3.1. Extraction Conditions
In order to minimize the interference of substances in the matrices and increase recovery, the optimization of extraction conditions is essential [
18]. The protein precipitation method is used to precipitate the protein and centrifuge the supernatant to determine the content of synthetic cannabinoids in biological samples. Organic reagents destroy structures of proteins in the plasma and precipitate the protein to extract synthetic cannabinoids from plasma. Thus, suitable sample preparation is important to reduce ion suppression of compounds in the matrices [
13]. The matrix effect is critical in establishing reliable methods, and ignoring this effect may adversely affect the reliability of determination of analyte concentration [
17]. The matrix effect is used as the evaluation parameter for the optimization of protein precipitation in plasma. A matrix effect value greater than one hundred percent indicates ion enhancement, whereas a matrix effect value less than one hundred percent indicates ion suppression [
19]. Concurrently, recovery is used as an indicator for the extraction of synthetic cannabinoids in urine. Low recovery may be ascribed to incomplete extraction and elution of synthetic cannabinoids from the SPE cartridge.
The present experiment was conducted to investigate the effects of methanol and acetonitrile on protein precipitation. As shown in
Figure 2, when acetonitrile was used, the matrix effect values of the three synthetic cannabinoids were higher than with the use of methanol. Thus, acetonitrile was selected as the protein precipitation reagent. We also further explored the effect of different acetonitrile volumes on the matrix effect. The volume ratios of plasma to acetonitrile of 1:1, 1:2, 1:3, 1:4, and 1:5 were used to evaluate the matrix effect for three synthetic cannabinoids (
Figure 3). Collectively, the matrix effect was significantly enhanced with the increasing volume of acetonitrile for each synthetic cannabinoid (
p < 0.05), and the matrix effect value tended to be stable when the volume ratio of plasma to acetonitrile was 1:3. The matrix effect did not change significantly (
p > 0.05) when the volume ratio of plasma to acetonitrile was greater than 1:3, as the volume of acetonitrile increased further. Finally, given the environmental protection and cost, the volume ratio of plasma to acetonitrile was selected as 1:3.
Three different SPE cartridges, including the Waters Oasis HLB column (3cc/60 mg), Oasis WCX column (3cc/60 mg), and Oasis MCX column (3cc/60 mg), were harnessed for extraction. The Waters Oasis HLB column is a hydrophilic–lipophilic balanced extraction column, which is suitable for acid, alkali, and neutral compounds and thus can remove approximately 95% of matrix interferences (such as phospholipids, fats, salts, and proteins) in all biological matrices [
20,
21]. The Waters Oasis WCX (weak cation exchange) column provides high selectivity and recovery for strong bases and quaternary and withstands elution with high concentration solvents [
22]. The Waters Oasis MCX, a mixed-mode cation-exchange sorbent, provides high selectivity and recovery for alkaline compounds [
23]. The type of solid phase extraction column significantly affected the extraction recovery of the three synthetic cannabinoids (
Figure 4). The extraction recovery with the Waters Oasis HLB column, which was 92.45 ± 2.29%, 96.95 ± 4.39%, and 99.33 ± 1.03% for JWH-122, 5F-AMB, and AMB-FUBINACA, respectively, was all significantly higher than the other two solid phase extraction columns (
p < 0.05). Therefore, the Waters Oasis HLB column was finally selected for the extraction of these synthetic cannabinoids.
Furthermore, we investigated the effect of the ratio of acetonitrile to water for the extraction recovery of synthetic cannabinoids in urine. Ultrapure water causes the incomplete transfer of synthetic cannabinoids into SPE cartridges. The addition of a certain volume of acetonitrile during extraction can effectively dissolve the synthetic cannabinoids. However, the synthetic cannabinoids are directly eluted by an SPE column under a high ratio of acetonitrile to water. Therefore, optimizing the ratio of the extract is crucial for the extraction of synthetic cannabinoids [
13]. As shown in
Figure 5, the total volume of the extract was maintained at 2 mL, while the ratio of acetonitrile to water changed. The volume ratio of acetonitrile to water was set to 0 (0 µL), 5% (100 µL), 10% (200 µL), 20% (400 µL), 30% (600 µL), and 40% (800 µL). As the proportion of acetonitrile increased, the recovery rate increased significantly for JWH-122 (
p < 0.05). When the proportion of acetonitrile increased to 20%, the recovery rate stabilized and then no longer increased significantly (
p > 0.05). Similarly, the recovery in the “20%” group was significantly higher than that in the “0”, “5%”, or “10%” groups for 5F-AMB (
p < 0.05). Whereas the recoveries no longer increased significantly (
p > 0.05) when the volume of acetonitrile continuously increased for AMB-FUBINACA, the recoveries in the “20%” group and the “30%” group were significantly higher than those in the other groups (
p < 0.05). Therefore, the final volume of acetonitrile was 30% (600 µL) according to the optimum recoveries of the three synthetic cannabinoids.
The influence of the volume of the elution reagent methanol on the extraction recovery of three synthetic cannabinoids was examined. The methanol (2 mL, 3 mL, 4 mL, 5 mL, and 6 mL) was harnessed for the complete elution of three synthetic cannabinoids by the SPE column. With the increase in methanol volume, the extraction recovery increased significantly for JWH-122 (
p < 0.05) (
Figure 6), and the administration of 4 mL of methanol reached the maximum recovery rate. For 5F-AMB and AMB-FUBINACA, the recoveries almost reached the maximum value when the volume of methanol was 4 mL. Therefore, 4 mL of methanol was employed by considering a high extraction recovery for synthetic cannabinoids.
3.2. Method Validation
All synthetic cannabinoids were confirmed to be linear within the calibration curve ranges (
Figure 7 and
Table 3). The correlation coefficients (R
2) were more than 0.99. The LODs of synthetic cannabinoids covered the range of 0.003–0.004 ng/mL and 0.00125–0.002 ng/mL for plasma and urine samples, respectively. Concurrently, the LOQs of synthetic cannabinoids ranged 0.012–0.016 ng/mL and 0.003–0.005 ng/mL for plasma and urine samples, respectively (
Table 3), which are lower than in previous studies (0.05–1.0 ng/mL and 0.1–1.0 ng/mL) [
16,
18,
24,
25]. The intra-day and inter-day precisions were 1.3–9.0% and 3.0–8.6% for plasma samples and were 2.8–6.7% and 3.9–8.8% for urine samples, respectively (
Table 4).
The concentration of synthetic cannabinoid in the spiked matrix, these from A to I are 0.4, 4, 40, 0.05, 0.5, 5, 0.1, 1, 10 ng/mL.
The extraction recovery rate is used to evaluate the reproducibility of the extraction efficiency of the target analytes by the protein precipitation method and the solid phase extraction method [
26,
27]. The extraction recoveries of synthetic cannabinoids were 95.4–106.8% and 92–102% at three concentrations in plasma and urine samples, respectively (
Table 4). Results from the matrix effect experiment are shown in
Table 4, which fell in the ranges of 82–96% and 93–119% for plasma and urine samples, respectively. Slight ion enhancement was observed in urine, while less impact for the matrix effects was found in plasma. Taken together, the current method showed robust results of reproducibility and recovery rate and proved quite potent for the simultaneous analysis of synthetic cannabinoids in urine and blood samples.
3.3. Sample Analysis of Synthetic Cannabinoids
The LC-MS/MS method has been widely harnessed for detecting synthetic cannabinoids since 2014 [
23]. The profiling methods should be continuously updated because new synthetic cannabinoids appear in the market all the time [
28,
29]. Therefore, the current study established a robust method for three emerging synthetic cannabinoids including JWH-122, 5F-AMB, and AMB-FUBINACA and confirmed the suitability of the methods via the plasma and urine samples of rats exposed to synthetic cannabinoids.
Figure 8 shows the typical chromatograms of both the blank sample and plasma and urine samples spiked with the synthetic cannabinoids. After 72 h of ingestion for three synthetic cannabinoids, AMB-FUBINACA and JWH-122 could be detected in plasma, while 5F-AMB was not detected. Concurrently, all synthetic cannabinoids could be simultaneously detected in urine. In detail, the concentrations of JWH-122 and AMB-FUBINACA were 0.08 ng/mL and 0.05 ng/mL, respectively, at 72 h in rat plasma. The relative standard deviation (RSD) values in all plasma samples were less than 20%, indicating qualified results. On the other hand, the concentrations of JWH-122, 5F-AMB, and AMB-FUBINACA were 0.20 ng/mL, 8.68 ng/mL, and 3.38 ng/mL, respectively, in rat urine after 2 h from oral gavage (
Table 5). The concentrations of JWH-122, 5F-AMB, and AMB-FUBINACA then decreased to 0.12 ng/mL, 1.11 ng/mL, and 0.41 ng/mL, respectively, at 4 h. After 48 h, the concentrations of JWH-122, 5F-AMB, and AMB-FUBINACA attenuated to 0.02 ng/mL, 0.03 ng/mL, and 0.03 ng/mL, respectively, while the concentrations of JWH-122, 5F-AMB, and AMB-FUBINACA in rat urine were 0.01 ng/mL, 0.01 ng/mL, and not detected, respectively, at 72 h. The RSD values in all plasma samples were less than 20%, indicating credible results (
Table 5). Collectively, the concentrations of the three synthetic cannabinoids gradually decreased during 0–72 h. The data revealed that a detection window of 2–3 days was achievable with low concentrations of JWH-122, 5F-AMB, and AMB-FUBINACA (the dose for the tail vein was 1 mg/kg) (
Table 5). Our method is consistent or even superior with related studies, as samples could be stably profiled for 36 h or more with the detection concentration of 2–25 ng/mL and as it achieved the detection of three synthetic cannabinoids with lower content [
18].