Determination of ciprofloxacin levels in chinchilla middle ear effusion and plasma by high-performance liquid chromatography with fluorescence detection

An isocratic high-performance liquid chromatographic method has been developed to determine ciprofloxacin levels in chinchilla plasma and middle ear fluid. Ciprofloxacin and the internal standard, difloxacin, were separated on a Keystone ODS column (100 × 2.1 mm I.D., 5 μm Hypersil) using a mobile phase of 30 mM phosphate buffer (pH 3), 20 mM triethylamine, 20 mM sodium dodecyl sulphate—acetonitrile (60:40, v/v). The retention times were 3.0 min for ciprofloxacin and 5.2 min for difloxacin. This fast, efficient protein precipitation procedure together with fluorescence detection allows a quantification limit of 25 ng/ml with a 50 μl sample size. The detection limit is 5 ng/ml with a signal-to-noise ratio of 5:1. Recoveries (mean ± S.D., n = 5) at 100 ng/ml in plasma and middle ear fluid were 89.4 ± 1.2% and 91.4 ± 1.6%, respectively. The method was evaluated with biological samples taken from chinchillas with middle ear infections after administering ciprofloxacin.     

Determination of saquinavir in human plasma by high-performance liquid chromatography

We developed and characterized a high-performance liquid chromatography (HPLC) assay for the determination of saquinavir, an HIV protease inhibitor, in human plasma samples. Extraction of plasma samples with diethyl ether resulted in quantitative recovery of both saquinavir and its stereoisomer Ro 31-8533 which was used as an internal standard. The assay was performed isocratically using 5 mM H₂SO₄ (pH 3.5) and acetonitrile (75.5:24.5, v/v) containing 10 mM tetrabutylammonium hydrogen sulfate (TBA) as a mobile phase, a Nucleosil 3C8 column kept at 45°C and UV detection at 240 nm. Using this method, saquinavir and Ro 31-8533 can be separated from endogenous substances, and in the concentration range of 5–110 ng/ml the relative standard deviations for the determination of saquinavir were below 5%. The detection limit of saquinavir in human plasma was 1 ng/ml. The usefulness of the method was demonstrated by quantification of saquinavir in plasma of human subjects treated with 600 mg of saquinavir per os or 12 mg intravenously.     

High-performance liquid chromatographic determination of trimebutine and its major metabolite, N-monodesmethyl trimebutine, in rat and human plasma

A rapid, selective and very sensitive ion-pairing reversed-phase HPLC method was developed for the simultaneous determination of trimebutine (TMB) and its major metabolite, N-monodesmethyltrimebutine (NDTMB), in rat and human plasma. Heptanesulfonate was employed as the ion-pairing agent and verapamil was used as the internal standard. The method involved the extraction with a n-hexane–isopropylalcohol (IPA) mixture (99:1, v/v) followed by back-extraction into 0.1 M hydrochloric acid and evaporation to dryness. HPLC analysis was carried out using a 4-μm particle size, C₁₈-bonded silica column and water–sodium acetate–heptanesulfonate–acetonitrile as the mobile phase and UV detection at 267 nm. The chromatograms showed good resolution and sensitivity and no interference of plasma. The mean recoveries for human plasma were 95.4±3.1% for TMB and 89.4±4.1% for NDTMB. The detection limits of TMB and its metabolite, NDTMB, in human plasma were 1 and 5 ng/ml, respectively. The calibration curves were linear over the concentration range 10–5000 ng/ml for TMB and 25–25000 ng/ml for NDTMB with correlation coefficients greater than 0.999 and with within-day or between-day coefficients of variation not exceeding 9.4%. This assay procedure was applied to the study of metabolite pharmacokinetics of TMB in rat and the human.     

Determination of a new antimalarial drug, benflumetol, in blood plasma by high-performance liquid chromatography

A rapid and selective high-performance liquid chromatographic assay for determination of a new antimalarial drug (benflumetol, BFL) is described. After extraction with hexane-diethyl ether (70:30, v/v) from plasma, BFL was analysed using a C₁₈ Partisil 10 ODS-3 reversed-phase stainless steel column and a mobile phase of acetonitrile-0.1 M ammonium acetate (90:10, v/v) adjusted to pH 4.9 with ultraviolet detection at 335 nm. The mean recovery of BFL over a concentration range of 50–400 ng/ml was 96.8±5.2%. The within-day and day-to-day coefficients of variation were 1.8–4.0 and 1.8–4.2%, respectively. The minimum detectable concentration in plasma for BFL was 5 ng/ml with a C.V. of less than 10%. This method was found to be suitable for clinical pharmacokinetic studies.     

Solid-phase extraction and determination of ranitidine in human plasma by a high-performance liquid chromatographic method utilizing midbore chromatography

An improved high-performance liquid chromatographic (HPLC) method utilizing solid-phase extraction (SPE) and midbore chromatography was developed for the determination of ranitidine in human plasma. A mobile phase of 20 mM K₂HPO₄-acetonitrile-triethylamine (87.9:12.0:0.1, v/v) pH 6.0 was used with a phenyl analytical column and ultraviolet detection (UV). The method demonstrated linearity from 25 to 1000 ng/ml in 500 μl of plasma with a detection limit of 10 ng/ml. The method was utilized in a pharmacokinetic study evaluating the effects of pancreatico-biliary secretions on ranitidine absorption.     

Alternative method for determination of pyrimethamine in plasma by high-performance liquid chromatography

A rapid, selective, sensitive and reproducible reversed-phase high-performance liquid chromatography (HPLC) procedure for the quantitative determination of pyrimethamine (PYR) in plasma is described. The procedure involved the two-step extraction of PYR and the internal standard, quinine (QN) with acetonitrile and dichloromethane at basic pH. Chromatographic separation consisted of the mobile phase (methanol-water containing 0.005 M octanesulfonic acid, 50:50, v/v) running through the column (Techopak-10 C₁₈) at a flow-rate of 1.6 ml/min. Detection was at UV wavelength of 240 nm. The mean recoveries of PYR and QN at a concentration range of 50 and 500 ng/ml were 98.9 and 89%, and 94.7 and 96% for PYR and QN. The within-day coefficients of variation were 2.1–5.1% for PYR and 5.9% for QN. The day-to-day coefficients of variation were 2.1–4.1% for PYR and 5% for QN. The minimum detectable concentrations for PYR and QN in plasma were 3 and 10 ng/ml. The method was found to be suitable for use in clinical pharmacokinetic study.     

High-performance liquid chromatographic determination of vinorelbine in human plasma and blood: application to a pharmacokinetic study

A sensitive and specific high-performance liquid chromatographic method with fluorescence detection (excitation wavelength: 280 nm; emission wavelength: 360 nm) was developed and validated for the determination of vinorelbine in plasma and blood samples. The sample pretreatment procedure involved two liquid–liquid extraction steps. Vinblastine served as the internal standard. The system uses a Spherisorb cyano analytical column (250×4.6 mm I.D.) packed with 5 μm diameter particles as the stationary phase and a mobile phase of acetonitrile–80 mM ammonium acetate (50:50, v/v) adjusted to pH 2.5 with hydrochloric acid. The assay showed linearity from 1 to 100 ng/ml in plasma and from 2.5 to 100 ng/ml in blood. The limits of quantitation were 1 ng/ml and 2.5 ng/ml, respectively. Precision expressed as RSD was in the range 3.9 to 20% (limit of quantitation). Accuracy ranged from 92 to 120%. Extraction recoveries from plasma and blood averaged 101 and 75%, respectively. This method was used to follow the time course of the concentration of vinorelbine in human plasma and blood samples after a 10-min infusion period of 20 mg/m² of this drug in patients with metastatic cancer.     

Determination of cisapride and norcisapride in human plasma using high-performance liquid chromatography with ultraviolet detection

A simple, rapid and reproducible high-performance liquid chromatographic assay for cisapride and norcisapride in human plasma is described. Samples of plasma (150 μl) were extracted using a C₁₈ solid-phase cartridge. Regenerated tubes were eluted with 1.0 ml of methanol, dried, redissolved in 150 μl of methanol and injected. Chromatography was performed at room temperature by pumping acetonitrile–methanol–0.015 M phosphate buffer pH 2.2–2.3 (680:194:126, v/v/v) at 0.8 ml/min through a C₁₈ reversed-phase column. Cisapride, norcisapride and internal standard were detected by absorbance at 276 nm and were eluted at 4.3, 5.3 and 8.1 min, respectively. Calibration plots in plasma were linear (r>0.998) from 10 to 150 ng/ml. Intraday precisions for cisapride and norcisapride were 3.3% and 5.4%, respectively. Interday precisions for cisapride and norcisapride were 9.6% and 9.0%, respectively. Drugs used which might be coadministered were tested for interference.     

Stereoselective high-performance liquid chromatographic determination of ketamine and its active metabolite, norketamine, in human plasma

A stereoselective high-performance liquid chromatographic method for the determination of the enantiomers of ketamine and its active metabolite, norketamine, in human plasma is described. The compounds were extracted from plasma by liquid–liquid extraction three times in a combination of cyclohexane with 2.5 M NaOH, 1 mM HCl and 1 M carbonate buffer. Stereoselective separation was achieved on a Chiralcel OD column with a mobile phase of n-hexane–2-propanol (98:2, v/v). The detection wavelength was 215 nm. The lower limits of the determination of the method were 5 ng/ml for ketamine and 10 ng/ml for norketamine. The intra- and inter-day coefficients of variation ranged from 2.9 to 9.8% and from 3.4 to 10.7% for all compounds, respectively. The method was sensitive and sufficiently reproducible for stereoselective monitoring of ketamine and norketamine in human plasma during pharmacokinetic studies after the administration of ketamine for analgesia.     

A high-performance liquid chromatography assay with ultraviolet detection for olanzapine in human plasma and urine

Olanzapine is a commonly used atypical antipsychotic medication for which therapeutic drug monitoring has been proposed as clinically useful. A sensitive method was developed for the determination of olanzapine concentrations in plasma and urine by high-performance liquid chromatography with low-wavelength ultraviolet absorption detection (214 nm). A single-step liquid–liquid extraction procedure using heptane-iso-amyl alcohol (97.5:2.5 v/v) was employed to recover olanzapine and the internal standard (a 2-ethylated olanzapine derivative) from the biological matrices which were adjusted to pH 10 with 1 M carbonate buffer. Detector response was linear from 1–5000 ng (r²>0.98). The limit of detection of the assay (signal:noise=3:1) and the lower limit of quantitation were 0.75 ng and 1 ng/ml of olanzapine, respectively. Interday variation for olanzapine 50 ng/ml in plasma and urine was 5.2% and 7.1% (n=5), respectively, and 9.5 and 12.3% at 1 ng/ml (n=5). Intraday variation for olanzapine 50 ng/ml in plasma and urine was 8.1% and 9.6% (n=15), respectively, and 14.2 and 17.1% at 1 ng/ml (n=15). The recoveries of olanzapine (50 ng/ml) and the internal standard were 83±6 and 92±6% in plasma, respectively, and 79±7 and 89±7% in urine, respectively. Accuracy was 96% and 93% at 50 and 1 ng/ml, respectively. The applicability of the assay was demonstrated by determining plasma concentrations of olanzapine in a healthy male volunteer for 48 h following a single oral dose of 5 mg olanzapine. This method is suitable for studying olanzapine disposition in single or multiple-dose pharmacokinetic studies.     

Determination of clozapine, desmethylclozapine and clozapine N-oxide in human plasma by reversed-phase high-performance liquid chromatography with ultraviolet detection

An isocratic high-performance liquid chromatography (HPLC) method with ultraviolet detection for the simultaneous determination of clozapine and its two major metabolites in human plasma is described. Analytes are concentrated from alkaline plasma by liquid–liquid extraction with n-hexane–isoamyl alcohol (75:25, v/v). The organic phase is back-extracted with 150 μl of 0.1 M dibasic phosphate (pH 2.2 with 25% H₃PO₄). Triprolidine is used as internal standard. For the chromatographic separation the mobile phase consisted of acetonitrile–0.06 M phosphate buffer, pH 2.7 with 25% phosphoric acid (48:52, v/v). Analytes are eluted at a flow-rate of 1.0 ml/min, separated on a 250×4.60 mm I.D. analytical column packed with 5 μm C₆ silica particles, and measured by UV absorbance detection at 254 nm. The separation requires 7 min. Calibration curves for the three analytes are linear within the clinical concentration range. Mean recoveries were 92.7% for clozapine, 82.0% for desmethylclozapine and 70.4% for clozapine N-oxide. C.V. values for intra- and inter-day variabilities were ≤13.8% at concentrations between 50 and 1000 ng/ml. Accuracy, expressed as percentage error, ranged from −19.8 to 2.8%. The method was specific and sensitive with quantitation limits of 2 ng/ml for both clozapine and desmethylclozapine and 5 ng/ml for clozapine N-oxide. Among various psychotropic drugs and their metabolites, only 2-hydroxydesipramine caused significant interference. The method is applicable to pharmacokinetic studies and therapeutic drug monitoring.     

Use of post-column fluorescence derivatization to develop a liquid chromatographic assay for ranitidine and its metabolites in biological fluids

Ranitidine and its main metabolites, ranitidine N-oxide and ranitidine S-oxide, were determined in plasma and urine after separation using reversed-phase liquid chromatography. The mobile phase consisted of an initial isocratic step with 7:93 (v/v) acetonitrile–7.5 mM phosphate buffer (pH 6) for 8 min, followed by a linear gradient up to a 25:75 (v/v) mixture over 1 min. Detection was carried out by a post-column fluorimetric derivatization based on the reaction of the drugs with sodium hypochlorite, giving rise to primary amines that reacted with o-phthalaldehyde and 2-mercaptoethanol to form highly fluorescent products. The calibration graphs, based on peak area, were linear in the range 0.1–4 μg/ml for all drugs. The detection limits were 30, 41 and 32 ng/ml (8.6, 12.5 and 9.1 pmol) for ranitidine S-oxide, ranitidine N-oxide and ranitidine, respectively. Chromatographic profiles obtained for plasma and urine samples showed no interference from endogenous compounds.     

Determination of a new non-narcotic analgesic, DA-5018, in plasma, urine and bile by high-performance liquid chromatography

A high-performance liquid chromatographic method was developed for the determination of a new non-narcotic analgesic, DA-5018 (I), in rat plasma, urine and bile samples, using propranolol for plasma samples and protriptyline for urine and bile samples as internal standards. The method involved extraction followed by injection of 100 μl of the aqueous layer onto a C₁₈ reversed-phase column. The mobile phases were 5 mM methanesulfonic acid with 10 mM NaH₂PO₄ (pH 2.5)-acetonitrile, 70:30 (v/v) for plasma samples and 75:25 (v/v) for urine and bile samples. The flow-rates were 1.0 ml/min for plasma samples and 1.2 ml/min for urine and bile samples. The column effluent was monitored by a fluorescence detector with an excitation wavelength of 270 nm and an emission wavelength of 330 nm. The retention time for I was 4.8 min in plasma samples and 10.0 min in urine and bile samples. The detection limits for I in rat plasma, urine and bile were 20, 100 and 100 ng/ml, respectively. There was no interference from endogenous substances.     

High-performance liquid chromatographic–electrochemical assay for the quantitation of BMS-181885 in monkey plasma

A high-performance liquid chromatographic–electrochemical assay was developed and validated for the quantitation of BMS-181885 (I), an anti-migraine agent, in monkey plasma. The assay involved a solid-phase extraction of I and BMY-46317 (internal standard; I.S.) on a 1-ml cyano cartridge using the automatic solid-phase extraction cartridge (ASPEC) system. Immediately following the conditioning of the cyano column (3 ml of methanol and 2 ml of 1% glacial acetic acid), plasma (0.25 ml) was loaded on to the column. The column was then washed with a 3 ml of 0.1 M ammonium acetate buffer (pH 6). The final elution of the analytes was performed using 2 ml of methanol. The eluate was then evaporated to dryness (gentle stream of nitrogen at 40°C) and the residue was dissolved in the mobile phase and injected on to a YMC basic column (15 cm×4.6 mm; 5 μm particle size) at a flow-rate of 1 ml/min. A mixture of 0.1 M ammonium acetate at pH 6–acetonitrile–methanol (70:20:10, v/v) was used as the mobile phase. Standard curves, with a lower limit of quantitation of 2 ng/ml of I were linear (r²≥0.998; range: 2–50 ng/ml). Based on the analysis of the quality control (QC) samples, the assay was both accurate and precise. The stability of I was established following freeze–thaw cycles and storage at or below −20°C. The extraction recovery of I from monkey plasma was about 82%. The validated assay method was applied to determine the pharmacokinetics of I in monkeys following a single 1 mg/kg intravenous dose.     

Determination of 8-oxoguanine in human plasma and urine by high-performance liquid chromatography with electrochemical detection

A highly sensitive and selective method for determining 8-oxoguanine in plasma and urine was developed by high-performance liquid chromatography with electrochemical detection. The compound was separated by gradient elution on a C₁₈ reversed-phase column with a mobile phase of acetonitrile and 0.1 M sodium acetate, pH 5.2. 8-Hydroxy-2′-deoxyguanosine was used as internal standard. 8-Oxoguanine was detected electrochemically by setting the potential to +300 mV vs. Pd reference. The sensitivity of the assay was 22 ng/ml with a signal-to-noise ratio of 7:1. The within-day relative standard deviations for 8-oxoguanine quality control samples with concentrations of 3340, 1340 and 84 ng/ml were 3.6, 4.3 and 5.7% for plasma, and 4.1, 4.6 and 6.2% for urine, respectively. The day-to-day relative standard deviations for the same samples were 3.8, 6.8 and 7.1% for plasma, and 3.9, 7.0 and 7.9% for urine, respectively. The method is designed to study the pharmacokinetics and metabolic fate of O⁶-benzylguanine in a phase I clinical trial. Previously, O⁶-benzyl-8-oxoguanine was identified as the primary metabolite of O⁶-benzylguanine in humans. We now demonstrate that 8-oxoguanine is a further metabolite of O⁶-benzylguanine.     

Enantioselective analysis of disopyramide and mono-N-dealkyldisopyramide in plasma and urine by high-performance liquid chromatography on an amylose-derived chiral stationary phase

An enantioselective high-performance liquid chromatography method was developed for the simultaneous determination of disopyramide (DP) and mono-N-dealkyldisopyramide (MND) enantiomers in plasma and urine. The drugs were extracted from plasma samples by liquid–liquid extraction with dichloromethane after protein precipitation with trichloroacetic acid; the urine samples were processed by liquid–liquid extraction with dichloromethane. The enantiomers were resolved on a Chiralpak AD column using hexane–ethanol (91:9, v/v) plus 0.1% diethylamine as the mobile phase and monitored at 270 nm. Under these conditions the enantiomeric fractions of the drug and of its metabolite were analyzed within 20 min. The extraction procedure was efficient in removing endogenous interferents and low values for the relative standard deviations were demonstrated for both within-day and between-day assays. The method described in this paper allows the determination of DP and MND enantiomers at plasma levels as low as 12.5 ng/ml and can be used in clinical pharmacokinetic studies.     

High-performance liquid chromatographic assay of propofol in human and rat plasma and fourteen rat tissues using electrochemical detection

This paper describes a sensitive HPLC-electrochemical detection analytical method for determining the concentration of the intravenous anesthetic, propofol, in human or rat plasma or serum and a variety of rat tissues. Internal standard and drug are extracted from serum or plasma and other tissues with pentane. 2,6-tert.-Butylmethylphenol is used as internal standard. It includes a novel steam distillation procedure for separating the highly lipophilic propofol from skin and fat. The plasma/serum assay has a precision of 1–4% (C.V.) in the range 10 ng/ml to 1 μg/ml and permits the assay of 5 ng/ml from 0.1 ml of plasma/serum. The tissue procedure allows the estimation of 50 ng/g in 0.1 g of tissue for most of the major organs with less than 2% (C.V.) precision. This assay was used to measure propofol concentrations in plasma/serum and tissue samples in support of a project to develop a physiological pharmacokinetic model for propofol in the rat.     

High-performance liquid chromatographic determination of levodropropizine in human plasma with fluorometric detection

The present describes a new high-performance liquid chromatographic method with fluorescence detection for the analysis of levodropropizine [S-(−)-3-(4-phenylpiperazin-1-yl)-propane-1,2-diol] (Levotuss), an anti-tussive drug, in human serum and plasma. A reversed-phase separation of levodropropizine was coupled with detection of the native fluorescence of the molecule, using excitation and emission wavelengths of 240 nm and 350 nm respectively. The analytical column was packed with spherical 5 μm poly(styrene-divinylbenzene) particles and the mobile phase was 0.1 M NaH₂PO₄ pH 3-methanol (70:30, v/v), containing 0.5% (v/v) tetrahydrofuran. For quantitation, p-methoxylevodropropizine was used as the internal standard. Samples of 200 μl of either serum or plasma were mixed with 200 μl of 0.1 M Na₂HPO₄ pH 8.9 and extracted with 5 ml of chloroform-2-propanol (9:1, v/v). The dried residue from the organic extract was redissolved with distilled water and directly injected into the chromatograph. The limit of detection for levodropropizine, in biological matrix, was about 1–2 ng/ml, at a signal-to-noise ratio of 3. The linearity was satisfactory over a range of concentrations from 3 to 1000 ng/ml (r² = 0.99910); within-day precision tested in the range 5–100 ng/ml as well as day-to-day reproducibility proved acceptable, with relative standard deviations better than 1% in most cases. Interferences from as many as 91 therapeutic or illicit drugs were excluded.     

Determination of a new thymidine phosphorylase inhibitor, TPI, in dog and rat plasma by reversed-phase ion-pair high-performance liquid chromatography

A high-performance liquid chromatographic method for the determination of a new thymidine phosphorylase inhibitor, TPI, in dog and rat plasma is described. TPI was isolated from biological samples by solid-phase extraction on Bond Elut PRS columns. Chromatographic separation was achieved on a C₁₈ column using a mobile phase consisting of acetonitrile–10 mM acetate buffer (pH 4.3) including hexanesulfonate, with UV detection at 276 nm. This method has been validated across the range of 50–50 000 ng/ml using a 0.1-ml plasma volume. The mean recoveries from spiked plasma were 93% for dog and 94% for rat, respectively. The accuracy, precision and specificity of the method were demonstrated to be acceptable, and it was applied to the toxicokinetic study of TPI in rats.     

Determination of morphine by high-performance liquid chromatography with electrochemical detection: application to human and rabbit pharmacokinetic studies

A rapid, sensitive, precise and accurate high-performance liquid chromatographic assay with coulometric electrochemical detection was developed for the determination of morphine in human, rabbit, pig and dog plasma. It includes a one-step extraction procedure with hexane–isoamyl alcohol (1:1, v/v) at pH 8.9 (adjusted with phosphoric acid) and reversed-phase liquid chromatography on a μPorasil column. The mobile phase was composed of 5 mM sodium acetate buffer (pH 3.75)–acetonitrile (25:75, v/v). A flow-rate of 1.2 ml/min at 20°C was used. The working potentials for the electrochemical detector were +0.20 V for detector cell 1, +0.55 V for detector cell 2 and +0.75 V for the guard cell. The limit of detection of morphine was 100 pg/ml of plasma. Repeatability, precision and accuracy were also determined concomitantly. The calibration graphs were linear in the concentration range 0.25–250 ng/ml with correlation coefficients of 0.998±0.01 and with a minimum intercept of 0.05±0.08. The precision in plasma was acceptable, with coefficients of variation less than 11%. The absolute recoveries of morphine and nalbuphine (internal standard) were between 86 and 89% and independent of morphine concentration. Pharmacokinetics after oral morphine [MST Continus™ (morphine sulphate tablets) 30 mg, Bard Pharmaceutical, Cambridge, UK] in humans revealed a one-compartment first-order absorption model with one absorption phase and one elimination phase. The absorption and elimination half-lives were 2.46 and 1.80 h, respectively. Pharmacokinetics after intravenous morphine (3 mg/kg) in rabbits showed a linear two-compartment open model with one distribution phase and one elimination phase. The distribution and elimination half-lives were 0.5 and 33.8 h, respectively.