A high-throughput LC–MS/MS method for the quantitation of posaconazole in human plasma: Implementing fused core silica liquid chromatography

A rapid and robust liquid chromatographic tandem mass spectrometric (LC–MS/MS) method for the determination of posaconazole concentrations in human plasma was validated. Posaconazole was extracted from human plasma using mixed-mode cation exchange solid phase extraction in a 96-well plate format followed by gradient separation on a fused-core Halo C18 column. The analyte and its corresponding internal standard were detected using a Sciex API 4000 triple quadrupole LC–MS/MS system equipped with a TurboIonSpray™ ionization source operated in the positive ion mode. The calibration range of the method was 5.00–5000 ng/mL using a 50 μL aliquot of plasma. The assay inter-run accuracy and precision were—4.6–2.8% and 2.3–8.7%, respectively (n = 18). The results from method validation indicate the method to be sensitive, selective, accurate, and reproducible. The method was successfully applied to the routine analysis of clinical samples with the fused-core silica columns providing excellent reproducibility for greater than 1000 injections per column.     

液相色谱-串联质谱法测定人血浆中瑞舒伐他汀钙浓度及临床药代动力学研究

目的：建立测定人血浆中瑞舒伐他汀钙的液相色谱．串联质谱法，考察瑞舒伐他汀钙在中国健康志愿者体内的药代动力学。方法：含有瑞舒伐他汀钙和氟伐他汀钠的血浆样品经液-液提取后，进行色谱分析，在三重四极杆串联质谱仪上，以多反应离子监测方式进行定量分析。结果：瑞舒伐他汀钙的定量下限为0．03ng／mL，线形范围为0．03～60ng／mL，精密度与准确度符合生物样品分析要求。结论：该方法操作简便、快速、灵敏度高，适合瑞舒伐他汀钙的临床药代动力学研究。     

Simultaneous determination of dextromethorphan and dextrophan in rat plasma by LC-MS/MS and its application to a pharmacokinetic study

A highly selective and sensitive liquid chromatography-tandem mass spectrometric (LC-MS/MS) method was developed and validated for quantitating dextromethorphan (DXM) and its metabolite dextrophan (DXO) in rat plasma using pirfenidone as an internal standard. Protein precipitation with acetonitrile was employed for the sample preparation. Chromatographic separation was achieved on a SB-C18 column at 25 degrees C, with a gradient elution programme of which acetonitrile-0.1% formic acid in water as mobile phase. The flow rate was 0.4 mL/min. Detection is carried out by multiple reaction monitoring (MRM) on a ion-trap LC-MS/MS system with an electrospray ionization interface. The assay is linear over the range 1-500 ng/mL for DXM and 1-250 ng/mL for DXO, with a lower limit of quantitation of 1 ng/mL for both. Intra- and inter-day precision of the assay were less than 9.80% and the accuracy were in the range 96.35-106.39%. The developed method was successfully applied to analyze the drug in samples of rat plasma for pharmacokinetic study.     

LC-MS/MS 法测定人血浆中倍他米松

建立测定人血浆中倍他米松的LC-MS/MS方法。采用Venusil XBP C₈ (200 mm×3.9 mm ID, 5 μm)色谱柱，流动相为甲醇-水(含甲酸铵5 mmol·L^-1)(80∶20)，流速0.4 mL·min^-1；质谱仪离子源为电喷雾离子源(ESI)，正离子模式检测，监测离子为393.3→355.2(倍他米松)和361.3→343.2(泼尼松龙,内标)。血浆样本用乙酸乙酯处理。倍他米松在0.5~80.0 ng·mL^-1线性关系良好(r=0.999 2)， 血浆低、 中、 高3种浓度(1.0， 10.0， 60.0 ng·mL^-1)平均提取回收率为88.24%，定量限为0.5 ng·mL^-1。本方法操作简便、准确、灵敏，适用于复方倍他米松注射液人体药代动力学研究。     

LC-MS/MS测定人血浆中哌甲酯

目的建立LC-MS/MS测定人血浆中盐酸哌甲酯的浓度。方法待测血浆1.0 mL,经1 mol.L-1的氢氧化钠40μL碱化后,用4 mL乙醚萃取处理,采用Eclipse XDB-C18（4.6 mm×150 mm,5μm）色谱柱,以甲醇和水为流动相梯度洗脱,流速1.0 mL·min^-1。以电喷雾正离子源离子化,检测离子对盐酸哌甲酯为234.0/84.0,内标卡马西平为237.0/194.0。结果血浆中内源性物质对测定无干扰,最低定量限为0.1 ng·mL^-1,在1-100 ng·mL^-1内盐酸哌甲酯线性关系良好（r=0.999 1）,日内、日间RSD均小于8%,样品分析时间为10 min。结论该法专属性强、灵敏度高,操作简便快速,测定结果可靠,适于进行盐酸哌甲酯血药浓度监测。     

Determination of arbidol in human plasma by LC-ESI-MS

A sensitive, specific and accurate method for determination of arbidol in human plasma was developed. Arbidol and internal standard were extracted from plasma samples by liquid-liquid extraction with diethyl ether. The chromatographic separation was accomplished on a Shiseido C18 3 microm analytical column (100 mm x 2.0 mm i.d.) at a flow rate of 0.3 mL/min isocratically. Detection was performed on a single quadrupole mass spectrometer by selected ion monitoring (SIM) mode via electrospray ionization (ESI) source. The method had a chromatographic run time of 6 min and a good linear relationship over the range 1-1000 ng/mL. The limit of quantitation for arbidol in plasma was 1 ng/mL. The intra-day and inter-day precision (R.S.D.%) was lower than 7% and accuracy ranged from 95 to 105%. The proposed method enables unambiguous identification and quantification of arbidol in vivo and has been successfully applied to study the pharmacokinetics of arbidol in healthy male Chinese volunteers.     

Rapid and simultaneous measurement of midazolam, 1'-hydroxymidazolam and digoxin by liquid chromatography/tandem mass spectrometry: application to an in vivo study to simultaneously measure P-glycoprotein and cytochrome P450 3A activity

In order to simultaneously determine in vivo P-glycoprotein (P-gp) and Cytochrome P450 3A (CYP3A) activity, a new, rapid and sensitive liquid chromatography/tandem mass spectrometry (LC-MS/MS) method has been developed and fully validated to simultaneously determine midazolam (MDZ, as CYP3A substrate), 1'-hydroxymidazolam (1'-OHMDZ) and digoxin (DG, as P-gp substrate) in rat plasma using digitoxin as the internal standard (IS). After a single step liquid-liquid extraction with tert-butyl methyl ether/dichloromethane (75:25, v/v), analytes were subjected to LC-MS/MS analysis using positive electro-spray ionization (ESI(+)) under selected reaction monitoring mode (SRM). Chromatographic separation was performed on an XTerra MS C18 column (50mm×2.1mm, i.d. 3.5μm). The MS/MS detection was conducted by monitoring the fragmentation of 326.05 → 244.00 (m/z) for MDZ, 342.02 →168.01 (m/z) for 1'-OHMDZ, 798.33 → 651.36(m/z) for DG and 782.67 → 635.24 (m/z) for IS. The method had a chromatographic running time of 3min and linear calibration curves over the concentrations of 2-400ng/mL for MDZ and 1'-OHMDZ and 0.5-100ng/mL for DG. The recoveries of the method were 86.8-96.3% for MDZ, 84.6-86.4% for 1'-OH MDZ, and 81.7-85.1% for DG. The lower limit of quantification (LLOQ) of the method was 2ng/mL for MDZ and 1'-OHMDZ and 0.5ng/mL for DG. The intra- and inter-batch precision were less than 15% for all quality control samples at concentrations of 5, 50 and 320ng/mL for MDZ and 1'-OHMDZ and 1, 10 and 80ng/mL for DG. The validated LC-MS/MS method has been successfully used to analyze the concentrations of MDZ, 1'-OH MDZ and DG in rat plasma for simultaneous measurement of in vivo P-gp and CYP 3A activity.     

LC-MS／MS法测定人血浆中利培酮及其代谢物9-羟基利培酮的浓度

目的建立LC-MS／MS法测定人血浆中利培酮及其代谢物9-羟基利培酮浓度的方法。方法血浆经特丁基甲醚提取,采用同位素内标（氘4-利培酮、氘4-9-羟基利培酮）进行测定。色谱柱：CAPCELL PACK C18Ⅲ（100mm×2．0mm,5μm）,流动相为乙腈-5mmol·L^-1醋酸铵氨水缓冲液（pH=8）（50：50,11／V）,等度洗脱;流速0．4mL·min^-1;进样体积lO皿;电喷雾离子化,正离子MRM扫描。结果利培酮及9-羟基利培酮线性范围均为0．1—50μg·L^-1（r〉0．9999）,定量下限均为0．1μg·L^-1,平均提取回收率均〉80％,批内、批间精密度RSD均〈10％。结论本方法灵敏度高、专一性好、操作简单,适用于利培酮的药动学研究。     

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。     

Simultaneous determination of GFA and its active metabolites in human plasma by liquid chromatography electrospray ionization mass spectrometry and its application to pharmacokinetic studies

A sensitive and rapid liquid chromatography electrospray ionization mass spectrometry (LC-ESI-MS) method has been developed and validated for simultaneous quantification of guanfu base A (GFA) and its metabolites guanfu base I (GFI) and guanfu alcohol-amine (AA) in human plasma with phenoprolamine hydrochloride (DDPH) as the internal standard. The analytes were extracted from human plasma by using liquid-liquid extraction with ethyl acetate and the LC separation was performed on a Diamonsil C(18) analytical column (150 mm x 2.1 mm i.d., 5 microm). The MS acquisition was performed in selected ion monitoring (SIM) mode of positive ions. Analysis was carried out in SIM mode at m/z 430.25 for GFA [M+H](+), m/z 388.25 for GFI [M+H](+), m/z 346.25 for AA [M+H](+) and m/z 344.20 for the IS DDPH [M+H](+). The calibration curves were linear over the range of 50-5000 ng/mL for GFA and 5-1000 ng/mL for GFI and AA, with coefficients of correlation above 0.999. The lower limit of quantification for GFA was 1 ng/mL, while for GFI and AA were both 5 ng/mL. The intra- and inter-day precisions (CV) of analysis were within 9%, and the accuracy ranged from 91% to 108%. The overall recoveries for GFA, GFI and AA were about 94.2%, 87.8% and 80.6%, respectively. The total LC-MS run-time was only 5.5 min. This quantitation method was successfully applied to the simultaneous determination of GFA and its metabolites in human plasma for the metabolic study and pharmacokinetic evaluation.     

LC-MS/MS测定大鼠血浆中乌头碱的浓度

目的:建立LC-MS/MS法检测大鼠血浆中乌头碱的浓度。方法:以维拉帕米作内标,血浆样品经MTBE液液萃取后,采用HyPURITYCyano(150mm×2.1mm,5μm)柱分离,流动相为乙腈:10mmol/L甲酸铵=(70∶30,V/V),流速为0.30mL/min。然后采用电喷雾离子源(ESI源)正离子多反应监测(MRM)扫描分析,乌头碱和维拉帕米的离子选择通道分别为:m/z646.4→586.4和455.2→164.9。结果:乌头碱的线性范围为9.3～2390pg/mL,最低检测浓度为9.3pg/mL,日内和日间变异均<15%。结论:本方法灵敏度高,适用于乌头碱在鼠内的药物代谢动力学研究。     

LC-MS-MS法测定血浆中多西他赛和紫杉醇浓度及在乳腺癌患者中的应用

目的：建立一种快速、灵敏的液相色谱－串联质谱（LC-MS/MS）法检测乳腺癌患者血浆中多西他赛、紫杉醇的浓度。方法：多西他赛和紫杉醇互为内标,血浆样品100μL加入1 mL叔丁基甲醚萃取,分离有机相,以氮气吹干后流动相复溶进样。色谱柱为Agilent Eclipse XDB-C18（2.1 mm×100 mm,3.5μm）,流动相为0.4%甲酸水溶液-0.4%甲酸乙腈溶液（20∶80,v/v）,流速0.3 mL·min-1,柱温为40℃。采用多反应监测（MRM）进行定量,电喷雾电离源（ESI）正离子方式进行检测,多西他赛与紫杉醇用于定量分析的检测离子对分别为m/z 808.5→m/z 527.2和m/z 854.3→m/z 569.4。结果：多西他赛和紫杉醇的线性范围分别为5.0~1000 ng·mL-1和1.0～500 ng· mL-1,最低检测浓度分别为5.0 ng·mL-1和1.0 ng·mL-1。两药低、中、高三个浓度的批内和批间RSD均<15%,平均提取回收率分别为65.9%~84.3%和90.4%~106.5%。结论：本法快速、准确、灵敏、专属性强,适用于同步测定多西他赛和紫杉醇血药浓度及其在中国乳腺癌患者中的药动学研究。     

Determination of a novel thrombin receptor antagonist (SCH 530348) in human plasma: evaluation of Ultra Performance Liquid Chromatography™-tandem mass spectrometry for routine bioanalytical analysis

SCH 530348 is a safe and effective oral anti-platelet agent for patients with acute coronary syndrome. Clinical study results suggest that SCH 530348 dosage at 20 mg or 40 mg is feasible to achieve rapid maximum platelet inhibition following an acute coronary event or intervention procedure. To permit accurate determinations of circulating SCH 530348 in plasma following dosing, a method for measuring SCH 530348 concentrations in human plasma was validated using liquid chromatography–tandem mass spectrometry (LC–MS/MS). The method utilized semi-automated 96-well protein precipitation with gradient chromatography using an ACQUITY™ UPLC BEH C₁₈ (2.1 mm × 50 mm, 1.7 μm) column. The retention time of SCH 530348 was approximately 1.5 min. This method was validated for routine quantitation of SCH 530348 over the concentration range of 1.00–1000 ng/mL. Inter-run accuracy based on mean percent theoretical for replicate quality control samples was better than 95.2%. Inter-run precision based on percent relative deviation for replicate quality control samples was ≤3.3%. SCH 530348 quality control samples were stable in human plasma for up to three freeze/thaw cycles, for at least 467 days when frozen at −20 °C and for at least 7 h when stored at room temperature. The lower limit of quantitation was 1.00 ng/mL for a 100 μL plasma aliquot.     

Liquid chromatography tandem mass spectrometry method for simultaneous determination of antidiabetic drugs metformin and glyburide in human plasma

A simple and rapid liquid chromatography/tandem mass spectrometry (LC-MS/MS) method has been developed and validated for the simultaneous quantitation of antidiabetic drugs metformin and glyburide in human plasma using glimepiride as internal standard (IS). After acidic acetonitrile-induced protein precipitation of the plasma samples, metformin, glyburide and IS were chromatographed on reverse phase C18 (50 mm x 4.6 mm i.d., 5 microm) analytical column. Quantitation was performed on a triple quadrupole mass spectrometer employing electrospray ionization technique and operating in multiple reaction monitoring (MRM) and positive ion mode. The total chromatographic run time was 3.5 min and calibration curves were linear over the concentration range of 20-2500 ng/ml for metformin and 5-500 ng/ml for glyburide. The method was validated for selectivity, sensitivity, recovery, linearity, accuracy and precision, dilution integrity and stability studies. The recoveries obtained for the analytes and IS (>or=69%) were consistent and reproducible. Inter-batch and intra-batch coefficient of variation across four validation runs (LLOQ, LQC, MQC and HQC) was less than 8%. The accuracy determined at these levels was within +/-8% in terms of relative error (RE). The method was applied to a bioequivalence study of 500 mg metformin and 5mg of glyburide tablet after oral administration to 28 healthy human subjects under condition of fasting.     

Simultaneous determination of lansoprazole and its metabolites 5'-hydroxy lansoprazole and lansoprazole sulphone in human plasma by LC-MS/MS: application to a pharmacokinetic study in healthy volunteers

A highly sensitive and specific liquid chromatography coupled with tandem mass spectrometric (LC-MS/MS) method has been developed and validated for the simultaneous determination of lansoprazole and its metabolites 5'-hydroxy lansoprazole and lansoprazole sulphone. The detection was operated with multiple reaction-monitoring (MRM) using the electrospray ionization technique. The assay procedure involved precipitation of plasma samples with acetonitrile after indapamide was added as internal standard (IS). The chromatographic separation was achieved with a mixture of methanol-0.2% ammonium acetate and 0.1% methanoic acid in water (75:25, v/v) as mobile phase on an Inertsil ODS-3 column. The method was proved to be accurate and precise with linearity ranges of 10-4,000 ng/ml, 5.0-400 ng/ml, and 1.0-400 ng/ml for lansoprazole, 5'-hydroxy lansoprazole and lansoprazole sulphone, respectively, with the correlation coefficients (r) better than 0.999. The lower limits of quantification (LLOQ) were 2.0 ng/ml, 2.0 ng/ml, and 0.5 ng/ml for lansoprazole, 5'-hydroxy lansoprazole and lansoprazole sulphone, respectively. The intra- and inter-day precision and accuracy values were found to be within the assay variability limits (R.S.D.% within +/-15) in accordance with FDA guidelines. The validated LC-MS/MS method has been successfully applied for the determination of lansoprazole and its metabolites in human plasma.     

Sensitive determination of a pharmaceutical compound and its metabolites in human plasma by ultra-high performance liquid chromatography-tandem mass spectrometry with on-line solid-phase extraction

This paper describes the determination of a drug candidate and two metabolites in human plasma by column-switching LC-MS/MS after protein precipitation. Starting from a standard method with a quantitation limit of 0.5 ng/mL, a highly sensitive assay was developed, employing UHPLC separation and detection on an API 5000 mass spectrometer. The injected plasma equivalent was increased from 6 to 20 μL; conventional column trapping for compound enrichment and removal of matrix constituents was combined with high-pressure analytical separation using small particle columns to improve resolution and signal-to-noise ratio. Quantitation limits were thus lowered to between 5 and 20 pg/mL, offering the possibility to provide bioanalytical support for microdosing studies in humans. Excellent assay quality and robustness were achieved by both methods.     

液相色谱-串联质谱法同时测定人血浆中二甲双胍和格列吡嗪

建立了快速、灵敏的液相色谱-串联质谱法测定人血浆中的二甲双胍和格列吡嗪。血浆样品经0.3%甲酸-乙腈(v/v)沉淀蛋白后，以乙腈-水-甲酸(70∶30∶0.3，v/v/v)为流动相，流速为0.50 mL·min^-1。Zorbax Extend C₁₈柱分离，采用大气压化学电离源；以选择反应监测(SRM)方式进行正离子检测。用于定量分析的离子反应分别为m/z 130→m/z 60(二甲双胍)，m/z 446→m/z 321(格列吡嗪)和m/z 256→m/z 167(内标，苯海拉明)。测定血浆中二甲双胍的线性范围为2.00～2 000 ng·mL^-1， 定量下限为2.00 ng·mL^-1； 格列吡嗪的线性范围为1.00～1 000 ng·mL^-1， 定量下限为1.00 ng·mL^-1。该方法专属性好，灵敏度高，准确快捷，适用于二甲双胍和格列吡嗪的临床药代动力学研究。     

液相色谱-串联质谱法测定人血浆中卡维地洛的浓度

目的建立测定血浆中卡维地洛的液相色谱-串联质谱(LC-MS/MS)方法。方法血浆样品中加入内标氘3-卡维地洛,直接沉淀法处理样品。色谱柱为CAPCELL PACK C_(18)Ⅲ(100.0mm×2.0mm,5μm),流动相为含0.1%甲酸的水溶液-含0.1%甲酸的乙腈溶液(70:30,V/V),流速0.5mL·min~(-1),等度洗脱,进样体积8μL,正离子多离子反应监测(MRM)扫描分析,离子通道分别为m/z 407.3→100.1(卡维地洛),m/z 410.3→100.1(氘3-卡维地洛)。结果卡维地洛线性范围为0.2～200μg·L~(-1)(r>0.999),定量下限为0.2μg·L~(-1),提取回收率在92.27%～104.0%,日内、日间精密度RSD均小于8%。结论本方法操作简便,特异性强,灵敏度高,可适用于卡维地洛的药动学研究。     

Liquid chromatography-tandem mass spectrometry assay for the quantitation of plagiochin E and its main metabolite plagiochin E glucuronides in rat plasma

Plagiochin E, a macrocyclic bisbibenzyl isolated from liverwort Marchantia polymorpha, was found to have antifungal activity. To evaluate the pharmacokinetics of plagiochin E in rats, a sensitive and specific liquid chromatography/tandem mass spectrometric (LC-MS/MS) method was developed and validated for the quantitation of plagiochin E and its total conjugated metabolites in rat plasma. For detection, a Sciex API 4000 LC-MS/MS with a TurboIonSpray ionization (ESI) inlet in the negative ion-multiple reaction monitoring (MRM) mode was used. The plasma samples were pretreated by a simple liquid-liquid extraction with ethyl acetate. The concentration of plagiochin E parent form was determined directly and the concentration of plagiochin E conjugated metabolites was assayed in the form of plagiochin E after treatment with beta-glucuronidase/sulfatase. The statistical evaluation for this method reveals excellent linearity, accuracy and precision for the range of concentrations 0.5-1000.0 ng/mL. The method had a lower limit of quantification (LLOQ) of 0.5 ng/mL for plagiochin E in 50 microL of plasma. The method was successfully applied to the characterization of the pharmacokinetic profile of plagiochin E in rats after an oral and an intravenous administration.     

液相质谱串联法同时测定舒尼替尼及其活性代谢产物SU12662在BABL/c裸鼠血浆中的浓度及其药物动力学研究(英文)

本研究建立并验证了一种灵敏、快速、简单的液质联用方法,用于同时测定BABL/c裸鼠血浆中舒尼替尼及其活性代谢产物SU12662的药物浓度。血浆样品采用蛋白沉淀方法处理,并使用帕唑帕尼作为内标。采用C18反相柱进行分离,流动相为10 mM甲酸胺–乙腈(65:35,v/v,pH 3.25),流速0.5 m L/min。所有化合物均采用电喷雾电离源,正离子方式检测。舒尼替尼及SU12662的最低定量下限均为0.5 ng/m L,线性范围均为0.5–1000 ng/m L(r>0.99)。该方法对舒尼替尼及SU12662的测定均具有良好准确度以及可靠的日内、日间精密度,方法稳定性良好,无明显基质效应。此方法成功用于BABL/c裸鼠口服20 mg/kg舒尼替尼的药物代谢动力学研究。