Rapid simultaneous determination of 11 synthetic cannabinoids in urine by liquid chromatography-triple quadrupole mass spectrometry

: Synthetic cannabinoids are a series of synthetic substances that mimic the effects of natural cannabinoids and produce a much stronger toxicity than natural cannabinoids, which have become the most abused family of new psychoactive substances. A solid-phase extractive-liquid chromatography-triple quadrupole/linear ion trap mass spectrometry method was developed to determine 11 synthetic cannabinoids in rat urine. The factors affecting recovery were optimized, and Oasis HLB was selected to extract synthetic cannabinoids simultaneously. The results showed that the linear correlation coefficients of the synthetic cannabinoids ranged from 0.993 to 0.999, and the limit of quantitation ranged from 0.01 to 0.1 ng/mL, and the spiked recoveries ranged from 69.90% to 118.39%. This method has the advantages of good purification ability, simple operation, and good reproducibility, and can be used for the high sensitivity analysis of various synthetic cannabinoids in urine.

Synthetic cannabinoids have many commercial names, such as Spice, Black Mamba, and Cold K2 [6]. SCs have emerged in many countries and regions worldwide, especially among young people. SCs have been the most widely monitored new psychoactive substances in Europe since 2008 [3]. The increase in consumption of SCs is particularly significant compared to other drugs on the market. Small changes in the original SCs structure will lead to the formation of new SCs, and thus many illegal manufacturers can easily produce new SCs. It is important to note that new SCs continue to emerge rapidly, making detection and analysis difficult.
Studies on the identification and toxicology of SCs have been carried out.
Ingestion of cannabinoids can cause many diseases, as appeared and described in the literature, including psychosis [7], intoxication [8], tachycardia [9], changes in blood pressure [10], and atrial fibrillation [11]. Some reports declared a fatality directly related to the toxic effects of synthetic cannabinoid use [12,13]. Tomiyama et al. reported that SCs induce cell apoptosis by regulating the cascade of cystatin and stressed that the abuse of cannabinoids could lead to neurological brain problems [14].
Davide et al. concluded SCs could activate CB1 receptors in cardiomyocytes, participate in the production of reactive oxygen species, ATP consumption, and cell death, and then induce cardiotoxicity [15]. However, SCs also have been reported to have specific therapeutic effects. Aleksandra et al. found that SCs could activate the death pathway of human glioma cells and had anti-tumor activity [16]. Due to new SCs constantly emerging in the market, there is an urgent need to update the synchronous detection method of SCs. After being metabolized in the body, the content of the primary SCs is very low, and the matrix composition of biological samples is complex. Therefore, it is necessary to develop a low limit of quantitation and high precision method to detect trace synthetic cannabinoids in biological samples.
Usually, blood and urine are the main biological samples tested for cannabinoids, while saliva and hair can also be used as critical biological samples for testing. The advantage of urine testing drugs is that the sample pretreatment process is simple, and the collection process of the urine sample is non-invasive. There are many types of SCs, and the structure of different types of synthetic cannabinoids varies greatly, which brings certain difficulties to the detection of synthetic cannabinoids. In recent years, the detection and analysis methods of synthetic cannabinoid drugs mainly include thin layer chromatography (TIC) [17], nuclear magnetic resonance (NMR) [18], infrared spectroscopy (IR) [19], gas chromatography-mass spectrometry (GC-MS) [20], in human urine, with LOD ranging from 0.01 to 0.5 ng/mL, and the extraction recovery rate exceeded 50%, and the matrix effect was between 59.4% and 100.1% [23]. LC-MS is mainly used to analyze compounds with high thermal instability, strong polarity, and high molecular weight, with the advantages of strong separation ability, low detection limit, high sensitivity, and high accuracy [23]. Therefore, LC-MS is widely used to analyze multiple SCs in biopages simultaneously.
This article presented an improved and fully validated LC-MS/MS method for detecting 11 synthetic cannabinoids in rat urine. The method validation process involves the assessment of linearity, precision, accuracy, and limits of quantification.
In addition, SCs concentrations in urine were measured at different time points after injection of synthetic cannabinoids in rats. This method provides a useful tool for the identification of SCs in rat urines and a reference for the assessment of SCs internal biological exposure.

Method Validation
The method's calibration curves, linear ranges, matrix effect, recovery, precision, and limit of quantification (LOQ) were determined according to Taverniers et al. for a robust method validation [24]. In simple terms, 11 synthetic cannabinoid standard compounds were added to pretreated blank rat urine to configure standard matrix curves. Blank rat urine samples were treated with solid-phase extraction method, and then mixed standard substances of 11 SCs were added to prepare 0.1 ng/mL, one ng/mL, and ten ng/mL spiked rat urine sample solution to calculate matrix effect. We

Optimization of sample pretreatment
For the pretreatment of urine matrix, liquid-liquid extraction or solid-phase extraction methods are usually adopted. As a conventional extraction method, liquidliquid extraction has the disadvantages of large consumption of organic solvents and unstable recovery [25]. Compared with liquid-liquid extraction, solid-phase extraction has advantages of high enrichment ratio, high accuracy, and clean matrix treatment, so it is widely used [26]. Yeter [28]. And the adsorbent does not require adjustment and balancing steps, and the recovery rate is high. Per Ole M. Gundersen compared different solid-phase extraction columns to quantify the SCs metabolites in the urine by UHPLC-QTOF-MS, and finally selected HLB solid-phase extraction columns [29]. Based on the physical and chemical properties of 11 synthetic cannabinoids, the Waters Oasis HLB column was selected as a solid-phase extraction column for pretreatment optimization.
We also investigated the effects of the acetonitrile ratio of loading solution on the extraction recovery of 11 SCs. Since most SCs are lipophilic and insoluble in water, the use of ultrapure water as the loading liquid will result in the incomplete transfer of SCs to the SPE column. We added a certain volume of acetonitrile to the sample solution to make the SCs dissolve better in the loading solution. However, a large proportion of acetonitrile in the loading solution could lead to elution of the tested substance during loading. Therefore, it is essential to select the appropriate loading solution. We kept the volume of the measured substance and rat urine constant, and changed the volume of acetonitrile and ultrapure water. We set the volume of acetonitrile as 400 µL, 600 µL, and 800 µL and added a certain volume of ultrapure water, respectively, so that the total volume of the final sample solution was 2 mL. The results showed that the proportion of acetonitrile in the loading solution significantly affected the extraction recovery of 11 SCs (Figure 2). When the volumes of acetonitrile were 400, 600, and 800 μL, respectively, the extraction recovery rates were 52. SCs and concluded that 20% V/V acetonitrile could produce better peak resolution [30].
We also investigated the effect of elution reagent volume on the extraction recovery of 11 SCs. To completely elute 11 SCs while reducing the amount of organic reagent, the volume of elution reagent was set as 2 mL, 3 mL, 4 mL, 5 mL, and 6 mL.
Results showed that the volume of elution reagent greatly influenced the extraction recovery of SCs ( Figure 3). Theoretically, as the elution reagent mentioned increased, the recovery rate would gradually increase. The results showed that when the volumes of elution reagents were 2 mL, 3 mL, 4 mL, 5 mL, and 6 mL, respectively, the recovery rates of 11 SCs were 15.5-20.2%, 50.4-79.5%, 79.6-104%, 81.4-103.1%, and 82.7-107.9%, respectively. It could be seen that when the recovery rate varied little when the elution reagent volume was 4 mL, 5 mL, and 6 mL. When the volume of the elution reagent was 4 mL, all the 11 SCs could achieve a good extraction recovery (Figure 3).

Establishment of standard curves
After pretreatment, 11 SCs standard compounds were added into blank rat urine to configure matrix standard curves and the concentrations of the 11 standard curves were 0.05 ng/mL, 0.1 ng/mL, 0.2 ng/mL, 0.5 ng/mL, 1 ng/mL, 5 ng/mL, 10 ng/mL and 20 ng/mL. The matrix standard curve and the prepared samples were analyzed by LC-MS/MS. Linear regression is performed with the peak area as the ordinate coordinate and the standard solution concentration as the abscissa. The linear regression equation was obtained with a linear range of 0.05-10 ng/mL ( Table 2). The results show that all 11 target analytes are linear in the range of 0.05-10 ng/mL, and the correlation coefficient is between 0.993-0.999. The linear fit complex correlation coefficient in the linear regression equation is greater than 0.99, indicating good linearity [32].

Method validation
Blank rat urine samples were treated with solid-phase extraction method, and then mixed standard substances of 11 SCs were added to prepare 0.1 ng/mL, one ng/mL, and ten ng/mL spiked rat urine sample solution. When diluting urine with acetonitrile reagent, other substances along with the analyte, including salts, amines, fatty acids, and other small molecules. The flow of these substances and analytes out of the spray needle can affect the atomization, volatilization, splitting, chemical reaction, and charging process of the analyte, resulting in a decrease or increase in ions entering the mass spectrometry, thereby affecting the reliability and accuracy of quantitative results. The final matrix effect range in our experiment was 76.7%-106.1% (Table 3). In addition to AB-PINACA, the matrix effects of the 11 SCs are all within 80-100%, indicating that the influence of the matrix is small [33]. It can be concluded that the optimized solid-phase extraction method can effectively reduce the matrix effect.
The blank rat urine was mixed with 11 kinds of SCs, and the concentrations were one ng/mL, ten ng/mL, and 100 ng/mL, respectively. Urine samples from rats (n=6) were treated with SPE to evaluate the recovery and precision of target analytes extracted by SPE pretreatment. The spiked recoveries of 11 SCs at three levels were 69.90-118.39% (Table 4). As for the accuracy in method verification, the generally acceptable recovery rate ranges from 80 to 120%, indicating that the recovery rate of this method is within the acceptable range [34]. And the intraday precision was 0.52-18.95%. The systematic and random errors of the illustrative method are minor so that they can meet the quantitative analysis. The LOQ was the concentration of the analyte when the signal-noise ratio was 10. LOQ were 0.01 ng/mL for JWH-122 and AMB-FUBINACA, 0.03 ng/mL for 5F-AMB, UR-144, RCS-4 and 5F-ADB, and 0.1 ng/mL for AB-CHMINACA, AB-PINACA, ADB-FUBINACA, MDMB-FUBINACA and 5F-MDMB-PICA (Table 2). This method meets the requirements of trace detection. LOQ is lower than most previous studies that detect SCs in urine [26,35,36].

Conclusion
In our study, the solid-phase extraction LC-MS/MS method was established to determine 11 synthetic cannabinoids in rat urine.    Table 3 Synthetic cannabinoids matrix effects in urine matrix Compound