De novo sequencing of highly modified therapeutic oligonucleotides by hydrophobic tag sequencing coupled with LC-MS.

Correct sequences are prerequisite for quality control of therapeutic oligonucleotides. However, there is no definitive method available for determining sequences of highly modified therapeutic RNAs, and thereby, most of the oligonucleotides have been used clinically without direct sequence determination. In this study, we developed a novel sequencing method called 'hydrophobic tag sequencing'. Highly modified oligonucleotides are sequenced by partially digesting oligonucleotides conjugated with a 5'-hydrophobic tag, followed by liquid chromatography-mass spectrometry analysis. 5'-Hydrophobic tag-printed fragments (5'-tag degradates) can be separated in order of their molecular masses from tag-free oligonucleotides by reversed-phase liquid chromatography. As models for the sequencing, the anti-VEGF aptamer (Macugen) and the highly modified 38-mer RNA sequences were analyzed under blind conditions. Most nucleotides were identified from the molecular weight of hydrophobic 5'-tag degradates calculated from monoisotopic mass in simple full mass data. When monoisotopic mass could not be assigned, the nucleotide was estimated using the molecular weight of the most abundant mass. The sequences of Macugen and 38-mer RNA perfectly matched the theoretical sequences. The hydrophobic tag sequencing worked well to obtain simple full mass data, resulting in accurate and clear sequencing. The present study provides for the first time a de novo sequencing technology for highly modified RNAs and contributes to quality control of therapeutic oligonucleotides. Copyright © 2016 John Wiley & Sons, Ltd.

Natural occurrence of aflatoxins and zearalenone in maize in Brazil. Part II.

The levels of aflatoxins and zearalenone were determined in 328 samples of corn from the States of Santa Catarina, Minas Gerais, São Paulo, Paraná, Rio Grande do Sul and Espírito Santo by thin-layer chromatography; the samples were obtained from April 1985 through to March 1986. In 12.3% of these samples aflatoxin B1 was detected in concentrations that varied from 10 to 900 micrograms/kg (ppb); 18 samples showed levels above those tolerated by Brazilian legislation. Zearalenone was found in 4.5% of the samples analysed in concentrations that varied from 653 to 9830 micrograms/kg (ppb). The limit of detection of the method for the determination of zearalenone was 260 micrograms/kg (ppb) and the recovery was 100%.