Determination of 2',3'-dideoxyinosine and 2',3'-dideoxyadenosine in rat plasma by high-performance liquid chromatography with precolumn fluorescence derivatization.

A high-performance liquid chromatographic method with precolumn fluorescence derivatization using 2-(5-chlorocarbonyl-2-oxazolyl)-5,6-methylenedioxybenzofu ran is described for the quantification of 2',3'-dideoxyinosine, a therapeutic drug for acquired immunodeficiency syndrome, and 2',3'-dideoxyadenosine, an anti-human-immunodeficiency-viral agent, in rat plasma. The dideoxyribonucleosides and 3'-deoxythymidine (internal standard) in rat plasma (0.1 ml) are cleaned up by a solid-phase extraction technique using an octadecyl silica (ODS) cartridge, Toyopak ODS M, and the dideoxyribonucleosides in the eluate are reacted with the reagent to produce the corresponding fluorescent esters. The esters are separated by chromatography on a reversed phase column, TSKgel ODS-80TM. The detection limits (signal-to-noise ratio = 3) for the dideoxyribonucleosides are 1.3-5.4 pmol on column. Plasma concentrations of 2',3'-dideoxyinosine after intra-jugular-venous administration to rat can be monitored by this method.     

High-performance liquid chromatographic assay of a new non-steroidal anti-inflammatory agent, 2-(10,11-dihydro-10-oxodibenzo[b,f]thiepin-2-yl)propionic acid, in human plasma and urine

A high-performance liquid chromatographic method for the quantitation of a new anti-inflammatory agent, 2-(10,11-dihydro-10-oxodibenzo[b,f]thiepin-2-yl)propionic acid (CN-100; I), has been developed. The assay consists in extracting samples containing I and mefenamic acid, the internal standard, under acidic conditions and analysis by reversed-phase chromatography using ultraviolet detection at 330 nm. Preliminary plasma concentration-time and cumulative urinary excretion profiles from a healthy subject following oral administration of the tablet formulation are presented. This method is simple, sensitive and reproducible and is applicable to studies of the pharmacokinetic behaviour of I in humans.     

Fluorescence determination of 5-fluorouracil and 1-(tetrahydro-2-furanyl)-5-fluorouracil in blood serum by high-performance liquid chromatography

Fluorescence derivatization of 5-fluorouracil (5-FU) and 1-(tetrahydro-2-furanyl)-5-fluorouracil (ftorafur, FT) with 4-bromomethyl-7-methoxycoumarin using 18-crown-6 as a catalyst is studied with aim of developing a sensitive and selective liquid chromatographic method. 5-FU and FT form virtually substituted derivatives which possess maxima in their fluorescence emission spectra near 400 nm. These derivatives are separated by thin-layer chromatography and high-performance liquid chromatography to confirm the completion of reaction. For the determination of 5-FU and FT in serum, the reversed-phase high-performance liquid chromatographic separation of the derivatives is studied with a C13 column. This chromatography is of importance for the accurate determination of 5-FU and FT, which are, respectively, an important antitumour agent for the treatment of solid tumours in clinical medicine and a masked form of 5-FU generated in vivo.     

Determination of HP 749, a potential therapeutic agent for Alzheimer's disease, in plasma by high-performance liquid chromatography.

N-(n-Propyl)-N-(4-pyridinyl)-1H-indol-1-amine hydrochloride (HP 749, I), a non-receptor-dependent cholinomimetic agent with noradrenergic activity, is a potential agent for the treatment of Alzheimer's disease. Pharmacokinetic studies in animals and humans showed that I was well absorbed and metabolized primarily to the N-despropyl metabolite (P7480, II) after oral administration. To facilitate the kinetic studies, a sensitive and selective high-performance chromatographic assay was developed. I and II are extracted from plasma by a mixture of cyclohexane-ethyl acetate and chromatographed on an isocratic reversed-phase high-performance liquid chromatographic system employing an analytical phenyl column with acetonitrile-ammonium formate as mobile phase. The concentrations of these two compounds, quantitated by internal standardization, are monitored by ultraviolet detection. The method is linear in the plasma assay over a concentration range of 0.5-500 ng/ml for both compounds with a quantitation limit of 0.5 ng/ml. The precision and accuracy of the calibration curves and/or method are less than 10%. The recovery of I and II from plasma is 63-74 and 63-68%, respectively, over a concentration range of 0.5-500 ng/ml.     

Determination of tertatolol enantiomers in biological fluids by high-performance liquid chromatography.

A stereospecific high-performance liquid chromatographic method for the quantification of (-)- and (+)-tertatolol in plasma and urine is described. The method involves solid-phase extraction followed by derivatization with S(+)-naphthylethylisocyanate to form the urea derivative, which is more sensitive to fluorescence detection. The separation of the diastereomeric derivatives was performed by reversed-phase high-performance liquid chromatography. Fluorimetric detection (lambda excitation = 220 nm, lambda emission = 320 nm) allows the quantification of tertatolol enantiomers down to 6 ng/ml. The assay was used to study the pharmacokinetic profile of tertatolol enantiomers following oral administration of racemic tertatolol; preliminary results suggest enantioselective absorption and/or disposition of tertatolol.     

Determination of the R-(-) and S-(+) enantiomers of the monohydroxylated metabolite of oxcarbazepine in human plasma by enantioselective high-performance liquid chromatography.

An enantioselective liquid chromatographic assay for the simultaneous determination of the S-(+) and R-(-) enantiomers of the monohydroxylated metabolite of oxcarbazepine in human plasma is described. The metabolite is the active principle. The method is based on the extraction of plasma with diethyl ether-dichloromethane (2:1, v/v), separation of the organic phase, evaporation of the solvent and dissolution of the residue in the mobile phase. The two enantiomers were resolved on a Chiralcel OD (250 mm x 4.6 mm I.D.) high-performance liquid chromatographic column. The separation was achieved by isocratic elution with n-hexane-2-propanol (77:23, v/v). The flow-rate of the mobile phase was 1.0 ml/min and the two enantiomers were detected by ultraviolet absorbance at 210 nm. The analytical method is suitable for the quantitative and simultaneous determination of the two enantiomers in plasma at concentrations down to 0.4 mumol/l after administration of oxcarbazepine.     

Determination of Ro 14-1761, a new third-generation cephalosporin, in the plasma and milk of cattle by column switching high-performance liquid chromatography

A sensitive and specific high-performance liquid chromatographic procedure was developed for the determination of the third-generation cephalosporin Ro 14-1761 in cow plasma and milk. The molecular structure of the new antimicrobial was very close to that of ceftriaxone, but the high-performance liquid chromatographic methods available for the latter could not be used as Ro 14-1761 adsorbed and/or degraded during the chromatographic process. Furthermore, the high-performance liquid chromatographic technique derived for ceftriaxone was not sensitive enough for our purposes. In the new assay, the plasma (milk) protein was precipitated with acetonitrile after dilution of the sample with water. For low concentrations (less than or equal to 10 micrograms/ml), the supernatant obtained after centrifugation was concentrated by extracting acetonitrile with methylene chloride. Quantification was performed by column switching high-performance liquid chromatography with UV detection (274 nm) using ion-pair reversed-phase chromatography. Ethylenediaminotetraacetic sodium salt had to be added to the mobile phase (1.2 mM) to prevent adsorption and/or degradation of the cephalosporin on the analytical column. The selectivity of the chromatographic separation was enhanced by heating the column to ca. 50 degrees C. The drug recovery was better than 85%. The limit for quantitative determination in both milk and plasma was 0.1 microgram of Ro 14-1761 per millilitre with an accuracy of 1% (coefficient of variation 10%). The overall accuracy and precision were 1-10% in the 0.1-100 micrograms/ml concentration range.     

HPLC Assay of 6-Methoxy-2-Naphthylacetic Acid,a Major Metabolite of Nabumetone,in Human Serum

Abstract

A modified high-performance liquid chromatographic method for the quantitative determination of 6-methoxy-2-naphthylacetic acid (6-MNA), a major metabolite of nabumetone, in human serum was developed and validated. The composition of the mobile phase in Daigneault & Ferslew's method was changed in this study to improve the separation of 6-MNA from serum components. The volume ratio of acetonitrile to 1.5% acetic acid solution was changed from 60: 40 to 25: 75 in this study. As a result of the modification, the separation of 6-MNA from 2-naphtol (internal standard) and serum components was greatly improved. At a flow rate of 3.0 ml/min of the mobile phase, retention times of 6-MNA and 2-naphtol were 13 and 9 min, respectively. The detection limit was 1 μg/ml. Intra- and interday variations of the assay were <10.0 and <7.28%, respectively. Intra- and interday relative errors were <7.82 and <6.76%, respectively. 6-MNA in human serum could be determined successfully after oral administration of nabumetone (1g). Thus, the modified UV-HPLC method was confirmed to be applicable to the pharmacokinetic study of 6-MNA after oral administration of nabumetone to human subjects.     

Simultaneous determination of lidocaine and its metabolites in plasma and myocardium

No validated method exists for measuring lidocaine and its metabolites in myocardial tissue. We modified a previously described high-performance liquid chromatographic assay and applied it to plasma and to homogenized myocardial samples obtained from dogs that had received lidocaine by a double-infusion technique. Recovery of lidocaine, monoethylglycylxylidide and glycylxylidide after homogenization and extraction is reported. Assay variability, sensitivity and linearity over a wide range of sample sizes are also described. The results obtained with high-performance liquid chromatographic analysis are compared to quantitation of 14C-labeled lidocaine plus metabolites measured by an oxidation-scintillation technique. Myocardium to plasma partition coefficients for lidocaine, monoethylglycylxylidide and glycylxylidide were 2.16, 4.27, and 2.91, respectively.     

Simultaneous determination of artificial sweeteners, preservatives, caffeine, theobromine and theophylline in food and pharmaceutical preparations by ion chromatography.

A novel ion chromatographic method was proposed for the simultaneous determination of artificial sweeteners (sodium saccharin, aspartame, acesulfame-K), preservatives (benzoic acid, sorbic acid), caffeine, theobromine and theophylline. The separation was performed on an anion-exchange analytical column operated at 40 degrees C within 45 min by an isocratic elution with 5 mM aqueous NaH2PO4 (pH 8.20) solution containing 4% (v/v) acetonitrile as eluent, and the determination by wavelength-switching ultraviolet absorbance detection. The detection limits (signal-to-noise ratio 3:1) for all analytes were below the sub-microg/ml level. Under the experimental conditions, several organic acids, including citric acid, malic acid, tartaric acid and ascorbic acid, did not interfere with the determination. The method has been successfully applied to the analysis of various food and pharmaceutical preparations, and the average recoveries for real samples ranged from 85 to 104%. The levels of all analytes determined by this method were in good agreement with those obtained by the high-performance liquid chromatographic procedure. The results also indicated that ion chromatography would be possibly a beneficial alternative to conventional high-performance liquid chromatography for the separation and determination of these compounds.     

High-performance liquid chromatographic determination of ambroxol in human plasma.

Ambroxol has been determined in biological fluids using a rapid and sensitive high-performance liquid chromatographic method. The samples prepared from plasma by liquid-liquid extraction were analysed on reversed-phase silica gel by competing-ion chromatography with ultraviolet detection. The method was applied to the determination of ambroxol levels in twelve healthy volunteers after oral administration of 90 mg of ambroxol in tablets of Mucosolvan and Ambrosan.     

Determination of amidepin in human plasma by reversed-phase high-performance liquid chromatography with fluorescence detection

An isocratic reversed-phase high-performance liquid chromatographic method for the determination of amidepin has been developed. The method is based on the extraction of alkaline plasma with diethyl ether-dichloromethane, and the injection into the Supelcosil LC-18 column of the evaporated and reconstituted organic phase. After separation, detection is carried out by a fluorescence detector (excitation at 195 nm with no filter). The limit of detection is 10 ng/ml of plasma. The mean coefficient of variation is 12%. The plasma levels after oral administration and after intravenous administration are shown.     

Determination of ceftriaxone, a novel cephalosporin, in plasma, urine and saliva by high-performance liquid chromatography on an NH2 bonded-phase column

A high-performance liquid chromatographic method has been developed for the determination of a new cephalosporin antibiotic in plasma, urine and saliva (mixed saliva) using normal-phase technique and an NH2 bonded-phase column. The eluent mixture was a combination of acetonitrile and an aqueous solution of ammonium carbonate. The rapid method involved precipitation of protein from fluids by means of acetonitrile followed by automatic injection of the supernatant. The detection limit was 0.4 micrograms/ml for plasma, 3 micrograms/ml for urine and 0.03 micrograms/ml for saliva using UV detection.     

High-performance liquid chromatographic determination of morphine, morphine-3-glucuronide, morphine-6-glucuronide and codeine in biological samples using multi-wavelength forward optical detection.

An isocratic high-performance liquid chromatographic method has been developed for the determination of morphine, morphine-3-glucuronide, morphine-6-glucuronide and codeine in plasma, urine and cerebrospinal fluid. The use of an efficient solid-phase extraction procedure together with a forward optical scanning detector allows a detection limit of 500 pg/ml. The method was evaluated by examination of biological samples taken from newborn infants following the intravenous administration of morphine sulfate.     

Separation,Identification, and Quantification of N-Acetyl Cilastatin in Human Urine

Abstract

A new high-performance liquid chromatographic method coupled with anion-exchange sample extraction has been developed for the isolation and quantification of N-acetyl cilastatin from human urine samples. N-Acetyl cilastatin, isolated from urine after I.V. administration of cilastatin, was converted to dimethyl esters, and characterized by thin-layer chromatography and mass spectrometry. The detection limit of the assay was 5 μg/ml in urine. The method was shown to be linear, reproducible and reliable for the quantification of N-acetyl cilastatin in urine from four human subjects given I.V. doses of cilastatin alone and together with 250 and 1,000 mg of imipenem.     

Enantiospecific high-performance liquid chromatographic analysis of 2-phenylpropionic acid, ketoprofen and fenoprofen

A high-performance liquid chromatographic method has been developed for the quantitation of the R- and S-enantiomers of 2-phenylpropionic acid, ketoprofen and fenoprofen. The assay consists of extracting the arylpropionic acid with an internal standard and measuring the total (R + S) concentration of enantiomers by reversed-phase chromatography, derivatising the chromatographic fraction corresponding to the enantiomers to form R- and S, R-2-phenylethylamide distereoisomers which are resolved by normal-phase chromatography in order to calculate the fraction of each enantiomer. The limits of sensitivity of the assay for 2-phenylpropionic acid, ketoprofen and fenoprofen are 6, 0.2 and 2.5 mg/l, respectively.     

Simultaneous determination of 2-deoxy-D-glucose and D-glucose in rat serum by high-performance liquid chromatography with post-column fluorescence derivatization

A simple high-performance liquid chromatographic method for the determination of 2-deoxy-D-glucose and D-glucose in rat serum is described; this method is based on a post-column fluorescence derivatization. The sugars are automatically converted into fluorescent derivatives by reaction with meso-1,2-bis(4-methoxyphenyl)ethylenediamine in an alkaline medium after their separation on a strong anion exchanger column (TSK gel Sugar AXG). The detection limits (S/N = 3) for 2-deoxy-D-glucose and D-glucose in rat serum are 0.52 and 0.56 nmol/ml, respectively.     

Analysis of preservatives in cosmetics—Methylisothiazolones

Summary Antimicrobially active formulations based on 2-methyl-3(2H)-isothiazolone and 5-chloro-2-methyl-3(2H)-isothiazolone (Kathon CG, Euxyl K 100) are incorporated into manufactured cosmetics as preservatives. In this paper we report on the analysis of these active components by means of liquid chromatography. It is conventional for the original methylisothiazolone components in cosmetics to be analyzed by reversed phase high-performance liquid chromatography after column chromatographic separation and flash chromatographic purification. In our new analytical method the methylisothiazolones are converted into the respective -thiosubstituted acrylamide derivatives, which are ionic substances, with the help of the nucleophilic reagent hydrogensulfite. An ion pair high-performance liquid chromatographic method has been developed for the separation and quantification of these derivatives.     

High-performance liquid chromatographic determination of the stereoselective biotransformation of the chiral drug praziquantel.

A selective reversed-phase high-performance liquid chromatographic method for the simultaneous quantification of praziquantel and its monohydroxylated metabolites in serum is described. The quantitative analysis was followed by determination of the enantiomeric ratio of praziquantel and trans-4-hydroxypraziquantel, the main metabolite of praziquantel in humans, on a cellulose tris-3,5-dimethyl-phenylcarbamate column (Chiralcel OD). Serum level data for five volunteers after oral administration of racemic praziquantel were compared with in vitro metabolism data for praziquantel, obtained with liver microsomes of different species.     

High-performance liquid chromatographic analysis of doxacurium, a new long-acting neuromuscular blocker

A rapid and sensitive high-performance liquid chromatographic procedure was developed for the analysis of the new, long-acting neuromuscular blocker doxacurium in the plasma and urine of dog and man and in the bile of dog. Samples were prepared on solid-phase extraction cartridges containing a methyl (C1) bonded phase and were chromatographed on a 15 cm reversed-phase column (C1) using a mobile phase of 0.05 M monobasic potassium phosphate-acetonitrile (30:70, v/v). The compound was detected at 210 nm with a lower limit of quantitation of 10 ng/ml. An inter-assay accuracy of 90-92% was obtained for the analysis of the drug from biological fluids. The method was applied to studies of doxacurium after intravenous administration to dog and man.