Figure 4 shows a rapid increase in CBD in brain tissue for all tested carriers; the maximum concentration, C_max, was reached within 1–2 h (T_max) after gavage administration; then, a slower decrease occurred until 4 h, and no notable changes were observed between 4 h and 6 h. The declared CBD content in each formulation was analytically confirmed prior to administration, as described in Section 3.6. The T_max value complies with the findings of other similar studies. In the review by Millar et al. (2018), which documented the dose dependence of C_max, reported T_max generally ranged between 1 and 4 h [9]. As longer measurements were not performed and matrices other than the brain were not available, a more detailed description of CBD distribution in the animals’ bodies and the way of excretion could not be obtained.

In our single-dose study (10 mg CBD/kg for all animals), the highest mean C_max of CBD in the brain was 259 ± 150 µg/kg, recorded 1 h after administration of carrier B (10% CBD oil). In this case, the highest AUC_0–6 (596 µg/kg × h) was calculated, indicating the highest exposure of brain tissue by CBD. Conversely, the lowest C_max of 59 ± 33 µg/kg and correspondingly relatively low AUC_0–6 (186 µg/kg × h) were observed for carrier E (CBD proliposomes-CC).

As shown in Figure 4 and Table 2, the pharmacokinetic profile of CBD in brain tissue was strongly influenced by the formulation used. The shortest T_max (1 h) was observed for carriers B (10% CBD oil) and C (SEDDS with MCT (Medium-Chain Triglyceride) oil), which also achieved the highest C_max values (259 and 226 µg/kg) and AUC_0–6 values (596 and 397 µg/kg x h), indicating rapid and efficient brain delivery. These results suggest that formulations promoting fast solubilization and absorption (such as oil-based and MCT-based SEDDS) enhance CBD uptake into brain tissue.

On the other hand, carriers D (SEDDS with sesame oil) and F (proliposomes—spray drying) reached T_max later (2 h), yet still achieved relatively high AUC_0–6 values (412 and 255 µg/kg x h), pointing to a more sustained CBD release. Interestingly, although their C_max values were lower compared to B and C, the prolonged exposure time may be beneficial in maintaining CBD levels over time. Carrier E (proliposomes with chitosan coating) showed both low C_max (59 µg/kg) and AUC_0–6 (186 µg/kg x h), indicating less efficient delivery to brain tissue.

These findings highlight that a delayed T_max does not necessarily indicate poor performance; on the contrary, it may reflect sustained drug release, which can be advantageous depending on the therapeutic context. In summary, carriers A, D, E, and F showed a slower decrease in CBD concentration over time compared to carriers B (10% CBD oil) and C (SEDDS with MCT oil) which might be desirable for therapeutic purposes.

The impact of the type of oil used in the CBD-formulated SEDDS on C_max was also assessed. Higher C_max (226 ± 118 µg/kg) was achieved by carrier C (SEDDS with MCT oil) compared to carrier D (SEDDS with sesame oil) for which the C_max was 124 ± 45 µg/kg. Interestingly, the highest C_max was achieved when triacylglycerols with the shortest fatty acid chains (caprylic/capric, C8:0/C10:0, in the case of carrier B) were used for CBD formulation. Generally, an increase in the molecular weight of triacylglycerols used for this purpose resulted in a decrease in C_max. MCT oil (carrier C) contains medium-chain fatty acids such as lauric acid (C12:0) and myristic acid (C14:0), while sesame oil (carrier D) predominantly contains long-chain fatty acids such as linoleic acid (C18:2) and oleic acid (C18:1). While the influence of excipients on CBD pharmacokinetics has been explored in previous studies, research specifically addressing its distribution in the brain remains limited. Nevertheless, the dependence of CBD intestinal lymphatic transport and systemic bioavailability on the composition of vegetable oils as a delivery system has been documented in a recent study [30].

A more detailed comparison of the PK parameters reveals that oil-based and MCT-based carriers (B and C) result in faster absorption and higher brain exposure, as reflected by elevated AUC_0–6 values and C_max. In contrast, proliposomal systems (carriers E and F) show a slower onset and lower peak levels, suggesting a sustained-release effect. Such differences underscore the importance of formulation type in modulating CBD pharmacokinetics and brain availability, which is critical for developing targeted CNS (central nervous system) delivery strategies.

Unfortunately, plasma data were not collected at this stage, as the primary objective of the present study was to focus on brain pharmacokinetics. This limitation is acknowledged, and future work will include plasma profiling and release studies of CBD formulations for a better understanding of formulation effects on CBD pharmacokinetics.

In the next phase, two major phase I CBD metabolites originating through processes catalyzed by cytochrome P450 enzymes were monitored. While the primary metabolite, 7-hydroxycannabidiol (7-OH-CBD), is pharmacologically active, 7-carboxycannabidiol (7-COOH-CBD) does not exhibit such activity anymore [31,32]. Like in the case of CBD, the time-dependent profile of these metabolites differed depending on the carrier system used. For carrier C, the T_max values of both metabolites were identical to that of CBD (1 h). Shorter T_max values of 7-OH-CBD were observed for carriers A, D, E, and F (0.5, 1, 0.5, and 1 h, respectively). For carrier B, the T_max of 7-COOH-CBD was delayed to 2 h, whereas for carrier F, it was substantially prolonged to 6 h, compared to 2 h for CBD. This delay, together with a relatively high AUC_0–6, may be linked to slower CBD release from proliposomal matrices and high inter-individual variability at later time points. The measured data are illustrated in Figure 5 and Figure 6 and summarized in Table 2.

In some cases, the concentration–time curves appeared biphasic, with a secondary rise. This pattern may reflect formulation-dependent absorption, delayed release, or redistribution mechanisms [33].

In the final phase of CBD metabolites investigation, we inspected carefully the records of the targeted UHPLC-MS/MS analysis and in each of them, the presence of an additional peak with identical dMRM (dynamic multiple-reaction-monitoring) transitions to those of 7-OH-CBD was found (see Figure 7). Regarding the identification of the ‘unknown’ peak, this was rather complicated when using MS with a triple quadrupole mass analyzer. Therefore, high-resolution mass spectrometry (HRMS) was employed to obtain more structural information. Nonetheless, the detected compound is likely one of the 7-OH-CBD isomers formed during CBD metabolism. Several studies have also reported signals corresponding to monohydroxylated CBD metabolites [25,34].

For further investigation, the UHPLC-HRMS/MS method described in Section 3.8 was used. The fragmentation spectra of 7-OH-CBD and the ‘unknown’ peak shown in Figure 8 were not identical; nevertheless, they appear to be isomers (C₂₁H₃₀O₃). Although no database match was found for either spectrum, the protonated molecular ion (theoretical m/z 331.2268) and ion corresponding to a loss of water (theoretical m/z 313.2162) were practically identical.

Interestingly, the same ‘unknown’ metabolite was also detected in the study by Citti et al., along with other unidentified compounds with molecular ions corresponding to carboxy CBD or dihydroxylated CBD. It is worth noting that the CBD dose used in our study was five times lower than in the study by Citti et al., which may explain the absence of these minor metabolites in our results [25].