Abstract
This paper describes a quick determination method of benzo(a)pyrene in yeast. This method consists of the following procedures; i) solvent extraction of aromatic compounds in yeast, ii) extraction of polynuclear hydrocarbons by liquid-liquid partitions, iii) isolation of benzo(a) pyrene by one-dimensional dual band thin layer chromatography, and iv) spectrofluo-rometrical determination of benzo(a)pyrene.
Aromatic compounds in yeast were extracted by benzene with the help of Soxhlet's extractor. The benzene extract was dried under reduced pressure at about 40°C, and the dried extract was dissolved in cyclohexane.
Polynuclear hydrocarbons in the cyclohexane solution were extracted selectively by a series of liquid-liquid partition of cyclohexane-dimethyl sulfoxide(DMSO), (DMSO + 20 vol% HCl; 1: 1, v/v)-cyclohexane, cyclohexane-70% H2SO4, cyclohexane-water, cyclohexane-5% NaOH, and cyclohexane-water. The cyclohexane solution thus obtained was evaporated to dryness under reduced pressure at low temperature (ca. 40°C). Polynuclear hydrocarbons in the residue were dissolved in ethyl ether and filtered. The filtrate was evaporated to small volume at low temperature (ca. 30°C) by rotary evaporator. The net amount of polynuclear hydrocarbons in the concentrated solution was applied repeatedly with intermittent drying onto the aluminum oxide G layer of thin-layer plate which was composed of aluminum oxide G layer and 26% acetylated cellulose layer. The plate was then inserted into a developing chamber with freshly prepared ethyl alcohol-ethyl ether-water (4: 4: 1, v/v). Development was continued until the front of the developer reached 10 cm from the layer boundary. After the development, the plate was withdrawn from the developing chamber and dried in a dark room. The plate was again developed to the 10 cm mark with methyl alcohol-ethyl ether-water (4: 4: 1, v/v). Benzo(a)pyrene thus separated was detected on ace-tylated cellulose layer as a narrow fluorescent spot under ultraviolet ray (365 nm). The benzo(a)pyrene spot was scraped off into a small centrifuge-tube, and benzo(a)pyrene in it was extracted with benzene by means of centrifugation. Benzo(a)pyrene in the benzene solution was analysed by spectrofluorometry.
We applied this analytical method to the quanti-tative determination of benzo(a)pyrene in seven kinds of yeasts and found the presence of benzo(a)pyrene in all yeasts tested. The contents of benzo(a)pyrene were very small and ranged from 0.32 ppb to 1.19 ppb.
