Simultaneous determination of clozapine and its N-desmethyl and N-oxide metabolites in plasma by liquid chromatography/electrospray tandem mass spectrometry and its application to plasma level monitoring in schizophrenic patients

A liquid chromatography tandem mass spectrometry (LC-MS-MS) assay method for the simultaneous determination of clozapine and its N-desmethyl (norclozapine) and N-oxide metabolites in human plasma is described. The compounds were extracted from plasma by a single step liquid-liquid extraction procedure and analyzed using a high performance liquid chromatography electrospray tandem mass spectrometer system. The compounds were eluted isocratically on a C-18 column, ionized using positive ion atmospheric pressure electrospray ionization method by a TurboIonspray source and analyzed using multiple reaction monitoring mode. The ion transitions monitored were m/z 327 --> m/z 270 for clozapine, m/z 313 --> m/z 192 for norclozapine, m/z 343 --> m/z 256 for clozapine-N-oxide and m/z 421--> m/z 201 for internal standard. The standard curves of clozapine, norclozapine and clozapine-N-oxide were linear over the range of 1 ng/ml to 1000 ng/ml when 0.5 ml of plasma was used for the analysis (r(2) >0.998). Three pooled plasma samples collected from patients who were treated with clozapine were used as long-term quality control samples to check the validity of spiked standard curve samples made at various times. The intra- and inter-assay variations for the spiked standard curve and quality control samples were less than 14%. These variations for the long-term patient quality control samples were less than 11%. The LC-MS-MS assay for simultaneous determination of clozapine, norclozapine and clozapine-N-oxide reported here is highly specific, sensitive, accurate and rapid. This method is currently being used for the plasma level monitoring of clozapine and its N-desmethyl and N-oxide metabolites in patients treated with clozapine. The plasma levels of clozapine, norclozapine and clozapine-N-oxide varied widely within and among patients. The data revealed that the norclozapine and clozapine N-oxide metabolites were present at about 58%+/-14% and 17%+/-6% of clozapine concentrations in plasma, respectively.     

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for simultaneous determination of tenofovir and emtricitabine in human plasma and its application to a bioequivalence study

A rapid and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method has been developed and validated for simultaneous quantification of Tenofovir (TEN) and Emtricitabine (EMT) in human plasma using Chromolith Speed Rod RP18. The mass transition ion-pair has been followed as m/z 288.10-->176.10 for TEN, m/z 248.20-->130.20 for EMT and m/z 230.10-->112.10 for Lamivudine (LAM). The method involves solid phase extraction from plasma, simple isocratic chromatographic conditions and mass spectrometric detection using an API 5000 instrument that enables detection at nanogram levels. Lamivudine was used as the internal standard. The proposed method has been validated with a linear range of 10-600 ng/ml for TEN and 25-2,500 ng/ml for EMT. The intrarun and interrun precision values are within 12.0% for TEN and 15.6% for EMT at their respective LOQ levels. The overall recoveries for TEN and EMT were 84.3% and 68.5%, respectively. Total elution time was as low as 2 min.     

High performance liquid chromatography-tandem mass spectrometric determination of rupatadine in human plasma and its pharmacokinetics

A simple, rapid, sensitive and selective liquid chromatographic-tandem mass spectrometric (LC-MS/MS) method was developed and validated for the quantification of rupatadine in human plasma using estazolam as internal standard (IS). Following liquid-liquid extraction, the analytes were separated using a mobile phase of methanol-ammonium acetate (pH 2.2; 5mM) (50:50, v/v) on a reverse phase C18 column and analyzed by a triple-quadrupole mass spectrometer in the positive ion and multiple reaction monitoring (MRM) mode, m/z 416-->309 for rupatadine and m/z 295-->267 for the IS. The assay exhibited a linear dynamic range of 0.1-100 ng/ml for rupatadine in human plasma. The lower limit of quantification (LLOQ) was 0.1 ng/ml with a relative standard deviation of less than 20%. Acceptable precision and accuracy were obtained for concentrations over the standard curve range. The validated LC-MS/MS method has been successfully applied to study the pharmacokinetics of rupatadine in healthy volunteers.     

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.     

Liquid chromatography/tandem mass spectrometry for the determination of carbamazepine and its main metabolite in rat plasma utilizing an automated blood sampling system

A liquid chromatography/tandem mass spectrometry (LC/MS/MS) method for the simultaneous determination of carbamazepine and its main metabolite carbamazepine 10,11-epoxide in rat plasma is described. The method consists of a liquid-liquid extraction procedure and electrospray LC/MS/MS analysis. The chromatographic separation was achieved within 5 min using a C(8) (150 mm x 2.1mm) 5 microm column with a mobile phase composed of water/acetonitrile/acetic acid (69.5:30:0.5, v/v/v) at a flow rate of 0.4 ml/min. D(10)-carbamazepine is used as the internal standard for all compounds. Analytes were determined by electrospray ionization tandem mass spectrometry in the positive ion mode using selected reaction monitoring (SRM). Carbamazepine was monitored by scanning m/z 237-->194, carbamazepine 10,11-epoxide by m/z 253-->210 and d(10)-carbamazepine by m/z 247-->204. The lower limit of quantitation (LLOQ) is 5 ng/ml for each analyte, based on 0.1 ml aliquots of rat plasma. The extraction recovery of analytes from rat plasma was over 87%. Intra-day and inter-day assay coefficients of variations were in the range of 2.6-9.5 and 4.0-9.6%, respectively. Linearity is observed over the range of 5-2000 ng/ml. This method was used for pharmacokinetic studies of carbamazepine and carbamazepine 10,11-epoxide in response to two different blood sampling techniques (i.e., manual sampling versus automated sampling) in the rat. Several differences between the two sampling techniques suggest that the method of blood collection needs to be considered in the evaluation of pharmacokinetic data.     

Liquid/liquid extraction using 96-well plate format in conjunction with hydrophilic interaction liquid chromatography-tandem mass spectrometry method for the analysis of fluconazole in human plasma

A bioanalytical method using automated sample transferring, automated liquid/liquid extraction (LLE) and hydrophilic interaction liquid chromatography-tandem mass spectrometry was developed for the determination of fluconazole in human plasma. Samples of 0.05 ml were transferred into 96-well plate using automatic liquid handler (Multiprobe II). Automated LLE was carried out on a 96-channel programmable liquid handling workstation (Quadra 96) using methyl-tetra butyl ether as the extraction solvent. The extract was evaporated to dryness, reconstituted, and injected onto a silica column using an aqueous-organic mobile phase. The chromatographic run time was 2.0 min per injection, with retention times of 1.47 and 1.44 min for fluconazole and internal standard (IS) ritonavir, respectively. The detection was by monitoring fluconazole at m/z 307-->238 and IS at m/z 721-->296, respectively. The standard curve range was 0.5-100 ng ml(-1). The inter-day precision and accuracy of the quality control samples were <7.1% relative standard deviation and <2.2% relative error.     

LC-ESI-MS法测定人血浆中紫杉醇的浓度及其在药代动力学中的应用(英文)

建立了一种灵敏度高、特异性好的高效液相色谱-电喷雾质谱法 (LC-ESI-MS) 测定人血浆中紫杉醇浓度的方法。采用一步液液萃取法进行血浆样品预处理, 提取液为甲基叔丁基醚, 内标选用炔诺酮。色谱柱为Zorbax SB-C18 柱 (100 mm×2.1 mm, 3.5 μm, Agilent), 流动相为甲醇-0.2 mmol/L甲酸铵缓冲盐溶液 (包含0.1%甲酸), 采用梯度洗脱。选择离子监测 (SIM) 的目标离子为紫杉醇的[M+Na]+ m/z 876.5和内标的[M+H]+ m/z 299.4。方法学验证表明线性范围是1.0-400 ng/mL (r>0.998), 最低定量限为1.0 ng/mL, 方法的批内和批间精密度都小于9.0%, 准确度在6.8%以内。此方法已成功应用于紫杉醇脂质体注射液在患者体内的药动学研究。     

Simultaneous quantitation of 17alpha-hydroxyprogesterone caproate, 17alpha-hydroxyprogesterone and progesterone in human plasma using high-performance liquid chromatography-mass spectrometry (HPLC-MS/MS)

A sensitive and specific assay method for the simultaneous quantitation of 17alpha-hydroxyprogesterone caproate (17-OHPC), 17alpha-hydroxyprogesterone (17-OHP), and progesterone (P) in human plasma using high-performance liquid chromatography and tandem mass spectrometry (LC-MS/MS) was developed and validated. Plasma samples were processed by a solid phase extraction (SPE) procedure using Oasis((R)) HLB extraction cartridge prior to chromatography. Medroxyprogestrone acetate (MPA) was used as the internal standard. The compounds were separated using Waters C18 Symmetry analytical column (3.5 microm, 2.1 mm x 50 mm) using a gradient elusion with a mobile phase consisting of 5% methanol in water [A] and methanol [B], with ammonium acetate (2mM) and formic acid (0.1%) being added to both [A] and [B], at a flow rate 0.3 ml/min. The retention times for 17-OHPC, 17-OHP, P and MPA were 4.5, 1.5, 2.5 and 2.2 min, respectively, with a total run time of 7 min. The analytes were detected by a Micromass Quattro Micro triple quadrupole mass spectrometer in positive electron spray ionization (ESI) mode using multiple reaction monitoring (MRM). The extracted ions monitored following MRM transitions were m/z 429.10-->313.10 for 17-OHPC, m/z 331.17-->97.00 for 17-OHP, m/z 315.15-->109.00 for P and m/z 387.15-->327.25 for MPA (IS). The assay was linear over the range 1-200 ng/ml for 17-OHPC and 17-OHP, and 2-400 ng/ml for P, when 0.4 ml of plasma was used in the extraction. The overall intra- and inter-day assay variation was <15%. No significant variation in the concentration of 17-OHPC, 17-OHP or P was observed with different sample processing and/or storage conditions. This method is simple, allows easy, accurate and reproducible measurement of 17-OHPC, 17-OHP and P simultaneously in human plasma, and is used to evaluate the pharmacokinetics of 17-OHPC in pregnant subjects.     

Simultaneous determination of pioglitazone and its two active metabolites in human plasma by LC-MS/MS

A liquid chromatography/tandem mass spectrometry (LC-MS/MS) method was developed and validated for the simultaneous determination of pioglitazone (PIO) and its two metabolites: M-III (keto-derivative) and M-IV (hydroxy-derivative) in human plasma. Human plasma samples of 0.2 ml were extracted by a single step liquid-liquid extraction procedure and analyzed using a high performance liquid chromatography (HPLC) electrospray tandem mass spectrometer system. The compounds were eluted isocratically on a C-18 column, ionized using a positive ion atmospheric pressure electrospray ionization source and analyzed using multiple reaction monitoring mode. The ion transitions monitored were m/z 357-->134 for PIO, m/z 371-->148 for M-III, m/z 373-->150 for M-IV and m/z 413-->178 for the internal standard. The chromatographic run time was 2.5 min per injection, with retention times of 1.45, 1.02 and 0.95 min for PIO, M-III and M-IV, respectively. The calibration curves of pioglitazone, M-III and M-IV were well fit over the range of 0.5-2000 ng/ml (r(2)>0.998759) by using a weighted (1/x(2)) quadratic regression. The inter-day precisions of the quality control samples (QCs) were 相似文献   

Determination of cetirizine in human plasma using high performance liquid chromatography coupled with tandem mass spectrometric detection: application to a bioequivalence study

A rapid, sensitive and selective HPLC-MS/ MS method was developed and validated for the quantification of cetirizine dihydrochloride (CAS 83881-51-0) in human plasma using mosapride citrate as internal standard (IS, CAS 112885-42-4). Following liquid-liquid extraction, the analytes were separated using a mobile phase consisting of methanol and aqueous ammonium acetate solution (10 mM) (60:40, v/v) on a reverse phase C18 column and analyzed by a triple-quadrupole mass spectrometer in the selected reaction monitoring (SRM) mode using the respective [M+H]+ ions, m/z 398 --> 201 for cetirizine and m/z 422 --> 198 for mosapride. The analysis time for each run was 8.0 min. The assay exhibited a linear dynamic range of 0.5-500 ng/ml for cetirizine dihydrochloride in human plasma. The lower limit of quantification (LLOQ) was 0.5 ng/ml with a relative standard deviation of less than 15% (all the concentration data in this study related to the salt (cetirizine dihydrochloride)). Acceptable precision and accuracy were obtained for concentrations over the standard curve range. It is the first time that the validated HPLC-MS/MS method has been successfully applied to a bioequivalence study in 20 healthy male Chinese volunteers.     

Liquid chromatography-tandem mass spectrometry method for the quantification of deglymidodrine in human plasma

A simple, rapid and sensitive liquid chromatography-tandem mass spectrometry method was developed for quantification of deglymidodrine in human plasma. The plasma samples were pretreated by protein precipitation with trichloroacetate. The chromatographic separation was performed on reversed phase Aquasil C18 column, and the plasma extraction was eluted with a mobile phase solution consisting of acetonitrile (containing 0.02% formic acid) and water (containing 0.02% formic acid). The molecular ion of analyte was detected in positive ionization by multiple reaction monitoring. The mass transitions of m/z 198.4--> 148.1 and m/z 212.4--> 162.3 were used for detection of deglymidodrine and its internal standard, respectively. The assay exhibited linear ranges from 0.25 to 32 ng/ml for the analyte in human plasma. Acceptable precision and accuracy were obtained for concentrations of quality control (QC) samples. The proposed method has been successfully used to analyze human plasma samples for application in oral pharmacokinetic study.     

Development and validation of an automated SPE-LC-MS/MS assay for valdecoxib and its hydroxylated metabolite in human plasma

A sensitive and specific liquid chromatography-tandem mass spectrometry assay was developed to quantitate valdecoxib (I) and its hydroxylated metabolite (II) in human plasma. The analytes (I and II) and a structurally analogue internal standard (IS) were extracted on a C(18) solid phase extraction (SPE) cartridge using a Zymark RapidTrace automation system. The chromatographic separation was performed on a narrow-bore reverse phase Zorbax XDB-C(8) HPLC column with a mobile phase of acetonitrile:water (50:50, v/v) containing 10 mM ammonium acetate. The analytes were ionized using negative electrospray mass spectrometry, then detected by multiple reaction monitoring (MRM) with a tandem mass spectrometer. The precursor to product ion transitions of m/z 313-->118 and m/z 329-->196 were used to measure I and II, respectively. The assay exhibited a linear dynamic range of 0.5-200 ng/ml of I and II in human plasma with absolute recoveries from plasma at 91 and 86%, respectively. The lower limit of quantitation was 0.5 ng/ml for I and II. Acceptable precision and accuracy were obtained for concentrations over the calibration curve ranges (0.5-200 ng/ml). Sample analysis time for each injection was 5 min, a throughput of 70 human plasma standards and samples per run was achieved. The assay has been successfully used to analyze human plasma samples to support clinical phase I and II studies.     

An automatic 96-well solid phase extraction and liquid chromatography-tandem mass spectrometry method for the analysis of morphine, morphine-3-glucuronide and morphine-6-glucuronide in human plasma.

A bioanalytical method using automated sample transferring, automated solid phase extraction (SPE) and liquid chromatography-tandem mass spectrometry (LC-MS-MS) was developed for morphine (MOR), and its metabolites morphine-3-glucuronide (M3G) and morphine-6-glucuronide (M6G) in human plasma. Samples of 0.25 ml were transferred into 96-well plate using automatic liquid handler (Multiprobe II). Automated SPE was carried out on a 96-channel programmable liquid handling workstation (Quadra 96) using a C(18) sorbent. The extract was injected onto a silica column using an aqueous-organic mobile phase. The chromatographic run time was 3.5 min per injection, with retention times of 1.5, 2.0 and 2.6 min for MOR, M6G, and M3G, respectively. The detection was by monitoring MOR at m/z 286-->152, M6G and M3G at m/z 462-->286. The deuterated internal standards were monitored at m/z 289-->152 for MOR-d(3), and m/z 465-->289 for M6G-d(3) and M3G-d(3). The standard curve range was 0.5-50 ng ml(-1) for MOR, 1.0-100 ng ml(-1) for M6G, and 10-1000 ng ml(-1) for M3G. The inter-day precision and accuracy of the quality control samples were <8% relative standard deviation (RSD) and <7% relative error (RE) for MOR, <5% RSD and <2% RE for M6G, and <2% RSD and <4% RE for M3G.     

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.     

大鼠血浆中奥昔布宁及其N-去乙基代谢物的UPLC-MS/MS法测定

建立了超高效液相色谱-串联质谱法同时测定大鼠血浆中的奥昔布宁及其主要代谢物N-去乙基奥昔布宁.用液-液萃取法处理血样,以盐酸奥昔布宁-D11为内标,采用ESI源正离子模式、多反应监测进行测定.检测离子对分别为m/z 358.2→124.1(奥昔布宁)、m/z 330.2→96.1 (N-去乙基奥昔布宁)和m/z 369.3→142.1(奥昔布宁-D11).奥昔布宁及N-去乙基奥昔布宁在0.5～100 ng/ml和0.2～40 ng/ml浓度范围内线性关系良好,日内、日间RSD均小于8％.     

Determination of morphine, morphine-3-glucuronide and morphine-6-glucuronide in monkey and dog plasma by high-performance liquid chromatography-electrospray ionization tandem mass spectrometry

A specific and simultaneous assay of morphine, morphine-3-glucuronide (M-3-G) and morphine-6-glucuronide (M-6-G) in monkey and dog plasma has been developed. These methods are based on rapid isolation using solid phase extraction cartridge, and high-performance liquid chromatography (HPLC)-electrospray ionization (ESI)-tandem mass spectrometric (MSMS) detection. Analytes were separated on a semi-micro ODS column in acetonitrile-formic (or acetic) acid mixed solution. The selected reaction monitoring for assay in monkey and dog plasma, as precursor-->product ion combinations of m/z 286-->286 for morphine, m/z 462-->286 for glucuronides and m/z 312-->312 for internal standard (IS, nalorphine) were used. The linearity of morphine, M-3-G and M-6-G was confirmed in the concentration range of 0.5-50, 25-2500, 2.5-250 ng/ml in monkey plasma, 0.5-100, 25-5000, 2.5-500 ng/ml in dog plasma, respectively. The precision of this assay method, expressed as CV, was less than 15% over the entire concentration range with adequate assay accuracy. Therefore, the HPLC-ESI-MSMS method is useful for the determination of morphine, M-3-G and M-6-G with sufficient sensitivity and specificity in pharmacokinetic studies.     

Development of a sensitive bioanalytical method for determination of PNU-83757 in rat,monkey and human plasma: from LC-UV to LC-MS/MS

To support pre-clinical pharmacokinetic/toxicokinetic (PK/TK) evaluation, a sensitive bioanalytical method for determination of N-cyano-N'-(tert-pentyl)-N"-(3-pyridinyl) guanidine (PNU-83757), in rat and monkey plasma was required. Although the UV response of PNU-83757 was quite decent and the extracts using solid phase extraction (SPE) were very selective and concentrated, the best limit of quantitation (LOQ) achieved was 0.4 ng ml(-1) using 0.5 ml plasma for extraction and 2 ng ml(-1) using 0.1 ml plasma for extraction, which was insufficient for PK/TK evaluation at lower doses. When using liquid chromatography with atmospheric pressure chemical ionization tandem mass spectrometric detection (LC-APCI-MS/MS, positive ions) and SPE, a LOQ of 0.045 ng ml(-1) for PNU-83757 was reached. Quantitation was accomplished using the precursor --> product ion combinations of m/z 232 --> 162 for PNU-83757 and m/z 236 --> 166 for the internal standard, [2H(4)]PNU-83757, in the multiple reaction monitoring mode. This method has been successfully utilized for PK/TK evaluation in pre-clinical studies and proved to have sufficient sensitivity to determine plasma concentrations for a dose level as low as 1 microg kg(-1) day(-1) in the rat and monkey. Further improvement of this method by using electrospray mass spectrometric detection (LC-ESI-MS/MS, positive ions) and automated membrane SPE, gave an LOQ of 0.008 ng ml(-1), and allowed analysis of large numbers of samples to support clinical PK studies in microg dose levels.     

A sensitive LC-ESI-MS method for the determination of indapamide in human plasma: method and clinical applications

A sensitive LC-ESI-MS method for the determination of indapamide in human plasma using glibenclamide as the internal standard (IS) was established. Following acidification with 1 M hydrochloric acid solution, plasma samples were extracted with ethyl acetate and separated on a C(18) column with a mobile phase of 10 mM ammonium acetate-methanol (22:78, v/v). Indapamide was determined using electrospray ionization in a single quadrupole mass spectrometer. LC-ESI-MS was performed in the selected ion monitoring (SIM) mode using target ions at m/z 364.3 for indapamide and m/z 492.4 for the IS. Calibration curves were linear over the ranges of 0.1-100 ng/ml for indapamide. The lower limit of quantification was 0.1 ng/ml. The intra- and inter-assay precisions were less than 9.5% and 10.6%, respectively. The mean plasma extraction recovery of indapamide was 90.5-93.9%. The method has been successfully applied to study the pharmacokinetics of indapamide in healthy male Chinese volunteers.     

Rapid determination of granisetron in human plasma by liquid chromatography coupled to tandem mass spectrometry and its application to bioequivalence study

A simple, sensitive and rapid method for analysis of granisetron in human plasma, utilizing liquid chromatography tandem mass spectrometry (LC-MS/MS), has been developed and validated to satisfy FDA guidelines for bioanalytical methods. The analyte and internal standard (IS) were isolated from 100microl plasma samples by liquid-liquid extraction (LLE). A Varian 1200l tandem mass spectrometer equipped with an electrospray ionization source was operated in selected reaction monitoring (SRM) mode with the precursor-to-product ion transitions m/z 313.4/138 for granisetron and m/z 270/201 for the IS used for quantitation. The assay exhibited a linear dynamic range of 0.02-20ng/ml for granisetron in human plasma. The lower limit of quantification (LLOQ) was 0.02ng/ml with a relative standard deviation of less than 15%. The mean extraction recovery from spiked plasma samples was 97.9%. The intra-day accuracy of the assay was within 10% of nominal and intra-day precision was better than 15% C.V. A run time of 2.0min for each sample made it possible for high-throughput bioanalysis. The method was employed in a bioequivalence study of two formulations of granisetron hydrochloride 1mg rapidly disintegrating tablets/1mg capsules.