Quantitative determination of alkylresorcinols in cereal grains: independence of the length of the aliphatic side chain

Two general spectrophotometric methods were used to analyze alkylresorcinols and determine their total amounts in rye, wheat and cereal products. Alkylresorcinols were extracted from rye bran with acetone, hydrogenated to obtain a mixture of saturated homologues, subjected to preparative high-performance liquid chromatography (HPLC) to obtain individual species with the side chain ranging from 15 to 25 carbon atoms, and finally quantified by colorimetric reaction with the diazonium salt Fast Blue B (2-methoxy-4-nitoraniline) and by HPLC. The intensity of the reaction between the same weight amounts of homologues with Fast Blue B decreased in the order C15:0 > C17:0 > C19:0 > C21:0 > C23:0 > C25:0. It was demonstrated that calibration curves based on molecular concentration instead of weight amount of the particular homologues were characterized by the same regression equation and showed an acceptable linearity in the range of 5-30 nmol of homologue in a sample. This confirmed that the colorimetric reaction of alkylresorcinols with Fast Blue B is independent of the length of the aliphatic side chain. Total alkylresorcinols were accordingly determined in cereal grain materials. Two extraction systems (acetone and ethyl acetate) were used. The results of the colorimetric method and those calculated from calibration curves based on the weight amounts of the homologues C15:0 and C19:0 were different. In contrast, the results calculated from the calibration curves based on the mole amounts of the particular homologues were very similar or even identical. The color and intensity of the reaction between alkylresorcinols and the diazonium salt Fast Blue B was independent of the length of the alkyl side chain. This feature could be helpful for the differential determination of the phenolic and resorcinolic lipids present in biological samples. Most cereal material and products contain a mixture of various homologues that is characteristic not only of the source (rye, wheat, triticate, barley), but also of the variety and the growth conditions. Therefore, for accurate calibration curves independent of the composition of homologues, they should be prepared on the basis of the mole fraction of the alkylresorcinols present, similar to the quantitation of phospholipids via phosphorus determination. This procedure will also allow the direct comparison of their amounts in different sources of alkylresorcinols. Nevertheless, simultaneous determination of the homologue composition in the samples is desirable (e.g., by reverse-phase high-performance liquid chromatography) as different homologues may have various impacts on the biological activity of alkylresorcinols from cereal products.