An improved method for determining the composition of FFA in red tide flagellates by RP‐HPLC with fluorescence detection

Abstract

An improved method for determining the composition of FFA in red tide flagellates by HPLC with fluorescence detection is described. For this purpose, total lipids from Heterosigma akashiwo, Chattonella antiqua, and C. marina were allowed to react with 9-anthryldiazomethane, then the resulting 9-anthrylmethyl esters of FFA were analyzed without any purification by RP-HPLC on a highly efficient C18 column (Superspher 100 RP-18e, 25 cm × 4 mm i.d., 4 µm particle size; Merck, Darmstadt, Germany). Clear separations of long-chain saturated and unsaturated FFA, including 14:0 and 16:1, which were major components in the flagellates and were unresolved on a previously used C18 column, were achieved by a stepwise gradient elution using acetonitrile, water, and propan-2-ol. Two characteristic FA, 18:5n-3 and trans-16:1n-13, whose behaviors on RP-HPLC had not been reported previously, were also clearly separated from the other FFA. The FFA compositions of the flagellates determined by HPLC were in good agreement with those obtained by GLC. The present method is simple and sensitive, and would be widely applicable for compositional analysis of microalgal FFA.FFA produced by marine microalgae causing red tides, such as the raphidophyte flagellates Heterosigma akashiwo, Chattonella antiqua, and C. marina, have been implicated in the causative substances that damage the epithelial tissues of fish gills (1,2). Recently, we found that the FFA in C. marina are released mainly from monogalactosylglycerols (MGDG) by galactolipase in the alga, where the FFA composition was determined by open-tubular GLC of the methyl esters (3). Although open-tubular GLC has been widely used for determining the composition of FFA in biological samples, it is usually necessary to isolate FFA from crude lipids before methyl esterification or to purify the methyl esters before GLC analysis, which is time-consuming and laborious. In addition, GLC has a limited sensitivity, which usually requires several hundred micrograms of FFA for the analysis.9-Anthryldiazomethane (ADAM) reacts highly specifically with FFA in crude lipids without preliminary sample purification, catalysts, or heating to give 9-anthrylmethyl ester derivatives, which are highly sensitive and clearly resolved on RP-HPLC with fluorescence detection (4,5). Recently, Suzuki and Matsuyama (6) reported the FFA compositions of several species of the red tide flagellates, including H. akashiwo and C. antiqua. The FFA were extracted with n-hexane from crude lipid extracts dissolved in 80% methanol, then derivatized with ADAM and separated on RP-HPLC on a C18 column (Develosil ODS-5, 25 cm × 4.6 mm i.d., 5 µm particle size; Nomura Chemical, Seto, Japan). Although good separations were achieved for 12 saturated and unsaturated FFA, no resolution of myristic (14:0) and hexadecenoic (16:1) acids, which were major components in the flagellates, was obtained under the conditions employed. Thus, the overlapped components were collected by preparative HPLC and then rechromatographed by RP-HPLC on a cyan...

Glycerolipid Acyl Hydrolase Activity in the Brown AlgaCladosiphon okamuranusTOKIDA

“…The released FFA was determined by highperformance liquid chromatography with ‰uores-cence detection after conversion to the 9-anthrylmethyl ester. 6,7) The value was corrected by subtracting the small quantity of FFA released from the crude enzyme preparation.…”

Influence of Hot Spring Water on Fatty Acid Composition of Skin Surface Lipids in Hairless Mouse Model of Atopic Dermatitis

“…18) The logarithmic values of RTs of FA homologues, such as saturated, monounsaturated, and branched-chain FAs, exhibit a linear relationship with the carbon number. 19) Based on these relationships and MS findings, peaks 10-18 were identified to be 19 : 0, 22 : 0b, 22 : 0, Table 1 also shows the FA composition of skin surface lipids of NCN24 mice, exhibiting a pattern similar to that in experimental mice previously reported. 20) Branched-chain FAs, not contained in serum (data not shown), accounted for approximately 20% of the total FA content and FAs with a carbon number of ≥20 accounted for approximately 50% of total FA content.…”

Validation of Thermally Assisted Hydrolysis and Methylation–Gas Chromatography Using a Vertical Microfurnace Pyrolyzer for the Compositional Analysis of Fatty Acid Components in Microalgae

“…Generally, fatty acid components in microalgae have been determined by gas chromatography (GC) with preliminary sample treatments such as solvent extraction, saponification, and/or derivatization. − A high-performance liquid chromatography (HPLC) system with a fluorescence detector was also used for compositional analysis of fatty acid components extracted from microalgae . However, these techniques require tedious and time-consuming sample pretreatments prior to the final analytical measurements.…”

“…9−11 A high-performance liquid chromatography (HPLC) system with a fluorescence detector was also used for compositional analysis of fatty acid components extracted from microalgae. 12 However, these techniques require tedious and time-consuming sample pretreatments prior to the final analytical measurements.…”