Interference in the Quantitation of Methylated Arsenic Species in Human Urine

Published: Journal of Analytical Toxicology, Volume 24, Number 1, January/February, pp 73-75

D⁹-Tetrahydrocannabivarin (THCV) as a Marker of Cannabis Use: Is the Methodology Forensically Acceptable?

In a recent Letter to the Editor (1), the authors presented certain data on the detection of 11-nor-9-carboxy-D⁹-tetrahydrocannabivarin (THCVCOOH) in urine specimens. It was implied, by analogy with the metabolism of D⁹-tetrahydrocannabinol (THC), that THCVCOOH is the primary metabolite of D⁹-tetrahydrocannabivarin (THCV) and, given that THCV occurs only in marijuana or related products, the identification of THCVCOOH in urine must imply the ingestion of marijuana in some form.

To the casual reader, familiar with the theoretical aspects of cannabinoid chemistry and metabolism, the authors’ findings and conclusions might seem logical, if not downright obvious. However, in view of the fact that there is absolutely no published literature on the metabolism of THCV in humans (or in any other animal, for that matter) and neither is there any previous publication on the identification of THCVCOOH in urine, it is important to review the actual data presented in the context of the specific claims being made by the authors, specifically the implication that the detection of this metabolite in urine is, to quote, “...a positive identification that marijuana (or a related product) has been ingested sometime (within days) prior to collection of the urine...”

What specific evidence is presented to support these claims? Figure 3 contains GC–MS data that show the ions expected from the methyl derivative of the THCV metabolite. The text and the caption to Figure 3 indicate that the specimen analyzed was “urine from a cannabis user”, but no basis is presented for this claim. If we assume that the urine was in fact from a user of cannabis, what were the alleged use habits of this person? How much and how frequently did he use? How long after the last alleged use was the specimen obtained? There are many other relevant questions that could be posed, but I will omit these in the interests of brevity. However, if the urine was obtained from an individual who was an alleged illegal recreational user, we must bear in mind that any answers to these questions that might have been obtained from the donor would need to be considered with the greatest circumspection. Later in the text we are informed that another urine specimen that showed the same characteristic ions came from a donor who had a prescription for Marinol, but later admitted that he had smoked marijuana. These two specimens, neither of which can be acceptably documented, appear to be the sole basis for the authors’ conclusions.

Where are the controlled-administration studies? The metabolism and excretion kinetics? The negative controls? The passive-exposure studies? The answer is that there are none. There is not a single published study that has been performed (by the present authors or by other researchers) that directly demonstrates that smoking, or otherwise ingesting, marijuana or related products leads to the excretion of detectable amounts of THCVCOOH in urine. There is not a single peer-reviewed publication that reports on the identification of this metabolite in urine and specifically correlates its presence with the documented ingestion of marijuana that was shown to contain a given level of THCV.

Most importantly, in the present report (1), there is absolutely no indication of the level (i.e., ng/mL) of THCVCOOH that was identified in the two urines that were tested. No data were presented on the THC metabolite that we are to assume was also present in the specimen referred to in Figure 3. What levels of THCCOOH were present, and what ratio of the two metabolites can be expected in such urines?

The statement that the detection of THCVCOOH implies use “within days” is extremely vague. If we assume that means within a few days, and we interpret few in its generally accepted context of between three and seven, then we might conclude that the claim is that this procedure detects marijuana use up to a week after last exposure. However, in the absence of any data on levels of THCVCOOH that can be detected in urine, and with no controlled-dose studies on the time course of metabolism and excretion, any claims relating to detection period are unscientific and unsubstantiated. It would appear that these two homologues are metabolized and excreted similarly, but the rate of conversion of homologues frequently differs markedly. Given the parallels being implied by the authors concerning the alleged similarity between the metabolism and excretion of THC and THCV, one could make the claim that mere detection of THC metabolite in urine at any level implies that marijuana use must have occurred within seven days prior to the specimen being taken. I am not going to cite the extensive literature on this subject, but will simply state that such a conclusion is clearly preposterous. Even after more than 20 years of research on THC metabolism and excretion in humans, studies are still being performed (2) on detection periods after last use. It is interesting that in that study, THC metabolite was detectable in urine for seven days after single exposure. I am wondering if that is the basis for the claim being made in the present study (1) concerning the detection period for THCV metabolite?

What has been presented in the present report (1) are the observations that THCVCOOH can be detected in urine by GC–MS and that there are indications that, as expected, this seems to correlate with exposure to marijuana at some prior time. As such, this report describes an approach that appears to be scientifically acceptable for research purposes. However, for a number of reasons outlined here, it is not yet forensically acceptable for the unequivocal identification of marijuana use, especially time of last use.

This conclusion was recently supported by a Court decision rendered in a case heard in the U.S. District Court for the Central District of California [Case no. CR 97-997(A)-GHK]. In his ruling, the judge found that this procedure did not satisfy the requirements under Daubert (Daubert v. Merrel Dow Pharmaceuticals, 509 U.S. 579, 1993), which standard has effectively replaced the old Frye standard in Federal Rules of Evidence. The finding referred to the nonacceptability of the method as presented for identifying marijuana use per se and did not even progress to consider the more problematic claim of its ability to determine exposure “within days”. Finally, the judge pointed out that the mere publication of a procedure in a scientific journal does not in and of itself satisfy the requirements that are inherent in Daubert acceptability, and that particularly applies to reports that may be hastily submitted for review using such routes as Letters to the Editor.

The Author’s Reply:
This is in response to Dr. Brian Sedgwick’s comments on our recent Letter to the Editor (1) and the forensic acceptability of the methodology used to detect the ingestion of a cannabis product with or without Marinol® through analysis of THCV metabolite in urine. Although Dr. Sedgwick has no problem with the science involved in the methodology, he is not ready to accept it for forensic purposes. Perhaps one of the main reasons for his being so reserved and his need to see the work of others to support our conclusion is his own lack of personal research experience with cannabis and the cannabinoids. Dr. Sedgwick did not acknowledge that Dr. Rodger Foltz has previously reported on the same issue (2).

Although I respect the judge’s conservative ruling on the issue, this author questions Dr. Sedgwick’s criticism of the data presented in our previous Letter (1), especially because Dr. Sedgwick wrote the following in his affidavit submitted for the same case mentioned in his Letter. In response to the presence of low levels of THCVCOOH in many urine specimens submitted weeks apart by the same individual, Dr. Sedgwick wrote “Given the total absence of information on the persistence of THCV in the body or of THCV-COOH excretion in the urine, these levels of around 1 ng/mL could represent a baseline level of THCV [sic] that persists indefinitely once an individual has been exposed directly to THCV.” I consider this to be a statement that no objective scientist should make.

Furthermore, in his criticism, Dr. Sedgwick failed to recognize that we have shown that THCV is a cannabinoid just like THC and only exists in the cannabis plant and products thereof; that Marinol does not contain THCV; that ingestion of Marinol does not produce THCVCOOH; that THCV is metabolized by human hepatocytes to produce THCVCOOH; that we did synthesize THCVCOOH and determine its structure and, therefore, did have a standard; that we showed that the THCVCOOH produced by the human hepatocytes was identical to the synthetic standard; and that we used negative controls in our testing. (In his Letter, he makes the reader believe that we did not use negative controls!)

Additional facts for the readers’ benefit are that THCV exists in just about all marijuana samples, but, just like other cannabinoids, its concentration relative to THC varies. However, drug-type cannabis samples have a low THCV:THC ratio (mostly < 1:20). Knowing that THCV is the C3 homologue of THC (C5) and, therefore, is less lipophilic, it follows that if the difference in lipophilicity is sufficient enough to influence excretion rate, one would expect C3 homologue to be excreted faster. Therefore, ingestion of a product containing THC and THCV in the relative amounts cited would lead to a much shorter detection time in urine after last ingestion for THCV metabolite relative to THC metabolite. If one assumes that THC metabolite is detectable in the urine of heavy marijuana users at LOD levels of 0.5–1 ng/mL for 6–8 weeks from last use, then the time of detection of THCV metabolite would be only for a “few days” given the much lower dose of THCV relative to THC. A few days would be defined as less than a week. That is the logic behind our conclusion that the detection of THCVCOOH (even at levels of approximately 1 ng/mL) by GC–MS would indicate the use of a cannabis-related product within days prior to specimen collection. Furthermore, if an individual stopped the use or ingestion of a cannabis-related product but started the use of prescribed Marinol, one would expect the ratio of THCVCOOH:THCCOOH to be lower and lower as the THCVCOOH continues to be excreted (and not replaced) while the THCCOOH continues to be formed in the body from the use of Marinol. The fact of the matter is that in the court case referenced by Dr. Sedgwick, the ratio of THCVCOOH:THCCOOH was essentially constant in several specimens collected from the subject over a period of 4–6 weeks. This is clearly inconsistent with the sole use of Marinol, which contains no THCV and was shown not to result in the formation of any THCVCOOH (1).

As far as passive inhalation goes, again, because of the relative amount of THCV to THC in the drug-type marijuana, it is unlikely that THCVCOOH would be detected even at 0.5 ng/mL when one considers that even under forced (unrealistic) passive inhalation conditions, THCCOOH was detectable by GC–MS at low nanogram-per-milliliter levels (3,4).

Regarding the peer review, I want the readers to know that in discussions with Journal of Analytical Toxicology staff, I was informed that our work presented in the previous Letter (1) was subjected to the normal peer-review process and that both reviewers recommended publication without revision. Furthermore, I submitted the information presented to the court to some of the most knowledgeable forensic toxicologists in the area of cannabinoids research (Drs. Rodger Foltz, Edward Cone, and Marilyn Huestis) for their comments, and none of them had any problem with the conclusion drawn from our work.

Again, I do not have a problem with the judge’s ruling in favor of the defendant, but I feel that Dr. Sedgwick should rethink his criticism of this work.

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