Stereospecific high-performance liquid chromatographic assay of metoprolol.

A valid, sensitive high-performance liquid chromatographic technique is reported for the separation of the two enantiomers of metoprolol in human plasma. The procedure involves pre-column derivatization with the homochiral reagent S-(+)-1-(1-naphthyl)ethyl isocyanate. Once formed, the diastereomers are separated using normal-phase high-performance liquid chromatography. Fluorescence detection (220 nm excitation; no emission filter) was utilized, resulting in baseline resolution (Rs greater than 1.5). The peaks corresponding to metoprolol enantiomers were free from interference throughout the examined range of 5-500 ng/ml; accuracy and precision were within approximately 10%. Analysis of a plasma sample collected from a healthy volunteer demonstrated that the assay is applicable to clinical studies.     

Normal-phase high-performance liquid chromatographic determination of phenols

Normal-phase high-performance liquid chromatography has been used for separation of phenol and its monoderivatives. Multi-component mixtures of hexane (non-polar component) with butan-1-ol, chloroform, butyl bromide, butyl chloride or diethyl ether (polar additives) were used as selective eluents. Silica gel "Silasorb 600" with specific surface area of about 600 m(2)/g and average particle size of $ 10 mum was used as the sorbent. Phenol and the o-, m-, p-isomers of cresol were concentrated by extraction with n-butyl acetate from aqueous solutions. A method for determination of microamounts of phenols in aqueous solutions in the presence of 160-fold amounts of aromatic hydrocarbons has been developed.     

Stereospecific high-performance liquid chromatographic assay of acebutolol in human plasma and urine

A sensitive high-performance liquid chromatographic technique is described for the separation of R- and S-acebutolol in human plasma and urine. The procedure involves derivatization with the chiral reagent S-(+)-1-(1-naphthyl)ethyl isocyanate. The resulting diastereoisomers are quantified using normal-phase high-performance liquid chromatography with fluorescence detection (220/389 nm). Virtual baseline separation, free from interference, with achieved (resolution factor = 1.45). Excellent linearity (r greater than 0.998) was observed throughout the range 10-500 ng/l and 2-100 mg/l in plasma and urine, respectively. Inter-assay variability was less than 5% for each enantiomer at concentrations of 10 ng/ml. This method is applicable for the determination of the pharmacokinetics, in man, of acebutolol enantiomers in plasma and urine.     

Isocratic high-performance liquid chromatographic determination of plasma adenosine

Summary Due to manifold physiological and cardioprotective actions of adenosine, the demand for a simple but accurate method to determine its concentration in plasma is increasing. The aim of this study was firstly to develop a simple isocratic method instead of the gradient elution or peak-shifting techniques used earlier and secondly to check conflicting data on the composition of stop-solution, added to the sample in order to prevent changes in adenosine concentration. Isocratic elution improved signal to noise ratio and concentrations of 100 mol L^–1 dipyridamole and 2.5 mol L^–1 erythro-9(2-hydroxy-3-nonyl)adenine in the blood sample effectively prevented both adenosine formation and degradation, even without the use of a 5-ecto-nucleotidase inhibitor. Lowering the concentration of dipyridamole to 25 mol L^–1 caused more than a tenfold increase of adenosine concentration in two out of five cases and even 100 mol L^–1 dipyridamole alone is not sufficient to inhibit adenosine deaminase in blood samples.     

Pre-column derivatization with fluorescamine and high-performance liquid chromatographic analysis of drugs. Application to tocainide

The quantitative determination of tocainide, a new antiarrhythmic agent, by high-performance liquid chromatography (HPLC) is reported. The drug and a chemically similar internal standard were extracted from blood plasma with acetonitrile under salting-out conditions obtained by saturation of the aqueous medium with sodium chloride-sodium carbonate. The organic extract, without evaporation, was treated with borate buffer (pH 8.2) and fluorescamine. The resulting derivatives were chromatographed on an ODS reversed-phase column using a methanol-phosphate buffer (pH 7.0) mixture as mobile phase and were detected fluorometrically by monitoring the emission at 485 nm, with excitation at 395 nm. The intra-assay coefficients of variation were 3.0 and 4.3% for ten replicate 0.25 and 1.00 microgram/ml samples, respectively, and the inter-assay coefficient of variation was 3.6% for ten replicate 1.00 microgram/ml samples. The procedure is simple, rapid, sensitive, and specific. Several other drugs and drug metabolites also were derivatized with fluorescamine and chromatographed successfully. Pre-column derivatization with fluorescamine followed by HPLC with fluorometric detection may have significant advantages in drug analysis.     

Sensitive high-performance liquid chromatographic determination of pirenzepine in plasma

A high-performance liquid chromatographic method for the determination of pirenzepine in human plasma is reported using imipramine as an internal standard. The assay has a lower limit of detection of 2.5 ng/ml. The calibration function is found to be linear in the range from 5 ng/ml up to at least 100 ng/ml. Two sets of chromatographic conditions are described, which provide different chromatographic selectivities for the separation of the compounds of interest from other material present in a sample.     

Micellar high-performance liquid chromatographic determination of folylpolyglutamate hydrolase activity

A rapid and sensitive method for the quantitative determination of folylpolyglutamate hydrolase activity in crude tissue extracts was developed. The procedure is based on high-performance liquid chromatographic separation of folate analogue mono- and polyglutamates on a reversed-phase column using sodium dodecyl sulfate in water as the mobile phase. Interfering substances in tissue extracts were removed by gel filtration on centrifugally-eluted mini-columns of Sephadex G-25 prior to incubation of polyglutamate substrate with tissue extract hydrolase. Reactions were terminated by denaturation of the enzyme in sodium dodecyl sulfate, which subsequently served as the micellar solvent system for chromatographic separation of substrate from reaction products.     

Stereospecific high-performance liquid chromatographic assay of pirprofen enantiomers in rat plasma and urine.

A stereospecific high-performance liquid chromatographic method was developed for the assay of pirprofen enantiomers in rat plasma and urine. Following addition of internal standard (ketoprofen) and acidifier (L-ascorbic acid) to biological fluids, pirprofen was extracted into an isopropanol-isooctane (5:95) mixture. Diastereomers of pirprofen enantiomers, which were formed using L-leucinamide, were separated on a reversed-phase column with ultraviolet detection at 275 nm using 0.06 M KH2PO4-acetonitrile-triethylamine (64:36:0.1) as mobile phase. The limit of quantitation was 0.1 microgram/ml for each enantiomer, based on 100 microliters of rat plasma. No spontaneous oxidation of pirprofen to its pyrrole metabolite occurred during sample preparation and analysis. In three female rats which were dosed with 10 mg/kg racemic pirprofen orally, plasma concentrations of the enantiomers could be followed for 24 h. Pirprofen enantiomers in plasma were virtually unconjugated, and negligible concentrations of pyrrole metabolites were observed. Less than 10% of the total dose was recovered in urine as intact drug and its glucuroconjugates. The assay was found suitable for the study of the pharmacokinetics of pirprofen enantiomers in the rat.     

A high-performance liquid chromatographic method for the determination of monofluoroacetate

A simple isocratic high-performance liquid chromatographic (HPLC) method for the quantitative analysis of monofluoroacetic acid (MFA), the toxic substance of Dichapetalum cymosum, in plant material, rumen contents (gastric contents), and liver samples is described. A suitable HPLC column that gives optimum sensitivity, accuracy, precision, and separation of MFA is identified. A C-610 organic acid analysis column at ambient temperature with 0.02M H3PO4 as an eluent and ultraviolet detection at 210 nm is utilized to quantitate MFA. Using this method, the average percentage recovery in plant material, bovine liver, and rumen samples is 94.8%, and a detection limit of 12 microg/L is achievable.     

Derivatization and high-performance liquid chromatographic determination of urinary glycolic acid

A high-performance liquid chromatographic method for the determination of urinary glycolic acid is proposed, based on pre-column derivatization with phenylhydrazine coupled with the enzymatic oxidation of glycolate to glyoxylate. The phenylhydrazone formed is separated by liquid chromatography and detected at 324 nm. The minimum detectable concentration of glycolate was 10.0 mumol/l. The recovery of glycolate added to urine averaged 96.1%. The day-to-day coefficients of variation calculated by analysis of two urine samples with normal and high glycolate contents were 4.6 and 7.5%, respectively. Results of analyses of urine samples from healthy persons, idiopathic calcium stone formers and Type I primary hyperoxaluria patients are reported.     

Ion-paired high-performance liquid chromatographic determination of mitoxantrone in physiological fluids

Mitoxantrone is one of the newer anthracenedione derivatives which has already been studied in phase I and II trials, where it has shown significant antitumor activity against a variety of human tumours. To determine the prolonged terminal half-life of mitoxantrone, we developed a sensitive high-performance liquid chromatographic method, providing a detection limit of 1 ng/ml of extracted serum. This system uses a C18 reversed-phase column. The mobile phase consists of a mixture of acetonitrile (30%, v/v) and an ammonium formate buffer (70%, v/v) with a pH of 2.7. Hexane sulphonic acid is added as an ion-pair former. Detection at a wavelength of 658 nm provides a highly selective system, showing no interfering peaks. Ametantrone, another anthracenedione derivative, is used as an internal standard. The extraction procedure for serum also uses hexane sulphonic acid in an ion-paired system. Because of the highly selective detection wavelength, urine samples can be injected without a sample clean-up procedure. This very sensitive method, combined with high selectivity and a fast and inexpensive serum clean-up procedure, has allowed us to document the prolonged terminal plasma half-life of mitoxantrone (levels of 2-5 ng/ml of plasma can still be detected six days after an intravenous infusion of 15 mg/m2 over 30 min). In urine an as yet unidentified metabolite was discovered.     

Simultaneous high-performance liquid chromatographic determination of phenazopyridine and nitrofurantoin in tablets

Summary A reversed-phase, High-performance Liquid chromatographic method for the simultaneous determination of phenazopyridine and nitrofurantoin in tablets is described. An aminopropyl-silica (APS-Hypensil) 5μm column and a mobile phase consisting of Methanol:H₂O: 0.05M sodium dihydrogen phosphate (50∶45∶5) were used. Calibration curves were linear over the concentration range 2–20μg/ml, with minimum detectability of 10ng/ml for both drugs. The method was applied to tablets containing the two species and the results obtained were compared to those given with a published method.     

An improved high-performance liquid chromatographic determination of chlorpropamide in human plasma

Summary Samples were extracted with dichloromethane and the organic layer evaporated to dryness. The residue was dissolved in methanol, and 10 μl aliquot injected onto the column. Tolbutamide was used as the internal standard for chlorpropamide. The UV detector response was linear over the range 0–200 μg ml⁻¹ with a correlation coefficient of 0.999; detection limit: 0.002 μg ml⁻¹. Within-day and between-day assay variation was generally ≤7%. No interference from endogenous constituents was observed. The utility of the method was demonstrated by determining chlorpropamide in samples from six healthy volunteers following a single oral dose of 250 mg. The procedure is simple and requires small volumes of plasma.     

Ion-pair-reversed-phase high-performance liquid chromatographic determination of porphyrins from red blood cells

Summary An extraction and ion-pair reversed-phase HPLC procedure is described for the separation and determination of protoporphyrin and its zinc chelate from red blood cells. The recoveries were greater than 90% with coefficients of variation between 8 and 10%. A linear response curve was obtained from 800 to 4000 g/l erythrocytes for the porphyrins.Presented at the 14th International Symposium on Chromatography London, September, 1982     

Fluorimetric and high-performance liquid chromatographic determination of D-lactate in biological samples

D-Lactate in biological samples was converted into a strongly fluorescent substance in a one-vial reaction. It was first converted into the pyruvate hydrazone in the presence of D-lactate dehydrogenase, an NADH-reoxidation system using diaphorase, D,L-6,8-thioctamide and hydrazine. This hydrazone was then converted into 2-hydroxy-6,7-dimethoxy-3-methylquinoxaline by 1,2-diamino-4,5-dimethoxybenzene in 1 M hydrochloric acid, and the quinoxaline was extracted and measured fluorimetrically at 432 nm (excitation at 365 nm). The calibration curve for D-lactate was linear up to at least 100 nmol/ml of the assay mixture, with a determination limit of 2 nmol/ml. The quinoxaline was also analysed by high-performance liquid chromatography with fluorimetric detection. The calibration curve for D-lactate was linear from 500 fmol to 75 nmol in the reaction mixture. This method was 4000 times more sensitive than the fluorimetric method, and could determine D-lactate in blood plasma volumes of less than 1 microliter.