In this series of posts we are going to exam­ine this seemly sim­ple question:

1. What is the goal and the pur­pose of test­ing of unknowns gen­er­ally? How do we best design a test for marijuana?
2. How is most mar­i­juana test­ing con­ducted in the United States?
3. What is micro­scopic mor­pho­log­i­cal exam­i­na­tion? Is it a “good” test?
4. What is the mod­i­fied Duquenois-Levine test? Is it a “good” test?
5. What is Thin Layer Chro­matog­ra­phy? Is it a “good” test?
6. Is the com­bi­na­tion of all three tests cre­ate a “good” test­ing scheme?
7. Is there a bet­ter way to test for marijuana?

Part 4: What is the mod­i­fied Duquenois-Levine test? Is it a “good” test?

Mod­i­fied Duquenois-Levine test­ing

What is it?

It is tech­ni­cally referred to as a col­ori­met­ric test. In short, it is a color test. A totally dif­fer­ent selec­tion of the unknown is sam­pled and sub­jected to this test. A reagent is added to the unknown. The reagent is made up of a com­bi­na­tion of vanillin, acetalde­hyde, and ethanol in a spe­cific ratio of these com­po­nent materials.

Mechan­i­cally how is it preformed?

This totally sep­a­rate sam­ple from the unknown is placed into typ­i­cally a test tube. A cer­tain amount of the Duquenois reagent is added (typ­i­cally about 10 drops). The tube is closed. The tube is agi­tated (shaken) for an unspec­i­fied period. The tube is reopened. Con­cen­trated hydrochlo­ric acid is then added (usu­ally about 20 drops). The tube is closed. It is agi­tated (shaken) again. Any color change is then noted. The tube is reopened. Chlo­ro­form is added. The tube is closed. It is again agi­tated (shaken) or vor­texed (mixed). The ana­lyst is look­ing for a color change (thought to be vio­let or pur­ple) and a sep­a­ra­tion into two layers.

This is the end result of the mod­i­fied Duquenois-Levine test. This pic­ture is after the HCl and the chlo­ro­form are added. There is a deep pur­ple color at the top and then a pink color at the bottom.

How is the typ­i­cal crime lab­o­ra­tory ana­lyst trained to con­duct this test?

They are trained in the process and the steps in the mechan­i­cal process. No crime lab­o­ra­tory ana­lyst is ever instructed by a doc­toral level ana­lyt­i­cal chemist as to why this process results in any sort of color change or the way the phys­i­cal sep­a­ra­tion occurs in the col­ors with the addi­tion of chlo­ro­form or why it these changes hap­pen at all.

In essence, it is a sub­jec­tive test as it is based upon per­cep­tion of color by the ana­lyst and the per­cep­tion of this devel­op­ing of a sep­a­rate layer once chlo­ro­form is added.

It is a sec­ond sam­ple that is tested. Prior to the test­ing by mod­i­fied Duquenois-Levine tech­nique, there is no micro­scopic mor­pho­log­i­cal exam­i­na­tion con­ducted on this sam­ple. There is no TLC analy­sis on this sample.

It is tested one time and then dis­carded. No other test­ing is per­formed on this sample.

The ana­lysts are not aca­d­e­m­i­cally trained in the the­ory of the reagent use, and are not taught about cross-reactivity and false pos­i­tives or other sources of errors.

Is this a ver­i­fi­able test?

Poten­tially, yes. Dig­i­tal cam­eras exist. Pho­tographs can be taken just like the one above. Heck, even video can be taken to show how it is con­ducted on a par­tic­u­lar sam­ple from the unknown. How­ever, the mod­ern prac­tice is to not take pho­tos. They video­tape noth­ing.  The lab­o­ra­tory pro­duces noth­ing ver­i­fi­able in court that the test was even con­ducted or that the analyst’s per­cep­tion of the change in color did hap­pen and was cor­rect or that there was this sep­a­ra­tion once the chlo­ro­form is added.

Again, there are no crime lab­o­ra­to­ries that I am aware of that use the ACE-V (Analy­sis, Com­par­i­son, Eval­u­a­tion, and Ver­i­fi­ca­tion) tech­nique that one would find in fin­ger­print iden­ti­fi­ca­tion with a dou­ble check in real time by a fel­low bench ana­lyst. In essence, it is checked one time, by one per­son with no dou­ble check by another, and noth­ing pro­duced that proves that the analy­sis was done or that the fea­tures that are reported as present were in fact objec­tively present.

Is there empir­i­cal valid­ity stud­ies that prove that this is a spe­cific and con­fir­ma­tory test?

Nope. In fact the empir­i­cal stud­ies clearly show the oppo­site. It is not spe­cific for THC. The reac­tion is not unique to THC. In fact, the stud­ies clearly show that it is not even mean­ing­fully selec­tive for THC. Chi­nese moth­er­wort if tested, by this method will turn vio­let. But that is not all. There are a great many plants that yield sim­i­lar color results when Duquenois-Levine test­ing is applied. Yet ana­lysts are taught that is color change is diag­nos­tic of THC. Naka­mura him­self pub­lished and acknowl­edges that M. J. de Faubert Maun­der listed 25 species of plants which exhib­ited vio­let to pur­ple col­ors in the Duqenois test, and were extractable in chlo­ro­form, which is the dis­tin­guish­ing fea­tures of the Duquenois Levine test. (502) Naka­mura tested 23 of those species noted by M. J. de Faubert Maun­der. Using the Duquenois-Leine test, he found that the vio­let to pur­ple color when test­ing the leaves of the fol­low­ing (502):

• cof­fee
• a species of gum copal called Caplafer conjugata
• gum Kawri
• wood sage
• Thuja occi­den­talis
• San­darac

He pub­lished that he tested and found vio­let to pur­ple color reac­tions using the Duquenois-Levine test in other plants as well, and not just the leafy por­tions of these plants. (502). That list includes:

• cala­mus
• cul­ver root
• gin­ger
• gum animi
• gum copal
• gum myrrh
• henna
• let­tuce opium
• san­dal wood
• tolu
• wood betony
• liquorice
• nut­meg
• poi­son flag

The United Nations study on mar­i­juana con­cludes that Duquenois-Levine test on sub­stances com­pletely unre­lated to mar­i­juana will give false pos­i­tives too. This includes Arthemisia Gran­cunulus, Euca­lyp­tus Glab­u­lus Labill and sev­eral other herbs that are very com­mon such as rose­mary, thyme, and sage.

None of these items above con­tain THC. Yet, they fea­ture these color changes and some even have the sep­a­ra­tion when chlo­ro­form is added that the ana­lysts are trained to look for when apply­ing these reagents.

While we have exam­ined the pro­pri­ety and suit­abil­ity of this mod­i­fied Duquenois-Levine test­ing in the iden­ti­fi­ca­tion of THC, we can­not judge this Duquenois-Levine test in iso­la­tion. We must remem­ber and in fair­ness this is a 3 test process and tech­nique. What com­pounds the issue of the pos­si­bil­ity of error is that as the ana­lyst is not using the same sam­ple through­out each of these 3 tests. It is in fact, three dif­fer­ent sam­ples are taken of the unknown and each test gets its own sam­ple with no sam­ple receiv­ing the ben­e­fit of all three tests. The ana­lyst pre­sumes that the sam­pling and the sam­ple selec­tion of the unknown is homo­ge­neous mean­ing that each sam­ple will be exactly iden­ti­cal. This is not a jus­ti­fied sci­en­tific assump­tion (An assump­tion is not drawn from evi­dence; it is a hypoth­e­sis {my assump­tion can be tested by look­ing at the dic­tio­nary}. A pre­sump­tion implies a basis in evi­dence {the legal pre­sump­tion of inno­cence}) Just because the sought after fea­tures of the micro­scopic mor­pho­log­i­cal exam­i­na­tion were present in the first sam­ple, that doesn’t nec­es­sar­ily mean that it will be present in the next sam­ple that the ana­lyst now sub­jects to mod­i­fied Duquenois-Levine testing.

Naka­mura tested and found that mace and nut­meg, which yielded col­ors sim­i­lar to that obtained with mar­i­juana with the Duquenois-Levine test, could be “cred­i­bly con­fused” with mar­i­juana based on micro­scopic appear­ance. (502)