Sensitive high-performance liquid chromatographic determination of nifedipine in cat plasma following improved sample treatment

A simple, easy and accurate reversed-phase high-performance liquid chromatographic method is described for the determination of nifedipine in cat plasma. The procedure involves extraction of nifedipine from plasma using a Sep-Pak C₁₈ cartridge and ultraviolet detection at 350 nm. The present method provides the required reproducibility and sensitivity for the determination of low concentrations of nifedipine without interference from plasma components or photodegradation products. The method was validated over the range 1–50 ng/ml nifedipine. Accuracy and precision were, respectively, 97% or more and 5% or less over the concentration range examined. The minimum quantifiable concentration of nifedipine was found to be 1 ng/ml.     

Simultaneous determination of nifedipine and dehydronifedipine in human plasma by liquid chromatography–tandem mass spectrometry

Quantitative analysis of therapeutic compounds and their metabolites in biological matrix (such as plasma, serum or urine) nowadays requires sensitive and selective methods to allow the determination of concentrations in the ng/ml range. A new on-line LC–MS–MS method using atmospheric pressure chemical ionisation (APCI) as interface for the simultaneous determination of nifedipine (NIF) and its metabolite in human plasma, dehydronifedipine (DNIF) has been developed. The compounds were extracted from plasma using solid-phase extraction (SPE) on disposable extraction cartridges (DECs). The SPE operations were performed automatically by means of a sample processor equipped with a robotic arm (ASPEC system). The DEC filled with phenyl modified silica was first conditioned with methanol and water. The washing step was performed with water. Finally, the analytes were successively eluted with methanol and water. The liquid chromatographic (LC) separation of NIF and DNIF was achieved on a RP-18 stationary phase (4 μm). The mobile phase consisted of methanol–50 mM ammonium acetate solution (50:50, v/v). The LC was then coupled to tandem mass spectrometry with an APCI interface in the positive ion mode.

The method developed was validated. The absolute recoveries evaluated over the whole concentration range were 95±2% and 95±4% for NIF and DNIF, respectively. The method was found to be linear in the 0.5–100 ng/ml concentration range for the two analytes (r²=0.999 for both NIF and DNIF). The mean R.S.D. values for repeatability and intermediate precision were 2.9 and 3.0% for NIF and 2.2–4.7% for the metabolite.The method developed was successfully used to investigate the plasma concentration of NIF and DNIF in the pharmacokinetic studies.     

Liquid chromatographic method for the simultaneous determination of caffeine and fourteen caffeine metabolites in urine

An HPLC method has been developed for the separation and the determination of caffeine and its metabolites in urine samples using a one extraction–analysis run and UV detection. The compounds were extracted by liquid–liquid extraction using chloroform–isopropylalcohol (85:15, v/v). Chromatographic separation was accomplished on an ODS analytical column with a mobile phase containing 0.05% acetic acid/methylalcohol (92.5:7.5, v/v). Compounds were monitored at 280 nm. The method was validated for the determination of AFMU, 1X, 1U, 17X and 17U caffeine metabolites required to assess the metabolic activity of the enzymes subject to in vivo caffeine testing. The validated assay was applied to urine samples from ten healthy volunteers. The method was proved to be suitable to assess simultaneously the enzymatic activity of cytochrome P450 CYP1A2 and CYP2A6, as well as N-acetyltransferase and xanthine oxidase.     

Determination of the anticancer agent CI-980 in plasma by achiral liquid chromatography on a Pirkle-type stationary phase

A high-performance liquid chromatographic assay has been developed and validated for the determination of the anticancer agent CI-980 in plasma samples from pediatric patients. After ether extraction from alkaline plasma, the CI-980 was chromatographed on a (R)-N-(3,5-dinitrobenzoyl)phenylglycine stationary phase using a mobile phase composed of hexane–isopropanol (70:30, v/v) modified with 1% acetonitrile. The method was linear over a 0.25 to 25.00 ng/ml range and the intra- and inter-day coefficients of variation (C.V.s) were less than 15%. The method was applied to the determination of the plasma concentration–time profile for a pediatric patient receiving 3.5 mg/m² of CI-980 per day as a continuous 72-h infusion.     

A combination of methods for the on-line determination of alcohols in microbial cultures

Summary A new method for the continuous on-line determination of methanol (range 0.2 to 10 gl^–1) and ethanol (0.2 to 120 gl^–1) is described. The rate limiting step is diffusion of the alcohol through the walls of a silicone tube immersed in the culture broth. A sintered SnO₂ sensor was used instead of a Flame Ionization Detector for alcohol determination. Measurement is not affected by bioreactor aeration or agitation rates, dissolved oxygen, carbon dioxide, ammonia or the concentration of cells in the medium. The assay system was tested in extended batch cultivation of Methylomonas sp. with methanol as the sole carbon source (final biomass concentration, 35 gl^–1). Sensor readings agreed well with simultaneous off-line gas chromatographic methanol determination.     

Determination of the antitumor agent depsipeptide in plasma by liquid chromatography on serial octadecyl stationary phases

A high-performance liquid chromatographic assay has been developed and validated for the determination of the antitumor agent depsipeptide (FR-901228) in plasma samples from patients with advanced cancer. After the plasma proteins were precipitated with acetonitrile, the supernatant was extracted with ethylacetate. Depsipeptide was chromatographed on two serial octadecylsilica stationary phases using a mobile phase consisting of acetonitrile–potassium phosphate buffer (0.03 M, pH 3) (27:73, v/v), at a flow-rate of 2.0 ml/min and at ambient temperature. The method was linear over a 50 to 2000 ng/ml range and the intra- and inter-day coefficients of variations were less than 8%. The method was applied to the determination of the plasma concentration–time profile for 14 patients with advanced cancer receiving from 1 to 7.5 mg/m² of depsipeptide per day as a continuous 4-h infusion.     

A sensitive fluorometric assay for the determination of DNA

A sensitive fluorometric assay for DNA determination using m-diaminobenzoic acid dihydrochloride for the reaction with deoxyribose liberated by perchloric acid is described. Fluorescence is proportional to the amount of DNA over the range 0.05–5 μg of DNA when the reaction is conducted in a volume of 400 μl and over the range 0.01–0.5 μg when the reaction is conducted in a volume of 40 μl. The assay is suitable for estimating DNA both in perchloric acid extracts and in complex mixtures with protein and RNA. The determination of DNA in cells grown as a monolayer by the described method is simple and rapid.     

Sensitive high-performance liquid chromatographic determination of nifedipine in dog plasma using an automated sample preparation system with laboratory robot

Nifedipine, a calcium-channel blocking drug was analysed in dog plasma after oral dosing with two different formulations. Sample preparation was automated with a laboratory robot. Quantitative determination of the drug was performed on a reversed-phase HPLC system with electrochemical detection (ED) using an internal standard. Validation of the analytical method showed that the system is well suited for pharmacokinetic studies on dogs. The assay was linear in the range 1–50 ng/ml. Inter-day and intra-day variability were between 6.43–18.15% C.V. and 1.57–5.53% C.V., respectively.     

On-line solid-phase extraction of ceftazidime in serum and determination by high-performance liquid chromatography

A column-switching high-performance liquid chromatographic assay is described for the determination of ceftazidime (a third-generation cephalosporin) in human serum. The method does not require prior sample pretreatment. Serum is directly injected in a first chromatographic column for sample clean-up and extraction. Thereafter, using an on-line column-switching system, the drug is quantitatively transferred and separated on a second, analytical column followed by determination using ultraviolet absorption at 258 nm. The technique allows direct, rapid, precise, and simple determination of ceftazidime in serum over the range of 1–250 μg/ml using 12.5 μl of serum. This method was applied to study the pharmacokinetics of the drug in patients undergoing vascular surgery.     

Interlaboratory study of the analysis of benzylpenicillin by liquid chromatography

A liquid chromatography method for analysis of benzylpenicillin was examined in a collaborative study involving seven laboratories. The method comprised an isocratic part, which is used in the assay. The isocratic part corresponds to the assay method for benzylpenicillin used by a manufacturer. When the isocratic part is combined with gradient elution, the method is suitable for purity control. Five samples of benzylpenicillin (sodium and potassium salts) were analysed. The main component and the impurities were determined. An analysis of variance proved the absence of consistent laboratory bias. The laboratory–sample interaction was not significant. Estimates for the repeatability and reproducibility of the method, expressed as standard deviations (S.D.) of the result of the determination of benzylpenicillin, were calculated to be 0.71 and 0.80, respectively.     

Determination of a novel selective inhibitor of type 1 5α-reductase in human plasma by liquid chromatography with atmospheric pressure chemical ionization tandem mass spectrometry

A sensitive and specific assay of human plasma for the determination of (5α,7β,16β)-16[(4-chlorophenyl)oxy]-4,7-dimethyl-4-aza-andronstan-3-one (I), a selective inhibitor of human type 1 5α-reductase, has been developed. The method is based on high-performance liquid chromatography (HPLC) with tandem mass spectrometric (MS–MS) detection. The analyte (I) and internal standard, Proscar (II), were isolated from the basified biological matrix using a liquid–liquid extraction with methyl-tert.-butyl ether (MTBE). The organic extract was evaporated to dryness, the residue was reconstituted in mobile phase and injected into the HPLC system. The MS–MS detection was performed on a PE Sciex API III Plus tandem mass spectrometer using a heated nebulizer interface. Multiple reaction monitoring using the precursor→product ion combinations of m/z 430→114 and 373→305 was used to quantify I and internal standard (II), respectively. The assay was validated in the concentration range of 0.5 to 500 ng/ml in human plasma. The precision of the assay, expressed as coefficient of variation (C.V.), was less than 7% over the entire concentration range, with adequate assay specificity and accuracy. The HPLC–MS–MS method provided sufficient sensitivity to completely map the 24 h pharmacokinetic time-course following a single 0.5 mg dose of I.     

Determination of picogram levels of heptylphysostigmine in human plasma using high-performance liquid chromatography with fluorescence detection

A sensitive (50 pg/ml) method for the determination of heptylphysostigmine in human plasma is described. The procedure is based on liquid—liquid extraction of the drug from buffered plasma, and analysis of the concentrated organic extract using high-performance liquid chromatography on a silica column, under normal-phase chromatographic conditions, with fluorescence detection. Physostigmine was used as an internal standard. The assay has been fully validated in the concentration range 50–2000 pg/ml and utilized for the analysis of clinical samples from subjects dosed with heptylphysostigmine.     

Determination of chlorpromazine in human breast milk and serum by high-performance liquid chromatography

A high-performance liquid chromatographic (HPLC) assay was developed for the determination of chlorpromazine in serum and human breast milk. Chlorpromazine in serum and human breast milk was extracted by a rapid and simple procedure based on C₁₈ bonded-phase extraction, and a reversed-phase HPLC separation technique was developed. Chlorpromazine and levomepromazine as the internal standard were detected by ultraviolet absorbance at 254 nm. Determination was possible for chlorpromazine in the concentration range 10–300 ng/ml. The recoveries of chlorpromazine added to serum and human breast milk were 80.1–87.6 and 80.3–84.4%, respectively, with coefficients of variation of less than 10.2 and 7.8%. The method is applicable to drug level monitoring in the serum and human breast milk of patients treated with chlorpromazine.     

Development of an HPLC method for the determination of nifedipine in human plasma by solid-phase extraction

Nifedipine, a dihydropyridine calcium channel antagonist, is widely used in the treatment of hypertension and other cardiovascular disorders. A selective, sensitive and accurate high-performance liquid chromatographic method has been developed, validated and applied for determination of nifedipine in human plasma samples. A series of studies were conducted in order to investigate the effects of mobile phase composition, buffer concentration, mobile phase pH and concentration of organic modifiers, and to develop a convenient and easy-to-use method for quantitative analysis of nifedipine. The method involves solid-phase extraction on C18 cartridges. The chromatographic separation was accomplished on a Lichrocart Lichrospher 60 RP selectB column with a mobile phase composed of 0.020 mol/L KH2PO4 (pH 4.8) and acetonitrile (42:58, v/v). UV detection was set at 240 nm. The calibration curve was linear in the concentration range of 5.0-200.0 ng/mL for nifedipine in plasma and the limit of quantification was 5.0 ng/mL.     

Simultaneous determination of catecholamines and dobutamine in human plasma and urine by high-performance liquid chromatography with fluorimetric detection

We report a reliable fluorimetric assay for the simultaneous determination of norepinephrine, epinephrine, dopamine and dobutamine in human plasma and urine, based on liquid—liquid extraction and derivatization with the fluorogenic agent 1,2-diphenylethylenediamine prior to chromatography. The method is sensitive (detection limit 0.3–0.8 pg injected) and reproducible (coefficients of variation 1–10%), and shows good accuracy (93–98%). The method should also be used when one only wants to measure the concentrations of the natural catecholamines, in order to avoid interference by metabolites of dobutamine and by the late-eluting dobutamine itself.     

Simultaneous determination of theophylline, enoxacin and ciprofloxacin in human plasma and saliva by high-performance liquid chromatography

A simple reversed-phase high-performance liquid chromatographic method has been developed for the simultaneous determination of theophylline, ciprofloxacin and enoxacin in plasma and saliva. The biological fluid samples were extracted with methylene chloride-isopropyl alcohol prior to isocratic chromatography on a Waters C₁₈ μBondapak column. Ultraviolet detection was carried out at 268 nm. The assay in linear for ciprofloxacin and enoxacin (0.05–10 μg/ml), and theophylline (0.1–20 μ/ml). The assay can be used to investigate the interaction of these two fluoroquinolones with theophylline.     

Protein determination of cells immobilized in cross-linked synthetic gels

Summary An assay for the determination of the protein content of whole cells immobilized in cross-linked synthetic gels was developed. The assay is based on a three step procedure: a) methanol dehydration, b) protein extraction by 1.0 M alkali at 125°C c) colorimetric assay of the extracted protein according to Bradford's procedure (Bradford M. M. (1976), Anal. Biochem. 72:248–254). The procedure worked out was found adequate for the determination of the protein content of microbial cells immobilized in synthetic and native polymer-gel-systems.     

Determination of cibenzoline in plasma and urine by high-performance liquid chromatography

A rapid, sensitive and selective high-performance liquid chromatographic (HPLC) assay was developed for the determination of cibenzoline (Cipralan ^TM) in human plasma and urine. The assay involves the extraction of the compound into benzene from plasma or urine buffered to pH 11 and HPLC analysis of the residue dissolved in acetonitrile---phosphate buffer (0.015 mol/1, pH 6.0) (80:20). A 10-μ ion-exchange (sulfonate) column was used with acetonitrile—phosphate buffer (0.015 mol/1, pH 6.0) (80:20) as the mobile phase. UV detection at 214 nm was used for quantitation with the di-p-methyl analogue of cibenzoline as the internal standard.The recovery of cibenzoline in the assay ranged from 60 to 70% and was validated in human plasma and urine in the concentration range of 10–1000 ng/ml and 50–5000 ng/ml, respectively. A normal-phase HPLC assay was developed for the determination of the imidazole metabolite of cibenzoline. The assays were applied to the determination of plasma and urine concentrations of cibenzoline and trace amounts of its imidazole metabolite following oral administration of cibenzoline succinate to two human subjects.     

Sensitive high-performance liquid chromatographic method for the determination of methyl N-[5-[[4-(2-pyridinyl)-1-piperazinyl]carbonyl]-1H-benzimidazol-2-yl] carbamate in rat blood

A sensitive high-performance liquid chromatographic assay has been developed and validated for the determination of methyl N-[5-[[4-(2-pyridinyl)-1-piperazinyl]carbonyl]-1H-benzimidazol-2-yl] carbamate (CDRI compound 81/470) in normal rat blood. The method described herein is simple, with improved selectivity and sensitivity over a previously reported HPLC method. The limit of quantitation is 10 ng/ml (method 1) and 2.5 ng/ml (method 2) in blood, as compared with 40 ng/ml for the previous method. The standard curve in blood is linear over the concentration range 10–1000 ng/ml in method 1 and 2.5–1000 ng/ml in method 2 and the extraction recovery is higher than 80% for both methods.     

Determination of olopatadine, a new antiallergic agent, and its metabolites in human plasma by high-performance liquid chromatography with electrospray ionization tandem mass spectrometry

A rapid, sensitive and specific assay method has been developed to determine plasma concentrations of olopatadine hydrochloride (A) and its metabolites, M1 (B), M2 (C) and M3 (D), using high-performance liquid chromatography with electrospray ionization tandem mass spectrometry (LC–ESI-MS–MS). Olopatadine, its metabolites, and internal standard, KF11796 (E), were separated from plasma using solid-phase extraction (Bond Elut C₁₈ cartridge). The eluate was dried, reconstituted and injected into the LC–ESI-MS–MS system. The calibration curves showed good linearity over the ranges 1–200 ng/ml for olopatadine and M3, and 2–100 ng/ml for M1 and M2, and the method was thoroughly validated and applied to the determination of olopatadine and its metabolites in plasma collected during Phase I clinical trials. Furthermore, the assay values were compared with those determined by the radioimmunoassay method, which has been routinely used to determine olopatadine in plasma.