Simple, sensitive and rapid LC-MS/MS method for the quantitation of cerivastatin in human plasma--application to pharmacokinetic studies

A simple and sensitive liquid chromatography-tandem mass spectrometry method was developed and validated for estimation of cerivastatin (I) in human plasma, a potent hydroxy-methylglutaryl-coenzyme A reductase inhibitor. The analyte and internal standard (atorvastatin, II) were extracted by liquid/liquid extraction with diethyl ether/dichloromethane (70/30, v/v). The chromatographic separation was performed on reverse phase Xterra ODS column with a mobile phase of water/acetonitrile (30/70, v/v) with 0.03% formic acid. The protonated analyte was quantitated in positive ionization by multiple reaction monitoring with a mass spectrometer. The mass transitions m/z 460.4 --> 356.3 and 559.2 --> 440.3 were used to measure I and II, respectively. The lower limit of quantitation was 10pg/mL with a relative standard deviation of less than 15%. Acceptable precision and accuracy were obtained for concentrations over the calibration curve ranges (0.01-10ng/mL). Sample analysis time of 2min for each sample made it possible to analyze a throughput of more than 400 human plasma samples per day. The assay can be used to analyze human plasma samples to support phase I and II clinical studies.     

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.     

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。     

Simultaneous determination of amoxicillin and ambroxol in human plasma by LC-MS/MS: validation and application to pharmacokinetic study

A rapid, simple and sensitive LC-MS/MS method was developed for simultaneous determination of amoxicillin and ambroxol in human plasma using clenbuterol as internal standard (IS). The plasma samples were subjected to a simple protein precipitation with methanol. Separation was achieved on a Lichrospher C(18) column (150 mm x 4.6mm ID, dp 5 microm) using methanol (containing 0.2% of formic acid) and water (containing 0.2% of formic acid) as a mobile phase by gradient elution at a flow rate of 1.0 mL/min. Detection was performed using electrospray ionization in positive ion multiple reaction monitoring (MRM) mode by monitoring the ion transitions from m/z 365.9-->348.9 (amoxicillin), m/z 378.9-->263.6 (ambroxol) and m/z 277.0-->203.0 (IS). Calibration curves were linear in the concentration range of 5-20,000 ng/mL for amoxicillin, and 1-200 ng/mL for ambroxol, with the intra- and inter-run precisions of <9% and the accuracies of 100+/-7%. The method has been validated and applied to pharmacokinetic studies of compound amoxicillin and ambroxol hydrochloride tablets in healthy Chinese volunteers.     

Determination of fexofenadine in human plasma using 96-well solid phase extraction and HPLC with tandem mass spectrometric detection

A fast and sensitive HPLC-MS/MS method, utilizing atmospheric pressure chemical ionization, for the determination of fexofenadine in human plasma is described. A deuterated analog, d6-fexofenadine is used as the internal standard (IS). Plasma samples are prepared using 96-well solid phase extraction with plates containing Waters Oasis HLB sorbent. The analytes are chromatographed on a Restek Ultra IBD column (3.2 mm x 50 mm, 3 microm) using a mobile phase consisting of a mixture of 90% acetonitrile and 10% 10 mM ammonium acetate buffer and 0.1% formic acid. Quantitation of the analyte is based on the response from the multiple reaction monitoring of the precursor to product ion pairs for fexofenadine (m/z 502 --> 466) and d6-fexofenadine (m/z 508 --> 472). The assay has been validated over the concentration range of 1-200 ng/ml based on the analysis of 0.5 ml aliquots of plasma. Within-day assay accuracy was between 97 and 102% of nominal, while within-day precision was better than 3.5% CV at all points on the standard curve. Analyte extraction recovery was better than 70% over the range of the standard curve. The method was found to be suitable for the analysis of human plasma samples obtained 24 h following the administration of a single 60 mg dose of fexofenadine.     

Liquid chromatography-negative ion electrospray tandem mass spectrometry method for the quantification of ezetimibe in human plasma

A simple, reliable and sensitive liquid chromatography-tandem mass spectrometry method (LC-MS/MS) was developed and validated for quantification of free and total ezetimibe in human plasma. The analyte and internal standard (13C6-ezetimibe) were extracted by liquid-liquid extraction with methyl tert-butyl ether. The reversed-phase chromatographic separation was performed on a Capcell C18 column, and the plasma extract was eluted with a gradient consisting of acetonitrile and 5 mM ammonium acetate. The analyte was detected using negative ionization by multiple reaction monitoring mode. The mass transition pairs of m/z 408.5-->270.8 and m/z 414.5-->276.8 were used to detect ezetimibe and internal standard, respectively. The assay exhibited linear ranges from 0.02 to 20 ng/ml for free ezetimibe and 0.25 to 250 ng/ml for total ezetimibe in human plasma. Acceptable precision and accuracy were obtained for concentrations of the calibration standard and quality control. The validated method was successfully used to analyze human plasma samples for application in a pharmacokinetic study.     

A fast and sensitive liquid chromatographic-tandem mass spectrometric method for assay of lorazepam and application to pharmacokinetic analysis

A fast and sensitive method of coupled high-performance liquid chromatography-electrospray tandem mass spectrometry for the assay of lorazepam in human plasma was developed. Plasma samples were simply treated with acetonitrile to precipitate and remove proteins and the isolated supernatants were directly injected into the HPLC/MS/MS system. Chromatographic separation was performed on a Zorbax C(18) (100 x 2.1 mm I.D.) column with a 65:35 (v/v) mixed solution of acetonitrile and 10mM aqueous formic acid being used as mobile phase. With diazepam as an internal standard, quantification was performed by selected reaction ion monitoring of the transitions of m/z 321--> m/z 275 for lorazepam and m/z 285--> m/z 193 for the internal standard. The assay was validated in the concentration range of 0.71-71.3 ng/ml in human plasma. A detection limit of 0.10 ng/ml for lorazepam was achieved, and inter- and intra-run precisions of better than 4.4% (R.S.D.) were observed. The developed method has been successfully applied for pharmacokinetic study of the drug in man.     

高效液相色谱-质谱/质谱联用法测定人血浆中莫沙必利

建立测定人血浆中莫沙必利的高效液相色谱-质谱/质谱联用法。取血浆样品经液-液萃取后，以乙腈为有机相，0.3%甲酸水溶液为水相，采用梯度洗脱的方式，用C₁₈柱分离，通过电喷雾离子化，以多反应监测(MRM)方式进行正离子检测。莫沙必利线性范围为0.17~68.00 ng·mL^-1，定量下限为0.17 ng·mL^-1，每个样品测试时间仅2.8 min，日内、日间精密度(RSD)均小于13%，准确度(RE)在±6.3%范围内。应用此法研究了20名志愿者单剂量口服枸橼酸莫沙必利片后的药代动力学特点。该方法、灵敏、准确、快速，适用于莫沙必利的药代动力学及生物等效性研究。     

Rapid quantification of gabapentin in human plasma by liquid chromatography/tandem mass spectrometry

A simple, sensitive and rapid liquid chromatography/tandem mass spectrometry (LC-MS/MS) method was developed and validated for the quantification of gabapentin, a new antiepileptic drug, in human plasma using its structural analogue, 1,1-cyclohexane diacetic acid monoamide (CAM) as internal standard. The method involved a simple protein precipitation by means of acetonitrile followed by a rapid isocratic elution with 10mM ammonium formate buffer/acetonitrile (20/80, v/v, pH 3.0) on Waters Symmetry C(18 reversed phase chromatographic column and analyzed by mass spectrometry in the multiple reaction monitoring mode. The precursor to product ion transitions of m/z 172-->154 and m/z 200-->182 were used to measure the analyte and the IS, respectively. The assay exhibited a linear dynamic range of 40-10000 ng/mL for gabapentin in human plasma. The limit of detection and lower limit of quantification in human plasma were 10 and 40 ng/mL, respectively. Acceptable precision and accuracy were obtained for concentrations over the standard curve ranges. A run time of 2 min for each sample made it possible to analyze a throughput of more than 400 human plasma samples per day. The validated method has been successfully used to analyze human plasma samples for application in pharmacokinetic, bioavailability or bioequivalence studies.     

Sensitive bioassay for the simultaneous determination of pseudoephedrine and cetirizine in human plasma by liquid-chromatography-ion trap spectrometry

A liquid chromatography-ion trap mass spectrometry coupled with electrospray ionization (HPLC-ESI-ion trap mass spectrometry) method for simultaneous determination of cetirizine and pseudoephedrine in human plasma is presented. Chromatographic separation was performed on a Hypurity C18 column (Thermo Hypersil-Keystone 2.1 mm x 150 mm, 5 microm, USA), The mobile phase was composed of 65% methanol and 35% water (contained 0.1% formic acid, 10 mM ammonium formate), which was run with a flow-rate of 0.2 ml/min at 40 degrees C. Quantitation was achieved by monitoring the product ions at m/z 166-->m/z 148 (pseudoephedrine), m/z 389.9-->m/z 201.1 (cetirizine), m/z 264-->m/z 246 (tramadol, IS). The calibration curve of pseudoephedrine and cetirizine was established with standard solutions. The limit of detection for pseudoephedrine and cetirizine each was 5 ng/ml. This simplified analytical method is sensitive, specific and accurate enough for simultaneous determination of pseudoephedrine and cetirizine in human plasma and is successfully applied to the pharmacokinetic study of pseudoephedrine and cetirizine.     

Development and validation of a sensitive LC/MS-MS method for the determination of letrozole in nude mice plasma and its application to a pharmacokinetic study

A sensitive, rapid and simple liquid chromatography-tandem mass spectrometric (LC-MS/MS) method was developed and validated for the determination of letrozole (LTZ) in nude mouse plasma in the current study, which was successfully applied to a pharmacokinetic study. Using anastrozole as internal standard (IS), plasma samples went through a one-step protein precipitation with acetonitrile before determination. The analyte and IS were analyzed on a reversed-phase ZORBAX-SB-C₁₈column (4.6 mm×250 mm, 5 μm) with an isocratic mobile phase consisting of acetonitrile and water containing 0.1% formic acid (v/v) at a flow rate of 1.0 mL/min. The analyte and IS were detected by a triple-quadrupole tandem mass spectrometer, and electrospray and multiple reaction monitoring (MRM) were employed to select LTZ at m/z 286.4/217.1 and IS at m/z 294.1/225.3 simultaneously in the positive ion mode. The calibration curve showed good linearity ranging from 0.8–2000.0 ng/mL (r>0.99). The intra-day and inter-day precisions of LTZ were 4.0%–8.4%, with an accuracy of 98.6%–104.9%. Using this method, we successfully characterized the pharmacokinetics (PK) of LTZ by a one-compartment model with first-order absorption in female BALB/c nude mice.     

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。     

HPLC-MS/MS测定人体血浆中克拉霉素浓度的方法及方法学确证

目的建立测定人体血浆中克拉霉素浓度的高效液相色谱-串联质谱联法（HPLC-MS/MS）法。方法血浆样本用甲醇沉淀蛋白后,选用色谱柱Agilent-XDB-C8柱（2.1mm×150mm,5μm）,以甲醇（A相）：水（含0.1%甲酸）=85：15（V：V）的比例为流动相,流速为0.2ml/min,选用岛津（SHIMADZU）高效液相色谱系统,串联API3200型三重四极杆质谱仪,采用多重反应监测（MRM）扫描方式进行监测,电喷雾离子化源（ESI源）,正离子方式,用于定量分析的离子反应对分别为质荷比m/z748.4→m/z158.2（克拉霉素）和m/z515.3→m/z276.2（替米沙坦,内标）。结果本方法血浆中克拉霉素的线性范围为10～3000ng/ml（r〉0.99）,定量下限为10ng/ml,批内和批间相对标准偏差均〈15%,准确度（相对回收率）相对标准偏差也均〈15%。稳定性试验中,血浆中克拉霉素在方法学要求的各种贮存条件下均较稳定。结论该方法快速、灵敏、专属性强、重现性好,适用于人体内克拉霉素的药代动力学研究。     

液相色谱串联质谱法测定人血浆中文拉法辛及其代谢物的浓度

目的建立测定血浆中文拉法辛及其代谢物O-去甲基文拉法辛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6-文拉法辛和氘6-O-去甲基文拉法辛),直接沉淀法处理样品。色谱柱为CAPCELL PAK C18 MGⅢ分析柱(100 mm×2.0 mm,5μm),流动相为含0.3%甲酸的水溶液-含0.3%甲酸的乙腈溶液(78∶22,V/V),流速为0.3 mL.min-1。正离子多离子反应监测(MRM)扫描分析,离子通道分别为m/z 278→58(文拉法辛)、m/z 264→58(O-去甲基文拉法辛)、m/z 284→58(氘6-文拉法辛)、m/z 270→58(氘6-O-去甲基文拉法辛)。结果文拉法辛和O-去甲基文拉法辛的线性范围均为2～1 000μg.L-1,定量下限均为2μg.L-1,提取回收率在90.14%～97.33%,批内、批间RSD均小于8%。结论本方法操作简便,特异性强,灵敏度高,可用于人血浆内文拉法辛和O-去甲基文拉法辛的含量测定研究。     

盐酸坦洛新缓释胶囊在犬体内的药动学

目的建立犬血浆中坦洛新浓度的测定方法,并应用于盐酸坦洛新缓释胶囊中坦洛新犬体内的药动学研究。方法液相色谱-串联质谱法(LC-MS/MS)测定犬血浆中的坦洛新浓度。坦洛新血浆样品经乙酸乙酯萃取,Agilent ZORBAX SB-C18柱(150 mm×2.1 mm,5μm)分离,阿立哌唑为内标,流动相A为体积分数为0.1%的甲酸水溶液,B为乙腈,线性梯度洗脱,电喷雾电离源(ESI),以多反应离子监测(multiple reaction monitoring,MRM)方式进行正离子检测,用于分析的定量离子分别为m/z409→m/z228(坦洛新),m/z 447.5→m/z 284.8(内标:阿立哌唑)。结果犬血浆中坦洛新的线性为0.1～20.0μg.L-1,定量下限为0.1μg.L-1,日内和日间精密度(RSD)均小于13.59%,准确度(relative error,RE)为-2.54%～4.27%。结论本方法适用于盐酸坦洛新缓释胶囊在犬体内的药动学研究。     

HPLC—MS／MS测定人体血浆和尿液中帕洛诺司琼的浓度

目的建立一种简便、灵敏的测定人体血浆和尿液中帕洛诺司琼浓度的高效液相色谱一串联质谱（HPLC-MS／MS）方法。方法血浆、尿液样品分别采用甲醇沉淀处理后,选样分析。采用Agilent-ZORBAX-C18色谱柱（2．1mm×50mm,5fμm）．以乙腈-0．1％甲酸溶液为流动相,采用正离子,多反应监测方式测定样品浓度。用于定量分析的检测离子质荷电（m／z）297．2→m/z110．1（帕洛诺司琼）和DI／Z285．0→M／z193．0（内标）。结果帕洛诺司琼血浆样品在0．02～10ng·mL^-1与峰面积线性关系良好（r=0．9975）;定量下限（LLOQ）为0．02ng·L^-1;日内与日间RSD均〈10％;回收率在89．6％～114．0％。尿样在2．5～100ng·mL^-1与峰面积线性关系良好,7—0．9974;定量下限（LLOQ）为2．5ng·mL^-1;日内与日间RSD均〈10％;回收率在96．4％～113．4％。结论本方法简便快速、灵敏准确,适用于帕洛诺司琼在人体体内的药物动力学研究。     

Quantification of vincristine and its major metabolite in human plasma by high-performance liquid chromatography/tandem mass spectrometry

An analytical method using electrospray ionization and high-performance liquid chromatography/tandem mass spectrometry (LC/ESI-MS/MS) was developed to quantify vincristine and M1, the CYP3A-mediated metabolite of vincristine, in human plasma. Vinblastine (internal standard), vincristine, and M1 in plasma were extracted in methylene chloride after acidification with TCAA. The analytes were separated on an Inertsil ODS-3 C18 column (2.1 x 150 mm) with a 5-mum particle size using a gradient elution with a run time of 20 min. The initial mobile phase composition was 0.2% formic acid/water (80:20, v/v) with a final composition of 0.2% formic acid/water (20:80, v/v). Detection was accomplished with multiple reaction monitoring for vinblastine (m/z 406.3--> 271.7), vincristine (m/z 413.2--> 362.2), and M1 (m/z 397.3 --> 376.2). At three concentrations of vincristine and M1, the inter-day and intra-day accuracy and precision were within the acceptable limits for validation (106.8 +/- 9.6% for intra-day, n = 5 each concentration; 90.9 +/- 10.9% for inter-day, n = 4 each concentration). For both vincristine and M1, the concentration limits of quantification and detection were 12 pg/mL and 6 pg/mL, respectively. Stability studies indicated that 80% of M1 degraded in plasma after 15 hours at room temperature (n = 3, high and low QC concentrations). Therefore, short plasma processing times (<30 min) are recommended. The assay was used successfully to quantify vincristine and M1 in pediatric plasma samples up to 24 hours after vincristine administration. Vincristine and M1 concentrations were within the limits of quantification for all patient plasma samples.     

液相色谱-串联质谱法测定人血清中苯磺酸氨氯地平的浓度

目的:建立测定体内苯磺酸氨氯地平的液相色谱-串联质谱(LC-MS/MS)方法。方法:血清样品中加入内标苯海拉明,直接沉淀蛋白法处理样品。色谱柱为Atlantis C_(18)(100 mm×2.1 mm,3μm),流动相为含0.1%甲酸的乙腈-0.1%甲酸水溶液(42:58,V:V),流速为0.25 mL·min~(-1)。MRM扫描分析,苯磺酸氨氯地平和内标苯海拉明的离子通道分别选择为m/z 409.1→238.1和256.3→167.0。结果:苯磺酸氨氯地平的线性范围为0.2～32μg·L~(-1),最低定量限为0.2μg·L~(-1),提取回收率均大于95%,日内、日间RSD均小于15%。结论:本方法灵敏、准确、专一、操作简便,适用于苯磺酸氨氯地平的体内药动学研究。     

Ultra-performance liquid chromatography-tandem mass spectrometry method for the determination of nitrendipine in dog plasma and its application to a pharmacokinetic study of a solid self-emulsifying pellet formulation

A rapid, sensitive and selective ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed and validated for the quantitative determination of nitrendipine (NTD, CAS 39562-70-4) in dog plasma. Using propranolol hydrochloride (CAS 318-98-9) as an internal standard (IS), plasma samples pretreatment adopted a simple liquid-liquid extraction process with diethyl ether. Separation was carried out by a gradient elution on an Acquity UPLC BEH C18 column with a mobile phase consisting of water (containing 0.1% formic acid) and acetonitrile. Detection was performed by a triple-quadrupole mass spectrometry with positive electrospray ionization (ESI) as source ionization in multiple-reaction monitoring (MRM) mode at m/z 361.0 --> 315.0 for NTD and m/z 260.2 --> 116.0 for IS. The method demonstrated good linearity at the concentrations ranged from 0.1-200 ng/mL and the lower limit of quantification (LLOQ) of NTD was 0.1 ng/mL. The intra- and inter-day relative standard deviations (RSD) were less than 10%. The mean extraction recoveries of NTD and IS were 90.2% and 82.4%, respectively. Finally, the method was successfully applied to a pharmacokinetic study of home-made solid self-emulsifying pellets and conventional NTD tablets in beagle dogs following a single oral administration.     

Sensitive bioassay of bupivacaine in human plasma by liquid-chromatography-ion trap mass spectrometry

A sensitive high-performance liquid chromatography-tandem mass spectrometric (HPLC-MS-MS) method, using an ion trap spectrometer, was developed for quantitation of bupivacaine in human plasma. Bupivacaine and an internal standard (ropivacaine) were extracted in a single step from 100 microL of alkalinized plasma with diethyl-ether. The mobile phase consisted of acetonitrile with 0.1% formic acid (50:50, v/v), and was delivered at a flow rate of 0.3 mL/min. The effluent was detected by MS-MS in positive ion mode. Ionisation was performed, using an electrospray ion source, operating at 200 degrees C. The selected reaction monitoring transitions m/z 289-->m/z 140 and m/z 275-->m/z 126 were chosen for bupivacaine and ropivacaine, respectively. Calibration curves were linear over the concentration range of 3.90-500 microg/L with determination coefficients >0.996. The method is accurate (bias <10%) and reproducible (intra-assay and inter-assay precision <15%), with a quantitation limit of 3.90 microg/L, using only 100 microL of plasma. The high specificity and sensitivity, achieved by this fast method (total run-time <3 min), allowed the determination of bupivacaine plasma levels in pediatric patients, following epidural administration of bupivacaine.