Through­out this series of posts we have exam­ined the Thorton-Nakumura pro­to­col that is used through­out the United States for the pros­e­cu­tion of ille­gal pos­ses­sion mar­i­juana. A fair exam­i­na­tion of the ques­tion reveals that there is no valid­ity to the notion that the 3 test reg­i­men pro­duces a valid con­clu­sion that the unknown exam­ined in fact con­tains THC.

Here are those series of posts:

  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?

There is a bet­ter way: Mod­ern Instru­men­ta­tion

There is an instru­ment dri­ven way that is very spe­cific and pro­duces ver­i­fi­able data. This instru­ment dri­ven tech­nique has been val­i­dated and if use in the val­i­dated man­ner with a prop­erly trained oper­a­tor uti­liz­ing proper sam­ple col­lec­tion, proper sam­ple selec­tion, proper sam­ple prepa­ra­tion, per­fect instru­men­ta­tion, and legit­i­mate inter­pre­ta­tion of the data will arrive at a valid result. This mod­ern day instru­men­ta­tion is called the Gas Chro­matog­ra­phy with Mass Spec­trom­e­ter (GC-MS).

Gas Chromatography with Mass Spectrometer

Gas Chro­matog­ra­phy with Mass Spectrometer

When the lab­o­ra­tory ana­lyst is pro­fi­ciency tested on unknowns and then graded to see whether or not they can con­duct a proper analy­sis of an unknown, the ana­lyst uses GC-MS. When the lab­o­ra­tory wants to check that the known that they have pur­chased from a third party ven­dor that is used and needed in Thin Layer Chro­matog­ra­phy (TLC) to com­pare against the unknown, the lab­o­ra­tory requires that it be ver­i­fied by GC-MS.

So when the lab­o­ra­tory really wants to know or really needs to know whether or not some­thing con­tains delta 9 THC, it uses the most spe­cific device avail­able that pro­duces ver­i­fi­able data. The ver­i­fi­able data are the print­outs that result from the analy­sis. This is called a Total Ion Cur­rent (TIC) chro­matogram and the result­ing spec­trum that is com­pared against an adju­di­cated known that is pro­duced by the National Insti­tutes of Stan­dards and Tech­nol­ogy (NIST).

Here are other blog posts on the GC-MS process:

  1. Introduction-The dif­fer­ent con­fig­u­ra­tions and the Elec­tron Impact process
  2. What types of mass ana­lyz­ers are there?
  3. What type of detec­tors are there?
  4. What types of analy­sis can be done?
  5. How do you read the output?
  6. How do they come to a qual­i­ta­tive mea­sure using software?
  7. How do they quan­ti­tate the results?
  8. Do you need chro­matog­ra­phy if you are using Mass Spectrometry?
  9. Other top­ics of inter­est about GC-MS

In the analy­sis of  unknowns that are seized, the process of deriva­ti­za­tion can be used to volatilize the sam­ple for intro­duc­tion the the GC-MS. For exam­ple, the ana­lyst can use MTBSTFA (N-methyl-N-(tert-butyldimethylsilyl)-trifluoroacetamide) or BSTFA/TMCS N,O-bis (trimethylsi­lyl) trifluoroacetamide/ Trimethylchlorosi­lane or MSTFA: N-methyl-N-trimethylsilyltrifluoroacetamide to deriv­a­tive the unknown.

A pop­u­lar tech­nique includes:

A sam­ple prepa­ra­tion that includes 500 mg of dry and homog­e­nized herbal cannabis are extracted with 5 ml methanol : chlo­ro­form (9:1 v/v) by the fol­low­ing pro­ce­dure: 10 sec­onds on a vor­tex, 15 min. ultra­sonic bath includ­ing again vor­tex­ing after 5, 10 and 15 min­utes, then cen­trifu­ga­tion. The sam­ple then needs to go through decar­boxy­la­tion. A 200 μl of the above extract are trans­ferred into a deriva­ti­za­tion ves­sel. The sol­vent is evap­o­rated under nitro­gen gas to dry­ness. The sam­ple is decar­boxy­lated for 15 min­utes at 210°C. The residue is dis­solved in 200 μl methanol : chlo­ro­form (9:1 v/v). The prepa­ra­tion of the final solu­tion next involves tak­ing the above decar­boxy­la­tion solu­tion and dilut­ing it with methanol by a fac­tor of 100 (in two steps, each 100 μl + 900 μl) and is then used for the analysis.

Also, THC is very much amenable to Head­space Solid Phase Microex­trac­tion (HS-SPME). It is a non-derivatization-based tech­nique where there is a extrac­tion from the solid dose itself. It is a more direct mea­sure as one is not chem­i­cally chang­ing the sam­ple. Dif­fer­ent types of fil­ters and fibers can be used such as poly­di­methyl­silox­ane 100 μm.

GC based test­ing for THC has been dis­cussed in  the sci­en­tific lit­er­a­ture since 1971. In 1991 the UNDCP study dis­cussed both GC-based meth­ods and HPLC meth­ods to iden­tify THC.

Other for­eign gov­ern­ments require much more test­ing than we do here in the United States. For exam­ple, Canada requires four tests includ­ing two instrument-based analy­sis that pro­duce ver­i­fi­able infor­ma­tion such as a spectrum.

There is a bet­ter way, but it is not used. This cer­tainly can­not be fair and just. It is time that we as cit­i­zens demand proof in the court­room and end the myth of spe­cific iden­ti­fi­ca­tion of mar­i­juana in crim­i­nal courtrooms.

The way to close this series is by an apt obser­va­tion made by Dr. Fred­eric White­hurst, PhD JD who wrote:

Many a defense attor­ney has looked in amaze­ment at the court­room “iden­ti­fi­ca­tion” of Cannabis Sativa L. by a law enforce­ment offi­cer sport­ing a gilded pot metal badge and a high school diploma who has writ­ten a report that would turn a ninth grade Eng­lish teacher’s hair gray and deny that same offi­cer a high school diploma. Obvi­ously the award­ing of a Basic Law Enforce­ment Train­ing (BLET) diploma ele­vates such high school grad­u­ates to the doc­toral level in botany, enabling them to con­duct a visual leaf archi­tec­tural analy­sis and the court accepted alchemy of the Duquenois Levine test to reach a con­clu­sion that the green veg­etable mate­r­ial in that lit­tle baggy can be iden­ti­fied as mar­i­juana to the exclu­sion of all other plant material.

The mod­ern day pros­e­cu­tion for the unlaw­ful pos­ses­sion of mar­i­juana is based upon a three test régime involv­ing micro­scopic mor­pho­log­i­cal exam­i­na­tion, mod­i­fied Duquenois-Levine col­ori­met­ric test­ing, and Thin Layer Chro­matog­ra­phy. Each of these three tests are non-specific for THC which is the phar­ma­co­dy­nam­i­cally active ingre­di­ent which makes mar­i­juana ille­gal. The ques­tion becomes is this three test bat­tery col­lec­tively con­clu­sively spe­cific to arrive at a valid con­clu­sion that the unknown seized and tested is in fact mar­i­juana (con­tains THC) and there is no pos­si­bil­ity of a false positive?

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 6: Is the com­bi­na­tion of all three tests cre­ate a “good” test­ing scheme?

The gov­ern­ment would choose to argue that the com­bi­na­tion of these three tests results in a valid con­clu­sion that this tested unknown is mar­i­juana (con­tains THC). All  three roads leads, so they say, lead to THC. The more sophis­ti­cated ver­sion of this argu­ment is the Venn dia­gram. Per­haps some­thing like the below:

Venn diagram that the prosecution argues
Venn dia­gram that the pros­e­cu­tion argues

This is sim­ply not proven or sup­ported by the pub­lished empir­i­cal research. Even if it were true there is no evi­dence how wide or big that “D” area is. It could be very wide and large con­tain­ing many com­pounds such as this below exam­ple below where the result of this type of test­ing would be a pos­i­tive for O, T, H, P, M, A, B, X, K, Y and not for B alone:

What could be in there? We don't know.
What could it be? We don’t know.

There is no evi­dence that these dis­tinct tests ever intercept.

Maybe these three tests have nothing in common
Maybe these three tests have noth­ing in common

While in ear­lier posts, we have exam­ined the pro­pri­ety and suit­abil­ity of these three sep­a­rate and dis­tinct tests in the iden­ti­fi­ca­tion of THC, we can­not prop­erly judge them in iso­la­tion. We must remem­ber and in fair­ness this is a 3 test process and technique.

There are no mean­ing­ful or robust stud­ies pub­lished that truly val­i­dates this three test pro­ce­dure as result­ing in a spe­cific qual­i­ta­tive mea­sure for THC.

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 of the orig­i­nal unknown. The ana­lyst pre­sumes that the unknown is homoge­nous. Fur­ther assumed is that the sam­pling and the sam­ple selec­tion of the unknown con­ducted by the ana­lyst result in iden­ti­cal homo­ge­neous sam­ples. These are not jus­ti­fied sci­en­tific assump­tions (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})

Each of these tests are wholly destruc­tive in nature by their very process.

  • Just because the sought after fea­tures of the micro­scopic mor­pho­log­i­cal exam­i­na­tion were present in the first sam­ple doesn’t nec­es­sar­ily mean that that this sam­ple will con­tain the sought after fea­tures in the mod­i­fied Duquenois-Levine and the TLC exam­i­na­tion because they are not exam­ined for in this sample.
  • Just because the sought after fea­tures of the mod­i­fied Duquenois-Levine exam­i­na­tion were present in the sec­ond sam­ple doesn’t nec­es­sar­ily mean that the fea­tures sought after in the micro­scopic mor­pho­log­i­cal exam­i­na­tion are there (because it was not exam­ined) and the sought after fea­tured of the TLC exam­i­na­tion are there (because it was not exam­ined) in this sec­ond sample.
  • Just because the sought after fea­tures of the TLC exam­i­na­tion were present in the third sam­ple doesn’t nec­es­sar­ily mean that the fea­tures sought after in the micro­scopic mor­pho­log­i­cal exam­i­na­tion are there (because it was not exam­ined) and the sought after fea­tured of the mod­i­fied Duquenois-Levine exam­i­na­tion are there (because it was not exam­ined) in this third sample.

All pros­e­cu­tions for the unlaw­ful pos­ses­sion of mar­i­juana requires as an essen­tial ele­ment of the crime for the gov­ern­ment to advance proof that the unknown sub­mit­ted for test­ing is in fact mar­i­juana (con­tains THC). Can the gov­ern­ment actu­ally do that based upon its typ­i­cal test­ing method?

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 5: What is Thin Layer Chro­matog­ra­phy? Is it a “good” test?

Thin Layer Chro­matog­ra­phy test­ing

What is it?

Thin Layer Chro­matog­ra­phy (TLC) is a chro­mato­graphic tech­nique. It is a com­bi­na­tion of a col­ori­met­ric test and is mea­sured in terms of separation.

Mechan­i­cally how is it preformed?

TLC depends on the sta­tion­ary phase, often a glass plate coated with sil­ica (it must be prop­erly  des­ic­cated or dried) and the mobile phase which is com­prised of a sol­vent mix­ture made of toluene and diethy­lamine typ­i­cally. When placed on a prop­erly dried plate and using a prop­erly mixed and pre­pared sol­vent, the sol­vent (the mobile phase) will be wicked up by the sil­ica with cap­il­lary action and travel up the plate. A sam­ple from the unknown is selected. It is mashed up in some mechan­i­cal process and in some processes call for it to be dis­solved. It is placed in the des­ig­nated spot. A test is com­pared by per­form­ing the reac­tion of the plate of a known sam­ple from an adju­di­cated source. This com­par­i­son is made with the analyst’s eyes. There will be dif­fer­ent spots on the plate at dif­fer­ent posi­tions from the ori­gin with var­i­ous inten­si­ties of color. The height and the color change are visu­al­ized. The spots typ­i­cally need to be visu­al­ized with a chem­i­cal spray such as Fast Blue B Salt (50 mg in 20 ml of NaOH (0.1 N)) or par­tic­u­lar lighting.

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

Once again, the ana­lyst is not trained in the fun­da­men­tals of how or why this process works. If you handed them a pen and paper and ask them to dia­gram and explain with specifics the chro­mato­graphic process, they would likely be baf­fled. Also for­eign to them would be the specifics as to why the sol­vent used to elute up the plate has to be in a spe­cific ratio and not another. In essence, it is another sub­jec­tive test as it is based upon per­cep­tion of color by the ana­lyst and the per­cep­tion of this height devel­op­ing on the plate ver­sus the adju­di­cated known.

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

Is this a ver­i­fi­able test?

Much like the mod­i­fied Duquenois-Levine test, TLC is poten­tially ver­i­fi­able. Dig­i­tal cam­eras exist. Pho­tographs can be taken. Heck, even video can be taken to show how it is con­ducted on a par­tic­u­lar sam­ple from the unknown and this com­par­i­son to the adju­di­cated known. How­ever, the mod­ern prac­tice is to not take a sin­gle pho­to­graph, to not take advan­tage of video tech­nol­ogy and pro­duce noth­ing ver­i­fi­able in court that the test was even con­ducted or that the analyst’s per­cep­tion of the change did hap­pen and was cor­rectly interpreted.

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?

Once again, this type of test­ing has not been proven to be a val­i­dated method to test specif­i­cally for THC. There are well-known and dis­cov­ered false pos­i­tives which includes cof­fee, basil and even tobacco products.

Once again, the same issues that are dis­cussed prove true with this test­ing. This TLC test is pre­formed on a totally dif­fer­ent sam­ple from the unknown that is not sub­jected to the micro­scopic mor­pho­log­i­cal exam­i­na­tion or the mod­i­fied Duquenois-Levine col­ori­met­ric test.

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.

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)

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

  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 3: What is micro­scopic mor­pho­log­i­cal exam­i­na­tion? Is it a “good” test?

Micro­scopic mor­pho­log­i­cal examination

What is it?

The micro­scopic mor­pho­log­i­cal exam­i­na­tion in short is an exer­cise of botan­i­cal iden­ti­fi­ca­tion using a microscope.

Mechan­i­cally how is it preformed?

A very small amount of the dried unknown is selected. This becomes the sam­ple. The sam­ple is placed on a micro­scopic slide. A drop or two of water is then added to the slide. The slides are exam­ined at vary­ing lev­els of mag­ni­fi­ca­tion and under dif­fer­ent light con­di­tions. What the ana­lyst is look­ing for is two dis­tinct mor­pho­log­i­cal fea­tures. They are look­ing for micro­scopic “hairs” on the unknown. These are cys­tolithic hairs and glan­du­lar hairs. Cys­tolithic hairs are often likened to like lit­tle bear claws in their appearance.

photomicrographs of cystolithic hairs

pho­tomi­cro­graphs of cys­tolithic hairs

The sec­ond type of hair is called a glan­du­lar hair. These are fre­quently remarked as look­ing like mushrooms.

photomicrographs of glandular hairs

pho­tomi­cro­graphs of glan­du­lar hairs

Some tech­niques call for the use of hydrochlo­ric acid after they look for these hairs. A few drops of HCL are added by the ana­lyst. The ana­lyst then looks to see if there is some unspec­i­fied effer­ves­cence under the light of the microscope.

How is the typ­i­cal crime lab­o­ra­tory ana­lyst trained to con­duct this form of testing?

The ques­tion becomes what expe­ri­ence level in botany and tax­on­omy and microscopy does the ana­lyst truly have? Very few under­grad­u­ate pro­grams exist in botany in the United States. Most ana­lysts have on the job train­ing where another per­son who like­wise have no for­mal train­ing in botany or tax­on­omy that instructs them. It also involves the use of a micro­scope. For­mal train­ing in microscopy is required in order to use a micro­scope prop­erly and to prop­erly inter­pret what the human eye sees through var­i­ous pow­ers and light­ing con­di­tions of the micro­scope. At the end of the in-house train­ing, the typ­i­cal ana­lyst can­not typ­i­cally express the fam­ily, the genus and the species that is “mar­i­juana” or at what power and under what light­ing con­di­tions they saw the mor­pho­log­i­cal characteristics.

This sam­ple that is exam­ined under the micro­scope is then dis­carded. All future or addi­tional test­ing is con­ducted on totally dif­fer­ent sam­ples from the unknown.

Is this a ver­i­fi­able test?

It poten­tially is. There is a device that can be linked to the micro­scope to take pic­tures of what the ana­lyst thinks he or she sees. This is called a pho­tomi­cro­graph. In fact the pic­tures above come from just such a micro­scope that is equipped with one. A dig­i­tal cam­era attached to a micro­scope is very com­monly used in sci­ence. They are very mod­er­ately priced. As they are dig­i­tal cam­eras, the cost of pro­duc­tion and print­ing and data stor­age is neg­li­gi­ble. It is fre­quently used in other types of com­par­a­tive exam­i­na­tions such as some higher lev­els of foren­sic firearm or tool­mark iden­ti­fi­ca­tion. I know of no lab­o­ra­tory in the United States that relies upon micro­scopic mor­pho­log­i­cal exam­i­na­tion that uses mod­ern tech­nol­ogy and pro­duces pho­tomi­cro­graphs. In fact, few, if any, crime lab­o­ra­to­ries use the ACE-V (Analy­sis, Com­par­i­son, Eval­u­a­tion, and Ver­i­fi­ca­tion) tech­nique that one would find in fin­ger­print iden­ti­fi­ca­tion using a stereo-microscope and a dou­ble check in real time by a fel­low bench ana­lyst. In essence, the unknown is checked one time, by one per­son with no dou­ble check by another, and noth­ing is pro­duced that proves that the analy­sis was done or that the fea­tures that are reported as present were in fact objec­tively present. There is no proof.

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

No. There are no empir­i­cal and robust val­i­da­tion stud­ies that con­clude that this form of micro­scopic mor­pho­log­i­cal exam­i­na­tion even when the two botan­i­cal fea­tures (cys­tolithic and glan­du­lar hairs) are objec­tively present yield a valid opin­ion that the plant exam­ined is def­i­nitely con­tains THC. There are no stud­ies that say the two fea­tures means that there is THC present. In fact, what stud­ies that are out there con­clude that this form of mor­pho­log­i­cal exam­i­na­tion using a micro­scope is per­haps not even selec­tive. In the orig­i­nal stud­ies by Naka­mura, he indi­cated that cys­toliths of var­i­ous types are found in the leaves of a num­ber of dicots. (497). He also indi­cated that the pres­ence of cys­toliths is not diag­nos­tic for a fam­ily, let alone a genus of plants. (497) Naka­mura specif­i­cally noted that cys­toliths are found on a great num­ber of plants includ­ing but not lim­ited to: hops plants (500), oregano (500), lemon thyme (501), sil­ver thyme (501), and rose­mary (501). Naka­mua he specif­i­cally noted 63 “rep­re­sen­ta­tive” species in 13 plant gen­era that con­tain cys­toliths in table 5 of his arti­cle (501) Naku­mura indi­cated that he made NO attempt to pre­pare a com­pre­hen­sive list­ing of species bear­ing cys­tolith hairs sim­i­lar to those found in cannabis “because of the sheer enor­mity of the task to exam­ine 31,874+ dicotyle­dons.” (500). For instance, in one genus found in Table 5 of his arti­cle, the Loasa, he specif­i­cally noted 9 species that had cys­toliths; how­ever, he went on to say that there were actu­ally some 80 species of that genus known to have sim­i­lar hairs. (501). He fully acknowl­edged that his list­ing was not com­pre­hen­sive. So it is accu­rate and very fair to say that the 63 “rep­re­sen­ta­tive” species that have cys­toliths that were noted by Naka­mura in Table 5 of his arti­cle are not an exhaus­tive list. Other stud­ies agree that at least 6 other sub­stances also have hairs that con­tain these two fea­tures (cys­tolithic and glan­du­lar hairs). In terms of the addi­tional step of adding HCL to the sam­ple and exam­in­ing for effer­ves­cence under the light of the micro­scope, it is quite clear that other sub­stances can pro­duce the same effer­ves­cence when a few drops of hydrochlo­ric acid are added to them. For exam­ple net­tles and cat­nip do exactly that.

Some folks main­tain and tes­tify under oath every day in the United States that this unver­i­fied micro­scopic mor­pho­log­i­cal exam­i­na­tion is diag­nos­tic of iden­ti­fi­ca­tion of THC pres­ence in an unknown. There is no sci­en­tific sup­port for this type of testimony.

One thing that every ana­lyst should agree with is that sim­ply because these hairs are present and if they con­duct HCL addi­tion and if there is effer­ves­cence that does not mean that the unknown con­tains THC. This is why they have to do addi­tional test­ing, mean­ing the mod­i­fied Duquenois-Levine and Thin Layer Chro­matog­ra­phy testing.

What is fre­quently not part of any mor­pho­log­i­cal exam­i­na­tion for cannabis is what botanists have noted to be other fea­tures con­sis­tent with cannabis. The sim­pli­fied exam­i­na­tion for the typ­i­cal foren­sic sci­ence iden­ti­fi­ca­tion is pur­pose­fully designed to make this exam­i­na­tion and con­clu­sions from it eas­ier to per­form by non-botanists. As with every­thing in life, the more cri­te­rion attached to qual­ify some­thing the least likely there will be a qual­i­fi­ca­tion. The exam­i­na­tion of cannabis and espe­cially a mor­pho­log­i­cal exam­i­na­tion by untrained botanists should not be made easy. All of the fea­tures that are known to be diag­nos­tic by the world of botany should be used not sim­ply the easy ones. For exam­ple, botanists have noted that that cannabis has ses­sile glands as well as con­tain­ing ser­rated edges of the leaves and com­pound palmate struc­ture mean­ing sev­eral leaflets arise from the same point. The addi­tion of all of the known mor­pho­log­i­cal fea­tures known to true botanists as diag­nos­tic of cannabis would make this exam­i­na­tion more robust and the result more selec­tive than the sim­plis­tic exam­i­na­tion that now per­me­ates the foren­sic sci­ence world.

In 2006, more than 829,000 peo­ple were arrested in this coun­try for marijuana-related offenses alone. Since 1937 with the pas­sage and adop­tion of the Mar­i­huana Tax Act, mar­i­juana has been effec­tively pro­hib­ited in the United States.

Lit­er­ally mil­lions upon mil­lions of peo­ple have been accused of pos­sess­ing mar­i­juana. The ques­tion becomes are these con­vic­tions sci­en­tif­i­cally supported?

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 2: How is most mar­i­juana test­ing con­ducted in the United States?

So what hap­pens in Amer­ica, here, now and today in the iden­ti­fi­ca­tion of marijuana?

  • Are the tests pre­sump­tive or confirmatory?
  • Are the meth­ods used verifiable?
  • Does it result in a spe­cific or selec­tive identification?

Let’s take a look at how mar­i­juana is tested in the United States today in the super-majority of cases. It all begins with a police offi­cer or another per­son who seizes an unknown sub­stance. It’s green. It’s veg­etable like. It looks like mar­i­juana to them. Per­haps they do some sort of quick and dirty test on the side of the road using a fast col­ori­met­ric test and there is a change in color. This is a true unknown. We don’t know what it is. We can­not con­clude what it is based upon sim­ply look­ing at it with our own two eyes or even by our sense of smell. This is why we have foren­sic sci­en­tists and fur­ther test­ing because those types of obser­va­tions may lead to an improper or incor­rect result.

attempts at identifying marijuana at the gross or macro level can be misleading

Attempts at iden­ti­fy­ing mar­i­juana at the gross or macro level can be misleading

A basic fun­da­men­tal ques­tion becomes: What makes mar­i­juana ille­gal to pos­sess? What makes mar­i­juana ille­gal is that it con­tains the phar­ma­co­dy­nam­i­cally sub­stance known as Delta-9 Tetrahy­dro­cannabi­nol (THC).

Given that THC is what makes mar­i­juana ille­gal, then log­i­cally what would be best is if we were design tests that react exclu­sively to THC and noth­ing else and pro­duces ver­i­fi­able data that iden­ti­fies that the green veg­etable sub­stance in fact specif­i­cally con­tains THC.

Because we can’t tell what it is just by look­ing at it or just by smelling it, then we must sub­ject this unknown to test­ing. Largely in the United States there is a régime or process of how crime lab­o­ra­to­ries con­duct this test­ing. The triad of test­ing that is con­ducted are:

  1. Micro­scopic mor­pho­log­i­cal examination
  2. Mod­i­fied Duquenois-Levine testing
  3. Thin Layer Chromatography

This is gen­er­ally referred to as the Thorton-Nakumura protocol.

In order to answer our ques­tions of speci­ficity, ver­i­fi­ca­tion, and con­fir­ma­tory we need to look at each of these three test­ing tech­niques. In our next blog posts over the next sev­eral weeks each of these tests will be scrutinized.

In many court­rooms all across the United States today, a state ana­lyst opines under oath that a unknown leafy green veg­etable like sub­stance is in fact mar­i­juana. The ques­tion becomes, in the typ­i­cal case, is this a valid con­clu­sion? Is it the whole sci­en­tific truth?

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

  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 1: What is the goal and the pur­pose of test­ing of unknowns gen­er­ally? How do we best design a test for marijuana?

Remem­ber that the goal of all ana­lyt­i­cal test­ing is to pro­duce a valid result. A valid result is the epit­ome of good science.

A valid result is made up of three dis­tinct features:

  1. It is a spe­cific measurement,
  2. using a tech­nique or series of tech­niques that are part of a val­i­dated procedure
  3. that is verifiable.

Fea­tures of a spe­cific test

The first step is that the test­ing must be unique and spe­cific qual­i­ta­tive mea­sure­ment. The dif­fer­ence between speci­ficity and selec­tiv­ity is very impor­tant. To be spe­cific in terms of qual­i­ta­tive mea­sure­ment means that there is an iden­ti­fi­ca­tion of a par­tic­u­lar com­pound and exclu­sively that one spe­cific com­pound to the exclu­sion of every other com­pound or sub­stance in the uni­verse. It is unequiv­o­cal iden­ti­fi­ca­tion based upon objec­tive cri­te­rion and val­i­dated meth­ods or tech­niques that are ver­i­fi­able. In short, it is what it is and could be noth­ing else.

On the other hand, if the iden­ti­fi­ca­tion is selec­tive, then it is not spe­cific. The iden­ti­fi­ca­tion that results from selec­tive test­ing or meth­ods means that it could be that sub­stance but it also could be some­thing else. There could be cross-reactivity, co-elution or just poor sen­si­tiv­ity. There could be false pos­i­tives. In essence and fun­da­men­tally, we do not know what it is defin­i­tively, uniquely, and specif­i­cally. We may not know what it is, but we can learn what it is not.

Gen­er­ally, the dif­fer­ence between a spe­cific test­ing régime and that of a selec­tive one is the dif­fer­ence between con­fir­ma­tory data and pre­sump­tive data and the dif­fer­ence between the sci­en­tific weight of the opin­ions expressed based upon them. A pre­sump­tive test is some­thing that is char­ac­ter­ized by a high rate of false pos­i­tives and cross-reactivity. It is not at all spe­cific and may or may not be use­fully selec­tive. A con­fir­ma­tory test is one that results in a near spe­cific mea­sure­ment with a very, very low rate of false pos­i­tives. A con­fir­ma­tory test or even a com­bi­na­tion of con­fir­ma­tory tests can never be truly 100% cor­rect. There is still room for error. Noth­ing is perfect.

Selective versus Specific is kind of like Sesame Street's "one of these things is not like the other things"

Selec­tive ver­sus Spe­cific is kind of like Sesame Street’s “one of these things is not like the other things”

The Inter­na­tional Union of Pure and Applied Chem­istry (IUPAC), which is the world author­ity on chem­i­cal nomen­cla­ture, ter­mi­nol­ogy, stan­dard­ized meth­ods for mea­sure­ment, atomic weights and other crit­i­cally eval­u­ated data and oth­ers have defined the dif­fer­ence between these often con­fused terms as follows:

A spe­cific reac­tion or test is one that occurs only with the sub­stance of inter­est, while a selec­tive reac­tion or test is one that can occur with other sub­stances but exhibits a degree of pref­er­ence for the sub­stance of inter­est.  Few reac­tions are spe­cific, but many “exhibit selectivity”.

Other com­mon def­i­n­i­tions include:

Selec­tiv­ity gives an indi­ca­tion of how strongly the result is affected by other com­po­nents in the sample.

and also

Selec­tiv­ity refers to the extent to which the method can be used to deter­mine par­tic­u­lar ana­lytes in mix­tures or matri­ces with­out inter­fer­ences from other com­po­nents of sim­i­lar behavior.

A selec­tive test may be not a spe­cific test due to cross-reactivity, inter­fer­ence, coelu­tion, or codetermination.

Fea­tures of a val­i­dated tech­nique or series of techniques

We wrote a whole 6 part series on method val­i­da­tion and what it takes to be valid result.

  1. Part 1: Introduction-Is it valid, invalid or non-validated?
  2. Part 2: What is method validation?
  3. Part 3: Can we use some­one else’s val­i­dated method?
  4. Part 4: What trig­gers ver­i­fi­ca­tion, re-validation or out right new val­i­da­tion of a method?
  5. Part 5: What are the essen­tial terms in method validation?
  6. Part 6: What is qual­ity assur­ance and qual­ity control?

Fea­tures of a ver­i­fi­able test

A ver­i­fi­able test is one in which oth­ers who were not present for the test­ing can look at some­thing to basi­cally dou­ble check what test­ing was pre­formed and its result. With­out ver­i­fi­ca­tion, then there can­not be a val­i­dated result.

The end result in the courtroom

If we, as a soci­ety, sin­cerely mean the prin­ci­ples of the pre­sump­tion of inno­cence, proof beyond a rea­son­able doubt, and the bur­den of pro­duc­tion and per­sua­sion, then in the case of iden­ti­fi­ca­tion of an unknown when it is pre­sented in court and if the gov­ern­ment can only prove selec­tiv­ity, then the jury’s result should always be not guilty.

a typical seized unknown

a typ­i­cal seized unknown

In courts all across the United States evi­dence is pre­sented in the pros­e­cu­tion of those charged with pos­ses­sion (either mis­de­meanor or felony) that the green veg­etable sub­stance seized is in fact mar­i­juana. The way that this is typ­i­cally pre­sented in the court­room is as an absolute and as a spe­cific qual­i­ta­tive mea­sure­ment. This series of posts seeks to ask the ques­tion: Is this in fact true?

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

  1. What is the goal and the pur­pose 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?
What the heck is this?

What the heck is this?