Simultaneous Determination of Pregabalin, Sildenafil and Its Active Metabolite in Rat Plasma Utilising SPE Followed by LC�CMS�CMS

A sensitive and selective analytical method for the quantification of pregabalin, sildenafil and the active desmethyl metabolite of sildenafil (UK-103320) has been developed. The method can simultaneously quantify the three analytes within the expected in vivo concentration ranges using 50 ??L of rat plasma. It utilises solid-phase extraction followed by high performance liquid chromatography coupled with tandem mass spectrometry. Quantitation in rat plasma demonstrated good accuracy and precision over the following dynamic ranges for each analyte: pregabalin (70?C10,000 ng mL^?1), sildenafil (1?C2,000 ng mL^?1) and UK-103320 (1?C2,000 ng mL^?1). For each analyte, the following lower limits of quantitation were obtained: 70 ng mL^?1 for pregabalin and 1 ng mL^?1 for sildenafil and UK-103320, respectively. The method was successfully used to analyse plasma samples from rats when pregabalin and sildenafil were administered in combination.     

HPLC�CMS�CMS for the Simultaneous Determination of Atorvastatin and Amlodipine in Plasma of Hypertensive Patients

Combination drug products containing amlodipine and atorvastatin are widely marketed and used in the treatment of concomitant hypertension and dyslipidemia. A rapid, simple and sensitive high performance liquid chromatography?Ctandem mass spectrometry (HPLC?CMS?CMS) method for determination of atorvastatin and amlodipine in plasma of hypertensive patients has been developed and validated to be used for therapeutic drug monitoring. The plasma samples were subjected to methanol protein precipitation. Chromatographic separation was performed on a C18 column using a gradient elution. The mobile phase consisted of 0.1% of formic acid in water and 0.1% of formic acid in acetonitrile and was pumped at a flow rate of 0.4 mL min^?1. Detection of analytes was achieved by tandem mass spectrometry with electrospray ionization (ESI) interface in positive ion mode. The calibration curves were linear over the range of 0.46?C1,000 ng mL^?1. The intra- and inter-day precisions were within 12.2%, while the accuracy ranged from 92.7 to 108.1%. The validated LC?CMS?CMS method was successfully applied for the determination of atorvastatin and amlodipine in plasma of hypertensive patients.     

LC�CMS�CMS Quantitative Determination of Brivudine in Human Plasma and Its Application to Pharmacokinetic Studies

A sensitive and specific liquid chromatography?Celectrospray ionization?Ctandem mass spectrometry method has been developed and validated for the identification and quantification of brivudine in human plasma using diclofenac as an internal standard. The method involves extraction with ethyl acetate. The analyte was separated on a C₁₈ column and analyzed in multiple reaction monitoring mode with a negative electrospray ionization interface using the [M?CH]^? ions, m/z 332.8??m/z 80.9 for brivudine, m/z 293.6??m/z 249.5 for diclofenac. The method was validated over the concentration range of 5.54?C2,836 ??g L^?1 for brivudine. The intra-and inter-day precisions were less than 8.91% in terms of relative standard deviation (RSD), and the accuracy was within ?4.22% in terms of relative error (RE). The lower limit of quantification (LLOQ) was 5.54 ??g L^?1 with acceptable precision and accuracy. There were almost no matrix effects. Recovery of brivudine spiked in drug-free plasma was higher than 77.17%. The method was used to study the pharmacokinetic profile of brivudine in human plasma after oral administration of brivudine tablets.     

Determination of Imidol in Rat Plasma by UPLC�CMS�CMS and Its Application in a Pharmacokinetic Study

A rapid, sensitive and accurate ultra-performance liquid chromatography/tandem mass spectrometry method was developed and validated for the quantitative determination of imidol in rat plasma for the first time. The analyte and internal standard were extracted from plasma by liquid?Cliquid extraction with diethyl ether. The separation was performed on a BEH C₁₈ column (50 mm × 2.1 mm, 1.7 ??m). The detection was carried out by electrospray ionization mass spectrometry in positive ion mode with multiple reaction monitoring. Linear calibration curves were obtained in the concentration range of 2.5?C2,500 ng mL^?1, with the lower limit of quantification of 2.5 ng mL^?1. The intra- and inter-day precision (RSD) values were below 8% and accuracy (RE) was from ?7.9 to 6.3%. After strict validation, the method was applied successfully to the pharmacokinetic study of imidol in rats after oral and intravenous administration, respectively.     

Determination of Pilocarpine in Human Plasma by LC�CAPCI�CMS�CMS and Application to a Pharmacokinetic Study

A sensitive, specific and rapid high performance liquid chromatography?Catmospheric pressure chemical ionization source-tandem mass spectrometry (LC-APCI-MS-MS) method for the determination of pilocarpine in human plasma was developed and validated. The method is based on liquid?Cliquid extraction, followed by a reversed-phase liquid chromatographic separation, and detected by means of tandem mass spectrometry. The linear calibration curve covered a concentration range of 2?C500 ??g L^?1. The intra- and inter-day precisions for pilocarpine were <10% and the accuracies were between 90 and 110%. The method was applied successfully to a pharmacokinetic study involving 20 healthy Chinese male volunteers after oral administration of 6 mg pilocarpine.     

Simultaneous Determination of Pethidine and Atropine in Rabbit Plasma by LC�CMS�CMS and Its Application to a Pharmacokinetic Study after Intramuscular Administration

A sensitive and selective liquid chromatography?Ctandem mass spectrometry method for the determination of pethidine and atropine in rabbit plasma was developed and validated. The analytes and internal standard (IS) are extracted from plasma by liquid?Cliquid extraction using ethyl acetate, and separated on a Zorbax SB-Aq column (2.1 × 150 mm, 3.5 ??m) using acetonitrile?C0.1% formic acid as mobile phase with gradient elution. Electrospray ionization source was applied and operated in positive ion mode, and multiple reaction monitoring mode was used for quantification using target fragment ions m/z 247.8 ?? 219.7 for pethidine, m/z 289.9 ?? 123.8 for atropine and m/z 295.0 ?? 266.8 for IS, respectively. The assay is linear over the range of 5?C1,000 ng mL^?1 for pethidine and atropine, with a lower limit of quantification of 3 ng mL^?1 for pethidine and 5 ng mL^?1 for atropine. Intra-day and inter-day precision are less than 11% and the accuracy are in the range of 90.4?C106.3%. Furthermore, the newly developed method is successfully used for the determination of pethidine and atropine in rabbit plasma for pharmacokinetic study.     

Determination of Revaprazan in Human Plasma by LC�CMS�CMS and Application to a Pharmacokinetic Study in Chinese Volunteers

A sensitive, specific, and rapid liquid chromatography?Celectrospray ionization?Ctandem mass spectrometry (LC?CESI?CMS?CMS) method was developed for determination of revaprazan in human plasma. Plasma samples were simply treated with methanol to precipitate, and then isolated supernatants were directly injected into the LC?CESI?CMS?CMS system. A Thermo Hypurity C18 column (150 × 2.1 mm, 5 ??m) with mobile phase of methanol?Cwater (70:30, v/v) containing 0.05% formic acid was used for chromatographic separation. Mass-spectrometric quantification was carried out in multiple reaction monitoring (MRM) mode, monitoring the m/z transitions 363.1 ?? 245.1 for revaprazan and 531.2 ?? 489.2 for ketoconazole (internal standard, IS) in positive ion mode. The linear calibration curves covered a concentration range of 2?C1,000 ??g L^?1. The intra- and interday precisions (percentage relative standard deviation, RSD%) for revaprazan at three quality control levels were all <5%, and the accuracies were between 90% and 110%. The method has been successfully applied to a pharmacokinetic study involving 12 Chinese volunteers, and the main pharmacokinetic parameters of revaprazan in Chinese population are reported for the first time.     

Rapid and Simultaneous Determination of Efavirenz, 8-Hydroxyefavirenz, and 8,14-Dihydroxyefavirenz Using LC�CMS�CMS in Human Plasma and Application to Pharmacokinetics in Healthy Volunteers

We developed a rapid and sensitive method for determining efavirenz, 8-hydroxyefavirenz, and 8,14-dihydroxyefavirenz in human plasma simultaneously using liquid chromatography?Ctandem mass spectrometry (LC?CMS?CMS). Three compounds and ritonavir, an internal standard, were extracted from plasma using ethyl acetate in the presence of 0.1 M sodium carbonate after incubation of ??-glucuronidase (500 U). After drying the organic layer, the residue was reconstituted in mobile phase (acetonitrile:20 mM ammonium acetate, 90:10, v/v) and injected onto a reversed-phase C₁₈ column. The isocratic mobile phase was eluted at 0.2 mL min^?1. The ion transitions monitored in multiple reaction-monitoring mode were m/z 314 ?? 244, 330 ?? 258, 346 ?? 262, and 721 ?? 296 for efavirenz, 8-hydroxyefavirenz, 8,14-dihydroxyefavirenz, and ritonavir, respectively. The retention time is 1.93, 1.70, 1.52, and 1.82 min for efavirenz, 8-hydroxyefavirenz, 8,14-dihydroxyefavirenz, and ritonavir, respectively. The coefficients of variation of the assay precision were less than 10.7%, and the accuracy was 90?C111%. The lower limits of quantification (LLOQ) were 5 ng mL^?1 for efavirenz and 8-hydroxyefavirenz. This method was used to measure the plasma concentrations of efavirenz and its metabolites from healthy volunteers after a single 600 mg oral dose of efavirenz. This analytical method is a very rapid, sensitive, and accurate to determine the pharmacokinetic profiles of efavirenz including its metabolites.     

Rapid Simultaneous Determination of Eight Benzimidazoles in Animal Feed by LC�CMS�CMS

A rapid, sensitive and reliable LC?CMS?CMS method for the determination of eight benzimidazoles in animal feed was developed and validated. Samples were extracted with acidic acetonitrile. The extract was diluted with 0.1% formic acid in water, and analyzed by LC?CMS?CMS on a Waters XBridge? C₁₈ column with acetonitrile/0.1% formic acid in water as mobile phase. The samples were quantified with the matrix standard calibration curve method. Good linearity was obtained for eight benzimidazoles at a concentration of 0.005?C2.5 ??g mL^?1 with a linear relative coefficient more than 0.990. Recoveries of 84.0?C104.0% with CVs of 2.50?C7.50% were obtained. Limit of detection was 2.1?C63.0 ??g kg^?1. The method demonstrated to be suitable for the determination of eight benzimidazoles in animal feed samples.     

Simultaneous Determination of Ramipril and Its Active Metabolite Ramiprilat in Human Plasma by LC–MS–MS

A selective, rapid and sensitive liquid chromatography tandem mass spectrometry method has been developed for the simultaneous determination of ramipril and ramiprilat in human plasma using enalapril as the internal standard via one-step extraction with ethyl acetate under acidic condition. The analysis was carried out on a Diamonsil C₁₈ column (150 mm × 4.6 mm i.d., 5 μm) with a mobile phase consisting of 1% formic acid-acetonitrile (25:75, v/v) at a constant flow rate of 0.5 mL min^?1. The detection was performed on a triple-quadruple tandem mass spectrometer by selective reaction monitoring mode via electrospray ionization. Linear calibration curves of ramipril and ramiprilat were obtained in the concentration range of 0.107–107.0 and 0.262–105.0 ng mL^?1, respectively. The intra- and inter-day precision (RSD) values were below 8.2 and 4.8% for ramipril, 10.4 and 12.3% for ramiprilat, and accuracy (RE) were within ±5.5 and ±3.2%, respectively at all QC levels. The method was utilized to support clinical pharmacokinetic studies in healthy volunteers following oral administration of ramipril tablets.     

LC�CMS�CMS Quantitative Determination of Gefitinib in Human Serum and Cerebrospinal Fluid

A specific, sensitive, and rapid method based on high-performance liquid chromatography coupled to tandem mass spectrometry (LC?CMS?CMS) was developed for determination of gefitinib in human serum and cerebrospinal fluid (CSF). The analyte was detected by tandem mass spectrometry operating in positive electrospray ionization mode with multiple reaction monitoring (MRM). Gefitinib was extracted from serum or CSF samples with ethyl acetate using icotinib as internal standard. The method was validated over the concentration range of 1.00?C1,000 ng mL^?1 in human serum and 0.05?C50.0 ng mL^?1 in CSF. For both matrices, inter- and intraday precision (CV%) were less than 15% and accuracy was within 85?C115%. Average extraction recoveries were 78.9 and 61.8% in human serum and CSF, respectively. Linearity, recovery, matrix effects, and stability were validated in the two matrices. The method was successfully used for analysis of clinical samples from lung cancer patients with brain metastases treated with gefitinib in the dosage range of 250?C500 mg day^?1.     

Simultaneous LC−UV Analysis of Mirodenafil and Its Two Main Metabolites in Rat Plasma and Urine, and in Tissue Homogenates

A simple, rapid, and reproducible isocratic reversed-phase LC method has been established for simultaneous analysis of mirodenafil and its two main metabolites, SK3541 and SK3544, in rat plasma, urine, and tissue homogenates. Samples were deproteinized with acetonitrile containing sildenafil (internal standard). The compounds were separated on a C₁₈ column with 52:48 (v/v) 0.02 m ammonium acetate buffer (pH 6)—acetonitrile as mobile phase at a flow rate of 1.4 mL min^?1. UV detection was at 254 nm and detection limits of mirodenafil, SK3541, and SK3544 in plasma were 0.03, 0.05, and 0.1 μg mL^?1, respectively. The method is applicable to pharmacokinetic studies of mirodenafil and its metabolites in rats.     

Simultaneous Determination of Tamsulosin and Dutasteride in Human Plasma by LC–MS–MS

A sensitive and selective liquid chromatographic tandem mass spectrometric (LC–MS–MS) method was developed for simultaneous identification and quantification of tamsulosin and dutasteride in human plasma, which was well applied to clinical study. The method was based on liquid–liquid extraction, followed by an LC procedure with a Gemini C-18, 50 mm × 2.0 mm (3 μm) column and using methanol:ammonium formate (97:3, v/v) as the mobile phase. Protonated ions formed by a turbo ionspray in positive mode were used to detect analytes and internal standard. MS–MS detection was by monitoring the fragmentation of 409.1 → 228.1 (m/z) for tamsulosin, 529.3 → 461.3 (m/z) for dutasteride and 373.2 → 305.3 (m/z) for finasteride (IS) on a triple quadrupole mass spectrometer. The lower limit of quantification for both tamsulosin and dutasteride was 1 ng mL^?1. The proposed method enables the unambiguous identification and quantification of tamsulosin and dutasteride for clinical drug monitoring.     

Simultaneous Determination of Atenolol and Chlorthalidone by LC–MS–MS in Human Plasma

A simple, sensitive, selective and rapid liquid chromatography–tandem mass spectrometry method was developed and validated for the simultaneous separation and quantitation of atenolol and chlorthalidone in human plasma using metoprolol and hydrochlorothiazide as internal standard. Following solid phase extraction, the analytes were separated by an isocratic mobile phase on a reversed-phase C₁₈ column and analyzed by MS in the multiple reaction-monitoring mode (atenolol in positive and chlorthalidone in the negative ion mode). The limit of quantitation for this method was 10 and 15 ng mL^?1 and the linear dynamic range was generally 10–2,050 ng mL^?1 and 15–3,035 ng mL^?1 for atenolol and chlorthalidone, respectively.     

Simultaneous Determination of Paracetamol and Caffeine in Human Plasma by LC–ESI–MS

A rapid, selective and convenient liquid chromatography–mass spectrometric method for the simultaneous determination of paracetamol and caffeine in human plasma was developed and validated. Analytes and theophylline [internal standard (I.S.)] were extracted from plasma samples with diethyl ether-dichloromethane (3:2, v/v) and separated on a C₁₈ column (150 × 4.6 mm ID, 5 μm particle size, 100 Å pore size). The mobile phase consisted of 0.2% formic acid–methanol (60:40, v/v). The assay was linear in the concentration range between 0.05 and 25 μg mL^?1 for paracetamol and 10–5,000 ng mL^?1 for caffeine, with the lower limit of quantification of 0.05 μg mL^?1 and 10 ng mL^?1, respectively. The intra- and inter-day precision for both drugs was less than 8.1%, and the accuracy was within ±6.5%. The single chromatographic analysis of plasma samples was achieved within 4.5 min. This validated method was successfully applied to study the pharmacokinetics of paracetamol and caffeine in human plasma.     

Determination of Quinine and Doxycycline in Rat Plasma by LC�CMS�CMS: Application to a Pharmacokinetic Study

A fast, sensitive, and specific LC?CMS?CMS method for determination of quinine (QN) and doxycycline (DOX) in rat plasma has been developed and validated. QN, DOX, and cimetidine (internal standard, IS) were extracted from the plasma by protein precipitation. The compounds were separated on a C₁₈ column with methanol?C0.1% aqueous formic acid 70:30 (v/v) as mobile phase at a flow rate of 0.5 mL min^?1 (split 1:3). Detection was by positive electrospray ionization (ESI+) in multiple reaction monitoring (MRM) mode, monitoring the transitions 325.0 ?? 307.0, 445.0 ?? 428.1, and 252.8 ?? 159.0, for QN, DOX, and IS, respectively. The analysis was carried out in 2.0 min and the method was linear in the plasma concentration range 5?C5,000 ng mL^?1. The mean extraction recoveries for QN, DOX, and IS from plasma were 89.4, 90.5, and 86.3%, respectively. The method was validated for linearity, precision, accuracy, specificity, and stability; the results obtained were within the acceptable range. The proposed method was successfully applied to the determination of QN and DOX in rat plasma samples to support pharmacokinetic studies.     

Simultaneous Determination of Escin Ia and Its Isomer Isoescin Ia by LC�CMS�CMS: Application to a Pharmacokinetic Study of Escin Ia in Rats

A rapid and sensitive LC?CMS?CMS method for the simultaneous determination of escin Ia and isoescin Ia in rat plasma, urine, feces and bile samples was developed and validated. Analytes and telmisartan [internal standard (IS)] were extracted by solid-phase extraction on C₁₈ cartridges. Components in the extract were separated on an HC-C₁₈ column (5 ??m, 150 × 4.6 mm i.d.) using 10 mM ammonium acetate?Cmethanol?Cacetonitrile (40:30:30, v/v/v) as the mobile phase. The method demonstrated good linearity from 5 ng mL^?1 (LLOQ) to 1,500 ng mL^?1 for both escin Ia and isoescin Ia. Intra- and inter-day precision measured as RSD was within ±15%. Recoveries and matrix effects of both escin Ia and isoescin Ia were satisfactory in all four matrices examined. The method was successfully applied to a pharmacokinetic study in Wistar rats after a single intravenous administration of escin Ia at the dose of 1.0 mg kg^?1.     

Extraction and Determination of Two Antidepressant Drugs in Human Plasma by Dispersive Liquid–Liquid Microextraction‒HPLC

In this study, an effective method of ultrasound-assisted ionic liquid based dispersive liquid–liquid microextraction (UA?IL?DLLME) coupled with HPLC was applied for extraction and determination of two antidepressant drugs: venlafaxine hydrochloride and amitriptyline hydrochloride from human plasma samples. Three ionic liquids were studied: 1-butyl-3-methyl imidazolium hexafluorophosphate, 1-hexyl-3- methyl imidazolium hexa-fluoro-phosphate, and 1-octyl-3-methyl imidazolium hexafluorophosphate [C8MIM][PF6]. Various factors affect the stages and efficiency of extraction, some of which are pH of sample solution, type and volume of ionic liquid, the time of ultrasonication, centrifuging time and rate, and the ionic strength of solution. In this research, optimum conditions were obtained as 55 μL of [C8MIM][PF6] selected as ionic liquid, pH 11, 2% NaCl, 4 min ultrasonication and 5 min centrifuging at 3500 rpm. Under the optimized conditions, the linearity was obtained in the range of 0.2 to 250 μg/L. The limits of detection were 0.5 μg/L for venlafaxine and 0.8 μg/L for amitriptyline. Pre-concentration factors were 1.3 × 10³ for venlafaxine and 1.2 × 10³ for amitriptyline. The UA?IL?DLLME method coupled with HPLC was successfully used for the determination of venlafaxine and amitriptyline spiked into the real samples of human plasma.     

Determination of Fulvestrant in Rat Plasma by LC�CMS�CMS: Application to a Pharmacokinetic Study

A reversed-phase liquid chromatography coupled to tandem mass spectrometry (LC?CMS?CMS) method was developed and validated for the determination of fulvestrant in rat plasma. Sample preparation involved a liquid-liquid extraction using 1.0 mL of n-hexane?Cisopropanol (90:10, v/v) to extract the analyte from 0.1 mL of rat plasma. The analytes were separated on a phenyl-based column using the mobile phase consisting of methanol/water containing 5 mM ammonium acetate at the flow rate of 0.3 mL min^?1. The analytes were monitored by tandem mass spectrometry under electrospray negative ionization mode. Linear calibration curves were generated over the fulvestrant concentration ranges of 0.05?C10.0 ng mL^?1 in rat plasma. The accuracy and within- and between-day precisions were within the generally accepted criteria for bioanalytical methods (<15%). This developed and validated assay method was successfully employed to characterize the plasma concentration-time profile of fulvestrant after its intramuscular administration in rats at a dose of 10 mg kg^?1.