Does HHC-COOH in Blood Prove HHC Consumption? What 308 Forensic Cases Reveal

Answer: No. An HHC-COOH finding in whole blood, even when analytically identified, is not specific proof that the person consumed hexahydrocannabinol (HHC). In a 2023 primary investigation of 308 Danish forensic traffic-case blood specimens, 186 of 222 specimens positive for delta-9-tetrahydrocannabinol (Δ9-THC) but negative for parent HHC nevertheless contained the 9R epimer of HHC-COOH. Complementary experiments demonstrated that pooled human liver microsomes could form HHC-COOH while metabolizing Δ9-THC and 11-hydroxy-THC. The presence of a product compatible with more than one exposure pathway should not be converted into a categorical conclusion about which cannabinoid a particular person used.

The distinction matters whenever an allegation depends on identifying an HHC-containing product rather than ordinary THC exposure. The laboratory may be right about the chemical identity and still lack a uniquely attributable source. Falck Jørgensen and colleagues’ original 2023 study in Metabolites supplies unusually direct evidence: genuine forensic whole-blood samples, chromatographic separation of related cannabinoid compounds, and a controlled microsomal experiment examining alternative metabolic origins.

What forensic question did the researchers test?

The central question was whether human metabolism of Δ9-THC can generate compounds previously associated with HHC metabolism. HHC differs structurally from Δ9-THC by reduction of a double bond. The resulting HHC-related metabolites include 11-hydroxy-hexahydrocannabinol (11-OH-HHC) and 11-nor-9-carboxy-hexahydrocannabinol (HHC-COOH). These labels identify molecules and metabolic relationships; they do not themselves document the identity of a product consumed by an individual.

Earlier experimental observations raised a possibility that THC could undergo biological conversion to HHC-related compounds. The Copenhagen team tested that possibility in two complementary ways. First, they examined authentic forensic traffic-case blood specimens for Δ9-THC, parent HHC and the corresponding hydroxylated and carboxylated metabolites. Second, they placed selected THC compounds into an enzyme-containing human liver microsome preparation to see which products could appear without administering HHC.

Those components answer related but different questions. The blood survey shows what co-occurred in case specimens. The microsomal experiment tests whether a plausible biochemical source exists. Neither component reveals a driver’s exact drug history, contemporaneous driving behavior or individualized impairment.

Were these real blood tests, or calculated THC estimates?

They were analyses of actual whole-blood samples from 308 consecutive forensic traffic cases received from the Danish capital region in June and July 2023. The clinical and roadside reasons for obtaining each specimen were not controlled experimental exposures. A specimen’s inclusion as a forensic traffic case does not mean that the researchers independently established an offense, observed impaired driving, or knew which commercial product the person used.

The laboratory extracted cannabinoids from whole blood using protein precipitation and solid-phase extraction, followed by ultraperformance liquid chromatography–tandem mass spectrometry (UPLC-MS/MS). The instrument monitored analyte-specific precursor/product ion transitions. Measured chromatographic signals were compared with standards and internal-standard responses. These are chemical measurements in a sampled physical matrix, not a breath-alcohol conversion, an estimated blood alcohol concentration, or a pharmacokinetic prediction of THC at an earlier time.

The distinction between whole blood and other matrices is fundamental. Serum, plasma, oral fluid, urine and hair are not interchangeable with whole blood merely because an analyte has the same name. For the broader meaning of those compartments, see the site’s guide to biological matrices in forensic toxicology.

How were the 308 cases divided?

The original study divided the 308 specimens according to whether parent Δ9-THC and parent HHC were found at the specified analytical identification criteria. Of all cases, 222 were THC-positive/HHC-negative, ten were positive for both parent compounds, and 76 were negative for both. No specimen was parent-HHC-positive while parent-THC-negative.

That last detail is consequential. The study includes no parent-HHC-only comparison group in which one could empirically determine how commonly a particular HHC metabolite appears after exposure that excludes THC. Nor does parent-HHC negativity prove that a person never consumed HHC: parent drug may disappear while metabolites persist, depending on timing, exposure and measurement limits.

What did the study actually detect?

The table below reproduces the study’s counts, denominators and rounded proportions for the principal chemical identifications. Each percentage belongs to its own row. Positivity means that a chromatographic peak satisfied the authors’ identification criteria, not that a specific behavior or level of impairment was demonstrated.

The 9S form of 11-OH-HHC was identified in none of the 222 THC-positive/HHC-negative samples, one of the ten dual-positive samples, and none of the 76 parent-negative samples. The results therefore do not support treating all epimers or all metabolites as if they had identical detectability, prevalence or evidentiary significance.

Most notably, the 186 cases with 9R-HHC-COOH in the THC-positive/HHC-negative group are the basis of the study’s reported 84% figure. It is not an 84% prevalence across all 308 cases, not a predictive probability for an arbitrary member of the public, and not a sensitivity estimate for confirmed HHC ingestion.

Why does parent HHC negativity not settle the exposure history?

Parent compounds and metabolites may have different concentration–time profiles. A person may have encountered a substance earlier without its parent compound remaining detectable at collection. The study did not experimentally document each driver’s ingestion route, amount, product ingredients, elapsed time, or prior exposure. Consequently, a THC-positive/HHC-negative result is not a controlled proof of THC-only use in that one person.

But that limitation does not erase the more important biochemical result. The investigators separately showed the production of HHC-COOH under conditions in which Δ9-THC or 11-OH-THC—not administered parent HHC—was the supplied experimental substrate. The two components reinforce a source-attribution problem: the measured HHC-COOH molecule need not have a uniquely HHC-origin exposure history.

What concentrations were measured, and which were estimated?

For the 222 THC-positive/HHC-negative cases, the authors reported medians for major cannabinoids in whole blood, in ng/mL. They calibrated Δ9-THC, 11-OH-THC and THC-COOH with a six-point curve extending from 0.53 to 53 ng/mL. HHC-related quantitative results were less direct: at the time of analysis, calibration for those compounds used another analyte’s response curve and a response-correction factor, rather than compound-specific calibration throughout. The study explicitly labels their HHC-COOH concentrations as estimates.

These summary medians do not establish a fixed metabolite-to-parent ratio in every individual. Dividing the reported median 9R-HHC-COOH concentration of 1.4 by the median THC-COOH concentration of 21 gives approximately 7%; that is a comparison of two group medians, not a validated individual conversion factor. The source also says the minimum concentration for every compound was below its relevant lower limit of quantification.

The Δ9-THC and 11-OH-THC lower limits of quantification were 0.53 ng/mL, and the THC-COOH limit was 1.06 ng/mL. The authors extended corresponding limits to their HHC-related measurements under the specified response-factor approach. A reported non-detection, a below-quantitation observation and an accurately established zero are different analytical claims.

What did the human liver microsome experiments add?

The laboratory incubated Δ9-THC, 11-OH-THC, or THC-COOH separately with pooled human liver microsomes under an NADPH-supported reaction system. The microsomes came from a preparation pooled from 28 mixed-gender donors; this was not a study in which 28 volunteers ingested cannabis. Each substrate was introduced at 5 micromolar, incubations were performed in duplicate, and samples were taken at 20, 120 and 180 minutes. Parallel incubations lacking the NADPH-regenerating system served as negative controls.

At 120 and 180 minutes, both the Δ9-THC and 11-OH-THC incubations yielded an identifiable 9R-HHC-COOH signal. The 9S-HHC-COOH signal met the stated identification criteria in the 11-OH-THC incubation but not in the Δ9-THC incubation. Neither HHC-COOH epimer appeared at 20 minutes or in negative controls, and THC-COOH incubation did not yield HHC-COOH. Crucially, the investigators did not detect parent HHC in any incubation.

These findings support biochemical conversion along an oxidative pathway rather than requiring parent HHC as the exposure source. They are stronger mechanistic evidence than a correlation observed in routine casework alone. They still do not specify which reductase enzyme performed the reaction, establish how much conversion occurs in every living person, or reproduce a complete person’s physiology.

Did the experiment prove the formation of 11-OH-HHC in vitro?

Not to the same degree as HHC-COOH. This is an important technical limit often lost when the study is reduced to a headline. In Δ9-THC incubations, peaks at the anticipated retention time for 11-OH-HHC appeared at later timepoints, but the qualifier-ion evidence was insufficient for full confirmation. The reference material used for the 11-OH-THC incubations also contained 11-OH-HHC impurities, including in negative controls. Those impurities prevented a clean experimental conclusion about newly formed 11-OH-HHC in that incubation series.

The forensic specimens independently showed a smaller subset containing identified 9R-11-OH-HHC, particularly 33 of the 222 THC-positive/HHC-negative cases. Taken together, the study supports a metabolic pathway, but it does not justify claiming that every step was experimentally proven with equal certainty. Chemical identifications, qualified mechanistic inference and an unproven intermediate should remain separate statements.

What did the chromatograms establish about identity?

The authors used reference compounds for the 9R and 9S HHC-related epimers, compound-specific retention behavior and two monitored mass transitions. Their target identification required a retention time within 0.02 minute and a quantifier-to-qualifier ion ratio within 20% relative to standard. Those details matter because an analyte label is only as trustworthy as the method’s selectivity, calibration, signal quality and quality-control criteria.

The published chromatograms also show why the distinction is not merely semantic. HHC-COOH shares a nominal mass with the naturally occurring ¹³C₂ isotopic form of THC-COOH. In insufficiently resolving methods, a peak attributed to an HHC-related species might instead involve this interfering isotope signal. The paper’s Figure 3 depicts chromatographically separated 9R-HHC-COOH, the isotope peak and 9S-HHC-COOH. A court-facing explanation should specify which signal was identified and how the laboratory excluded a plausible interference.

For a general account of why reference standards, qualifiers and matrix-specific validation matter, see confirmatory drug testing. Identification of a molecule and inference of an exclusive source remain separate propositions even when the identification itself is sound.

Can any one marker distinguish HHC use from THC use?

Not on the evidence of this study alone. HHC-COOH was highly prevalent among specimens positive for THC and negative for parent HHC. The investigators proposed that HHC-COOH and 11-OH-HHC can both arise during Δ9-THC metabolism. They did not recruit a controlled HHC-only dose group and did not obtain reliable product histories for the forensic cases. The study therefore supplies no validated cutoff, ratio, or multianalyte decision rule that uniquely classifies prior HHC consumption in an unidentified person.

A complete forensic evaluation can still consider parent HHC, the distribution of relevant epimers, corresponding THC compounds, competing drug exposures, product records, time since possible intake and the performance characteristics of the actual laboratory method. Those may improve or constrain source attribution in a particular record. But adding several correlated metabolites to a report does not automatically create an individually validated HHC-use signature.

Conversely, an absence of parent HHC is not proof of never having used it, and an absence of a particular HHC-related metabolite is not proof that THC was never present. Collection timing and the analytical decision limits remain essential context.

What statistical uncertainty matters here?

The study’s percentages are descriptive proportions from submitted forensic specimens, not prospective estimates of clinical test sensitivity or specificity. The 222-case subgroup was defined by measured parent compounds; the researchers did not randomize people to THC-only, HHC-only or placebo. Without an HHC-only validation group, the specificity of 9R-HHC-COOH for HHC ingestion cannot be estimated. Without documented dosing and sampling times, the authors also could not establish an individualized rate of formation or elimination.

The number 186/222 is informative about the chemical pattern observed in those Danish case specimens, but it does not permit a probability statement such as “this person was 84% likely to have used THC rather than HHC.” Nor does the group’s median 7% comparison allow correction of an individual HHC-COOH result into a known THC dose. In a legal setting, those would be transformations of a descriptive dataset into a classification rule the experiment did not validate.

The microsome observations were duplicated in a pooled human-liver preparation, not replicated as independent controlled human exposure studies. A supported pathway is not synonymous with a precisely quantified population frequency or a universal person-specific conversion curve.

Does a positive metabolite establish impairment or driving causation?

No. HHC-COOH and THC-COOH are metabolite measurements. Parent THC is a distinct analyte, and even a correctly quantified parent THC concentration does not, by itself, establish a particular driver’s functional condition at a disputed moment. These 308 forensic cases were not an experimental driving-performance cohort. The investigators reported no standardized lane keeping, reaction time, sobriety examination, vehicle-event reconstruction, or individual impairment classification derived from the metabolites under examination.

Attributing a crash, medical event or legally significant act to HHC exposure requires independent evidence of exposure identity, chronology, relevant concentration when applicable, pharmacological effect and the particular event. The original study neither supplies that evidentiary chain nor authorizes reverse inference from a metabolite to state of mind, driving ability or crash responsibility.

Specimen acquisition, timing, labeling and custody also matter. A carefully established collection record allows the reported concentration to be located at the time of sampling, not silently at the earlier time of driving. The site’s specimen-collection guide outlines those foundational distinctions.

What should be requested when an HHC metabolite is disputed?

The starting point is the complete analytical record rather than the face of a certificate listing “HHC metabolite positive.” Determine which compound and epimer were actually reported, whether it was simply detected or quantitatively established, and which specimen was analyzed. The original chromatograms, retention-time windows, monitored qualifier and quantifier transitions, integration data, reference standards, isotopic interference evaluation, calibrators, quality controls, internal-standard recoveries and reporting thresholds determine what the laboratory measurement can support.

Next, identify whether the interpretation rests on direct HHC parent detection or on a metabolite shared with a THC pathway. Confirm whether the method was validated for HHC-COOH rather than estimating its concentration with another cannabinoid’s calibration. Obtain the product and usage evidence actually available; do not insert an assumed HHC-only exposure history where the records contain none. Separate a defensible analytical opinion from a narrower but still uncertain conclusion about origin.

Finally, keep source identification, timing and impairment in distinct questions. The prosecution or defense may have additional case evidence, but none of it may be invented from the chemistry alone. The scientific problem is not whether the metabolite exists; it is what its existence can identify with sufficient specificity.

Frequently asked forensic questions

1. Does 9R-HHC-COOH always mean someone took HHC?

No. The original 308-case study identified 9R-HHC-COOH in 186 of 222 specimens positive for Δ9-THC but negative for parent HHC. Human liver microsomal incubations with THC-related substrates also produced HHC-COOH without parent HHC being added. The marker cannot by itself establish exclusive HHC ingestion.

2. Did the researchers show that THC is chemically the same as HHC?

No. They are different molecules. The finding concerns a metabolic transformation capable of producing an HHC-related downstream compound from THC and its oxidative metabolite. Sharing a downstream metabolite does not make the parent drugs chemically identical.

3. Were 84% of all 308 blood specimens HHC-COOH positive?

No. The 84% was 186 positive specimens within the specific 222-case Δ9-THC-positive/parent-HHC-negative group. Ten further cases had both parent drugs identified, and 76 had neither identified. Mixing denominators would misstate the study.

4. Can the median 1.4 ng/mL HHC-COOH result be used to back-calculate a THC dose?

No. That is a group median based on estimated HHC-COOH concentrations, not an individualized pharmacokinetic calibration. Amount used, timing, product composition and person-specific kinetics were unknown; the paper validated no inverse dosing formula.

5. Is a positive HHC-COOH result proof of intoxication or impaired driving?

No. A carboxylated metabolite is evidence about an analyte in the sampled matrix, not a direct measurement of contemporaneous driving performance, impairment or collision causation. Those conclusions need separate evidence and an appropriately validated interpretive method.

6. Why do 9R and 9S epimers matter?

They are stereochemical forms that the investigators separated and identified at different frequencies. In the THC-positive/HHC-negative subgroup, 9R-HHC-COOH appeared in 186 specimens and 9S-HHC-COOH in 112. An unspecified “HHC metabolite” report can conceal meaningful identity and selectivity information.

7. Does the microsome study prove every proposed conversion step?

No. HHC-COOH was identified after THC and 11-OH-THC incubations, but some 11-OH-HHC signals did not satisfy full identification requirements, and 11-OH-THC reference material contained HHC-related impurities. The investigators did not identify the exact reducing enzyme. The supported pathway is stronger than a purely speculative proposal but is not a complete quantitative account of human metabolism.

Bottom line for forensic interpretation

HHC-COOH is a real analytical target, but it is not necessarily an HHC-exclusive exposure marker. The original forensic traffic-case data and enzyme experiment demonstrate why compound identity, metabolic origin and an individual’s drug-taking history are different propositions. One can accept an appropriately confirmed whole-blood result while rejecting the unsupported leap from that result to exclusive HHC ingestion, personal intoxication or legal causation.

Primary scientific source: Falck Jørgensen C, Schou Rasmussen B, Linnet K, Thomsen R. “Evidence of 11-Hydroxy-hexahydrocannabinol and 11-Nor-9-carboxy-hexahydrocannabinol as Novel Human Metabolites of Δ9-Tetrahydrocannabinol.” Metabolites. 2023;13(12):1169. doi:10.3390/metabo13121169.