Effects of flow rate on high-throughput quantitative analysis of protein-precipitated plasma using liquid chromatography/tandem mass spectrometry

The effects of flow rate and column length on analyte response (peak area and height), total cycle time, column backpressure, and elution volume are presented. Rapid chromatographic separations and tandem mass spectrometric (MS/MS) detection are applied to the supernatant of protein-precipitated plasma standards containing four compounds from a drug discovery screen. The plasma samples were injected onto three C-18 columns (2 x 10,2.1 x 30 and 2.1 x 50 mm) at flow rates of 0.25, 0.50, 1.00 and 1.50 mL/min. The plasma samples were detected using a Sciex API 3000 tandem mass spectrometer operated in the Turbo Ionspray mode. A post-column split was used to maintain a flow rate of 0.25 mL/min into the mass spectrometer source to avoid differences in nebulization efficiency. The data show that diluted protein-precipitated plasma supernatants show average matrix effects (i.e. suppression) of 60.0% (2 x 10 mm), 89.3% (2 x 30 mm), and 76.7% (2 x 50 mm) of expected response at 10 ng/mL. Average matrix effects of 70.2% (2 x 10 mm), 88.9% (2 x 30 mm), and 81.2% (2 x 50 mm) of expected response at 1000 ng/mL plasma. The data also show if peak widths remain relatively constant, analytes are less sensitive as flow rates are increased. These data are consistent with the concentration-dependent relationship of ionspray in the range of flow rates studied. The data show that, while analyte response decreased proportionately to increases in flow rate, the analysis cycle times did not decrease proportionately.     

高效液相色谱-质谱法分析菊芋叶中的绿原酸类化合物

建立了菊芋叶中绿原酸类化合物的高效液相色谱-紫外检测-质谱(HPLC-UV-MS)定性分析方法。液相色谱条件:Inertsil ODS-3色谱柱(250 mm×4.6 mm,5 μm);甲醇和水(含1%乙酸)梯度洗脱,流量1.0 mL/min;柱温35 ℃;检测波长327 nm。质谱条件:Thermo公司TSQ三级四极杆质谱仪;电喷雾电离(ESI)接口;负离子检出模式。采用该方法得到了菊芋叶提取物的紫外检测的色谱图、负离子监测的总离子流图以及相应色谱峰的紫外光谱图和一级、二级质谱图,对其进行解析,鉴别出菊芋叶中的7个绿原酸类成分。该方法简便、快速、灵敏度高,可以很好地对菊芋叶中的绿原酸类化合物进行定性分析。     

Highly sensitive determination of Schisandrin and Schisandrin B in plasma of rats after administration of Wurenchun (Fructus Schisandrae Chinensis Extracts) preparations by LC‐ESI‐MS/MS

A sensitive and specific method based on liquid chromatography‐tandem mass spectrometry using electrospray ionization (LC‐ESI‐MS/MS) has been developed for the determination of Schisandrin and Schisandrin B in rat plasma. A 100 μL plasma sample was extracted by methyl tert‐butyl ether after spiking the samples with nimodipine (internal standard) and performed on an XTerra®MS‐C₁₈ column (150 mm × 2.1 mm, 3.5 μm) with the mobile phase of acetonitrile–water–formic acid (80:20:0.2, v/v) at a flow rate of 0.2 mL/min in a run time of 8.5 min. The lower limit of quantification of the method was 40 ng/mL for Schisandrin and 20 ng/mL for Schisandrin B. The method showed reproducibility with intra‐day and inter‐day precision of less than 13.8% RSD, as well as accuracy, with inter‐ and intra‐assay accuracies between 93.5 and 107.2%. Finally, the LC‐ESI‐MS/MS method was successfully applied to study the pharmacokinetics of Schisandrin and Schisandrin B in rats after administration of Wurenchun commercial formulations to rats. Copyright © 2009 John Wiley & Sons, Ltd.     

Simultaneous determination of metformin and gliclazide in human plasma by liquid chromatography-tandem mass spectrometry: application to a bioequivalence study of two formulations in healthy volunteers

A rapid and sensitive liquid chromatography/tandem mass spectrometry (LC/MS/MS) method was developed and validated to simultaneously determine gliclazide and metformin in human plasma using huperzine A as the internal standard (IS). After acetonitrile-induced protein precipitation of the plasma samples, gliclazide, metformin and the IS were subjected to LC/MS/MS analysis using electro-spray ionization (ESI). Chromatographic separation was performed on a Hypersil BDS C18 column (50 mm x 2.1 mm, i.d., 3 microm). The method had a chromatographic running time of 2.0 min and linear calibration curves over the concentration ranges of 10-10,000 ng ml(-1) for gliclazide and 7.8-4678.9 ng ml(-1) for metformin. The recoveries of the method were found to be 71-104%. The lower limits of quantification (LOQ) of the method were 10.0 and 7.8 ng ml(-1) for gliclazide and metformin, respectively. The intra- and interday precision was less than 15% for all quality control samples at concentrations of 100, 500, and 2000 ng ml(-1). The validated LC/MS/MS method has been used to study bioequivalence in healthy volunteers. These results indicate that the method was efficient with a very short running time (2.0 min) for metformin and gliclazide compared to the methods reported in the literature. The presented method had acceptable accuracy, precision and sensitivity and was used in clinical bioequivalence study.     

槲寄生中高圣草素-7-O-β-D-葡萄糖苷的分离及含量测定

对槲寄生中的有效成分高圣草素-7-O-β-D-葡萄糖苷进行了分离和结构鉴定,并对其含量进行了分析。色谱条件：C18柱(200 mm×4.6 mm i.d., 5 μm),流动相为乙腈-0.5%冰醋酸水溶液(体积比为18∶82),流速为1.0 mL/min,柱温为30 ℃,检测波长为284 nm,进样量为10 μL。结果表明,高圣草素-7-O-β-D-葡萄糖苷的峰面积与其质量浓度有良好的线性关系,相关系数r为0.9997;方法的加标回收率为96.0%~100.1%。该方法简便、快速、准确,精密度好,可作为槲寄生质量控制的一个有效方法。     

Ultra-low flow nanospray for the normalization of conventional liquid chromatography/mass spectrometry through equimolar response: standard-free quantitative estimation of metabolite levels in drug discovery

Nanospray experiments were performed on an ensemble of drug molecules and their commonly known metabolites to compare performance with conventional electrospray ionization (ESI) and to evaluate equimolar response capabilities. Codeine, dextromethorphan, tolbutamide, phenobarbital, cocaine, and morphine were analyzed along with their well-known metabolites that were formed via hydroxylation, dealkylation, hydrolysis, and glucuronidation. Nanospray exhibited a distinct trend toward equimolar response when flow rate was reduced from 25 nL/min to less than 10 nL/min. A more uniform response between the parent drug and the corresponding metabolites was obtained at flow rates of 10 nL/min or lower. The largest discrepancy was within +/-50% for plasma samples. Nanospray was used as a calibrator for conventional ESI liquid chromatography/tandem mass spectrometry (LC/MS/MS) and normalization factors were applied to the quantitation of an acyl-glucuronide metabolite of a proprietary compound in rat plasma. A nanospray calibration method was developed with the standard curve of the parent drug to generate quantitative results for drug metabolites within +/-20% of that obtained with reference standards and conventional ESI. The nanospray method provides a practical solution for the quantitative estimation of drug metabolites in drug discovery when reference standards are not available.     

反相高效液相色谱法分离测定墨旱莲中的鳢肠醛

对墨旱莲中的有效成分鳢肠醛进行了分离和结构鉴定,并对其含量进行了分析。色谱条件:Kromasil C18柱(200 mm×4.6 mm,5 μm),流动相为乙腈-0.1%磷酸水溶液(体积比为65∶35),流速为1.0 mL/min,柱温为30 ℃,检测波长为365 nm,进样量为10 μL。结果表明,鳢肠醛的峰面积与其质量浓度有良好的线性关系(r=0.9993)。方法的加样回收率为96.7%～100.0%。该方法简便、快速、准确,可作为墨旱莲质量控制的一个有效方法。     

Simultaneous determination of mifepristone and monodemethyl-mifepristone in human plasma by liquid chromatography-tandem mass spectrometry method using levonorgestrel as an internal standard: application to a pharmacokinetic study

A rapid and sensitive liquid chromatography/tandem mass spectrometry (LC-MS/MS) method was developed and validated to simultaneously determine mifepristone and monodemethyl-mifepristone in human plasma using levonorgestrel as the internal standard (IS). After solid-phase extraction of the plasma samples, mifepristone, monodemethyl-mifepristone and the IS were subjected to LC-MS/MS analysis using electro-spray ionization (ESI) in the multiple reaction monitoring (MRM) mode. Chromatographic separation was performed on an XTERRA MS C(18) column (150 x 2.1 mm i.d., 5 microm). The method had a chromatographic run time of 4.5 min and linear calibration curves over the concentration ranges of 5-2000 ng/mL for mifepristone and monodemethyl-mifepristone. The recoveries of the method were found to be 94.5-103.7% for mifepristone and 70.7-77.3% for monodemethyl-mifepristone. The method had a lower limit of quantification (LLOQ) of 5.0 ng/mL and a lower limit of detection (LOD) of 1.0 ng/mL for both mifepristone and monodemethyl-mifepristone. The intra- and inter-batch precision was less than 15% for all quality control samples at concentrations of 10, 100 and 1000 ng/mL. These results indicate that the method was efficient with a short run time (4.5 min) and acceptable accuracy, precision and sensitivity. The validated LC-MS/MS method was successfully used in a pharmacokinetic study in healthy female volunteers after oral administration of 25 mg mifepristone tablet.     

Application of low-pressure gas chromatography-ion-trap mass spectrometry to the analysis of the essential oil of Turnera diffusa (Ward.) Urb

Turnera diffusa Willd. var. afrodisiaca (Ward) Urb. (syn. T. aphrodisiaca) belongs to the family of Turneraceae and is an aromatic plant growing wild in the subtropical regions of America and Africa. It is widely used in the traditional medicine as e.g. anti-cough, diuretic, and aphrodisiac agent. This work presents a 3 min chromatographic analysis using low-pressure (LP) gas chromatography (GC)-ion-trap (IT) mass spectrometry (MS). The combination of a deactivated 0.6 m x 0.10 mm i.d., restrictor with a wide-bore CP-Wax 52 capillary column (10 m x 0.53 mm i.d., 1 microm) reduces the analysis time by a factor of 3-7 in comparison to the use of a conventional narrow bore column. Chromatographic conditions have been optimized to achieve the fastest separation with the highest signal/noise ratio in MS detection. These results allow fast and reliable quality control of the essential oil to be achieved.     

分散固相萃取-超快速液相色谱-串联质谱法测定牛蛙全血中6种酚类环境雌激素

建立了一种快速、灵敏、准确的同时测定牛蛙全血中双酚A、己烯雌酚、己二烯雌酚、己烷雌酚、4-叔辛基酚和4-壬基酚等6种酚类环境雌激素的分散固相萃取-超快速液相色谱-串联质谱(dSPE-UFLC-MS/MS)分析方法。牛蛙全血样品经含0.1%(v/v)甲酸的甲醇溶液沉淀蛋白后,利用自制的氨基功能化Fe3O4磁性高分子复合微粒(EDA-MPs)作为dSPE吸附剂进行净化,着重考察了沉淀剂、吸附净化时间、吸附剂用量等因素对6种酚类环境雌激素回收率的影响。采用Shim-pack XR-ODSII(100 mm×2.0 mm, 2.2 μm)反相液相色谱柱进行分离,在电喷雾离子源(ESI)负离子多反应监测(MRM)模式下进行检测。结果表明: 6种酚类环境雌激素在0.5～100.0 μg/L范围内具有良好的线性关系(r2≥0.9996),方法的定量限(信噪比大于10)为0.075～0.40 μg/L,方法的精密度为0.6%～6.3%,空白样品中3个不同水平的添加回收率为95.0%～110.0%。本方法适用于牛蛙全血中6种酚类环境雌激素的同时测定。     

Determination of terbinafine (Lamisil) in human hair by microbore liquid chromatography/tandem mass spectrometry

An analytical method for the determination of terbinafine (Lamisil(R)) in human hair was developed and validated. Human hair (10 mg) was hydrolyzed in 0.50 mL of 5.0 N sodium hydroxide for 1.5 h. The aqueous layer was extracted with 1.5 mL of n-hexane. The organic layer was separated and re-extracted with 0.20 mL of formic acid (12.5%)/2-propanol (85:15, v/v). The aqueous layer was separated and 0.010 mL of the aqueous extract was injected onto a reversed-phase microbore (50 x 1.0 mm i.d.) column for analysis by liquid chromatography/tandem mass spectrometry (LC/MS/MS). The instrument was equipped with an electrospray ionization (ESI) interface and operated in the positive ion mode of detection. Interday and intraday accuracy and precision were assessed from the relative recoveries of spiked samples analyzed on three different days. The method showed excellent specificity and ruggedness with a lower limit of quantitation of 10 ng/g (i.e., 10 ppb) using 10 mg of human hair.     

超高效液相色谱-串联质谱法快速测定鸡蛋中氟虫腈及其代谢物残留

建立了超高效液相色谱-串联质谱（UPLC-MS/MS）快速测定鸡蛋中氟虫腈及其代谢物的方法。在2 g鸡蛋中加入2 mL水后,用4 mL乙腈提取,然后加入1 g NaCl,于4℃以9000 r/min离心10 min,稀释后过有机膜。采用C18色谱柱（100 mm×2.1 mm,1.7 μm）分离,在电喷雾电离源、负离子模式下进行多反应监测（MRM）采集。结果表明,氟虫腈及其代谢物在3个添加水平下的回收率为77.4%~112.1%,相对标准偏差为4.0%~13.6%,检出限为0.10~0.43 μg/kg。该法简单、高效,可用于实际样品检测。     

Capillary electrophoresis-mass spectrometry of basic proteins using a new physically adsorbed polymer coating. Some applications in food analysis

A new physically adsorbed capillary coating for capillary electrophoresis-mass spectrometry (CE-MS) of basic proteins is presented, which is easily obtained by flushing the capillary with a polymer aqueous solution for two min. This coating significantly reduces the electrostatic adsorption of a group of basic proteins (i.e., cytochrome c, lysozyme, and ribonuclease A) onto the capillary wall allowing their analysis by CE-MS. The coating protocol is compatible with electrospray inonization (ESI)-MS via the reproducible separation of the standard basic proteins (%RSD values (n = 5) < 1% for analysis time reproducibility and < 5% for peak heights, measured from the total ion electropherograms (TIEs) within the same day). The LODs determined using cytochrome c with total ion current and extracted ion current defection were 24.5 and 2.9 fmol, respectively. Using this new coating lysozymes from chicken and turkey egg white could be easily distinguished by CE-MS, demonstrating the usefulness of this method to differentiate animal species. Even after sterilization at 120 degrees C for 30 min, lysozyme could be detected, as well as in wines at concentrations much lower than the limit marked by the EC Commission Regulation. Adulteration of minced meat with 5% of egg-white could also be analysed by our CE-MS protocol.     

分散固相萃取净化与液相色谱/串联质谱法测定牛奶中8类禁用药物残留

建立了牛奶中8类禁用药物的液相色谱-串联质谱( LC- MS/MS)检测方法.分析物包括5种硝基咪唑、7种β-受体激动剂、9种雄性激素、7种糖皮质激素、3种雌性激素、2种镇静剂、1种氯霉素以及6种二羟基苯甲酸内酯共40种禁用药物.样品以β葡萄糖苷醛酶/芳基硫酸酯酶在乙酸铵缓冲液中酶解,用氨化和酸化乙腈各提取一次.提取液经改良的分散固相萃取(QuEChERS)净化,浓缩后采用C18色谱柱分离(150 mm×2.1 mm i.d.,3.0 um).以甲醇和水(含0.1％甲酸)、乙腈和水分别作为正、负电喷雾离子化模式的色谱分离流动相进行梯度洗脱,多反应监测模式进行定性和定量分析.7种药物以内标法定量;33种药物以基质标准曲线外标法定量,氯霉素在0.02～0.4μg/kg; 39种药物在0.20～ 10.0 μg/kg范围内相关系数(r)均大于0.99;定量限(S/N=10)在0.07～0.93 μg/kg之间.分别以各个药物0.5,1和2倍MRPL( Minimum required performance limits)浓度水平加标验证实验,回收率范围60.3％～119.3％范围;相对标准偏差小于18.9％.     

Ultra-performance liquid chromatography/tandem mass spectrometry method for the determination of lercanidipine in human plasma

A simple, sensitive and rapid ultra-performance liquid chromatography/positive electrospray ionization tandem mass spectrometry (UPLC/ESI-MS/MS) method has been developed and validated for the determination of lercanidipine in human plasma. Lercanidipine and the internal standard, nicardipine, were extracted from plasma by liquid-liquid extraction using tert-butyl methyl ether as the extraction solvent. UPLC analysis was performed isocratically on an AcQuity UPLC BEH C18 analytical column (2.1 x 50.0 mm i.d., particle size 1.7 microm). The mobile phase consisted of 70% acetonitrile in water containing 0.2% v/v formic acid and pumped at a flow rate of 0.30 mL/min. ESI in positive ion mode, with multiple reaction monitoring (MRM), was chosen for the detection of the analytes. The assay was linear over a concentration range of 0.05-30 ng/mL for lercanidipine with a limit of quantitation of 0.05 ng/mL. Quality control samples (0.05, 0.15, 15 and 25 ng/mL) in five replicates from five of analytical runs demonstrated intra-assay precision (% CV < or =7.3%), inter-assay precision (% CV < or =6.1%) and an overall accuracy (% relative error) of less than 6.2%. A run time of less than 1.0 min for each sample made it possible to analyze a large number of human plasma samples per day. The method can be used to quantify lercanidipine in human plasma covering a variety of pharmacokinetic or bioequivalence studies.     

Electrospray ionization stability and concentration sensitivity in capillary electrophoresis-mass spectrometry using a flow-through microvial interface

Concentration sensitivity is a key performance indicator for analytical techniques including for capillary electrophoresis-mass spectrometry (CE–MS) with electrospray ionization (ESI). In this study, a flow-through microvial interface was used to couple CE with MS and improve the ESI stability and detection sensitivity. By infusing a peptide mixture through the interface into an MS detector at a typical flow rate for CE-MS analysis, the spatial region near the interface was mapped for MS signal intensity. When the sprayer tip was within a 6 × 6.5 × 5 mm region in front of the MS inlet, the ESI was stable with no significant loss of signal intensity for ions with m/z 239. Finite element simulations showed that the average electric field strength at the emitter tip did not change significantly with minor changes in emitter tip location. Experiments were conducted with four different mass spectrometer platforms coupled to CE via the flow-through microvial interface. Key performance indicators, that is, limit of detection (LOD) and linearity of calibration curves were measured for nine amino acids and five peptides. Inter- and intraday reproducibility were also tested. The results were shown to be suitable for quantification when internal standards were used.     

等离子体质谱法测定珊瑚锶和钙

研究了珊瑚中Ｓｒ（～８０００ｕｇ／ｇ）、Ｃａ（～４００ｍｇ／ｇ）在同一份溶液中的等离子体质谱的同时测定。所有样品均加入Ｓｃ和Ｙ作内标元素分别控制测定Ｃａ和Ｓｒ时的仪器波动。本方法Ｓｒ、Ｃａ测量精度（ＲＳＤ％）分别～０．３％、～１％。９０个珊瑚样品Ｓｒ分析结果中,仅有７个样品Ｓｒ数据与同位素稀释热电离质谱法数据相比,相对偏差较大（～１％）,其它数据相互吻合。本方法适合珊瑚样中Ｓｒ的快速测定,并可以此     

液相色谱-串联四极杆飞行时间质谱和超高效液相色谱-串联三重四极杆质谱用于检测牛肉中的刚果红

建立了牛肉中刚果红的检测方法。定性方法采用液相色谱-串联四极杆飞行时间质谱对未知物进行质谱谱图库匹配,定量分析采用超高效液相色谱-串联三重四极杆质谱。牛肉样品中的刚果红经液液萃取净化后,采用Agilent ZORBAX Eclipse Plus C₁₈ Rapid Resolution HD色谱柱(50 mm×2.1 mm, 1.8 μm)进行分离,流动相为95%(体积分数)甲醇,流速为0.2 mL/min。AB 4000⁺三重四极杆质谱仪在电喷雾负离子化(ESI)及MRM模式下定量。结果显示,刚果红在0.03~1 mg/L浓度范围内,线性关系良好(相关系数为0.9998),精密度良好(RSD小于5%),回收率为88%~91%,检出限约为0.01 mg/L。本方法快速简便,重现性好,可以为牛肉及其他肉制品中刚果红的定量提供良好的解决方案。     

微波消解-ICP-MS测定中成药胶囊中15种金属元素

采用硝酸-过氧化氢体系微波消解,以钪、铟作内标,电感耦合等离子体质谱法(ICP-MS)同时测定几种中成药及空心胶囊中的15种金属元素,各元素方法检出限在0.000 7~0.047μg/g之间,相对标准偏差小于10%,标准物质加标回收率在82.3%~116%之间.方法简便快捷,灵敏度高,重现性好,是测定中成药及其空心胶囊中多元素高效、准确的方法.     

Liquid chromatography/tandem triple-quadrupole mass spectrometry for determination of paclitaxel in rat tissues

A liquid chromatography/tandem triple-quadrupole mass spectrometry assay to quantify paclitaxel in rat tissue homogenates containing taxol or paclitaxel nanoliposome (PTX-NLP) was developed and validated. Liquid-liquid extraction with tert-butyl methyl ether was used for tissue sample preparation and docetaxel was used as the internal standard. Paclitaxel and docetaxel were separated on a 200 mm x 4.6 mm x 5 microm C(18) column and quantified using a triple-quadrupole mass spectrometer operating in positive ion electrospray selective reaction monitoring mode (ESI(+)-SRM) with a total run time of 6.0 min. The peak area of the m/z 876.3 --> 307.9 transition of paclitaxel is measured versus that of the m/z 830.3 --> 549.1 transition of docetaxel to generate the standard curves. The standard curves were linear over the concentration range of 0.2008-2008 ng/mL for different tissues. The method had high extraction recovery (>90%) and accuracy (>90%) with the intra-day and inter-day precision <15%. Frozen stability, freeze/thaw stability, extraction stability and solution stability at ambient temperature were examined, which indicated the tissue samples should be extracted within 5 days and avoid being frozen and thawed repeatedly over 5 times. Extracted samples after evaporation could be stored at -20 degrees C for 20 days without drug degradation and no degradation was also observed after solution samples were left to stand at ambient temperature for 24 h. This assay was used to support an in vivo biodistribution study of PTX-NLP in rats.