Simultaneous separation and determination of active components in Cordyceps sinensis and Cordyceps militarris by LC/ESI-MS

A simple and rapid isocratic LC/MS coupled with electrospray ionization (ESI) method for simultaneous separation and determination of adenine, hypoxanthine, adenosine and cordycepin in Cordyceps sinensis (Cs) and its substitutes was developed. 2-Chloroadenosine was used as internal standard for this assay. The optimum separation for these analytes was achieved using the mixture of water, methanol and formic acid (85:14:1, v/v/v) as a mobile phase and a 2.0×150 mm Shimadzu VP-ODS column. Selective ion monitoring (SIM) mode ([M+H]⁺ at m/z 136, 137, 268, 252 and 302) was used for quantitative analysis of above four active components. The regression equations were liner in the range of 1.4–140.0 μg ml⁻¹ for adenine, 0.6–117.5 μg ml⁻¹ for hypoxanthine, 0.5–128.5 μg ml⁻¹ for adenosine and 0.5–131.5 μg ml⁻¹ for cordycepin. The limits of quantitation (LOQ) and detection (LOD) were, respectively 1.4 and 0.5 μg ml⁻¹ for adenine, 0.6 and 0.2 μg ml⁻¹ for hypoxanthine, 0.5 and 0.1 μg ml⁻¹ for adenosine and cordycepin. The recoveries of four constituents were from 93.5 to 107.0%. The nucleoside contents of various types of natural Cs and its substitutes were determined and compared with this developed method.     

Simultaneous LC determination of paracetamol and related compounds in pharmaceutical formulations using a carbon-based column

A simple, rapid and convenient high performance liquid chromatographic method, which permits the simultaneous determination of paracetamol, 4-aminophenol and 4-chloracetanilide in pharmaceutical preparation has been developed. The chromatographic separation was achieved on porous graphitized carbon (PGC) column using an isocratic mixture of 80/20 (v/v) acetonitrile/0.05 M potassium phosphate buffer (pH 5.5) and ultraviolet detection at 244 nm. Correlation coefficient for calibration curves in the ranges 1–50 μg ml⁻¹ for paracetamol and 5–40 μg ml⁻¹ for 4-aminophenol and 4-chloroacetanilide were >0.99. The sensitivity of detection is 0.1 μg ml⁻¹ for paracetamol and 0.5 μg ml⁻¹ for 4-aminophenol and 4-chloroacetanilide. The proposed liquid chromatographic method was successfully applied to the analysis of commercially available paracetamol dosage forms with recoveries of 98–103%. It is suggested that the proposed method should be used for routine quality control and dosage form assay of paracetamol in pharmaceutical preparations. The chromatographic behaviour of the three compounds was examined under variable mobile phase compositions and pH, the results revealed that selectivity was dependent on the organic solvent and pH used. The retention selectivity of these compounds on PGC was compared with those of octadecylsilica (ODS) packing materials in reversed phase liquid chromatography. The ODS column gave little separation for the degradation product (4-aminophenol) from paracetamol, whereas PGC column provides better separation in much shorter time.     

Determination of naltrexone and its major metabolite, 6-β-naltrexol, in human plasma using liquid chromatography with electrochemical detection

A sensitive and specific high-performance liquid chromatographic method with electrochemical detection was developed for the simultaneous determination of naltrexone and its major metabolite, 6-β-naltrexol, in human plasma. After alkalinizing 2 ml plasma samples with pH 9 sodium carbonate buffer, naltrexone and 6-β-naltrexol were extracted into dichloromethane and then back-extracted into 0.017 M phosphoric acid. A portion of the acid extract was chromatographed on a YMC phenyl column using a mobile phase of methanol-phosphoric acid (50 mM) (20:80, v/v) (pH* 3.2) at a flow-rate of 1.2 ml min⁻¹. Quantification was performed using an ESA Coulometric electrochemical detector. Acceptable intra-day and inter-day assay precision (RSD < 10%) and accuracy (< 16%) for both compounds were observed over concentration ranges of 0.25–50.0 ng ml⁻¹ for naltrexone and 0.5–100 ng ml⁻¹ for 6-β-naltrexol. No degradation of either naltrexone or 6-β-naltrexol was observed in frozen human plasma stored at − 20°C over an 8 month period. The method is sufficiently sensitive and selective to quantify plasma concentrations of naltrexone and 6-β-naltrexol after oral doses of 50 mg of naltrexone to healthy subjects or alcoholic patients.     

Simultaneous determination of valsartan and hydrochlorothiazide in tablets by first-derivative ultraviolet spectrophotometry and LC

First-derivative ultraviolet spectrophotometry and high-performance liquid chromatography (HPLC) were used to determine valsartan and hydrochlorothiazide simultaneously in combined pharmaceutical dosage forms. The derivative procedure was based on the linear relationship between the drug concentration and the first derivative amplitudes at 270.6 and 335 nm for valsartan and hydrochlorothiazide, respectively. The calibration graphs were linear in the range of 12.0–36.1 μg ml⁻¹ for valsartan and 4.0–12.1 μg ml⁻¹ for hydrochlorothiazide. Furthermore, a high- performance liquid chromatographic procedure with ultraviolet detection at 225 nm was developed for a comparison method. For the HPLC procedure, a reversed phase column with a mobile phase of 0.02 M phosphate buffer (pH 3.2)-acetonitrile (55: 45; v/v), was used to separate for valsartan and hydrochlorothiazide. The plot of peak area ratio of each drug to the internal standard versus the respective concentrations of valsartan and hydrochlorothiazide were found to be linear in the range of 0.06–1.8 and 0.07–0.5 μg ml⁻¹, respectively. The proposed methods were successfully applied to the determination of these drugs in laboratory-prepared mixtures and commercial tablets.     

Pharmacokinetics of thrombin-like enzyme from venom of Agkistrodon halys ussuriensis Emelianov determined by ELISA in the rat.

A thrombin-like enzyme (TLE) was separated and purified from the venom of a northeast Chinese snake Agkistrodon halys ussuriensis Emelianov. Experiments were performed in rats to determine the pharmacokinetic parameters following an intravenous (i.v.) or a subcutaneous (s.c.) injection of the thrombin-like enzyme. The plasma levels of TLE were estimated by enzyme-linked immunosorbent assay. The method exhibited high reproducibility and accuracy in correlating optical densities with TLE concentrations (0.2–30 ng ml⁻¹, r=0.99). The plasma concentration-time course after i.v. administration of 50 μg kg⁻¹ TLE was well fitted by a two-compartment open model. The half-life of the -phase was 18.0±3.2 min, and that of the β-phase 3.9±0.7 h. The apparent volume of distribution was 1.8±0.5 l kg⁻¹, and clearance was 5.4±0.5 ml min⁻¹ kg⁻¹. When the TLE was injected s.c. at a dose of 0.75 mg kg⁻¹, the changes in plasma concentration were best described by a two-compartment model with a first-order absorption. The maximal plasma level of 51±2.7 ng ml⁻¹ was reached at 5.2±0.5 h. The absorption rate constant was 0.3±0.03 h⁻¹. The area under the plasma concentration-time curve (AUC) was 2.8±0.8 μg h⁻¹ ml⁻¹.     

LC determination of dinitrosopiperazine in simulated gastric juice

A simple and specific reversed phase HPLC method for the determination of dinitrosopiperazine in simulated gastric juice using UV detection was reported. The chromatographic resolution of the analyte and the internal standard isosorbide dinitrate was performed without extraction from the gastric juice on a reversed phase ODS column. Isocratic elution was carried out with methanol–0.02 M sodium dihydrogen phosphate (60:40 v/v, pH 3.0) at a flow rate of 1.0 ml min⁻¹ with UV detection at 238 nm. The calibration graph was linear over the concentration range 0.072–2.88 μg ml⁻¹ of dinitrosopiperazine with minimum detectability (S/N=2) of 0.01 μg ml⁻¹ (8×10⁻⁸ M). Inter-day and intra-day precisions calculated as% RSD were in the range 0.32–0.38% and 0.19–0.25% respectively. Inter-day and intra-day accuracies calculated as% error were in the range 0.18–0.21 and 0.08–0.11% respectively. The proposed method was successfully applied to the study of the possible in–vivo production of DNPZ under the standard nitrosation conditions recommended by WHO.     

Liquid chromatographic determination of hydralazine in human plasma with 2-hydroxy-1-naphthaldehyde pre-column derivatization

A selective and sensitive high-performance liquid chromatographic method is described for determination of hydralazine and its metabolites in human plasma. The method involves pre-column derivatization with 2-hydroxy-1-naphthaldehyde at pH 1.2. The reaction product and Methyl Red used as internal standard are extracted into dichloromethane and chromatographed in the reversed-phase mode on an ODS-2 column using acetonitrile—aqueous triethylamine phosphate buffer (80:20, v/v) at pH 3 as eluent.

The plasma calibration curve of hydralazine is linear in the concentration range 10–500 ng ml⁻¹. The detection limit is 1 ng ml⁻¹ and the relative standard deviation is <2.4. In vivo pharmacokinetics of hydralazine in two volunteers after oral administration of 50 mg of the drug is studied using the proposed LC method.     

Determination of ethylenediamine tetraacetic acid in injection forms by ion-pair chromatography

A high performance liquid chromatographic method was developed for the determination of ethylenediamine tetraacetic acid (EDTA) in injection forms. The method consists of direct extraction of the samples with ethyl acetate; the organic layers were evaporated to dryness and further diluted to a 0.025% (w/v) copper nitrate in order to achive the formation of the EDTA–copper solution complex. The chromatographic separation was performed on a C8 Hypersil column. The mobile phase consisted of a mixture of acetonitrile–0.015 M tetrabutylammonium hydroxide (10:90, v/v), (pH* 7.0) pumped at a flow rate of 1.5 ml min⁻¹. The UV detector was operated at 300 nm. Correlation coefficients of the calibration graphs were better than 0.9995, relative standard deviation was less than 2.5%. Detection limit of EDTA was found to be 1.97 μg ml⁻¹.     

Determination of cisapride, its oxidation product, propyl and butyl parabens in pharmaceutical dosage form by reversed-phase liquid chromatography

A simple, rapid and reproducible high-performance liquid chromatography (HPLC) assay for cisapride, its oxidation product (OP), propyl and butyl parabens in a pharmaceutical formulation is described. Chromatography was performed at room temperature by pumping acetonitrile–20 mM phosphate buffer pH 7 (50:50, v/v) at 1.5 ml min⁻¹ through C₈ reversed-phase column. Cisapride, OP, propyl and butyl parabens were detected at 276 nm and were eluted at 9.7, 3.1, 5.1 and 7.1 min, respectively. Calibration plots were linear (r>0.999) for all compounds from 0.5 to 200 μg ml⁻¹ for cisapride and OP and 0.1–200 μg ml⁻¹ for propyl and butyl parabens. Detection limits for cisapride, OP, propyl and butyl parabens were 40, 46, 48 and 54 ng ml⁻¹, respectively. Forced degradation investigations showed that cisapride does not undergo degradation under heat, acidic and basic conditions but it was susceptible to oxidation. The proposed method was successfully applied to the assay of cisapride in the presence of preservatives and OP in a commercial suspension.     

Development and validation of a reversed-phase liquid chromatographic method for the assay of lidocaine in aqueous humour samples

A simple, fast and reliable reversed-phase liquid chromatographic method was developed for the assay of lidocaine in human aqueous humour samples. The samples were analysed without any preliminary treatment on a C8 column with UV detection at 225 nm. The mobile phase consisted of methanol/sodium dihydrogen phosphate (30 mM) containing sodium pentansulphonate (10 mM) adjusted to pH 2.5 with phosphoric acid (50:50 v/v). Validation of the method showed it to be precise, accurate and linear over the concentration range of analysis with a limit of detection of 0.2 μg ml⁻¹. The limit of quantitation was 2.5 μg ml⁻¹ with a relative standard deviation of 2.5%. Linear regression analysis in the range 2.5–60 μg ml⁻¹ gave correlation coefficients higher than 0.999. No interference from three commonly co-administered drugs was observed. The method developed was applied to the analysis of lidocaine in aqueous humour samples in order to evaluate and compare the efficacy of two different forms of administration of lidocaine for topical anaesthesia in cataract surgery.     

Development and validation of a novel LC non-derivatization method for the determination of amikacin in pharmaceuticals based on evaporative light scattering detection

A novel method for the direct determination of the aminoglycoside antibiotic amikacin and its precursor component kanamycin was developed and validated, based on reversed phase LC with evaporative light scattering detector (ELSD). ELSD response to amikacin was found to be enhanced by: (a) use of ion-pairing acidic reagents of increased molecular mass, (b) increase of mobile phase volatility and (c) decrease of peak width and asymmetry (obtained by controlling the mobile phase acidity and/or ratio of organic solvent to water). Utilizing a Thermo Hypersil BetaBasic C₁₈ column, the selected optimized mobile phase was water–methanol (60:40, v/v), containing 3.0 ml l⁻¹ nonafluoropentanoic acid (18.2 mM) (isocratic elution with flow rate of 1.0 ml min⁻¹). ELSD experimental parameters were: nitrogen pressure 3.5 bar, evaporation temperature 50 °C, and gain 11. Amikacin was eluted at 8.6 min and kanamycin at 10.4 min with a resolution of 1.5. Logarithmic calibration curves were obtained from 7 to 77 μg ml⁻¹ (r > 0.9995) for amikacin and 8 to 105 μg ml⁻¹ (r > 0.998) for kanamycin, with a LOD equal to 2.2 and 2.5 μg ml⁻¹, respectively.

In amikacin sulfate pharmaceutical raw materials, the simultaneous determination of sulfate (t_R = 2.3 min, LOD = 1.8 μg ml⁻¹, range 5–40 μg ml⁻¹, %R.S.D. = 1.1, r > 0.9997), kanamycin and amikacin was feasible. No significant difference was found between the results of the developed LC–ELSD method and those of reference methods, while the mean recovery of kanamycin from spiked samples (0.5%, w/w) was 97.3% (%R.S.D. ≤ 2.0, n = 6). Further, the developed method was applied for the determination of amikacin in pharmaceutical formulations (injection solutions) without any interference from the matrix (recovery from spiked samples ranged from 95.6 to 103.8%).     

Development and validation of two chromatographic methods for the quantification of E-6087 and one of its metabolites, E-6132, in rat plasma

E-6087 is a nonsteroidal anti-inflammatory compound under development that selectively inhibits cyclooxygenase-2. In vitro studies have shown that one of its metabolites, E-6132, also inhibits this enzyme. Due to chromatographic reasons, two reverse phase HPLC methods were developed and validated in order to elucidate which compound is responsible for the pharmacological activity in vivo. Chromatographic separation of E-6087 was achieved using acetonitrile–phosphate buffer (pH 2.5; 25 mM) (60:40, v/v) as mobile phase and two 4.6×150 mm×5 μm Inertsil ODS-2 columns. For E-6132, two Inertsil ODS-3 columns and 52% of acetonitrile were used instead. Internal standards and fluorescence detection differed between both methods. The same on-line solid-phase extraction method was used. Mean retention times for E-6087 and E-6132 were 15.2 (±1.3) and 36.1 (±0.6) min, respectively. The methods were selective and linear over the concentration range of 10–500 ng ml⁻¹ (r²>0.996) for E-6087 and 5–200 ng ml⁻¹ (r²>0.997) for E-6132. The limits of quantitation were 10 ng ml⁻¹ (E-6087) and 5 ng ml⁻¹ (E-6132) with a precision and accuracy <16% (E-6087) and <11% (E-6132). Mean recoveries from plasma were 43.2–61.9% (E-6087) and 60.4–65.2% (E-6132). For both compounds, both inter-assay and intra-assay precision and accuracy were within acceptable limits (<15%). As an example of the suitability of these methods, the results from a pharmacokinetic study are reported. After single oral administration of 5 mg kg⁻¹ of E-6087 to rats, plasma concentrations of E-6087 at peak time were higher than those of E-6132, suggesting that activity is mainly due to E-6087.     

Determination of 5-fluorouracil in human plasma by a simple and sensitive reversed-phase HPLC method

A simple and sensitive reversed-phase HPLC method with UV detection was developed and validated for the quantitation of 5-fluorouracil (5-FU) in human plasma. After acidification and salting out, 5-FU was extracted into ethyl acetate and back-extracted into a basic buffer. The extract was adjusted to neutral pH before being injected onto the HPLC column. 5-FU was separated from the matrix components on a YMC ODS-AQ column at 40°C using an aqueous mobile phase of 10 mM potassium phosphate at pH 5.5. A linear gradient of 0–25% methanol wash eluted late peaks, maintained column performance, and increased column stability. The run time was 20 min. The linear range was 25–300 ng ml⁻¹ (r²>0.999). The limit of quantitation was 25 ng ml⁻¹, with a signal-to-noise ratio of 23:1. Interday precision and accuracy of quality control samples were 6.2–8.4%, relative standard deviation and −0.1– + 1.9% relative error.     

Simultaneous determination of cinnarizine and domepiridone maleate from tablet dosage form by reverse phase ion pair high performance liquid chromatography

A new simple, precise, rapid and selective reverse phase ion pair high performance liquid chromatography (HPLC-RP) method has been developed for the simultaneous determination of cinnarizine (CINN) and domepiridone maleate (DOME) from tablets using acetonitrile–methanol–water–0.1 N sulfuric acid (37:10:48:5 v/v/v/v) containing sodium lauryl sulfate (0.01 M), as a mobile phase and a Machery Nagel nitrile column (10 μ, 25 cm×4.0 mm i.d.) as the stationary phase. The flow of mobile phase through the column was kept at 1.0 ml min⁻¹ through out the analysis. Detection was carried out using a UV detector at 225 nm. The retention times for CINN and DOME were 4.73 and 9.41 min, respectively. The linearity range and percentage recoveries for CINN and DOME were 4–1000 and 60–750 μg ml⁻¹ and 99.90 and 99.60%, respectively.     

Analysis of flurbiprofen, ketoprofen and etodolac enantiomers by pre-column derivatization RP-HPLC and application to drug-protein binding in human plasma

A stereoselective reversed-phase high-performance liquid chromatography (HPLC) assay to determine the enantiomers of flurbiprofen, ketoprofen and etodolac in human plasma was developed. Chiral drug enantiomers were extracted from human plasma with liquid–liquid extraction. Then flurbiprofen and ketoprofen enantiomers reacted with the acylation reagent thionyl chloride and pre-column chiral derivatization reagent (S)-(−)--(1-naphthyl)ethylamine (S-NEA), and etodolac enantiomers reacted with S-NEA using 1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide (EDC) and 1-hydroxybenzotriazole (HOBT) as coupling agents. The derivatized products were separated on an Agilent Zorbax C₁₈ (4.6 mm × 250 mm, 5 μm) column with a mixture of acetonitrile–0.01 mol·L⁻¹ phosphate buffer (pH 4.5) (70:30, v/v) for flurbiprofen enantiomers, acetonitrile–0.01 mol·L⁻¹ phosphate buffer (pH 4.5) (60:40, v/v) for ketoprofen enantiomers and methonal–0.01 mol·L⁻¹ potassium dihydrogen phosphate buffer (pH 4.5) (88:12, v/v) for etodolac enantiomers as mobile phase. The flow of mobile phase was set at 0.8 mL·min⁻¹ and the detection wavelength of UV detector was set at 250 nm for flurbiprofen and ketoprofen enantiomers and 278 nm for etodolac enantiomers. The assay was linear from 0.5 to 50 μg·mL⁻¹ for each enantiomer. The inter- and intra-day precision (R.S.D.) was less than 10% and the average extraction recovery was more than 87% for each enantiomer. The limit of quantification for the method was 0.5 μg·mL⁻¹ (R.S.D. < 10%, n = 5). The method developed was used to study the drug–protein binding of flurbiprofen, ketoprofen and etodolac enantiomers in human plasma. The results showed that the stereoselective binding of etodolac enantiomer was observed and flurbiprofen and ketoprofen enantiomers were not.     

Development and validation of an LC-MS-MS method for the determination of terfenadine in human plasma

A sensitive LC-MS-MS method capable of quantifying terfenadine at levels down to 100 pg ml⁻¹ in human plasma is reported. The method was validated over a linear range from 0.1 to 5.0 ng ml⁻¹ using a liquid-liquid extraction with a deuterium-labelled internal standard. The between-run precision and accuracy of the calibration standards were 2.6–6.0% RSD and −2.0 to +2.2% relative error (RE). The between-run and within-run precision and accuracy of quality control samples (0.3, 1.5 and 3.5 ng ml⁻¹) were 1.0–5.9% RSD and +1.7 to +6.3% RE. This method was applied to the analysis of human plasma samples.     

A time-resolved fluorescence immunoassay for the determination of a novel respiratory therapeutic agent, AR-C68397XX (Viozan) in human plasma

A dissociation-enhanced lathanide fluorescence immunoassay (DELFIA™) method has been developed for the determination of AR-C68397XX, a novel respiratory therapeutic agent, in human plasma. The method is a ‘direct’ immunoassay and provides an alternative to the solid phase extraction RIA described in a previous publication, which employs the same specific antiserum. The DELFIA method is suitable for the determination of the analyte at pg ml⁻¹ concentrations. The non-isotopic label was prepared by complexation of a DTPA derivative of AR-C68397XX with free europium cation (Eu³⁺). Plasma samples were diluted at least 5-fold prior to analysis to eliminate matrix interference. The calibration range is 10–2000 pg ml⁻¹, and the LOQ of the method is 50 pg ml⁻¹ using 50 μl of diluted human plasma sample.     

Fully automated methods for the determination of hydrochlorothiazide in human plasma and urine

LC assays utilizing fully automated sample preparation procedures on Zymark PyTechnology™ Robot and BenchMate™ Workstation for the quantification of hydrochlorothiazide (HCTZ) in human plasma and urine have been developed. After aliquoting plasma and urine samples, and adding internal standard (IS) manually, the robot executed buffer and organic solvent addition, liquid—liquid extraction, solvent evaporation and on-line LC injection steps for plasma samples, whereas, BenchMate™ performed buffer and organic solvent addition, liquid—liquid and solid-phase extractions, and on-line LC injection steps for urine samples. Chromatographic separations were carried out on Beckman Octyl Ultrasphere column using the mobile phase composed of 12% (v/v) acetonitrile and 88% of either an ion-pairing reagent (plasma) or 0.1% trifluoroacetic acid (urine). The eluent from the column was monitored with UV detector (271 nm). Peak heights for HCTZ and IS were automatically processed using a PE-Nelson ACCESS*CHROM laboratory automation system. The assays have been validated in the concentration range of 2–100 ng ml⁻¹ in plasma and 0. 1–20 μg ml⁻¹ in urine. Both plasma and urine assays have the sensitivity and specificity necessary to determine plasma and urine concentrations of HCTZ from low dose (6.25/12.5 mg) administration of HCTZ to human subjects in the presence or absence of losartan.     

Determination of clotrimazole in mice plasma by capillary electrophoresis

In addition to its antifungal activity, clotrimazole attracts interest as an anti-inflammatory drug. In order to correlate this effect with plasma concentrations in mice, a capillary electrophoretic method was developed. Sample preparation was carried out by protein precipitation using methanol. Quantification of clotrimazole was achieved by means of capillary electrophoresis using ketoconazole as an internal standard (IS). The background electrolyte (BGE) composed of a Tris buffer solution (100 mM, pH 3.0, adjusted with acetic acid) and methanol (8:2, v/v). Injection was carried out electrokinetically with 10 kV over a time period of 20 s. A special rinsing procedure utilizing a sequence of a SDS/methanol solution, a sodium hydroxide solution, water and BGE, was applied to enhance the reproducibility. With this procedure, an intermediate precision (day-to-day precision) of the area ratios of clotrimazole and IS of 5.0% for 0.5 μg ml⁻¹ and 2.6% for 10 μg ml⁻¹ was obtained. In summary, with the described capillary zone electrophoresis (CZE) method it is possible to handle small sample volumes of 60 μl, to detect clotrimazole concentrations of 0.3 μg ml⁻¹ (limit of detection), and to quantify clotrimazole down to concentrations of 0.5 μg ml⁻¹ (limit of quantification).     

LC/MS/MS analysis of vesnarinone and its principal metabolites in plasma and urine

An LC/MS/MS assay was developed and successfully used to quantitate vesnarinone and its principal metabolites (OPC-8230, OPC-18136, and OPC-18137) in human plasma and urine. Samples were pre-treated with liquid–solid extraction followed by simultaneous monitoring of primary and daughter ions which were used for the identification and quantitation of the analytes on LC/MS/MS. This assay offers advantages of specificity, speed and greater sensitivity over the previously developed HPLC-UV assay. The lower limit of quantitation is 500 ng ml⁻¹ for vesnarinone and 20 ng ml⁻¹ for OPC-8230, OPC-18137, and OPC-18136 in plasma. Methodology is similar for the estimation of these analytes in urine with the lower limit of quantitation being 500 ng ml⁻¹ for vesnarinone and 100 ng ml⁻¹ for each metabolite. Ascorbic acid was added to stabilize the analytes from degradation. This LC/MS/MS method was developed to overcome many practical problems associated with the HPLC method. The LC/MS/MS method offers the flexibility of analyzing additional metabolites and changing the linearity range to accommodate the differences in linear range (200–10 000 ng ml⁻¹ for vesnarinone and 20–1000 for metabolites) for the analytes.