50 
"Trsmsfer 5 cc. of the material to a large test tube, add 
2 cc. of concentrated ritric acid and shake for 30 to 30 seconds, 
. dilute immediately with 20 cc. of v.'ater. Close the tube and 
shake to disperse the oil in the aqueous solution; allow the oil 
to -separate, which should not require more than 30 to 40 seconds, 
and add 1 or 2 cc- of amnonf.a poured dov.Ti the side of- the tube. 
The presence of rotenone or derris extract is indicated by the 
formation of a fugitive blue color." 
Quantities of rotenone as small as 2.5 mg. in 5 cc. of a mineral oil-pyre thrum 
extract were said to have been detected oy this method. 
Guillaume and Herve {^) in 1939 described a modification of the Durham 
test which rendered the color slightly more peruanent r.nd permitted a ro-ughly 
quantitative estim?tion. Tnis schame involved r.dding only a few drops of nitric 
acid to the acetone solution of rot»='none or exoract, covering with a layer of 
toluene and allowing to str.nd in vacuo at -8° tc-J.O<^ for 1 ■. hour. The blue 
color then obtained by addin-^ a fe'-f drops of £-?jaonia persisted unchanged for 
about 1 minute. Coloro produced s-r-multaneously from the s-ample and from stand- 
ard soliitions of rotenone v;ere compared rrpicly in tert tub^s.' In this form 
the method was said to be sufficiently precise end sensitive for many purposes. 
The G-ross and Smith Test 
A more useful color rea-:;tion than that of Durhr.m from the quantitative 
standpoint was discovered in 1934 by G-ross and Smith (50), It involved treat- 
ment of an acetone extract of the sa.nple with alcoholic alkali followed by 
nitric acid containing nitrite. A rather permanent red color was given by 
both rotenone and degaelin, and also by din^dro rotenone. Briefly the proce*- 
(iire was as follows; 
To 2 cc. of an acetone solution or extract containing 0.05 
to 0.30 mg, of rotenone rjer cubic centimeter, add 2 cc. of 10* 
percent alcoholic potassi-um hydx-oxi-de solution, and allov; to 
stand at 20^ C. for 2 minutes. Then add 6 cc. of a nitric acid- 
sodium nitrite mixture containing 1 volume of concentrated nitric 
acid to 1 vol-ume of aqueous sodium nitrite having 0.25 gm. of 
sodium' nitrite por liter. Mix, cool to 20° C, and allow to stand 
at this temperature for 15 minutes. Comp,?re visually with standi 
ards containing pure rotenone prepared at the srjne time. 
When applied to samples containing only rotenone, the results agreed with those 
by the gravimetric methods. Applied to derris a:id cube samples, the results 
were 50 to 100 percent higher than for rotenone alone, owing to the presence of 
deguolin. The method was applied to rotenone spray residues on fruits and 
foliage. 
In using the Gross and Smith test for total rotenone and deguelin in 
several sajruples of derris root, Tattersfield and Martin (igO) employed two 
methods of extraction. In one method the saraples were extracted with acetone 
and aliquots of this extract were diluted to the proper concentration for the 
