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1.
Alzheimers Dement ; 16(11): 1493-1503, 2020 11.
Artículo en Inglés | MEDLINE | ID: mdl-32755010

RESUMEN

INTRODUCTION: Reference materials based on human cerebrospinal fluid were certified for the mass concentration of amyloid beta (Aß)1-42 (Aß42 ). They are intended to be used to calibrate diagnostic assays for Aß42 . METHODS: The three certified reference materials (CRMs), ERM-DA480/IFCC, ERM-DA481/IFCC and ERM-DA482/IFCC, were prepared at three concentration levels and characterized using isotope dilution mass spectrometry methods. Roche, EUROIMMUN, and Fujirebio used the three CRMs to re-calibrate their immunoassays. RESULTS: The certified Aß42 mass concentrations in ERM-DA480/IFCC, ERM-DA481/IFCC, and ERM-DA482/IFCC are 0.45, 0.72, and 1.22 µg/L, respectively, with expanded uncertainties (k = 2) of 0.07, 0.11, and 0.18 µg/L, respectively. Before re-calibration, a good correlation (Pearson's r > 0.97), yet large biases, were observed between results from different commercial assays. After re-calibration the between-assay bias was reduced to < 5%. DISCUSSION: The Aß42 CRMs can ensure the equivalence of results between methods and across platforms for the measurement of Aß42 .


Asunto(s)
Péptidos beta-Amiloides/líquido cefalorraquídeo , Inmunoensayo/normas , Calibración , Humanos , Inmunoensayo/métodos , Estándares de Referencia
2.
Bioanalysis ; 12(1): 53-65, 2020 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-31849266

RESUMEN

Aim: Developing LC-MS methods for biomolecules is often challenging due to issues with molecular size and complexity, nonspecific binding, protein binding, solubility and sensitivity. As a result, complex sample preparation workflows, including immune-affinity and/or protein digestion and lengthy analysis potentially using nano-flow LC, may be needed to achieve the required sensitivity. This work aims to provide a simple, sensitive, fast and robust method for quantification of intact IGF-I from human serum using UPLC-MS/MS. Methods: IGF-I serum samples were denatured with sodium dodecyl sulfate, followed by organic protein precipitation to effectively disrupt protein binding and subsequent SPE of the resulting supernatant for sample cleanup and enrichment prior to LC-MS/MS analysis. Separation was performed on an analytical scale LC using a reversed-phase column containing <2 µm solid core particle followed by detection on a tandem quadrupole MS in multiple reaction monitoring mode. Results: Intact IGF-I was quantified from serum using the method described above at a LLOQ of 5 ng/ml with a dynamic range 5-1000 ng/ml (r2>0.99) and mean accuracy of 101.76%. Accuracies for quality control samples were between 93.9-107.7% with RSD <7%. Conclusion: The analytical sensitivity, linear dynamic range and excellent reproducibility of this method reliably measures endogenous and elevated serum IGF-I levels, demonstrating its utility in discovery, bioanalysis and clinical research.


Asunto(s)
Cromatografía Líquida de Alta Presión/métodos , Factor I del Crecimiento Similar a la Insulina/análisis , Espectrometría de Masas en Tándem/métodos , Biomarcadores/sangre , Humanos , Factor I del Crecimiento Similar a la Insulina/química , Factor I del Crecimiento Similar a la Insulina/aislamiento & purificación , Límite de Detección , Relación Señal-Ruido , Dodecil Sulfato de Sodio/química , Extracción en Fase Sólida
3.
Alzheimers Dement ; 12(1): 55-9, 2016 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-26206625

RESUMEN

INTRODUCTION: Cerebrospinal fluid (CSF) amyloid-ß 1-42 (Aß42) is an important biomarker for Alzheimer's disease, both in diagnostics and to monitor disease-modifying therapies. However, there is a great need for standardization of methods used for quantification. To overcome problems associated with immunoassays, liquid chromatography-tandem mass spectrometry (LC-MS/MS) has emerged as a critical orthogonal alternative. METHODS: We compared results for CSF Aß42 quantification in a round robin study performed in four laboratories using similar sample preparation methods and LC-MS instrumentation. RESULTS: The LC-MS results showed excellent correlation between laboratories (r(2) >0.98), high analytical precision, and good correlation with enzyme-linked immunosorbent assay (r(2) >0.85). The use of a common reference sample further decreased interlaboratory variation. DISCUSSION: Our results indicate that LC-MS is suitable for absolute quantification of Aß42 in CSF and highlight the importance of developing a certified reference material.


Asunto(s)
Péptidos beta-Amiloides/líquido cefalorraquídeo , Biomarcadores/líquido cefalorraquídeo , Cromatografía Liquida/métodos , Fragmentos de Péptidos/líquido cefalorraquídeo , Espectrometría de Masas en Tándem/métodos , Enfermedad de Alzheimer/líquido cefalorraquídeo , Calibración , Ensayo de Inmunoadsorción Enzimática/normas , Humanos , Estándares de Referencia
4.
Bioanalysis ; 7(7): 857-67, 2015.
Artículo en Inglés | MEDLINE | ID: mdl-25932520

RESUMEN

BACKGROUND: Increased pressure to obtain more, higher sensitivity data from less sample is especially critical for large peptides, whose already optimized LC-MS methods are heavily challenged by traditional ligand-binding assays. RESULTS: Critical bioanalytical assays were adapted to integrated microscale LC to reduce sample volumes while increasing sensitivity. Assays for teriparatide, glucagon and human insulin and five analogs were transferred from 2.1 mm analytical scale LC to a 150 µm scale system. This resulted in a 15-30 fold overall improvement in sensitivity derived from increased signal to noise, three to six fold reduction in injection volumes, and a two to five fold reduction in sample consumption. CONCLUSION: Integrated microscale LC reduces sample consumption while enabling single picomolar quantification for therapeutic and endogenous peptides.


Asunto(s)
Análisis Químico de la Sangre/métodos , Dispositivos Laboratorio en un Chip , Péptidos/sangre , Integración de Sistemas , Análisis Químico de la Sangre/instrumentación , Cromatografía Liquida , Humanos , Inyecciones , Modelos Lineales , Espectrometría de Masas , Factores de Tiempo
5.
Artículo en Inglés | MEDLINE | ID: mdl-24076523

RESUMEN

Teriparatide, the 1-34 fragment of human parathyroid hormone, is used to treat osteoporosis patients with a high risk of fracture by stimulating new bone formation. Routinely teriparatide is quantified using radioimmunoassay however the LC-MS/MS described here has the potential to achieve greater accuracy and precision, higher specificity, and is readily implemented in routine bioanalytical laboratories. Hence a complete method combining effective sample prep with appropriate LC separation and selected reaction monitoring (SRM) MS detection was developed to selectively separate teriparatide from closely related endogenous peptides and to reduce interferences. Samples were concentrated without evaporation, minimizing the risk of adsorptive losses. Chromatography was performed on a sub 2µm particle charged surface hybrid column, which provided significantly higher peak capacity than a traditional C18 column when formic acid was used as the mobile phase modifier. Total LC cycle time was 6min. An LOD of 15pg/mL (3.6fmol/mL) from 200µL of human plasma was readily achieved and standard curves were accurate and precise from 15pg/mL to 500pg/mL. Mean QC accuracies ranged from 90% to 106%. Mean QC precision was better than 7%. The CV of matrix factors across 6 sources of human plasma was 5%. The assay presented here is the first LC-MS method which reaches clinically relevant detection limits for teriparatide.


Asunto(s)
Cromatografía Liquida/métodos , Espectrometría de Masas en Tándem/métodos , Teriparatido/sangre , Femenino , Humanos , Límite de Detección , Masculino , Reproducibilidad de los Resultados , Extracción en Fase Sólida , Teriparatido/química
6.
Anal Biochem ; 419(2): 133-9, 2011 Dec 15.
Artículo en Inglés | MEDLINE | ID: mdl-21888888

RESUMEN

Critical events in Alzheimer's disease (AD) involve an imbalance between the production and clearance of amyloid beta (Aß) peptides from the brain. Current methods for Aß quantitation rely heavily on immuno-based techniques. However, these assays require highly specific antibodies and reagents that are time-consuming and expensive to develop. Immuno-based assays are also characterized by poor dynamic ranges, cross-reactivity, matrix interferences, and dilution linearity problems. In particular, noncommercial immunoassays are especially subject to high intra- and interassay variability because they are not subject to more stringent manufacturing controls. Combinations of these factors make immunoassays more labor-intensive and often challenging to validate in support of clinical studies. Here we describe a mixed-mode solid-phase extraction method and an ultra-performance liquid chromatography tandem mass spectrometry (SPE UPLC-MS/MS) assay for the simultaneous quantitation of Aß(1-38), Aß(1-40), and Aß(1-42) from human cerebrospinal fluid (CSF). Negative ion versus positive ion species were compared using their corresponding multiple reaction monitoring (MRM) transitions, and negative ions were approximately 1.6-fold greater in intensity but lacked selectivity in matrix. The positive ion MRM assay was more than sufficient to quantify endogenous Aß peptides. Aß standards were prepared in artificial CSF containing 5% rat plasma, and quality control samples were prepared in three pooled CSF sources. Extraction efficiency was greater than 80% for all three peptides, and the coefficient of variation during analysis was less than 15% for all species. Mean basal levels of Aß species from three CSF pools were 1.64, 2.17, and 1.26 ng/ml for Aß(1-38); 3.24, 3.63, and 2.55 ng/ml for Aß(1-40); and 0.50, 0.63, and 0.46 ng/ml for Aß(1-42).


Asunto(s)
Péptidos beta-Amiloides/líquido cefalorraquídeo , Cromatografía Líquida de Alta Presión/métodos , Espectrometría de Masas en Tándem/métodos , Secuencia de Aminoácidos , Péptidos beta-Amiloides/química , Humanos , Datos de Secuencia Molecular , Fragmentos de Péptidos/líquido cefalorraquídeo , Fragmentos de Péptidos/química , Control de Calidad , Estándares de Referencia , Extracción en Fase Sólida
8.
Anal Biochem ; 382(2): 147-9, 2008 Nov 15.
Artículo en Inglés | MEDLINE | ID: mdl-18706385

RESUMEN

Matrix-assisted laser desorption mass spectrometry successfully analyzes mixed populations of amyloid-beta (Abeta) peptides, providing a profile in which changes caused by drug action are directly observed. A spectrum of Abeta immunocaptured from guinea pig brain included a novel component with monoisotopic [M+H]+ at 4511.22, close to the monoisotopic value of [M+H]+ for Abeta(1-42) of 4512.27 and overlapping and interfering with the authentic Abeta(1-42) peak. Hypothesis and experiment led to the conclusion that modification of Abeta(1-40) by the protease inhibitor aminoethylbenzenesulfonyl fluoride generates a product with monoisotopic [M+H]+ at 4511.19, and that this accounts for the interfering peak.


Asunto(s)
Péptidos beta-Amiloides/química , Fragmentos de Péptidos/análisis , Fragmentos de Péptidos/química , Inhibidores de Proteasas/farmacología , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Sulfonas/farmacología , Péptidos beta-Amiloides/análisis , Péptidos beta-Amiloides/metabolismo , Animales , Cobayas , Peso Molecular , Fragmentos de Péptidos/metabolismo
9.
Drug Metab Dispos ; 35(6): 848-58, 2007 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-17344339

RESUMEN

2-(3-Chlorobenzyloxy)-6-(piperazin-1-yl)pyrazine (3) is a potent and selective 5-HT(2C) agonist that exhibits dose-dependent inhibition of food intake and reduction in body weight in rats, making it an attractive candidate for treatment of obesity. However, examination of the genotoxicity potential of 3 in the Salmonella Ames assay using tester strains TA98, TA100, TA1535, and TA1537 revealed a metabolism (rat S9/NADPH)- and dose-dependent increase of reverse mutations in strains TA100 and TA1537. The increase in reverse mutations was attenuated upon coincubation with methoxylamine and glutathione. The irreversible and concentration-dependent incorporation of radioactivity in calf thymus DNA after incubations with [14C]3 in the presence of rat S9/NADPH suggested that 3 was bioactivated to a reactive intermediate that covalently bound DNA. In vitro metabolism studies on 3 with rat S9/NADPH in the presence of methoxylamine and cyanide led to the detection of amine and cyano conjugates of 3. The mass spectrum of the amine conjugate was consistent with condensation of amine with an aldehyde metabolite derived from hydroxylation of the secondary piperazine nitrogen-alpha-carbon bond. The mass spectrum of the cyano conjugate suggested a bioactivation pathway involving N-hydroxylation of the secondary piperazine nitrogen followed by two-electron oxidation to generate an electrophilic nitrone, which reacted with cyanide. The 3-chlorobenzyl motif in 3 was also bioactivated via initial aromatic ring hydroxylation followed by elimination to a quinone-methide species that reacted with glutathione or with the secondary piperazine ring nitrogen in 3 and its monohydroxylated metabolite(s). The metabolism studies described herein provide a mechanistic basis for the mutagenicity of 3.


Asunto(s)
Piperazinas/toxicidad , Pirazinas/toxicidad , Salmonella typhimurium/efectos de los fármacos , Agonistas del Receptor de Serotonina 5-HT2 , Biotransformación , Pruebas de Mutagenicidad , Obesidad , Piperazinas/síntesis química , Piperazinas/metabolismo , Pirazinas/síntesis química , Pirazinas/metabolismo , Salmonella typhimurium/genética , Salmonella typhimurium/metabolismo
10.
J Am Soc Mass Spectrom ; 17(3): 363-75, 2006 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-16442304

RESUMEN

We report herein, facile metabolite identification workflow on the anti-depressant nefazodone, which is derived from accurate mass measurements based on a single run/experimental analysis. A hybrid LTQ/orbitrap mass spectrometer was used to obtain accurate mass full scan MS and MS/MS in a data-dependent fashion to eliminate the reliance on a parent mass list. Initial screening utilized a high mass tolerance ( approximately 10 ppm) to filter the full scan MS data for previously reported nefazodone metabolites. The tight mass tolerance reduces or eliminates background chemical noise, dramatically increasing sensitivity for confirming or eliminating the presence of metabolites as well as isobaric forms. The full scan accurate mass analysis of suspected metabolites can be confirmed or refuted using three primary tools: (1) predictive chemical formula and corresponding mass error analysis, (2) rings-plus-double bonds, and (3) accurate mass product ion spectra of parent and suspected metabolites. Accurate mass characterization of the parent ion structure provided the basis for assessing structural assignment for metabolites. Metabolites were also characterized using parent product ion m/z values to filter all tandem mass spectra for identification of precursor ions yielding similar product ions. Identified metabolite parent masses were subjected to chemical formula calculator based on accurate mass as well as bond saturation. Further analysis of potential nefazodone metabolites was executed using accurate mass product ion spectra. Reported mass measurement errors for all full scan MS and MS/MS spectra was <3 ppm, regardless of relative ion abundance, which enabled the use of predictive software in determining product ion structure. The ability to conduct biotransformation profiling via tandem mass spectrometry coupled with accurate mass measurements, all in a single experimental run, is clearly one of the most attractive features of this methodology.


Asunto(s)
Microsomas Hepáticos/metabolismo , Espectrometría de Masa por Ionización de Electrospray/métodos , Triazoles/farmacocinética , Antidepresivos/análisis , Antidepresivos/farmacocinética , Células Cultivadas , Humanos , Piperazinas , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Triazoles/análisis
11.
Chem Biol Interact ; 155(1-2): 10-20, 2005 Jun 30.
Artículo en Inglés | MEDLINE | ID: mdl-15978881

RESUMEN

Therapy with the antidepressant trazodone has been associated with several cases of idiosyncratic hepatotoxicity. While the mechanism of hepatotoxicity remains unknown, it is possible that reactive metabolites of trazodone play a causative role. Studies were initiated to determine whether trazodone undergoes bioactivation in human liver microsomes to electrophilic intermediates. LC/MS/MS analysis of incubations containing trazodone and NADPH-supplemented microsomes or recombinant P4503A4 in the presence of glutathione revealed the formation of conjugates derived from the addition of the sulfydryl nucleophile to mono-hydroxylated- and hydrated-trazodone metabolites. Product ion spectra suggested that mono-hydroxylation and sulfydryl conjugation occurred on the 3-chlorophenyl-ring, whereas hydration and subsequent sulfydryl conjugation had occurred on the triazolopyridinone ring system. These findings are consistent with bioactivation sequences involving: (1) aromatic hydroxylation of the 3-chlorophenyl-ring in trazodone followed by the two-electron oxidation of this metabolite to a reactive quinone-imine intermediate, which reacts with glutathione in a 1,4-Michael fashion and (2) oxidation of the pyridinone ring to an electrophilic epoxide, ring opening of which, by glutathione or water generates the corresponding hydrated-trazodone-thiol conjugate or the stable diol metabolite, respectively. The pathway involving trazodone bioactivation to the quinone-imine has also been observed with many para-hydroxyanilines including the structurally related antidepressant nefazodone. It is proposed that the quinone-imine and/or the epoxide intermediate(s) may represent a rate-limiting step in the initiation of trazodone-mediated hepatotoxicity.


Asunto(s)
Antidepresivos de Segunda Generación/farmacocinética , Benzoquinonas/metabolismo , Sistema Enzimático del Citocromo P-450/metabolismo , Compuestos Epoxi/metabolismo , Microsomas Hepáticos/metabolismo , Trazodona/farmacocinética , Biotransformación , Cromatografía Líquida de Alta Presión , Citocromo P-450 CYP3A , Humanos , Iminas/metabolismo , Hígado/efectos de los fármacos , Hígado/metabolismo , Hígado/patología , Microsomas Hepáticos/efectos de los fármacos , Proteínas Recombinantes , Espectrometría de Masa por Ionización de Electrospray
12.
Drug Metab Dispos ; 33(2): 243-53, 2005 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-15523046

RESUMEN

The therapeutic benefits of the antidepressant nefazodone have been hampered by several cases of acute hepatotoxicity/liver failure. Although the mechanism of hepatotoxicity remains unknown, it is possible that reactive metabolites of nefazodone play a causative role. Studies were initiated to determine whether nefazodone undergoes bioactivation in human liver microsomes to electrophilic intermediates. Following incubation of nefazodone with microsomes or recombinant P4503A4 in the presence of sulfydryl nucleophiles, conjugates derived from the addition of thiol to a monohydroxylated nefazodone metabolite were observed. Product ion spectra suggested that hydroxylation and sulfydryl conjugation occurred on the 3-chlorophenylpiperazine-ring, consistent with a bioactivation pathway involving initial formation of p-hydroxynefazodone, followed by its two-electron oxidation to the reactive quinone-imine intermediate. The formation of novel N-dearylated nefazodone metabolites was also discernible in these incubations, and 2-chloro-1,4-benzoquinone, a by-product of N-dearylation, was trapped with glutathione to afford the corresponding hydroquinone-sulfydryl adduct. Nefazodone also displayed NADPH-, time-, and concentration-dependent inactivation of P4503A4 activity, suggesting that reactive metabolites derived from nefazodone bioactivation are capable of covalently modifying P4503A4. A causative role for 2-chloro-1,4-benzoquinone and/or the quinone-imine intermediate(s) in nefazodone hepatotoxicity is speculated. Although the antianxiety agent buspirone, which contains a pyrimidine ring in place of the 3-chlorophenyl-ring, also generated p-hydroxybuspirone in liver microsomes, no sulfydryl conjugates of this metabolite were observed. This finding is consistent with the proposal that two-electron oxidation of p-hydroxybuspirone to the corresponding quinone-imine is less favorable due to differences in the protonation state at physiological pH and due to weaker resonance stabilization of the oxidation products as predicted from ab initio measurements.


Asunto(s)
Antidepresivos de Segunda Generación/metabolismo , Benzoquinonas/metabolismo , Sistema Enzimático del Citocromo P-450/metabolismo , Microsomas Hepáticos/enzimología , Microsomas Hepáticos/metabolismo , Triazoles/metabolismo , Antidepresivos de Segunda Generación/análisis , Antidepresivos de Segunda Generación/química , Benzoquinonas/análisis , Benzoquinonas/química , Biotransformación , Citocromo P-450 CYP3A , Inhibidores Enzimáticos del Citocromo P-450 , Humanos , Iminas/análisis , Iminas/química , Iminas/metabolismo , Piperazinas , Triazoles/análisis , Triazoles/química
13.
Drug Metabol Drug Interact ; 20(1-2): 11-24, 2004.
Artículo en Inglés | MEDLINE | ID: mdl-15283300

RESUMEN

The pharmacokinetics and metabolism of the antioxidant and reactive oxygen scavenger alpha-phenyl-N-tert-butyl nitrone (PBN) was examined in the male cynomolgus monkey after intravenous administration. Following an i.v. bolus dose of 5 mg/kg, plasma concentrations of PBN declined in a bi-exponential fashion. PBN demonstrated a moderate plasma clearance (CL(p) = 27.02 +/- 6.46 ml/min/kg) and a moderate volume of distribution at steady state (Vd(ss) = 1.70 +/- 0.23 l/kg), resulting in a terminal elimination half-life of 0.76 +/- 0.25 h. The corresponding area under the curve (AUC(0-infinity)) was 3.20 +/- 0.77 microg-h/ml. Scale-up of the in vitro microsomal intrinsic clearance data for PBN afforded a blood clearance (CLb) value of 22 ml/min/kg, which was in reasonable agreement with the observed in vivo CLb. Monkey liver microsomes catalyzed the NADPH-dependent monohydroxylation of PBN to the corresponding alpha-4-hydroxyphenyl-N-tert-butylnitrone (4-HOPBN) metabolite. The formation of 4-HOPBN and its corresponding O-glucuronide was also discernible upon qualitative analysis of pooled (0-24 h) monkey plasma and urine samples. Less than 5% of the administered dose was excreted as unchanged PBN in the urine, suggesting that P450-catalyzed metabolism constituted the major route of PBN clearance in the primate. In conclusion, the pharmacokinetic attributes and the clearance mechanism of PBN in the cynomolgus monkey is similar to that observed in the Sprague-Dawley rat.


Asunto(s)
Antioxidantes/farmacocinética , Depuradores de Radicales Libres/farmacocinética , Fármacos Neuroprotectores/farmacocinética , Óxidos de Nitrógeno/farmacocinética , Animales , Antioxidantes/administración & dosificación , Antioxidantes/metabolismo , Biotransformación , Proteínas Sanguíneas/metabolismo , Óxidos N-Cíclicos , Depuradores de Radicales Libres/administración & dosificación , Depuradores de Radicales Libres/metabolismo , Inyecciones Intravenosas , Macaca fascicularis , Masculino , Tasa de Depuración Metabólica , Microsomas Hepáticos/metabolismo , Fármacos Neuroprotectores/administración & dosificación , Fármacos Neuroprotectores/metabolismo , Óxidos de Nitrógeno/administración & dosificación , Óxidos de Nitrógeno/metabolismo , Ratas , Ratas Sprague-Dawley
14.
Bioorg Med Chem Lett ; 14(13): 3389-95, 2004 Jul 05.
Artículo en Inglés | MEDLINE | ID: mdl-15177439

RESUMEN

The SAR of a series of sterically hindered sulfonamide hydroxamic acids with relatively large P1' groups is described. The compounds typically spare MMP-1 while being potent inhibitors of MMP-13. The metabolically more stable compounds in the series contain either a monocyclic or bicyclic pyran ring adjacent to the hydroxamate group. Despite the sparing of MMP-1, pre-clinical and clinical studies revealed that fibrosis in rats and MSS in humans is still produced.


Asunto(s)
Metaloproteinasa 1 de la Matriz/metabolismo , Inhibidores de la Metaloproteinasa de la Matriz , Inhibidores de Proteasas/síntesis química , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Humanos , Ácidos Hidroxámicos/química , Metaloproteinasa 13 de la Matriz , Metaloproteinasas de la Matriz/metabolismo , Inhibidores de Proteasas/farmacología , Piranos/química , Relación Estructura-Actividad , Sulfonamidas/química
15.
Drug Metab Dispos ; 31(2): 147-52, 2003 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-12527695

RESUMEN

The pharmacokinetics of the spin-trap alpha-phenyl-N-tert-butylnitrone (PBN) was investigated in male Sprague-Dawley rats. Plasma concentrations after i.v. administration (10 mg/kg) declined monoexponentially with a terminal half-life of 2.01 +/- 0.35 h and total plasma clearance (CL(p)) and volume of distribution at steady state (Vd(ss)) averaged 12.37 +/- 3.82 ml/min/kg and 1.74 +/- 0.5 l/kg, respectively. The observed CL(p) was in close agreement with the blood clearance (CL(b)) value (11.5 ml/min/kg) predicted from in vitro liver microsomal incubations suggesting that PBN CL(p) in rats is predominantly due to hepatic metabolism. Peak plasma concentration (C(max)) following p.o. (20 mg/kg) and s.c. (30 mg/kg) PBN administration was 7.35 +/- 1.92 and 3.56 +/- 0.66 microg/ml, whereas the area under the concentration-time curve from 0 to infinity was 23.89 +/- 5.84 and 15.96 +/- 3.10 microg-h/ml, respectively. The mean oral bioavailability of PBN was 85.63 +/- 20.93%. Biotransformation studies indicated the P450 2C11-catalyzed hydroxylation of PBN to M1. Potential sites of hydroxylation included the benzylic carbon resulting in phenyl-N-tert-butylhydroxamic acid or the phenyl ring that would afford alpha-hydroxyphenyl-N-tert-butylnitrone (HOPBN). The structure of M1 was established as alpha-4-Hydroxyphenyl-N-tert-butylnitrone (4-HOPBN) on the basis of: 1) obvious LC R(t) differences between M1 and the authentic hydroxamate standard, 2) P450 catalyzed hydroxylation of [(2)H]PBN that contained a deuterium instead of a hydrogen atom on its benzylic position and which afforded [(2)H]M1, and 3) comparison of the liquid chromatography-tandem mass spectrometry properties with a synthetic 4-HOPBN standard. We speculate that 4-HOPBN is an "active" PBN metabolite that provides an additive effect to the pharmacological action of PBN in vivo.


Asunto(s)
Depuradores de Radicales Libres/farmacocinética , Microsomas Hepáticos/metabolismo , Óxidos de Nitrógeno/farmacocinética , Especies Reactivas de Oxígeno/farmacocinética , Animales , Óxidos N-Cíclicos , Depuradores de Radicales Libres/sangre , Masculino , Óxidos de Nitrógeno/sangre , Ratas , Ratas Sprague-Dawley , Especies Reactivas de Oxígeno/metabolismo
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