It is shock­ing but very true state­ment that most sophis­ti­cated instru­ments such as a Gas Chro­mato­graph (GC) with var­i­ous detec­tors whether it is a mass spec­trom­e­ter (MS) or flame ion­iza­tion detec­tor (FID) when pro­duced and man­u­fac­tured are inca­pable of pro­duc­ing mean­ing­ful results “straight out of the box.” These machines have to be “taught” what it is look­ing for and how much there is. Inher­ently and orig­i­nally they “know” noth­ing. They have to be taught what it is ana­lyz­ing and also taught how much there is. This method of teach­ing is gen­er­ally referred to as Qual­ity Con­trol (QC). This series of teach­ing when it comes to GC-FID is really a process of teach­ing the machine what it is look­ing for (the tar­get ana­lyte which, for exam­ple, in GC-FID for EtOH deter­mi­na­tion for pur­poses of Blood Alco­hol Con­tent is ethanol oth­er­wise known as EtOH) and then once it has iso­lated (hope­fully) the tar­get ana­lyte, it then has to be taught to mea­sure how much there is. This process of teach­ing it what it is look­ing for in the case of GC-FID is really a process of teach­ing it what is not the tar­get analyte.

The eas­i­est way of think­ing about this process from a global per­spec­tive is to think about colors.

When we are born we do not know our col­ors. This is some­thing that needs to be taught. Just like all things in life, our teacher is vitally impor­tant. Every time we are asked “What color is this?,” in real­ity that “this” that is shown to us is an unknown that we are asked to ana­lyze. We know what red is because we were taught through some sort of process that the hue that our eyes detect (our eyes are a detec­tor just like a FID or MS) is this “red” and there­fore from that point on we use this “red” that we were taught to become our stan­dard against which all future unknowns are com­pared to see if it is “red.” We fur­ther “know” that some­thing is “red” because it is not “yel­low,” “orange,” “blue” or “vio­let.” The more col­ors we learn, the stronger our con­fi­dence in the con­clu­sion that “red” is “red” and not some­thing else.

But what if our teacher, taught us “red” wrong when we were lit­tle? Let’s say that our teacher taught us what we believe is “red,” based upon show­ing us what is objec­tively in real­ity green (the com­ple­men­tary or oppo­site color of “red”). Then, we would learn incor­rectly or wrongly that what is objec­tively and in real­ity is green is in fact “red.” All of this results in the cir­cum­stance of when we are pro­duced an unknown and asked “what is this?” and this “this” (which is our unknown hue that we are asked to iden­tify) is objec­tively and in real­ity green but is hon­estly but mis­tak­enly inter­preted as “red” and reported out with con­fi­dence as “red” because we were taught incor­rectly and wrong. When we do so (incor­rectly but hon­estly report the green as “red”), we are not lying or inten­tion­ally deceiv­ing the per­son who is ask­ing us, we are just taught incor­rectly and wrongly. So the moral of the color exam­ple is if we are taught the wrong way, then the result we report will be wrong.

Well, a GC is not any dif­fer­ent. If you teach the machine wrong, it will report the wrong result. The machine does not inher­ently know any­more what any­thing is qual­i­ta­tively than an infant does who is being taught his/her colors.

The goal of all ana­lyt­i­cal chem­istry is to pro­duce a valid result. A valid result is really com­prised of two sep­a­rate, dis­tinct and impor­tant aspects: to pro­duce a spe­cific qual­i­ta­tive result and to pro­duce as close as pos­si­ble to a true quan­ti­ta­tive result as can be pro­duced, mean­ing it is as free from cal­i­bra­tion and bias related error as possible.

We cov­ered the con­cept of Qual­ity Con­trol (QC) before, but let’s do so in more detail with this post. QC is strictly speak­ing a process that is used to try to insure valid­ity of the results. QC is the pro­ce­dure or the method used to demon­strate that the machine has achieved the two chro­mato­graphic commandments.

The 2 commandments of chromatography
The 2 com­mand­ments of chromatography

This nec­es­sary act of QC is best pre­formed by a series of tests that (1) proves through ver­i­fi­able data the qual­i­ta­tive strength of the test­ing régime, and (2) the quan­ti­ta­tive sen­si­tiv­ity of the method. As there are no uni­ver­sal nomen­cla­ture for foren­sic chro­matog­ra­phy, this qual­i­ta­tive test­ing régime should be com­prised at a min­i­mum the following:

  1. A vial is pre­pared which pur­pose­fully con­tains sev­eral dif­fer­ent com­pounds. This addi­tion is called spik­ing (adding) the com­pounds into the vial. The vial is then sam­pled and injected into the instru­ment to see what pro­duces. If the method is a valid method, then the pur­pose­fully spiked com­pounds must be shown to have com­plete chro­mato­graphic sep­a­ra­tion in the resul­tant chro­matogram. Oth­er­wise, com­mand­ment num­ber one (thou shall sep­a­rate) has not been sat­is­fied. The prover­bial Achilles heel of any ana­lyt­i­cal chem­istry method is in this step. It is a test of speci­ficity. In the case of GC-FID, some crime lab­o­ra­to­ries skip this step alto­gether and oth­ers use only a sin­gle col­umn analy­sis and only prove sep­a­ra­tion between 4 or 5 ana­lytes. Four or five ana­lytes is a foren­si­cally inde­fen­si­ble amount in the con­text of test­ing EtOH in human blood. Lab­o­ra­to­ries have dif­fer­ent nam­ing schemes for this proof of res­o­lu­tion. Some lab­o­ra­to­ries call it a volatile mix, sep­a­ra­tion matrix, sep­a­ra­tion con­trol, res­o­lu­tion mix, res­o­lu­tion con­trol, or res­o­lu­tion matrix.
  2. A series of blanks must be used. Blanks are an essen­tial com­po­nent of QC. There are dif­fer­ent types of blanks. There are true blanks, inter­nal stan­dard blanks, and tar­get ana­lyte blanks. There are two essen­tial pur­poses of blanks. First is to prove the reten­tion time of a given ana­lyte to serve as a stan­dard and serves an essen­tial part of this teach­ing process. The sec­ond rea­son, is to demon­strate that there is no carry-over. Carry-over is a form of con­t­a­m­i­na­tion where the con­di­tions of one injec­tion car­ries over to another. It is con­trib­u­tory error that leads to an invalid result. (For more on the carry-over effect, see gen­er­ally: The Car­ry­over Effect: Lack of Blanks between tests leads to false pos­i­tive or inflated BAC results, Car­ry­over effect part Deux: Autodi­lu­tion may be part of the prob­lem for false blood results in DUI and Car­ry­over effect part 3: Flush­ing of inert gas is not enough to prove there is no carryover)
  • A true blank is a sam­ple in a vial that is designed to have noth­ing mean­ing it is one in which there are no detectable ana­lytes. If the method is valid and the prepa­ra­tion of the sam­ple is per­fect and accord­ing to design, then the result­ing chro­matogram is sup­posed to be just the base­line sig­nal with noth­ing else. If there is any sub­stances detected at all, then the true blank is invalid.
  • An inter­nal stan­dard blank is a sam­ple in a vial that is designed to have only the inter­nal stan­dard (usu­ally n-propanol) mean­ing it is one in which there is only one detectable ana­lyte. The result­ing chro­matogram is sup­posed to fea­ture one peak at a reten­tion time that is char­ac­ter­is­tic to the inter­nal stan­dard accord­ing to the method and the chro­mato­graphic con­di­tions. If the method is valid and the prepa­ra­tion of the sam­ple is per­fect and accord­ing to design, then the result­ing chro­matogram is sup­posed to be just the base­line sig­nal with one peak and noth­ing else. If there is any other sub­stances detected at all, then the inter­nal stan­dard blank is invalid.
  • A tar­get ana­lyte blank is one in which there is only one detectable ana­lyte. The result­ing chro­matogram is sup­posed to fea­ture one peak at a reten­tion time that is char­ac­ter­is­tic to the true tar­get ana­lyte (in the case of GC-FID for EtOH deter­mi­na­tion this would be EtOH oth­er­wise known as ethanol) accord­ing to the method and the chro­mato­graphic con­di­tions. If the method is valid and the prepa­ra­tion of the sam­ple is per­fect and accord­ing to design, then the result­ing chro­matogram is sup­posed to be just the base­line sig­nal with one peak and noth­ing else. If there is any other sub­stances detected at all, then the tar­get ana­lyte blank is invalid.

The sec­ond part of QC is the proper con­struc­tion of  the cal­i­bra­tion curve that our knowns are tested and then an unknowns are tested against. This is the teach­ing com­po­nent of the quan­ti­ta­tive mea­sure­ment. This is typ­i­cally per­formed in the begin­ning of the run. If not, then there are legit­i­mate issues about the valid­ity of the quan­ti­ta­tion. See gen­er­ally, Is it legit­i­mate for a crime lab­o­ra­tory to use ‘his­tor­i­cal data’ to prove its test results are valid? For infor­ma­tion gen­er­ally about cal­i­bra­tion and cal­i­bra­tion curves, I offer to you the fol­low­ing posts: When is a straight line a curve: Cal­i­bra­tion curve and Why do instru­ments need to be cal­i­brated? The only metro­log­i­cally accept­able method for estab­lish­ing the cal­i­bra­tion of any device is to employ the 5x5 and 120% method. The 5x5 and 120% method is to test at least five dif­fer­ent con­cen­tra­tions five times each time at those spe­cific con­cen­tra­tion points along a wide lin­ear dynamic range that sets as low as pos­si­ble limit of quan­tifi­ca­tion based upon a legit­i­mate val­i­da­tion stud­ies and a last cal­i­bra­tor that is 120% of the high­est expected value in the unknown sam­ples that will be tested.

The most impor­tant aspect of all of QC is that the mate­ri­als that are used for it must be derived and orig­i­nate from cer­ti­fied ref­er­ence mate­ri­als (CRMs) or United States Phar­ma­copeia (USP) grade or Amer­i­can Chem­i­cal Soci­ety (ACS) grade raw mate­ri­als. (See gen­er­ally our posts on the def­i­n­i­tions of CRMs and USP grade cal­i­bra­tors, stan­dards and con­trols: Stan­dards, Con­trols, Cal­i­bra­tors, and Ver­i­fiers, Oh my…) Even if pur­chased from rep­utable third party ven­dors such as the NIST SRM line of third party prod­ucts, these mate­ri­als when pur­chased or made must be ver­i­fied before they are placed into the QC process in the test­ing of unknowns.

Mr. Fletcher vs. Mr. Daubert: A Jour­ney into the Surreal

By:  Fred­eric White­hurst, J.D., Ph.D.

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. On the face of it, this iden­ti­fi­ca­tion screams to our com­mon senses as not being pos­si­ble. How­ever, we find that in State v. Fletcher, 92 N.C. App. 50, 373 S.E. 2d 681 (N.C. App. 1988), our courts in North Car­olina are will­ing to accept this tes­ti­mony as evi­dence. Though State v. Ward, 364 N.C. 133 (2010) began a foray into rec­og­niz­ing the ridicu­lous­ness of the legit­i­macy of visual iden­ti­fi­ca­tion of con­trolled sub­stances in a world full of coun­ter­feit mate­ri­als, State v. Gar­nett, ___ N.C. App. ___, 706 S.E.2d 280 (2011) backed away from Ward, thus allow­ing an in-court iden­ti­fi­ca­tion of cannabis sativa to be admis­si­ble. This all flies in the face of a long list of sci­en­tific lit­er­a­ture and even State v. Tate, 300 N.C. 180 (1980), which found that the then known list of mate­ri­als caus­ing false pos­i­tives with this Duquenois chem­i­cal spot test ren­dered the test not sci­en­tif­i­cally reli­able or admis­si­ble as an means of iden­ti­fy­ing mar­i­juana. In North Car­olina we are now given a new stan­dard for admis­si­bil­ity of scientific/expert tes­ti­mony in courts of law. Rule 702 has entered the mod­ern era. As of Octo­ber 1, 2011, the amend­ments to Chap­ter 8C, Rule 702(a) read:

If sci­en­tific, tech­ni­cal or other spe­cial­ized knowl­edge will assist the trier-of-fact to under­stand the evi­dence or to deter­mine a fact in issue, a wit­ness qual­i­fied as an expert by knowl­edge, skill, expe­ri­ence, train­ing, or edu­ca­tion, may tes­tify thereto in the form of an opin­ion, or oth­er­wise, if all of the fol­low­ing apply:
1. The tes­ti­mony is based upon suf­fi­cient facts or data.
2. The tes­ti­mony is the prod­uct of reli­able prin­ci­ples and meth­ods.
3. The wit­ness has applied the prin­ci­ples and meth­ods reli­ably to the facts of the case.

So let us now con­sid­ered this “expert iden­ti­fi­ca­tion” of Cannabis Sativa L. by a BLET “Ph.D. botanist”. The expert must be qual­i­fied by knowl­edge, skill, expe­ri­ence, train­ing or edu­ca­tion. The law enforce­ment offi­cer who has seized green veg­etable mate­r­ial on ten or ten thou­sand occa­sions is not test­ing his qual­i­fi­ca­tions or his “iden­ti­fi­ca­tion pro­to­col”. He is sim­ply seiz­ing green veg­etable mate­r­ial. Let’s con­sider the data that he has at the point of seizure.
The most preva­lent form of seized and “iden­ti­fied as mar­i­juana” mate­r­ial that we see is the mate­r­ial that has been finely crushed. What this means is that the law enforce­ment offi­cer has seized a mate­r­ial which no longer has the macro­scopic char­ac­ter­is­tics of the orig­i­nal plant (if indeed the seized mate­r­ial is plant mate­r­ial). But is it plant mate­r­ial? What is a plant and there­fore veg­etable mate­r­ial? Ask any dic­tio­nary. The Amer­i­can Her­itage Dic­tio­nary defines a plant as “an organ­ism of the veg­etable king­dom, char­ac­ter­is­ti­cally hav­ing cel­lu­lose cell walls, grow­ing by syn­the­sis of inor­ganic sub­stances and lack­ing loco­mo­tion.” Good­ness gra­cious, look it up. We all have dic­tio­nar­ies. And at that point we are see­ing the flaw in the law enforce­ment officer’s qual­i­fi­ca­tions. We ask, “Do you carry a micro­scope?” I’m not being ridicu­lous. A plant mate­r­ial (veg­etable mate­r­ial) con­tains cells. With­out a micro­scope one can not see those cells. So what expe­ri­ence does the offi­cer have in con­duct­ing micro­scopic analy­sis? If none, then we must con­clude that he does not have the req­ui­site expe­ri­ence, train­ing or skill needed to qual­ify as an expert in iden­ti­fy­ing plant mate­r­ial. Of course he can opine that the mate­r­ial he has seized lacks loco­mo­tion but so does road kill, so do rocks, so does a fake plas­tic plant. At a recent CLE sem­i­nar at which I taught, before my lec­ture I went out into the hotel where the CLE was held and broke off a small part of a dec­o­ra­tive plas­tic pot­ted plant. Dur­ing the lec­ture I pulled this pur­loined “veg­etable” mat­ter from my coat pocket and handed it to the man in front of me and asked him what kind of plant it was. He advised that he knew it was a plant but did not know what kind of plant.

Let us get beyond the issue of whether the law enforce­ment offi­cer car­ries a micro­scope as well as or instead of a gun. Let’s sup­pose that the offi­cer in Gar­nett actu­ally had a micro­scope on the wit­ness stand and was able to see cell struc­ture. Let’s give him the ben­e­fit of the doubt that he was not will­ing to give to our client. What would be the next step in this “iden­ti­fi­ca­tion” that is being con­ducted by the offi­cer who is qual­i­fied by train­ing, skill, edu­ca­tion, etc.. We know that mar­i­juana is a seed bear­ing plant. What did the offi­cer see that indi­cated to him that this seized mate­r­ial was a seed bear­ing plant? Did he see any seeds at all? And if he did see objects that he thought were seed what qual­i­fi­ca­tions does he have to rec­og­nize these objects as seeds. Has he con­ducted any research what-so-ever in the visual iden­ti­fi­ca­tion of mar­i­juana seeds? The answer to that ques­tion is absolutely not. In fact there was no research in this area at all any­where until about 2009 when Jenna Fussell, John Thorn­ton and Fred­eric White­hurst authored “The Visual Char­ac­ter­i­za­tion and Iden­ti­fi­ca­tion of Cannabis sativa (Mar­i­juana) Seeds pub­lished in the Jour­nal of Foren­sic Iden­ti­fi­ca­tion 59 (5) 2009. So that leaves out the seeds issue and the offi­cer can not tes­tify that he has skill in iden­ti­fy­ing mar­i­juana seeds. Very likely at this point we have an offi­cer on the stand or at the point of seizure who has no expe­ri­ence or train­ing or skill at “iden­ti­fy­ing” mar­i­juana based upon the char­ac­ter­is­tics we shall call “plant” and “seeds.”

Let’s give the offi­cer the ben­e­fit of the doubt that he did not give our clients. Let’s assume that he has con­ducted research and has the skills needed to iden­tify a mate­r­ial as a plant based upon an accepted def­i­n­i­tion of the word “plant” and he has con­ducted the research and has the skills needed to iden­tify the “seeds” that he sees as seeds from a mar­i­juana plant. So next we reach the issue of flow­ers. Mar­i­juana has flow­ers. Those of us who have been involved in legal cases where “mar­i­juana” was seized have a com­mon sense that mar­i­juana has flow­ers. So we must ask the offi­cer what expe­ri­ence and skill he has in “iden­ti­fy­ing” mar­i­juana flow­ers. And not nec­es­sar­ily even the flow­ers on the grow­ing plant but the flow­ers in the end prod­uct, that which on the street is rec­og­nized as the qual­ity prod­uct because of its high tetrahy­dro­cannabi­nol (THC) con­tent. Who wants leaves and stems and stalks when one can pur­chase the bud/flower? But what is a flower? Can the offi­cer iden­tify the parts of a flower? What skill and train­ing has he got to make him qual­i­fied to ren­der an opin­ion that the mate­r­ial he is observ­ing is even flower mate­r­ial much less mar­i­juana flower mate­r­ial? “I just know it when I see it” is a great response but the obvi­ous query is “Just what is it that you are see­ing and how does that com­pare with the struc­ture of the mar­i­juana flower?” In “The Struc­ture of Eco­nomic Plants”, Pro­fes­sor Her­man Hay­ward tells us that “Hemp is dioe­cious and the num­ber of sta­mi­nate and pis­til­late plants is rel­a­tively con­stant under nor­mal con­di­tions.” And describ­ing the sta­mi­nate inflo­res­cence and flower, Hay­ward tells us that ”the flow­ers develop in small, droop­ing, branched pan­i­cles, which arise in the axils of foliage leaves. The flow­ers of the pan­i­cle may occur singly on slen­der pedicels or in groups, and usu­ally the ter­mi­nal branches bear three flow­ers, a median one and two lat­er­als which are sub­tended by bracts or stip­ules. The indi­vid­ual flow­ers are petalous with a deeply parted calyx hav­ing five greenish-yellow or red lobes that are wide­spread at matu­rity. There are five sta­mens, and the anthers are sus­pended from long thread-like fil­a­ments.” This, of course describes the flow­ers in their grow­ing state, not in the state that one finds them on the street. The expe­ri­enced law enforce­ment offi­cer who has the skill, train­ing, knowl­edge, etc. to qual­ify him as an expert who can offer an opin­ion that what he is look­ing at is mar­i­juana flower, of course knows all of this and more and will be able to describe all of those parts that he is see­ing that fit Hayward’s very clear descrip­tion of grow­ing mar­i­juana plant flow­ers. If not then he does not have the req­ui­site expe­ri­ence, train­ing or skill. And if the offi­cer can rec­og­nize all the parts of mar­i­juana flower are present within the mate­r­ial he has seized and is call­ing mar­i­juana, we must still ask if he has the req­ui­site expe­ri­ence and train­ing to not only rec­og­nize the parts but tell us that no other plants has such parts, not no other plant he has seen, but no other plant. He has been asked to iden­tify the plant as mar­i­juana, mean­ing that he is say­ing that the plant is mar­i­juana to the exclu­sion of all other plants or at least a rea­son­able por­tion of the other plants on the planet.

This is not cannabis. It is Kenaf of Kanap that contaons no THC

This is not cannabis. It is Kenaf of Kanap that con­taons no THC

But again, let’s give this offi­cer the ben­e­fit of the doubt that he was not will­ing to extend to our clients and progress to the next level of inquiry about his skill and expe­ri­ence and train­ing. Mar­i­juana is a dicotyle­don as opposed to being a mono­cotyle­don. Does the offi­cer know what this means? Surely with the proper train­ing, skill and expe­ri­ence he does. In fact, if he remem­bers his ninth grade biol­ogy class he will real­ize what these terms mean. A cotyle­don is the part of the plant that nour­ishes the embry­onic plant until it can suc­ceed on its own. In North Car­olina, an agri­cul­tural state, all of us should know about open­ing up peanuts or lima beans. Two halves, fat leaves, inside of which exists a tiny lit­tle plant. (If you haven’t opened up a lima bean you haven’t lived. Go do it now.) Those two halves are cotyle­dons and because there are two of them we call plants with two of them, dicotyle­dons. Plants such as corn have only one lit­tle fat leaf, one cotyle­don, the ker­nel of corn, that feed the lit­tle corn plant until it grows into the stalk on which the corn is pro­duced. So, assum­ing the offi­cer remem­bers his ninth grade biol­ogy class, he will have the req­ui­site knowl­edge and skill needed to rec­og­nize if this plant is a dicotyle­don, which mar­i­juana is. But how many other plants are there that are dicotyle­dons? Does he know this? Can he prop­erly edu­cate a trier-of-fact to the poten­tial num­ber of plants that might be mis­taken for mar­i­juana based on the char­ac­ter­is­tics we have con­sid­ered thus far?

Please remem­ber that what the offi­cer seizes most often is crushed mate­r­ial, no longer hav­ing all the char­ac­ter­is­tics of the grow­ing plant. Dur­ing the prepa­ra­tion for dis­tri­b­u­tion, the flow­ers are left whole but the leaves are crushed into very small pieces. A “leaf archi­tec­tural analy­sis” is no longer pos­si­ble. Go out into the woods right now or out into your yard. Pick leaves off a num­ber of plants. Let them dry a bit and then crush them up. Ask your­self if you can then tell from which plant those crushed up leaves orig­i­nated. You might be able to dif­fer­en­ti­ate crush yard grass or corn leaves from oak leaves but can you dif­fer­en­ti­ate live oak leaves from red oak leaves from maple? Can you dif­fer­en­ti­ate oregano leaves from tea leaves just by look­ing at them? Does the offi­cer have the skill, train­ing and edu­ca­tion to do this? Hickey in the Jour­nal of Botany, 60(1): 17–33 (1973) describes for us this leaf archi­tec­tural analy­sis. Has the offi­cer the skill, knowl­edge or train­ing in this area? Leaves are clas­si­fied accord­ing to leaf ori­en­ta­tion, orga­ni­za­tion, shape, mar­gin, tex­ture, gland posi­tion, peti­ole, types of vena­tion, and ele­ments of tooth archi­tec­ture. When we fol­low Hicky we being to look at the form of leaf mar­gin; we see clas­si­fi­ca­tion con­cepts such as entire, lobed, toothed, cre­nate, erose, rev­o­lute or enrolled, sinuses, spac­ing and series. Does the offi­cer know what these con­cepts even mean? But wait! These are gen­er­ally not seized plants that we have in evi­dence but crushed up mate­r­ial. So how do we begin to con­duct Hickey’s sug­gested leaf archi­tec­tural analy­sis? What train­ing, expe­ri­ence or skill can the offi­cer tes­tify to to be qual­i­fied in the “iden­ti­fi­ca­tion” of mar­i­juana from a leaf archi­tec­tural analy­sis when the leaves are no longer in their nat­ural state? In his paper “Foren­sic Aspects of Cys­tolith Hairs of Cannabis and Other Plants”, Jour­nal of AOAC (Vol. 52, No.1, 1969), George Naka­mura noted that even with a micro­scopic analy­sis he could not dif­fer­en­ti­ate 82 dif­fer­ent seed bear­ing plants out of 31874 he con­sid­ered. He never con­sid­ered a leaf archi­tec­tural analy­sis because, obvi­ously once the leaves are crushed up into prod­uct for street sales, such an analy­sis would not be pos­si­ble. So how can a law enforce­ment offi­cer visu­ally iden­tify green veg­etable mate­r­ial as mar­i­juana even with a microscope?

The response this author has heard in the past has been that one need not be a botanist in order to under­stand botan­i­cal con­cepts. OK, but Rule of Evi­dence 702 now requires that

If sci­en­tific, tech­ni­cal or other spe­cial­ized knowl­edge will assist the trier-of-fact to under­stand the evi­dence or to deter­mine a fact in issue, a wit­ness qual­i­fied as an expert by knowl­edge, skill, expe­ri­ence, train­ing, or edu­ca­tion, may tes­tify thereto in the form of an opin­ion, or oth­er­wise, if all of the fol­low­ing apply:
1. The tes­ti­mony is based upon suf­fi­cient facts or data.
2. The tes­ti­mony is the prod­uct of reli­able prin­ci­ples and meth­ods.
3. The wit­ness has applied the prin­ci­ples and meth­ods reli­ably to the facts of the case.

Cannabis Sativa L. is an extremely com­plex organ­ism which shares many fea­tures in com­mon with many other plants. We are informed by Ivan Ross in “Med­i­c­i­nal Plants of the World, Chem­i­cal Con­stituents, Tra­di­tional and Mod­ern Med­i­c­i­nal Uses” Vol­ume 3 that over 500 known chem­i­cal con­stituents have been iso­lated and iden­ti­fied in mar­i­juana. We are informed by Robert Thorne in “How Many Speci­cies of Seed Plants Are There”, Taxon 51, August 2002, that there are con­ser­v­a­tively 258,650 known seed bear­ing plants. If we refer to the Ency­clo­pe­dia Bri­tan­nica we see esti­mates of 200,000 to 500,000 known seed bear­ing plants. When we iden­tify mar­i­juana we are say­ing that the data we have col­lected is suf­fi­cient to iden­tify the mate­r­ial to the exclu­sion of any other plant or at least to the exclu­sion of a rea­son­able num­ber of other plants. We are say­ing that the com­bi­na­tion of fea­tures we have observed are only seen in mar­i­juana. Does the iden­ti­fy­ing offi­cer have the skill, edu­ca­tion, knowl­edge or train­ing to be able to artic­u­late why the fea­tures he is observ­ing would not be observed in that com­bi­na­tion in any other plant mate­r­ial? And has he col­lected suf­fi­cient data to deter­mine the mate­r­ial he has iden­ti­fied as mar­i­juana is in fact mar­i­juana. In Gar­nett the offi­cer sim­ply looked at the green veg­etable mate­r­ial and did not deter­mine if it was a plant mate­r­ial in a reli­able man­ner. The opin­ion does not address the pro­to­col the offi­cer fol­lowed in deter­min­ing if the mate­r­ial was a seed bear­ing mate­r­ial, if the mate­r­ial was a flow­er­ing plant or a dicotyle­don, if the offi­cer con­ducted a leaf archi­tec­tural analy­sis or an analy­sis of any poten­tial flower mate­r­ial in the evi­dence. In the words of Rule 702, is it true that “The tes­ti­mony is based upon suf­fi­cient facts or data?”, to say that this com­bi­na­tion of fea­tures would be observed in no other plant. And what fea­tures are we talk­ing about. Green veg­etable mate­r­ial? Unique odor?

How does one deter­mine if the odor the offi­cer is detect­ing is unique to mar­i­juana? Does the offi­cer have the expe­ri­ence, train­ing, skills or edu­ca­tion to say that the odor that he is detect­ing (what­ever that is) is unique to mar­i­juana. The offi­cer may very well tes­tify that he has never smelled any­thing else that smells like mar­i­juana. How does one test that state­ment? The officer’s olfac­tory senses can be tested but have they been tested? Is there suf­fi­cient data from the olfac­tory analy­sis from the offi­cer that the court can deter­mine the admis­si­bil­ity of the evi­dence or even the weight of the evi­dence? In other words, is the tes­ti­mony the prod­uct of reli­able prin­ci­ples and meth­ods and did the offi­cer prop­erly apply those prin­ci­ples and meth­ods to the facts of the case. It would be vir­tu­ally impos­si­ble for the trial court to test the officer’s olfac­tory senses and I am not aware of any police agency that tests for olfac­tory senses in its offi­cers. Flem­ming et. al. in “Top­ics in Het­e­ro­cyclic Chem­istry”, Vol­ume 10, 1–42 (2007) inform us that there are 120 susquiter­penoid and ter­penoid com­pounds which com­pose the mate­ri­als which are detected as the “odor” of mar­i­juana. Any Google search of sites that pur­port to sell mar­i­juana or its seeds for pro­duc­tion adver­tise their prod­ucts accord­ing to effect on the body, appear­ance and odor. It is rea­son­able to believe that envi­ron­men­tal impact will alter the qual­i­ta­tive and quan­ti­ta­tive enve­lope of the chem­i­cal com­po­si­tion of Cannabis Sativa L.. One need only look at the results of the use of such prod­ucts as Mir­a­cle Grow on a home gar­den to real­ize that envi­ron­men­tal fac­tors play a very big role in plant com­po­si­tion. The Court may very well be left with no way of deter­min­ing if the odor detec­tion part of the mar­i­juana analy­sis pro­to­col is reli­able? And the Court would do well to ask this, “Why does or did the North Car­olina Crime Lab ever con­duct a mar­i­juana analy­sis if an offi­cer need only smell the green veg­etable mate­r­ial to prove it is mar­i­juana”? It defies logic.

D.W. John­son and J.W. Gunn in Dan­ger­ous Drugs, Adul­ter­ants, Dilu­ents and Decep­tion in Street Sam­ples” , Jour­nal of Foren­sic Sci­ence, 1972 found that up to 20% of the alleged mar­i­juana sam­ples sub­mit­ted to the US Depart­ment of Justice’s Bureau of Nar­cotics and Dan­ger­ous Drugs lab were not in fact mar­i­juana. Though I have not found any more recent data describ­ing the occur­rence of coun­ter­feit mar­i­juana being sub­mit­ted by law enforce­ment offi­cers as mar­i­juana to crime labs, North Car­olina Gen­eral Statutes accept that coun­ter­feit con­trolled sub­stances are in fact a very real prob­lem and are deemed to be ille­gal. We must ask how many times law enforce­ment offi­cers sub­mit mate­r­ial they sus­pect of being mar­i­juana to a crime lab and the mate­r­ial is found to not be mar­i­juana. In other words, has any­one deter­mined the error rate for an analy­sis of sus­pected mar­i­juana con­ducted by a law enforce­ment offi­cer visu­ally and chem­i­cally ana­lyzed mar­i­juana or worse, sim­ply visu­ally deter­min­ing the green veg­etable mate­r­ial is marijuana.

In sum­mary, Fletcher meets Daubert (or in North Car­o­line Rule of Evi­dence 702) and leaves us very only with ques­tions, ques­tions that should be answered before a North Car­olina Court can accept an officer’s “iden­ti­fi­ca­tion” of mar­i­juana sim­ply by a visual or olfac­tory test. If a defen­dant wishes to stip­u­late that the mate­r­ial that was in his pos­ses­sion was mar­i­juana then the ques­tion is closed. How­ever, do we know even if defen­dants are capa­ble of deter­min­ing that they have not been sold coun­ter­feit mate­r­ial? Remem­ber that there is a new test of reli­a­bil­ity now in North Car­olina which must be sat­is­fied before sci­en­tific, tech­ni­cal or other spe­cial­ized knowl­edge will be admit­ted as evidence.

If sci­en­tific, tech­ni­cal or other spe­cial­ized knowl­edge will assist the trier-of-fact to under­stand the evi­dence or to deter­mine a fact in issue, a wit­ness qual­i­fied as an expert by knowl­edge, skill, expe­ri­ence, train­ing, or edu­ca­tion, may tes­tify thereto in the form of an opin­ion, or oth­er­wise, if all of the fol­low­ing apply:
1. The tes­ti­mony is based upon suf­fi­cient facts or data.
2. The tes­ti­mony is the prod­uct of reli­able prin­ci­ples and meth­ods.
3. The wit­ness has applied the prin­ci­ples and meth­ods reli­ably to the facts of the case.”
Street and/or court­room “iden­ti­fi­ca­tion” of green veg­etable mate­r­ial being mar­i­juana in no way meets that test.

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.

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.

A fre­quently asked ques­tion on the list­servs that I belong to basi­cally asks the fol­low­ing question:

In GC-FID use to quan­tify Blood Alco­hol Con­tent where EtOH is the tar­get ana­lyte, how does the machine arrive at the reported number?

The machine is called a Gas Chro­mato­graph with a Flame Ion­iza­tion Detec­tor (GC-FID). Typ­i­cally the sam­ple is intro­duced by way of head­space. The most fre­quently used tech­nique is tech­ni­cally called Sta­tic Head­space Isother­mal Wall Coated Open Tubu­lar Gas Chro­matog­ra­phy with Flame Ion­iza­tion Detector.

First a crash course on the Flame Ion­iza­tion Detec­tor (FID).

The FID is the part of the appa­ra­tus that quan­ti­tates the result of the chro­mato­graphic efflu­ent. Remem­ber that the num­ber one rule of GC-FID is that you must demon­strate with data (actu­ally prove) proper res­o­lu­tion first. The mea­sure of qual­i­ta­tive selec­tiv­ity (sep­a­ra­tion) must be proven before we can validly quantitate.

The FID actu­ally is a destruc­tive, mass count­ing device. It lit­er­ally burns all carbon-hydogen bonds (C-H bonds) from what comes off the col­umn. The cur­rent is sensed by an elec­trom­e­ter, con­verted to a dig­i­tal form and sent to an out­put device that gives us the peak. It counts the increase in the num­ber of ions between a cath­ode and a diode. A polar­iz­ing volt­age attracts these ions to a col­lec­tor located near the flame. This cur­rent is mea­sured with a high-impedance picoam­me­ter. An elec­trom­e­ter is an elec­tri­cal instru­ment for mea­sur­ing elec­tric charge or elec­tri­cal poten­tial dif­fer­ence. The response is pro­por­tional to the num­ber of C-H bonds. So, there­fore, iden­ti­cal amounts of methanol, ethanol, butanol and hexa­nol would not give equal area count responses. The FID itself emits an ana­log (con­stant) sig­nal; yet, it is reported through a com­puter sys­tem that is dig­i­tal in nature.

There are two main meth­ods of deter­min­ing quan­ti­ta­tion when it comes to GC-FID: (1) Peak height, and (2)Peak area.

Peak Height versus Peak Area

Peak Height ver­sus Peak Area

Peak height is an anti­quated method of quan­tifi­ca­tion that is not used a lot. It harkens back to a time when there were no com­puter pro­grams. Lab­Corp and cer­tain labs in Cal­i­for­nia still use the peak height method. Both are based upon the stan­dard dose response cal­i­bra­tion curve.

In order to deter­mine either peak height or peak area, the cru­cial bound­ary is the baseline.

The baseline and its determination is a crucial bound

The base­line and its deter­mi­na­tion is a cru­cial bound

It is essen­tial to remem­ber that all detec­tors have ana­lyt­i­cal noise. There is no way of record­ing zero. No machine can mea­sure true zero. There is always going to be an off­set. As an aside, this is why on a cal­i­bra­tion curve zero (the ori­gin) can­not be used as a legit­i­mate data point in deter­min­ing the slope which gives us the quantitation.

While there is a peak, there is, of course, sig­nal. As a result dur­ing the act of detec­tion there is no base­line per se. Instead, the com­puter uses an algo­rithm based upon prior input before the peak and “guesses” what the base­line would be if there was no sig­nal. As this is the computer’s guess, is it is sub­ject to inter­pre­ta­tion and error.

When we use the peak area method of quan­ti­ta­tion, then the ques­tion becomes not only where is the base­line, but where does the com­puter begin to cal­cu­late the begin­ning and end­ing of a peak. This is so very important.

As you can see in the above there are ver­ti­cal hash marks that are labeled “start” and “stop,” these are called “tick marks.” This is the other perime­ter that deter­mines peak area.

So in sum the deter­mi­na­tion of the peak area is a func­tion of 3 bounds: (1) the base­line and (2) start­ing tick mark, and (3) end­ing tick mark. Think of them as bound­ary marks.

There are two types of inte­gra­tion: (1) auto-integration and (2) man­ual integration.

If the but­ton pusher uses auto-integration, then the deter­mi­na­tion of the base­line will be cal­cu­lated the same through­out the run. This is what should be done (pro­vided that the use of the auto-integration events is sta­tic and equally applies to the cal­i­bra­tors and the unknowns that are tested and is part of a truly val­i­dated method).

On the other hand, one can use man­ual inte­gra­tion events to manip­u­late the data. This is a func­tion of the end user. This is typ­i­cally a one-time event in that only one chro­matogram is manip­u­lated. It may be some­thing that can be dis­cov­ered by look­ing at the chro­matogram or it may be utterly undis­cov­er­able unless you have the raw data. (Here is a video that shows this con­cept: The case for raw data: “Inte­gra­tion” in Gas Chro­matog­ra­phy: How to make an inno­cent per­son guilty in a DUI case by manip­u­lat­ing the soft­ware Actu­ally at Axion, we dis­cov­ered another 3 ways of manip­u­lat­ing the raw data so it would be undetectable.)

The key func­tion of all of this is that every­thing remain the same in order for the result to be valid. The inte­gra­tion events must be sta­tic in order for the quan­tifi­ca­tion to be valid.

When look­ing at the EtOH peak:

The unknown that is your client’s sam­ple must use the same inte­gra­tion perime­ters as the cal­i­bra­tors. The bound­ary marks used to deter­mine the peak area for the cal­i­bra­tors in their indi­vid­ual chro­matograms must match that of the unknown. If the base­line remains the same, but the tick marks are wider for the cal­cu­la­tion of the unknown than the cal­i­bra­tors, then this will over-report the Blood Alco­hol Con­tent (BAC). The con­verse to this is true as well. If the tick marks for the cal­i­bra­tor and the unknown remain the same, but the base­line for the unknown is lower than the cal­i­bra­tor, it will over-report the BAC. The con­verse is true as well.

Auto-integration versus manual integration

Auto-integration ver­sus man­ual integration

Above is an exam­ple of how the auto-integration leads to less area and how the man­ual inte­gra­tion leads to more area. More area equates a higher BAC if this is per­formed on the EtOH peak.

What fur­ther com­pli­cates this is the use of the Inter­nal Stan­dard (ISTD). The inte­gra­tion events for the ISTD must also remain sta­tic. A peak is a peak is a peak. The same bound­ary marks apply. How­ever, with respect to the ISTD, the ISTD amount is inversely related to the EtOH. If there is too much ISTD cal­cu­lated, then the BAC will be ratio reduced. The con­verse is true as well.

How can you tell if any of this is done? Well, maybe you can and maybe you can’t as dis­cussed above and in the video. If you trust the lab­o­ra­tory, and if they report the base­line and the tick marks on all of the chro­matograms, you can sim­ply use a light­box or even hold it up to a light with the cal­i­bra­tors and the unknowns on top of one another and see if the bound­ary marks are all treated the same.

Bot­tom line take away: Con­sis­tent and appro­pri­ate inte­gra­tions are sci­en­tif­i­cally defen­si­ble. Incon­sis­tent or inap­pro­pri­ate inte­gra­tions are dif­fi­cult to defend. All QC, Ver­i­fiers and Unknowns must be treated in a con­sis­tent (same) man­ner and per a val­i­dated method. Oth­er­wise, you have a non-validated result.

The take away ques­tion: What does your lab­o­ra­tory do when it reports out it’s BAC?

In the past, we have blogged on the severe lim­i­ta­tions on pat­tern recog­ni­tion as a foren­sic sci­ence dis­ci­pline. (Pat­tern Recog­ni­tion is it Sci­ence or an Art?). In fact, the National Research Coun­cil of the National Acad­emy of Sci­ences pointed to one form a pat­tern recog­ni­tion as being most prob­lem­atic: foren­sic odontology.

Most broadly defined, it is the prac­tice of apply­ing den­tal prin­ci­ples to the legal world. It can be used in mass dis­as­ter events to help iden­tify the dead. In the court­room, foren­sic odon­tol­ogy is pre­dom­i­nantly in the form of bite mark evidence.

Foren­sic odon­tol­ogy has been with us as admis­si­ble evi­dence in the United States since 1849 which is nearly 50 years longer than fin­ger­prints. J.W. Web­ster was con­victed for the mur­der of George Park­man after his incin­er­ated remains were iden­ti­fied by Nathan Coo­ley. Coo­ley was a den­tist who had made par­tial den­tures for Park­man. Coo­ley came into court and iden­ti­fied the charred remains of the body and con­cluded source because he phys­i­cally took the den­tures and placed them into the casts. He thought they were a “per­fect match.” It was the iden­ti­fi­ca­tion of the body that was accom­plished by the com­par­i­son of these den­tures to the casts alone that lead to con­vic­tion. Web­ster was put to death. A more famous case of where foren­sic odon­tol­ogy was used is that of ser­ial killer Ted Bundy who left a bite mark on the but­tock of a vic­tim, which helped secure his con­vic­tion in 1978. He too was exe­cuted by the government.

Like the appli­ca­tion of most pat­tern recog­ni­tion dis­ci­plines there are large issues.

  • In the court­room there are no spe­cific require­ments for prac­tic­ing foren­sic odon­tol­ogy. In fact, one does not even need to be a prac­tic­ing dentist.
  • The analy­sis under­taken is largely arbi­trary and sub­ject to the whim of each exam­iner. There are no uni­ver­sally accepted or prac­ticed pro­to­cols or instruc­tions as to how the analy­sis must be under­taken (no standards).
  • There is insuf­fi­cient study that the tech­niques used can cor­rectly iden­tify spe­cific or unique source, mean­ing that the marks left (the unknown) can be traced and attrib­uted uniquely to one spe­cific source.
  • A person’s den­tal pro­file changes over time.
  • Fre­quently there is unequal appli­ca­tion of force in the real world. The bite itself made in the real world and in uncon­trolled con­di­tions is totally dif­fer­ent than the exem­plars that are taken with equal appli­ca­tion of force and under con­trolled conditions.
Wax Exemplar of Ted Bundy

Wax Exem­plar of Ted Bundy

  • If the impres­sion that is the unknown is left on skin, the medium (the skin) can change over time and as the bite-mark heals or the body decom­poses, and there­fore dis­tort the orig­i­nal impres­sion left. What few stud­ies that have been com­pleted were not done on humans, but rather on pigskin. Pigskin and human skin behave in dynam­i­cally dif­fer­ent ways due to dif­fer­ences in elasticity.
Bite mark impressions can change over time

Bite mark impres­sions can change over time

  • Each den­ti­tion can pro­duce vari­able impres­sions and can change based on pres­sure and sur­face of contact.
Different impressions left based upon application of force

Dif­fer­ent impres­sions left based upon appli­ca­tion of force

  • It is not highly reg­u­lated or mon­i­tored and has vir­tu­ally non-existent Qual­ity Assur­ance safeguards.

In fact, as reported in the New York Times story “Evi­dence From Bite Marks, It Turns Out, Is Not So Ele­men­tary,” the rate of false pos­i­tives is alarm­ing (up to 65 per­cent as ref­er­enced in the arti­cle that refers to a study)

Crit­ics of bite mark com­par­i­son cite the case of Ray Krone, an Ari­zona man con­victed of mur­der on bite mark evi­dence left on a woman’s breast. He was 100% pos­i­tively iden­ti­fied as being the only source for the bite mark. He was later exon­er­ated by DNA. Sim­i­larly, Roy Brown was con­victed of mur­der due in part to bite-mark evi­dence, and freed after DNA test­ing of the saliva left in the bite wounds matched some­one else.

The very basic tech­nique and analy­sis employed by most foren­sic odon­tol­o­gists is as follows:

  • Bite marks are pho­tographed with a scale
  • Bite marks on skin are taken over repeated intervals
  • Casts of impres­sion are taken
  • Impres­sions are traced onto transparencies
  • Casts of sus­pects teeth are taken
  • Com­par­i­son between sus­pect cast and bite mark

And then there are cases of down­right fail­ures of human integrity where fraud is com­mit­ted such as this story:

Video Shows Con­tro­ver­sial Foren­sic Spe­cial­ist Michael West Fab­ri­cat­ing Bite Marks

Accord­ing to that report:

On Aug. 9, The Huff­in­g­ton Post reported on the case of Leigh Stubbs, a Mis­sis­sippi woman serv­ing a 44-year sen­tence for assault and drug charges. Stubbs was con­victed in large part due to the tes­ti­mony of Michael West, a dis­graced bite mark spe­cial­ist. Though West has been largely dis­cred­ited, pros­e­cu­tors and state offi­cials in Mis­sis­sippi (and to a lesser extent in Louisiana) con­tinue to defend con­vic­tions won based on his testimony.

In Stubbs’ case, West pre­sented two key pieces of evi­dence. The first involved the bite mark wiz­ardry that made him famous, and then infa­mous: West claimed to have found bite marks on alleged vic­tim Kim Williams that med­ical per­son­nel hadn’t seen. He then used a den­tal mold of Stubbs’ teeth to per­form an analy­sis on the marks, and would later tes­tify that it was a “prob­a­bil­ity” that Stubbs had bit­ten Williams.

FABRICATING EVIDENCE

On Wednes­day, foren­sic spe­cial­ists Mike Bow­ers and David Aver­ill posted a video record­ing of West’s exam­i­na­tion of Williams on their site, Bitemarks.org. In his ini­tial exam­i­na­tion, West claims to have “missed” the evi­dence of a bite mark. He tes­ti­fied he found it in a a sub­se­quent exam­i­na­tion per­formed days later. That exam­i­na­tion is depicted in the video below. Note that at the 50-second mark, a bite mark appears in Williams’ skin, seem­ingly out of nowhere.

 
Lions and Tigers and Bears... Verifiers, Calibrators and Controls... Oh my!
Lions and Tigers and Bears… Ver­i­fiers, Cal­i­bra­tors and Con­trols… Oh my!

Some­times a crim­i­nal defense attor­ney can at times feel like Dorothy in the Wiz­ard of Oz in that we are trans­ported from the rel­a­tive safety of home (the court­room) to the weird world of Oz (the lab­o­ra­tory). There are unusual and often times con­flict­ing phrases and words that seem to defy com­mon sense. Some­times, words and phrases are used inter­change­ably and with lit­tle appar­ent dis­tinc­tion. In this post we will exam­ine a decon­vo­lute some of these impor­tant terms.

Qual­ity Con­trol- We cov­ered the con­cept of Qual­ity Con­trol (QC) before. QC is strictly speak­ing a process that is used to con­struct the cal­i­bra­tion curve that our knowns are tested and then an unknowns are tested against. This is typ­i­cally per­formed in the begin­ning of the run.

Standards/Controls- is a ref­er­ence solu­tion or test solu­tion used for assess­ment of the per­for­mance of an ana­lyt­i­cal pro­ce­dure. A rig­or­ously tested and high qual­ity known ana­lyte at a cer­tain con­cen­tra­tion is known as a Cer­ti­fied Ref­er­ence Mate­r­ial (CRM). CRMs are gen­er­ally gov­erned by ISO Guide 34:2009. NIST makes its own ser­vice marked brand of CRMs named Stan­dard Ref­er­ence Mate­ri­als (SRMs). CRMs and SRMs should have state­ments of cal­i­bra­tion (pre­ci­sion) and boas (accuracy).

Cal­i­bra­tor- Often it is in the begin­ning of a run. What makes a stan­dard or con­trol a cal­i­bra­tor is that it is placed before the unknowns are tested. The cal­i­bra­tors are run in a series over the hoped for lin­ear dynamic range. The response from the test­ing of the cal­i­bra­tors is plot­ted on a sig­nal ver­sus con­cen­tra­tion y-x axis graph. A line is drawn along the data points with the R2 cal­cu­lated to deter­mine if the response is lin­ear. Cal­i­bra­tors are used to con­struct the cal­i­bra­tion curve. It is the QC of a quan­ti­ta­tive process. A cal­i­bra­tor is a solu­tion hope­fully from a trace­able source and hope­fully a CRM/SRM with a known amount (con­cen­tra­tion) of ana­lyte of inter­est that is hope­fully pure and only con­tains that ana­lyte of inter­est. It is placed within the batch of the run as part of the QC pro­ce­dures to insure that the ana­lyt­i­cal instru­ment is detect­ing the known within an estab­lished stated and often times arbi­trary range of values.

Ver­i­fier- Often it is in the mid­dle of a run or at an end of the run. What makes a stan­dard or con­trol a ver­i­fier is that it is placed among the unknowns are tested after the run. It too con­tains a known ana­lyte at a known con­cen­tra­tion. It is placed within or at the end of the run to insure that the ana­lyt­i­cal instru­ment is detect­ing the known within an estab­lished tol­er­ance through­out the test­ing of unknowns. Think of it as a check. If the test­ing method has a scheme where the lab­o­ra­tory places a ver­i­fier amongst the unknowns tested, then this is not a func­tion of qual­ity con­trol, but rather an act of verification.

There is a big dif­fer­ence. QC data (where a series of cal­i­bra­tors are used over a range of con­cen­traions) is used to estab­lish a cal­i­bra­tion curve. The data is inputted and R2 value is cal­cu­lated and then the data adjusted to make it fit. Ver­i­fi­ca­tion data sim­ply tests at one point on the cal­i­bra­tion curve already estab­lished, that data is then eval­u­ated by a human, not a machine, and no adjust­ment is made to cor­rect for bias if the ver­i­fier data result does not per­fectly fit against the cal­i­bra­tion curve expected result. The cal­i­bra­tion curve is not altered based upon this new data point. Test­ing con­tin­ues if the ver­i­fi­ca­tion data is within an arbi­trary range. Again, even if the ver­i­fi­ca­tion data shows appre­cia­ble bias, noth­ing is done about it. Unfor­tu­nately, a lot of crim­i­nal lab­o­ra­to­ries in an effort to save money (which really is not that much) make their own in-house ver­i­fier solu­tions. I call this home brew or lab­o­ra­tory moon-shining. The dif­fi­culty with mak­ing it on your own is two fold: (1) You may make it wrong (impu­ri­ties or impre­cise or inac­cu­rate con­cen­tra­tion lev­els), and (2)it’s like the fox guard­ing the hen house in that you are trust­ing the lab­o­ra­tory to guard itself.

Home brew is not a good idea

Home brew is not a good idea

You must under­stand the dif­fer­ence between cal­i­bra­tors and verifiers.

 

There is a large dif­fer­ence between a sin­gle col­umn analy­sis and a dual col­umn analy­sis when it comes to the abil­ity to most cor­rectly iden­tify and quan­ti­tate an unknown in the sci­en­tific world.

In foren­sic sci­ence, we are con­stantly test­ing unknowns. What is meant by this is that we have a sam­ple that is seized from a crime scene or from a per­son, but we don’t know what it con­tains. For exam­ple, in blood analy­sis for EtOH in an alleged DUI case, we have a sam­ple of blood that is taken from the accused, but just by look­ing at it, we can­not know if there is even ethanol in it, and even if present, how much there is. We need to ana­lyze it using instru­men­ta­tion in a sci­en­tific manner.

We can't tell if there is EtOH in this sample just by looking at it
We can’t tell if there is EtOH in this sam­ple just by look­ing at it

When we are look­ing to be sci­en­tific about our analy­sis we are look­ing to be as spe­cific as pos­si­ble, and try­ing not to be merely selec­tive. There is a large and impor­tant sci­en­tific dif­fer­ence between being selec­tive and spe­cific. As we wrote before on this blog: Metrol­ogy in Quan­ta­tive Mea­sure: Is it Spe­cific or Selec­tive or Neither…

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, or codetermination.

So, we search for he most spe­cific form of analy­sis. In the world of DUI for EtOH, the gov­ern­ment typ­i­cally set­tles for Head­space gas Chro­matog­ra­phy with Flame Ion­iza­tion Detec­tor (HS-GC-FID). GC-FID is not the most spe­cific test avail­able for EtOH exam­i­na­tion as there is Gas Chro­matog­ra­phy with Mass Spec­trom­e­try (GC-MS) for exam­ple which is much more selec­tive and bor­ders on spe­cific when it comes to EtOH analy­sis, but for what­ever pol­icy rea­son, the gov­ern­ment chooses not to do the most scein­tific thing which is to use the most spe­cific assay avail­able. There is no sci­en­tific rea­son not to test for EtOH on the most spe­cific assay avail­able. In fact, it could be legit­i­mately argued that rely­ing on GC-FID instead of GC-MS for EtOH deter­mi­na­tion and quan­tifi­ca­tion is not sci­en­tific as GC-MS exists and is read­ily avail­able. How­ever, that is a post for another day.

As we are seemly inex­plic­a­bly stuck with the sci­en­tific step-sister of analy­sis in GC-FID as opposed to GC-MS, we must look at ways that the gov­ern­ment chooses to employ GC-FID to see whether or not as an assay it is valid. As our last series of posts “Method Val­i­da­tion for Lawyers” revealed, there is power in the words “valid” and “valid­ity.” With­out hav­ing a truly valid method that has been proven to be suit­able for its intended pur­pose, we can­not have a valid result.

Some foren­sic lab­o­ra­to­ries choose to use a con­fig­u­ra­tion in GC-FID that is known as a sin­gle col­umn, sin­gle injec­tion setup. In this set up there is one installed col­umn and the ana­lyst makes one injec­tion (or the autosam­pler does) to test the sample.

An installed single column GC-FID setup
An installed sin­gle col­umn GC-FID setup

With­out any sci­en­tific doubt, a sin­gle col­umn method of analy­sis is not foren­si­cally or sci­en­tif­i­cally defen­si­ble or acceptable.

Remem­ber that when we use GC-FID, we can never achieve true speci­ficity, the most we can hope for is the pos­si­bil­ity of being merely selec­tive as demon­strated and proven through the res­o­lu­tion stan­dard (sep­a­ra­tion matrix/standard mix). The qual­i­ta­tive result is only based upon one cri­te­ria which is the reten­tion time. Reten­tion times through any given col­umn are not unique to one spe­cific volatile organic com­pound (VOC) to the exclu­sion of every thing else in the uni­verse. Hence, we have the often repeated phrase that all legit­i­mate tech­ni­cally trained chro­matog­ra­phers know and can recite in their sleep—the lim­i­ta­tion of GC-FID is that the reten­tion time is merely char­ac­ter­is­tic of a com­pound and cer­tainly not adju­dica­tive or con­fir­ma­tory of the speci­ficity of that com­pound— mean­ing a peak at a given reten­tion time is not a unique qual­i­ta­tive mea­sure (to the exclu­sion of every other com­pound in the universe).

Don’t take just my word for it con­sider the following:

As ven­er­ated Pro­fes­sor Harold McNair, PhD writes in his book, Basic Gas Chro­matog­ra­phy,

Reten­tion times are char­ac­ter­is­tic of a GC sys­tem, but they are not unique, so GC reten­tion times can­not be used for qual­i­ta­tive confirmation.

He fur­ther writes:

Iden­ti­fi­ca­tion of an unknown by com­par­i­son to reten­tion times using stan­dards that forms the basis of the qual­i­ta­tive analy­sis [in GC-FID analysis].

He con­cludes:

Unfor­tu­nately, GC sys­tems can­not con­firm the iden­tity or struc­ture of any peak. Reten­tion times are related to par­ti­tion coef­fi­cients (Chap­ter 3); and while they are char­ac­ter­is­tic of a well-defined sys­tem, they are not unique.

How do we acknowl­edge this lim­i­ta­tion in the lack of speci­ficity and try to mit­i­gate it?

We can add a dif­fer­ent col­umn and ana­lyze the sam­ple con­cur­rently on both columns. As we learned in our post What is a Gas Chro­matog­ra­phy col­umn and why should I care?, the col­umn, if prop­erly selected and prop­erly installed, is what pri­mar­ily causes the sep­a­ra­tion of the var­i­ous VOCs to occur. What we do is select two dif­fer­ent columns with two dif­fer­ent sta­tion­ary phases. We attach a y-splitter that will take the sin­gle injec­tion made into the injec­tor port and divide the sam­ple into two dif­fer­ent path­ways with one part of the sam­ple going to one col­umn for analy­sis and the sec­ond part going to another col­umn for analysis.

A y-splitter
A y-splitter splits the same sam­ple injec­tion and sends the parts to two dif­fer­ent columns for analysis

The strength of a well-designed dual col­umn analy­sis method where the sta­tion­ary phase is dif­fer­ent between the two columns is that this dif­fer­ence in the sta­tion­ary phase will cause dif­fer­ent sep­a­ra­tion of the ana­lytes (both in terms of reten­tion time and pos­si­bly even elut­ing order) as proven by the chro­matograms of the analy­sis of the res­o­lu­tion stan­dard (sep­a­ra­tion matrix/standard mix). This dif­fer­ent elut­ing order and dif­fer­ent reten­tion times only min­i­mizes, but does not entirely elim­i­nate the pos­si­bil­ity of co-elution as again, the resolv­ing (sep­a­rat­ing) power of the method is only deter­mined by one non-unique mea­sure which is the two reten­tion times. Even though there is a change in the elut­ing order poten­tially and the reten­tion times are dif­fer­ent based upon the sta­tion­ary phase com­po­si­tion, again, it must be empha­sized that the basic lim­i­ta­tion of GC-FID remains in that a reten­tion time is merely char­ac­ter­is­tic of the ana­lyte, but is cer­tainly not confirmatory.

A schematic of a dual column GC-FID. Note the change in the eluting order and retention time among the columns
A schematic of a dual col­umn GC-FID. Note the change in the elut­ing order and reten­tion time among the columns

This is why dual col­umn is referred to as the poor man’s Mass Spec as it has a more orthog­o­nal approach towards the qual­i­ta­tive mea­sure than does a sin­gle column.

How the cur­rent trend of the crime lab­o­ra­tory using a dual col­umn GC-FID is alarm­ingly unscientific.

What is most alarm­ing to me is the trend that is devel­op­ing across the United States where the sec­ond col­umn is not being used for quan­tifi­ca­tion at all. In this trend that I see sweep­ing all across the US, the sec­ond col­umn is merely being used as a “con­fir­ma­tory” col­umn in that if the reten­tion time matches with the standards/controls in the Qual­ity Con­trol sam­ples, then it is pre­sented as “ver­i­fied” in terms of the qual­i­ta­tive mea­sure by sim­ply that sec­ond col­umn match­ing reten­tion time with the knowns that act as the standards/controls. As we explain above, that is a dan­ger­ous and unsci­en­tific approach.

Installed dual column GC-FID

Installed dual col­umn GC-FID

The rea­son that this is so impor­tant given the above con­text (there are other rea­sons that it is alarm­ing, but I want to stick to this ref­er­ence) is that with­out the sec­ond col­umn giv­ing a quan­ti­ta­tive mea­sure, we can­not fairly elim­i­nate co-elution (where two com­pounds elute at the same time, but only get iden­ti­fied and quan­ti­tated as one com­pound) because if the sec­ond col­umn is used to also quan­tify it will serve as an indi­rect deter­mi­na­tion of whether or not there is co-elution. If the sec­ond col­umn also pro­vides a quan­tifi­ca­tion of the unknown, we would exam­ine the pre­ci­sion of the quan­ti­ta­tive results (how closely the num­bers agree among those given by col­umn A and those of col­umn B– do the A’s match the B’s?). If there is co-elution that was “dis­cov­ered” by the dual col­umn approach, then we would expect to see impre­ci­sion between these num­bers (the A’s don’t match the B’s).

Now that they have decided as an orga­ni­za­tion to not quan­ti­tate on the sec­ond col­umn, we have lost a vital part of qual­ity con­trol and the lab super­vi­sor has lost a pow­er­ful tool of qual­ity assur­ance. It is just bad science.

Fur­ther, there is no legit­i­mate sci­en­tific rea­son for quan­ti­at­ing on the sec­ond col­umn. If you are doing good qual­ity work ad your meth­ods and instru­ments are in con­trol, then your num­bers should agree.

It’s not a time thing.

As it is a sin­gle injec­tion y-splitter dual col­umn analy­sis any­way, the amount of time it takes to make a cal­i­bra­tion curve, eval­u­ate it and then incor­po­rate it into the soft­ware for one col­umn is vir­tu­ally the same amount of time to do the same on col­umn 2. There is vir­tu­ally no added time. It makes no sense from a sci­ence point-of-view.

So in con­clu­sion, we can fairly con­clude the following:

  • Sci­en­tif­i­cally, we always want to test our unknowns on the most spe­cific assay available.
  • GC-FID is not the most spe­cific assay available.
  • Lab­o­ra­to­ries that use sin­gle col­umn GC-FID as their method of analy­sis do not pro­duce foren­si­cally or sci­en­tif­i­cally defen­si­ble or accept­able results.
  • Lab­o­ra­to­ries who use dual col­umn GC-FID that do not quan­ti­tate on both their columns do not pro­duce foren­si­cally or sci­en­tif­i­cally defen­si­ble or accept­able results.
  • Good sci­ence is not always prac­ticed in the mod­ern state crime laboratory.