-MS/MS定量测定人体血浆中阿托伐他汀浓度

目的:建立定量测定人体血浆中阿托伐他汀浓度的HPLC-MS/MS的方法.方法:以吲哚美辛为内标,采用Shim-packVP-ODS柱(150× 2.0 mm I.D.,5μm,日本Shimadzu Technologies Inc.公司)为固定相;乙腈-0.5％甲酸溶液(90:10,v/v)为流动相,流速为0.3 ml/min;通过电喷雾离子源(ESI),以正离子多反应监测模式进行检测.阿托伐他汀与内标用于检测的离子对分别为m/z 559.4 m/z 250.3和m/z 358.3 rn/z 139.2.结果:阿托伐他汀在0.10～20.00 ng/ml范围内与峰面积比值线性范围良好(r=0.9962),定量下限为0.10 ng/ml,日内日间精密度的RSD均小于12％,平均回收率大于71％.结论:所建方法准确度高,方法灵敏,专属性强且操作简便,可适用于阿托伐他汀的血药浓度测定和临床药代动力学研究.     

LC-MS/MS法检测人头发中利培酮及其代谢物9-羟利培酮含量的实验研究

目的:建立高效液相色谱-三重四级杆质谱联用(LC-MS/MS)法检测人头发中利培酮(RIP)及其代谢物9-羟利培酮(9-OH-RIP)含量的方法。方法:采用同位素内标氘4-利培酮(RIP-d4)及氘4-9-羟利培酮(9-OH-RIP-d4),流动相A为10 mmol/L醋酸胺溶液(甲酸调p H值为4.0),流动相B为乙腈,A/B=70/30,流速为0.3 m L/min,等度洗脱4.00 min。色谱柱为安捷伦Zorbax SB C18(2.1×50 mm,1.8μm),柱温30℃。准确称取20 mg丙酮清洗过晾干剪碎成粉末的头发样本,加1N氢氧化钠(Na OH)超声2 h,等量酸中和后,加200μL1N氢氧化钠溶液及5.0 m L的甲基叔丁基醚(MTBE)提取涡旋1分钟,3000×g离心5 min后取上清液在40度水浴下氮气吹干后用100μL流动相复溶,进样2μL经LC-MS/MS检测。MRM监测离子对:RIP:m/z 411.2→191.0,9-OH-RIP:m/z 427.2→207.1,RIP-d4:m/z 415.2→195.2,9-OH-RIP-d4:m/z 431.2→211.1。结果:利培酮及9-羟利培酮线性范围分别为0.5-25 ng/mg,0.0025-0.15 ng/mg,提取回收率均70.0%,方法回收率均在85.0%-115.0%之间,线性r均0.999,精密度和重现性RSD均15%。结论:本研究建立了采用LC-MS/MS法检测人头发中利培酮及9-羟利培酮含量的方法,该法快速、简单、准确、重现性好。     

LC-MS/MS法测定大鼠血浆中的野黄芩苷含量

目的:建立LC-MS/MS的分析方法测定大鼠血浆中的野黄芩苷,研究灯盏生脉胶囊中野黄芩苷在大鼠体内的药动学行为。方法:以噻氯匹定为内标,血浆样品经1%甲酸乙腈沉淀蛋白处理后,用LC-MS/MS法测定血浆中的野黄芩苷浓度。结果:野黄芩苷线性范围为1.31～670.00 ng·mL-1(γ0.999),最低定量浓度为1.31 ng·mL-1,回收率、日内、日间考察均符合生物样品分析要求。实验结果显示,野黄芩苷在大鼠体内出现多峰现象。结论:建立的LC-MS/MS定量分析方法灵敏、准确,可用于大鼠血浆中野黄芩苷的测定及其药代动力学研究。     

LC-MS/MS测定小鼠血浆中多靶点抗AD bis-MEP-乙二酰胺杂合物ZLA浓度及药动学研究

目的:建立检测小鼠血浆内新型多靶点抗阿尔茨海默病(Alzheimer's disease,AD)药物双美普他酚-乙二酰胺杂合物(ZLA)浓度的高效液相色谱-质谱联用法(LC-MS/MS),并研究其在小鼠体内的药代动力学。方法:样品经甲醇沉淀去蛋白,应用Waters Xbridge C18色谱柱(2.1×100 mm,3.5μm),以甲醇-水(含5 m M甲酸铵,p H 9.8)(85:15,v/v)为流动相,流速0.25 m L/min;采用电喷雾(ESI)离子源,选择正离子模式多反应监测,待测物分别为m/z 304.3→107.0(ZLA)和m/z 621.7→232.1(内标)。分别给予KM小鼠腹腔和尾静脉注射ZLA 5mg/kg,不同时间点采集血浆用于ZLA定量分析。结果:ZLA和内标保留时间分别为3.2 min和2.5 min。血浆中ZLA线性范围为1-1000 ng/m L。血浆中提取回收率超过91%,日内和日间精密度RSD均小于6%。药动学研究结果显示,腹腔注射时ZLA可快速分布到血浆中,在10.2 min达到峰值,且能达到良好的生物利用度(47.6%)。结论:本研究建立的ZLA血药浓度测定方法快速、灵敏,特异性好,并成功应用于小鼠血浆中ZLA的药代动力学研究。本研究资料将为ZLA在AD治疗中的进一步临床前评估提供依据。     

小型猪血浆中二丙酸倍他米松及其代谢物倍他米松的LC-MS/MS 同时测定方法的建立及在药动学研究中的应用

目的：建立同时测定小型猪血浆中二丙酸倍他米松及其代谢物倍他米松的LC-MS/MS 法,并研究小型猪皮肤外用二丙酸倍他米松乳膏后,二丙酸倍他米松及倍他米松的药动学特征。方法：血浆样品经酸化后以乙醚- 环己烷（4 ∶ 1）提取,LC-MS/MS 分析,以Hedera ODS-2（150 mm×2.1 mm,5 μm）为分析柱,流动相为5 mmol?L-1 醋酸铵水溶液（含0.1% 乙酸）– 甲醇,梯度洗脱。二丙酸倍他米松、倍他米松及内标布地奈德的监测离子对分别为m /z 563.2（[M+CH3COO]-）→ 483.1、m /z 451.2（[M+CH3COO]-）→ 361.0 和m /z 489.3（[M+CH3COO]-）→ 357.1。对小型猪外用二丙酸倍他米松乳膏后其血浆中二丙酸倍他米松及代谢物倍他米松的浓度进行测定,并计算主要药动学参数。结果：二丙酸倍他米松血药浓度在26.85~644.4 ng?L-1 范围内线性关系良好,倍他米松血药浓度在10.62~637.2 ng?L-1 范围内线性关系良好。结论：本方法专属性强、简便、灵敏,可用于血浆样本中二丙酸倍他米松及其代谢物倍他米松的测定及药动学研究。     

基于LC-MS/MS分析马缨杜鹃花代谢物的变化

为分析马缨杜鹃(Rhododendron delavayi)花开花至凋谢过程中的代谢产物差异及其通路,该文采用LC-MS/MS技术对其花苞期、开裂期、传粉期、盛开期、衰老期和凋谢期的化学成分进行非靶向代谢组学分析。结果表明:(1)共鉴定到973种代谢物,主要包含黄酮类、有机酸、酚酸类、氨基酸及其衍生物、脂类、生物碱等。(2)主成分分析(PCA)表明样本间代谢物存在差异,结合正交偏最小二乘判别分析(OPLS-DA)、t检验的P值和单变量分析的差异倍数(fold-change)筛选差异代谢物(VIP>1,P<0.05,Fc>2或Fc<0.5),涉及591种,在马缨杜鹃花期进入衰老期和凋谢期后差异代谢物数量和表达量显著上升,其中花苞期至开裂期差异代谢物的表达主要呈现下调,而进入衰老期和凋谢期后差异代谢物的表达主要呈现上调。(3)KEGG注释到68条代谢通路,其中差异代谢物极显著富集(P < 0.01)通路3条,包括苯丙素类生物合成、植物激素的生物合成和类黄酮生物合成。(4)结合苯丙素类、黄酮类等有效成分生物合成通路共筛选到10种代谢物包括苯丙氨酸(L-phenylalanine)、反式肉桂酸(trans-cinnamic acid)、查耳酮(chalcone)、柚皮素(naringenin)、对香豆酰基莽草酸(p-coumaroyl shikimic acid)、阿魏酸(ferulic acid)、松柏醇(coniferyl alcohol)、芥子酸(sinapic acid)、紫丁香苷(syringin)、槲皮素(quercetin)。此外,有效成分的差异代谢物表明苯丙素类生物合成代谢活动随马缨杜鹃花的发育逐渐增强,而黄酮类化合物生物合成逐渐减弱,这些关键差异代谢物可能对马缨杜鹃花的发育有重要的调控作用。该研究为马缨杜鹃花开花至凋谢进程中的有效成分代谢途径活性物质的研究提供了代谢组学基础,为进一步研究马缨杜鹃花花期调控的分子机理提供参考。     

GC/MS分析血浆中丁丙诺啡

目的:建立血浆中丁丙诺啡GC/MS分析方法。方法:血浆中丁丙诺啡,加入内标长春西汀,加pH 7缓冲溶液,用三氯甲烷提取,提取物经BSTFA衍生化后进行GC/MS分析。结果:方法的线性范围为2～100 g·L~(-1),检出限为1g·L~(-1)。结论:该方法灵敏度高,可用于涉毒案件血浆中丁丙诺啡的分析。     

LC-MS/MS测定单抗药动学参数的样品前处理方法研究进展

随着生物技术药物研发的大力开展,单抗类药物的药动学评价越来越受到重视。对近年来应用LC-MS/MS检测单抗类药物药动 学特征的文献进行归纳,总结其样品前处理的不同方法及各自优缺点和适用范围,为单抗类药物LC-MS/MS检测方法的进一步研究提供 参考。     

胃内容物中甲卡西酮LC-MS/MS检验方法研究

目的:建立胃内容物中甲卡西酮及其代谢物卡西酮的LC-MS/MS检验方法。方法:以双苯戊二氨酯(SKF525A)为内标,Column Eclipse Plus C18色谱柱(1.8μm, 100 mm×3 mm)为固定相,流动相为0.1%甲酸水溶液和乙腈,梯度洗脱,流速为0.3m L/min,进样量为1μL。通过电喷雾离子源(ESI),利用正离子多反应监测模式(MRM)进行检测。结果:胃内容物中甲卡西酮检验方法线性范围为1-5000 ng/g,标准曲线方程Y=0.026X+0.4501(R2=0.9998),检出限为0.5 ng/g,定量限为1.0 ng/g;卡西酮的检验方法线性范围为2-5000 ng/g,标准曲线方程Y=0.0099X+0.1576(R2=0.9997),检出限为1.0 ng/g,定量限为2.0 ng/g。结论:该方法操作简便,检测灵敏度较高,可用于甲卡西酮中毒案件胃内容物中甲卡西酮及其代谢物卡西酮的检验。     

LC-MS/MS分析人支气管上皮细胞与烟曲霉共培养模型中胶霉毒素的含量

目的：采用液相色谱-串联质谱法（LC-MS/MS）分析人支气管上皮细胞与烟曲霉共培养模型中胶霉毒素的含量。方法建立人支气管上皮细胞与烟曲霉共培养模型并于不同时间段检测共培养模型中胶霉毒素的含量。以支气管上皮细胞单独培养为对照组,分别于12h、24h、36h收集对照组、AF293（烟曲霉标准株）共培养组、AFB5233WT（烟曲霉野生株）及AFB5233ΔGlip（烟曲霉胶霉毒素基因敲除株）共培养组细胞培养上清液,采用LC-MS/MS检测胶霉毒素的含量。结果共培养模型中胶霉毒素水平随培养时间增加而逐渐升高（P〈005）,胶霉毒素回收率为687%~726%。结论该方法快速、灵敏、结果准确,适用于测定人支气管上皮细胞与烟曲霉共培养模型中胶霉毒素的含量。     

LC-MS/MS 法测定人血浆中伊伐布雷定浓度及药动学

目的:建立人血浆中伊伐布雷定的液相色谱-质谱-质谱联用测定方法,研究健康人体药代动力学.方法:以地西泮为内标物,采用液相色谱-质谱-质谱联用法,电喷雾电离源选择性正离子峰检测.测30名健康志愿者单剂量口服盐酸伊伐布雷定片的体内血药浓度,获得药动学参数.结果:伊伐布雷定在0.101-101 ng·mL-1浓度范围内呈良好的线性关系(r=0.998),最低检测浓度为0.101 ng·mL-1.高、中、低浓度的方法提取回收率分别为93.2%、86.6%、87.5%,日内、日间精密度RSD均小于15%.结论:LC-MS/MS方法灵敏度高,专属性强,准确,简便,适用于盐酸伊伐布雷定片的人体药代动力学研究.     

LC-MS/MS法测定人血浆中伊伐布雷定浓度及药动学

目的：建立人血浆中伊伐布雷定的液相色谱-质谱-质谱联用测定方法,研究健康人体药代动力学。方法：以地西泮为内标物,采用液相色谱-质谱-质谱联用法,电喷雾电离源选择性正离子峰检测。测30名健康志愿者单剂量口服盐酸伊伐布雷定片的体内血药浓度,获得药动学参数。结果：伊伐布雷定在0.101-101 ng·mL-1浓度范围内呈良好的线性关系（r=0.998）,最低检测浓度为0.101 ng·mL-1。高、中、低浓度的方法提取回收率分别为93.2%、86.6%、87.5%,日内、日间精密度RSD均小于15%。结论：LC-MS/MS方法灵敏度高,专属性强,准确,简便,适用于盐酸伊伐布雷定片的人体药代动力学研究。     

Quantitative analysis of acrylamide labeled serum proteins by LC-MS/MS

Isotopic labeling of cysteine residues with acrylamide was previously utilized for relative quantitation of proteins by MALDI-TOF. Here, we explored and compared the application of deuterated and (13)C isotopes of acrylamide for quantitative proteomic analysis using LC-MS/MS and high-resolution FTICR mass spectrometry. The method was applied to human serum samples that were immunodepleted of abundant proteins. Our results show reliable quantitation of proteins across an abundance range that spans 5 orders of magnitude based on ion intensities and known protein concentration in plasma. The use of (13)C isotope of acrylamide had a slightly greater advantage relative to deuterated acrylamide, because of shifts in elution of deuterated acrylamide relative to its corresponding nondeuterated compound by reversed-phase chromatography. Overall, the use of acrylamide for differentially labeling intact proteins in complex mixtures, in combination with LC-MS/MS provides a robust method for quantitative analysis of complex proteomes.     

Quantitative analysis of thymosin alpha1 in human serum by LC-MS/MS

A high performance liquid chromatography with tandem mass spectrometry (LC-MS/MS) method was developed to measure the thymosin alpha 1 (Talpha1) concentration in human serum. Tá1 in human serum was determined by solid phase extraction and reverse phase LC-MS/MS. The high-performance liquid chromatography (HPLC) system interfaced with the MS/MS system with a Turbo Ion spray interface. Positive ion detection and multiple reaction monitoring (MRM) mode were used for this human serum quantitation. Eight different concentration standards were used to establish the detection range. Six quality control (QC) and 2 matrix blanks were checked by calibration curves performed on the same day. The lower quantitation limit was 0.5 ng/mL Talpha1 in human serum. Calibration curves were established between 0.5 to 100 ng/mL by weighted linear regression. The correlation coefficients for different days were 0.9955 or greater. Quantitation of Talpha1 by the LC-MS/MS method is fast, accurate, and precise.     

Sensitive Determination of Fentanyl in Low-Volume Serum Samples by LC-MS/MS

Fentanyl is a widely used drug in the management of pain. Present LC-MS/MS methods for analysis of fentanyl require a large volume of serum, but yet the sensitivity was at about 50 pg/mL. Here, we report a modified liquid-liquid extraction method for the analysis of fentanyl in serum. The method is very sensitive with a LLOQ of 5 pg/mL while using only 0.175 mL of serum for analysis. The separation was performed on a Zorbax XDB-C18 column (4.6?×?50 mm, 1.8 μm, 600 bar) using a mobile phase of water: acetonitrile (70:30 v/v) with 0.1% formic acid that was pumped isocratically at a flow rate of 0.5 mL per minute. The calibration curve was found to be linear over a range of 5–10,000 pg/mL. The inter-day and intra-day accuracy and precision were tested using low (20 pg/mL), medium (1000 pg/mL), and high (5000 pg/mL) quality control samples of fentanyl prepared in blank human serum and were within ±?15% of the nominal value. Fentanyl was also found to be stable in various storage and sample preparation conditions, including short-term bench-top storage (for 5 h), freeze-thaw cycling (three cycles), long-term frozen condition (4.5 months at -?70°C), and post-preparative storage (for 48 h).     

Determination of macrocyclic trichothecenes in mouldy indoor materials by LC-MS/MS

Stachybotrys occurring in mouldy indoor environments is associated with the so called “sick building syndrome” in humans or cases of idiopathic pulmonary hemorrhages. Samples of mouldy materials from indoor environments (n=15) were analysed for the occurrence of this fungus and its secondary metabolites by a sensitive LC-MS/MS method. In four samples,Stachybotrys and macrocyclic trichothecenes have been detected. Maximum values for Satratoxin G and H in wallpaper were determined with 9.7 μg/cm² and 12.0 μg/cm², respectively.     

HPLC-MS/MS法测定人血浆中草乌甲素的浓度及生物等效性研究(英文)

本文建立了一种快速、高灵敏的HPLC-MS/MS法用于检测人血浆中的草乌甲素浓度。血浆样品采用沃特斯HLB小柱进行固相萃取,汉邦C18色谱柱(150 mm×4.6 mm,5μm)进行分离,流动相为甲醇∶水(85∶15,v/v),水相含10 mmol/L的醋酸铵和0.1%的甲酸。采用ESI源和多反应监测(MRM)的方式进行检测,草乌甲素及内标的反应离子对分别为644.4/584.4和237.2/194.2,草乌甲素血药浓度在0.010～1.0 ng/mL范围内线性关系良好,最低定量限为0.010 ng/mL可以满足口服0.4 mg草乌甲素后血药浓度的检测,日内日间及质控样品精密度及准确度均在允许范围内。本检测方法被成功的应用在中国健康志愿者生物等效性研究中,20名志愿者口服0.4 mg草乌甲素试验制剂和参比制剂后主要药代动力学参数分别如下:Cmax(0.325±0.110),(0.323±0.115)ng/mL;AUC0-16(1.627±0.489),(1.732±0.556)ng.h/mL;AUC0-∞(1.730±0.498),(1.831±0.562)ng.h/mL;t1/2(4.26±0.95),(3.80±0.90)h;Tmax(1.34±0.54),(1.83±0.99)h。     

Quantitative protein determination for CYP induction via LC-MS/MS

The Cytochrome P450 (CYP) proteins are a family of membrane bound proteins that function as a major metabolizing enzyme in the human body. Quantification of CYP induction is critical in determining the disposition, safety and efficacy of drugs in humans. Described is a gel-free, high-throughput LC-MS approach to quantitate the CYP isoforms 1A2, 2B6, 3A4 and 3A5 by measuring isoform specific peptides released by enzymatic digestion of the hepatocyte incubations. The method uses synthetic stable isotope-labeled peptides as internal standards and allows both relative and absolute quantification to be performed from hepatic microsomal preparations. CYP protein determined by this LC-MS method correlated well with the mRNA and activity for induced levels of CYP1A2, CYP2B6 and CYP3A4. Interestingly, a small fold change was observed for the induction of 3A5 with phenobarbital. The results were reproducible with an average CV less then 10% for repeat analysis of the sample. This LC-MS method offers a robust assay for CYP protein quantitation for use in CYP induction assays.