Determination of the major urinary metabolite of flurazepam in man by high-performance liquid chromatography

A high-performance liquid chromatographic method for the determination of N-1-hydroxyethylflurazepam, the major urinary metabolite of flurazepam, in human urine is described. Urine specimens were incubated enzymatically to deconjugate N-1-hydroxyethylflurazepam glucuronide (metabolite) and were then extracted at pH 9.0 to extract the metabolite. The extracts were chromatographed on a microparticulate silica gel column using automatic sample injection, isocratic elution at ambient temperature and UV monitoring at 254 nm. The internal stanard was 7-chloro-5-(2′-chlorophenyl)-1,3-dihydro-1-2-dimethylaminoethyl-2H-1,4-benzodiazepine-2-one. The recovery from urine, in the 0.5–25.0 μg/ml range, was 96.5 ± 11.5% (S.D.), and the sensitivity limit was 0.5 μg/ml. The method was found to be specific for N-1-hydroxyethylflurazepam in the presence of intact flurazepam and other possible urinary metabolites of flurazepam. The method was successfully applied to urine specimens collected from human subjects following the administration of 30-mg single oral doses of flurazepam dihydrochloride.     

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 sulfinpyrazone and two of its metabolites in human plasma and urine by gas chromatography and selective detection

A selective and sensitive gas chromatographic method for simultaneous determination of sulfinpyrazone and two of its metabolites (the para-hydroxylated metabolite and the sulfone metabolite) in biological fluids using alkali flame ionization detection (AFID), electron capture detection (ECD) and mass fragmentographic detection is described. The compounds are extracted from the samples, methylated and separated on 2% OV-17 or 8% OV-225 columns. Phenylbutazone is used as internal standard. Standard curves are linear. The coefficient of variation at 10 μg/ml of sulfinpyrazone in plasma was shown to be 1.8% (AFID), and the detection limits were 0.1 μg/ml (AIFD) and 10 ng/ml (ECD). Mass spectra of the methylated compounds are shown and serum concentration curves after oral administration of 100 mg sulfinpyrazone to two persons are determined together with the excreted amounts of drug and metabolites.     

Determination of the antiallergenic agent, N-[4-(1H-imidazol-1-YL)butyl]-2-(1-methylethyl)-11-oxo-11H-pyrido[2,1,-b]quinazoline-8-carboxamide, in plasma by reversed-phase high-performance liquid chromatographic analysis using fluorometric detection

A rapid, sensitive and selective high-performance liquid chromatographic (HPLC) assay was developed for the determination of the antiallergenic compound N-[4-(1H-imidazol-1-yl)butyl]-2-(1-methylethyl)-11-oxo-11H-pyrido[2,1-b] quinazoline-8-carboxamide (I), and its major metabolite, 2-(1-methylethyl)-11-oxo-11H-pyrido[2,1-b] quinazoline-8-carboxylic acid (I-A), in plasma. The assay involves precipitation of the plasma proteins with aceto-nitrile—methanol (9:1), followed by the analysis of an aliquot of the protein-free filtrate by reversed-phase ion-pair HPLC with fluorescence detection for quantitation. The analogous compound, N-[6-(1H-imidazol-1-yl)hexyl]-2-(1-methylethyl)-11-oxo-11H-pyrido[2,1-b]-quinazoline-8-carbonxamide (II), is used as the internal standard. The overall recovery of compounds I and I-A from plasma is 107.0 ± 8.6% and 107.0 ± 10.0%, respectively. The sensitivity limits of quantitation are 20 ng of I, and 10 ng of I-A per ml of plasma using a 0.5-ml aliquot. The assay was used to monitor the plasma concentrations of I and of I-A in a dog following a 5 mg/kg intravenous infusion of I · 2HCl, a 10mg/kg oral dose of I · 2HCl and of metabolite I-A.     

Determination of yohimbine and its two hydroxylated metabolites in humans by high-performance liquid chromatography and mass spectral analysis

The existence of at least two metabolites of yohimbine (YO) in humans is demonstrated. Combined high-performance liquid chromatographic (HPLC), NMR and mass spectral analyses permitted them to be identified as hydroxylated metabolites at the C-10 and C-11 positions. A normal-phase HPLC method allowing the simultaneous determination of YO and its main metabolite, 11-hydroxyyohimbine (11-OHYO), in biological samples is described. This assay was performed using a LiChrosorb Si 60 column and a mobile phase consisting of 0.02 M sodium acetate (pH 5)—methanol (5:95, v/v) at a flow-rate of 1 ml/min. Detection was achieved by a fluorimetric method (excitation at 280 nm and emission at 320 nm). The extraction yields of YO, 10-OHYO and 11-OHYO from plasma were 91.8, 45.3 and 17.8%, respectively, and their respective within-day reproducibilities were 3.8, 1.4 and 5.9%. The between-day reproducibility for YO at the concentrations of 1 and 10 ng/ml were 8.9 and 6.4%, respectively. The accuracy of the method for YO at concentrations of 1 and 10 ng/ml were 5.1 and 2.3%, respectively. The limits of determination of YO, 10-OHYO and 11-OHYO were 0.1, 0.5 and 1 ng/ml, respectively. The method was used in bioavailability study of YO following oral and intravenous administration in humans.     

Determination of the anti-platelet-activating factor BN-50727 and metabolites in human urine by high-performance liquid chromatography using solid-phase extraction

A sensitive and selective HPLC solid-phase extraction procedure was developed for the determination of platelet-activating factor antagonist BN-50727 and its metabolites in human urine. The procedure consisted in a double solid-phase extraction of the urine samples on cyanopropyl and silica cartridges, followed by an automated solid-phase extraction of the drug and metabolites on CBA cartridges and posterior elution on-line to the chromatographic system for its separation. The method allowed quantitation in the concentration range 10–2400 ng/ml urine for both BN-50727 and the main metabolite, the O-demethylated BN-50727 product. The limit of quantitation for both compounds was 10 ng/ml. The inter-assay precision of the method, expressed as relative standard deviation, ranged from 1.9 to 4.5% for BN-50727 and from 2.5 to 9.0% for the metabolite. The accuracy, expressed as relative error, ranged from −2.4 to 4.2% and from 0.2 to 6.2%, respectively. This paper describes the validation of the analytical methodology for the determination of BN-50727 in human urine and also for its metabolites. The method has been used to follow the time course of BN-50727 and its metabolites in human urine after single-dose administration.     

Oral platelet aggregation inhibitor Ro 48-3657: Determination of the active metabolite and its prodrug in plasma and urine by high-performance liquid chromatography using automated column switching

A sensitive and highly automated high-performance liquid chromatography (HPLC) column-switching method has been developed for the simultaneous determination of the active metabolite III and its prodrug II, both derivatives of the oral platelet inhibitor Ro 48-3657 (I), in plasma and urine of man and dog. Plasma samples were deproteinated with perchloric acid (0.5 M), while urine samples could be processed directly after dilution with phosphate buffer. The prepared samples were injected onto a pre-column of a HPLC column switching system. Polar plasma or urine components were removed by flushing the precolumn with phosphate buffer (0.1 M, pH 3.5). Retained compounds (including II and III) were backflushed onto the analytical column, separated by gradient elution and detected by means of UV detection at 240 nm. The limit of quantification for both compounds was 1 ng/ml (500 μl of plasma) and 25 ng/ml (50 μl of urine) for plasma and urine, respectively. The practicability of the new method was demonstrated by the analysis of about 6000 plasma and 1300 urine samples from various toxicokinetic studies in dogs and phase 1 studies in man.     

Simultaneous determination of a novel angiotensin II receptor blocking agent, losartan, and its metabolite in human plasma and urine by high-performance liquid chromatography

A sensitive and selective high-performance liquid chromatographic method for the simultaneous determination of a new angiotensin II receptor blocking agent, losartan (DuP 753, MK-954, I), and its active metabolite, EXP3174 (II), in human plasma or urine is described. The two analytes and internal standard are extracted from plasma and urine at pH 2.5 by liquid—liquid extraction and analyzed on a cyano column with ultraviolet detection at 254 nm. The mobile phase is composed of acetonitrile and phosphate buffer at pH 2.5. The limit of quantification for both compounds in plasma is 5 ng/ml. The limit in urine is 20 and 10 ng/ml for I and II, respectively. The assay described has been successfully applied to samples from pharmacokinetic studies.     

Analysis of procarbazine and metabolites by gas chromatography—mass spectrometry

Twelve compounds representing procarbazine, seven metabolites, and an internal standard were analyzed by gas chromatography—mass spectrometry on a 3% OV-1 column. Procarbazine and four metabolites were derivatized with acetic anhydride.A sensitive, specific and quantitative assay was established by selected ion monitoring using a synthetic analogue of the drug as an internal standard. The limits of detection were approximately 1 ng/ml of plasma while the limits of quantitation were 10 ng/ml of plasma.Studies on the degradation of procarbazine - HCl in 0.05 M phosphate buffer (pH 7.4) were compared to in vivo studies. At 1 h after incubation of procarbazine - HCl in buffer, the azo and aldehyde metabolites were detected in the highest concentrations representing 27.2% and 20.3% of total drug and metabolites. In the in vivo studies, analyses of rat plasmas indicated that 1 h after an oral dose of procarbazine - HCl, the aldehyde metabolite represented 72% of the total drug and metabolites, and that relatively little of the azo metabolite was present.     

Analysis of methadone and metabolites in biological fluids with gas chromatography—mass spectrometry

The analysis of methadone and its metabolites in biological fluids by gas chromatography—mass spectrometry is described with deuterated methadone and metabolites as internal standards. The method allowed the determination of 20 ng methadone in 0.5 ml of plasma or saliva. Mean saliva to plasma ratio of methadone for two patients was determined to be 0.51 ± 0.13. Methadone and 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) in urine were measured by selected ion monitoring. Gas chromatography—mass spectrometry was found to have advantages over conventional gas chromatographic methods in terms of ratio analysis. 1,5-Dimethyl-3,3-diphenyl-2-pyrrolidone previously reported as a metabolite was shown to result primarily from the decomposition of EDDP free base.     

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.     

Gas chromatographic determination of phentolamine (regitine®) in human plasma and urine

A method for the determination of unconjugated phentolamine at concentrations down to 6 ng/ml in human plasma, and of free and total (free plus conjugated) phentolamine down to 25 ng/ml in urine is described. After addition of 2-[N-(p-chlorophenyl)-N-(m-hydroxyphenyl)-aminomethyl]-2-imidazoline as internal standard, both compounds are extracted into benzene—ethyl acetate (1:1, v/v) at pH 10, transferred into an acidic aqueous solution and back-extracted at pH 10 into benzene—ethyl acetate. They are then derivatized with N-heptafluorobutyrylimidazole. The derivatives are determined by gas chromatography using a ⁶³Ni electron-capture detector. In urine, total (free plus conjugated) phentolamine is determined after enzymatic hydrolysis. The technique was applied for the study of the plasma concentrations and urinary elimination after oral administration to man.     

Selective high-performance liquid chromatographic assays for hydralazine and its metabolites in plasma of man

Selective high-performance liquid chromatographic assays for hydralazine (I), hydralazine pyruvic acid hydrazone (II) and the acetylation metabolites, namely s-triazolo[3,4-a]-phthalazine (V) and 3-hydroxymethyl (VI) and 3-methyl-s-triazolo[3,4-a]phthalazine (VII) in human plasma were developed. Utilizing the fluorescence of these compounds or their derivatives the limits of detection could be extended down to 5 nmole/l (1 ng/ml) for I, 1 nmole/l (0.2 ng/ml) for II and 0.5 nmole/l (0.1 ng/ml) for V–VII. The intra-assay coefficients of variation for the assays ranged from 2 to 7% over the concentration range 5.0 to 0.05 μmole/l and the inter-assay variability in the slope of the standard curves ranged from 4 to 8%. An improved method for measuring the sum of I plus all its hydrazones (apparent I) was also developed. On addition of I to fresh plasma at 37°, half the added I was converted to II within 15 min and there was no detectable level of I, 2 h after the addition. The plasma level—time course of I, and its metabolites in a healthy volunteer (slow acetylator) following separate oral and intravenous administrations of I indicated that I contributed only a small fraction (4.3 and 4.7% respectively) to the area under the plasma level—time curve of apparent hydralazine.     

Determination of the anti-platelet-activating factor BN-50727 and its metabolites in human plasma by high-performance liquid chromatography-solid-phase extraction

A sensitive and selective HPLC-solid-phase extraction procedure was developed for the determination of platelet-activating factor antagonist BN-50727 and its metabolites in human plasma. The procedure consisted of an automated solid-phase extraction of the drug and metabolites on disposable propylcarboxylic acid cartridges, followed by on-line chromatographic separation. The method was linear from 3.75 to 2400 ng/ml and the limit of quantitation for BN-50727 in plasma samples was 3.75 ng/ml. The within-run precision of the method, expressed as relative standard deviation, ranged from 2.1 to 8.1%. The accuracy, expressed as relative error, ranged from −3.5 to 4.0%. For the main metabolite, the O-demetthylated BN-50727 product, the method was linear from 7.5 to 2400 ng/ml and the limit of quantitation in plasma was 7.5 ng/ml. The within-run precision ranged from 2.1 to 11.0% and the accuracy from −5.3 to 1.1%. This paper describes the validation of the analytical methodology for the determination of BN-50727 in human plasma and also of its metabolites. The method has been used to follow the time course of BN-50727 and its metabolites in human plasma after administration of single and multiple doses.     

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.     

Analytical method development and validation of mianserin hydrochloride and its metabolite in human plasma by LC-MS

Mianserin is a tetracyclic antidepressant drug and administered as racemate of R (-) and S (+) mianserin hydrochloride in a dose of 30-90 mg/day in divided doses. Liquid chromatography-mass spectroscopy (LC-MS) is a tool, which is widely used for determination of drug and their metabolites in biological fluids because of its high sensitivity and precision. Here we describe a liquid chromatography mass spectroscopy method for simultaneous determination of mianserin and its metabolite, N-desmethylmianserin, from human plasma using a liquid-liquid extraction with hexane:isoamylalcohol (98:2) and back extraction with 0.005 M formic acid solution. This method is specific and linear over the concentration range of 1.00-60.00 ng/ml for mianserin and 0.50-14.00 ng/ml for N-desmethylmianserin in human plasma. The lowest limits of quantification (LLQ) is 1.00 ng/ml for mianserin and 0.50 ng/ml for N-desmethylmianserin. Intraday and interday precision (%C.V.) is <10% for both mianserin and N-desmethylmianserin. The accuracy ranges from 94.44 to 112.33% for mianserin and 91.85-100.13% for N-desmethylmianserin. The stability studies showed that mianserin and N-desmethylmianserin in human plasma are stable during short-term period for sample preparation and analysis. The method was used to assay mianserin and its metabolite, N-desmethylmianserin, in human plasma samples obtained from subjects who had been given an oral tablet of 30 mg of mianserin.     

Development and validation of column-switching high-performance liquid chromatographic methods for the determination of a potent AII receptor antagonist, TCV-116, and its metabolites in human serum and urine

Column-switching HPLC methods have been developed and validated for the determination of a new antihypertensive prodrug, TCV-116 (I), and its metabolites, CV-11974 (II) and CV-15959 (III), in human serum and urine. Initial sample cleanup was achieved by extracting the analytes into an organic solvent. After chromatographing on an ODS column with a mobile phase consisting of acetonitrile and an acidic phosphate buffer, the zone of the analyte's retention was heart-cut onto a second ODS column with a mobile phase of acetonitrile and a phosphate buffer at a higher pH. Complete separation of the analytes and the endogenous peaks was accomplished by the two-dimensional chromatography. Good precision and linearity of the calibration standards, as well as the inter-day and intra-day precision and accuracy of quality control samples, were achieved. The limit of quantitation (LOQ), using 0.5 ml of serum, was 2 ng/ml for I, 0.8 ng/ml for II, and 0.5 ng/ml for III. The LOQ for urine sample was 10 ng/ml for II and III. Stability of the analytes during storage, extraction, and chromatography processes was established. The results illustrate the versatile application of column switching to method development of multiple analytes in various biological matrices. The methods have been successfully used for the analyses of I and its metabolites in thousands of clinical samples to provide pharmacokinetics data.     

Determination of oxpentifylline and a metabolite, 1-(5′-hydroxyhexyl)-3,7-dimethylxanthine, by gas—liquid chromatography using a nitrogen-selective detector

A gas chromatographic method for the determination of oxpentifylline and a metabolite, 1-(5′-hydroxyhexyl)-3,7-dimethylxanthine is described. Oxpentifylline, metabolite and internal standard are extracted from basified plasma into dichloromethane, then the metabolite and internal standard are converted to their O-trifluoroacetates. Analysis by gas—liquid chromatography using a nitrogen-selective detector allows quantification of oxpentifylline and 1-(5′-hydroxyhexyl)-3,7-dimethylxanthine down to levels of 3 ng/ml and 3–10 ng/ml, respectively. The assay had been applied to plasma samples from volunteers after both intravenous and oral administration of oxpentifylline. The need to separate plasma from erythrocytes immediately after venipuncture sampling to prevent further metabolism of oxpentifylline is emphasized.     

Analysis of benzphetamine and its metabolites in rat urine by liquid chromatography–electrospray ionization mass spectrometry

An analytical method to identify and determine benzphetamine (BMA) and its five metabolites in urine was developed by liquid chromatography–electrospray ionization mass spectrometry (LC–ESI–MS) using the solid-phase extraction column Bond Elut SCX. Deuterium-labeled compounds, used as internal standards, were separated chromatographically from each corresponding unlabeled compound in the alkaline mobile phase with an alkaline-resistant ODS column. This method was applied to the identification and determination of BMA and its metabolites in rat urine collected after oral administration of BMA. Under the selected ion monitoring mode, the limit of quantitation (signal-to-noise ratio 10) for BMA, N-benzylamphetamine (BAM), p-hydroxybenzphetamine (p-HBMA), p-hydroxy-N-benzylamphetamine (p-HBAM), methamphetamine (MA) and amphetamine (AM) was 700 pg, 300 pg, 500 pg, 1.4 ng, 6 ng and 10 ng in 1 ml of urine, respectively. This analytical method for p-HBMA, structurally closer to the unchanged drug of all the metabolites, was very sensitive, making this a viable metabolite for discriminating the ingestion of BMA longer than the parent drug or other metabolites in rat.     

Improved solid-phase extraction and HPLC measurement of torasemide and its important metabolites

Torasemide is a "loop type" diuretic drug. For pharmacokinetic studies sensitive analytic methods are essential for authentic qualitative and quantitative information. A robust, selective and sensitive HPLC method is described for the simultaneous determination of torasemide, its major metabolite M5 and its active metabolites M1 and M3 and an internal standard within 17 min. Solid-phase extraction with C(2)-cartridges was used for the clean-up of plasma samples. The chromatographic separation was carried out on a CN-column with a mobile phase consisting of perchloric acid (0.02 M, pH 2.5)/acetonitrile (90/10, v/v)). The calibration range used reached from 20 to 1000 ng/ml for all analytes. Coefficients of variation were less than 10% at every calibration point for each analyte. Plasma concentrations in samples obtained from volunteers in the course of a clinical study could be reliably measured with this method. Median maximum concentrations in plasma after a 10mg oral dose during a 24h study interval were located at 1h for torasemide, 1h for M1 and 2h for M5. Concentrations between 2226 and <20 ng/ml for torasemide, between 159 and <20 ng/ml for M1 and between 420 and <20 ng/ml for M5 were observed.