Simultaneous Determination of Clopidogrel and Its Carboxylic Acid Metabolite (SR26334) in Human Plasma by LC–ESI–MS–MS: Application to the Therapeutic Drug Monitoring of Clopidogrel

A sensitive and specific liquid chromatography-tandem-mass spectrometry method was developed and validated for the simultaneous determination of clopidogrel and its carboxylic acid metabolite (SR26334) in human plasma using nateglinide and pioglitazone as internal standards. Analytes were extracted from 0.50 mL of plasma using diethyl ether–n-hexane (4:1, v/v). Chromatographic separation was performed on a Teknokroma C₁₈ column with a mobile phase of methanol–water (containing 0.1% formic acid) (80:20, v/v) at a flow rate of 0.20 mL min^?1 within 5.6 min. Linearity was established over the concentration range of 0.005–5 ng mL^?1 for clopidogrel and 20–2,500 ng mL^?1 for SR26334. Intra- and inter-batch standard deviations were less than 9.2% and the accuracy of this assay was found to fall within an acceptable range ≤10.0%. The method was successfully applied to the therapeutic drug monitoring of clopidogrel.     

LC-MS-MS Method for Simultaneous Determination of Icariin and Its Active Metabolite Icariside II in Human Plasma

An LC-MS-MS method was revised and validated for simultaneous determination of icariin and its active metabolite icariside II in human plasma. The analytes and daidzein (IS) were extracted by liquid–liquid extraction and analyzed by LC-MS-MS. The separation was performed by a Zorbax SB-C₁₈ column (3.5 μm, 2.1 × 100 mm) with an isocratic mobile phase consisting of methanol–water–formic acid (65:35:0.035, v/v/v) at a flow rate of 0.25 mL min^?1. Detection was performed on a triple quadrupole tandem mass spectrum by multiple reaction monitoring mode using the electrospray ionization technique in positive mode. The method had lower limits of quantitation 0.2 and 0.1 ng mL^?1 for icariin and icariside II, respectively, using 500 μL plasma sample. The linear calibration curves were obtained in the concentration range of 0.2–100 ng mL^?1 for icariin and 0.1–100 ng mL^?1 for icariside II. The RSD values of intra- and inter-day precision calculated from quality control (QC) samples were less than 7.2% for icariin and less than 6.5% for icariside II. The accuracy as determined from QC samples was within 3.8% for each analyte. The method has been applied to determine and evaluate the pharmacokinetic of icariin and its metabolite icariside II in volunteers following oral administration of icariin and extract of Epimedium, respectively.     

Sensitive Quantification of Carboxylic Acid Metabolite of Clopidogrel in Human Plasma by LC with UV Detection

A rapid LC method with UV detection was developed for the quantification of carboxylic acid metabolite of clopidogrel in human plasma. Following a simple protein precipitation using a mixture of methanolic solution of ZnSO₄, the analyte and commercially available internal standard were separated using a mobile phase of water–acetonitril (85:15, v/v) adjusted to pH 3.5 on a Chromolith C18 column at a flow rate of 2.5 mL min^?1 with a total retention time of 4 min. Linearity was verified over the range of 20–3,000 ng mL^?1 where the LOQ was 20 ng mL^?1. This method was applied in a pharmacokinetic study.     

An LC Method for the Determination of Bupropion and Its Main Metabolite, Hydroxybupropion in Human Plasma

A new LC method has been developed and validated for the direct determination of bupropion and its main metabolite, hydroxybupropion in human plasma. Plasma samples were analyzed after a simple, one step protein precipitation with trichloroacetic acid using a C₈ column and mobile phase, consisting of methanol/acetonitrile/phosphate buffer (10 mM, pH 3.0) (40:10:50, v/v/v) and 20 mM 1-heptane sulfonic acid sodium salt with carbamazepine as the internal standard. UV detection was performed at 214 and 254 nm. The method was validated over the concentration range of 60–2,400 and 150–4,700 ng mL^?1 for bupropion and hydroxybupropion, respectively. The intra- and inter-day assay variability was less than 15% for the two analytes. Limit of detection values were 24.8 and 63.4 ng mL^?1 for bupropion and hydroxybupropion, respectively. The method developed was applied to quantification of bupropion and hydroxybupropion in human plasma.     

Simultaneous Quantitation of Paracetamol, Pseudoephedrine and Chlorpheniramine in Dog Plasma by LC-MS-MS

A simple, rapid and sensitive liquid chromatography/electrospray tandem mass spectrometry quantitative detection method, using amantadine as internal standard, was developed for the simultaneous analysis of paracetamol, pseudoephedrine and chlorpheniramine concentrations. Analytes were extracted from plasma samples by liquid–liquid extraction with n-hexane–dichloromethane–2-propanol (2:1:0.1, v/v), separated on a C18 reversed-phase column with 0.1% formic acid–methanol (40:60, v/v) and detected by electrospray ionization mass spectrometry in positive multiple reaction monitoring mode. Calibration curves for plasma were linear over the concentration range 10–10,000 ng mL^?1 of paracetamol, 2–2,000 ng mL^?1 of pseudoephedrine and 0.2–200 ng mL^?1 of chlorpheniramine. The method has a lower limit of quantitation of 10 ng mL^?1 for paracetamol, 2.0 ng mL^?1 for pseudoephedrine and 0.2 ng mL^?1 for chlorpheniramine. Recoveries, precision and accuracy results indicate that the method was reliable within the analytical range, and the use of the internal standard was very effective for reproducibility by LC-MS-MS. This method is feasible for the evaluation of pharmacokinetic profiles of a novel multicomponent sustained release formulation containing 325 mg of paracetamol, 30 mg of pseudoephedrine hydrochloride and 2 mg of chlorpheniramine maleate. It is the first time the pharmacokinetic evaluation of a novel sustained-action formulation containing paracetamol, pseudoephedrine and chlorpheniramine has been elucidated in vivo using LC-MS-MS.     

LC–ESI–MS Determination of Imperatorin in Rat Plasma After Oral Administration and Total Furocoumarins of Radix Angelica dahuricae and its Application to a Pharmacokinetic Study

A simple, rapid, sensitive and reliable liquid chromatography–electrospray ionization mass spectrometry method for the quantification of imperatorin in rat plasma after oral administration and total furocoumarins of Radix Angelica dahuricae has been established. The plasma samples were deproteinized by adding internal standard (IS) osthole solution, which was prepared by acetonitrile. The analysis was performed on a Shim-pack C₁₈ column (150 × 2.0 mm i.d., 5 μm) using acetonitrile and 0.5% formic acid solution (70:30, v/v) as a mobile phase. The detection was performed on a quadrupole mass spectrometer detector with an ESI interface operated in the selected ion monitoring mode. The linear quantification range of the method was 2–4000 ng mL^?1 in rat plasma with a correlation coefficient greater than 0.99, the limit of detection (LOD) was 0.5 ng mL^?1 and the lower limit of quantification (LLOQ) 2 ng mL^?1. The intra- and inter-day relative standard deviations (RSD) were less than 2.5 and 3.5%, respectively. The recoveries were above 90%. The validated method was successfully applied to a pharmacokinetic study of imperatorin in rats after oral administration and total furocoumarins of Radix Angelica dahuricae.     

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.     

An Accurate Assay for Simultaneous Determination of Irinotecan and Its Active Metabolite SN-38 in Rat Plasma by LC with Fluorescence Detection

An accurate LC method was developed and validated for simultaneous determination of irinotecan (CPT-11) and its active metabolite SN-38 in rat plasma. Plasma samples were pretreated with 0.4 g mL^?1 sodium dodecyl sulfate to inactive the carboxylesterase and avoid the conversion of CPT-11 to SN-38. Chromatographic separation was achieved on a Diamaonsil C₁₈ column using acetonitrile–50 mM phosphate buffered solution (30:70, v/v) at pH 4.0 as the mobile phase with the flow rate of 1 mL min^?1. The linear quantitation ranges for CPT-11 and SN-38 were 5.05–3,030 and 3.15–315 ng mL^?1 with r ² > 0.99, respectively. The lower limit of quantification (LLOQ) was 2.33 ng mL^?1 for CPT-11 and 0.26 ng mL^?1 for SN-38 with intra- and inter-day relative standard deviation of <12% and the accuracy values of >90%. The method was proved to be accurate and sensitive enough and was successfully applied to a pharmacokinetic study of CPT-11 in rats.     

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.     

Rapid LC-APCI-MS-MS Method for Simultaneous Determination of Phenacetin and Its Metabolite Paracetamol in Rabbit Plasma

A highly sensitive liquid chromatographic-atmospheric pressure chemical ionization-tandem mass spectrometric method is developed to quantitate phenacetin and its metabolite paracetamol in rabbit plasma. The analytes and internal standard oxazepam are extracted from plasma by liquid–liquid extraction using ethyl acetate, and separated on a Zorbax SB-C₁₈ column (2.1 mm × 150 mm, 5 μm) using acetonitrile–0.1% formic acid in water (40:60 v/v) at a flow of 0.4 mL min^?1. Detection is carried out by multiple reaction monitoring on a ion-trap LC-MS-MS system with an atmospheric pressure chemical ionization interface. The assay is linear over the range 4–1,600 ng mL^?1 for phenacetin and 3–2,000 ng mL^?1 for paracetamol, with a lower limit of quantitation of 4 ng mL^?1 for phenacetin and 3 ng mL^?1 for paracetamol. Intra- and inter-day precision are less than 7.1% and the accuracy are in the range 97.3–103.5%. The validated method is successfully used to analyze the drug in samples of rabbit plasma for pharmacokinetic study.     

LC Determination and Pharmacokinetic Study of Gemfibrozil in Human Plasma

A simple and sensitive LC method for the quantitative determination of gemfibrozil in human plasma samples is described. Mometasone furoate was used as the internal standard. Plasma samples were pretreated by protein precipitation using methanol. Separation was performed at 40 °C on a YMC^® ODS-A reverse phase column (5 μm particle size, 150 mm × 4.6 mm i.d.) using 0.2% (v/v) triethylamine in water (adjusting to pH 4.0 with phosphoric acid) and acetonitrile (45:55, v/v) as mobile phase which was delivered at 1.5 mL min^?1. Ultraviolet detection was performed at 230 nm. The linear concentration range for gemfibrozil was 0.25–50 μg mL^?1. The detection limit of this method was 0.1 μg mL^?1. Intra- and inter-assay RSD ranged from 0.63 to 2.04% and 1.37 to 4.27%, respectively. The method was sensitive, simple and repeatable enough to be used in pharmacokinetic studies.     

Simultaneous Determination of Clopidogrel and Its Carboxylic Acid Metabolite (SR26334) in Human Plasma by LC–ESI–MS–MS: Application to the Therapeutic Drug Monitoring of Clopidogrel

A sensitive and specific liquid chromatography-tandem-mass spectrometry method was developed and validated for the simultaneous determination of clopidogrel and its carboxylic acid metabolite (SR26334) in human plasma using nateglinide and pioglitazone as internal standards. Analytes were extracted from 0.50 mL of plasma using diethyl ether–n-hexane (4:1, v/v). Chromatographic separation was performed on a Teknokroma C₁₈ column with a mobile phase of methanol–water (containing 0.1% formic acid) (80:20, v/v) at a flow rate of 0.20 mL min⁻¹ within 5.6 min. Linearity was established over the concentration range of 0.005–5 ng mL⁻¹ for clopidogrel and 20–2,500 ng mL⁻¹ for SR26334. Intra- and inter-batch standard deviations were less than 9.2% and the accuracy of this assay was found to fall within an acceptable range ≤10.0%. The method was successfully applied to the therapeutic drug monitoring of clopidogrel.

     

LC–MS Determination of p-(1-Dimethylamino ethylimino)aniline: a Metabolite of Tribendimidine in Human Plasma

A sensitive and selective liquid chromatography–mass spectrometric method was developed and validated for the determination of p-(1-dimethylamino ethylimino)aniline (dADT), a metabolite of tribendimidine, in human plasma. The analyte was separated on a Hypersil C₁₈ column (250 × 4.6 mm ID, 5 μm) by isocratic elution with 10 mM ammonium acetate (containing 0.1% triethylamine)-acetonitrile (20:80, v/v) at a flow rate of 1.0 mL min^?1 and measured by electrospray ionization source in positive selective ion monitoring mode at m/z 178. The weighted (1/x ²) calibration curve was linear within a concentration range of 0.5–1,000 ng mL^?1 and displayed a correlation coefficient (r) of 0.9996. The lower limit of quantification was determined to be at 0.5 ng mL^?1. The inter and intra-day precisions (%RSD) were less than 8% and the extraction recoveries ranged from 84.21 to 85.20%. The developed method was successfully applied to the determination of dADT in human plasma as part of a clinical pharmacokinetic study.     

Development and Validation of LC-UV for Simultaneous Estimation of Rosiglitazone and Glimepride in Human Plasma

A simple, sensitive and specific liquid chromatographic method with UV detection (228 nm) was developed for the simultaneous estimation of rosiglitazone and glimepride in human plasma. Rosiglitazone and glimepride were extracted from plasma using liquid–liquid extraction. Separation was achieved with an RP C₁₈ Column using a mixture of phosphate buffer (50 mM) with octane sulfonic acid (10 mM), methanol and acetonitrile as a mobile phase (55:10:35, v/v). pH was adjusted to 7.0. Amlodipine was used as an internal standard (IS). LOD of the method was found to be 20 ng mL^?1 for both drugs. Results were linear over the studied range 40.994–2007.556 ng mL^?1 for rosiglitazone (r ² ≥ 0.99) and 41.066–2094.84 ng mL^?1 for glimepride( r ² ≥ 0.99). The method was found to be simple, selective, precise and reproducible for the estimation of both drugs from spiked human plasma.     

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.     

Development and Validation of an RP–LC–UV Method for the Determination of Ondansetron: Application to Pharmaceutical Dosage Forms

A new, simple, rapid, sensitive and specific isocratic RP–LC–UV method was developed and validated for the determination of ondansetron in pharmaceutical dosage forms of orally disintegrating tablets, oral solution and injection. The LC separation was achieved on a Hypersil C4 column (250 × 4.6 mm, 5 μm) using a mobile phase of 50 mM potassium dihydrogen phosphate anhydrous adjusted to pH 3.5 with orthophosphoric acid and acetonitrile (30:70, v/v) at a flow rate of 1.0 mL min^?1 and UV detection at 310 nm. The method was validated for specificity, linearity, precision, accuracy, limit of quantification, limit of detection, robustness and solution stability. The calibration curve was linear over a concentration range of 100–1,000 ng mL^?1 (r ²  = 0.9996) with limit of detection and limit of quantification 50 and 100 ng mL^?1, respectively. The intra-day and inter-day precision and accuracy were between 0.79 and 2.37% and ?0.64 and 1.65%, respectively. The method was successfully applied for analysis of ondansetron in the presence of excipients in commercially available pharmaceutical dosage forms.     

Highly sensitive simultaneous quantification of buprenorphine and norbuprenorphine in human plasma by magnetic solid-phase extraction based on P<Emphasis Type="Italic">p</Emphasis>PDA/Fe<Subscript>3</Subscript>O<Subscript>4</Subscript> nanocomposite and high-performance liquid chromatography

A rapid and sensitive method based on magnetic solid-phase extraction coupled to high-performance liquid chromatography with ultraviolet detection was developed for the simultaneous determination of buprenorphine (BPN) and its major metabolite, norbuprenorphine (N-BPN), in human plasma samples. Poly (para-phenylenediamine)-modified Fe₃O₄ nanoparticles (PpPDA/Fe₃O₄) were synthesized and used as a magnetic adsorbent for the extraction and preconcentration of BPN and N-BPN in biological samples. The synthesized nanocomposites were characterized by X-ray diffraction, Fourier transform infrared spectroscopy, field emission scanning electron microscopy, transmission electron microscopy, energy-dispersive X-ray spectroscopy and vibrating sample magnetometery. An isocratic separation was achieved on a Nova-Pak C18 reversed-phase column using a mobile phase consisting phosphate buffer (pH 3.4) and acetonitrile (50:50, v/v) at a flow rate of 1.0 mL min^?1. The detection was conducted at 280 nm. Under the optimum conditions, the calibration curves for BPN and N-BPN were linear in the ranges 3.0–150.0 and 1.0–120.0 ng mL^?1, respectively. The sensitivity was also high with limit of detection of 0.8 and 0.3 ng mL^?1 for BPN and N-BPN in plasma, respectively. The method was successfully applied to the extraction and determination of BPN and N-BPN in human plasma samples with an average recovery of 98.10 and 96.41%, respectively.     

Development and Validation of an Accurate LC Method for the Quantitative Determination of Voriconazole in a New Emulsion Formulation

A simple, sensitive and accurate liquid chromatographic method with UV detection was developed and validated to determine voriconazole in a new emulsion formulation. Chromatographic separation was achieved on a Diamonsil C₁₈ column (250 × 4.6 mm I.D., 5 μm) using a mobile phase consisting of acetonitrile-water-acetic acid (40:60:0.25, v/v/v) at a flow rate of 1.0 mL min^?1. The UV detection wavelength was set at 256 nm. The linear calibration curves were obtained in the concentration range of 1.00–100 μg mL^?1 with the limit of quantification of 1.00 μg mL^?1. The within- and between-run precisions in terms of percentage relative standard deviation were lower than 7.4 and 7.1%, respectively. The accuracy in terms of percentage relative error ranged from ?1.5 to 1.4%. This validated method was successfully applied to the determination of the content of voriconazole in a new emulsion formulation.     

Dissipation dynamics of fenoxaprop-p-ethyl and fenoxaprop acid under Indian rice field conditions

Dissipation dynamics of fenoxaprop-p-ethyl (FPPE) and fenoxaprop acid (FPA) (metabolite) in rice field conditions was investigated for two consecutive seasons. FPPE dissipated rapidly in soil with average half life of 1.42–2.19 days. Dissipation followed first-order kinetics. The method was validated in terms of accuracy, linearity, specificity and precision. Linearity was in the range of 0.005–5 µg mL^?1 with limit of detection as 0.002 and 0.001 µg mL^?1 for fenoxaprop-p-ethyl and fenoxaprop acid, respectively. Quantitation limit in soil, grain, straw and husk were 0.005, 0.008, 0.01 and 0.01 µg g^?1 for fenoxaprop-p-ethyl, and 0.005, 0.01, 0.01 and 0.01 µg g^?1 for fenoxaprop acid, respectively. Recovery in soil, rice grains, straw and husk ranged from 81.60–93.40, 77.85–87.00, 75.20–84.40 and 76.00–87.20% for FPPE and 82.50–88.20, 76.25–83.00, 74.80–83.60 and 75.00–85.40% for FPA, respectively. Below detectable limit of residues of FPPE and metabolite were observed in soil, rice grain, husk and straw samples at harvest. FPPE and FPA were of short persistence under field conditions and residues were below European Union-Maximum Residue Limits in all matrices that would cause adverse effect on environment and human/animal health.     

Analysis of Isopropyl 3-(3,4-Dihydroxyphenyl)-2-hydroxypropanoate, a Metabolite of Danshensu, from Salvia miltiorrhiza, in Rabbit Plasma

Isopropyl 3-(3,4-dihydroxyphenyl)-2-hydroxypropanoate (IDHP), a metabolite of Danshensu, from Salvia miltiorrhiza, has been proved to have potential as a novel drug for regulation of vasomotor activity in small-resistance vascular circulation. In this presentation we report a new specific method for analysis of IDHP in rabbit plasma. Plasma samples were pretreated with 1.5% formic acid in acetonitrile to remove the protein, and the resulting supernatant was extracted with ethyl acetate. Chromatographic separation was achieved on a C₁₈ column with 15.0% acetonitrile in 0.3% aqueous formic acid (pH 2.2) as mobile phase. Multiple-reaction-mode ion-trap mass spectrometry was selected for accurate analysis of IDHP. The calibration plot was linear in the range 0.1–200.0 ng mL^?1 for plasma samples. The detection limit was 0.02 ng mL^?1. Intra-day and inter-day coefficients of variation were <13.0% and intra-day and inter-day accuracy was within ±8.0% of known concentrations. Finally, the method was used to investigate the pharmacokinetics of IDHP in rabbits; the results indicated IDHP was eliminated rapidly after oral administration.