Rapid and Sensitive RP-LC Method for the Quantification and Pharmacokinetic Study of p-Hydroxyphenethyl Anisate in Rat Plasma

A rapid, sensitive and specific reversed-phase liquid chromatographic method was developed and validated for the quantification of p-hydroxyphenethyl anisate (HPA), which is one of the main constituents of Notopterygium Radix (underground parts of Notopterygium incisum and N. forbesii), in rat plasma, and study its pharmacokinetics after the intravenous administration of 40 mg kg^?1 HPA to rats. The method involves a plasma clear-up step using liquid–liquid extraction by ethyl acetate, followed by RP-LC separation and detection. Separation of HPA was performed on an analytical Diamonsil ODS C₁₈ column equipped with a Dikma ODS C₁₈ EasyGuard column using a mobile phase consisting of MeOH–H₂O (75:25, v/v) at a flow-rate of 1.0 mL min^?1. The UV detection was performed at a wavelength of 256 nm. The linear calibration curves were obtained in the concentration range of 0.05–5.0 μg mL^?1 (r = 0.9992, n = 5) in rat plasma with the lower limit of detection of 0.01 μg mL^?1 and the lower limit of quantification of 0.04 μg mL^?1, and the extraction recovery of HPA was calculated to be the range of 82.01–86.66%. The intra- and inter-day precisions in terms of % relative standard deviation were lower than 2.33 and 3.99% in rat plasma, respectively, with accuracies ranging from 91.22 to 110.5%. The developed method was suitable for the determination and pharmacokinetic study of HPA in rat plasma.     

LC Method for Quantification of Lutein in Rat Plasma: Validation, and Application to a Pharmacokinetic Study

A reversed-phase LC method has been developed for quantitative analysis of lutein in rat plasma and applied to a study of the pharmacokinetics of lutein in rats. From a variety of compounds and solvents tested, astaxanthin was selected as the internal standard. n-Hexane was found to be the best solvent for extracting lutein from plasma. LC analysis of the extracts was performed on a C₁₈ column equipped with a guard pre-column. Linearity was good (r > 0.99) over the range 10–100 ng mL^?1. Recovery from plasma was 82.7–92.9% the intra-day and inter-day precision were always better than 3%. The limits of detection (LOD) and quantification (LOQ) were 2.5 and 8.3 ng mL^?1, respectively. The LC method was used to quantify lutein and zeaxanthin in rat plasma in a 36-h pharmacokinetic study in which experimental rats received a single oral dose of lutein (20 mg kg^?1). The results are presented.     

Sensitive and Selective LC-MS-MS Assay for the Quantification of Palonosetron in Human Plasma and Its Application to a Pharmacokinetic Study

A highly sensitive and selective liquid chromatography-tandem mass spectrometry method was developed for the determination of palonosetron in human plasma samples. Chromatographic conditions and mass spectral parameters were optimized in order to achieve a limit of quantification of approximately 0.03 ng mL^?1. Palonosetron and citalopram (internal standard) were extracted by liquid–liquid extraction under alkaline conditions using saturated sodium bicarbonate. Separation was achieved with a Hanbon Lichrospher C₁₈ column and detection was carried out by tandem mass spectrometry using positive electrospray ionization in selected reaction monitoring mode. The target ions of palonosetron and citalopram were to m/z 297.00 → 297.00 and 325.00 → 325.00 respectively. Calibration curves were linear over the range of approximately 0.03–10 ng mL^?1. Precision and accuracy of this method was acceptable. The method was successfully applied to a pharmacokinetic study with healthy Chinese volunteers after intravenous administration of a single dose of 0.125, 0.25 or 0.5 mg palonosetron hydrochloride.     

Rapid Quantification of Astilbin in Rat Plasma by Liquid Chromatography-tandem Mass Spectrometry and Its Application to Pharmacokinetic Study

Astilbin is a potential immunosuppressive agent with minor cytotoxicity. Its oral bioavailability is supposed to be rather low and therefore a sensitive analytical method is required for its pharmacokinetic study after oral administration. A simple, sensitive and rapid liquid chromatography-tandem mass spectrometry（LC-MS/MS） method was developed and validated for the determination of astilbin in rat plasma. Plasma samples were subjected to liquid-liquid extraction with ethyl acetate and separated by reversed phase high performance liquid chromatography（HPLC） with methanol-0.01%（volume fraction） formic acid（50：50, volume ratio） as mobile phase. Quantitive determination was achieved on negative LC-MS/MS by a multiple reaction moitoring method with transitions m/z 449.1→150.9（quantifier） and m/z 449.1→284.9（qualifier） for astilbin and m/z 128.9→42.0 for internal standard（IS）. A lower limit of quantification（LLOQ） of ng/mL was achieved within a short cycle time of 3.4 min. The method was successfully applied to a pharmacokinetic study involving oral and intravenous administrations of 6 mg/kg astilbin to six rats.     

A Simple and Sensitive LC Method for Quantification of Cefteram in Human Plasma

A simple and sensitive liquid chromatographic method was developed for quantification of cefteram in human plasma. Amoxicillin was used as an internal standard. The present method used protein precipitation for extraction of cefteram from human plasma. Separation was carried out on a reversed-phase C₁₈ column. The column effluent was monitored by UV detection at 262 nm. The mobile phase was a mixture of methanol and water containing 0.3% v/v triethylamine and 0.6% v/v glacial acetic acid (35:65:0.3:0.6 v/v) at a flow rate of 0.30 mL min^?1. The column temperature was 20 °C. This method was linear over the range of 47.5–4,750.0 ng mL^?1 with determination coefficient greater than 0.99. The mean extraction recovery of cefteram and IS was ≥76.82 and ≥76.49%, respectively, and the method was found to be precise, accurate, and specific during the study. The method was successfully applied for a pharmacokinetic study of cefteram in human.     

Sensitive HPLC Assay for Ketoprofen in Human Plasma and its Application to Pharmacokinetic Study

Abstract

A selective and sensitive HPLC method was developed for the analysis of ketoprofen in human plasma. The assay involves an extraction of the drug and the internal standard (piroxicam) into diethyl ether from acidified plasma and then back-extracted into a small volume of alkaline aqueous solution before injection onto the HPLC column. A microbore column (2 mm I.D. × 10 cm) packed with a C18 reversed-phase material (5 pm ODS Hypersil) was used. The chromatographic separation was accomplished with a mobile phase comprising a mixture of acetonitrile-methanol-water (15 :20 : 65, v/v) containing 10 mM Na2HP04 buffer, pH 4. The mobile phase was pumped at a flow rate of 0.5 dmin. The eluant was monitored at 258 nm. With this procedure coefficients of variation were less than 10%. The detectionlimit was 0.05 μg/ml (i.e., 50 ng/ml) of plasma. The highly sensitive nature of this method was applied successfully to the dewmination of ketoprofen in human plasma for phmacokinetic studies.     

Establishment of an LC-Based Assay to Determine Caffeic Acid Tetramer in Rat Plasma and Its Application to a Pharmacokinetic Study

A facile, sensitive, and accurate liquid chromatographic method with ultraviolet detection was developed for the determination of caffeic acid tetramer in rat plasma. Chromatographic separation was performed on an YMC C18 10 μm column (250 × 4.6 mm) using acetonitrile and phosphate buffer (19:81, v/v) as mobile phase at a flow rate of 1 mL min^?1. The UV detection wavelength was set at 252 nm. The method showed good linearity in the range of 1–150 μg mL^?1 with a satisfactory correlation coefficient (r) of 0.997. The limit of detection was 20 ng mL^?1 while inter- and intra-day precisions were less than 5.39 and 5.48%, respectively. Furthermore, the accuracy ranged from 98.27 to 103.76% with high extraction recoveries of caffeic acid tetramer from plasma greater than 88.0%. This practical methodology opens a facile and effective pathway for a pharmacokinetic study.     

Development and Validation of an HPLC–UV Method for the Determination of Insulin in Rat Plasma: Application to Pharmacokinetic Study

A simple, sensitive high performance liquid chromatographic method with UV detection was developed and validated for determination of insulin in rat plasma, using methyl paraben as an internal standard. Insulin was extracted from plasma by a liquid–liquid extraction with a mixture of dichloromethane and n-hexane (1:1, v/v) followed by an acidic back extraction. Chromatographic separation was achieved isocratically with a Phenomenex® C₁₈ analytical column (150 × 4.6 mm ID, 5 μm) at ambient room temperature. The calibration curves were linear within a concentration range of 0.7–8.4 μg mL^?1 (r ² = 0.9994). The inter-day and intra-day accuracy and precision were ≤3.33 and ≤5.55%. The limit of detection (LOD) and limit of quantification (LOQ) were 0.35 and 0.7 μg mL^?1. The average recovery was 87.86% for insulin and 83.52% for methyl paraben. Insulin containing plasma samples were stable at ?20 °C for 7 days. Validated HPLC method was successfully applied to a pharmacokinetic study of insulin in streptozotocin induced diabetic rats.     

LC Method for the Determination of Rhaponticin in Rat Plasma, Faeces and Urine for Application to Pharmacokinetic Studies

A simple liquid chromatography (LC) method has been developed and validated to determine rhaponticin in rat plasma, faeces and urine. Chromatographic separation was achieved through mobile phase consisting of acetonitrile and water at a flow rate of 1.0 mL min^?1. Rhaponticin was quantified using UV detection at 324 nm. The assay was linear over the concentration range of 50–4,000 ng mL^?1 for plasma, faeces and urine. The intra- and inter-day RSD were less than 10%. The plasma, faeces and urine rhaponticin levels were monitored in rats after oral administration. This simple LC method appears to be useful in the pharmacokinetic investigation of rhaponticin.     

LC-MS/MS Method for the Quantitation of Cefotetan in Human Plasma and Its Application to Pharmacokinetic Study

A rapid and sensitive liquid chromatography-tandem mass spectrometry（LC-MS/MS） method for the de- termination of cefotetan in human plasma was developed and validated. After the protein precipitation of sample with acetonitrile, the analyte and internal standard（IS）, tramadol, were separated on a Zorbax XDB C8 column using ace- tonitrile/1%（volume fraction） formic acid（volume ratio 35：65, pH=2.5） as mobile phase at a flow rate of 1.0 mL/min with a 1 ： 1 split. The detection was performed by electrospray ionization with positive ion mode, followed by multiple reaction monitoring of the transitions for cefotetan at m/z 576.3→460.2（quantifier） and m/z 576.3→432.2（qualifier） and for IS at m/z 264.1→58.1. Cefotetan and IS were eluted at 1.86 and 1.87 rain, respectively. The assay was linear over the concentration range of 0.1-100 gg/mL for 20 μL of human plasma only with intra- and inter-day preci- sions（expressed as the relative standard deviation） of less than 6.62% and accuracies（as relative error） of ＋1.31%. The method was applied to the pharmacokinetic study of a l-h intravenous infusion of 1.0 g of cefotetan disodium for human volunteers（n=6）.     

Determination of Chlorzoxazone in Rat Plasma by LC-ESI-MS/MS and Its Application to a Pharmacokinetic Study

A sensitive LC-ESI-MS/MS method for determination of chlorzoxazone in rat plasma has been developed. Chromatographic separation was achieved on a Zorbax SB-C₁₈ column, with 45:55 (v/v) acetonitrile–water as the mobile phase. A LC-ESI-MS/MS was performed in a multiple reactions monitoring (MRM) mode using target ions m/z 167.5→131.6 for chlorzoxazone and m/z 230.7→185.6 for phenobarbital (internal standard). The calibration plots were linear over the range of 10.0–2,000 ng/mL. Intra-day and inter-day precisions were better than 5.1% and 6.8%, respectively. The validated method was successfully used to analyze the drug in samples of rat plasma for pharmacokinetic study.     

Simple, Sensitive and Rapid LC–ESI–MS Method for the Quantitation of Olprinone in Rat Plasma

Olprinone is a phosphodiesterase (PDE)-3 inhibitor. This paper describes a simple, selective and sensitive method for the quantification of olprinone in rat plasma using a liquid–liquid extraction procedure followed by liquid chromatography mass spectrometric (LC–MS) analysis. The method had an advantage of high sensitivity. Analyses were conducted at a flow rate of 0.25 mL min⁻¹ by a gradient elution. The detection utilized selected ion monitoring in the positive ion mode at m/z 251.0 and 344.0 for the protonated molecular ions of olprinone and the internal standard, respectively. The quantitation limit for olprinone in rat plasma was 0.5 ng mL⁻¹. The linearity was also excellent over the concentration range of 0.5–100 ng mL⁻¹ of olprinone. The intra- and inter-day precision (relative standard deviation (RSD) %) was lower than 10%, and accuracy ranged from 90 to 110%. This developed method was successfully applied to analysis of olprinone in biological fluids.     

LC Method for Determination of Ginkgolic Acids in Mice Plasma and Its Application to a Pharmacokinetic Study

A sensitive and simple LC method for the quantification of ginkgolic acids in mice plasma has been developed. Following acetonitrile deproteinization, samples were separated on a SinoChrom ODS-AP C₁₈ column. The mobile phase was 3% (v/v) acetic acid water solution–methanol (8:92, v/v) at a flow rate of 1.0 mL min⁻¹. Detection was at 310 nm. Calibration curve was linear over the range of 0.25–50 μg mL⁻¹ with intra- and inter-day precisions (RSD%) of less than 9.5%. The extraction recovery ranged from 87.0 to 90.2% (RSD 2.4–6.4%) for ginkgolic acids. The method was successfully applied to the pharmacokinetic study of ginkgolic acids in mice after oral dosing of 1.0 g kg⁻¹.

     

A Simple and Sensitive HPLC Method for Antipyrine in Plasma

Abstract

A rapid, simple and sensitive high-performance liquid chromatographic (HPLC) method was developed for the determination of antipyrine in small volume (50 μl) of plasma samples. Aminopyrine was employed as the internal standard. The sample preparation is a direct plasma protein precipitation procedure so is less tedious and rapid. The assay employs a column packed with a C₁₈ reversed-phase material (5 μm Nucleosil) with an isocratic mixture of acetonitrile and water (25:75, v/v) as the mobile phase. The eluant was detected at 254 nm. The assay achieves the level of sensitivity (0.5 μg/ml) and accuracy required to obtain meaningful data about the single-dose pharmacokinetics of antipyrine in guinea pig and rat. The method gave high reproducibility with coefficients of variation less than 5%.     

A GC-SIM-MS Method for the Determination of Butylidenephthalide in Rat Plasma and Tissue: Application to the Pharmacokinetic and Tissue Distribution Study

Abstract

Butylidenephthalide is one of the major active components isolated from Rhizoma Chuanxiong. This paper describes a simple, rapid, specific and sensitive method for the quantification of butylidenephthalide in rat plasma and tissue distribution using a liquid-liquid extraction procedure followed by capillary gas chromatography-selected ion monitoring mode-mass spectrometry (GC-SIM-MS) analysis. The calibration curves were linear over the concentration ranging from 0.02–10.0 µg/mL (r > 0.99) for plasma samples and 0.18–7.25 µg/g (r > 0.99) for the tissue samples. The limit of quantification (LOQ) was 1.0 ng/mL or 1.0 ng/g (ten times signal/noise ratio). Within- and between-day precisions expressed as the relative standard deviation (RSD) for the method were 2.39–2.98% and 2.97–4.26%, respectively. The methods of recovery for all samples were greater than 80% at the low, medium, and high concentrations. The method has been successfully applied to a pharmacokinetics study in rats after an oral administration of Butylidenephthalide with a dose of 20.0 mg/kg. The main pharmacokinetic parameters obtained were T _max  = (0.22 ± 0.06) h, C _max = (3 ± 1) µg/mL, AUC = (32 ± 6) h?µg/mL, and K _a  = (8.5 ± 0.8)/h. The results showed that the butylidenephthalide was easily absorbed. The concentrations of butylidenephthalide in rat kidney, lung, heart, and cerebellum were higher than those in other organs. To determine free fraction in serum, samples were filtered using ultrafiltration membranes with a molecular weight cut-off of 10,000 Da and extracted using liquid-liquid extraction. The extracts were evaporated and analyzed by GC-MS. The protein binding in rat plasma, human plasma, and human serum albumin were 83 ± 4%, 94 ± 3%, and 89 ± 3%, respectively.     

Simultaneous Quantification of Oroxylin A and Its Metabolite Oroxylin A-7-O-Glucuronide: Application to a Pharmacokinetic Study in Rat

A sensitive liquid chromatography?Celectrospray ionization?Ctandem mass spectrometry (LC?CESI?CMS?CMS) method was developed and validated for the quantification of cepharanthine (CEP) in beagle dog plasma. The chromatographic separation was performed on an Agilent-C18 column and the mobile phase was composed of methanol:water with 10 mM ammonium acetate (20:80, v/v). Detection was operated in the positive ion mode and the tandem mass spectrometer was tuned in the multiple reactions monitoring mode (MRM) to monitor m/z transitions 607 ?? 365 for CEP and 285 ?? 193 for the internal standard (IS) diazepam. This method exhibited a linear range of 5?C2,500 ng mL^?1. The precision (RSD%) and accuracy (RME%) of the assay were <8.7 and 2.4%, respectively. The limit of quantification was 5 ng mL^?1 and no significant matrix effect was observed. The validated method has been successfully applied to pharmacokinetic study of CEP in beagle dog.     

高效液相色谱法测定大鼠血浆中芳香异羟肟酸二丁基锡的含量以及它在动力学研究中的应用

[(n-Bu)2Sn[{4-ClC6H4C(O)NHO}2] (DBDCT) 是课题组自主设计合成的一种新型芳香异羟肟酸二丁基锡化合物(已获国际国内发明专利),有较高的体内和体外抗肿瘤活性,小鼠急毒实验揭示其具有较低的毒性作用,初步动物实验提示DBDCT还具有升高白细胞的功能,在肿瘤化疗治疗中将产生重要的影响。本文首次建立了HPLC法测定化合物在血浆中的动力学参数。用甲醇直接沉淀血浆蛋白,乙酰苯胺为内标, Diamonsil ODS（4.6 mm × 200 mm, 5 μm）色谱柱,甲醇:0.5%三氟乙酸水溶液（30:70,pH 3.0,v/v）为流动相,检测波长238 nm。方法在0.1～25 µg·mLl-1范围内线性关系良好(r = 0.9992),定量限和检测限分别为50 和10 ng·mL-1。该方法用来测定单次静脉注射不同剂量(2,5,12mg·kg-1) DBDCT后大鼠体内的浓度-时间曲线,并采用3p97软件对动力学参数和房室模型进行估计,结果表明DBDCT在大鼠体内的动力学符合二室模型,方法简便快速,专属性好,其动力学研究中的应用为制剂的质量控制和临床前动物合理用药以及临床研究提供了实验基础。     

Simple, Sensitive, and Rapid LC–ESI-MS Method for Quantification of Mitiglinide in Human Urine

A sensitive and specific liquid chromatographic method with electrospray ionization mass spectrometry (LC–ESI-MS) has been developed and validated for identification and quantification of mitiglinide in human urine. A simple liquid–liquid extraction procedure was followed by separation on a C₁₈ column with gradient elution, and detection using a single-quadrupole mass spectrometer in selected-ion-monitoring (SIM) mode. The method was tested using six different batches of urine. Linearity was established for the mitiglinide concentrations in the range 0.005–1.0 μg mL⁻¹, with a coefficient of determination (r) of 0.9998 and good back-calculated accuracy and precision. Intra- and inter-day precision (as RSD, %) was below 10% and accuracy for mitiglinide ranged from 85 to 115%. The lower limit of quantification was reproducible at 0.002 μg mL⁻¹ for 500 μL urine. The proposed method enables unambiguous identification and quantification of mitiglinide in pre-clinical and clinical studies.     

UPLC-MS-MS Method for the Determination of N-(2,6-Dimethoxypyridine-3-yl)-9-methylcarbazole-3-sulfonamide in Rat Plasma and Its Application to a Pharmacokinetic Study

The purpose of the study is first to develop a sensitive and rapid ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS-MS) method for the determination of a new synthesized tubulin ligand, N-(2,6-dimethoxypyridine-3-yl)-9-methylcarbazole-3-sulfonamide (IG-105), in rat plasma. The analyte and internal standard (carbamazepine) were extracted by liquid/liquid extraction with petroleum ether/diethyl ether (2:1, v/v). The chromatographic separation was performed on an Acquity UPLC BEH C₁₈ column with a mobile phase gradient consisting of methanol and water. The mass spectrometric detection was performed by triple-quadrupole mass spectrometry with multiple reaction monitoring (MRM) via an ESI source operating in positive ionization mode. The mass transitions m/z 398??153 and m/z 237??194 were used to assay the analyte and IS, respectively. The method demonstrated good linearity over a concentration range of 0.67?C333.33 ng mL^?1, and the lower limit of quantitation (LLOQ) of IG-105 was 0.67 ng mL^?1. The intra- and inter-day precision (relative standard deviation) values were <6%, and the accuracy (relative error) was <5% at three quality control levels. The extraction recovery of IG-105 and IS was 84.45 and 88.5%, respectively. Finally, the validated method was successfully applied to a pharmacokinetic study of IG-105 in rat plasma.     

Validated LC-ESI-MS/MS Method for the Quantitation of Neopanaxadiol: a Novel Neuroprotective Agent from Panax ginseng and Its Application to a Pharmacokinetic Study in Rat Plasma

Neopanaxadiol (NPD), a new panaxadiol first obtained from the acid hydrolysate of the total ginsenosides of Panax ginseng C. A. Meyer (Araliaceae) in our laboratory, showed neuroprotective effect against glutamate-induced neurotoxicity in primary cultures of rat cortical cells (Tao et al. Chin Chem Lett 20:687–689, 2009). In this research, a sensitive and specifical method of ultra-performance liquid chromatography–electrospray mass spectrometry (UPLC-ESI-MS/MS) for the quantitative analysis of NPD in rat plasma was developed. The NPD and internal standard of panaxadiol were extracted from 200 μL of rat plasma samples with methanol, then the samples were injected in an UPLC (Waters BEH) with C18 column (50 mm × 2.1 mm i.d., 1.7 μm) and eluted with the mobile phase consisting of methanol–water–formic acid (80:20:0.2, v/v/v). Excellent linearity was found between 5 and 160 ng mL^?1 with a limit of quantitation of 4 ng mL^?1. Intra- and inter-day precision values (RSD) of QC samples were both below 7 %. This study was successfully utilized for the pharmacokinetic profiles of NPD in rats after oral administration and provided an experimental basis for the further pharmacology and clinical application of ginseng products.