RESUMEN
The separation of novel permanently charged oligomers was studied using paired-ion reversed-phase liquid chromatography. The polyionene studied is less than 5 kDa in size, but contains three oligomer series with different end-group chemistries. The complexity of this polyionene makes development of a single-dimension separation quite challenging. Separation under critical conditions was employed to fractionate the end-group conformations and then the chain length of the oligomers in each series was confirmed by LC-MS. The oligomers were then used to optimize a single-dimension HPLC separation. Precise modulation of the hydrophobicity of the ion-pair reagent and the stationary-phase chemistry yielded very high resolution one-dimensional separations.
Asunto(s)
Polímeros/química , Compuestos de Piridinio/química , Compuestos de Amonio Cuaternario/química , Cromatografía Líquida de Alta Presión , Espectrometría de MasasRESUMEN
A 25-mer phosphorothioate oligodeoxynucleotide (GEM 91) complementary to the gag gene mRNA of HIV-1 virus was administered intravenously (i.v.) at a dose of 10 mg/kg/day for 8 weeks or 25 mg/kg single dose subcutaneously (SC) to adult Rhesus monkeys. No radioactive markers were used. A capillary gel electrophoresis (CGE) method with UV detection was used to determine the concentration of GEM 91 in plasma and the metabolite profile. The metabolite profile was virtually the same following a single dose of either 10 mg/kg i.v. or 25 mg/kg SC. A different metabolite profile was observed after 4 or 8 weeks of multiple i.v. doses of 10 mg/kg/day. The extract was subjected to matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOFMS) for positive identification. Mass spectrometry confirmed the major metabolic pathway in vivo to be via 3'-end exonuclease activity. The extract was then subjected to a hybridization-assisted ligation reaction in which only 5'-end intact metabolites were labeled. Analysis by CGE with laser-induced fluorescence (LIF) detection allowed each of these metabolites to be quantified with a limit of detection of 1 ppb (ng/ml). MALDI-TOFMS identified components digested from both ends of the DNA. This study demonstrates that the combination of quantitative CGE-LIF and MALDI-TOFMS yields a powerful and unique approach to study the metabolism of phosphorothioate oligonucleotides.
Asunto(s)
Oligodesoxirribonucleótidos Antisentido , Oligonucleótidos Antisentido/administración & dosificación , Oligonucleótidos Antisentido/metabolismo , Tionucleótidos/administración & dosificación , Tionucleótidos/metabolismo , Animales , Esquema de Medicación , Genes gag/efectos de los fármacos , Genes gag/genética , VIH-1/genética , VIH-1/metabolismo , Humanos , Inyecciones Intravenosas , Macaca mulatta , Oligonucleótidos Antisentido/sangre , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Tionucleótidos/sangreRESUMEN
The analysis of antisense phosphorothioate DNA (SODN) in human plasma via direct injection using anion-exchange high-performance liquid chromatography (AE-HPLC) is presented. The method relies on the ability to selectively extract phosphorothioate DNA from undigested serum, plasma and urine on anion-exchange resins. The automated HPLC method can analyze a sample every 5 min with a limit of detection of 50 ng/ml (ppb). The DNA was collected, desalted and analyzed by capillary gel electrophoresis. Due to the high resolving power of this technique, a qualitative assessment of enzymatic degradation of the antisense oligonucleotide can be made.
Asunto(s)
Oligonucleótidos Antisentido/análisis , Tionucleótidos/análisis , Cromatografía por Intercambio Iónico , ADN/análisis , Electroforesis , Humanos , Oligonucleótidos Antisentido/sangre , Oligonucleótidos Antisentido/orina , Solventes , Espectrofotometría Ultravioleta , Tionucleótidos/sangre , Tionucleótidos/orinaRESUMEN
Polymeric reagents have been developed for performing off- and on-line derivatizations of numerous organic analytes in HPLC-detection modes. Such reagents utilize ionic or covalent attachment of labile tags that possess specific detector enhancement properties: ultraviolet, electrochemical, fluorescence, and so forth. Specific synthetic procedures have evolved to generate various linkages of the tag to the underlying, polymeric support, usually involving activated ester connections (leashes). The polymer itself may play a number of roles in the nature of the overall reactions, such as hydrophobic-hydrophillic exclusion, pore size restriction, stabilization of the attachment leashes, and protection of the tags from hydrolysis in aqueous media. The basic, underlying chemistry of polymeric reagents has evolved to the point where it is possible to engineer the polymer support itself, the attachment leash, and the various tags that are then transferred to the analyte molecules. These procedures have now reached the stage of commercialization and practical applicability for real-world drugs and bioorganics in complex biofluid type samples. Polymer supported reagents can now be used for direct injection of biofluids with solid-phase (hydrophobic) extraction of the analytes of interest, followed by sample cleanup, derivatization, elution onto the HPLC column, peak compression, gradient HPLC elution, multiple detection, and final data interpretation with quantitation. This review summarizes much or most of what has been described in the scientific literature over the past decade in the various areas where polymeric reagents are being used for derivatization in HPLC and in capillary electrophoresis as well.
Asunto(s)
Cromatografía Líquida de Alta Presión/métodos , Técnicas de Química Analítica , Indicadores y Reactivos , Polímeros , EstereoisomerismoRESUMEN
An automated system is described for the simultaneous extraction and derivatization of nucleophilic compounds from various biological media. The method includes the use of a solid-phase reagent containing a 9-fluorenylacetate activated ester. The reagent is based on a controlled pore, polystyrene divinylbenzene support prepared through a silica template procedure. An X-Y-Z robotic arm equipped with a needle is used in conjunction with a syringe pump for aspirating and dispensing samples and standards into the HPLC system. A precolumn cartridge containing the solid-phase reagent is put on-line in place of the fixed-volume injection loop. Injections of biological fluids such as urine or plasma with minimal sample treatment and handling are made directly into this reactor. The analytes are derivatized as they are extracted, allowing virtually unlimited sample volumes to be injected. The polymeric cartridge can be used for up to 100 injections without accruing unacceptable reductions in sensitivity. A detection limit of 500 p.p.t. (parts per trillion) of amphetamine in urine was achieved with this system.
Asunto(s)
Anfetamina/análisis , Cromatografía Líquida de Alta Presión/métodos , Metanfetamina/análisis , Detección de Abuso de Sustancias/métodos , Acetatos , Acetonitrilos , Anfetamina/sangre , Anfetamina/orina , Autoanálisis , Dimetilformamida , Dioxanos , Fluorenos , Hidróxidos , Indicadores y Reactivos , Metanfetamina/sangre , Metanfetamina/orina , Nitrobencenos , Fenoles , Polímeros , Compuestos de Potasio , Hidróxido de Sodio , Solventes , TemperaturaRESUMEN
The effect of cross-linking, surface area, and porous nature of modified polystyrene-divinylbenzene (STY-DVB) reagents has been investigated. The supports were prepared via two techniques and modified to contain various chemical functionalities. These reagents were used in an on-line reactor for automated derivatization of amines in HPLC. The reproducibility of the response vs the physical nature of the porous support and the chemical functionality was determined. The ability to stabilize highly reactive acylating reagents toward high concentrations of aqueous base was found to be a complex interaction of pore size distribution, percent cross-linking, surface area, and absolute loading of the analytical reagent on the porous support.
Asunto(s)
Resinas de Intercambio Iónico/química , Poliestirenos/química , Cromatografía Líquida de Alta Presión , Reactivos de Enlaces Cruzados/química , Radicales Libres , Tamaño de la Partícula , Porosidad , Reproducibilidad de los ResultadosRESUMEN
Phosphorothioate oligonucleotides are potentially useful as anti-viral drugs. Classical DNA extraction methods are not as effective on short single-stranded DNA as with longer double-stranced chains. The classical method of phenol-chloroform extraction followed by ethanol precipitation is difficult to quantify, thus monitoring of the pharmacological disposition of these compounds is subject to error. A method has been devised and validated for extraction and analysis of modified oligonucleotides from biological fluids such as urine and serum based on protein kinase digestion and phenol-chloroform extraction. Due to the high native ultraviolet absorbance of the oligomers, detection limits in the low ppb range were obtained without derivatization.
Asunto(s)
Cromatografía por Intercambio Iónico/métodos , Oligonucleótidos/análisis , Tionucleótidos/análisis , Humanos , Oligonucleótidos/sangre , Oligonucleótidos/orina , Espectrofotometría Ultravioleta , Temperatura , Tionucleótidos/sangre , Tionucleótidos/orinaRESUMEN
Solid-phase derivatization reagents containing a 3,5-dinitrophenyl moiety for the derivatization of amines are described. The reagents are useful for Pirkle-type recognition of stereochemical composition of amines and related nucleophiles. The ability to stabilize 3,5-dinitrophenylisocyanate by covalent immobilization on a polymeric support varied greatly with the support chosen. The kinetics of the activated dinitrophenylcarbamate and the use of this reagent for enhanced recognition of optical purity is shown and compared to previous findings with a similar polymeric activated ester.
Asunto(s)
Dinitrobencenos/química , Isocianatos/química , Fenetilaminas/química , Alcoholes/química , Amino Alcoholes/química , Cromatografía Liquida , Cinética , Fenetilaminas/análisis , Polímeros , Espectrofotometría Ultravioleta , EstereoisomerismoRESUMEN
The concentration of amphetamine was determined in urine using solid-phase extraction, polymeric-reagent derivatization, and reversed-phase high-performance liquid chromatography with ultraviolet detection. To remove a majority of acidic and neutral compounds in urine, a solid-phase extraction was first performed on a sample spiked with the internal standard, 1-methyl-3-phenyl-propylamine. Because amphetamine has a relatively low molar absorptivity, the base was derivatized with a polymeric 1-hydroxybenzotriazole reagent containing a 3,5-dinitrobenzoate active ester. The limit of detection is 14 ng/ml, and the limit of quantification is 47 ng/ml. The calibration curve is linear from 0.01 to 4.0 micrograms/ml. The pooled relative standard deviation is +/- 5.5% for eight urine samples measured in duplicate. The average relative error (bias) is +2.2% when compared to gas chromatography-mass spectrometry.
Asunto(s)
Anfetamina/orina , Cromatografía Líquida de Alta Presión/métodos , Artefactos , Humanos , Indicadores y Reactivos , Polímeros , Reproducibilidad de los Resultados , Espectrofotometría UltravioletaRESUMEN
Microporous and macroporous polystyrene beads are modified to contain various activated centers to which are attached a detector-sensitive label useful for analytical-scale derivatization of nucleophilic species. The effect of surface area, porosity, and percent cross-linkage of the polymeric support containing an o-nitro activated ester is investigated. The polymers are characterized by a loading determination to calculate the amount of active acylating reagent incorporated per gram of dry polymer. The kinetics of reaction with substrates of varying steric hindrance vs. solvent strength are determined with the optimized polymeric support and a second polymeric reagent is synthesized based on that support. The second polymer contains a hydroxybenzotriazole functionality. This polymer has increased electrophilicity and is used to prepare a much stronger acylating reagent than the polymeric o-nitrobenzophenol when labeled with 3,5-dinitrobenzoyl chloride. The polymeric hydroxybenzotriazole (HoBTA-DNB) is able to derivatize much weaker nucleophiles such as alcohols, thiols, and phenols at room temperature in less than 30 seconds.
Asunto(s)
Cromatografía Líquida de Alta Presión/instrumentación , Polímeros , Alcoholes/química , Aminas/química , Reactivos de Enlaces Cruzados , Indicadores y Reactivos , Cinética , Fenoles/química , Espectrofotometría Ultravioleta , Compuestos de Sulfhidrilo/químicaRESUMEN
A solid-phase reaction technique is described for improved derivatization of aliphatic amines, amino alcohols and amino acids. A polymeric activated ester is used for the immobilization of the 3,5-dinitrobenzoyl group, which can then be used for derivatizations of strong or weak nucleophiles, while avoiding solution-phase derivatization conditions. The reagent is easily prepared and can be regenerated after use to attain its original reactivity. The resulting chromatograms are free of system peaks due to excess derivatizing reagent, and sample handling is kept to a minimum. The reagent can be used in conjunction with both reversed- and normal-phase chromatography and can be used for off-line gas chromatographic or high-performance liquid chromatographic (HPLC) derivatizations. In addition, the reagent can be used on-line for derivatization in HPLC. Since the labelling reagent is a strong pi-acid, chiral substrates can be derivatized and separated on a Pirkle-type pi-donor column. The confirmation and quantitation of amphetamine in urine was accomplished using a polymer containing two labelling moieties, p-nitrobenzoyl and 3,5-dinitrobenzoyl. The derivatization and separation of chiral and achiral amines, amino alcohols and amino acids is described.