Development and validation of a liquid chromatography with tandem mass spectrometry (LC-MS/MS) method for the determination of sunitinib in rabbit plasma

A liquid chromatography with tandem mass spectrometry (LC-MS/MS) method has been developed and validated for the measurement of sunitinib in rabbit plasma. After protein precipitation with acetonitrile, samples were analyzed on a Zorbax Extend-C₁₈ column (150 mm×4.6 mm, 5μm). The mobile phase consisted of a mixture of acetonitrile and deionized water (containing 0.05% formic acid)at a ratio of 27:73 (v/v), and the flow rate was set at 0.8 mL/min.The column temperature was maintained at 30 ^oC. The LC eluate was detected by an electrospray ionization (ESI) source operated in the positive ion mode, and quantification was conducted using MRM of the transitions m/z 399.24→283.01 and m/z 415.19→178.00 for sunitinib and internal standard (IS, diltiazem hydrochloride), respectively. The calibration curve was linear in the range of 2–600 ng/mL. The lower limit of quantification was 2 ng/mL. The method also exhibited satisfactory results in terms of sensitivity, specificity, accuracy (with relative error ranging from –4.0% to 1.1%), precision (with intra- and inter-day relative standard deviations ranging from 2.8% to 9.5%),matrix effect, recovery as well as stability. Taken together, our newly developed method was reliable to monitor sunitinib concentrations in rabbit plasma.     

液相色谱-串联质谱法测定大鼠血浆中脱水穿心莲内酯琥珀酸半酯的含量（英文）

目的建立了测定大鼠血浆中脱水穿心莲内酯琥珀酸半酯（DAS）的液相色谱-串联质谱法。方法血浆样品经液-液萃取后,以甲醇-水（70∶30,V/V）为流动相,通过Restek PinnacleⅡC18柱分离,格列吡嗪为内标,选择负离子扫描方式,以多反应监测（MRM）方式进行检测。用于定量分析的离子反应分别为m/z 531→m/z 431（DAS）和m/z 444→m/z319（格列吡嗪,内标）。结果DAS血浆浓度测定方法的线性范围为5～2500 ng/ml,定量下限为5 ng/ml。日内、日间精密度（RSD）均小于9.51%,准确度（RE）在-0.18%~1.93%。样品提取回收率为79.73%~85.99%,每个样品的测试时间为3 min。应用此法测试了大鼠口服或静注穿琥宁（DAS的单钾盐）后DAS的血药浓度,计算出其绝对生物利用度为3.69%。结论本方法灵敏度高、专属性强,适合于DAS的临床前药动学研究。     

A highly sensitive ultra-high performance liquid chromatography/tandem mass spectrometry method with in-source fragmentation for rapid quantification of raspberry ketone

Raspberry ketone (RK) is the characteristic aromatic compound in raspberry (Rubus idaeus L.) with wide applications as food additive and anti-obesity agent. However, quantification of RK has presented difficulties in MS detection and reliable LC-MS method for RK analysis in literature is in limit to date. In order to facilitate quality control of raspberry derived products and RK metabolomics study, this study aimed to develop a validated and sensitive UHPLC-MS/MS method. Strong in-source fragmentation was noted and the fragmental ion of 107 m/z produced was selected as the precursor ion for MRM detection, and as such the electrospray ionization performance was optimized by fractional factorial design to accommodate such ion-source dissociation behavior as well as its moderate volatility. A pathway involving the formation of quinone-like structure with strong conjugation was proposed to explain the intense in-source fragmentation. The MRM transition was optimized with product ion of 77 m/z selected as the quantifier ion. The method featured low limit of quantification of ～2 ng/mL and allowed for rapid detection of RK in fresh raspberries following direct sample preparation. RK contents were found to be higher from locally grown and harvested farm sources compared to commercial products shipped into the state, and higher in those at late-stage compared with early-stage maturity. No correlations in RK content between organic and non-organic labels were noted.     

LC-MS/MS法同时测定洛匹那韦和利托那韦的血浆浓度

建立液相色谱-串联质谱法 (LC-MS/MS) 测定人血浆中洛匹那韦 (LPV)、利托那韦 (RTV) 的浓度。血浆样品经碱化沉淀蛋白后, 经乙酸乙酯液-液萃取, 以甲醇-0.1%甲酸水溶液 (80∶20) 为流动相, Agilent ZORBAX Eclipse XDB-C₁₈ (150 mm × 4.6 mm ID, 5 μm) 柱分离; 采用电喷雾电离源, 以多反应监测 (MRM) 方式进行正离子检测, 用于定量分析的离子对LPV为629.6→155.2, RTV为721.4→268.2, 内标替米沙坦 (TEL) 为515.2→276.2。测定血浆中LPV线性范围为62.5～10 000 ng·mL⁻¹, 检测限为15 pg·mL⁻¹, RTV的线性范围为12.5～2 000 ng·mL⁻¹, 检测限为8 pg·mL⁻¹, r均大于0.99。日内和日间精密度均小于15%, 提取回收率均大于75%。该法选择性强、灵敏度高、重现性好, 能同时快速、准确测定人血浆LPV和RTV浓度, 为临床治疗药物浓度监测 (TDM) 奠定基础。     

Simultaneous determination of creatine and guanidinoacetate in plasma by liquid chromatography-tandem mass spectrometry (LC-MS/MS)

Background

Guanidinoacetate (GAA) and creatine are reliable biochemical markers for primary and secondary creatine defects. We describe a method by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) for simultaneous determination of plasma GAA and creatine. We analyzed 283 healthy subjects from 0 to 63 years old to obtain age-related control values.

Methods

Plasma samples were extracted with acetonitrile containing 13C2-GAA and d3-creatine. Samples were analyzed by LC-MS/MS in positive ionisation mode, after derivatization to butyl-esters. Optimal chromatographic separation was achieved using a column Supelcosil™ LC-4.6 mm with isocratic elution in 5 min.

Results

Run time was 5 min. Standard curves were linear from 0.05 to 200 μmol/L for creatine and from 0.02 to 40 μmol/L for GAA. Limit of detection (LOD) and limit of quantitation (LOQ) were respectively 0.005 and 0.05 μmol/L for creatine; LOD and LOQ were 0.002 and 0.02 μmol/L respectively for GAA. Intra and inter-assay CVs for creatine and GAA were <8%. Recovery experiments adding 50 and 100 μmol/L creatine and 10 and 20 μmol/L GAA were 102.1% and 101.2%, for creatine; 102.95% and 96.45% for GAA. The method was applied to 283 plasma controls from healthy subjects to obtain control values in three specific age ranges: 0-12, 13-20, >20 years old.

Conclusion

A rapid and high sensitive LC-MS/MS method was developed and validated for determination of creatine and GAA in plasma and it could also be applied to other biological materials, such as CFS and urines. This method is useful for diagnoses of primary and also for secondary creatine defects that may occur in inherited metabolic diseases in which precursors of creatine biosynthesis are involved.     

LC-MS/MS法测定血浆中甲氯噻嗪的浓度

目的:建立血浆中甲氯噻嗪的LC-MS/MS定量测定方法.方法:以双氢克尿噻为内标,血浆样品经碱化后用含5%异丙醇的乙酸乙酯溶液提取,采用液质联用色谱法进行MRM扫描分析,色谱柱为CAPCELL PAK C18 MG(50mm×2.0mm,5μm),流动相为20mmol/L乙酸胺水-乙腈(64:36),离子选择通道分别为甲氯噻嗪:358.2/321.9amu;双氢克尿噻:296.3/268.9amu.甲氯噻嗪、双氢克尿噻的保留时间分别为1.6min和1.0min.结果:本文所建立的血浆样品中甲氯噻嗪液质联用色谱测定方法,血浆内源性物质不干扰样品峰,相对回收率为93.7%～105.4%;绝对回收率为71.2%～78.4%;日间和日内相对标准差均小于6.04%.血浆中的最低定量限为0.2ng/mL(S/N≥20:1),线性范围为0.2ng/mL～51.2ng/mL.结论:本方法操作简便,特异性强,灵敏度高,取血量少,符合生物样品的分析要求,可以用于甲氯噻嗪药代动力学研究及临床测定.     

快速灵敏的LC-MS/MS法测定人血浆中缬沙坦浓度

目的：建立一种快速、灵敏的高效液相色谱-串联质谱法（LC-MS/MS）测定人血浆缬沙坦浓度。方法：200μL血浆样品经乙腈一步沉淀蛋白后,在Inertsil ODS-色谱柱（2.1mm×150mm,5μm）上分离,流动相由乙腈和1‰甲酸水溶液（70∶30）组成。采用电喷雾离子源（ESI源）正离子多反应监测（MRM）扫描分析,缬沙坦和厄贝沙坦的离子选择通道分别为：m/z436.3→235.2和429.4→207.2。结果：缬沙坦的线性范围为24.2～3100.0μg/L,日内和日间相对标准差均小于15%。结论：本法操作快速、灵敏,适用于缬沙坦的临床药动学研究。     

A liquid chromatography tandem mass spectrometry method for the quantification of indocyanine green in dog plasma and bile

A sensitive and specific liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS) method was developed and validated for the determination of indocyanine green (ICG) in dog plasma and bile. An ICG analog (IR-820) was used as an internal standard. A protein precipitation method was used with acetonitrile for plasma sample preparation, whereas bile samples were diluted with water (120-fold) prior to analysis. Using MS/MS in the multiple reaction monitoring mode, ICG and IR-820 were detected in both matrices without interference. The lower limit of quantitation for ICG in dog plasma was 3 ng/mL, with an intra- and inter-day accuracy (%Bias) and precision (%CV) of less than 15%, so it was possible to study the pharmacokinetics of ICG in bile duct-cannulated dogs and assess their liver function after surgery. The method described herein is sensitive, selective and faster than other existing methods (e.g., spectrophotometry, HPLC/UV–vis detection, or HPLC/fluorescence detection).     

LC-MS/MS法测定人血清中亮丙瑞林的浓度

目的:建立人血清中亮丙瑞林浓度的LC-MS/MS测定方法。方法:采用乙腈沉淀蛋白提取血清中目标成分。选用Agela Venusil ASB C18色谱柱(150 mm×4.6 mm,5μm),以乙腈-5 mmol.L-1醋酸铵-甲酸(30∶70∶0.1)为流动相,采用多反应离子监测(MRM)模式进行正离子检测,选择监测离子反应为m/z605.6→248.9[M+2H]2+(亮丙瑞林)和151.8→110.1[M+H]+(对乙酰氨基酚)。结果:血清中内源性物质不干扰亮丙瑞林的测定;亮丙瑞林的线性范围为0.1～10.0 ng.mL-1,最低定量限为0.1 ng.mL-1。方法准确度为88.5%～111.5%,日内和日间精密度均<15%。结论:本文建立的LC-MS/MS法,简单快速,专属性强,灵敏度高,可用于亮丙瑞林缓释制剂在人体内的药代动力学研究。     

LC-MS／MS法测定人血浆中氯沙坦及其代谢物E-3174的浓度

目的建立LC-MS／MS法测定人血浆中氯沙坦及其代谢物E-3174血药浓度的方法。方法血浆酸化后用乙醚提取,采用同位素内标（氘3-B3174）进行测定。色谱柱：CAPCELLPACKC18Ⅲ（100mm×2．0mm,5μm）,流动相：0．02％甲酸乙腈-水溶液（53：47,v／v）;等度洗脱;流速0．3mL·min-1;进样体积5μL;电喷雾离子化,正离子MRM扫描。结果氯沙坦和E-3174线性范围均为5—500μg·L-1（r〉0．999）,最低定量限均为5μg·L-1,平均提取回收率均〉50％,批内、批间精密度RSD均〈8％。结论本方法灵敏度高、专一性好、操作简单,适用于氯沙坦的药动学研究。     

LC-MS/MS法分析人血浆中利培酮浓度及健康人体的药动学研究

目的 建立一种简便、灵敏的液相串联质谱（LC-MS/MS）法测定人血浆中利培酮的浓度,并应用于健康人体的药动学研究。方法 血浆样品经乙腈沉淀蛋白后,使用ZORBAX Eclipse XDB-C₁₈ （50 mm×4.6 mm,5 μm）色谱柱分离,以60%乙腈-40%甲醇、0.1%甲酸-5%乙腈-10 mmol/L乙酸铵水溶液作为流动相,梯度洗脱;在电喷雾离子化源（ESI）正离子检测条件下,采用多反应离子监测模式（MRM）对利培酮及内标利培酮-d4进行定量分析,检测离子对分别为m/z 411.3→191.2、m/z 415.3→195.2;进行专属性、系统适用性、准确度、精密度、基质效应、提取回收率、稳定性等方法学验证;8名健康成年受试者（无脱落）空腹单次口服利培酮片1 mg后,分别于给药前0 h和给药后10、20、30、45 min,1.00、1.25、1.50、2.00、3.00、4.00、5.00、6.00、8.00、12.00、16.00、24.00、36.00、48.00 h采集血样至含有肝素钠的抗凝管中,分离血浆样品,进行LC-MS/MS分析。结果 建立的LC-MS/MS法专属性良好,系统适用性良好,内标与待测物之间不存在交叉影响,人血浆中利培酮的线性范围为0.1～20.0 ng/mL,定量下限（LLOQ）为0.1 ng/mL,利培酮在空腹血浆、餐后血浆及溶血血浆中经内标归一化的基质效应分别为0.991～1.00、1.00～1.01和0.994～0.999,利培酮在人血浆中的平均提取回收率为96.8%～99.7%,准确度、精密度以及稳定性等均符合有关要求。健康受试者单次口服利培酮片1 mg后,主要药动学参数t_max、C_max、AUC_0~t、t_1/2分别为（0.969±0.248）h、（7.83±2.24）ng/mL、（25.5±12.0）h·ng/mL、（3.31±1.74）h。结论 建立的LC-MS/MS法前处理简便快速,灵敏度高,满足生物分析的法规要求,可应用于利培酮在健康人体中的药动学研究。     

Rapid and sensitive LC-MS/MS method for determination of Pravastatin in human plasma

快速灵敏的LC-MS/MS方法检测人血浆中普伐他汀的浓度@张敏$Institute of Clinical Pharmacology, Central South University!Changsha 410078,Hunan,China @谭志荣$Institute of Clinical Pharmacology, Central South University!Changsha 410078,Hunan,China @周宏灏$Ins     

LC-MS/MS法测定人血浆中加巴喷丁的浓度及其药动学研究

建立液相色谱串联质谱(LC-MS/MS)法测定人血浆中加巴喷丁的浓度并将其应用于人体药动学研究。取血浆样品经甲醇沉淀蛋白后,以甲醇0.2%甲酸水溶液(80∶20)为流动相,用Inertsil ODS-3 C18柱(50 mm×2.1 mm ID,3μm)分离,采用电喷雾离子源,以多反应监测(MRM)方式进行正离子检测,定量分析的离子反应分别为m/z 172→m/z 154(加巴喷丁)和m/z 130→m/z 71(内标二甲双胍)。加巴喷丁线性范围为40.8～8.16×103 ng.mL 1,定量限为40.8 ng.mL 1,每个样品测试时间仅2.2 min,日内、日间精密度(RSD)均小于12%,准确度(RE)在±6.4%范围内。应用此法研究了20名健康志愿者单剂量口服加巴喷丁胶囊600 mg后的药动学特点。该方法快速、专属、灵敏、适用性强,可应用于加巴喷丁的人体药动学研究。     

A selective and rapid method for the quantification of captopril in human plasma using liquid chromatography/selected reaction monitoring mass spectrometry

A specific hyphenated high performance liquid chromatography–mass spectrometric (LC–MS/MS) assay was developed for the determination of captopril in plasma. The drug was extracted from plasma using liquid–liquid extraction with a mixture of diethylether:dichloromethane. After the addition of the internal standard, samples were applied to a prepacked C₈ Waters Symmetry column. The ion trap MS/MS detector was equipped with electrospray ionization (ESI) source operating in the positive ion mode. Drug determination was accomplished monitoring captopril at molecular ion m/z 218 and MS/MS (daughter) at m/z 171.6. The method was applied to captopril determination in human plasma after the administration of captopril 50 mg tablets to healthy volunteers who have participated in a pharmacokinetic study.

The method was proved to be specific and precise by testing six different plasma batches. Linearity was established for the range of concentrations 25–3000 ng/ml with a regression factor of 0.9995. Intra-day accuracy ranged from 90.16 to 96.18%, while the intra-day precision ranged from 2.60 to 9.66% at the concentrations of 75, 1440 and 2500 ng/ml. Inter-day precision of the method ranged from 5.04 to 10.10%. This validated method of analysis was successfully applied to human plasma analyses after the administration of a single dose of 50 mg captopril tablets to healthy volunteers.     

LC-MS／MS法测定人血浆中米格列醇

目的:建立测定人血浆中米格列醇的 LC-MS/MS 法。方法:以盐酸格拉司琼为内标,以 Kromasil CN 柱(2.1 mm×150mm,3.0μm)为分析柱;采用0.05%三氟乙酸乙腈溶液-0.05%三氟乙酸水溶液(80:20)为流动相;流速0.2 mL·min~(-1);柱温45℃。质谱条件为电喷雾电离源(ESI~ ),以选择反应离子监测(SRM)方式进行检测,用于定量分析的反应离子分别为 m/z208.1→146.1(米格列醇),m/z 313.1→138.0(盐酸格拉司琼)。结果:米格列醇在0.01～4.0μg·mL~(-1)浓度范围内线性良好;最低检测限为2.0 ng·mL~(-1)(S/N=4);日内、日间精密度(RSD)均小于7%;低、中、高3个浓度的方法回收率均大于90%。结论:本法专属性强,灵敏度高,样品处理简单,可用于米格列醇临床药物动力学研究。     

LC-MS/MS法测定人血浆中西布曲明代谢物(M2)

西布曲明代谢物M2(N-双脱甲基西布曲明)是减肥药西布曲明(N-[1-[1-(4-氯苯基)环丁基]-3-甲基丁基]-N,N-二甲基胺,sibutramine)的主要活性代谢产物,其体内浓度较低,难以用常规的HPLC-UV法进行测定.国内至今未有西布曲明血药浓度测定的相关报道,为了解西布曲明制剂的体内过程,我们参考有关文献[1],采用液相色谱-质谱/质谱(LC-MS/MS)法建立了西布曲明主要活性代谢产物M2的血药浓度分析方法,可较好地满足研究工作的需要. 1 材料和方法 1.1 仪器 Waters 2690高效液相色谱仪(美国Waters公司);QuattroLC质谱仪(英国质谱公司,micromass).XW-80A旋涡混合器(上海医科大学仪器厂);CQ50超声波清洗器(上海超声波仪器厂);80-2型离心机(上海手术器械厂);LNG-T83台式快速离心浓缩干燥器(江苏太仓医用器械厂);SHB-Ⅲ循环多用水泵(郑州长城仪器厂);TGL.16G台式高速离心机(上海医用分析仪器厂).     

Analysis of carvedilol in human plasma using hydrophilic interaction liquid chromatography with tandem mass spectrometry

A rapid, sensitive and selective method for the determination of carvedilol in human plasma was developed using hydrophilic interaction liquid chromatography with tandem mass spectrometry (HILIC-MS/MS). Carvedilol and cisapride (internal standard) were extracted from human plasma with methyl tert-butyl ether at basic pH and analyzed on an Atlantis HILIC Silica column with the mobile phase of acetonitrile-ammonium formate (50 mM, pH 4.5) (90:10, v/v). The analytes were detected using an electrospray ionization tandem mass spectrometry in the multiple-reaction-monitoring mode. The standard curve was linear (r=0.9998) over the concentration range of 0.1-200 ng/ml. The lower limit of quantification for carvedilol was 0.1 ng/ml using 50 microl plasma sample. The coefficient of variation and relative error for intra- and inter-assay at four QC levels were 1.6-4.5% and -6.4 to 4.8%, respectively. The absolute and relative matrix effect for carvedilol and cisapride were practically absent. The extraction recoveries of carvedilol and cisapride were 81.6 and 85.2%, respectively. This method was successfully applied to the bioequivalence study of carvedilol in humans.     

同时定量检测水胺硫磷和水胺氧磷的液相色谱-串联质谱法及应用

目的建立同时测定大鼠全血中水胺硫磷及其活性代谢产物水胺氧磷的液相色谱-串联质谱（LC-MS/MS）分析方法,用于毒代动力学研究中血药浓度的测定。方法采集大鼠全血样品后即刻用4倍体积冰冷乙腈沉淀蛋白。待测物用Zorbax SB C18柱（2.1 mm×50 mm,1.8μm）分离,以含有0.1%甲酸的水和甲醇为流动相系统进行梯度洗脱。采用ESI源正离子多反应监测模式分析,水胺硫磷、水胺氧磷和内标三唑磷的定量离子对分别为m/z 231→121、274→215和314→162。结果水胺硫磷和水胺氧磷在5μg/L～5 mg/L浓度范围内线性良好,最低定量限为5μg/L,回收率在63.1%～77.8%的范围内,准确度和精密度符合生物样品的检测要求。大鼠静脉注射3 mg/kg水胺硫磷后,水胺硫磷在血中很快下降,消除半衰期为22.2 min。活性代谢产物水胺氧磷的生成也较快,10～15 min达到（257.91±42.00）ng/ml的平均峰值,消除半衰期为17.6 min。水胺硫磷和水胺氧磷的AUC0~t分别为（528.62±49.15）和（191.11±33.23）h.μg/L。结论本研究首次建立了同时定量测定大鼠全血中水胺硫磷和水胺氧磷的LC-MS/MS分析方法,方法的专属性好、灵敏度高,适用于水胺硫磷及水胺氧磷在大鼠体内的毒物代谢动力学研究。     

液相色谱-质谱联用测定人血浆中罗哌卡因的浓度

目的建立测定人血浆中罗哌卡因浓度的液相色谱质谱联用(LCMS/MS)法。方法血浆样品加入布比卡因内标,经沉淀液(甲醇∶0.1%甲酸水溶液=9∶1)处理后,以甲醇-0.1%甲酸水溶液(70∶30,V/V)为流动相,在0.2mL·min-1的流速下,用ZorbaxC18柱(5cm×2.1mm,5μm)分离。样品经电喷雾离子源(ESI)正离子化后,通过三级四极杆串联质谱仪,以N2为碰撞气,采用多反应离子检测方式测定罗哌卡因(m/z275.2→126.1)和内标布比卡因(m/z289.2→140.3)浓度。结果线性范围为50～2000μg·L-1,最低定量浓度为50μg·L-1,方法的相对回收率在85%～115%之间,日内、日间RSD均<15%。结论该方法快速、简便,特异性强。     

LC-MS/MS法测定人血浆中甘草次酸及其临床药代动力学研究

目的建立人血浆中甘草次酸的LC-MS/MS测定方法,研究男性健康志愿者单剂量服用甘草酸二铵胶囊,其代谢产物甘草次酸体内药代动力学行为。方法健康男性志愿者单剂量口服甘草酸二铵胶囊150 mg,血浆样品经乙酸乙酯提取,进行LC-MS/MS分析。色谱柱为Agilent ZORBAXSB C18(3.0×100 mm,5μm),流动相为甲醇∶乙腈∶醋酸铵缓冲液(5 mmol.L-1醋酸铵,0.2%冰醋酸)(15∶60∶25,V/V/V),检测离子为m/z469.4/355.2(甘草次酸)、m/z358.9/279.9(内标泼尼松龙)。测定甘草次酸血药浓度,计算其药代动力学参数。结果在1.5～192μg.L-1内,甘草次酸与内标的峰面积比值与浓度的线性关系良好,定量限为1.5μg.L-1,提取回收率为77.14%～83.64%。人体中甘草次酸药代动力学参数:Cmax为(73.85±25.25)μg.L-1,Tmax为(11.50±3.07)h,T21β为(11.82±3.56)h,AUC0-60为(1252.49±489.06)μg.h.L-1。结论建立的LC-MS/MS分析方法准确灵敏,适于临床药代动力学研究。口服甘草酸二铵胶囊,其代谢产物甘草次酸在体内的药代动力学特点是达峰时间长,约占受试者总人数50%的药时曲线有双峰现象。