RESUMO
A gradient elution high-performance liquid chromatographic method is described for the analysis of the beta-lactamase inhibitor tazobactam (YTR-830H) and a semi-synthetic parenteral penicillin, piperacillin, in human plasma, serum, bile and urine. The assay for plasma, serum and bile involves deproteinization with acetonitrile and the removal of lipids with dichloromethane; urine is diluted with buffer. Separation and quantitation are achieved using a mobile phase based on ion-suppression chromatography on a C18 reversed-phase column with ultraviolet detection at 220 nm. The limit of quantitation for both compounds is 1.0 microgram/ml in plasma, serum and bile using a 0.2-ml sample and 50.0 micrograms/ml in urine using a 0.1-ml sample. The method has been validated by preparing and analyzing a series of fortified samples (range 1.0-200 micrograms/ml for each compound in plasma, serum and bile and 50.0-10,000 micrograms/ml for each compound in urine). Excellent linearity, accuracy, precision and recovery were obtained. The method was not interfered with by other endogenous components, nor by other commonly administered antibiotics such as amoxicillin, mezlocillin, cefometazole and cefotaxime. The assay has been successfully applied to the analysis of samples from pharmacokinetic studies in man and animals.
Assuntos
Antibacterianos/metabolismo , Bile/análise , Ácido Penicilânico/metabolismo , Piperacilina/metabolismo , Antibacterianos/sangue , Antibacterianos/urina , Cromatografia Líquida de Alta Pressão , Humanos , Ácido Penicilânico/sangue , Ácido Penicilânico/urina , Piperacilina/sangue , Piperacilina/urina , Espectrofotometria Ultravioleta , TazobactamRESUMO
A rapid, sensitive and selective high-performance liquid chromatographic (HPLC) assay was developed for the determination of (E)-1,2,3,4-tetrahydro-1,1,4,4-tetramethyl-6-(1-methyl-2- phenylethenyl)naphthalene (I) and its phenolic metabolite, (E)-4-[2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-2- methylethenyl]phenol (II) in plasma. The assay for both compounds involves precipitation of the plasma proteins with acetonitrile, followed by extraction of the entire mixture into methyl tert.-butyl ether and subsequent analysis by reversed-phase HPLC. The overall recovery of I and II was 96.9 +/- 5.3 and 96.2 +/- 5.8% for dog plasma, 80.0 +/- 4.0 and 93.5 +/- 5.0% for human plasma and 97.4 +/- 2.9 and 97.6 +/- 9.6% for rat plasma, respectively. The sensitivity limit is 20 ng/ml of plasma for both compounds I and II using UV detection at 280 nm. The HPLC assay was used in studies in the dog and in the rat.
Assuntos
Acne Vulgar/tratamento farmacológico , Anti-Infecciosos Locais/sangue , Naftalenos/sangue , Retinoides , Tetra-Hidronaftalenos/sangue , Animais , Biotransformação , Cromatografia Líquida de Alta Pressão , Cães , Estabilidade de Medicamentos , Feminino , Humanos , Indicadores e Reagentes , Masculino , Ratos , Fatores Sexuais , Especificidade da Espécie , Espectrofotometria Ultravioleta , TemperaturaRESUMO
A rapid, sensitive and selective high-performance liquid chromatographic (HPLC) assay was developed for the determination of 6-(2-chlorophenyl)-4-hydroxy-4H-imidazo[1,5-a]-[1,4]benzodiazepine- 3-carboxamide (I) and its benzophenone carboxylic acid metabolite, 1-[2-(2-chlorobenzoyl)phenyl]-4-(aminocarbonyl)-1H-imidazole-5-car boxylic acid (II) in plasma. The assay involves the extraction of both compounds into benzene from buffered plasma (pH 5.4) and subsequent analysis by reversed-phase HPLC. The overall recovery of I and II is 98.3 +/- 9.4 and 59.7 +/- 15.7% for dog plasma, 86.0 +/- 14.7 and 52.8 +/- 15.1% for rat plasma and 98.1 +/- 9.3 and 66.9 +/- 18.0% for human plasma, respectively. The sensitivity limit of the assay for I and II is 20.0 and 40.0 ng/ml of plasma using ultraviolet detection at 254 nm. The assay was used in studies in dog and rat.
Assuntos
Ansiolíticos/farmacocinética , Midazolam/análogos & derivados , Administração Oral , Animais , Ansiolíticos/sangue , Ansiolíticos/urina , Cromatografia Líquida de Alta Pressão , Cães , Estabilidade de Medicamentos , Humanos , Injeções Intravenosas , Midazolam/farmacocinética , Ratos , Especificidade da Espécie , Espectrofotometria UltravioletaRESUMO
A rapid, sensitive and selective high-performance liquid chromatographic (HPLC) assay was developed for the determination of midazolam, 8-chloro-6-(2-fluorophenyl)-1-methyl-4H-imidazo[1,5-a][1,4]- benzodiazepine (I), and its four metabolites in plasma. The assay involves extraction into diethyl ether--methylene chloride (7:3) from plasma buffered to pH 9 and subsequent analysis by reversed-phase HPLC with ultraviolet detection at 254 nm. The overall recovery of I from dog plasma was 94.5 +/- 7.1% and greater than 89.0% for its metabolites. The sensitivity limit of the assay was 50 ng/ml of plasma for all compounds. The HPLC assay was used to determine plasma concentrations of I and its metabolites from selected samples taken from an oral toxicity study in the dog.
Assuntos
Benzodiazepinas/sangue , Animais , Benzodiazepinas/metabolismo , Biotransformação , Cromatografia Líquida de Alta Pressão , Cães , Feminino , Masculino , MidazolamRESUMO
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.
Assuntos
Quinazolinas/sangue , Animais , Biotransformação , Cromatografia Líquida de Alta Pressão/métodos , Cães , Humanos , Quinazolinas/metabolismo , Soluções , Espectrometria de Fluorescência/métodos , Espectrofotometria Ultravioleta/métodosRESUMO
A rapid and selective high-performance liquid chromatographic (HPLC) assay was developed for the determination of the antiallergenic compound, trans-3-[6-(methylthio)-4-oxo-4H-quinazolin-3-yl]-2-propenoic acid, [I], in plasma. The assay involves acetonitrile protein precipitation followed by the analysis of an aliquot of the protein-free fraction by reversed-phase HPLC with fluorescence detection (excitation at 245 nm, with emission greater than 418 nm). The overall recovery of [I] from plasma was 103 +/- 10%. The sensitivity limit of the assay was 0.125 microgram/ml of plasma. The analogous compound, trans-3-[6-[(1-methylethyl)thio]-4-oxo-4H-quinazolin-3-yl]-2-propenoic acid, [II], is used as the internal standard. The assay was used to monitor the plasma concentration--time fall-off profile of [I] in the dog and in man. The stability of [I] was demonstrated in dog plasma on long-term storage for up to 180 days at -17 degrees C and -70 degrees C.
Assuntos
Hipersensibilidade/tratamento farmacológico , Animais , Biofarmácia , Cromatografia Líquida de Alta Pressão/métodos , Cães , Humanos , Cinética , Manejo de Espécimes , TemperaturaRESUMO
Electron-capture gas--liquid chromatographic and reversed-phase high-performance liquid chromatographic assays are described for the quantitation of the compound, 9-chloro-7-(2-chlorophenyl)-5H-pyrimido [5,4-d] [2]-benzazepine, [I], a member of the benzazepine class of compounds undergoing clinical evaluation as anxiolytic agents. Studies on the biotransformation of [I] in the rat and dog showed that the compound was metabolized mainly by hydroxylation to yield the 5-hydroxy compound, [II], 9-chloro-7-(2-chlorophenyl)-5H-pyrimido [5,4-d] [2]-benzazepin-5-ol (major metabolite), along with the formation of lesser amounts of the N-oxide, [III], 9-chloro-7-(2-chlorophenyl)-5H-pyrimido [5,4-d] [2]-benzazepine 3-oxide, and the phenolic analogue, [IV], 3-chloro-4-(9-chloro-5H pyrimido-[5,4-d] [2] benzazepin-7-yl)phenol. This report describes the quantitation of [I] and [II] (major metabolite) in plasma using the above analytical techniques, both in preclinical studies in the dog and in clinical pharmacokinetic studies in man.
Assuntos
Ansiolíticos , Benzazepinas/isolamento & purificação , Animais , Benzazepinas/sangue , Benzazepinas/metabolismo , Biotransformação , Cromatografia Gasosa , Cromatografia Líquida de Alta Pressão , Cães , Humanos , Cinética , Estatística como AssuntoRESUMO
A high-performance liquid chromatographic (HPLC) assay was developed for the determination of the ratios of the (S)-(+) and (R)-(-) enantiomers of the anti-inflammatory drug carprofen in blood, urine, and feces. The procedure relies on: (a) extraction and purification of carprofen from biological fluids, (b) reaction of carprofen with (S)-(-)-alpha-methylbenzylamine to form the two diastereomeric (S)-(-)-alpha-methylbenzylamides via the 1,1'-carbonyldiimidazole intermediate, (c) purification of the reaction mixture by extraction of the diastereomeric derivatives into hexane at pH 11, and (d) analysis of the diastereomeric derivatives by HPLC with UV detection. The (S)-(+): (R)-(-) ratios in the blood of three subjects receiving single 100-mg oral doses of carprofen were greater than unity up to 16 hr after dosing. The mean +/- SD of the ratios in the early blood samples (0.5, 1, and 2 hr) was 1.21 +/- 0.09, while the mean of the ratios in the later blood samples (4,6,8,12, and 16 hr) was slightly higher (1.48 +/- 0.17). The blood level fall off curves for the (S)-(+) and (R)-(-) enantiomers were similar in each of the three subjects for the 4-16 hr period. The carprofen enantiomers were excreted stereoselectively by humans. An excess of the (S)-(+) enantiomer relative to the (R)-(-) enantiomer was excreted in the urine as the ester glucuronide, while unchanged (R)-(-) enantiomer predominated in the feces. The total urinary plus fecal excretion of the enantiomers (0-96 hr) revealed only a slight excess of the (S)-(+) enantiomer over the (R)-(-) enantiomer, which amounted to 2.1-49% of the dose. Since the amount of carprofen (free and glucuronide) excreted in 96 hr by the three subjects only accounted for 62-72% of the dose, no definitive statement could be made relative to the possible inversion of the carprofen chiral center.
Assuntos
Anti-Inflamatórios não Esteroides/metabolismo , Carbazóis/metabolismo , Cromatografia Líquida de Alta Pressão/métodos , Fezes/análise , Humanos , Estereoisomerismo , Fatores de TempoRESUMO
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].
Assuntos
Ansiolíticos/sangue , Benzodiazepinas/sangue , Midazolam/análogos & derivados , Animais , Ansiolíticos/administração & dosagem , Ansiolíticos/urina , Benzodiazepinas/administração & dosagem , Benzodiazepinas/urina , Cromatografia Líquida de Alta Pressão/métodos , Cães , Injeções Intravenosas , Valores de Referência , Fatores de TempoRESUMO
A rapid, sensitive and specific high-performance liquid chromatography (HPLC) assay was developed for the determination of diazepam, and its major metabolites, oxazepam, temazepam and nordiazepam in plasma, blood, and urine of humans and cats. The assay for the compounds involves extraction into benzene--methylene chloride (90:10) from plasma, blood or urine buffered to pH 9.0. In both species the overall recovery of diazepam and its major metabolites from plasma or blood ranged from 60 +/- 3.2 to 89 +/- 13% (S.D.) and for urine from 79 +/- 7.9 to 93 +/- 10.5% (S.D.). The sensitivity limit of the assay using UV detection at 254 nm was 50 ng/ml of plasma and blood in both species except for human urine (post-Glusulase) which was 200 ng/ml. The HPLC assay was used to monitor the plasma concentration--time profile in humans following a 10-mg oral dose of diazepam and the blood concentration time profile of diazepam and nordiazepam in cats following a 10 mg/kg intravenous dose of either diazepam or nordiazepam. The HPLC assay data correlated well with data generated by an electron-capture--gas--liquid chromatography assay.
Assuntos
Diazepam/análise , Animais , Gatos , Cromatografia Líquida de Alta Pressão/métodos , Diazepam/metabolismo , Humanos , Hidroxilação , CinéticaRESUMO
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.
Assuntos
Antineoplásicos/sangue , Tretinoína/análogos & derivados , Tretinoína/sangue , Vitamina A/análogos & derivados , Administração Oral , Animais , Cromatografia Líquida de Alta Pressão/métodos , Cães , Humanos , Injeções Intravenosas , Isomerismo , Masculino , Tretinoína/urinaRESUMO
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.
Assuntos
Ansiolíticos/análise , Animais , Ansiolíticos/sangue , Ansiolíticos/urina , Benzodiazepinas , Cromatografia Gasosa/métodos , Cães , Humanos , Polarografia/métodos , Fatores de TempoRESUMO
A rapid method was developed for the determination of diazepam and nordiazepam (N-desmethyldiazepam) in human plasma using electron capture gas--liquid chromatography (GLC--ECE). The concentration of diazepam and nordiazepam is determined using 0.5 ml of plasma extracted with 1.0 ml of benzene containing 25 ng/ml of methylnitrazepam as the internal standard. The benzene extract is removed and an aliquot is subjected to automated GLC-ECD analysis. The method has a sensitivity limit of 5 ng diazepam and 10ng nordiazepam per milliliter of plasma. The method was used to determine the plasma levels in man following the first 5-mg diazepam dose, as well as during chronic oral administration of 5 mg diazepam three times daily and 15 mg diazepam once a day.
Assuntos
Diazepam/análogos & derivados , Diazepam/sangue , Nordazepam/sangue , Autoanálise , Cromatografia Gasosa , Cromatografia Líquida , Diazepam/administração & dosagem , Humanos , Nordazepam/administração & dosagemRESUMO
A rapid, sensitive, and specific high-pressure liquid chromatographic (HPLC) assay was developed for the determination of (D,L)-6-chloro-alpha-methylcarbazole-2-acetic acid (carprofen) in blood. The assay involves extraction into diethyl ether from blood buffered to pH 6. The overall recovery of carprofen from blood is 97.3 +/- 5.3% (S.D.), and the sensitivity limit of detection is 100-200 ng/ml of blood using a fluorescence detector with excitation at 240 nm and emission at wavelengths greater than 350 nm. The HPLC assay is amenable to rapid routine analysis of clinical specimens, and the data obtained using this assay showed an excellent correlation coefficient (0.99) compared with a previously published spectrofluorometric assay. The method was used to monitor the blood level-time fall-off profiles in four subjects following single and multiple dose administration of carprofen.
Assuntos
Anti-Inflamatórios/sangue , Carbazóis/sangue , Anti-Inflamatórios/administração & dosagem , Carbazóis/administração & dosagem , Cromatografia Líquida de Alta Pressão/métodos , Humanos , Espectrometria de Fluorescência , Espectrofotometria UltravioletaRESUMO
The benzofuran analog bufuralol, a beta-adrenergic blocker, was determined in blood and urine by a specific and sensitive spectrofluorometric assay. The compound was extracted into ether from blood or urine adjusted to pH 10. The ether extract was separated by TLC to resolve the parent drug from any basic metabolites present, and the spots were eluted off the silica gel and quantitated fluorometrically in 0.1 N HCl. The overall recovery of the assay was 85 +/- 3.0%; the sensitivity limit was 2-4 ng/ml of blood or urine, using a 2.5-ml specimen/analysis. The method was applied to the determination of blood levels in a dog following a single 10-mg/kg oral dose and in two human subjects administered a single 20-mg oral dose.
Assuntos
Benzofuranos/análise , Antagonistas Adrenérgicos beta/sangue , Antagonistas Adrenérgicos beta/urina , Animais , Benzofuranos/sangue , Benzofuranos/urina , Cães , Etanolaminas/sangue , Etanolaminas/urina , Humanos , Métodos , Espectrofotometria Ultravioleta , Fatores de TempoRESUMO
A sensitive and specific high-pressure liquid chromatographic assay was developed for the determination of phytoene in blood with an overall recovery of 86 +/- 6.0% and a limit of detection of 50-100 ng per ml of blood. This method provides for rapid and simple quantitation of phytoene using 1 ml or less of blood. The assay was used in the determination of phytoene blood levels in the dog following intravenous and oral administration of 10-mg/kg doses.