Fluorimetrische bestimmung von propranolol und seines metaboliten n-desisopropylpropranolol in plasma und urin durch direkte auswertung von dünnschichtchromatogrammen

Fluorometric determination of propranolol and its metabolite N-desisopropylpropranolol in plasma and urine by direct measurement of thin-layer chromatographic platesThe quantitative analysis of propranolol and its metabolite N-desisopropylpropranolol in plasma and urine is described. The drugs are extracted into 2-pentanol-heptane, and the solvent is concentrated. The whole residue is chromatographed on silica gel plates. The compounds are determined directly on the thin-layer plates without derivatization. The recovery of propranolol from plasma is 70%, with a standard deviation of ± 4%.     

Thin-layer chromatographic determination of furosemide and 4-chloro-5-sulfamoyl anthranilic acid in plasma and urine

A method is described for the assay of furosemide based on thin-layer chromatography and measurement of fluorescence directly on the plates. Conditions are specified for stabilizing fluorescence over the time of measurement. As little as 10 ng can be accurately measured and fluorescence is linear up to 160 ng. The metabolite or decomposition product 4-chloro-5-sulfamoyl anthranilic acid is well separated and measured quantitatively in the procedure. Application of the method to human plasma and urine is demonstrated.     

Fluorimetrische bestimmung von hydrochlorothiazid in körperflüssigkeiten durch direkte auswertung von dünnschichtchromatogrammen

Fluorometric determination of hydrochlorothiazide in body fluids by direct measurement of thin-layer chromatographic platesTwo fluorometric methods for analysis of hydrochlorothiazide (HCT) are described utilizing direct measurement of thin-layer plates. The first method employs a modification of the Bratton—Marshall reaction and is therefore applicable to all aromatic primary amines. Following diazotation and azocoupling of the HCT hydrolysis product, a fluorescent group is added to the compound. For this purpose N-(1-naphthyl)ethylenediamine is first coupled with 4-chloro-7-nitrobenzo-2,1,3-oxadiazole. In the second method, the intrinsic fluorescence of underivatized HCT, following its extraction from plasma, urine or saliva, is used. It is shown that the sensitivity of this method is sufficient for estimating the kinetics following oral administration of 25 mg HCT.     

Determination of (E)-5-(2-bromovinyl)-2′-deoxyuridine in plasma and urine by capillary electrophoresis

(E)-5-(2-Bromovinyl)-2′-deoxyuridine is an antiviral drug used for treatment of infections with Herpes simplex virus type 1 as well as Varicella zoster virus. Two fast methods for the determination of the drug and its metabolite in plasma and urine by capillary electrophoresis have been developed. The plasma method can be used for measurement of total as well as unbound drug and metabolite. Plasma and urine samples are prepared for measuring by liquid/liquid extraction resulting in a limit of quantification of 40 ng/ml for total and 10 ng/ml for free BVdU in plasma and 170 ng/ml in urine. Inter- as well as intra-day precision were found to be better than 10% and both methods have been used for drug monitoring of patients.     

Determination of candesartan cilexetil, candesartan and a metabolite in human plasma and urine by liquid chromatography and fluorometric detection

Liquid chromatographic methods are described for the determination of a new effective anti-hypertensive drug candesartan (CV-11974), its prodrug candesartan cilexetil (TCV-116) and a metabolite, CV-15959 in human plasma and urine. The assays comprise liquid–liquid extraction and separation on a phenyl column with fluorometric detection. The methods give absolute recoveries of 70, 83 and 78% for candesartan cilexetil, candesartan and CV-15959, respectively, and the limit of quantification is 5, 1 and 3 nM of plasma (RSD<20%), respectively. The methods were applied to plasma and urine samples from biopharmaceutical and clinical studies in man.     

High-performance liquid chromatographic determination of 3′-hydroxy-5′-(4-isobutyl-1-piperazinyl)benzoxazinorifamycin (KRM-1648) and its deacetyl metabolite in plasma, whole blood, urine and tissue samples in rats

A reversed-phase high-performance liquid chromatographic method was developed for the determination of 3′-hydroxy-5′-(4-isobutyl-1-piperazinyl)benzoxazinorifamycin (KRM-1648, I), a new rifamycin derivative, and its 25-deacetyl metabolite (KRM-1671, II) in plasma, whole blood, tissues and urine from rats. I and II were coextracted with an internal standard from each sample matrix by solid-phase extraction (Bond Elut). Plasma and urine were directly loaded onto Bond Elut, while whole blood and tissues were homogenized and extracted with methanol or dichloromethane—chloroform prior to Bond Elut extraction. The extracts were chromatographed on Shim-pack CLC-ODS(M) using acetonitrile—0.02 M citrate buffer containing 0.1 M sodium perchlorate (2:1, v/v), and peaks were detected at 643 nm. The validation data showed that the assays for I and II in plasma, whole blood, tissues and urine were selective, accurate and reproducible.     

Gaschromatographische analyse von sauren urinmetaboliten nach trennung auf kieselgelfertigsäulen

Gas chromatographic estimation of acidic urinary metabolites after separation on prepacked silica gel columnsThe acidic ethylacetate extracts of 24-h urine specimens are evaporated and redissolved in chloroform—methanol—acetic acid. The resulting solution is transferred to a prepacked silica gel column. Elution takes 160 min using a specially designed chloroform—methanol—acetic acid gradient. The eluate is divided into fractions (16 min each) which are evaporated to dryness. The residues are silylated and determined quantitatively by gas chromatography. The capacity of the silica gel column allows analysis of 30% of a 24-h urine specimen. In consequence, metabolites can be quantitated at concentrations less than 1 mg per 24 h. The method is suitable to obtain more detailed metabolic profiles of the carboxylic acids in urine.     

Fluorodensitometric evaluation of gentamicin from plasma and urine by high-performance thin-layer chromatography

High-performance thin-layer chromatographic (HPTLC) analysis of gentamicin by in situ fluorodensitometric evaluation of its 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) derivative is presented. The aminoglycoside components separated on silica gel plates using chloroform–methanol–20% ammonium hydroxide (2.4:2.2:1.5, v/v/v) as the mobile phase were reacted with NB-Cl to yield highly fluorescent derivatives. The calibration curves of gentamicin in water, plasma and urine were linear in the range 40–200 ng. The mean values of intercept, slope and correlation coefficient were 16.82±0.473, 6.83±0.015 and 0.9968±0.0017 for standard curves in water, 17.35±0.375, 6.85±0.018 and 0.9941±0.0012 for standard curves in plasma and 14.35±0.286, 6.86±0.002 and 0.9933+0.0011 for standard curves in urine respectively. The analytical technique was validated for within-day and day-to-day variation. The results indicate that HPTLC, coupled with in situ fluorodensitometry, is a reliable and valuable technique for quantitative analysis of the bulk drug gentamicin and gentamicin from urine and plasma.     

High-performance liquid chromatographic determination of (S)- and (R)-propanolol in human plasma and urine with a chiral β-cyclodextrin bonded phase

The determination of propanolol enantiomers in microsamples of human plasma and urine by HPLC using a chiral stationary phase is described. After extraction from 200 μl of plasma or urine with racemic alprenolol as internal standard (I.S.), the enantiomers are separated on a β-cyclodextrin column with a polar organic mobile phase and determined by fluorescence detection. The retention times of I.S. and propranolol enantiomers are about 12–13 min and 16–18 min, respectively. Peak resolutions are 1.4 for I.S. and 2.2 for propranol. The use of alprenolol as I.S. improves significantly the coefficients of variation (C.V.: 0.6–4.2%). Sensitivity is approximately 1.5 ng/ml per propranolol enantiomer. The assay is applied to pharmacokinetic studies of racemic propranolol in human biological fluids. The (S)-propranolol levels are always higher than the (R)-antipode concentrations in plasma and urine.     

Simultaneous determination of blood concentrations of methohexital and its hydroxy metabolite by gas chromatography and identification of 4′-hydroxymethohexital by combined gas—liquid chromatography—mass spectrometry

A simple, sensitive and selective method is described for the simultaneous determination of low concentrations (less than 50 ng/ml) of underivatized methohexital and its hydroxy metabolite in small (0.1 ml) samples of human and rat plasma or whole blood by gas chromatography with nitrogen-selective detection.Moreover, the main metabolite in rat and man was identified as 4′-hydroxymethohexital by comparison of chromatograms from gas—liquid chromatography (GLC) with data obtained from GLC—mass spectrometry and ¹H-nuclear magnetic resonance spectrometry of this metabolite, produced both by incubating methohexital with isolated rat liver microsomes and by isolating this metabolite from rat urine.     

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.     

Analysis of the metabolites of the sodium salt of 6-hydroxy-5-(phenylazo)-2-naphthalenesulfonic acid in Sprague–Dawley rat urine

The sodium salt of 6-hydroxy-5-(phenylazo)-2-naphthalenesulfonic acid (SS-AN), which is a subsidiary color present in Food Yellow No. 5 [Sunset Yellow FCF, disodium salt of 6-hydroxy-5-(4-sulfophenylazo)-2-naphthalenesulfonic acid], was orally administered to Sprague–Dawley rats. Metabolite A, metabolite B, and unaltered SS-AN were detected as colored metabolites in the rat urine. Analysis of the chemical structures showed that metabolite A (major peak) was 6-hydroxy-5-(4-sulfooxyphenylazo)-2-naphthalenesulfonic acid, the sulfuric acid conjugate of SS-AN, and metabolite B (minor peak) was 6-hydroxy-5-(4-hydroxyphenylazo)-2-naphthalenesulfonic acid (SS-PAP), which is a derivative of metabolite A without the sulfuric acid. The colorless metabolites p-aminophenol, o-aminophenol, and aniline present in the urine were analyzed by liquid chromatography–mass spectrometry. The orally administered SS-AN had been metabolized to the colorless metabolites (p-aminophenol 45.3%, o-aminophenol 9.4%, aniline 0.4%) in the 24-h urine samples. Analysis of the colored metabolites by high-performance liquid chromatography with detection at 482 nm indicated the presence of metabolite A (0.29%), SS-PAP (0.01%), and SS-AN (0.02%) were detected in the 24-h urine samples. Approximately 56% of SS-AN was excreted into the urine and the rest is probably excreted into feces.     

Identification of two Thormählen-positive compounds from melanotic urine by gas chromatography—mass spectrometry

The isolation of two Thormählen-positive compounds from the urine of a patient with malignant melanoma and the elucidation of their structure by gas chromatography—mass spectrometry is described. The compounds were isolated using a poly-N-vinylpyrrolidone column and separated by preparative thin-layer chromatography. After elution they were analyzed by gas chromatography and gas chromatography—mass spectrometry as their trimethylsilyl derivatives and after hydrolysis also as their tert.-butyldimethylsilyl derivatives. The results showed the main Thormählen-positive compound A to be the glucuronide of 5-hydroxy-6-methoxyindole, whereas the minor compound AX appeared to be the glucuronide of its isomer 6-hydroxy-5-methoxyindole.     

Determination of the naturally occurring monoacetyl derivatives of di- and polyamines

A method is described for the determination of pmol quantities of monoacetylputrescine, N¹-acetylspermidine, N⁸-acetylspermidine and related compounds. The method is based on the derivatization of these compounds with 5-dimethylaminonaphthalene-1-sulphonyl-chloride, followed by thin-layer chromatographic separation. Cleanup steps allow the application of the method to urine analyses. From the repeated determination of acetylated polyamines in the urine of healthy individuals it can be concluded that these conjugates are the major excretory form of di- and polyamines.The cleanup steps used in this procedure and the method described for the stabilization of 5-dimethylaminonaphthalene-1-sulphonyl derivatives on thin-layer plates are advantageous also for the analyses of total polyamines in urine hydrolysates, and in related applications of the dansylation method.     

Determination of 8-oxoguanine in human plasma and urine by high-performance liquid chromatography with electrochemical detection

A highly sensitive and selective method for determining 8-oxoguanine in plasma and urine was developed by high-performance liquid chromatography with electrochemical detection. The compound was separated by gradient elution on a C₁₈ reversed-phase column with a mobile phase of acetonitrile and 0.1 M sodium acetate, pH 5.2. 8-Hydroxy-2′-deoxyguanosine was used as internal standard. 8-Oxoguanine was detected electrochemically by setting the potential to +300 mV vs. Pd reference. The sensitivity of the assay was 22 ng/ml with a signal-to-noise ratio of 7:1. The within-day relative standard deviations for 8-oxoguanine quality control samples with concentrations of 3340, 1340 and 84 ng/ml were 3.6, 4.3 and 5.7% for plasma, and 4.1, 4.6 and 6.2% for urine, respectively. The day-to-day relative standard deviations for the same samples were 3.8, 6.8 and 7.1% for plasma, and 3.9, 7.0 and 7.9% for urine, respectively. The method is designed to study the pharmacokinetics and metabolic fate of O⁶-benzylguanine in a phase I clinical trial. Previously, O⁶-benzyl-8-oxoguanine was identified as the primary metabolite of O⁶-benzylguanine in humans. We now demonstrate that 8-oxoguanine is a further metabolite of O⁶-benzylguanine.     

Determination of 3-hydroxy-guanfacine in biological fluids by electron-capture gas—liquid chromatography

An electron-capture gas—liquid chromatographic method was developed for measuring 3-hydroxy-guanfacine, the main metabolite of guanfacine in human plasma and urine. After extraction, the metabolite was derivatized by condensing the amidino group with hexafluoroacetylacetone and by methylating the NH and OH groups with methyl iodide. The obtained derivative possessed good bioanalytical gas chromatographic properties, using a capillary column. The O-glucuronide was measured after enzymatic hydrolysis. Unchanged guanfacine could be determined in urine together with its 3-hydroxy metabolite by this method.     

Identification of a pyrovalerone metabolite in the rat by gas chromatography-mass spectrometry and determination of pyrovalerone by gas chromatography-nitrogen-phosphorus detection

Pyrovalerone and its hydrolated metabolite have been identified by gas chromatography-mass spectrometry in rat urine and plasma. A sensitive gas chromatographic method for the quantitative analysis of pyrovalerone in rat urine and plasma is described. The method also permits the quantitative monitoring of the urinary excretion of the drug and its metabolite. Pyrovalerone and its hydroxylated metabolite are detected up to 18 h after a single oral administration to the rat at a dose of 20 mg/kg.     

Direct analysis of salicylic acid,salicyl acyl glucuronide,salicyluric acid and gentisic acid in human plasma and urine by high-performance liquid chromatography

A method for the simultaneous direct determination of salicylate (SA), its labile, reactive metabolite, salicyl acyl glucuronide (SAG), and two other major metabolites, salicyluric acid and gentisic acid in plasma and urine is described. Isocratic reversed-phase high performance liquid chromatography (HPLC) employed a 15-cm C₁₈ column using methanol-acetonitrile-25 mM acetic acid as the mobile phase, resulting in HPLC analysis time of less than 20 min. Ultraviolet detection at 310 nm permitted analysis of SAG in plasma, but did not provide sensitivity for measurement of salicyl phenol glucuronide. Plasma or urine samples are stabilized immediately upon collection by adjustment of pH to 3–4 to prevent degradation of the labile acyl glucuronide metabolite. Plasma is then deproteinated with acetonitrile, dried and reconstituted for injection, whereas urine samples are simply diluted prior to injection on HPLC. m-Hydroxybenzoic acid served as the internal standard. Recoveries from plasma were greater than 85% for all four compounds over a range of 0.2–20 μg/ml and linearity was observed from 0.1–200 μg/ml and 5–2000 μg/ml for SA in plasma and urine, respectively. The method was validated to 0.2 μg/ml, thus allowing accurate measurement of SA, and three major metabolites in plasma and urine of subjects and small animals administered salicylates. The method is unique by allowing quantitation of reactive SAG in plasma at levels well below 1% that of the parent compound, SA, as is observed in patients administered salicylates.     

Assay of moguisteine metabolites in human plasma and urine: conventional and chiral high-performance liquid chromatographic methods

Moguisteine is a novel peripheral non-narcotic antitussive agent. Pharmacokinetic studies in animal and in man showed that no unchanged drug is present in plasma, urine and faeces after oral administration. The main active metabolite, M1, is the free carboxylic acid of moguisteine, which maintains a stereogenic centre and consists of R(+)-M1 and S(−)-M1 enantiomers. M1 is partly metabolized to M2, its sulfoxidation derivative. A conventional HPLC method is described for the simultaneous determination of M1 and M2 in human plasma and urine after administration of therapeutic moguisteine doses. Plasma samples, previously acidified with phosphoric acid, are extracted with dichloromethane; urine samples are analyzed after appropriate dilution with methanol. Chromatography is performed using a Lichrosorb RP2 column and a linear gradient. M1 enantiomers can be determined in plasma extracts and urine samples by a chiral HPLC method using a β-cyclodextrin column. The analytical characteristics of both HPLC procedures proved to be adequate to analyze samples of subjects treated with therapeutic doses of moguisteine during clinical pharmacokinetic studies.     

High-performance liquid chromatographic analysis of sulfinpyrazone and its metabolites in biological fluids

A rapid, sensitive, and specific high-performance liquid chromatographic method is described for the quantitative analysis of sulfinpyrazone and its sulfone and p-hydroxy metabolites in plasma and urine. The method uses two different procedures for sample preparation: (1) a rapid and convenient procedure using a single extraction with 1-chlorobutane and subsequent back-extraction into sodium hydroxide solution for the analysis of sulfinpyrazone and its sulfone metabolite, and (2) a more time consuming procedure using triple extraction with ethylene dichloride, a buffer wash, and back extraction into the base for the additional analysis of the p-hydroxy metabolite. The lower limit of sensitivity for sulfinpyrazone is 50 ng/ml. Concentrations of sulfinpyrazone between 0.05 to 0.1 and 50 μg/ml were measured with an average coefficient of variation of 3.9%, ranging from 1.5 to 6.1%.