A fluorometric method is described for determining indole in shrimp. The indole is extracted with n-hexane, partitioned into a methanol- saturated sodium chloride solution ( 9 + 1 ), and determined fluoromctrically. The detection limit of the method is 0.04 μg indole/g shrimp. Quantitative analytical data are presented for indole in samples of shrimp determined by the fluorometric method and the AOAC official colorimetric and gas-liquid chromatographic procedures. The correlation coefficient between the data of the fluorometric and colorimetric methods was 0.96. Indole recovered from 25 g samples of fresh shrimp spiked with 6 and 12 μg indole/25 g ranged from 97 to 106%. The fluorescence response is linear in the range of 1 to 25 μg indole/100 ml methanol solution and no significant change is noted in solutions kept in the dark 18 hr.
Diethylstilbestrol (DES) and its monoglucuronide conjugate (DES-G) are extracted from homogenized beef liver by blending with acetonitrile containing triethylamine sulfate. An alkaline solution saturated with sodium chloride extracts DES and DES-G from the acetonitrile. The alkaline extract is acidified with sulfuric acid, triethylamine is added, and DES and DESG are extracted with 1-heptanol. DES is cleaved from DES-G by refluxing the 1-heptanol extract after adding ammonium chloride and monobasic ammonium phosphate. Solvent partitioning is used to isolate the DES, which is irradiated in a buffer solution with ultraviolet light to form the phenanthrenedione derivative. The dione is converted to the phenanthrenediol derivative in an acidified bisulfite solution. The diol derivative is isolated by solvent partitioning and determined fluorometrically in methanol. Thin layer chromatography is used for confirmation of DES. Recovery on 5 samples spiked with DES-G equivalent to 1, 2, and 4 ppb DES ranged from 82.5 to 104%.
Experimental extractions of diethylstilbestrol from animal feed, containing alfalfa meal and other rich sources of chlorophyll, indicated that a reaction may occur between the drug and a component of the vegetable material. Recovery data, obtained by a new 1.5 hour acid-acetone-chloroform extraction, indicate that an acid-base reaction occurs with the magnesium ion in chlorophyll. Phosphoric acid frees the diethylstilbestrol and permits complete recovery with organic solvents. The drug is separated from the vegetable extracts by elution with ethyl ether from a barium hydroxide column. The final determination is made by a modification of the AOAC irradiation method. Recoveries are comparable to those of the longer AOAC method.
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