Determination of neomycin in plasma and urine by high-performance liquid chromatography. Application to a preliminary pharmacokinetic study.

A reversed-phase high-performance liquid chromatographic (HPLC) method has been developed for the determination of neomycin in plasma and urine. The plasma was deproteinated with trichloroacetic acid and centrifuged. The supernatant was mixed with ion-pair concentrate and centrifuged again. The resultant supernatant was analyzed by HPLC. Urine was centrifuged to remove debris, if any, mixed with ion-pair concentrate and analyzed directly by HPLC. The HPLC conditions consisted of an ion-pairing mobile phase, a reversed-phase column, post-column derivatization with o-phthalaldehyde (OPA) reagent and fluorescence detection. The overall average recovery of neomycin was 97 and 113% from plasma spiked at 0.25-1.0 micrograms/ml, using standard curves prepared in plasma extract and in water, respectively, and 94% for urine spiked at 1-10 micrograms/ml using a standard curve prepared in water. The method was used to detect neomycin in plasma and urine obtained from animals injected intramuscularly with neomycin. Various pharmacokinetic parameters of neomycin were also determined from its profile of plasma concentration versus time.     

High-performance liquid chromatographic assay of sulbactam using pre-column reaction with 1,2,4-triazole

A high-performance liquid chromatographic (HPLC) method for the determination of sulbactam in human and rat plasma and urine has been developed. Sulbactam was reacted with 1,2,4-triazole to yield a product having an ultraviolet absorption maximum at 326 nm. The product was separated using reversed-phase HPLC from the regular components of plasma and urine with an ion-pair buffer at 50 degrees C and detected at the ultraviolet maximum. The limits of accurate determination were 0.2 and 1.0 micrograms/ml in plasma and urine, respectively. The coefficients of variation of inter- and intra-assays in human plasma spiked at 4.0 micrograms/ml (n = 5) were 1.02 and 3.05%, respectively. Coexisting cefoperazone, penicillins, or the alkaline degradation product(s) of sulbactam did not interfere in the sulbactam assay. The pharmacokinetic behaviour of sulbactam and cefoperazone coadministered to rats was estimated by moment analysis.     

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.     

Spectrophotometric method for the determination of sorbic acid in various food samples with iron(III) and 2-thiobarbituric acid as reagents

A simple, rapid and accurate spectrophotometric method has been developed for the determination of sorbic acid in various food samples based on the oxidation of sorbic acid by iron(III) at 100 degrees C to malonaldehyde, which then reacts with 2-thiobarbituric acid to form a reddish brown product. The optimum experimental conditions for colour development have been assessed. Absorbance measurements were made at 529 nm in the presence of 0.4% m/V citric acid. The calibration graph was linear for 0-6 micrograms ml-1 of sorbic acid with a slope of 0.131 A micrograms-1 ml. The recoveries of sorbic acid at concentrations of 164-557 micrograms ml-1 ranged from 96 to 103%. The relative standard deviations of ten replicate determinations of sorbic acid in a synthetic cream soda sample spiked with 573 micrograms ml-1 of sorbic acid and in an onion juice sample containing 82 micrograms ml-1 of sorbic acid were 1.6 and 1.9%, respectively. Interferences from several common food additives can be minimised by extracting sorbic acid with diethyl ether and then back-extracting the acid with sodium hydrogen carbonate. The method has been applied successfully to the determination of sorbic acid in a wide range of food samples including beverages, cake, cake mate, garlic bread sprinkle, onion juice, oyster flavoured sauce and grenadine syrup.     

Extractive spectrophotometric determination of tungsten(VI) in alloy steels using ethopropazine hydrochloride.

Ethopropazine hydrochloride (EPH) has been proposed as a sensitive reagent for the spectrophotometric determination of tungsten(VI). The method is based on the formation of a chloroform-soluble yellow-colored ternary complex by the interaction of EPH and thiocyanate with tungsten(V). The complex exhibits the absorption maximum at 404 nm with Sandell's sensitivity value of 20.03 ng cm-2. The complex obeyed Beer's law in the concentration range of 1-15 micrograms ml-1 with an optimum concentration range of 2.3-12.9 micrograms ml-1. The effects of foreign ions in the determination of tungsten(VI) were investigated. The method has also been successfully applied to the analysis of alloy steels.     

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.     

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.     

Electrochemical oxidation at carbon paste electrode of tacrine and 1-hydroxytacrine and differential pulse voltammetric determination of tacrine in pharmaceuticals and human urine

The electrochemical oxidation of tacrine and its 1-OH-metabolite, has been studied by cyclic voltammetry and differential pulse voltammetry by using carbon paste electrodes. The peak current-concentration relationship was found to be linear up to 20 micrograms ml-1 with detection limits of 0.06 microgram ml-1 for tacrine and 0.18 microgram ml-1 for 1-OH-tacrine and quantitation limits of 0.20 microgram ml-1 for tacrine and 0.37 microgram ml-1 for 1-OH-tacrine. A method for determining tacrine by differential pulse voltammetry in pharmaceuticals and human urine, in the presence of 1-OH-tacrine, has been developed.     

Determination of imipenem (N-formimidoyl thienamycin) in human plasma and urine by high-performance liquid chromatography, comparison with microbiological methodology and stability

High-performance liquid chromatographic (HPLC) methods using ultraviolet (UV) detection have been developed for the assay of the antibiotic imipenem (N-formimidoyl thienamycin) in human plasma and urine. A reversed-phase analytical column is employed in the plasma assay method and a cation-exchange column is used in the urine assay method. Both methods use borate buffer in the mobile phase. The method of preparation of human fluid samples for HPLC injection has been optimized with respect to the stability of imipenem in aqueous buffers, in morpholine buffer--ethylene glycol stabilizer, and in urine and plasma. Preparation of the samples before injection into the HPLC systems involves deproteination/filtration of the plasma/urine samples. The open lactam metabolite and the coadministered dehydropeptidase inhibitor, cilastatin sodium, do not interfere with the 313-nm detection of imipenem in either the plasma or the urine assay. Thienamycin, the precursor of imipenem and an impurity in imipenem formulations, is separated from the drug using both of these methods. Concentrations generated from the HPLC analysis of plasma and urine samples from two healthy volunteers compare favorably with results using a microbiological assay method. Correlation of the two methods gives r greater than or equal to 0.990 for both fluids.     

Neurotoxicity of tetraphenylporphinesulfonate (TPPS4) and a hematoporphyrin derivative (Photosan) in organotypic cultures of chick embryonic dorsal root ganglia

The neurotoxic effect of tetraphenylporphinesulfonate (TPPS4) and a hematoporphyrin derivative (HPD, Photosan) has been studied in organotypic cultures of chick dorsal root ganglia maintained in a semi-solid culture medium. The changes in two characteristics of neurite outgrowth, the mean radial length of neurites growing out from the ganglia and the area of neurite outgrowths, are used as parameters to evaluate the toxic effect. The porphyrins are tested over the concentration range 10-160 micrograms ml-1. TPPS4 is slightly more toxic than the HPD Photosan. The median inhibitory concentration (IC50) for TPPS4 is 45-50 micrograms ml-1 and for the HPD Photosan 50-60 micrograms ml-1, respectively. Nevertheless, the toxicity of the two drugs is relatively low compared to that of commonly used anticancer drugs, such as cisplatin or taxol.     

Direct determination of codeine-6-glucuronide in plasma and urine using solid-phase extraction and high-performance liquid chromatography with fluorescence detection

A sensitive and selective method was developed for the direct determination of codeine-6-glucuronide in plasma and urine using high-performance liquid chromatography (HPLC) with fluorescence detection. Codeine-6-glucuronide was synthesised and its purity estimated using acid and enzyme hydrolysis. The hydrolysis of codeine-6-glucuronide by beta-glucuronidase was incomplete and urine reduced the extent of hydrolysis. Codeine-6-glucuronide was recovered from plasma using a solid-phase extraction column and separated on a reversed-phase C18 HPLC column. The assay showed good reproducibility and accuracy (within 10%), and standard curves were linear between 32 and 1600 ng/ml in plasma and between 0.32 and 160 micrograms/ml in urine. The assay has been applied to the study of the pharmacokinetics and metabolism of codeine in patients.     

A New Method for the HPLC Analysis of Pt(II) in Urine

Abstract

An analytical method has been developed to measure Pt(II) in urine via derivatization and UV or HPLC analysis. A measured quantity of urine is heated briefly with diethyl ammonium diethyl-dithiocarbamate, and the resulting Pt(Et₂NCS₂)₂ is extracted into a measured volume of chloroform. Concentrations of Pt(II) are determined by UV absorption at 346 nm or by reverse phase HPLC analysis. The detection limit for Pt(II) as its dithiocarbamate is ～ 1 ng by HPLC; the concentration limit for HPLC analysis by direct extraction was ～ 25 ng/ml. Chromatographic response was linearly related to Pt(II) concentration over the range 100-4, 000 ng/ml; dilution of more concentrated samples has extended this range to at least 30, 000 ng/ml. This method has been applied to the analysis of Pt(II) in the urine of patients who have received cis-dichlorodiamniineplatinum(II) (CDDP) chemotherapy.     

Comparison of HPLC, capillary electrophoretic and direct spectrofluorimetric methods for the determination of temoporfin-poly(ethylene glycol) conjugates in plasma.

High-performance liquid chromatographic (HPLC), capillary electrophoretic (CE) and direct spectrofluorimetric methods for the determination of temoporfin-poly(ethylene glycol) 2000 conjugate (m-THPC-PEG 2000) in plasma are described and compared. m-THPC-PEG 2000 in plasma was quantitatively extracted (recovery 101-107%) with CH3OH-DMSO (4 + 1 v/v). The supernatant after centrifugation was used for HPLC, CE or direct spectrofluorimetric determination. The major drawback of the HPLC method was that it gave a broad and split peak even under gradient elution conditions, resulting in difficulty in detection and quantification. This is because m-THPC-PEG 2000 consists of a group of compounds with an average molecular mass of approximately 8680 Da owing to the wide molecular mass distribution of PEG 2000 used in the synthesis of the drug. m-THPC-PEG 2000 gave a single and relatively sharp peak when separated by CE with sodium tetraborate buffer (pH 9.45) in the presence of sodium dodecyl sulfate as the running buffer. However, this method lacks the necessary sensitivity for detecting the drug in plasma extract because of the limited sample volume that can be injected. Direct spectrofluorimetry is the method of choice because of its simplicity, specificity and sensitivity. Using an excitation wavelength of 423 nm and the specific emission maximum of 657 nm, the fluorescence intensity could be sensitively measured. The calibration curve constructed by plotting fluorescence intensity against concentration was linear within the range 1.32-1056 ng ml-1. The detection limit (S/N = 3) was 1.32 ng ml-1 and the limit of quantification (S/N = 10) was 2.24 ng ml-1. The precision and reproducibility were assessed by repeated analysis (n = 24) of spiked plasma samples at 350.8 and 699.3 ng ml-1. The RSD was 4.5% and 1.6%, respectively.     

Determination of (S)-(-)-cathinone and its metabolites (R,S)-(-)-norephedrine and (R,R)-(-)-norpseudoephedrine in urine by high-performance liquid chromatography with photodiode-array detection.

A high-performance liquid chromatographic (HPLC) procedure with photodiode-array detection (DAD) is described for the determination of (S)-(-)-cathinone (S-CA) and its metabolites (R,S)-(-)-norephedrine (R-NE) and (R,R)-(-)-norpseudoephedrine (R-NPE) in urine. Extraction and clean-up of 1-ml urine samples were performed on a cyano-bonded solid-phase column using (+/-)-amphetamine as internal standard. The concentrated extracts were separated on a 3-microns ODS-1 column with acetonitrile-water-phosphoric acid-hexylamine as the mobile phase. Peak detection was done at 192 nm. The detection limits for S-CA and R-NE/R-NPE in urine were 50 and 25 ng/ml, respectively. The differentiation of the enantiomers of cathinone and norephedrine was achieved by derivatization with (S)-(-)-1-phenylethyl isocyanate to the corresponding diastereomers followed by HPLC-DAD on a 5-microns normal-phase column. The R and S enantiomers of norpseudoephedrine were determined by gas chromatography-mass spectrometry after on-column derivatization with (S)-(-)-N-trifluoroacetylprolyl chloride. Following a single oral dose of 0.5 mg/kg of S-CA, the concentrations found in urine ranged from 0.2 to 3.8 micrograms/ml of S-CA, from 7.2 to 46.0 micrograms/ml of R-NE and from 0.5 to 2.5 micrograms/ml of R-NPE.     

Exposure to 4,4'-methylenediphenyl diisocyanate (MDI) during moulding of rigid polyurethane foam: determination of airborne MDI and urinary 4,4'-methylenedianiline (MDA)

Occupational exposure to 4,4'-methylenediphenyl diisocyanate (MDI) was measured during moulding of rigid polyurethane foam. The aim of the study was to find out whether an MDI-derived urinary amine metabolite could be detected in the urine of workers exposed to apparently low levels of MDI. Airborne MDI was sampled on 1-(2-methoxyphenyl)-piperazine (2MP)-impregnated glass fibre filters and determined by high-performance liquid chromatography (HPLC) using ultraviolet (UV) and electrochemical (EC) detection. The limit of detection of MDI was 3 ng ml-1 for a 20 microliters injection. The precision of sample preparation, expressed as relative standard deviation (RSD), was 1.3% with UV detection and 2.1% with EC detection at a concentration of 70 ng MDI ml-1 (n = 6). The 2MP-MDI derivative was stable at +4 degrees C up to eight weeks. The accuracy of the method was validated in an international quality control programme. Workers (n = 57) from three different factories participated in the study. Urinary 4,4'-methylenedianiline (MDA) metabolite was determined after acid hydrolysis as heptafluorobutyric anhydride derivatives by gas chromatography-mass spectrometry using chemical ionisation and monitoring negative ions. The limit of detection in urine was 0.2 nmol l-1. The precision of six analyses for a urine sample spiked to a concentration of 1 nmol l-1 was 29% (RSD). The MDI concentrations were below the limit of detection in most (64%) of the air samples collected in the worker's breathing zone. Still, detectable amounts of MDA were found in 97% of the urine samples. Monitoring of urinary MDA appears to be an appropriate method of assessing MDI exposure in work environments with low or undetectable MDI concentrations in the workplace air.     

Analysis of delta-L-alpha-aminoadipyl-L-cysteinyl-D-valine by ion chromatography and pulsed amperometric detection

A novel high-performance liquid chromatography (HPLC) method is presented for the detection and trace level determination of the tripeptide delta-L-alpha-aminoadipyl-L-cysteinyl-D-valine (ACV). The tripeptide, an intermediate in penicillin production, is derived from fungal fermentation. The technique relies on ion-exchange separation of the tripeptide on an anion-exchange column followed by detection by reduction on a gold electrode using pulsed amperometry. The sensitivity of direct determination of ACV is increased by employing pulsed amperometric detection (PAD) over direct ultraviolet detection. Choice of the working electrode and optimisation of electrode potentials was based on cyclic voltammograms recorded for the tripeptide in the mobile phase. A linear regression equation was obtained over the range 0-100 micrograms ml-1. The detection limit in fermentation broths was found to be 0.1 micrograms ml-1 whereas in buffer the detection limit was found to be 10 ng ml-1. A good correlation coefficient was observed when ACV concentrations determined by ion chromatography-PAD were compared with measurements obtained by pre-column derivatisation with fluoromethylorthochloroformate followed by HPLC separation on a reversed-phase C18 silica column with UV detection. The procedure has been applied to the measurement of natural levels of ACV in fermentation broths of selected strains of Aspergillus nidulans and Penicillin chrysogenum.     

Chromatographic analysis of methylmercaptopurine riboside in human plasma and urine.

An isocratic reversed-phase high-performance liquid chromatographic (HPLC) method for the determination of methylmercaptopurine riboside (MMPR) in human plasma and urine is reported. Plasma samples were prepared for analysis by addition of internal standard (6-dimethylaminopurine 9-riboside) followed by extraction using disposable C18 cartridges. Urine samples were filtered through a 0.22-micron membrane prior to HPLC separation. The column effluent was monitored at 289 nm and quantitation performed using peak heights. The linear range for MMPR determination was from 10 to 500 ng/ml in plasma and from 0.25 to 50 micrograms/ml in urine. The reported method is convenient, sensitive, and reproducible, illustrating its usefulness for application in pharmacokinetic studies.     

Determination of nucleic acids with crystal violet by a resonance light-scattering technique

For the first time, Crystal Violet (CV) was used to determine nucleic acid concentrations using the resonance light-scattering (RLS) technique. Based on the enhancement of the RLS of CV by nucleic acids, a new quantitative determination method for nucleic acids in aqueous solutions has been developed. At pH 5.03 and ionic strength 0.005 mol kg-1, the interaction of CV with nucleic acids results in three characteristic RLS peaks at 344.0, 483.0 and 666.0 nm. With 4.0 x 10(-5) mol l-1 of CV, linear relationships were found between the enhanced intensity of RLS at 666.0 nm and the concentration of nucleic acids in the range 0-2.5 micrograms ml-1 for herring sperm DNA, 0-4.0 micrograms ml-1 for calf thymus DNA and 0-4.5 micrograms ml-1 for yeast RNA. The limits of determination were 13.8 ng ml-1 for herring sperm DNA, 36.8 ng ml-1 for calf thymus DNA and 69.0 ng ml-1 for yeast RNA. The assay is convenient, rapid, inexpensive and simple.     

Extraction--spectrophotometric determination of oxalate in urine and blood serum

An extraction--spectrophotometric method is described for the determination of oxalate, based on the formation of a mixed ligand vanadium (V)--mandelohydroxamic acid--oxalate complex. The complex was extracted into a solution of trioctylmethylammonium chloride (Adogen 446) in toluene and the absorbance measured at 535 nm. The experimental variables and interferences in this determination were studied. The detection limit is 0.5 microgram ml-1 and the range of application is between 2 and 8 micrograms ml-1. The method was applied to the determination of oxalate in urine and blood serum.     

Pharmacokinetics of clenbuterol in the ostrich.

The aim of this study was to investigate the pharmacokinetics of clenbuterol in the ostrich as no such data is available. Clenbuterol (2 mg) was given as a single oral dose to nine ostriches. Blood samples were collected over a period of 96 h after administration and urine for a period of 5 d. Plasma and urine samples were frozen at -20 degrees C pending analysis. Clenbuterol was quantified using a gas chromatograph-mass selective detector. The method for quantification of clenbuterol in plasma was validated by analysing spiked quality control samples at different concentrations. The limit of quantification was determined to be 0.75 ng ml-1 with an absolute recovery of more than 80%. The geometric mean maximum plasma clenbuterol concentration was 4.40 ng ml-1 with 3.0 h as the median time for maximum concentration. The plasma elimination half-life was 19.7 h. The clenbuterol concentration was above 0.75 ng ml-1 in plasma for 48 h and above 1.0 ng ml-1 in urine for 5 d. These data can be useful in residue analysis for clenbuterol in ostriches.