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.     

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.     

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.     

Rapid and Sensitive LC−MS–MS Method for the Determination of Sarpogrelate in Human Plasma

A rapid and sensitive liquid chromatographic–tandem mass spectrometric method has been developed and validated for the estimation of sarpogrelate in human plasma. Sarpogrelate was extracted from human plasma by solid-phase extraction. Temocapril was used as the internal standard. Heated electron spray ionization mass spectrometry was performed on a TSQ Quantum Ultra MS system. The LC column was a Hypurity C₁₈ and the mobile phase was 2 mM ammonium formate (pH 3.00 ± 0.05):acetonitrile (30:70 v/v). A flow rate of 0.250 mL min^?1 was used. The quantitative analyses were carried out in the positive ion and full scan mode over the mass range m/z 60–500. The capillary, vaporiser temperatures were 325 and 200 °C respectively. The sheath gas pressure, spray voltage, collision energy and tube lense were 40, 3,500 V, 19 V, 198 V, respectively, and the mass spectra of the drugs were recorded by total ion monitoring. Retention times and characteristic mass fragments were recorded and the chosen diagnostic mass fragments were monitored in the mass chromatography mode. Signal intensities of each of the mass fragments: m/z 477 [M + H]⁺ for temocapril, m/z 430 [M + H]⁺ for sarpogrelate, were used for quantification. The calibration curves (the ratio between the peak areas as signal intensities of the drug analyzed and that of the internal standard (temocapril: m/z 477 [M + H]⁺) vs. the concentration of drug) exhibited linearity over the concentration range 5.00–2,500.00 ng mL^?1 human plasma. The recovery and the accuracy were calculated by comparing the peak areas as the signal intensities of each mass fragment for the drug in spiked samples after solid-phase extraction from human plasma to the peak area as the signal intensity of the mass fragment of internal standard sample. The method involves a rapid solid phase extraction from plasma, simple isocratic chromatography conditions and mass spectrometric detection that enables detection up to picogram levels with a total run time of 3.0 min only. The method was validated over the range of 5.0–2,500.0 ng mL^?1. The absolute recoveries for sarpogrelate (93.72%) and IS (91.42%) achieved from spiked plasma samples were consistent and reproducible.     

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 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.     

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.     

LC–MS Method for the Quantification of Clonazepam in Rat Plasma

A sensitive and specific high-performance liquid chromatography–tandem mass spectrometry method has been developed and validated for the determination of clonazepam in rat plasma. Clonazepam and internal standard diazepam were extracted from plasma samples by a single-step protein precipitation. The chromatographic separation was performed on a Dikma ODS-C18 reversed-phase column at 40 °C. The mobile phase composed of a premix of solvent A (0.1% formic acid–4 mM ammonium acetate–water)–solvent B (acetonitrile) (13:87, v/v) at a flow-rate of 0.7 mL min^?1. Positive electrospray ionization was utilized as the ionization source. Clonazepam and the internal standard were determined using multiple reaction monitoring of precursor → product ion transitions at m/z 316.0 → 270.0 and m/z 285.1 → 193.2, respectively. The lower limit of quantification was 0.25 ng mL^?1 using 50 μL plasma samples and the linear calibration range was from 0.25 to 128 ng mL^?1. The within- and between-batch RSDs were lower than 15% and the relative recoveries of clonazepam ranged from 97.4 to 104.7%. The mean extraction recoveries of clonazepam and IS were 79.7 and 77.6%, respectively. The method has been successfully applied to the pharmacokinetic studies in rat after oral administration of clonazepam.     

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.     

LC Method for Determination and Pharmacokinetic Study of 5,6,7,8,3′,4′-Hexamethoxy-3-sulfonyl Flavone in Rat Plasma

A sensitive, specific and rapid high-performance liquid chromatography method was developed for determination of 5,6,7,8,3′,4′-hexamethoxy-3-sulfonyl flavone in rat plasma. A simple methanol-induced protein precipitation was applied to extract 5,6,7,8,3′,4′-hexamethoxy-3-sulfonyl flavone and Picroside II (the internal standard) from rat plasma. Chromatographic separation was achieved on a Hypersil ODS₂ analytical column (200 mm × 4.6 mm, 5 μm) with acetonitrile–0.04% triethylamine solution (adjusted to pH 5.8 using phosphoric acid) (24:76, v/v) as mobile phase. The calibration curves were linear over the range of 0.2–40 μg mL^?1. Absolute recoveries of 5,6,7,8,3′,4′-hexamethoxy-3-sulfonyl flavone were 82.7–95.9% from rat plasma. The intra- and inter-day relative standard deviation precisions were less than 5 and 9%, respectively. The method was successfully applied to the pharmacokinetic study of 5,6,7,8,3′,4′-hexamethoxy-3-sulfonyl flavone in rats after intravenous administration.     

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.     

Development and Validation of a Sensitive LC–Tandem-MS Method for the Quantitative Determination of Picroside II in Rat Plasma

A rapid and sensitive method for the quantitative determination of picroside II in rat plasma was developed and validated using liquid chromatographic separation with tandem mass spectrometric detection. The analytes of interest were extracted from rat plasma samples by ethyl acetate after acidification with 1.0% acetic acid solution. Chromatographic separation was achieved on a Hypersil GOLD column (50 × 2.1 mm I.D., 5 μm) using a mobile phase consisting of acetonitrile–0.1% formic acid solution (30:70, v/v) at a flow rate of 0.2 mL min^?1. Detection was performed on a triple quadrupole tandem mass spectrometer by selected reaction monitoring (SRM) mode via electrospray ionization (ESI). The calibration curve was linear in the concentration range of 1.00–400 ng mL^?1 in rat plasma, with a 1.00 ng mL^?1 lower limit of quantification (LLOQ). Satisfactory results were achieved for intraday repeatability [relative standard deviation (RSD) = 6.4–12.4%] and inter-day precision (RSD = 6.8–14.7%). The accuracy in terms of relative error ranged from ?2.1 to 10.0%. The extraction recoveries of picroside II and icariin (internal standard) were 80.0 and 89.3%, respectively. The developed method was successfully employed to determine picroside II plasma concentrations after oral administration to Wistar rats.     

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.     

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.     

LC–ESI-MS Determination of Hydroxycamptothecin in Rat Plasma

A rapid and sensitive method using liquid chromatography with electrospray ionization mass spectrometric detection (LC–ESI-MS) was developed and validated for the determination of hydroxycamptothecin in rat plasma. Plasma samples were extracted with ether and separated on a C₁₈ column interfaced with a single quadrupole mass spectrometer, with mobile phase consisting of 0.1% formic acid–methanol (45:55, v/v). Detection was carried out by positive electrospray ionization (ESI) in selected ion recording (SIR) mode at m/z 321 and 305 for hydroxycamptothecin and camptothecin (internal standard), respectively. The linearity was obtained ranged from 2.5 to 1,000 ng mL^?1 and the lower limit of quantification (LLOQ) was determined to be at 2.5 ng mL^?1. The intra- and inter-day precision (%RSD) was less than 5.5% and accuracy (% RE) ranged from 3.8 to 5.3%. This method was applied successfully to a preliminary pharmacokinetic study following the intravenous administration of hydroxycamptothecin to rats.     

Quantification of Doxorubicin in Pegylated Polymer Micelles in Rat Plasma by Methanol Precipitation�CUltrasonic Emulsion Breaking Method and UPLC�CMS�CMS

This study established a new methanol precipitation?Cultrasonic emulsion breaking method for extraction of doxorubicin from polymeric micelles and developed a UPLC?CMS/MS method for the determination of doxorubicin in rat plasma. The emulsion breaking efficiency of methanol is up to 99.2%. The plasma samples were analyzed by UPLC/MS/MS using positive electrospray ionization mode in the multiple-reaction monitoring (MRM) mode. The calibration curves were linear over the range 5?C1,000 ng mL^?1 with the lower limit of quantification of 5 ng mL^?1. The intra- and inter-day precisions of QC samples were all <10.0%. The chromatographic separation was 2.5 min. The developed method was successfully applied to a pharmacokinetic study of doxorubicin in rats following intravenous administration.     

LC�CMS�CMS Method for Quantification of Hydralazine in BALB/C Mouse Plasma and Brain: Application to Pharmacokinetic Study

A rapid, sensitive and accurate high performance liquid chromatography method using tandem mass spectrometry detection for hydralazine in BALB/C mouse plasma and brain was developed and validated. The method involved a derivatization with 2,4-pentanedione at 50 °C for 1 h, and a step of solid phase extraction to purify and concentrate hydralazine derivative. Chromatographic separation was carried out on an Agilent ZORBAX SB-C18 column by elution with methanol?C0.01 mol L^?1 ammonium acetate (60:40, v/v). The multiple reaction monitoring transition used for quantification was m/z 225.2 ?? 129.5 in the electrospray positive ionization mode. Good linearity was obtained over the concentration range of 10?C200 ng mL^?1. The limits of detection were 0.49 and 1.05 ng mL^?1 for hydralazine in mouse plasma and brain, respectively. The limits of quantitation were 1.5 and 3.18 ng mL^?1 for hydralazine in mouse plasma and brain, respectively. Sample analysis time was 6 min including sample separation. The method was successfully applied to a pharmacokinetic study following intraperitoneal injection of hydralazine in BALB/C mice at the dose of 20 mg kg^?1.