## 4 Discussions  
In the sample preparation methods presented previously for the serum, urine and saliva matrices, numerous semi-automated methods exist. The hurdle to complete automation is in many cases the subsequent evaporation step. We present a flexible system for fully automated sample preparation in the determination of THC and its metabolites. The system can easily be used for the determination in different matrices, such as serum, urine or saliva. Due to the use of solid-phase extraction, we can avoid time-consuming and error-prone evaporation steps.  
Limits of detection and quantification for serum sample preparation methods using LC-MS/MS ranges from 0.05-1.8 ng/mL for \(\Delta\)9-THC, 0.18-3.2 ng/mL for THC-OH, and 0.05-2.8 ng/mL for THC-COOH [8; 22; 24; 26; 27; 28; 29; 30]. The limit of detection and quantification determined by [PERSON] et al. for \(\Delta\)9-THC is 0.2 ng/mL and 0.5 ng/mL using LC-MS, which is within the limits determined using LC-MS/MS [21]. The methodological limits of detection and quantification obtained with our sample preparation method for serum are 0.156 ng/mL and 0.176 ng/mL for \(\Delta\)9-THC-D\({}_{3}\), 0.349 ng/mL and 0.384 ng/mL for THC-OH, and 0.896 ng/mL and 0.987 ng/mL for THC-COOH and are, therefore, comparable to the results reported in the literature.  
The limits of detection and quantification for THC from urine reported in the literature and previously introduced range from 0.1-22 ng/mL, for THC-OH from 0.2-25 ng/mL, and for THC-COOH from 0.2-7.8 ng/mL [10; 34; 37; 71; 72]. With the urine sample preparation method presented here, method detection and quantification limits are 0.04 ng/mL and 0.111 ng/mL for \(\Delta\)9-THC-D\({}_{3}\), 0.113 ng/mL and 0.279 ng/mL for THC-OH, and 0.235 ng/mL and 0.531 ng/mL for THC-COOH and are, thus, likewise within the range reported in the literature.  
An exception is THC-OH from saliva, for which the limit of detection we achieved is 0.328 ng/mL, and the limit of quantification is 0.773 ng/mL, which is higher than the limits of detection and quantification reported by [PERSON] et al. using MEPS with 0.12 ng/mL and 0.4 ng/mL due to a higher concentration factor (2.5 compared to 2.05). The Substance Abuse and Mental Health Services Administration (SAMSHA) recommend cutoff limits of 1 and 2 ng/mL for driving under the influence of drugs, alcohol, and medicines (DRUID). The values can be reached by our automated method [61]. The same applies for the analytes \(\Delta\)9-THC-D\({}_{3}\) and THC-COOH, which are within the values reported in the literature of 0.005-5 ng/mL for \(\Delta\)9-THC and 0.002-3.5 ng/mL for THC-COOH with methodological LODs and LOQs of 0.037 ng/mL and 0.082 ng/mL and 0.942 ng/mL and 2.247 ng/mL, respectively [10; 62; 63]. Therefore, we demonstrated that sensitive detection of the analytes \(\Delta\)9-THC-D\({}_{3}\), THC-OH, and THC-COOH is possible even without a final evaporation step using the presented automated sample preparation methods for the matrices' serum, urine and saliva.  
The specific use of SPE for concentration is achieved by using SPE plates with a very low sorbent bed volume of 2 mg, which also requires a very small elution volume (at least 25 \(\mu\)L) for the elution of the retained analytes, thus avoiding additional dilution and then, necessary evaporation and reconstitution. Furthermore, another advantage of reducing the solvent volume is the reduction of toxic waste and solvent vapors, contributing to a greener and healthier laboratory of the future.  
The few fully automated methods use either SLE or SPE to prepare whole blood, urine or saliva samples. The SLE technique is very fast with an average of 1.5 h sample preparation time for 96 samples but shows low recovery values (23-51%) for THC [19; 28; 35]. In the SPE with previous PPT or hydrolysis, the recovery rates are higher (66-75%) [21; 35] but also the time required is significantly higher, e.g., 2 h 50 min for 15 samples [21].