Determination of the anxiolytic agent 8-chloro-6-(2-chlorophenyl)-4H-imidazo-[1,5-alpha] [1,4]-benzodiazepine-3-carboxamide in whole blood, plasma or urine by high-performance liquid chromatography

A rapid, sensitive and specific high-performance liquid chromatographic (HPLC) assay was developed for the determination of 8-chloro-6-(2-chlorophenyl)-4H-imidazo-[1,5-alpha]-[1,4]-benzodiazepine-3-carboxamide [I] and its 4-hydroxy metabolite, 8-chloro-6-(2-chlorophenyl)-4-hydroxy-4H-imidazo-[1,5-alpha] [1,4]-benzodiazepine-3-carboxamide [II] in whole blood, plasma or urine. The assay for both compounds involves extraction into diethyl ether-methylene chloride (70:30) from blood, plasma, or urine buffered to pH 9.0. The overall recoveries of [I] and [II] are 92.0 +/- 5.4% (S.D.) and 90.3 +/- 4.9% (S.D.), respectively. The sensitivity limit of detection is 50 ng/ml of blood, plasma, or urine using a UV detector at 254 nm. The HPLC assay was used to monitor the blood concentration-time fall-off profiles, and urinary excretion profiles in the dog following single 1 mg/kg intravenous and 5 mg/kg oral doses, and following multiple oral doses of 100 mg/kg/day of compound [I].     

Determination of water soluble imidazo-1,4-benzodiazepines in blood by electron- capture gas--liquid chromatography and in urine by differential pulse polaragraphy.

A sensitive and specific electron-capture gas--liquid chromatographic (GLC--ECD) assay was developed for the determination of 8-chloro-6-(2'-fluorophenyl)-1-methyl-4H-imidazo(1,5a)(1,4)benzodiazepine (I) or 8-chloro-1,4-dimethyl-6-(2'-fluorophenyl)-4H-imidazo (1,5a)(1,4)benzodiazepine (II) in blood. The assay for both compounds involves extraction into benzene--methylene chloride (9:1) from blood buffered to pH 12.6 The overall recovery of I and II from blood is 86% +- 5.0 (S.D.) and the sensitivity limit of detection is of the order of 2 to 3 ng of I or II per milliltre of blood. The major urinary metabolite of I is 8-chloro-6-(2'-fluorophenyl)-1-hydroxymethyl-4H-imidazo(1,5a)(1,4)benzodiazepine, (IA) present as a glucuronide conjugate while 8-chloro-6-(2'-fluorophenyl)-4-hydroxyl-1-methyl-4H-imidazo(1,5a)(1,4)benzodiazepine, (IB) and 8-chloro-6-(2'-fluorophenyl)-4-hydroxy-1-hydroxymethyl-4H-imidazo(1,5a)(1,4) benzodiazepine, (IC) are minor metabolites. The major metabolite IA is extracted into benzene--methylene chloride (9:1) from urine buffered to pH 11.0 (after incubation with glucuronidase--sulfatase as pH 5.0), and analyzed by differential pulse polarography (DPP) in 0.1 M phosphate buffer PH 3). The overall recovery of IA is 84 +- 3.0% (S.D.) with a sensitivity limit of 50 ng per millilitre of urine. The metabolites of compound II have not as yet been elucidated. The GLC--ECD and DPP assays were applied to the determination of blood levels and urinary excretion in dogs following single 10 mg/kg intravenous and oral doses of I and following single 6 mg/kg intravenous and 10 mg/kg oral doses of II. Blood levels of compound I were also evaluated in man following intravenous infusion of single 10 mg doses.     

Determination of clorazepate and its major metabolites in blood and urine by electron capture gas-liquid chromatography.

A sensitive and specific blood level method employing differential extraction was developed for the determination of clorazepate and its N-desmethyldiazepam metabolite by electron capture gas-liquid chromatography (GLC-ECD). The assay requires the initial extraction of N-desmethyldiazepam, the major metabolite, into benzene-methylene chloride (90:10) from the biological sample made alkaline with 0.1 N NaOH. The samples is then acidified with 2 N HCl to decarboxylate clorazepate to N-desmethyldiazepam, which is then extracted into benzene-methylene chloride (90:10) after adjusting the pH to 12.8 with NaOH. The two extracts are evaporated and the residues are dissolved in benzene which contains griseofulvin as the reference standard. These solutions are assayed by GLC-ECD. The overall recovery and sensitivity limit of the assay for clorazepate is 60+/-5% (S.D.) and 4.0 ng/ml blood, respectively, while that for N-desmethyldiazepam is 95+/-5% (S.D.) and 4.0 ng/ml blood, respectively. The urinary excretion of clorazepate was determined by the measurement of the levels of N-desmethyldiazepam and oxazepam, the major urinary metabolites of clorazepate, both prior to and after enzymatic deconjugation. These methods were applied to the measurement of clorazepate and its metabolites in blood and urine following a single 15-mg dose of clorazepate dipotassium.     

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 acetonitrile--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-carboxamide (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 10 mg/kg oral dose of I . 2HCl and of metabolite I-A.     

Determination of midazolam and its α-hydroxy metabolite in plasma by gas chromatography with electron capture detection

A sensitive GC-ECD assay has been developed for the simultaneous determination of midazolam (I) and its α-hydroxy metabolite (II) in plasma. The assay involves extraction of both compounds into ether at alkaline pH (pH 12), followed by silylation of the α-hydroxy metabolite with N,O-bis(trimethylsilyl) trifluoroacetamide (BSTFA). On extracting 0.5 ml of plasma, the sensitivity limits are 4ng/ml for I and 3ng/ml for II. If present, the minor urinary metabolites, the 4-hydroxy (III) and the α,4-dihydroxy compound (IV), can also be determined by this method.     

Determination of the anti-allergenic agent, 2-methoxy-11-oxo-11H-pyrido[2,1-b]quinazoline-8-carboxylic acid, in biological fluids by high-performance liquid chromatography.

A rapid, sensitive, and specific high performance liquid chromatographic (HPLC) assay was developed for the determination of 2-methoxy-11-oxo-11H-pyrido-[2,1-b]quinazoline-8-carboxylic acid (I) from biological fluids. The overall recovery from blood and plasma is 69 +/- 10% (S.D.) and 84 +/- 6% (S.D.), respectively, and the sensitivity limit of quantitation is 100 ng/ml by UV absorption and 5 ng/ml by fluorescence detection using a 1 ml specimen. The assay was used in the determination of blood levels of compound in the Rhesus monkey following intravenous administration of a 10 mg/kg dose, and of blood and urine levels of compound I in a dog following intravenous and oral administration of a 1 mg/kg dose.     

The determination of 2-hydroxynicotinic acid and its major metabolite in blood and urine by spectro-photofluorimetry and differential pulse polarography

A sensitive and specific spectrofluorimetric assay was developed for the determination of 2-hydroxynicotinic acid (1) and its major metabolite, the N-1-riboside (11) in blood and urine. Both compounds exhibit strong fluorescence in acidic media. Thin-layer chromatography is employed to separate the drug from its metabolite; the compounds are eluted from the silica gel into methanol—0.1 M hydrochloric acid (80+20). The sensitivity is 0.04–0.05 μg of I and 0.10–0.12 μg of II per ml of blood or urine. The two compounds can also be determined by differential pulse polarography, which is especially suitable for urine. The fluorimetric method was applied to the determination of blood levels and the urinary excretion of the drug in man after single oral 500-mg doses.     

Enantiomeric separation and discrimination of 2-hydroxy acids as O-trifluoroacetylated (S)-(+)-3-methyl-2-butyl esters by achiral dual-capillary column gas chromatography

An efficient method is described for the simultaneous enantiomeric separation of 18 different racemic 2-hydroxy acids for the determination of their absolute configurations. It involves the conversion of each enantiomer into a diastereomeric O-trifluoroacetylated (S)-(+)-3-methyl-2-butyl ester for the direct separation by achiral dual-capillary column gas chromatography with subsequent identification and determination of its chirality by retention index (I) library matching. The enantiomers of each acid were well separated with high resolution values (R > or = 1.4) on DB-5 and DB-17 columns of different polarity. When temperature-programmed I values of 2-hydroxy acid enantiomers as their diastereomeric derivatives were measured on both columns, the I values were characteristic of each enantiomer. Simple I matching with the reference values was thus useful in cross-checking each acid enantiomer for the identification and chiral discrimination. When applied to urine samples, the present method allowed positive identification of most of the spiked 2-hydroxy acids from normal urine and for endogenous (S)-lactic acid and (S)-2-hydroxybutyric acid from a clinical urine specimen.     

Determination of retinoic acid (13-cis- and all-trans-) and aromatic retinoic acid analogs possessing anti-tumor activity, in biological fluids by high-performance liquid chromatography

A sensitive and specific high-performance liquid chromatographic assay was developed for the determination of 13-cis- and all-trans-retinoic acid in blood or urine with an overall recovery of 90 +/- 5.0% and a limit of detection of 10-20 ng/ml of sample. The method provides for rapid and simple quantitation of the compounds using 1 ml of blood. The assay was applied in the determination of blood levels of 13-cis-retinoic acid in the dog following intravenous and oral administration of 9.5 mg/kg and 2.0 mg/kg doses, and in man following a single 100-mg oral dose and following divided daily doses totalling 2 mg per kg of body weight. The assay is also applicable with minor modifications to the determination of a series of aromatic retinoic acid analogs of clinical interest as anti-tumor agents.     

Determination of an antipsychotic agent (ICI 204,636) and its 7-hydroxy metabolite in human plasma by high-performance liquid chromatography and gas chromatography-mass spectrometry.

ICI 204,636 (I) is an orally active antipsychotic agent under development for the treatment of schizophrenia in humans. It is partially converted in animals to an active 7-hydroxy metabolite (II). Methods were developed for the simultaneous determination of both analytes in human plasma using high-performance liquid chromatography (HPLC) and gas chromatography-mass spectrometry (GC-MS). The analytes were extracted from plasma using phenyl solid-phase extraction columns. Quantification by isocratic HPLC was performed in the reversed-phase mode with detection at 250 nm. Extracts were derivatized to trimethylsilyl ethers for quantification by GC-MS using selected-ion monitoring. Both assays were evaluated for consistency of response, precision, accuracy and specificity. Limits of quantification for I and II by HPLC were 15 and 20 ng/ml, respectively; limits of quantification for I and II by GC-MS were 2 and 5 ng/ml, respectively. Both methods were applied to the analysis of clinical samples from oral dosing studies with I.     

Determination of a new angiotensin-converting enzyme inhibitor (CS-622) and its active metabolite in plasma and urine by gas chromatography-mass spectrometry using negative ion chemical ionization

A sensitive and specific gas chromatographic-mass spectrometric method for the simultaneous determination of angiotensin-converting enzyme inhibitor (I, CS-622) and its active desethyl metabolite (II, RS-5139) in plasma and urine was developed. Compound D5-RS-5139 was used as an internal standard and measurements were made by electron-capture negative ion chemical ionization. Extraction from plasma and urine was carried out using Sep-Pak C18 and silica cartridges. The extract of plasma or urine was treated with diazomethane followed by trifluoroacetic anhydride to convert I and II into their methyl ester trifluoroacetyl derivatives. The detection limit of I and II was 0.5 ng/ml in plasma and 5 ng/ml in urine. The proposed method was satisfactory for the determination of I and II in plasma and urine with respect to accuracy and precision. Thus it is suitable for measurement of bioavailability and pharmacokinetics of I and II in body fluids.     

Simultaneous determination of zopiclone and its two major metabolites (N-oxide and N-desmethyl) in human biological fluids by reversed-phase high-performance liquid chromatography

A high-performance liquid chromatographic method has been developed for the simultaneous determination of zopiclone and its main metabolites (N-oxide and N-desmethyl derivatives) in biological fluids. After selective extraction (dichloromethane-2-propanol) these compounds are chromatographed on a column packed with Spherisorb ODS-2 (5 micron) using monobasic sodium phosphate-methanol (45:55, v/v). The eluted compounds are measured by fluorescence detection. The limit of detection of the method is 5 ng/ml for zopiclone in plasma and urine and 10 ng/ml for its two main metabolites (coefficient of variation less than 10%). This method has been successfully applied to pharmacokinetic studies of zopiclone and its two main metabolites in healthy subjects and patients with chronic renal failure.     

Pharmacokinetic and metabolism studies on girisopam by chromatographic and spectrometric methods in humans.

Girisopam possesses selective anxiolytic action without muscle relaxant and anticonvulsive activity. After a 100-mg oral dose of 14C-labelled girisopam to seven male subjects, the mean recovery of 14C radioactivity was 51% in urine and 33% in faeces. A high-performance liquid chromatographic method has been developed for studying girisopam in single-dose pharmacokinetic studies. The serum extract was chromatographed on a normal-phase column using a mobile phase of hexane-ethanol-diethyl ether (66:9:25, v/v) and ultraviolet detection at 235 nm. The recovery was 60% and the detection limit was 3 ng/ml, using 1 ml of serum. After a 20-min delay, girisopam is rapidly absorbed. After reaching a mean serum level of 178 ng/ml at a mean time of 2.0 h, the serum concentration of girisopam decreased with a mean elimination half-time of 22.2 h. The metabolites were separated by high-performance liquid chromatography, radio thin-layer chromatography and gas chromatography. Their structures were determined by liquid chromatography-mass spectrometry, mass spectrometry and gas chromatography-mass spectrometry. Their chemical structures were confirmed by comparison with synthesized reference compounds. The major urinary metabolites were 7-demethylgirisopam (I), 4'-hydroxygirisopam (II) and 4-hydroxymethyl-4-demethylgirisopam (III), which were in conjugated form, and 4-carboxy-4-demethylgirisopam (V), a compound with an open-chain structure (VII) and traces of 4-demethyl-4-oxogirisopam (VIII) and 4-hydroxymethyl-4-demethylgirisopam (III), which were in non-conjugated form. The metabolic profile in the serum consisted predominantly of the glucuronides of I, II and III. The non-conjugated metabolites were the metabolite with the open-chain structure (VII), III and V. Besides the parent compound, the faeces sample contained conjugates of I and II.     

Determination and identification of amiloride in human urine by high-performance liquid chromatography and gas chromatography-mass spectrometry.

A simple and sensitive high-performance liquid chromatographic method was developed to screen and determine amiloride (I) in human urine. The detection limit of the method is 0.12 micrograms/ml and the recovery of amiloride from urine was 80.4-85.5% at different concentrations. The coefficients of variation were less than 2.8 and 4.4% for intra- and inter-assays, respectively. Total urinary excretion of I in 24 h after oral administration of 5 mg or 15 mg of I ranged from 22.0 to 33.3% of the total dose for three different subjects. I could be detected in urine up to at least 44 h after a 5-mg dose and 72 h after a 15-mg dose. A gas chromatographic-mass spectrometric (GC-MS) confirmatory method was established based on the methanolysis of I to methyl 3,5-diamino-6-chloropyrazine-carboxylate (II). The di-N-trimethylsilyl derivative of II showed very good GC-MS properties and provided reliable structure information for confirmation analysis of I. This is the first time that a reliable GC-MS method has been reported for the detection of urinary I.     

Gas chromatographic quantitation of methoxyphenamine and three of its metabolites in plasma

Sensitive gas chromatographic procedures for the determination of methoxyphenamine and three of its metabolites in plasma have been developed. The metabolites were measured using an electron-capture detector. This simple procedure is based on the precipitation of protein from a 1-ml plasma sample with 10% trichloroacetic acid, followed by aqueous derivatization with pentafluorobenzoyl chloride at pH 9.2 and a single-step cyclohexane extraction. The lower limit of detection for the N-desmethyl, O-desmethyl and aromatic 5-hydroxy metabolites of methoxyphenamine were 1.6, 3.1 and 2.2 ng ml-1, respectively, with coefficients of variation less than 10%. The poor electron-capture response of fluorinated derivatives of methoxyphenamine necessitated the use of nitrogen-phosphorus detection. Extractive derivatization with pentafluorobenzoyl chloride, without the need for protein precipitation, enabled quantitation of methoxyphenamine down to 3.8 ng ml-1 from a 2-ml aliquot of plasma. In a pilot study involving healthy volunteers who received a single oral dose of methoxyphenamine hydrochloride plasma concentration could be followed in all three subjects for at least 24, 32, 12 and 4 h for methoxyphenamine and the O-desmethyl, 5-hydroxy and N-desmethyl metabolites, respectively.     

Intravenous administration of 2,7,8-trimethyl-2-(beta-carboxyethyl)-6-hydroxy chroman (gamma-CEHC) to rats and determination of its plasma concentration and urinary sodium excretion

A natriuretic hormone, 2,7,8-trimethyl-2-(beta-carboxyethyl)-6-hydroxy chroman (gamma-CEHC) was administered intravenously to male Sprague-Dawley rats and the plasma concentration of gamma-CEHC along with urinary sodium (Na+) excretion was investigated. The plasma gamma-CEHC concentrations were fluorimetrically determined by a column-switching HPLC method consisting of both phenyl and octadecyl silica columns, following a pre-column fluorescence derivatization with a fluorescence reagent, 4-N,N-dimethylaminosulfonyl-7-piperazino-2,1,3-benzoxadiazole (DBD-PZ). In rats fed with a high-NaCl (8.0%) diet, plasma gamma-CEHC concentrations rapidly decreased by 20% in 15-45 min after the administration of gamma-CEHC, while Na+ excretion gradually increased with time. Considering these results, the Na+ excretion effect appeared not to be associated with plasma gamma-CEHC concentration. In addition, attempts were made to examine a main urinary metabolite of gamma-CEHC, a large amount of 6-O-sulfated gamma-CEHC found to be present in the urine using an HPLC-tandem mass spectrometry. Thus, it is plausible that gamma-CEHC was easily metabolized to 6-O-sulfated metabolite and excreted into urine in rats.     

Simultaneous determination of a new dihydropyridine calcium antagonist (MPC-1304) and its metabolite in dog plasma by high-performance liquid chromatography with electrochemical detection.

A rapid and sensitive high-performance liquid chromatographic analysis, with electrochemical detection, has been developed for the simultaneous determination of a new calcium-channel antagonist, (+/-)-methyl 2-oxopropyl-1,4-dihydro-2,6-dimethyl-4-(2-nitrophenyl)-3,5-pyridinedi carboxylate (I, MPC-1304), and its active metabolite in dog plasma. The plasma extract with toluene was chromatographed on a reversed-phase column and detected by an electrochemical detector at + 0.92 V. Calibration curves were linear from 2.0 to 100.0 ng/ml, and the detection limit was ca. 0.25 ng/ml. This method is applicable to the simultaneous determination of I and its metabolite in dog plasma following the oral administration of I.     

Identification of major metabolites in rat urine and plasma of N(6) -(4-hydroxybenzyl) adenine riboside by LC/MS/MS

N(6) -(4-hydroxybenzyl) adenine riboside, a novel neuroprotective compound found in Gastrodia elata at trace level, is regarded as a potential drug for the treatment of neural degenerative disease. To understand the metabolism of this compound, the metabolites in rat urine and plasma of N(6) -(4-hydroxybenzyl) adenine riboside were analyzed by HPLC-ESI-MS/MS after oral administration of this compound. Beside the parent compound, six phase I metabolites and four phase II metabolites in urine were detected by scanning all possible metabolites in extracted ion chromatograms mode. By comparing their product ion spectra and retention times with those of parent compound, these metabolites were identified and proved to be mainly formed via hydrolysis or hydroxylation in phase I, N-sulfation or N-glucuronidation in phase II or their combinations. Similarly, the parent compound, one phase I metabolite and two phase II metabolites were also identified in rat plasma. Therefore, the in vivo metabolic pathways of N(6) -(4-hydroxybenzyl) adenine riboside in rat were proposed.     

Bioanalysis of (E)-5-(2-bromovinyl)-2'-deoxyuridine

(E)-5-(2-Bromovinyl)-2'-deoxyuridine is an antiviral drug that is experimentally used for modulation of the antitumour effect of fluoropyrimidines, such as ftorafur and 5-fluorouracil. The isolation of the analyte, in the presence of 5-fluorouracil, from the matrix is performed either by means of a simple protein precipitation (plasma) or by means of a liquid-liquid extraction with ethyl acetate (urine). Following pretreatment, the analyte is analysed by reversed-phase chromatography and quantified by absorbance detection at 307 nm. The minimum detectable concentration in plasma and urine samples is ca. 6 ng/ml. The recovery after deproteination of plasma samples is 75%, while after liquid-liquid extraction of urine the recovery amounts 92%. The degree of protein binding of the analyte, measured by ultrafiltration, is found to be 97%. These data allow the bioanalysis of (E)-5-(2-bromovinyl)-2'-deoxyuridine for pharmacokinetic studies.     

Metabolism and excretion of 5-ethyl-2-{5-[4-(2-hydroxyethyl)piperazine-1-sulfonyl]-2-propoxyphenyl}-7-propyl-3,5-dihydropyrrolo[3,2-d]-pyrimidin-4-one (SK3530) in rats

The in vitro and in vivo metabolism of a novel PDE 5 inhibitor, SK3530, was investigated in rats. Bile, plasma, feces, urine and liver samples were collected and analyzed using a high-performance liquid chromatography (HPLC) system equipped with ultraviolet (UV), mass spectrometric and radioactivity detectors. After a single oral administration, the mean radiocarbon recovery was 92.32+/-6.26%, with 91.25+/-6.25 and 1.07+/-0.21% in the feces and urine, respectively. The biliary excretion of radioactivity for the first 24 h period was approximately 38.82%, suggesting that SK3530 is cleared by hepatobiliary excretion. In vitro incubation of SK3530 with rat and human liver microsomes resulted in the formation of twelve and ten metabolites, respectively. SK3530 was extensively metabolized to twenty different metabolites, including three glucuronide and three sulfate conjugates in rats. The structures of these metabolites were elucidated based on MSn spectral analyses. Six major metabolic pathways were identified in the rat: N-dealkylation and oxidation of the hydroxyethyl moiety; N,N-deethylation and hydroxylation of the piperazine ring; hydroxylation of the propyl group and sulfate conjugation. An additional metabolite due to aromatic hydroxylation was also identified in hepatic microsomes.