51 
test. In the other procedure the samples were extracted vdth ether and the 
dried extract vias dissolved in acetone and treated as in the first method. 
Colors developed from extracts by the second method were more readily matched 
than those obtained from the first method. 
Ambrose and Eaag (j[) in 1936 shortened this test for use in detecting 
rotenone and deguelin in the excreta of animals fed rotenone and derris. The 
material was extracted with ether and the evaporated extract taken up in acetone. 
To this solution was added one-third its volume of freshly prepared 10-percent 
alcoholic potassi\ain hydroxide, and the resulting mixture warmed. A vane-red 
color developed to a maximum in about 15 minutes. The test was said to be 
sensitive to 1 part of rotenone in 35,000. 
Goodhue (4g) in 1936 described a more serviceable modification of the 
Gross and Smith test, which has Jbeen widely used in subsequent work. Sulfuric 
acid was substituted for nitric, the concentration of the alcoholic potassium 
hydroxide was reduced, and the nitrite necessary to produce the color v/as added 
in the alkali instead of in the acid. The method v;as as follows: 
Prepare the following reagents: (a) Kix 1 voliime of sulfuric 
acid (sp. gr. 1.84, free from nitrous acid) with 3 volumes of water, 
(b) Dissolve 1 gram of sodium nitrite in 10 cc. of water and dilute to 1 
liter with 95-percent alcohol, (c) Dissolve 40 grams of potassium 
hydroxide in 100 cc. of water, (d) Mix 1 folnme of -reagent (c) and 7 
volumes of reagent (b) . Prepare this solution fresh daily. 
Proceed as follows; Prepare an acetone extract of the sample containing 
from 0.005 to 0.25 mg. of rotenone per cubic centimeter and pipette 2 
cc. into a dry test tube. Add 2 cc of reagent (d) and place the 
tube in a water bath at about 25° C. for 5 minutes. Add 5 cc. of 
reagent (a), stojoer, shake, and place the tube back in the water 
bath. The color reaches a maximum after aoout 15 minutes, and then 
remains unchanged for 2 hours. Determine the amount of rotenone by 
comparing the color with standaiids prepared at the same time from 
known quantities of rotenone. 
The turbidity or brown color which sometimes developed during the analysis of 
crude plant extracts was removed upon extraction of the final mixture v/ith a 
small portion of ether. The red color due to rotenone and deguelin was not 
removed. In this work comparisons were made v/ith Lovibond color slides in a 
roulette comparator. The small amount of blue which accompanied the red was 
filtered out by a dichromate filter for easier matching. By this modified 
procedure the sensitivity of the original test was increased 20 times. The 
specificity for rotenone and deguelin remained the same. Deguelin was said to 
give the same amount of color as did rotenone. 
This method was criticized by Gouds'-vaard and Timmers (42) in 1937 on the 
same grounds as the Jones and Smith (75) test-namely, dependence of color in- 
tensity on the temperature and interference of various tints with the estimation 
