RESUMEN
Diabesity has become a popular term to describe the specific form of diabetes that develops late in life and is associated with obesity. While there is a correlation between diabetes and obesity, the association is not universally predictive. Defining the metabolic characteristics of obesity that lead to diabetes, and how obese individuals who develop diabetes different from those who do not, are important goals. The use of large-scale omics analyses (e.g., metabolomic, proteomic, transcriptomic, and lipidomic) of diabetes and obesity may help to identify new targets to treat these conditions. This report discusses how various types of omics data can be integrated to shed light on the changes in metabolism that occur in obesity and diabetes.
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Biología Computacional , Diabetes Mellitus Tipo 2/metabolismo , Adulto , Anciano , Animales , Enfermedades Cardiovasculares/etiología , Enfermedades Cardiovasculares/metabolismo , Comorbilidad , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Diabetes Mellitus Tipo 2/epidemiología , Diabetes Mellitus Tipo 2/etiología , Modelos Animales de Enfermedad , Descubrimiento de Drogas , Metabolismo Energético , Femenino , Glucosa/metabolismo , Humanos , Resistencia a la Insulina , Metabolismo de los Lípidos , Masculino , Síndrome Metabólico/complicaciones , Síndrome Metabólico/metabolismo , Ratones , Persona de Mediana Edad , Modelos Biológicos , Terapia Molecular Dirigida , Obesidad/complicaciones , Obesidad/epidemiología , Obesidad/metabolismo , Estado Prediabético/epidemiología , Estado Prediabético/metabolismo , Prevalencia , Proyectos de InvestigaciónRESUMEN
BACKGROUND: Recent developments in LC-MS have turned it into a viable and valid alternative to ligand-binding assays. Large molecule bioanalysis by LC-MS is generally performed by tryptic digestion, purification and detection of one or more small signature peptides. High-resolution MS instruments offer quantification of intact small proteins or peptides and are able to increase the selectivity, while maintaining sensitivity. RESULTS: Unlike multiple reaction monitoring assays, several factors affecting data processing were presented and the optimal parameters to consider during quantification method building were also demonstrated. MUC5AC-13 (MW 1709.8 Da), human hepcidin/LEAP-1 (MW 2797.4 Da), porcine calcitonin (MW 3604 Da) and chicken lysozyme (MW 14.3 kDa) were selected as model compounds and the possibility of intact peptide and small protein quantification, without tryptic digestion, was demonstrated. CONCLUSION: Selectivity and sensitivity were improved using different scan modes, such as TOF-MS and TOF-MS/MS.
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Péptidos Catiónicos Antimicrobianos/análisis , Calcitonina/análisis , Técnicas de Química Analítica , Espectrometría de Masas , Mucina 5AC/química , Muramidasa/análisis , Péptidos/análisis , Animales , Péptidos Catiónicos Antimicrobianos/química , Calcitonina/química , Hepcidinas , Humanos , Mucina 5AC/análisis , Mucina 5AC/sangre , Muramidasa/química , Péptidos/química , Ratas , PorcinosRESUMEN
It can be argued that the last true paradigm shift in the bioanalytical (BA) arena was the shift from high-performance liquid chromatography (HPLC) with ultraviolet (UV) detection to HPLC with tandem mass spectrometry (MS/MS) detection after the commercialization of the triple quadrupole mass spectrometer in the 1990s. HPLC-MS/MS analysis based on selected reaction monitoring (SRM) has become the gold standard for BA assays and is used by all the major pharmaceutical companies for the quantitative analysis of new drug entities (NCEs) as part of the new drug discovery and development process. While LC-MS/MS continues to be the best tool for drug discovery bioanalysis, a new paradigm involving high-resolution mass spectrometry (HRMS) and ultrahigh-pressure liquid chromatography (uHPLC) is starting to make inroads into the pharmaceutical industry. The ability to collect full scan spectra, with excellent mass accuracy, mass resolution, 10-250 ms scan speeds and no NCE-related MS parameter optimization, makes the uHPLC-HRMS techniques suitable for quantitative analysis of NCEs while preserving maximum qualitative information about other drug-related and endogenous components such as metabolites, degradants, biomarkers and formulation materials. In this perspective article, we provide some insight into the evolution of the hybrid quadrupole-time-of-flight (Qq-TOF) mass spectrometer and propose some of the desirable specifications that such HRMS systems should have to be integrated into the drug discovery bioanalytical workflow for performing integrated qualitative and quantitative bioanalysis of drugs and related components.
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Cromatografía Líquida de Alta Presión/métodos , Descubrimiento de Drogas/métodos , Espectrometría de Masas en Tándem/métodos , Descubrimiento de Drogas/instrumentación , Descubrimiento de Drogas/normas , Descubrimiento de Drogas/tendencias , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
Octadecenyl thiophosphate (OTP), a synthetic analogue of the lysophospholipid growth factor lysophosphatidic acid (LPA), significantly reduces mortality following a lethal dose of LD(80/30) radiation exposure in a mouse model of whole-body irradiation. To facilitate dose scaling between species, we developed a novel liquid chromatography/tandem mass spectrometry (LC-MS/MS) for the preclinical pharmacokinetic characterization of OTP in monkeys. Sample extraction was carried out using a butanol based liquid-liquid extraction method. A partially deuterated OTP analogue was used as internal standard (IS). OTP and IS were separated by reversed-phase liquid chromatography on a C-8 column using 10mM ammonium acetate and acetonitrile. A triple quadrupole mass spectrometer operating in the negative electrospray ionization mode with multiple reaction monitoring was used to detect OTP and IS transitions of m/z 363.1-->95.0 and 403.1-->95.0. The method was applied to determine pharmacokinetic parameters in monkeys receiving a single oral OTP dose (3mg/kg). OTP is readily absorbed with a relatively long half-life which supports further preclinical testing of OTP as a radioprotectant in monkeys.
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Cromatografía Liquida/métodos , Compuestos Organofosforados/farmacocinética , Protectores contra Radiación/farmacocinética , Espectrometría de Masas en Tándem/métodos , Animales , Butanoles/química , Fraccionamiento Químico , Estabilidad de Medicamentos , Femenino , Modelos Lineales , Macaca mulatta , Compuestos Organofosforados/sangre , Protectores contra Radiación/análisis , Estándares de Referencia , Reproducibilidad de los ResultadosRESUMEN
Quantification of alpha- and gamma-endorphins in rat brain using liquid chromatography-electrospray ionization-tandem mass spectrometry is described. [D-Ala(2)]-gamma-endorphin is used as an internal standard. The precursor-to-product ion MRM transitions for alpha-endorphin, gamma-endorphin, and [D-Ala(2)]-gamma-endorphin were m/z 873.6-->429.6; 929.6-->542.3; 936.6-->542.3, respectively. The method was validated in terms of linearity, specificity, sensitivity, recovery, precision, and accuracy. The assay was linear over a concentration range of 0.1-200 ng/mL with the limit-of-detection of 0.03 ng/mL and limit-of-quantification of 0.1 ng/mL. The endogenous concentrations of alpha- and gamma-endorphins in rat brains were 13.8+/-0.57 (mean+/-SD; n=5) and 2.5+/-0.43 ng/g of wet tissue weight, respectively.
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Química Encefálica , Cromatografía Liquida/métodos , Espectrometría de Masa por Ionización de Electrospray/métodos , Espectrometría de Masas en Tándem/métodos , alfa-Endorfina/análisis , gamma-Endorfina/análisis , Secuencia de Aminoácidos , Animales , Masculino , Datos de Secuencia Molecular , Ratas , Ratas Wistar , alfa-Endorfina/química , gamma-Endorfina/químicaRESUMEN
Our group has used the tetrahydroisoquinoline derivative EDL-155 to treat glioblastoma in animal models and it is currently being evaluated in the treatment of ocular cancers. The purpose of this study was to develop a rapid and sensitive liquid chromatography and tandem mass spectrometry (LC-MS/MS) method to study the plasma and vitreous humor disposition of EDL-155 in rats. Animals received a single periocular injection of EDL-155 (20 mg/kg). Animals were sacrificed at specified times (5, 60, 120, 240 and 360 min) and plasma and vitreous humor samples were obtained. EDL-155 was isolated by protein precipitation and the extracts were analyzed by reversed-phase high-pressure liquid chromatography (HPLC) with MS/MS detection. A structurally similar analog was used as internal standard (IS). The chromatographic run time was 3.5 min per injection. The mass spectrometer was operated in positive-ion, multiple reaction monitoring (MRM) mode. The mass transitions monitored were m/z332.2 --> 167.2 (EDL-155) and m/z391.2 --> 200.2 (IS). The lower limit of quantification (LLOQ) was 0.1 ng/ml in both vitreous humor and plasma. The method was validated for selectivity, linearity, accuracy and precision in rat vitreous humor and partially validated for accuracy and precision in rat plasma. The ion suppression, recovery and stability of the analyte in the biological matrix were also tested. The assay was rapid, sensitive and robust enough to support EDL-155 ocular penetration studies in a rodent model of intraocular cancer. Application of this method revealed that EDL-155 was rapidly passed into the vitreous humor following periocular administration. Further, vitreous humor exposure exceeded systemic exposure by approximately sevenfold. High local concentrations coupled with minimal systemic exposure supports further testing of EDL-155 as localized therapy for intraocular cancers.
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Antineoplásicos/farmacocinética , Cromatografía Líquida de Alta Presión/métodos , Espectrometría de Masa por Ionización de Electrospray/métodos , Tetrahidroisoquinolinas/farmacocinética , Cuerpo Vítreo/metabolismo , Animales , Antineoplásicos/administración & dosificación , Antineoplásicos/análisis , Antineoplásicos/sangre , Estabilidad de Medicamentos , Modelos Lineales , Ratas , Ratas Wistar , Estándares de Referencia , Reproducibilidad de los Resultados , Retinoblastoma/tratamiento farmacológico , Sensibilidad y Especificidad , Tetrahidroisoquinolinas/administración & dosificación , Tetrahidroisoquinolinas/análisis , Tetrahidroisoquinolinas/sangre , Cuerpo Vítreo/químicaRESUMEN
A rapid method for quantitative chiral analysis of phthaloylglutamic acid and its dimethyl ester by Cook's kinetic method is demonstrated using electrospray ionization (ESI) and matrix-assisted laser desorption techniques. Transition-metal-bound complex ions containing the chiral phthaloylglutamic acid and its dimethyl ester are generated by ESI mass spectrometry and subjected to collision-induced dissociation. The ratio of the two competitive dissociation rates is related to the enantiomeric composition of the drug mixture. A seven-point calibration curve, derived from the kinetic method, allowed rapid quantitation of the enantiomeric excess of drug mixtures. In this paper, matrix-assisted laser desorption/ionization (MALDI) coupled with the linear ion trap (LIT) technique is evaluated for its applicability as a complementary technique to ESI for chiral discrimination and quantitation.
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Glutamatos/análisis , Espectrometría de Masa por Ionización de Electrospray/métodos , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Espectrometría de Masas en Tándem/métodos , EstereoisomerismoRESUMEN
PURPOSE: Tetrahydroisoquinolines (THIs) have demonstrated anti-cancer activity in rodent models of glioma, a form of brain cancer refractory to therapeutic intervention. In this study, peripheral and cerebrospinal fluid (CSF) pharmacokinetics in rats were determined to assess the drug developability of the novel THI EDL-155 for the treatment of glioma. METHODS: Serial blood and CSF samples were collected from rats following intravenous bolus administration of EDL-155 (10-20 mg/kg). Samples were analyzed by LC/MS/MS. Pharmacokinetic analyses using compartmental and noncompartmental methods were performed using the computer program WinNonlin. Plasma protein binding was measured using the charcoal adsorption method. The in vivo efficacy of EDL-155 (i.p. 20 mg/kg twice daily for 7 days) was assessed in rats with stereotactically implanted C6 glioma cells into the caudate. RESULTS: EDL-155 plasma concentration data were described by a one-compartment model. EDL-155 demonstrated rapid clearance (342.5+/-49.9 ml/min/kg), high volume of distribution (13.0+/-1.2 l/kg) and a terminal half-life of 23.7+/-1.5 min. Dose-normalized CSF area under the curve (AUC(CSF)) as a percentage of peripheral exposure (AUC(Plasma)) was 1.4%. EDL-155 was highly bound to plasma proteins (>93%). Intracranial tumor volume at 7 days post-implantation was approximately 30% smaller in animals treated with EDL-155 when compared to vehicle control animals (13.2+/-5.3 mm(3) vs. 18.7+/-6.3 mm(3); P=0.04). CONCLUSION: High clearance and extensive protein binding limit the brain availability of EDL-155 following systemic administration. EDL-155 treatment resulted in reduced tumor size despite limited blood brain barrier penetrability, which suggests that analogs with increased metabolic stability and brain penetrability may provide a therapeutic option for primary central nervous system tumors such as glioma. On-going studies are focused on the design, synthesis, and testing of novel analogs based upon these findings.
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Antineoplásicos/farmacocinética , Tetrahidroisoquinolinas/farmacocinética , Animales , Antineoplásicos/sangre , Antineoplásicos/líquido cefalorraquídeo , Disponibilidad Biológica , Neoplasias Encefálicas/metabolismo , Cromatografía Liquida , Glioma/metabolismo , Semivida , Humanos , Inyecciones Intravenosas , Masculino , Espectrometría de Masas , Trasplante de Neoplasias , Ratas , Ratas Sprague-Dawley , Tetrahidroisoquinolinas/sangre , Tetrahidroisoquinolinas/líquido cefalorraquídeoRESUMEN
Detection of doping agents in urine frequently requires extensive separation prior to chemical analyses. Gas or liquid chromatography coupled to mass spectrometry has produced accurate and sensitive assays, but chromatographic separations require time and, sometimes, chemical derivatization. To avoid such tedious and lengthy procedures, vacuum matrix-assisted laser desorption ionization (vMALDI) coupled with the linear ion trap mass spectrometry (LIT/MS) technique is tested for its applicability as a rapid screening technique. Commonly used doping agents like nandrolone, boldenone, trenbolone, testosterone, and betamethasone were chosen as study compounds. Different MALDI matrixes like alpha-cyano-4-hydroxycinnamic acid (CHCA), dihyroxy benzoic acid (DHB) with and without cetyl trimethyl ammonium bromide (CTAB), a surfactant, and meso-tetrakis(pentafluorophenyl) porphyrin (F20TPP) were tested. Among them, F20TPP (MW 974.57 Da) was selected as the preferred matrix owing to the lack of interfering matrix peaks at the lower mass range (m/z 100-700). Urine samples spiked with study compounds were processed by solid-phase extraction (SPE) and consistently detected through a linear range of 0.1-100 ng/mL. The limit of detection and lower limit of quantification for all five analytes have been determined to be 0.03 and 0.1 ng/mL, respectively, in urine samples. Testosterone-d3 was used as an internal standard, and the quantitative measurements were achieved by the selective reaction monitoring (SRM) mode. The method was validated and showed consistency in the results. Hence, vMALDI-LIT/MS can be used as a rapid screening method to complement the traditional GC/MS and LC/MS techniques for simultaneous identification, confirmation, and quantification of doping agents in urine.
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Anabolizantes/orina , Doping en los Deportes , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Detección de Abuso de Sustancias/métodos , Benzoatos/química , Betametasona/orina , Ácidos Cumáricos/química , Alcoholes Grasos , Humanos , Nandrolona/orina , Porfirinas/química , Compuestos de Amonio Cuaternario/química , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Tensoactivos/química , Testosterona/análogos & derivados , Testosterona/orina , Acetato de Trembolona/orina , VacioRESUMEN
In the present work, we report conversion of fluoxetine (Prozac), a novel anti depressant to N-methyl fluoxetine in formalin fixed liver tissue. Earlier studies indicate that drugs containing secondary amino group will react with formalin to form corresponding N-methyl derivatives. Even though embalming cadavers is common, it may create problems for forensic toxicologists if a case was not previously suspected. In formalin solutions, fluoxetine is methylated producing N-methyl fluoxetine. N-Methyl fluoxetine standard was synthesized by treating fluoxetine in formaldehyde solution. The structure confirmed by (1)HNMR and gas chromatography-mass spectrometry in electron impact ionization mode. Randomly chosen rat liver pieces (200-250 mg) were injected with 100 microg of Fluoxetine. The liver pieces were covered with three different concentrations of formalin, 5%, 10%, and 20%, and at three different pHs, 3.0, 7.0, and 9.5. The reaction was studied for a total period of 30 days, and the reaction products were monitored on days 0, 4, 14, and 30 days. The study indicates that the rate of conversion of fluoxetine to its N-methyl derivative increased with increase in the concentration of formalin and pH of the solution. The conversion is rapid at higher pH values. Fluoxetine was totally converted to its N-methyl derivatives after 30 days in 20% formalin at pH 9.5. Therefore, analysis for parent drug or its N-methyl derivative in embalmed tissues may provide data that will reduce the likelihood of false negatives.
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Antidepresivos de Segunda Generación/química , Embalsamiento , Fijadores/química , Fluoxetina/química , Formaldehído/química , Animales , Medicina Legal , Hígado/química , Metilación , Ratas , Fijación del TejidoRESUMEN
A stability study has been initiated for propoxur (Baygon) in whole blood and urine samples stored over a period of 60 days at four different temperature conditions (room temperature, 4 degrees C, -20 degrees C, and -80 degrees C). Stability data was established on day 0, 1, 7, 14, 28, 42, and 60. Sample purification was done by solid-phase extraction using a weak cation exchange cartridge (Isolute CBA), and quantitation was carried out by a validated high-performance liquid chromatographic method with a photodiode-array UV detector. Propoxur was spiked at two different concentration levels in both blood and urine samples [low concentration (10 microg/L) and high concentration (100 microg/L)]. Isopropoxy phenol was observed as the major degradation product in blood and urine samples and confirmed by liquid chromatography-electrospray ionization-mass spectrometry. At room temperature, a substantial decrease in concentration of about 95% was observed at the end of the stability study in both blood and urine samples. However, at 4 degrees C, the concentration of propoxur observed after 60 days was around 60% in both samples. A decrease in temperature reduced the degradation, and finally propoxur was found to be stable at -80 degrees C and -20 degrees C for the whole observation period (60 days). The data collected suggests that knowledge about time-dependent decrease of propoxur in urine and blood samples is of considerable significance in forensic toxicology, and, therefore, forensic cases should be interpreted with caution.