Rapid quantification of nebivolol in human plasma by liquid chromatography coupled with electrospray ionization tandem mass spectrometry

A simple, sensitive and rapid liquid chromatographic/electrospray ionization tandem mass spectrometric method was developed and validated for the quantitation of nebivolol in human plasma. The method involved a simple single-step liquid–liquid extraction with diethyl ether/dichloromethane (70/30). The analyte was chromatographed on Waters symmetry^® C₁₈ reversed-phase chromatographic column by isocratic elution with water:acetonitrile:formic acid (30:70:0.03, v/v) and analyzed by mass spectrometry in the multiple reaction monitoring mode. The precursor to product ion transitions of m/z 406.4–151.5 and m/z 409.1–228.1 were used to measure the analyte and the internal standard (I.S.), respectively. The chromatographic runtime was 2 min and the weighted (1/x²) calibration curves were linear over the range 50–10,000 pg/mL. The method was validated in terms of accuracy, precision, absolute recovery, freeze-thaw stability, bench-top stability and re-injection reproducibility. The limit of detection and lower limit of quantification in human plasma were 10 and 50 pg/mL, respectively. The within- and between-batch accuracy and precision were found to be well within acceptable limits (<10%). The analyte was stable after three freeze-thaw cycles (deviation <10%). The average absolute recoveries of nebivolol and tamsulosin, used as an internal standard, from spiked plasma samples were 73.4 ± 3.7 and 72.1 ± 2.0%, respectively. The assay method described here was applied to study the pharmacokinetics of nebivolol.     

Simple and accurate quantitative analysis of ten antiepileptic drugs in human plasma by liquid chromatography/tandem mass spectrometry

A simple, accurate, and sensitive liquid chromatography (LC)-tandem mass spectrometry (MS/MS) method has been developed for the simultaneous quantification of 10 antiepileptic drugs (AEDs; gabapentin (GBP), levetiracetam (LEV), valproic acid (VPA), lamotrigine (LTG), carbamazepine-10,11-epoxide (CBZ-epoxide), zonisamide (ZNS), oxcarbazepine (OXC), topiramate (TPM), carbamazepine (CBZ), phenytoin (PHT)) in human plasma as a tool for drug monitoring. d₁₀-Phenytoin (d₁₀-PHT) and d_6-valproic acid (d₆-VPA) were used as internal standards for the positive- and negative-ionization modes, respectively. Plasma samples were precipitated by the addition of acetonitrile, and supernatants were analyzed on a C18 reverse-phase column using an isocratic elution. Detection was carried out in selected reaction monitoring (SRM) mode. The calibration curves were linear over a 50-fold concentration range, with correlation coefficients (r²) greater than 0.997 for all AEDs. The intra- and inter-day precision was less than 12%, and the accuracy was between 85.9 and 114.5%. This method was successfully used in the identification and quantitation of AEDs in patients undergoing mono- or polytherapy for epilepsy.     

Liquid chromatography/tandem mass spectrometry method for determination of olanzapine and N-desmethylolanzapine in human serum and cerebrospinal fluid

A validated, accurate and sensitive LC–MS/MS method for determination of olanzapine and its metabolite N-desmethylolanzapine has been developed. The analytes were quantified by tandem mass spectrometry operating in positive electrospray ionization mode with multiple reaction monitoring. Olanzapine and desmethylolanzapine were extracted from serum or cerebral spinal fluid samples, 200 μl, with tert-butyl methyl ether using olanzapine-D3 as internal standard. Calibrations for olanzapine and desmethylolanzapine were linear within the selected range of 0.2–30 ng/ml (6–96 nM) in cerebral spinal fluid and for olanzapine in plasma, in the range of 5–100 ng/ml (16–320 nM). The method was successfully used for the analysis of samples from patients treated with olanzapine in the dose range of 2.5–25 mg/day.     

LC-ESI-MS/MS法测定人血浆中卢帕他定的浓度（英文）

目的建立测定人血浆中卢帕他定浓度的LC-ESI-MS/MS方法。方法血浆样品加入内标,碱化后用二氯甲烷：乙酸乙酯（20：80）提取,在37℃真空干燥箱中干燥至干,残渣用200μL流动相溶解后进样。色谱条件为：色谱柱为Agilent Eclipse XDB-C18（4.6mm×150mm,5μL）;流动相为乙腈（含1%甲酸）：20mmol·L^-1醋酸铵（76：24,V/V）,流速为0.6mL·min^-1。质谱条件：采用美国安捷仑1100高效液相色谱系统和离子阱（Agilent MSD Trap XCT）检测仪,质谱条件为电喷雾离子源,检测方式为正离子电离、多离子反应监测（MRM）,用于定量分析的离子为卢帕他定m/z416→309,内标氯雷他定m/z383→337。结果该方法应用于检测20名健康志愿者服药后的血浆样品。线性范围为0.05～14ng·mL^-1（r=0.998）,日内和日间精密度均低于15%,方法回收率为85.1%～114.0%。最低检测限为0.05ng·mL^-1（当n=5时,RSD=9.22%）。结论该方法灵敏、准确、快速,可用于该药药代动力学和生物等效性研究。     

亲水作用色谱串联质谱法测定人血浆中的拉米夫定

目的:建立简便、快速的亲水作用色谱串联质谱法(HILIC-MS/MS)测定人血浆中的拉米夫定。方法:以13C1,15N2(拉米夫定为内标,血浆样品经乙腈沉淀蛋白后,采用Luna HILIC(100 mm×3.0 mm,3μm)柱分离。流动相为乙腈-5 mmol·L-1醋酸铵-甲酸(95∶5∶0.01,v/v/v),进样体积2μL,样品分析时间3 min。采用ESI源正离子模式、多反应监测(MRM),用于定量分析的离子反应分别为m/z230(112(拉米夫定)和m/z233(115(内标)。结果:测定人血浆中拉米夫定的线性范围为8.0～2000 ng·mL-1,定量下限为8.0 ng·mL-1,日内、日间精密度(RSD)均小于9.0%,准确度(RE)在-7.1%~2.7%之间。本法成功应用于健康受试者口服2种拉米夫定片后的生物等效性研究。结论:采用稳定同位素内标的HILIC-MS/MS法更为简便、快捷和准确,适用于人血浆样品中拉米夫定的测定。     

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.     

Quantification of artemisinin in human plasma using liquid chromatography coupled to tandem mass spectrometry

A liquid chromatographic tandem mass spectroscopy method for the quantification of artemisinin in human heparinised plasma has been developed and validated. The method uses Oasis HLB™ μ-elution solid phase extraction 96-well plates to facilitate a high throughput of 192 samples a day. Artesunate (internal standard) in a plasma–water solution was added to plasma (50 μL) before solid phase extraction. Artemisinin and its internal standard artesunate were analysed by liquid chromatography and MS/MS detection on a Hypersil Gold C18 (100 mm × 2.1 mm, 5 μm) column using a mobile phase containing acetonitrile–ammonium acetate 10 mM pH 3.5 (50:50, v/v) at a flow rate of 0.5 mL/min. The method has been validated according to published FDA guidelines and showed excellent performance. The within-day, between-day and total precisions expressed as R.S.D., were lower than 8% at all tested quality control levels including the upper and lower limit of quantification. The limit of detection was 0.257 ng/mL for artemisinin and the calibration range was 1.03–762 ng/mL using 50 μL plasma. The method was free from matrix effects as demonstrated both graphically and quantitatively.     

Liquid chromatography electrospray ionization tandem mass spectrometry（LC/ESI-MS/MS） method for quantitative estimation of moxifloxacin in human plasma

A rapid and sensitive liquid chromatography tandem mass spectrometry（LC-MS/MS） method has been developed and validated for the determination of moxifloxacin（MOXI） in human plasma. After a simple protein precipitation using acetonitrile, the post treatment samples were analysed on a C18 column interfaced with a Triple Quadropole Tandem Mass Spectrometer. Positive electrospray ionization was employed as the ionization source. The mobile phase consisted of 0.1% formic acid–acetonitrile（60：40, v/v）. Ciprofloxacin（CIPRO） was used as an internal standard. The analyte and internal standard（CIPRO） were monitored in the multiple reaction monitoring mode（MRM）. The mass transition ion-pair has been followed as m/z 402→358.2 for MOXI and 332→288.1 for CIPRO. The method was linear in the concentration range of 25–5000 ng/mL. The lower limit of quantification was 25 ng/mL. The intra- and inter-day precision（relative standard deviation） and accuracy（relative error） values were within 12.4%. Each plasma sample was analyzed within 3 min.     

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 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.     

超高效液相色谱串联质谱法快速测定尼扎替丁在人体血浆中的含量

目的: 建立超高效液相色谱串联质谱法测定人体血浆中尼扎替丁的含量。方法: 血浆以甲醇直接沉淀蛋白, 上清液用于目标物的检测;色谱柱为Waters ACQUITY UPLC BEH C₁₈;流动相为5 mmol·L^-1醋酸铵水溶液-甲醇 (65:35, V/V);流速:0.20 mL·min^-1;进样量:3 μL;柱温:35 ℃;样品室温度:6 ℃;采用电喷雾正离子模式电离, 尼扎替丁和内标雷尼替丁的监测离子分别为(m/z)332.2→155.0, (m/z)315.2→130.0。结果: 尼扎替丁的线性范围为10~3 000 ng·mL^-1, 平均回收率为93.2%~101.1%, 批内、批间RSD均小于15%。结论: 本研究建立的分析方法快速、准确、灵敏、简便, 适用于尼扎替丁人体药动学方面的研究。     

Quantitative determination of rosuvastatin in human plasma by ion pair liquid-liquid extraction using liquid chromatography with electrospray ionization tandem mass spectrometry

A simple and sensitive liquid chromatography/tandem mass spectrometry method was developed and validated for the quantification of rosuvastatin in human plasma. After being treated with acetic acid and tetrabutyl ammonium hydroxide, the analyte was extracted by simple one-step liquid-liquid extraction with the internal standard (IS: estrone). The chromatographic separation was performed on a Phenomenex Luna C18 column with a mobile phase consisting of 2% formic acid/methanol (20:90, v/v) at a flow rate of 1.00 mL/min with a split of 200 microL to mass spectrometer. The retention time of rosuvastatin and internal standard was 2.3 and 3.4 min, respectively. Triple-quadrupole MS/MS detection was operated in positive mode by monitoring the transition of m/z 482-->258 for rosuvastatin and m/z 271-->253 for IS. Validation results indicated that the lower limit of quantification (LLOQ) was 0.1 ng mL(-1) and the assay exhibited a linear range of 0.1-20 ng mL(-1) and gave a correlation coefficient (r) of 0.9990 or better. Inaccuracy was less than 8.4% and imprecision less than 12.8% at all tested concentration levels. The analyte was stable in human plasma following three freeze/thaw cycles and for up to 8 weeks following storage at -20 degrees C. The assay was successfully applied to the analysis of rosuvastatin in human plasma samples derived from clinical pre-trials.     

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。     

Development and validation of a simple and sensitive liquid chromatography–tandem mass spectrometry method for quantifying trimetazidine in human plasma

1. A simple and sensitive liquid chromatography–tandem mass spectrometry (LC‐MS‐MS) method for quantifying trimetazidine in human plasma was developed and validated. Sample preparation was based on deproteinating with acetonitrile. 2. Chromatography was performed on a C18 analytical column (5 μm; 150 × 2.1 mm i.d.) and the retention times for trimetazidine and cetirizine (used as the internal standard) were 1.8 and 3.0 min, respectively. The ionization was optimized using an electrospray ionization source and enhanced selectivity was achieved using tandem mass spectrometry. The calibration curve ranged from 0.1 to 200 ng/mL. The inter‐day precision, accuracy and the relative standard deviation (RSD) were all < 15%. The analyte was shown to be stable over the time‐scale of the entire procedure. 3. The robustness of the method was demonstrated by the good reproducibility of the results obtained during the analysis of clinical samples.     

Bioanalytical method for the quantification of sunitinib and its n-desethyl metabolite SU12662 in human plasma by ultra performance liquid chromatography/tandem triple-quadrupole mass spectrometry

A rapid and sensitive ultra performance liquid chromatography/tandem mass spectrometry (UPLC–MS/MS) method has been developed and validated for the quantitative determination of sunitinib and its n-desethyl metabolite SU12662, in 100 μl aliquots of human potassium EDTA plasma with deuterated sunitinib as internal standard. As sunitinib was found to be extremely sensitive to light causing rapid conversion of the Z (cis)-isomer to the E (trans)-isomer, the sample extraction and cleaning-up were performed under sodium-light and in amber vials. The extraction involved a simple liquid–liquid extraction with tert-butyl methyl ether. Chromatographic separations were achieved on an Aquity UPLC^® BEH C₁₈ 1.7 μm, 2.1 mm × 50 mm column eluted at a flow rate of 0.250 ml/min on a gradient of acetonitrile. The overall cycle time of the method was 4 min, with elution times of 1.05, 1.43, 0.95, and 1.34 min, for the E (trans)- and Z (cis)-isomers of sunitinib and the E (trans)- and Z (cis)-isomers of SU12662, respectively. The multiple reaction monitoring transitions were set at 399 > 326 (m/z), at 371 > 283 (m/z) and at 409 > 326 (m/z) for sunitinib, SU12662 and the internal standard, respectively. The calibration curves were linear over the range of 0.200 to 50.0 ng/ml with the lower limit of quantitation validated at 0.200 ng/ml for both sunitinib and SU12662. The within-run and between-run precisions were within 11.7%, while the accuracy ranged from 90.5 to 106.8%.     

Development and full validation of a sensitive quantitative assay for the determination of clemastine in human plasma by liquid chromatography-tandem mass spectrometry

A sensitive high-performance liquid chromatography-tandem mass spectrometry (LC/MS/MS) method was developed and validated for the determination of clemastine in human plasma. After having been extracted from plasma samples by ethyl acetate, clemastine and internal standard, diphenhydramine, were separated on a C(18) column. Detection was performed on Thermo Finnigan TSQ Quantum triple quadrupole mass spectrometer by selected reaction monitoring (SRM) mode via electrospray ionization (ESI) source. The method was linear in the concentration range of 5.0-1000.0 pg/ml for clemastine. The intra- and inter-day precisions were within 13.4% and the deviations were between -1.1% and 5.6%. The fully validated LC/ESI-MS/MS method has been successfully applied to the preliminary pharmacokinetic study in healthy male Chinese volunteers.     

Liquid chromatography/tandem mass spectrometry utilizing ion-molecule reactions and collision-activated dissociation for the identification of N-oxide drug metabolites

A liquid chromatography/tandem mass spectrometry (LC/MS³) method based on ion-molecule reactions and collision-activated dissociation (CAD) is presented for the identification of analytes with the N-oxide functional group directly in mixtures. Tri(dimethylamino)borane (TDMAB) rapidly and selectively derivatizes protonated N-oxides in a modified commercial linear quadrupole ion trap (LQIT) mass spectrometer to yield a distinct product ion (adduct—(CH₃)₂NH). The LQIT was outfitted with an external reagent-mixing manifold that allows TDMAB to be mixed with the helium buffer gas used in the trap. The derivatized analytes are readily identified on the basis of a shift of 98 Th (Thomson) relative to the m/z value of the protonated analyte. Further probing of the derivatized analytes via isolation followed by CAD can be used to confirm the presence of an N-oxide, and distinguish between aliphatic and aromatic tertiary N-oxides. Since the ion-molecule reaction is fast, these experiments can be accomplished on the same time scale as typical CAD-based MSⁿ experiments, thus maintaining the duty cycle of the instrument for this type of experiment. To demonstrate real world applicability, the method was tested on real active pharmaceutical ingredients and their derivatives.     

液相色谱-大气压化学电离-串联质谱法同时测定人血浆中氯苯那敏和咖啡因的含量

目的建立同时测定人血浆中氯苯那敏和咖啡因含量的液相色谱-大气压化学电离-串联质谱(LC-APCI-MS-MS)法。方法血浆样品经乙酸乙酯提取后,采用Diamonsil C18柱分离,流动相为甲醇-水-甲酸(体积比80∶20∶0.5),采用大气压化学电离(APCI)源,以选择反应监测(SRM)方式进行检测,用于定量分析的离子反应分别为m/z275→m/z230(氯苯那敏)、m/z195→m/z138(咖啡因)、m/z256→m/z167(内标,苯海拉明)。结果血浆中氯苯那敏和咖啡因的线性范围分别为0.2~40.0μg.L-1和20.0~4 000μg.L-1;日内和日间精密度均小于13.4%,相对误差在±8.2%以内。结论该法适用于人血浆中氯苯那敏和咖啡因的同时测定。     

Simultaneous determination of propranolol and 4-hydroxy propranolol in human plasma by solid phase extraction and liquid chromatography/electrospray tandem mass spectrometry

A very sensitive, reliable, reproducible and highly selective assay for the simultaneous determination of free and total (conjugated and unconjugated) propranolol and its equipotent hydroxyl metabolite, 4-hydroxy propranolol, in human plasma was developed and validated. The analytes were simultaneously extracted from 0.300 mL of human plasma using solid phase extraction and detected in positive ion mode by tandem mass spectrometry with a turbo ionspray interface. Deuterium-labeled propranolol and 4-hydroxy propranolol, propranolol-d7 and 4-hydroxy propranolol-d7, were used as internal standards. The method has a lower limit of quantitation (LOQ) of 0.20 ng/mL for both analytes with the limits of detection (LOD) 50 and 100 pg/mL for propranolol and 4-hydroxy propranolol, respectively, based on a signal-to-noise ratio of 5. The assay was linear over a range 0.20–135.00 ng/mL for free propranolol and 0.20–25.00 ng/mL for free 4-hydroxy propranolol and linear over range 1.00–500.00 ng/mL for total propranolol and 1.00–360.00 ng/mL for total 4-hydroxy propranolol, with coefficient of determination greater than 0.99 for both analytes. The extraction recoveries were >96 and >64% on an average for propranolol and 4-hydroxy propranolol, respectively. The analytes were found stable in human plasma through five freeze (−15 °C)–thaw (room temperature) cycles and under storage on bench-top for at least 6.5 h, and also in mobile phase at 10 °C for at least 48 h. The method has shown tremendous reproducibility, with intra- and inter-day precision <11.3% (RSD), and intra- and inter-day accuracy <11% of nominal values, for both analytes, and has proved to be highly reliable for the analysis of clinical samples.