RESUMEN
An automated online immobilized metal affinity chromatography/high-performance liquid chromatography mass spectrometric (IMAC-HPLC/MS/MS) method was developed to study cytidine 3',5'-cyclic monophosphate (cCMP)-specific protein phosphorylation, analogous to a previously successful offline IMAC method using microvolume IMAC pipette tips. The optimized method identified murine brain phosphoproteins selectively modified by challenge with cCMP, using manual interpretation of the results to confirm both phosphorylation and selectivity of response to cCMP. A number of proteins identified by this strategy have potential roles in hyperproliferation, a previously reported response to elevated levels of cCMP.
Asunto(s)
Química Encefálica/efectos de los fármacos , Encéfalo/efectos de los fármacos , Cromatografía de Afinidad/métodos , Citidina Monofosfato/farmacología , Fosfoproteínas/análisis , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Animales , Cromatografía Líquida de Alta Presión , Femenino , Ratones , Fosforilación , Proteómica , Espectrometría de Masas en TándemRESUMEN
A study of the interaction of phosphorylated organic compounds with the stainless components of a liquid chromatography-electrospray ionisation-mass spectrometry system (LC-ESI-MS) was carried out to disclose a (forgotten?) likely pitfall in the LC-ESI-MS analysis of phosphorylated compounds. The retention behaviour of some representative compounds of different important classes of phosphorylated biomolecules such as nucleotides, oligonucleotides, phosphopeptides, phospholipids and phosphorylated sugars was investigated during their passage through the injector and the stainless steel electrospray capillary. It became clear that the stainless steel components within the LC-ESI-MS setup were able to retain and trap phosphorylated compounds when these compounds were introduced under acidic conditions (0.1% acetic acid). Their release from these stainless steel parts was accomplished by applying an extreme basic mobile phase (25-50% ammonium hydroxide, ca. pH 12). From the data collected one could conclude that the availability of a primary phosphate group appeared imperative but was not always sufficient to realise adsorption on a stainless surface. Furthermore, the number of phosphate moieties seemed to enhance the adsorption properties of the molecules and hence roughly correlated with the analyte fraction lost. Corrosion of the inner surface caused by the mobile phase and the electrospray process was found to be an important factor in the course of these adsorption phenomena.
Asunto(s)
Compuestos Orgánicos/química , Espectrometría de Masa por Ionización de Electrospray/instrumentación , Adsorción , Secuencia de Aminoácidos , Microscopía Electrónica de Rastreo , Datos de Secuencia Molecular , Fosfopéptidos/química , FosforilaciónAsunto(s)
Monitoreo del Ambiente/estadística & datos numéricos , Fluorocarburos/análisis , Marsopas/metabolismo , Contaminantes Químicos del Agua/análisis , Animales , Océano Atlántico , Cromatografía Líquida de Alta Presión , Recolección de Datos , Europa (Continente) , Fluorocarburos/farmacocinética , Geografía , Hígado/metabolismo , Espectrometría de Masas , Músculo Esquelético/metabolismo , Mar del Norte , Especificidad de la Especie , Estadísticas no ParamétricasRESUMEN
For the first time estrogen DNA-adducts were identified in DNA human breast tumor tissue using nano-LC coupled to nano-Electrospray Tandem Mass Spectrometry. Normal breast tissue was analyzed analogously. The data obtained in the five breast tumor and five adjacent normal tissue samples were compared qualitatively, but no straightforward difference was observed. Prior to LC-MS analysis the DNA was enzymatically hydrolyzed to a nucleoside pool. The DNA-hydrolysates were directly injected onto a column switching system developed for on-line sample clean-up and subsequent analysis of the DNA-adducts. In four patients using Premarin, DNA-adducts of 4-hydroxy-equilenin (4OHEN) were detected. All except three samples contained DNA-adducts from 4-hydroxy-estradiol or 4-hydroxy-estrone. Also DNA isolated from eight alcohol fixed and paraffin embedded breast tumor tissue showed the presence of different estrogen DNA-adducts. Worthwhile mentioning is the presence of adducts responding to m/z 570 > m/z 454 transition. This is a well-known SRM-transition indicative for the presence of the 2'-deoxyguanosine (dGuo) adduct of Benzo[a]pyrene.
Asunto(s)
Neoplasias de la Mama/química , Neoplasias de la Mama/genética , Mama/metabolismo , Aductos de ADN/análisis , Estrógenos/análisis , Estrógenos/química , Espectrometría de Masa por Ionización de Electrospray/métodos , Cromatografía Líquida de Alta Presión , Aductos de ADN/química , Aductos de ADN/metabolismo , Femenino , Humanos , Hidrólisis , Microquímica/métodos , Estructura Molecular , Sensibilidad y EspecificidadRESUMEN
In the present study we evaluated the toxicological effects of a scarcely documented environmental pollutant, perfluorooctane sulfonic acid (PFOS), on selected biochemical endpoints in the common carp, Cyprinus carpio. Juvenile organisms were exposed to PFOS through a single intraperitoneal injection (liver concentrations ranging from 16 to 864 ng/g after 5 days of exposure) and after 1 and 5 days effects were assessed in liver and serum of the exposed organisms. The investigation of the hepatotoxicity of PFOS included the determination of the peroxisome proliferating potential (peroxisomal palmitoyl CoA oxidase and catalase activity) and the compounds influence on the average DNA basepair length (ABPL) by agarose gel electrophoresis. Total antioxidant activity (TAA), cholesterol and triglyceride levels were monitored in the serum. After 1 day of exposure the ABPL was significantly increased in the 270 and 864 ng/g treatment groups. After 5 days of exposure significant increases relative to the control were observed for the 16, 270 and 864 ng/g treatment groups. Enzyme leakage from the liver was investigated by measurement of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities in the serum. At 561, 670 and 864 ng/g PFOS a significant increase in serum ALT activity became apparent after 5 days of exposure with values ranging from 159 to 407% relative to the control. For serum AST activity a significant increase for the 864 ng/g treatment group was observed with a value of 112% relative to the control. Determination of the polymorphonuclear leukocyte migration into liver tissue as assessed through myeloperoxidase (MPO) activity in liver, was used as an indicator for inflammation. It appeared that inflammation was not involved in the observed membranous enzyme leakage for the 561, 670 and 864 ng/g PFOS treatment groups. The results of this study suggest that PFOS induces inflammation-independent enzyme leakage through liver cell membranes that might be related to cell necrosis. Furthermore, results show that PFOS does not significantly affects serum antioxidant levels nor does it clearly induce peroxisome proliferation in carp. This study also points out that PFOS might interfere with homeostasis of the DNA metabolism. The results of these biochemical analyses were used to perform an initial hazard assessment study indicating that PFOS levels observed in tissues of wildlife populations could induce a clear rise in serum transaminase levels indicative for disruption of hepatocyte membrane integrity.
Asunto(s)
Ácidos Alcanesulfónicos/toxicidad , Carpas/fisiología , Fluorocarburos/toxicidad , Inflamación , Proliferadores de Peroxisomas/farmacología , Contaminantes Químicos del Agua/toxicidad , Animales , Antioxidantes/análisis , Biomarcadores/análisis , Catalasa/farmacología , Colesterol/análisis , Inyecciones Intraperitoneales , Hígado/enzimología , Triglicéridos/análisisRESUMEN
A liquid chromatography/mass spectrometry (LC/MS) method for the analysis of complex mixtures of nucleoside mono-, di- and triphosphates has been developed. A short capillary column (35mm x 0.3mm i.d.) was operated under ion-pair high-performance liquid chromatography conditions and hyphenated to (negative) electrospray (tandem) mass spectrometry. As such, the separation of 12 nucleotides was performed by a binary gradient elution using CH(3)OH/H(2)O and N,N-dimethylhexylamine (N,N-DMHA) as ion-pairing agent. The influence of different N,N-DMHA concentrations on the chromatographic and mass spectrometric performance was evaluated to achieve optimal LC/MS conditions. In addition it was demonstrated that a controlled admission of ammonium dihydrogen phosphate (NH(4)H(2)PO(4)) improved both chromatographic performance and mass spectrometric detection. Because the system was hyphenated to an orthogonal designed electrospray interface (Z-spraytrade mark), long acquisition times were possible without loss of sensitivity.
RESUMEN
Qualitative and quantitative analyses of urinary nucleosides have diagnostic potential as tumour markers. We have developed separation techniques linked to mass spectrometric detection in order to overcome the problems associated with past identification and quantitation methods. The three methods of analysis utilised were: gas chromatography/mass spectrometry (GC/MS), high-performance liquid chromatography/ion-trap mass spectrometry (HPLC/ITMS) and capillary liquid chromatography/triple quadrupole mass spectrometry (CapLC/TQMS). Here we compare the relative effectiveness of each of the techniques for subsequent application in the systematic study of urinary nucleoside profiles in cancer patients. All three methods proved to be valuable techniques for such urinary nucleoside analyses, and a combination rather than one single choice is concluded as the ideal.
Asunto(s)
Nucleósidos/orina , Cromatografía Líquida de Alta Presión , Electroforesis Capilar , Cromatografía de Gases y Espectrometría de Masas , Humanos , Espectrometría de Masas , Neoplasias/orinaRESUMEN
The interaction of 4-hydroxy metabolites of estrogens with DNA leads to the formation of DNA adducts. These adducts are believed to play an important role in the incidence of breast and endometrial cancer. In order to be able to analyze these adducts in in vivo samples a method based upon the coupling of miniaturized liquid chromatography (LC) to electrospray tandem mass spectrometry (ES-MS/MS) was developed for the analysis of the adducts formed with 4-hydroxyequilenin. In vitro synthesized adducts obtained by the reaction of 4-hydroxyequilenin with the main 2'-deoxynucleosides were separated on a Hypersyl C(18) BDS nano-HPLC column (15 cm x 75 microm i.d.) at a flow-rate of 300 nl min(-1) using gradient elution with CH(3)OH--0.2% CH(3)COOH in H(2)O. The column was coupled, in combination with a column switching system, to a nano-electrospray interface. Analysis of the low- and high-resolution low-energy collision-activated dissociation product ion spectra of normal and deuterated adducts supported earlier data demonstrating equilenin to form different isomeric adducts, except with thymidine, for which no adducts were found. The nano-HPLC column-switching ES-MS system was tested for its sensitivity on a triple-quadrupole instrument, and detection limits down to 197 fg in the single reaction monitoring mode were obtained for semi-preparatively isolated equilenin--2'-deoxyguanosine adduct.
Asunto(s)
Cromatografía Líquida de Alta Presión/métodos , Aductos de ADN/biosíntesis , Desoxirribonucleósidos/metabolismo , Equilenina/metabolismo , Espectrometría de Masa por Ionización de Electrospray/métodos , Aductos de ADN/análisis , Desoxirribonucleósidos/análisis , Equilenina/análisis , Sensibilidad y EspecificidadRESUMEN
Nano liquid chromatography (nanoLC) coupled to electrospray mass spectrometry (ES-MS) was evaluated for the analysis of DNA adducts in melphalan-treated Jurkat cells. The detection limit of the nanoLC-ES-MS-MS system was assessed using a dAMP-melphalan adduct. Compared to capillary liquid chromatography (capLC) ES-MS the absolute detection limit could be improved by a factor 10, leading to the detection of 395 fg dAMP-melphalan adduct under single-ion monitoring conditions at a S/N of 14. Minor adducts such as cross-linked adducts could be detected in in vitro solutions of 2'-deoxynucleotides (dNMP) treated with melphalan using column-switching nanoLC-ES-MS. These adducts were not found using capLC-ES-MS. More detailed structural information of the alkylation sites was obtained by examining the nanoLC-ES-MS-MS data. Jurkat cells were treated with melphalan, the modified DNA was isolated and enzymatically hydrolyzed. Several modified dinucleotides were identified, the most abundant adducts were pdG(Mel(Cl))pdC (m/z=453, t(r)=17.0 min) and pdG(Mel(OH)) pdC ring opened (m/z=453, t(r)=39.5 min).
Asunto(s)
Cromatografía Liquida/métodos , Aductos de ADN/análisis , Melfalán/química , Espectrometría de Masa por Ionización de Electrospray/métodos , Animales , Bovinos , ADN/química , Aductos de ADN/química , Humanos , Células JurkatRESUMEN
1. The effect of ecto-nucleotide pyrophosphatase (ecto-NPPase; EC 3.6.1. 9) on the ATP- and ADP-mediated receptor activation was studied in rat C6 glioma cells. The P2-purinoceptor antagonists pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) and reactive blue (RB2) are potent inhibitors (IC(50)=12+/-3 microM) of the latter enzyme. 4,4'-diisothiocyanatostilbene-2,2' disulfonic acid (DIDS), 5'-phosphoadenosine 3'-phosphate (PAP) and suramin were less potent inhibitors with an IC(50) of 22+/-4, 36+/-7 and 72+/-11 microM respectively. 2. P1-purinoceptor antagonists CGS 15943, cyclo-pentyl theophylline (CTP) and theophylline did not affect the activity of the ecto-NPPase. 3. ATP- and ADP-mediated P2Y(1)-like receptor activation inhibited the (-)-isoproterenol-induced increase of intracellular cyclic AMP concentration. PPADS, an ineffective P2Y-antagonist in C6, potentiated the ATP and ADP effect approximately 3 fold due to inhibition of nucleotide hydrolysis by the ecto-NPPase. 4. We conclude that ecto-NPPase has a modulator effect on purinoceptor-mediated signalling in C6 glioma cell cultures.
Asunto(s)
Adenosina Trifosfato/metabolismo , AMP Cíclico/metabolismo , Pirofosfatasas/metabolismo , Receptores Purinérgicos P2/metabolismo , Animales , Inhibidores de Agregación Plaquetaria/farmacología , Antagonistas del Receptor Purinérgico P2 , Fosfato de Piridoxal/análogos & derivados , Fosfato de Piridoxal/farmacología , Pirofosfatasas/antagonistas & inhibidores , Pirofosfatasas/efectos de los fármacos , Ratas , Receptores Purinérgicos P2Y1 , Transducción de Señal , Células Tumorales CultivadasRESUMEN
A sensitive and specific method for the determination and quantitation of (despropionyl) bezitramide in postmortem samples using liquid chromatography combined with electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) is presented. The method is the result from a simple methodological transfer of a liquid chromatographic method with fluorescence detection (LC-FL) previously developed in our laboratory. A liquid-liquid back-extraction procedure using n-hexane isoamyl alcohol (93:7, v/v) as the extraction solvent is performed for a basic sample cleanup. N-Methyldespropionyl bezitramide is used as the internal standard. Chromatographic separation of the analytes of interest is achieved on a Hypersil ODS 5-micron column, using a 80:20 (v/v) mixture of 1.0 mM ammonium acetate and methanol/acetonitrile (50:50, v/v) and 1.0 mM ammonium acetate as the mobile phase. To obtain as high a sensitivity and selectivity as possible, a selected reaction-monitoring mass spectrometric technique is applied. In addition, low-energy collisional-activated dissociation (CAD) product ion spectra are recorded for a few samples. Calibration graphs are prepared for blood and urine, and good linearity is achieved over a concentration range of 1-150 ng/mL. The intra- and interassay coefficients of variation (CV%) for the analysis of quality control samples at 10 and 50 ng/mL concentration levels do not exceed 10.2% and percent of targets are within 12.1%. Postmortem samples (blood, urine, stomach contents, bile, liver, and kidney) from three fatalities, all suspected victims of drug overdoses, are analyzed, and the results are reported. The results obtained with LC-ESI-MS/MS are in close agreement with those obtained using the LC-FL method. Moreover, the isolates' identity and structure are confirmed by the CAD product ion spectra, thus allowing to make unequivocal conclusions about the prior intake of bezitramide by the three subjects.
Asunto(s)
Bencimidazoles/análisis , Cromatografía Líquida de Alta Presión/métodos , Espectrometría de Masas/métodos , Líquidos Corporales/química , Humanos , Cambios Post Mortem , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
The reaction between phenylglycidyl ether and 2'-deoxyguanosine or 2'-deoxyguanosine-5'-monophosphate yields a variety of different nucleoside and nucleotide adducts. The corresponding mixtures were analyzed by liquid chromatography/electrospray tandem mass spectrometry and the product ion spectra of the different isomers are discussed using the correct cone voltage and collision energy. The latter were selected by looking at the energy-resolved spectra. From these data the formation of N7 and N2 isomers was proposed. However, detailed NMR analysis of the latter proved this isomer to be the N(1)-alkylated adduct. The reaction between phenylglycidyl ether and 2'-deoxyguanosine or 2'-deoxyguanosine-5'-monophosphate yields a variety of different nucleoside and nucleotide adducts. The corresponding mixtures were analyzed by liquid chromatography/electrospray tandem mass spectrometry and the product ion spectra of the different isomers are discussed using the correct cone voltage and collision energy. The latter were selected by looking at the energy-resolved spectra. From these data the formation of N7 and N2 isomers was proposed. However, detailed NMR analysis of the latter proved this isomer to be the N(1)-alkylated adduct. 2'-Deoxyguanosine-5'-monophosphate was alkylated by phenylglycidyl ether at the 5'-phosphate position. If the nucleotide base moiety was alkylated, the corresponding tandem mass spectrometric data strongly suggested alkylation of N7. In the case of bis-PGE-dGMP adduct, evidence was found for simultaneous 5'-phosphate and N7-alkylation.2'-Deoxyguanosine-5'-monophosphate was alkylated by phenylglycidyl ether at the 5'-phosphate position. If the nucleotide base moiety was alkylated, the corresponding tandem mass spectrometric data strongly suggested alkylation of N7. In the case of bis-PGE-dGMP adduct, evidence was found for simultaneous 5'-phosphate and N7-alkylation.
Asunto(s)
Nucleótidos de Desoxiguanina/química , Desoxiguanosina/química , Éteres Fenílicos/química , Cromatografía Liquida/métodos , Isomerismo , Espectrometría de Masas , Conformación Molecular , Estructura Molecular , Espectrometría de Masa por Ionización de Electrospray/métodosRESUMEN
The interaction of dipeptidyl peptidase IV with structurally related proteins differing in chain length, namely vasostatin I and II and their precursor protein chromogranin A, was examined using high-performance liquid chromatography in combination with electrospray mass spectrometry. Suitable analytical procedures were developed involving the use of reversed-phase high-performance liquid chromatography for purification of the enzymatic degradation products and a peptide mapping procedure for evaluating the enzymatic degradation of the large precursor protein chromogranin A. While vasostatin I was found to be a substrate for dipeptidyl peptidase IV, no N-terminal cleavage of Leu-Pro could be noted for chromogranin A. With respect to vasostatin II, N-terminal degradation was only observed after degradation in the C-terminal domain to proteins containing < or = 78 amino acids. The specificity of the N-terminal release of Leu-Pro was proved by addition of a DPP IV specific inhibitor.
Asunto(s)
Cromograninas/química , Dipeptidil Peptidasa 4/química , Fragmentos de Péptidos/química , Secuencia de Aminoácidos , Animales , Catálisis , Bovinos , Cromatografía Líquida de Alta Presión , Cromogranina A , Espectrometría de Masas , Datos de Secuencia MolecularRESUMEN
The presence of a nucleotide pyrophosphatase (EC 3.6.1.9) on the plasma membrane of rat C6 glioma has been demonstrated by analysis of the hydrolysis of ATP labeled in the base and in the alpha- and gamma-phosphates. The enzyme degraded ATP into AMP and PPi and, depending on the ATP concentration, accounted for approximately 50-75% of the extracellular degradation of ATP. The association of the enzyme with the plasma membrane was confirmed by ATP hydrolysis in the presence of a varying concentration of pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS), a membrane-impermeable inhibitor of the enzyme. PPADS concentration above 20 microM abolished the degradation of ATP into AMP and PPi. The nucleotide pyrophosphatase has an alkaline pH optimum and a Km for ATP of 17 +/- 5 microM. The enzyme has a broad substrate specificity and hydrolyzes nucleoside triphosphates, nucleoside diphosphates, dinucleoside polyphosphates, and nucleoside monophosphate esters but is inhibited by nucleoside monophosphates, adenosine 3',5'-bisphosphate, and PPADS. The substrate specificity characterizes the enzyme as a nucleotide pyrophosphatase/phosphodiesterase I (PD-I). Immunoblotting and autoadenylylation identified the enzyme as a plasma cell differentiation antigen-related protein. Hydrolysis of ATP terminates the autophosphorylation of a nucleoside diphosphate kinase (NDPK/nm23) detected in the conditioned medium of C6 cultures. A function of the pyrophosphatase/PD-I and NDPK in the purinergic and pyrimidinergic signal transduction in C6 is discussed.
Asunto(s)
Adenosina Trifosfato/metabolismo , Espacio Extracelular/enzimología , Glioma , Pirofosfatasas/metabolismo , Animales , Astrocitos/química , Astrocitos/enzimología , Inhibidores Enzimáticos/farmacología , Guanosina Trifosfato/metabolismo , Hidrólisis , Nucleósido-Difosfato Quinasa/antagonistas & inhibidores , Nucleósido-Difosfato Quinasa/metabolismo , Radioisótopos de Fósforo , Fosforilación , Inhibidores de Agregación Plaquetaria/farmacología , Fosfato de Piridoxal/análogos & derivados , Fosfato de Piridoxal/farmacología , Ratas , Receptores Purinérgicos/fisiología , Células Madre/química , Células Madre/enzimología , Células Tumorales CultivadasRESUMEN
Melphalan is a bifunctional alkylating agent that covalently binds with intracellular nucleophilic sites. A methodology using electrospray mass spectrometry was developed to detect and identify DNA adducts. Alkylation sites within a particular nucleotide were examined using electrospray tandem mass spectrometry hyphenated to capillary liquid chromatography in combination with a column switching system. In the reaction mixtures resulting from the interaction of 2'-deoxynucleotides and melphalan several base-aklylated adducts were found. In the case of 2'-deoxyadenosine monophosphate, thymidine monophosphate and 2'-deoxyguanosine phosphate alkylation was observed in the mononucleotide reaction mixtures but not in the DNA-hydrolysates. Calf thymus DNA was reacted in vitro with melphalan. The DNA pellet was isolated and enzymatically hydrolyzed with the aid of Nuclease P1. In this hydrolysate both mono-alkylated 2'-deoxynucleotides and dinucleotides were found. The most important adduct found was identified as the N-7 alklylated dGMP adduct. The alkylated dinucleotides were identified as a pdApdT/melphalan and pdGpdC/melphalan the latter being the most important.
Asunto(s)
Cromatografía Líquida de Alta Presión/métodos , Aductos de ADN/análisis , Desoxirribonucleótidos/análisis , Espectrometría de Masas/métodos , Melfalán/metabolismo , Alquilación , Animales , Bovinos , ADN/metabolismo , Nucleótidos de Desoxiadenina/análisis , Desoxicitidina Monofosfato/análisis , Nucleótidos de Desoxiguanina/análisis , Melfalán/farmacología , Sensibilidad y Especificidad , Timidina Monofosfato/análisisRESUMEN
Calf thymus DNA was incubated in vitro with a new aminocoumarin platinum (II) complex in order to study its interaction with DNA. The platinated DNA was hydrolyzed enzymatically to the 5'-mononucleotide level using DNAase I and nuclease P1. Analysis of the DNA hydrolysate with capillary zone electrophoresis (CZE), using sample stacking, revealed the presence of unhydrolyzed oligonucleotides in the platinated DNA. A homemade system, using only some plastic pipet tips, was constructed to collect the oligonucleotide fraction during CZE analysis. The platinum content of this fraction was determined using graphite furnace atomic absorption with Zeeman background correction. This system proved to be a useful tool to detect platinated DNA species (with a quantifiable detection limit for the detection of platinum of 0.78 ng). Subsequent gel filtration experiments confirmed the presence of high molecular weight oligonucleotides that were platinated. This was proven by reversal of the platination using thiourea and subsequent enzymatic hydrolysis to 5'-mononucleotides.
Asunto(s)
ADN/análisis , ADN/metabolismo , Electroforesis Capilar/métodos , Compuestos Organoplatinos/metabolismo , Animales , Bovinos , Cromatografía en Gel , Hidrólisis , Peso Molecular , Endonucleasas Específicas del ADN y ARN con un Solo Filamento/metabolismo , Tiourea/farmacologíaRESUMEN
Calf thymus DNA was reacted in vitro with phenyl glycidyl ether (PGE) and was hydrolysed enzymatically, to the 5'-monophosphate nucleotides using deoxyribonuclease I (DNA-ase I) and nuclease P1. The adducts were concentrated using solid phase extraction (SPE), on a polystyrene divinylbenzene copolymer in order to remove the unmodified nucleotides. The adducts could be identified using capillary zone electrophoresis-electrospray tandem mass spectrometry (CZE ES-MS/MS), using sample stacking. In addition to the base alkylated 2'-deoxynucleotides present in the DNA-hydrolysate, also phosphate alkylated 2'-deoxynucleotide adducts were identified for TMP and dAMP. An additional adduct, dUMP alkylated on the uridine moiety was found originating from the hydrolytic deamination of dCMP alkylated on N3 of the cytosine moiety. Enzymatic hydrolysis using nuclease P1 was incomplete as shown by the presence of dinucleotides alkylated on the base moiety. They were successfully hydrolysed to the corresponding 2'-deoxynucleotides by snake venom phosphodiesterase (SVP). Data are shown indicating that alkylations on the pyrimidine bases were more resistant to enzymatic hydrolysis with nuclease P1 than the purine alkylated products.
Asunto(s)
Aductos de ADN/análisis , ADN/efectos de los fármacos , Electroforesis Capilar/métodos , Espectrometría de Masas/métodos , Éteres Fenílicos/farmacología , Alquilación , Animales , Bovinos , Fosfatos/metabolismo , Espectrofotometría Ultravioleta , Timo/efectos de los fármacos , Timo/metabolismoRESUMEN
A simple, but sensitive and specific, high-performance liquid chromatographic assay for cocaine, cocaethylene, and benzoylecgonine is described. Using direct fluorometric detection, the procedure is particularly interesting for the routine analysis of human hair samples. In the sample preparation part, the hair samples are cut and washed and two internal standards with close structural resemblance to benzoylecgonine and cocaine as well as to cocaethylene are added. Subsequently, the hair samples are homogenized, hydrolyzed overnight in a 0.1 M HCl solution at 56 degrees C, and extracted on IST Confirm HCX solid-phase extraction columns. Chromatographic separation is achieved on a narrow-bore Hypersil BDS C18 column (125 x 2.1 mm, 3 microns) by gradient elution with an ammonium acetate buffer-methanol/acetonitrile mixture. For the fluorometric detection, excitation and emission wavelengths of 242 and 315 nm, respectively, are used. This analysis protocol affords a method of high sensitivity and specificity which has been fully evaluated and validated. The data presented show good accuracy and linearity with excellent reproducibility and recovery. Because unequivocal identity confirmation is mandatory in forensic applications, an extension of the analysis protocol was accomplished toward mass spectrometric detection. We succeeded in a simple methodological transfer from LC/FL to LC/ESI-MS/MS, thus providing two complementary approaches after a single, common sample-processing step. Hair samples from 29 fatalities, all known drug users and suspected victims from a drug overdose, were analyzed in this way. Of the investigated samples, 12 were positive and the concentrations found range from 0.98 to 938 ng/mg of hair for cocaine and from 1.45 to 388 ng/mg of hair for benzoylecgonine. Traces of cocaethylene were also found in two of the hair samples. The results obtained with LC/ESI-MS/MS were in close agreement with those obtained with LC/FL, positively confirming the isolates' identity and structure by means of the resulting MS/MS spectra.
Asunto(s)
Cromatografía Líquida de Alta Presión/métodos , Cocaína/análisis , Cabello/química , Cocaína/análogos & derivados , Cocaína/metabolismo , Cabello/metabolismo , Humanos , Espectrometría de Masas , Reproducibilidad de los Resultados , Espectrometría de Fluorescencia , Trastornos Relacionados con Sustancias/diagnósticoRESUMEN
The in vitro adduct formation between phenyl glycidyl ether (PGE) and calf thymus DNA was investigated. Agarose slab gel electrophoresis of DNA incubated with PGE revealed that nearly all high-molecular-weight species were degraded after 10 h of incubation. After DNA precipitation the reaction products present in the supernatant were subjected to a solid-phase extraction on a polystyrene divinylbenzene copolymer, enabling analysis on capillary zone electrophoresis (CZE), using sample stacking. These reaction products were mainly produced during the first 10 h of incubation, indicating that these products result from the DNA degradation. On the other hand, analysis of the adducts present in the enzymatic digest of the DNA pellet revealed that these adducts were formed only after 10 h of incubation. The reaction products present in the DNA supernatant were identified by on-line coupling of CZE to electrospray tandem mass spectrometry. Three major reaction products resulted from phosphate alkylation, as proven by the analysis of the corresponding low-energy CAD product ion mass spectra. This phosphate alkylation results in phosphotriesters which readily hydrolyze, resulting in DNA strand breaks.
Asunto(s)
Daño del ADN , Electroforesis Capilar/métodos , Espectrometría de Masas/métodos , Éteres Fenílicos/toxicidad , Animales , Bovinos , ADN/química , ADN/efectos de los fármacos , Aductos de ADN , Hidrólisis , Quinonas/química , Espectrofotometría UltravioletaRESUMEN
In this work, the coupling of liquid nanochromatography to NanoFlow electrospray mass spectrometry was evaluated for the detection of DNA adducts. The NanoFlow ES LC/MS system was compared with the capillary and conventional ES LC/MS system by analyzing an in vitro reaction mixture resulting from the interaction of 2'-deoxyguanosine 5'-monophosphate with bisphenol A diglycidyl ether and by injecting 2'-deoxyadenosine. By using NanoFlow ES LC/MS, the mass sensitivity could be improved by a factor of 3300. Three different injection methods used in liquid nanochromatography, i.e., split, large-volume, and column-switching injections were compared in terms of sensitivity. Furthermore, NanoFlow ES LC/MS was used to detect 2'-deoxynucleotide adducts isolated from an in vitro mixture of calf thymus DNA and bisphenol A diglycidyl ether. Different 2'-deoxynucleotide adducts could be identified by monitoring typical product ions, diagnostic for the adducts.