Simultaneous Determination of Key Osmoregulants in Halophytes Using HPLC-ELSD

Osmoregulants are the substances produced by plants that assist in tolerating environmental stresses. Three commonly analysed osomoregulants include mannitol, betaine and proline. A simple, sensitive and rapid HPLC-ELSD method has been developed for the simultaneous analysis of these common osmoregulants in plant extracts. Osmoregulants were extracted using 80 % ethanol and separated on an NH2 column using 0.1 % formic acid and acetonitrile as the mobile phase. Retention time repeatability was 0.85, 1.50, and 0.93 % for mannitol, betaine and proline, respectively. The limit of detection (mu mol) was 1.43 x 10(-4), 7.81 x 10(-5) and 1.08 x 10(-4) for mannitol, betaine and proline, respectively. The developed method was applied to three different plant extracts, Stylosanthes guianensis, Atriplex cinerea and Rhagodia baccata. A second method using a C18 column with 0.1 % heptafluorobutyric acid and acetonitrile as the mobile phase proved to be a useful complementary method for verifying tentative peak identifications.