Abstract
A gradient elution high performance liquid chromatography method with detection at 230 nm for the analysis of perbenzoylated cerebrosides containing hydroxy and nonhydroxy fatty acids is described. The quantitative range of the method is 0.5-10 nmol of cerebrosides. The limit of detection for injected sample is about 10 picomol. The analysis time by liquid chromatography is less than 5 minutes and prior purification of the cerebrosides from other lipids in brain lipid extracts is not necessary. The cerebrosides are first perbenzoylated with 50 micronl of 10% benzoyl chloride in pyridine and then separated on a chromatographic column of Zipax. The gradient elution is with 2.8-5.5% of dioxane in hexane. This gradient elution micromethod is at least 10 times more sensitive than isocratic elution methods with detection at 280 nm. The method is applicable to other biological materials containing minute amounts of cerebrosides if the glycolipid fraction is first isolated from the lipid extracts. A further fourfold increase in the sensitivity is achieved by replacing air in the reference cell of the detector by gradient elution solvent.
Highlights
Supplementary key words: perbenzoylated cerebrosides . hydroxy and nonhydroxy fatty acids . lipid extracts of brain, plasma and tissue cultured cells
The analysis of benzoylated cerebrosides by high performance liquid chromatography with isocratic elution and detection at 280 nm has been described in a previous publication [1]
The isocratic elution system employed previously [1] for the separation of the benzoylated NFA- and HFAcontaining cerebrosides is not adequate for the analysis of subnanomol quantities of cerebrosides in tissue extracts because the benzoylated NFA cerebrosides are not completely separated from the solvent peak or from other products formed from the benzoylation of lipid extracts that elute with the solvent peak
Summary
Beef brain mixed cerebrosides and individual HFA and NFA cerebrosides were purchased from Supelco, Bellefonte, PA. HFA cerebrosides were purified from the mixed beef brain cerebrosides by benzoylation, HPLC, and debenzoylation as described previously [1]. The extracts were dried and redissolved in 1 ml of chloroform and chromatographed on a small column of silicic acid (50 mg). The vials were soaked in chromic acid and thoroughly washed with distilled water, methanol, and with chloroform-methanol 2: 1 (v/v) in order to remove possible trace contaminants. (Westwood, NJ) Model SF 770 variable-wavelength spectromonitor or with a Laboratory Data Control (LDC, Riviera Beach, FL) Model 1285 ultraviolet (280 nm) monitor, or with a LDC spectromonitor-I. These monitors have 8-p1 flow through cells. T h e peak areas were measured either with an Autolab Minigrator (Spectra Physics, Santa Clara, CA) or by the cut and weigh method
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