HPLC/MSN Analysis of Retinoids

This protocol describes a highly sensitive and selective method to quantify retinoids using normal-phase HPLC with onlineAPCI MS
N
. The retinoids are key regulators of gene expression, retinol being oxidized via a retinaldehyde intermediate to retinoicacid (RA) which activates specific nuclear receptors, the signalling of which is turned off by oxidative inactivation of theligand to 4-oxo-RA and other metabolites. Many of these retinoids are present only transiently at low concentrations in tissuesand during analysis are labile to heat, light, and oxygen. HPLC with online APCI MS
N
provides a rapid technique to quantify these retinoids simultaneously. Techniques to extract the retinoids and prevent theirdegradation are described, with an emphasis on transcriptionally active RA. RA controls patterning of gene expression in theembryo, organizing embryonic morphology in the central nervous system. Similarly, a patterned distribution of RA controlsfunction of the adult CNS, a tissue particularly difficult to analyse for RA because of its high lipid content. To understandhow these patterns are organized in the brain and change over time, it is essential to determine the concentration of RA insmall areas of tissues, and techniques of exquisite sensitivity are indispensable.