Measurement of bumetanide in plasma and urine by high-performance liquid chromatography and application to bumetanide disposition

A high-performance liquid chromatographic method for the measurement of bumetamide in plasma and urine is described. Following precipitation of proteins with acetonitrile, bumetanide was extracted from plasma or urine on a 1-ml bonded-phase C₁₈ column and eluted with acetonitrile. Piretanide dissolved in methanol was used as the internal standard. A C₁₈ Radial Pak column and fluorescence detection (excitation wavelength 228 nm; emission wavelength 418 nm) were used. The mobile phase consisted of methanol—water—glacial acetic acid (66:34:1, v/v) delivered isocratically at a flow-rate of 1.2 ml/min. The lower limit of detection for this method was 5 ng/ml using 0.2 ml of plasma or urine. Nafcillin, but not other semi-synthetic penicillins, was the only commonly used drug that interfered with this assay. No interference from endogenous compounds was detected. For plasma, the inter-assay coefficients of variation of the method were 7.6 and 4.4% for samples containing 10 and 250 ng/ml bumetanide, respectively. The inter-assay coefficients of variation for urine samples containing 10 and 2000 ng/ml were 8.1 and 5.7%, respectively. The calibration curve was linear over the range 5–2000 ng/ml.     

Simultaneous analysis of lignocaine and bupivacaine enantiomers in plasma by high-performance liquid chromatography

A sensitive analytical procedure is described for the simultaneous determination of lignocaine and the enantiomers of bupivacaine in biological fluids using diazepam as an internal standard. After solvent extraction into hexane, the local anaesthetics were separated using an α₁-acid glycoprotein (AGP) column and detected at 214 nm. Calibration curves were linear (r²>0.99) in the concentration range of 5 to 500 ng/ml for the enantiomers of bupivacaine and 12.5 to 1000 ng/ml for lignocaine. The corresponding limits of detection were 4 ng/ml and 10 ng/ml, respectively. The method was applied to the analysis of plasma from a healthy woman undergoing tubal ligation.     

High-performance liquid chromatographic method to screen and quantitate seven selective serotonin reuptake inhibitors in human serum

A high-performance liquid chromatographic screening method (HPLC) is described for the determination of seven selective serotonin reuptake inhibitors (SSRIs) (fluvoxamine, milnacipran, paroxetine, sertraline, fluoxetine, citalopram, venlafaxine) and for three pharmacologically active N-demethylated metabolites (desmethylcitalopram, didesmethylcitalopram and norfluoxetine). A tricyclic antidepressant, clomipramine, was used as an internal standard. The method consists of liquid extraction of serum after alcalinisation at pH 9.50, followed by chromatography on a Beckman C₁₈ reversed-phase column. Compounds were detected at 200.4 nm. The standard curves were linear over a working range of 50–1000 ng/ml for fluvoxamine, 15–1000 ng/ml for fluoxetine, 25–500 ng/ml for norfluoxetine, 50–500 ng/ml for sertraline, 20–500 ng/ml for paroxetine, 25–550 ng/ml for citalopram, 25–750 ng/ml for desmethylcitalopram, 25–800 ng/ml for didesmethylcitalopram, 25–650 ng/ml for milnacipran, and 25–500 ng/ml for venlafaxine. The quantitation limits of the method were 15 ng/ml for fluoxetine, 20 ng/ml for paroxetine, 25 ng/ml for venlafaxine, norfluoxetine and citalopram, and its metabolites, 40 ng/ml for sertraline and 50 ng/ml for fluvoxamine. No interferences were noted with this sensitive and specific method which can be used for therapeutic drug monitoring.     

Determination of epitestosterone and testosterone in urine by high-performance liquid chromatography

This paper describes an alternative HPLC method for the determination of testosterone and epitestosterone, which is simple, rapid, selective, sensitive and reproducible. Samples were prepared using a previous enzymatic hydrolysis with liquid-liquid extraction. The determination was carried out on a Hypersil BDS-C₁₈ reversed-phase column with gradient elution and UV absorbance detection (240 nm). The limits of quantification (signal-to-noise ratio = 6) were 20 ng/ml for testosterone and 30 ng/ml for epitestosterone.     

High-performance liquid chromatographic method for the determination of AG-331, a novel anti-cancer agent,in human serum and urine using solid-phase extraction and photodiode-array detection

A reversed-phase isocratic high-performance liquid chromatographic method has been developed for the determination of AG-331, a novel thymidylate synthase inhibitor, in human serum and urine. The method involves a solid-phase extraction from C₁₈ cartridges without addition of an internal standard. The methanol eluent is evaporated under nitrogen at 40°C, and reconstituted in mobile phase, acetonitrile-water (35:65, v/v) containing 25 mM ammonium phosphate. Separation of AG-331 was obtained on a C₁₈ column at a flow-rate of 1 ml/min. Chromatographic signals were monitored by a photodiode-array detector at a primary wavelength of 457 nm with a bandwidth of 4.8 nm. Standard curves are linear in the range of 22–2175 ng/ml in plasma and 44–2175 ng/ml in urine, respectively. The extraction recovery ranged from 92.9–102.4%. Intra-day coefficient of variation was less than 9.5%, and inter-day coefficient of variation was less than 14.3% for an AG-331 concentration of 44 ng/ml. This method has been used to characterize the pharmacokinetics of AG-331 in cancer patients as part of ongoing Phase I trials.     

Routine and sensitive method for determination of nicorandil in human plasma developed for liquid chromatography with ultraviolet and m0ass spectrometric detection

A rapid and sensitive method using HPLC has been developed for the quantification of nicorandil (SG-75) in human plasma samples for routine bioequivalence studies. The sample preparation needs two liquid–liquid extractions, first with CH₃Cl and HClO₄ as denaturation reagent and second with addition of ethyl acetate and Na₂CO_3(aq). Detection wavelength was 256 nm. The obtained correlation coefficient for weighted linear curve in the range from 5.0 to 300 ng/ml was higher than 0.9950. The limit of quantitation (LOQ) was established at 5.0 ng/ml. The HPLC separation was accomplished on Nucleosil Phenyl (5 μm) stainless steel column within 7 min. The mixture of 0.01 M ammonium acetate buffer (pH 6.2) and acetonitrile 10:3 (v/v) was used as the mobile phase. The same separation method was examined on HPLC–MS system. Using this system, the LOQ was established at 1.0 ng/ml and the linearity was obtained in the range from 1.0 to 150 ng/ml.     

Determination of piromidic acid residues in trout muscle tissue and in urine by liquid chromatography with post-column modification of pH and fluorimetric detection

A rapid high-performance liquid chromatographic method has been developed to determine piromidic acid in trout muscle tissue and in urine, in the presence of nalidixic, 7-hydroxymethylnalidixic, oxolinic and pipemidic acids and cinoxacin. A Nova-Pak C₁₈ column was used with acetonitrile–4·10⁻⁴ M oxalic acid (40:60, v/v) as the mobile phase. A post-column change of pH was made with NaOH. Fluorimetric detection at 456 nm (λ_ex 275 nm) was used. The instrumental detection limit was 5.91 ng/ml, based on height of peak. Pretreatment of the urine samples was not necessary and fish samples were extracted with sodium hydroxide solutions and cleaned by means of an extraction with chloroform. Detection limit was 147 ng/ml for urine and 5.91 ng/g for trout muscle. Good separation without interference from any other components was obtained. Recovery was better than 87% in urine and better than 72% in trout muscle tissue.     

Measurement of cefuroxime in human bronchoalveolar lavage fluid by high-performance liquid chromatography after solid-phase extraction

A sensitive and selective method for the determination of cefuroxime in bronchoalveolar lavage (BAL) fluid using high-performance liquid chromatography (HPLC) with UV detection at 280 nm after solid-phase extraction with C₁₈ cartridges was developed. A Waters symmetry C₁₈ column was used and the mobile phase was acetonitrile-0.05 M ammonium phosphate buffer (pH 3.2) (15:85, v/v). The method enabled the determination of cefuroxime at concentrations below 100 ng/ml, with a linear calibration curve at concentrations of 5–100 ng/ml for 400 μl of BAL. The intra- and inter-assay coefficient of variations for 10, 40 and 80 ng/ml were between 5.3 and 8.9%. Analytical recoveries were between 92.7 and 106.2%. The detection limit was 1 ng/ml at a signal-to-noise ratio of 3:1 using 400 μl of BAL. The method was successfully used for the analysis of BAL fluid from patients after oral administration of 500 mg cefuroxime axetil twice daily.     

Determination of mivacurium in plasma by high-performance liquid chromatography

An assay has been developed and validated for the routine monitoring of mivacurium in plasma. It consists of liquid-liquid extraction with dichloromethane and high-performance liquid chromatography with fluorometric detection (excitation and emission wavelengths 220 nm and 320 nm, respectively). A Spherisorb C₁ 5 μm column and a mobile phase containing acetonitrile, KH₂PO₄ and methanol are used. At a flow-rate of 1 ml/min, a concentration gradient is applied. The detection limit is approximately 1 ng/ml in plasma. For the separation of stereoisomeres, the Spherisorb SCX 10 μm column and acetonitrile-Na₂SO₄ as a mobile phase can be used. The assay shows good linearity over the range 1–1000 ng/ml. The accuracy and precision allows the utilisation in clinical pharmacokinetic studies.     

High-performance liquid chromatographic determination of peroxisomicine A1 (T-514) in genus Karwinskia

A chromatographic method was developed for the T-514 determination in Karwinskia leaves, stems and roots. A C₁₈ analytical column and a mobile phase consisting of methanol and McIlvaine buffer (pH 3) were used. T-514 was detected using a diode array detector and the chromatograms were recorded at 269 and 410 nm. A linear dependence of a peak area on the T-514 concentration (r=0.9991) was obtained in the range of 0.126–12.6 μg/ml. Limits of T-514 quantification (signal-to-noise ratio 10) in plant samples were 126 ng/ml at 410 nm and 28 ng/ml at 269 nm. T-514 was extracted from the plant material with ethyl acetate. Optimal extraction conditions were studied: number of extraction steps, volume of extracting agent and extraction time. The extracts were cleaned up using solid-phase extraction (SPE). SPE recoveries of 99.9% and 98.4% were achieved for the T-514 concentrations of 1.4 μg/ml and 0.26 μg/ml, respectively.     

High-performance liquid chromatographic method for the simultaneous determination of HIV-1 protease inhibitors indinavir,saquinavir and ritonavir in plasma of patients during highly active antiretroviral therapy

A new high-performance liquid chromatographic method for the simultaneous determination of indinavir, saquinavir and ritonavir in human plasma is described. Quantitative recovery following liquid–liquid extraction with diethyl ether from 500 μl of human plasma was achieved. Subsequently, the assay was performed with a linear gradient starting at 67 mM potassium dihydrogenphosphate–acetonitrile (65:35 to 40:60, v/v) as a mobile phase, a Phenomenex C₁₈ column and UV detection at 240 and 258 nm, respectively. Linear standard curves were obtained for concentrations ranging from 75 to 20 000 ng/ml for indinavir, from 10 to 6000 ng/ml for saquinavir, and from 45 to 30 000 ng/ml for ritonavir. The calculated intra- and inter-day coefficients of variation were below 6%.     

Quantitative determination of epinastine in plasma by high-performance liquid chromatography

A high-performance liquid chromatographic (HPLC) method for the quantitative determination of epinastine, a non-sedating histamine H₁ receptor antagonist, in rat plasma, was developed. A 100-μl volume of plasma sample was spiked with a solution of internal standard (diphenidol) and extracted with dichloromethane under alkaline conditions. The extract was applied onto the HPLC system and detected by ultraviolet absorption at a wavelength of 220 nm. The linearity of the calibration curve was preserved over the concentration range of 20--1000 ng/ml. Both intra-assay variation and relative error were less than 5% for the plasma sample containing 50 ng/ml or 1000 ng/ml of epinastine hydrochloride. The analytical method presented here should be useful for the investigation of the pharmacokinetic properties of epinastine, which is of clinical significance.     

Determination of temazepam and temazepam glucuronide by reversed-phase high-performance liquid chromatography

A rapid and sensitive method for extracting temazepam from human serum and urine is presented. Free temazepam is extracted from plasma and urine samples using n-butyl chloride with nitrazepam as the internal standard. Temazepam glucuronide is analyzed as free temazepam after incubating extracts with β-glucuronidase. Separation is achieved using a C₈ reversed-phase column with a methanol—water—phosphate buffer mobile phase. An ultraviolet detector operated at 230 nm is used and a linear response is observed from 20 ng/ml to 10 μg/ml. The limit of detection is 15.5 ng/ml and the limit of quantitation is 46.5 ng/ml. Coefficients of variation are less than 10% for concentrations greater than 50 ng/ml. Application of the methodology is demonstrated in a pharmacokinetic study using eight healthy male subjects.     

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

A new method for the determination of omeprazole in human plasma was developed. Omeprazole was extracted from plasma with toluene-isoamylalcohol (95:5, v/v), the organic phase was evaporated, dissolved in the mobile phase and injected into a reversed-phase C₁₈ column. Flunitrazepam was used as an internal standard. The mobile phase consisted of 47% methanol and 53% of 0.1 M dipotassium hydrogenphosphate, pH 7.8. The spectrophotometric detection was performed at 302 nm. Limit of quantitation was 9.7 ng/ml and the calibration curve was linear up to 1240 ng/ml.     

Stereoselective high-performance liquid chromatography determination of propranolol and 4-hydroxypropranolol in human plasma after pre-column derivatization

A stereoselective reversed-phase HPLC assay to quantify S-(−) and R-(+) enantiomers of propranolol and 4-hydroxypropranolol in human plasma was developed. The method involved liquid–liquid extraction for sample clean-up and employed 2,3,4,6-tetra-O-acetyl-β-glucopyranosyl isothiocyanate as a pre-column chiral derivatization reagent. The internal standard used was 4-methylpropranolol. The derivatized products were separated on an Altex C₁₈ column using a mixture of acetonitrile–water–phosphoric acid–triethylamine (58:42:0.1:0.06 and 50:50:0.15:0.06, v/v, for propranolol and 4-hydroxypropranolol, respectively) as mobile phase. The detection of propranolol derivatives was made at λ_ex=280 nm and λ_em=325 nm, and the corresponding 325 and 400 nm were used for 4-hydroxypropranolol derivatives. The assay was linear from 1 to 100 ng/ml and from 2 to 50 ng/ml using 0.5 ml of human plasma for propranolol and 4-hydroxypropranolol enantiomers, respectively. The present assay is used to quantify the enantiomers of propranolol and 4-hydroxypropranolol, respectively, in human plasma for pharmacokinetic studies.     

High-performance liquid chromatographic determination of GS4071, a potent inhibitor of influenza neuraminidase, in plasma by precolumn fluorescence derivatization with naphthalenedialdehyde

GS4071 is a potent inhibitor of influenza neuraminidase. A precolumn fluorescence derivatization HPLC method is described for the analysis of GS4071 in rat plasma. Plasma samples were subjected to solid-phase extraction on C₁₈ extraction columns. After extraction, GS4071 was derivatized with naphthalenedialdehyde in the presence of potassium cyanide to produce highly fluorescent cyano[f]benzoisoindole derivatives. Derivatized samples were stable for >24 h at 4°C. The samples were analyzed by an isocratic HPLC method using fluorescence detection at 420 nm excitation and 470 nm emission wavelength. The method was validated and applied to the analysis of plasma samples from pre-clinical pharmacokinetic studies in rats. The limit of detection for GS4071 was 20 ng/ml. For five replicate samples at 50, 400, and 1000 ng/ml, the within-day precision values were 16.9, 9.4 and 4.5%, respectively, and the between-day precision values were 16.9, 7.9, and 2.1%, respectively. The method was linear from 25 to 1600 ng/ml and the total recovery was >68% over this concentration range.     

High-performance liquid chromatographic analysis of mibefradil in dog plasma and urine

The objective of the study was to develop a sensitive and specific assay for studying the pharmacokinetics of a novel calcium antagonist, a benzimidazolyl-substituted tetraline derivative, mibefradil (I) in the dog. The assay involves liquid-liquid extraction of a biological sample, reversed-phase HPLC separation and fluorescence detection (λ_ex = 270 nm and λ_em = 300 nm) of a sample components. Each sample was eluted with a mobile phase pumping at a flow-rate of 2 ml/min. The mobile phase composition was a mixture of acetonitrile and aqueous solution (38:62, v/v). The aqueous solution contains 0.0393 M KH₂PO₄ and 0.0082 M Na-pentanesulphonic acid. The retention times were 10.7 min for I, and 12.2 min for internal standard Ro 40–6792. Calibration curves with concentrations of I ranging from 10 to 500 ng/ml were linear (r² > 0.99). The detection limit for I was 0.5 ng/ml when 0.5 ml of plasma or urine was used. Intra- and inter-day accuracy and precision were within 10%. The assay was successfully applied to the pharmacokinetic studies of I in dogs.     

Application of 2-halopyridinium salts as ultraviolet derivatization reagents and solid-phase extraction for determination of captopril in human plasma by high-performance liquid chromatography

A high-performance liquid chromatographic method was developed for the determination of captopril in human plasma. 1-Benzyl-2-chloropyridinium bromide (BCPB) was used as a precolumn derivatizing reagent. The mercapto group of captopril was arylated by the reagent to generate a stable UV-sensitive product. The derivative was solid-phase extracted (SPE), separated on a C₁₈ column using reversed-phase ion-paring chromatography and monitored by a spectrophotometric detector at 314 nm. The method enabled sensitive determination of captopril and its disulphides in human plasma in patients after oral administration. Disulphides of captopril with captopril itself and with endogenous thiol compounds are reduced with triphenylphosphine to form captopril, followed by derivatization with the same reagent. The quantification limit was 10 ng/ml. Calibration curves were prepared for human plasma samples spiked with captopril and captopril disulphide. The calibration curves were linear in the range of 10 to 500 ng/ml for captopril and 10 to 1000 ng/ml for captopril disulphide.     

Quantitative analytical methods for the determination of a new hypertension drug, CGS 25462, and its metabolites (CGS 25659 and CGS 24592) in human plasma by high-performance liquid chromatography

Two simple and sensitive reversed-phase high-performance liquid chromatography (HPLC) methods were developed and validated for the quantitative determination of a novel hypertension drug CGS 25462 and its major metabolites CGS 24592 and CGS 25659 in human plasma. CGS 25462 and CGS 25798 (internal standard) were purified by one-step liquid–liquid extraction with methylene chloride. The metabolites were analyzed on HPLC after plasma protein precipitation with 10% trichloroacetic acid (TCA). Separations were achieved on a Zorbax RX C₁₈ column. All compounds were detected by using a fluorescence detector. The excitation wavelength was 254 nm, and emission was monitored at 325±12.5 nm. Assessment of recovery and reproducibility indicated good accuracy and precision. Over the validation concentration range of 10 to 1000 ng/ml for CGS 25462 and 25 to 5000 ng/ml for both metabolites, overall mean relative recoveries were 96% for CGS 25462, 101% for CGS 25659 and 107% for CGS 24592, and the coefficients of variation were 4.6 to 13% for CGS 25462, 9.5 to 13% for CGS 25659 and 7.7 to 15% for CGS 24592. The limits of quantification (LOQs) were 10 ng/ml for CGS 25462 and 25 ng/ml for CGS 24592 and CGS 25659, which were of sufficient sensitivity to measure the concentrations of CGS 25462, CGS 25659 and CGS 24592 in plasma samples from normal volunteers following a single 800 mg oral dose.     

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.