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.     

Determination of fexofenadine enantiomers in human plasma with high-performance liquid chromatography

A simple and sensitive high-performance liquid chromatography (HPLC) method was developed as an assay for fexofenadine enantiomers in human plasma. Fexofenadine enantiomers were separated using a mobile phase of 0.5% KH(2)PO(4)-acetonitrile (65:35, v/v) on a Chiral CD-Ph column at a flow rate of 0.5 ml/min and measurement at 220 nm. Analysis required 400 microl of plasma and involved solid-phase extraction with an Oasis HLB cartridge, which gave recoveries for both enantiomers from 67.4 to 71.8%. The lower limit of quantification was 25 ng/ml for (R)- and (S)-fexofenadine. The linear range of this assay was between 25 and 625 ng/ml (regression line r(2)>0.993). Inter- and intra-day coefficients of variation were less than 13.6% and accuracies were within 8.8% over the linear range for both analytes. This method can be applied effectively to measure fexofenadine enantiomer concentrations in clinical samples.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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。     

A validated SPE-LC-MS/MS assay for Eplerenone and its hydrolyzed metabolite in human urine

An automated LC-MS/MS assay was validated to quantitate the first selective aldosterone blocker Eplerenone (I) and its hydrolyzed metabolite (II) in human urine. After the addition of the stable isotope labeled internal standards, human urine samples were extracted on a C(18) solid phase extraction (SPE) cartridge using a Zymark RapidTrace automation system. The extraction eluates were diluted with 20 mM ammonium acetate aqueous solution and directly injected onto the LC-MS/MS system. The chromatographic separation was performed on a reverse phase Zorbax XDB-C(8) HPLC column (2.1 x 50 mm, 5 microm) with a mobile phase of acetonitrile:water (40:60, v/v) containing 10 mM ammonium acetate (pH 7.4). I and II were ionized using positive and negative ionization mass spectrometry, respectively, to achieve the best sensitivity. The ionization polarity was switched during the run at approximately 2.5 min after the injection. Multiple reaction monitoring (MRM) with a tandem mass spectrometer was used to detect the analytes. The precursor to product ion transitions of m/z 415-->163 and m/z 431-->337 were used to measure I and II, respectively. The assay exhibited a linear dynamic range of 50-10000 ng/ml of urine for both of I and II. The lower limit of quantitation (LLOQ) was 50 ng/ml for I and II. Acceptable precision and accuracy were obtained for concentrations over the standard curve range. Sample analysis time for each injection was 5 min; a throughput of 100 human urine standards and samples per run was achieved.     

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.     

LC-MS-MS determination of exemestane in human plasma with heated nebulizer interface following solid-phase extraction in the 96 well plate format

A sensitive, specific and rapid analytical method for the quantitation of exemestane (EXE) in human plasma has been developed. EXE, 6-methylen-androsta-1,4-diene-3,17-dione, is an orally active irreversible steroidal aromatase inhibitor used for the therapy of metastatic postmenopausal breast cancer, with estrogen-dependent pathological conditions. The method involves extraction of EXE from human plasma by solid phase extraction using C2 endcapped sorbent in the 96 well plate format (50 mg/2 ml). After conditioning of the sorbent with 1 ml of acetonitrile (x2) the plates were rinsed with 1 ml of water (x2). The prepared samples (0.5 ml plasma, spiked with [13C3] EXE as internal standard (IS) and diluted with 0.5 ml water) were loaded and drawn through the plate with a minimum of vacuum. The plates were then washed with 1 ml acetonitrile:water (10:90) followed by a drying step for 30 min at full vacuum. Elution was by 0.15 ml of 0.1% trifluoracetic acid in acetonitrile (x2) under a minimum of vacuum. Aliquots of 80 microl were finally injected into the LC-MS-MS system. A Zorbax SB C8 column (4.6 x 150 mm, 5 microm) was used to perform the chromatographic separation; the mobile phase was 100% acetonitrile. MS detection used the heated nebulizer interface, with multiple reaction monitoring (MRM) (297-->121 m/z for EXE and 300-->123 m/z for IS) operated in positive ion mode. A weighed linear regression analysis (weighing factor 1/x2) was used to calculate EXE concentration in standard and unknown samples. The method was fully validated in the concentration range 0.05-25 ng ml(-1).     

Development and validation of a sensitive and robust LC-tandem MS method for the analysis of warfarin enantiomers in human plasma

A liquid chromatography-tandem mass spectrometry method (LC-MS-MS) was developed and validated for measuring warfarin (WAR) enantiomers (R-WAR and S-WAR) in human EDTA plasma. Liquid-liquid extraction using ethyl ether was used to extract the analytes from the plasma. Baseline resolution of S- and R-WAR as well as the internal standard enantiomers (S- and R-p-ClWAR, S-IS and R-IS) was achieved on a beta-cyclodextrin column with a mobile phase of acetonitrile-acetic acid-triethylamine (1000:3:2.5, v/v/v). The retention times are 6.9, 8.0, 7.0, and 7.9 min for S-WAR, R-WAR, S-IS and R-IS, respectively. The detection was by monitoring S- and R-WAR at m/z 307-->161 and S- and R-IS at m/z 341-->161 using (-) ESI. The standard curve range was 1-100 ng ml(-1) for both S- and R-WAR. The inter-day precision and accuracy of the quality control (QC) samples were <7.3% relative standard deviation (RSD) and <7.3% bias for S-WAR, and <6.5% RSD and <5.8% bias for R-WAR, respectively. Analyte stability during sample processing and storage were established. Method ruggedness was demonstrated by the reproducible performance from analysis of clinical samples.     

A sensitive LC/MS/MS method using silica column and aqueous-organic mobile phase for the analysis of loratadine and descarboethoxy-loratadine in human plasma

A sensitive method using liquid chromatography with tandem mass spectrometric detection (LC/MS/MS) was developed and validated for the simultaneous analysis of antihistamine drug loratadine (LOR) and its active metabolite descarboethoxy-loratadine (DCL) in human plasma. Deuterated analytes, i.e. LOR-d(3) and DCL-d(3) were used as the internal standards (I.S.). Analytes were extracted from alkalized human plasma by liquid/liquid extraction using hexane. The extract was evaporated to dryness under nitrogen, reconstituted with 0.1% (v/v) of trifluoroacetic acid (TFA) in acetonitrile, and injected onto a 50 x 3.0 mm I.D. 5 microm, silica column with an aqueous-organic mobile phase consisted of acetonitrile, water, and TFA (90:10:0.1, v/v/v). The chromatographic run time was 3.0 min per injection and flow rate was 0.5 ml/min. The retention time was 1.2 and 2.0 min for LOR and DCL, respectively. The tandem mass spectrometric detection was by monitoring singly charged precursor-->product ion transitions: 383-->337 (m/z) for LOR, 311-->259 (m/z) for DCL, 388-->342 (m/z) for LOR-d(3), and 316-->262 (m/z) for DCL-d(3). The low limit of quantitation (LLOQ) was 10 pg/ml for LOR and 25 pg/ml for DCL. The inter-day precision of the quality control (QC) samples was 3.5-9.4% relative standard deviation (R.S.D.). The inter-day accuracy of the QC samples was 99.0-107.9% of the nominal values.     

LC-MS法检测大鼠血中甲苯磺丁脲含量

目的建立用LC-MS法检测大鼠血中的甲苯磺丁脲的方法。方法 Agilent Zoxbax SB-C18色谱柱,30℃柱温;流动相:0.1%甲酸溶液和乙腈梯度洗脱,流速为0.4 mL·min-1。使用SIM测定甲苯磺丁脲,卡马西平(内标)m/z 237,采用峰面积法定量。结果甲苯磺丁脲在10～1000ng·mL-1浓度范围内线性关系良好(r=0.9988)。日内精密度RSD和日间精密度RSD均小于8%,回收率均大于80%。结论本方法灵敏度高、专属性强,且操作简单,达到生物样本分析的要求。     

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.     

Quantitative determination of paclitaxel in human plasma using semi-automated liquid-liquid extraction in conjunction with liquid chromatography/tandem mass spectrometry

This paper describes a high-throughput sample preparation procedure combined with LC-MS/MS analysis to measure paclitaxel in human plasma. Paclitaxel and an internal standard were extracted from plasma by a semi-automated robotic method using liquid-liquid extraction. Thereafter compounds were separated on a RP C18 column. Detection was by a PE Sciex API 3000 mass spectrometer equipped with a TurboIonSpray interface. The compounds were detected in positive ion mode using the mass transition m/z 854.6-->286.2 and m/z 831.6-->263.2 for paclitaxel and the internal standard, respectively. The limit of quantitation for paclitaxel was 1 ng/ml with an imprecision of 5.2% following extraction of 0.1 ml of plasma. Linearity was confirmed over the whole calibration range (1-1000 ng/ml) with correlation coefficients higher than 0.99 indicating good fits of the regression models. The inter and intra-day precision was better than 9.5% and the accuracy ranged from 90.3 to 104.4%. The assay was simple, fast, specific and exhibited excellent ruggedness.