It is not expected that the average reader will have either the expertise, resources, or inclination to perform this operation; it is provided for educational purposes only.

It should be noted that this method only detects the presence of thujone, with margin of error of 10ppm. Although there are much more accurate means now available to measure thujone content, the current regulations hinge on the margin of error for this particular method, hence, 10ppm is considered to be "thujone free".

9.129 Thujone (25)¹ Official First Action

    To 500 mL sample add 1 mL freshly distilled aniline and 1 mL H₃P0₄, and reflux 30 min on steam bath.   Distill two 100 mL portions; reject first and test second for thujone as follows:

    Add 0.5 g semicarbazide hydrochloride and 0.6g anhyd NaOAc (or 1.0 g crystd salt) and let mixt. stand overnight. Distil off alcohol at min. pressure.  Steam distil to remove essential oils and other volatile material; collect and reject first ca 15 mL distillate . Wash down condenser with little alcohol and with H20.  Cool sample, add 1 mL H₂S0₄ (1 + 1), and again steam distil, collecting 20 mL distillate in cylinder.  Pour distillate into small separator, and add 20 mL ether, using receiver as measure.  Shake and sep. ether soln. Add 10 mL 65% alcohol and let ether evap. spontaneously.   After all ether evaps, note odor of residue. Odor of thujone will be apparent if  > .2 mg is present in soln, provided it is not masked by presence of other odoriferous substances.  Make modified Legal test as follows:

    To soln obtained as above, add 1 mL 10% ZnS0₄ soln and 0.25 mL freshly prepd aq. Na nitroprusside soln (0.1 g/mL). Slowly, with const stirring, add 2 mL 5% NaOH soln. Let stand 1-2 min. Add 1.5 mL HOAc and mix. Ppt of raspberry red color (resembling alcohol ppt of red fruit juice) shows presence of thujone. Neg. test is shown by similar ppt having appearance similar to that of alcohol ppt from apple jelly or other light colored fruit.