Enantioselective analysis of (<Emphasis Type="Italic">R</Emphasis>)- and (<Emphasis Type="Italic">S</Emphasis>)-carvedilol in human plasma by high-performance liquid chromatography

Summary An HPLC column-switching method has been developed and validated for the enantioselective determination of (R)- and (S)-carvedilol in human plasma. Sample preparation was performed either off-line, by extraction with trichloromethane and back-extraction into 0.01m aqueous citric acid which was injected on to a LiChrosorb RP 8 column, or on-line, by injecting diluted (0.1m formic acid) plasma on to a LiChrosorb ADS column. In both instances separation was performed by gradient elution and on-line transfer of the fraction containing, the carvedilol on to an enantioselective Teicoplanin column. The enantiomers of carvedilol were separated isocratically by use of methanol-acetonitrile-triethylammonium acetate, 70:30:0.05 (v/v/w), as mobile phase. With fluorescence detection the limits of quantitation were 0.30 ng mL⁻¹ for (R)-carvedilol and 0.26 ng mL⁻¹ for (S)-carvedilol; these were sufficient to enable investigation of the effect of exercise on plasma concentrations of (R)- and (S)-carvedilol after oral administration of either the racemate or the pure enantiomers. Although the operating conditions were optimized for sample preparation by on-line deproteination on a LiChrospher RP 18 ADS column, the complete method was insufficiently rugged for analysis of large numbers of plasma samples—the enantioselectivity of the Teicoplanin column deteriorated too rapidly because of the transfer of enantioselectivity-poisoning interferences which could not be suppressed sufficiently. In contrast the liquid-liquid sample-extraction procedure combined with column switching resulted in a analytical method with long-term stability. Presented at Balaton Symposium '01 on High-Performance Separation Methods, Siófok, Hungary, September 2–4, 2001     

Determination of Oxolinic Acid Residues in Gilthead Seabream (Sparus aurata) Muscle Tissue and Plasma by High-Performance Liquid Chromatography

A high-performance liquid chromatographic method for the determination of oxolinic acid (OA) residues in muscle tissue and plasma of the cultured fish gilthead seabream (Sparus aurata L.), is described. OA was extracted with ethyl acetate and after centrifugation the combined extracts were evaporated. To the remaining residue 1 mL of the mobile phase was added and the extract was partitioned with n-pentane which then was rejected by aspiration. OA was chromatographed on a Zorbax^®SB-C₁₈ column at 50^oC and detected by fluorescence detection at λ_ex 327 nm and λ_em 369 nm. The mobile phase was a mixture of 0.1% trifluoroacetic acid (v/v) pH 2.0 and acetonitrile-methanol 3:2 (v/v) in a combination of 50:50 (v/v) and a flow rate of 1.0 mL min^?1, delivered isocratically. Method mean recovery (R%) achieved was 73.7 ± 4.4% (mean ± SD) for blank fortified samples (n=4) range at 50, 100 and 200 μg kg^?1 with a RSD=3.3%. The limit of detection (LOD) was 2.0 μg kg^?1 oxolinic acid in muscle tissue and plasma and the limit of quantification (LOQ) was 5.0 μg kg^?1. The method is fast and suitable to be used with safety and accuracy for the control of OA residues in cultured seabreams and a trained analyst could carry out ready for chromatography more than 50 samples per working day.     

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.     

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 Improved LC Method for the Simultaneous Determination of Pirfenidone and Its Carboxylic Acid Metabolite in Human Plasma

A sensitive and specific assay based on liquid chromatography with ultraviolet detection was developed for the simultaneous determination of pirfenidone (PFD), a novel antifibrotic agent, and its carboxylic acid metabolite in human plasma. The carboxylic acid metabolite was further identified by mass spectrometric analysis. PFD, its carboxylic acid metabolite and the internal standard methyl-p-aminobenzoate were extracted from plasma by a simple one-step liquid-liquid extraction with ethyl acetate and subsequently separated on a Zorbax SB-C18 column with a mobile phase of trifluoroacetic acid–triethylamine–acetonitrile–water (0.1:0.15:28:71.75, v/v/v/v) and monitored at 314 nm. Extraction recovery was over 70% in plasma. The calibration curves were linear over the concentration range of 0.05–25 μg mL^?1. The limit of detection (LOD) and lower limit of quantitation (LLOQ) in human plasma were 10 and 50 ng mL^?1, respectively. Intra- and inter-assay precision of the method were within 8.6%. The accuracy as expressed by the bias ranged between ?4.5 and 4.0%. The method was successfully applied to determine pharmacokinetic parameters of PFD and its carboxylic acid metabolite after a single oral dose of 200 mg of PFD in healthy volunteers.     

Development and Validation of LC Coupled with SPE for Simultaneous Determination of Lamivudine, Oxymatrine and its Active Metabolite Matrine in Dog Plasma

A simple and highly selective method, based upon solid-phase extraction (SPE), ion-pair HPLC and UV absorbance detection, was developed and validated to determine lamivudine, oxymatrine and its active metabolite matrine in dog plasma. The analytes and famotidine (internal standard) were simultaneously extracted from plasma samples by SPE, and separated on a C18 column. The mobile phase consisted of acetonitrile-water (13:87, v/v, 5 mmol L^?1 sodium heptanesulfonate, at pH 3.2). The lower limit of quantification of the method was 0.1 mg L^?1 for these analytes. The linear calibration curves of the analytes were obtained in the concentration range of 0.1–40 mg L^?1. This method was successfully applied to the quantitative determination of plasma concentration of lamivudine, oxymatrine and its active metabolite matrine in dogs after single oral co-administration of 5.0 mg kg^?1 lamivudine and 30.0 mg kg^?1 oxymatrine.     

HPLC–MS Analysis of Isoniazid in Dog Plasma

A simple, rapid, and sensitive liquid chromatography–mass spectrometric (LC–MS) method was developed and validated for the determination of isoniazid in dog plasma. Plasma samples were deproteined with methanol and separated on a C₁₈ column interfaced with a single quadrupole mass spectrometer, using 0.1% formic acid–acetonitrile (91:9 v/v) as mobile phase. Detection was performed by positive electrospray ionization with selected ion monitoring at m/z 138 for isoniazid and 152 for entecavir maleate internal standard. Linearity was obtained over the range of 25–5,000 ng mL^?1, with a lower limit of quantification of 25 ng mL^?1. The intra- and inter-day precision was less than 2.7% in terms of relative standard deviation. Accuracy, expressed as relative error, ranged from ?2.0 to 8.0%. Plasma samples were analysed within 5 min. The method was successfully applied to the evaluation of the pharmacokinetics of isoniazid in dog plasma.     

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.     

Stability-Indicating Chromatographic Methods for Simultaneous Determination of Mosapride and Pantoprazole in Pharmaceutical Dosage Form and Plasma Samples

Simple, sensitive, selective, precise, and stability-indicating thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC) methods for the determination of mosapride and pantoprazole in pharmaceutical tablets were developed and validated as per the International Conference on Harmonization guidelines. The TLC method employs aluminum TLC plates precoated with silica gel 60F₂₅₄ as the stationary phase and ethyl acetate/methanol/toluene (4:1:2, v/v/v) as the mobile phase to give compact spots for mosapride (R _f 0.73) and pantoprazole (R _f 0.45) separated from their degradation products; the chromatogram was scanned at 276 nm. The HPLC method utilizes a C18 column and a mobile phase consisting of acetonitrile/methanol/20 mM ammonium acetate (4:2:4, v/v/v) at a flow rate of 1.0 mL min^?1 for the separation of mosapride (t _R 11.4) and pantoprazole (t _R 4.4) from their degradation products. Quantitation was achieved with UV detection at 280 nm. The same HPLC method was successfully used in performing calibrations in lower concentration ranges for both drugs in human plasma using ezetimibe as internal standard. The methods were validated in terms of accuracy, precision, linearity, limits of detection, and limits of quantification. Mosapride and pantoprazole were exposed to acid hydrolysis and then analyzed by the proposed methods. As the methods could effectively separate the drugs from their degradation products, these techniques can be employed as stability-indicating methods that have been successively applied to pharmaceutical formulations without interference from the excipients. Moreover the HPLC method was successfully used in the determination of both drugs in spiked human plasma.     

An LC–MS–MS Method for the Simultaneous Quantitation of Acyclovir and Valacyclovir in Human Plasma

A simple, sensitive and selective LC–MS–MS method has been developed for the simultaneous determination of acyclovir and valacyclovir in human plasma. Acyclovir and valacyclovir in plasma were concentrated by solid phase extraction and chromatographed on a C18 column using a mobile phase of 0.1% formic acid: methanol (30:70% v/v). The method was validated over a linear range of 47–10,255 and 5–1,075 ng mL^?1 for acyclovir and valacyclovir respectively. The LOQs were 47.6 and 5.0 ng mL^?1. The validated method was applied for the quantitation of acyclovir and valacyclovir from plasma samples in a pharmacokinetic study.     

Semipreparative High-Performance Liquid Chromatographic Separation of Phosphatidylcholine Molecular Species From Soybean Leaves

Abstract

An improved high-performance liquid chromatographic (HPLC) method using UV detection at 205 nm is described for the semipreparative separation of the molecular species of phosphatidylcholine (PC) from soybean leaves. the separations of PC molecular species are achieved isocratically within ca. 75 min on C 18 reversed-phase column using the mobile phase, methanol:0.1 M ammonium acetate, pH 7.4 (95:5, v/v). Five molecular species for soybean PC are identified as 18:3/18:3, 18:2/18:3, 18:2/18:2, 16:0/18:3 and 16:0/18:2.     

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.     

LC Determination of Isosorbide-5-Mononitrate in Human Plasma

A simple, sensitive, selective and cost effective LC–UV method was developed for determination of isosorbide mononitrate in human plasma using guaifenesin as an internal standard. Isosorbide mononitrate in plasma was extracted by a single step liquid extraction using tert-butyl methyl ether and chromatographed on a C18 column using water and acetonitrile (80:20 v/v) as mobile phase. The method was validated and exhibited a linear range from 51.6 to 2064.4 ng mL^?1. The inter- and intra-assay accuracy ranged from 97.2–102.7 to 94.2–105.5%, respectively, with precision less than 10% in both the cases. The LLQ was 51.6 ng mL^?1. The validated method was applied to the quantitation of isosorbide mononitrate from plasma samples in a pharmacokinetic study.     

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.     

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 Flupentixol in Human Plasma

Flupentixol and an internal standard, loperamide were extracted from human plasma by liquid–liquid extraction and analyzed on a Thermo Hypersil HyPURITY C18 column, with 10 mM ammonium acetate–acetonitrile–methanol (26:62:12, v/v/v) as mobile phase, coupled with electrospray ionization mass spectrometry (ESI–MS). The protonated analyte was quantified by selected-ion monitoring (SIM) with a quadrupole mass spectrometer in a positive-ion mode. The calibration curve was linear (r = 0.9990) over the concentration range: 0.039–2.5 ng mL^?1. Intra-day and inter-day precision (RSD%) were less than 13.05%. The established method was successfully applied for the determination of pharmacokinetics of flupentixol in human plasma.     

Automated analysis of fluvoxamine in rat plasma using a column-switching system and ion-pair high-performance liquid chromatography

We have established a robust, fully automated analytical method for the analysis of fluvoxamine in rat plasma using a column-switching ion-pair high-performance chromatography system. The plasma sample was injected onto a precolumn packed with Shim-pack MAYI-ODS (50 microm), where the drug was automatically purified and enriched by on-line solid-phase extraction. After elution of the plasma proteins, the analyte was back-flushed from the precolumn and then separated isocratically on a reversed-phase C18 column (L-column ODS) with a mobile phase (acetonitrile-0.1% phosphoric acid, 36:64, v/v) containing 2 mM sodium 1-octanesulfonate. The analyte was monitored by a UV detector at a wavelength of 254 nm. The calibration line for fluvoxamine showed good linearity in the range of 5-5000 ng/mL (r > 0.999) with the limit of quantification of 5 ng/mL (RSD = 6.51%). Accuracy ranged from -2.94 to 4.82%, and the within- and between-day precision of the assay was better than 8% across the calibration range. The analytical sensitivity and accuracy of this assay is suitable for characterization of the pharmacokinetics of orally-administered fluvoxamine in rats.     

Determination of Palmatine in Rabbit Plasma by RP-HPLC with Solid-Phase Extraction

A rapid and specific reversed-phase high performance liquid chromatography (RP-HPLC) method for the determination of palmatine in rabbit plasma has been developed and validated. The chromatographic separation was performed on a C₁₈ column at 40 °C. The mobile phase, delivered at 1.0 mL min^?1, consisted of acetonitrile/phosphate buffer (pH 3.0) 40:60 (v/v). The detection wavelength was set at 345 nm. Palmatine and internal standard (IS) berberine were extracted from plasma by solid-phase extraction using C18 cartridges. Linearity was confirmed in the concentration range of 0.01 to 5 μg mL^?1, the inter-day and intra-day RSDs were within 10.0, the recoveries of palmatine ranged from 93.1 to 110.3, and the limit of detection (LOD, S/N > 3) was 0.002 μg mL^?1. The method is applicable to the determination of palmatine in rabbit plasma after intravenous administration of palmatine.     

Automated high-performance liquid chromatographic method for the simultaneous determination of cefotiam and delta 3-cefotiam in human plasma using column switching.

An automated high-performance liquid chromatographic method using column switching was established for the simultaneous determination of cefotiam (I) and delta 3-cefotiam (II) in human plasma after oral administration of cefotiam hexetil dihydrochloride. The method allowed the determination of analytes in plasma by the direct injection of diluted specimen with phosphate buffer. The analytes were enriched onto the C18 short pretreatment column by 0.05 M phosphate buffer (pH 7.7), while proteins and endogenous hydrophilic substances in plasma were washed off to waste. The enriched analytes were then back-flushed onto the analytical C18 column, separated by a mixture of 0.05 M phosphate buffer (pH 7.7)-acetonitrile (88:12, v/v) and detected by the ultraviolet absorbance at 254 nm. Recoveries from spiked plasma were quantitative, and the coefficients of variation were below 4%. The lower detection limits in plasma were 10 ng/ml for both I and II. Concentrations of I and II in plasma determined by the present method were in good agreement with those obtained by the conventional deproteinization method.     

Sunitinib LC–MS/MS Assay in Mouse Plasma and Brain Tissue: Application in CNS Distribution Studies

Sunitinib malate is a multi-targeted tyrosine-kinase inhibitor, currently in clinical trials for glioma. Previously developed methods for preclinical studies in species such as mice have either employed high-performance liquid chromatography (HPLC) or did not describe a detailed analytical method, which could be employed by other preclinical laboratories. In this paper, we have developed and validated a simple, sensitive high-performance liquid chromatography tandem mass-spectrometric method (LC–MS/MS) for the determination of sunitinib concentration in mouse plasma and brain tissue homogenate using dasatinib-free base as the internal standard. A single step liquid–liquid extraction method was used for both the matrices. Since sunitinib exhibits light-induced E/Z isomerism, all sample preparation was done in light-protected conditions. Separation was performed on a ZORBAX Eclipse XDB C18 column 4.6 × 50 mm, 1.8 μm. The mobile phase consisted of 20 mM ammonium formate (with 0.1 % formic acid): acetonitrile (70:30, v/v) pumped isocratically at a flow rate of 0.25 mL min^?1 with a total run-time of 13 min. The retention times of sunitinib and dasatinib were 7.8 and 5.5 min, respectively. The calibration curve was linear over the range from 1.95 to 500 ng mL^?1 in both plasma and brain tissue homogenate with 1.95 ng mL^?1 as the lower limit of quantification (LLOQ) for both the matrices. Inter- and intra-day accuracy and precision was <15 % for low QC, med QC and high QC and <20 % for LLOQ. The method was applied to a pharmacokinetic study in FVB wild-type mice to determine the plasma and brain concentrations after a single oral sunitinib malate dose of 20 mg kg^?1.