This report describes plasma cannabinoids after withdrawal from IH administration, with samples collected 14 d after the last IH dose. A previous study by Smith et al. (2023) evaluating depletion of cannabinoids sampled plasma up to 8 d following the last day of feeding hempseed meal. We detected a total of 11 cannabinoids, including 8 acidic cannabinoids. No cannabinoids were detected on days −7 or 0 in any group. We observed detectable concentrations of the cannabinoids CBD-7-acid 14 d after the final dose of IH was administered, whereas other cannabinoids were not detected by 7 d after the final dose of IH. We did not observe a significant effect of time on cannabinoid concentrations. There was only a significant effect of treatment (comparing HEMP vs. COMBO treatments) for CBD-7-acid. In general, cannabinoid concentrations were relatively static throughout the feeding period, with consistently, numerically lower concentrations on day 28. The cannabinoid CBD-7-acid was detected at low concentrations throughout the feeding period in steers not receiving IH, and CBDA was detected in 4 samples from steers not receiving IH; THCA and 9-THC were detected in a single animal in the CTC group.

The plasma cannabinoid profiles we described in this study are similar to previous reports that observed predominantly acidic cannabinoids in plasma (Kleinhenz et al., 2020a, 2022; Smith et al., 2023; Fruge et al., 2025). Studies evaluating IH flowers and leaves have detected CBCA, CBD-7-acid, CBD, CBDA, CBDVA, CBGA, THCA, and THCVA (Kleinhenz et al., 2020a, 2022; Fruge et al., 2025). In cattle receiving hempseed cake, Smith et al. (2023) reported detectable concentrations of CBCA, CBDA/THCA, CBDVA, and CBNA. Irawan et al. (2025) detected additional, non-acidic cannabinoids, including CBG, 9-THC, and THC-11-OH in cows fed spent hemp biomass (SHB). Compared to these studies, Wagner et al. (2022) detected mostly neutral cannabinoids, including CBD, CBDV, 9-THC, and THCV, along with THCA, in plasma of cows fed IH silage. This suggests that acidic cannabinoids are readily absorbed from the rumen but are eliminated more slowly from the plasma than neutral cannabinoids, potentially due to ion trapping, protein binding, or lower lipophilicity. Irawan et al. (2025) calculated increased absorption rates of CBCA and CBDA compared to their neutral forms and Wagner et al. (2022) reported a much lower milk-to-plasma ratio for THCA compared to 9-THC or the other neutral cannabinoids detected. Similarly, in dogs, CBDA and THCA appear to be more readily absorbed than CBD and 9-THC, respectively (Wakshlag et al., 2020).

Like the cannabinoid profiles we report, the cannabinoid elimination data from this study is consistent with prior literature. Fruge et al. (2025) found that CBD, CBDA, and THCA rapidly depleted to very low plasma concentrations within 96 hours of the last feeding of IH leaves. In a study evaluating hempseed cake, Smith et al. (2023) also reported undetectable plasma CBDA/THCA and CBDVA by day 2 of withdrawal. After a single dose of IH flowers, Kleinhenz et al. (2020a) observed rapid depletion of CBDA within 96 hours, calculating a mean residence time of 30.6 hours. In that study, CBCA and THCA were undetectable after 72 hours and CBDVA concentrations were approximately 1 ng/mL. In the subsequent 14-d feeding study, the authors reported all cannabinoids except CBDA were undetectable after day 2 of withdrawal; CBDA was undetectable on day 5 (Kleinhenz et al., 2022).

In the present study, most of the acidic and neutral cannabinoids were eliminated from the plasma by 7 d after final IH administration (the first blood collection after the last dose). However, CBD-7-acid was detected 7 and 14 d after the final dose. The FDA has a published interest in the inactive metabolite CBD-7-acid, as it reaches concentrations considerably higher than the parent, CBD, and has a much longer elimination half-life of up to 33 hours (FDA CDER, 2017; Taylor et al., 2018; FDA CDER, 2023). In horses, CBD-7-acid elimination is also prolonged, with one study reporting a terminal elimination half-life of 79.85 hours (Eichler et al., 2023). A recent report in cattle indicated that plasma CBD-7-acid did not reach steady state concentrations during a 14-d feeding period and had depleted minimally 5 d after the final dose was administered (Xu and Murphy, 2024). Similarly, Fruge et al. (2025) reported minimal changes in plasma CBD-7-acid in the 96 hours following the end of a 14-d period of IH administration.

Although 9-THC was not detected above the LLOQ in any sample from the HEMP or COMBO groups, one steer in the CTC group had detectable concentrations near the LLOQ (1.3 ng/mL detected vs. 1 ng/mL LLOQ). That same sample had THCA detected at concentrations up to 9 times higher than in the HEMP and COMBO groups (29 ng/mL vs. 3.2 ng/mL, the minimum non-zero value in the HEMP and COMBO groups). The unexpected and sporadic nature of the CBDA, 9-THC, and THCA findings begs the question whether the samples with detectable concentrations we identified were true positives or if they potentially represent sample contamination (e.g. during extraction) or detection of an unknown cannabinoid with the same transition and retention parameters. These cannabinoids were confirmed present based on the qualifier ion. However, statistical analysis revealed these samples were likely outliers, as CBDA, 9-THC, and THCA concentrations within the PLBO and CTC groups were not different than the associated LLOQ values. The authors believe compound interference or inadvertent sample contamination may be responsible for the CBDA, 9-THC, and THCA findings, as opposed to being a true positive. This theory is supported by the fact that concentrations of these cannabinoids were similar or higher in the PLBO and CTC steers compared to those receiving IH.

While the 9-THC, THCA, and CBDA findings are likely false positives, we believe the CBD-7-acid findings in the PLBO and CTC steers are reliable due to the precedent set by previous literature and the consistently detectable concentrations throughout the feeding period. Transference of residues in urine or feces to unexposed animals has been reported for non-steroidal anti-inflammatory drugs in pigs and horses (Popot et al., 2007; Hairgrove et al., 2019; Bates et al., 2020). Similar to human literature on disposition of cannabinoids (Grotenhermen, 2003), previous literature in cattle receiving IH has reported high levels of fecal and urine excretion (Addo et al., 2023; Smith et al., 2023; Fruge et al., 2025; Irawan et al., 2025). Specifically, Fruge et al. (2025) reported large standard deviations in their fecal cannabinoid measurements, indicating differences in individual animal’s cannabinoid metabolism. The authors believe that environmental contamination and residue transfer is the most plausible reason for consistent detection of CBD-7-acid in the PLBO and CTC steers. It is possible that residue transference was also responsible for the detectable CBDA, 9-THC, and THCA concentrations. However, the authors believe this situation is less likely than the compound interference or sample contamination scenarios, since there were no other samples with 9-THC concentrations above the LLOQ and no other CTC or PLBO samples with detectable THCA concentrations.

Based on the significant treatment effect for CBD-7-acid, it appears that concurrent administration of CTC may increase CBD-7-acid concentrations, potentially due to changes in cytochrome P450 (CYP) isoform levels or activity. The main CYP isoenzymes critical to CBD metabolism in humans are CYP3A4 and CYP2C19 (Jiang et al., 2011; Gaston and Friedman, 2017). While the role of CYP in CBD metabolism in bovines has not been established, four bovine CYP3A isoenzymes have been previously described, some of which correlate to human CYP3A4 (Zancanella et al., 2010). Furthermore, tetracycline has been shown to inhibit human CYP3A4 in vitro (Zhao and Ishizaki, 1997) and a study in pigs reported variable risk of CYP2 and CYP3 inhibition by oxytetracycline, chlortetracycline, and doxycycline (Hu et al., 2016). Future work should confirm our findings and investigate potential risks of coadministration of veterinary drugs and IH in cattle.

Although we detected CBD-7-acid up to 14 d after final administration, we cannot accurately estimate half-life from our results. Determining the half-life and other pharmacokinetic parameters would enable the application of CBD-7-acid for exposure time estimation. Development of a rapid analytical method for this cannabinoid could provide a useful tool for quickly identifying exposed cattle for further testing, as has been previously proposed by Xu and Murphy (2024). However, caution is warranted if an analytical method is developed for this purpose. The present study consistently identified CBD-7-acid in cattle not receiving IH, possibly secondary to exposure to urine and feces from cattle in the HEMP or COMBO groups. Thus, any future surveillance strategy should employ either a second, confirmatory test or should incorporate cutoff values that would exclude animals with detectable concentrations due to environmental contamination from exposed animals or should recommend confirmatory testing prior to any regulatory actions.

Limitations of this study include the small sample size and lack of follow-up past 14 d after final IH administration. There are no IH products approved in cattle and thus there is no tolerance for any cannabinoid in cattle tissues or plasma. If further regulatory discussions deem CBD-7-acid of importance for surveillance measures, additional studies should be performed with a longer follow-up period. Additionally, a relatively low dosage of cannabinoids was used in this study compared to previous feeding trials (Wagner et al., 2022; Irawan et al., 2025). Longer durations and higher inclusion levels than what was used in the present study may result in even more prolonged depletion of cannabinoids in plasma. The experimental vaccine confounding variable is another limitation. This effect was minimized, as we only compared the HEMP and COMBO groups, which both received the vaccine, and were merely reporting descriptive statistics for the CTC and PLBO groups. However, any differences in elimination between the HEMP and COMBO groups cannot be accurately ascribed to IH due to confounding vaccine status.

In the present study, we observed that acidic cannabinoids represented a majority of plasma cannabinoids. We detected the cannabinoid metabolite CBD-7-acid 14 d after final IH administration and believe that it could be a reliable indicator of prior IH exposure for use in future surveillance measures. These results will inform decisions regarding surveillance and interpretation of samples collected from cattle tested for cannabinoids or IH exposure.