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High-performance liquid chromatography–mass selective detection assay for adenine released from a synthetic RNA substrate by ricin A chain by Harry B Hines; Ernst E Brueggemann; Martha L Hale is a Biochemistry, Genetics and Molecular Biology article available to read on EtoBox.

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High-performance liquid chromatography (HPLC) and selected ion monitoring mass spectrometry (MS) were used to develop a quantitative assay for adenine released from a synthetic RNA substrate by ricin A chain, which contains the toxin's N-glycosidase activity. Because ricin and ricin A chain have potential applications as biotherapeutics and bioweapons, assays are needed to evaluate potency and potential inhibitors of activity. The detection limit for adenine was 0.02 M (2.4 ng/ml), and the standard curve was linear up to 27.3 M. The lower limit of quantitation was 0.27 M and was reproducible throughout this range. Reaction characterization showed that most adenine was released by 5 h and that the reaction could not be fully stopped with formic acid concentrations up to 0.75 mM (the maximum typically used for HPLC-MS). Injections were made at 2-min intervals, 10 injections could be performed before the column was backXushed, and no ricin A chain was observed in the column eZuent. This assay would also be useful for ricin since ricin A chain did not pass through the HPLC column. With minor modiWcations to this system, the assay should provide rapid, sensitive, selective, and quantita

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Author

Harry B Hines; Ernst E Brueggemann; Martha L Hale

Publisher

Elsevier Science; Elsevier ; Elsevier Inc.; Elsevier BV (ISSN 0003-2697)

Published

2004

Language

EN

Field

Biochemistry, Genetics and Molecular Biology (Life Sciences)