High-performance liquid chromatographic determination of cefonicid in human plasma and urine

A high-performance liquid chromatographic assay for determination of cefonicid concentrations in human plasma and urine samples has been developed using cefazolin as an internal standard. For the analysis of plasma samples two calibration curves were utilized covering the cefonicid concentration ranges of 0.05-1.0 microgram/ml and 1.0-50.0 micrograms/ml, respectively. Coefficients of variation of 7.4% or less were obtained for cefonicid concentrations of 0.05-50.0 micrograms/ml. Mean bias was +6.0% at 0.05 micrograms/ml cefonicid and between -2.1% and +1.6% for 1.0-50.0 micrograms/ml cefonicid. Plasma samples containing 30 ng/ml cefonicid could be well distinguished from blank plasma samples. Urine samples were analysed by using a calibration curve for cefonicid concentrations between 1.0 and 50.0 micrograms/ml. ranged from 8.6% at a cefonicid concentration of 1.0 microgram/ml to 0.5% at 50.0 micrograms/ml with a mean bias between -3.0% and +0.3%.     

Determination of the new fluoroquinolone fleroxacin and its N-demethyl and N-oxide metabolites in plasma and urine by high-performance liquid chromatography with fluorescence detection

A high-performance liquid chromatographic method is described for the determination of the new fluoroquinolone fleroxacin and its metabolites in plasma and urine. Plasma samples are deproteinized with acetonitrile, and, after evaporation and reconstitution of the supernatant, samples are analysed on a reversed-phase column. The limit of quantification is 10-20 ng/ml for the parent drug and 10 ng/ml for the metabolites, using a 0.2-ml sample. Urine samples are diluted with the mobile phase. An aliquot is then injected directly onto the column. The limits of quantification are 1 micrograms/ml for the parent drug and 0.5 micrograms/ml for the metabolites, using a 0.1-ml sample. The method has been successfully applied to pharmacokinetic studies of human volunteers and patients.     

Simultaneous determination of clarithromycin and 14(R)-hydroxyclarithromycin in plasma and urine using high-performance liquid chromatography with electrochemical detection.

A sensitive method for the simultaneous high-performance liquid chromatographic determination of clarithromycin and its active metabolite in plasma and urine is described. Alkalinized samples were coextracted with an internal standard and analyzed on a C8 column using electrochemical detection. Recoveries were greater than or equal to 85% and consistent. Standard curves for plasma were linear in the range 0-2 micrograms/ml for both compounds (r greater than 0.99), with limits of quantification of approximately 10.03 micrograms/ml (0.5-ml sample). Within-day and day-to-day precision were good, with coefficients of variation mostly within +/- 5%; accuracy for both compounds were routinely within 90-110% of theoretical values. Standard curves for urine were linear in the range 0-100 micrograms/ml with limits of quantification of 0.5 micrograms/ml (0.2-ml sample). Urine assays also had similar within-day and day-to-day precisions and accuracy.     

Quantitation of flupirtine and its active acetylated metabolite by reversed-phase high-performance liquid chromatography using fluorometric detection

A simple, selective and sensitive procedure is described for the quantitation of flupirtine maleate (FLU) and its active acetylated metabolite (Met. 1) in plasma and urine. Using a 0.5-ml sample, a sensitivity of 10 ng/ml is easily achieved with a reversed-phase octadecylsilane (C18) column, and a high-performance liquid chromatographic system with fluorescence detection. Quantitation from plasma involves addition of an internal standard, protein precipitation with acetonitrile and a sample concentrating step, while for urinalysis the samples are taken through a single extraction with methylene chloride. Analytical recoveries of FLU and Met. 1 from plasma averaged greater than or equal to 95%, while from urine only 60 and 50%, respectively, could be recovered. The overall, inter- and intra-day variability for both FLU and Met. 1 averaged 6, 5 and 3%, in plasma, respectively. Standard calibration plots in plasma were linear (r greater than or equal to 0.99) for FLU (range: 0.01-10.0 micrograms/ml) and Met. 1 (range: 0.5-25 micrograms/ml) over the extended range. A slightly modified elution system was employed for quantitation of FLU and Met. 1 in urine.     

Selective high-performance liquid chromatographic method for the determination of glycyrrhizin and glycyrrhetic acid-3-O-glucuronide in biological fluids: application of ion-pair extraction and fluorescence labelling agent

A selective high-performance liquid chromatographic method has been developed for the simultaneous determination of glycyrrhizin and glycyrrhetic acid-3-O-glucuronide in biological fluids of the rat. The procedure is based on the ion-pair formation using tetra-n-amylammonium bromide, extraction with ethyl acetate-n-heptane from the salt-saturated aqueous phase, labelling with 4-bromomethyl-7-methoxycoumarin, followed by chromatographic separation with fluorescence detection. Glycyrrhizin in plasma, bile and urine could be precisely determined in concentrations as low as 1, 1 and 2.5 micrograms/ml, respectively, in a 0.1-ml sample. The equivalent values for the glucuronide were 1, 2.5 and 2.5 micrograms/ml, respectively. The method is applicable in pharmacokinetic studies of glycyrrhizin in small animals.     

Determination of the monocyclic beta-lactam antibiotic carumonam in plasma and urine by ion-pair and ion-suppression reversed-phase high-performance liquid chromatography

A sensitive and selective high-performance liquid chromatography method has been developed for the determination of the new monocyclic beta-lactam antibiotic carumonam in plasma and urine. The method for plasma involves protein precipitation with acetonitrile and removal of lipids with dichloromethane; urine is diluted with buffer. Separation and quantification are achieved using a mobile phase based on either ion-suppression or ion-pair chromatography on a reversed-phase column with UV detection. The limit of determination is 0.5 micrograms/ml plasma, using a 0.5-ml specimen, and 25 micrograms/ml urine, using a 50-microliter specimen. The inter-assay reproducibility is generally better than 4% when an internal standard is used. Since beta-lactam antibiotics may degrade on storage, close attention must be paid to the stability of these drugs in biological fluids; novel measures to prevent degradation on storage are described. The assay has been successfully applied to the analysis of several thousand samples from pharmacokinetic studies, including a study involving patients with impaired renal function.     

High-performance liquid chromatographic determination of BRB-I-28, a novel antiarrhythmic agent, in dog plasma and urine.

A sensitive reversed-phase high-performance liquid chromatographic (HPLC) technique with ultraviolet detection has been developed to determine the concentration of BRB-I-28 (I), a novel antiarrhythmic agent, in dog plasma and urine. The mobile phase was acetonitrile-methanol-37.5 mM phosphate buffer, pH 6.8-triethylamine (50:50:75:0.1, v/v). The compound was extracted from dog plasma and urine with chloroform after alkalinization with sodium hydroxide. The extraction recovery was 83% from plasma and 84% from urine. Good linearity (r > 0.996) was observed throughout the ranges 0.1-12.0 micrograms/ml (plasma) and 0.1-8.0 micrograms/ml (urine). Intra- and inter-assay variabilities were less than 4%. The lower limit of quantitation was 0.08 microgram/ml in either plasma or urine. HPLC analysis of plasma and urine samples from a dog treated with I has demonstrated that the method was accurate and reproducible.     

Rapid analysis of ranitidine in biological fluids and determination of its erythrocyte partitioning

A reversed-phase ion-pair high-performance liquid chromatographic assay is described for the rapid and sensitive quantitation of the H2-receptor antagonist ranitidine in human plasma and urine. The method involves a single-step extraction of the alkalinized sample with methylene chloride and analysis of the evaporated extract on a cyano column. Detection was performed by ultraviolet absorbance monitored at 318 nm. The overall run time of the assay was 5 min at a flow-rate of 2.0 ml/min. The limit of sensitivity was 1 ng/ml ranitidine in human plasma. Urine and plasma samples collected from a subject after administration of an oral dose of 150 mg of ranitidine were analyzed by this method. Furthermore, the procedure was applied to determine the red blood cell partition coefficient of ranitidine in a concentration range up to 10 micrograms/ml.     

High-performance liquid chromatographic analysis of amphotericin B in plasma, blood, urine and tissues for pharmacokinetic and tissue distribution studies.

A sensitive and reproducible high-performance liquid chromatographic method was developed to assay ampherotericin B in plasma, blood, urine and various tissue samples. Amphotericin B was isolated from each sample matrix by solid-phase extraction (Bond-Elut). The eluate from Bond-Elut containing amphotericin B was injected onto a reversed-phase C18 column (Waters, mu Bondpak, 10 microns, 300 mm x 3.9 mm I.D.) with a mobile phase of 45% acetonitrile in 2.5 mM Na2EDTA at 1 ml/min. Detection of amphotericin B was by ultraviolet absorption at 382 nm. Blood and tissues were homogenized and extracted with methanol prior to Bond-Elut extraction. The extraction efficiencies of amphotericin B from plasma, blood and tissues were approximately 90, 70 and 75%, respectively. The sensitivity of the assay was less than or equal to 5 ng/ml for plasma, less than or equal to 25 ng/ml for blood, 2.5 ng/ml for urine and 50 ng/g for tissues. The linearity of the assay method was up to 2.5 micrograms/ml for plasma, 5 micrograms/ml for blood, 500 ng/ml for urine and 500 micrograms/g for tissues. The assay was reproducible with an intra-day coefficient of variation (C.V., n = 3) of less than 5% in general for plasma, blood and tissues. The inter-day C.V. of the assay was less than 5% for plasma (n = 5), less than 10% for blood (n = 4) and less than 5% for tissues (n = 3). The overall variability in the urine assay was generally less than 10%. This method has demonstrated significant improvement in the sensitivity and reproducibility in assaying amphotericin B in plasma and especially in blood, urine and tissues. We have employed this assay to compare the pharmacokinetic and tissue distribution profiles of amphotericin B in rats and dogs following administration of Fungizone and ABCD (amphotericin B-cholesteryl sulfate colloidal dispersion), a lipid-based dosage form. In addition, the assay method for plasma and urine samples can also be applied to pharmacokinetics studies of amphotericin B in man.     

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

A rapid, sensitive and specific high-performance liquid chromatographic (HPLC) assay was developed for the determination of amdinocillin (formerly mecillinam) in human plasma and urine. The assay is performed by direct injection of a plasma protein-free supernatant or a dilution of urine. A 10 micrometer muBondapak phenyl column with an eluting solvent of water--methanol--1 M phosphate buffer, pH 7 (70:30:0.5) was used, with UV detection of the effluent at 220 nm. Azidocillin potassium salt [potassium-6-(D-(-)-alpha-azidophenyacetamido)-penicillanate] was used as the internal standard and quantitation was based on peak height ratio of amdinocillin to that of the internal standard. The assay has a recovery of 74.4 +/- 6.3% (S.D.) in the concentration ranges of 0.1-20 microgram per 0.2 ml of plasma with a limit of detection equivalent to 0.5 microgram/ml plasma. The urine assay was validated over a concentration range of 0.025-5 mg/ml of urine, and has a limit of detection of 0.025 mg/ml (25 microgram/ml) using a 0.1-ml urine specimen per assay. The assay was applied to the determination of plasma and urine concentrations of amdinocillin following intravenous administration of a 10 mg/kg dose of amdinocillin to two human subjects. The HPLC and microbiological assays were shown to correlate well for these samples.     

High-performance liquid chromatographic determination of loracarbef, a potential metabolite, cefaclor and cephalexin in human plasma, serum and urine

A high-performance liquid chromatographic (HPLC) method is reported for the determination of a new carbacephem antibiotic, loracarbef, a hydroxylated analogue, and two cephalosporins, cefaclor and cephalexin, in plasma, serum, and urine. The antibiotics are extracted from plasma by means of C18 solid-phase cartridges. Urine samples are diluted with water and directly injected on the HPLC system. The HPLC system utilizes a Supelcosil LC-18-DB (250 mm x 4.6 mm I.D.) reversed-phase column and ultraviolet detection at 265 nm. The limit of quantitation is 0.5 micrograms/ml for each compound. Excellent correlation of plasma concentrations is shown between results determined by HPLC and those obtained by microbiological agar-well diffusion assays. Stability studies of loracarbef in human plasma show the antibiotic to be stable for at least 24 h at room temperature and for at least twelve months at -20 degrees C.     

Simultaneous determination of a new gastrointestinal prokinetic agent (HSR-803) and its metabolites in human serum and urine by high-performance liquid chromatography using automated column-switching.

A method based on high-performance liquid chromatography using column-switching is described for the simultaneous determination of HSR-803 and its metabolites in human serum and urine. The system uses a six-port valve with a Nucleosil CN pre-column for on-line sample clean-up, and direct injection of samples. The limits of quantitation in serum and urine were 5 and 20 ng/ml for HSR-803 and 50 and 200 ng/ml for the metabolites, respectively. The coefficients of variation for the intra- and inter-day accuracies were between 0.8 and 7.1% for each compound. This method was applied to the pharmacokinetic studies in humans after oral administration of HSR-803.     

High-performance liquid chromatographic determination of a new beta-lactamase inhibitor and its metabolite in combination therapy with piperacillin in biological materials

[2S-(2 alpha,3 beta,5 alpha)]-3-Methyl-7-oxo-3-(1H-1,2,3-triazol-1-yl- methyl)-4-thia-1-azabicyclo [3.2.0]-heptane-2-carboxylic acid 4,4-dioxide (YTR-830H) is a new beta-lactamase inhibitor and the combination therapy of this compound with piperacillin is now under study. For the determination of the beta-lactamase inhibitor and piperacillin in biological materials, plasma and visceral tissue homogenates were deproteinized, whereas diluted urine and filtered faeces homogenates were treated with a Sep-Pak C18 cartridge. In order to assay the inactive metabolite of beta-lactamase inhibitor, each sample was treated with a Sep-Pak C18 cartridge. Aliquots of each preparation were chromatographed using ion-pair and reversed-phase chromatographic techniques on a high-performance liquid chromatograph equipped with a UV detector, set at 220 nm. The detection limits of beta-lactamase inhibitor and piperacillin were 0.2 microgram/ml in plasma, 2.5-5.0 micrograms/ml in urine and 0.2-0.5 microgram/g in visceral tissue and faeces. Those of the metabolite were 1.0 microgram/ml in plasma, 2.5-5.0 micrograms/ml in urine and 1.0 microgram/g in visceral tissue and faeces. A precise and sensitive assay for the determination of the beta-lactamase inhibitor, its metabolite and piperacillin is described, and their stabilities in several media are reported.     

Body fluid analysis of a phosphonic acid angiotensin-converting enzyme inhibitor using high-performance liquid chromatography and post-column derivatization with o-phthalaldehyde

A method is described for the extraction of a phosphonic acid angiotensin-converting enzyme inhibitor from either urine or plasma, and subsequent quantitation using high-performance liquid chromatographic (HPLC) analysis and post-column o-phthalaldehyde reagent derivatization. The compound cannot be quantitatively extracted from the body fluids, but use of a fluorinated internal standard allowed for the computation of accurate results. With the use of an internal standard, excellent precision, linearity, and recovery were obtained for analyte response in both urine and plasma. In urine a working range of 0.2-10 micrograms/ml was found, with a limit of detection of 0.1 micrograms/ml. For plasma the working range was found to be 2-500 ng/ml, and the limit of detection was established as 1 ng/ml. Due to the non-polar character of the analyte at low pH values, it was possible to use novel extraction (solid-phase C8 column) and HPLC [poly(styrenedivinyl benzene) HPLC column] conditions to separate and quantitate the compound from plasma and urine.     

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

A high-performance liquid chromatographic method is described for the analysis of the anti-bacterial agent cefotaxime and desacetylcefotaxime in physiological fluids. Plasma or serum samples were mixed with chloroform--acetone to remove proteins and most lipid material. The aqueous phase was then freeze-dried, reconstituted in mobile phase and chromatographed on a reversed-phase column using UV detection at 262 nm. Urine was analysed directly after centrifugation to remove particulate matter. The detection limit was 0.5--1.0 micrograms/ml for serum and 5 micrograms/ml for urine. The method has been applied to the analyses of cefotaxime and desacetylcefotaxime in plasma, serum, urine, cerebrospinal fluid, saliva, and pus from infected wound secretions. Two additional metabolites, which are lactones in which the beta-lactam ring has been opened, could be separated by this method.     

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 beta-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 micrograms/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 morphine and 6-acetylmorphine in plasma by high-performance liquid chromatography with fluorescence detection

A method is described for the simultaneous determination of morphine and 6-acetylmorphine in small volumes of human plasma by normal-phase high-performance liquid chromatography using solid-phase extraction, dansyl derivatisation and fluorescence detection. The lower limits of quantitation in a 0.1-ml plasma sample are 10 ng/ml for morphine and 25 ng/ml for 6-acetylmorphine. The method has been applied to determine concentrations of morphine and 6-acetylmorphine in plasma samples from premature babies administered an intravenous infusion of diamorphine.     

High-performance liquid chromatographic method for the quantitation of quinidine and selected quinidine metabolites

A specific and sensitive assay for the separation and quantitation of quinidine, 3-hydroxyquinidine, quinidine-N-oxide, O-desmethylquinidine and dihydroquinidine is presented. The assay is shown to be sensitive to concentrations of 0.1 microgram/ml for all the above compounds when using a serum sample of 0.1 ml. The standard curve demonstrates linearity at concentrations from 0.1 to 5 micrograms/ml. The extraction procedure consists of adjusting the serum to an alkaline pH and extracting once with a mixture of methanol-dichloromethane (15:85). The organic extract is dried and the residue is solubilized in mobile phase. The chromatographic conditions are an isocratic delivery of the mobile phase 0.01 M K2HPO4-acetonitrile (96:4) through a C18 column at ambient temperature. Detection of the compounds of interest is by ultraviolet absorption at a wavelength of 210 nm. For each compound the inter-assay variation is less than 10% and the intra-assay variation is less than 15%. No interfering compounds were detected when a commercially prepared serum spiked with 28 commonly used therapeutic compounds was assayed by this method. The analytical method presented here for the isolation and quantitation of quinidine, several active metabolites, and dihydroquinidine has adequate sensitivity and specificity for monitoring the concentration of quinidine and quinidine metabolites in patient samples.     

Determination of cefodizime in biological materials by high-performance liquid chromatography

Cefodizime (THR-221) is a new semi-synthetic cephalosporin. A high-performance liquid chromatographic method has been developed for the determination of cefodizime in biological materials. A plasma or serum sample was deproteinized with methanol and the resulting methanol eluate was concentrated to a volume of 0.5 ml. Urine and bile samples were diluted with buffer and each diluted sample was filtered. Faeces samples were homogenized and the supernate obtained after centrifugation was filtered. Visceral tissue samples were homogenized, the centrifuged supernate was deproteinized with methanol, and the methanol eluate was concentrated to a volume of 0.5 ml. Aliquots of each preparation were chromatographed on a reversed-phase column with an ion-pair chromatographic technique on a high-performance liquid chromatograph equipped with an UV detector set at 264 nm. The detection limits for cefodizime were 0.1 microgram/ml in plasma or serum, 0.3 microgram/ml in bile, and 0.5 microgram/ml in urine, 0.5 microgram/g in faeces and visceral tissue. This precise and sensitive assay for the determination of cefodizime is described, and its stability in several media is reported.     

Determination of the cephalosporin Ro 13-99041 in plasma,urine, and bile by means of ion-pair reversed phase chromatography

An HPLC method has been developed for the determination of the cephalosporin antibiotic Ro 13-9904 in plasma, urine, and bile of dogs and of human volunteers using the technique of ion-pair chromatography with a LiChrosorb RP-18 column. The three mobile phases employed contained tetrapentyl-, tetraoctyl- and hexadecyltrimethyl-ammonium bromide, respectively, as lipophilic counterions. The chromatographic conditions chosen allowed simple and rapid sample preparation. Plasma was deproteinized with ethanol and the supernatant was directly injected onto the column; urine and bile were diluted with mobile phase and injected without any purification. The detection limit for the cephalosporin was about 0.5 μg/ml for plasma samples and approximately 5 μg/ml for bile and urine.