RESUMEN

Oligonucleotides have emerged as important therapeutic options for inherited diseases. In recent years, RNA therapeutics, especially mRNA, have been pushed to the market. Analytical methods for these molecules have been published extensively in the last few years. Notably, mass spectrometry has proven as a state-of-the-art quality control method. For RNA based therapeutics, numerous methods are available, while DNA therapeutics lack of suitable MS-based methods when it comes to molecules exceeding approximately 60 nucleotides. We present a method which combines the use of common restriction enzymes and short enzyme-directing oligonucleotides to generate DNA digestion products with the advantages of high-resolution tandem mass spectrometry. The instrumentation includes ion pair reverse phase chromatography coupled to a time-of-flight mass spectrometer with a collision induced dissociation (CID) for sequence analysis. Utilizing this approach, we increased the sequence coverage from 23.3% for a direct CID-MS/MS experiment of a 100 nucleotide DNA molecule to 100% sequence coverage using the restriction enzyme mediated approach presented in this work. This approach is suitable for research and development and quality control purposes in a regulated environment, which makes it a versatile tool for drug development.

Asunto(s)

Enzimas de Restricción del ADN , ADN , Oligonucleótidos , Espectrometría de Masas en Tándem , Espectrometría de Masas en Tándem/métodos , ADN/química , ADN/genética , Enzimas de Restricción del ADN/metabolismo , Oligonucleótidos/química , Nucleótidos/análisis , Nucleótidos/química , Cromatografía de Fase Inversa/métodos , Control de Calidad , Análisis de Secuencia de ADN/métodos

RESUMEN

A novel fiber optic spectrophotometric method for nitrite determination in different samples is suggested, based on the reaction of nitrite with Safranin O in acidic medium to form a diazo-safranin, which is subsequently coupled with pyrogallol in alkaline medium to form a highly stable, red azo dye, followed by cloud point extraction (CPE) using a mixed micelle of a nonionic surfactant, Triton X-114, with an anionic surfactant, sodium dodecyl sulphate (SDS). The reaction and extraction conditions (e.g., acidity for diazotization and alkalinity for pyrogallol coupling, and other reagent concentrations, time, and tolerance to other ions) were optimized. Linearity was obeyed in a concentration range up to 230 µg L(-1), and the detection limit of the method is 0.5 µg L(-1) of nitrite ion. The molar absorptivity for nitrite of the Safranin-diazonium salt (ɛ(610 nm) =4 × 10(3) L mol(-1)cm(-1)) existing in literature was greatly enhanced by pyrogallol coupling and CPE enrichment (ɛ(592 nm)=1.39 × 10(5) L mol(-1)cm(-1)). The method was applied to the determination of nitrite in tap water, lake water and milk samples with an optimal preconcentration factor of 20.