Subsequently, the SPE method was applied to pooled human urine and plasma samples to test applicability to analyze human samples. Sample treatment was further streamlined and transferred from SPE cartridges to 96-well plates to allow parallel sample extraction and maximize sample throughput. Furthermore, the applicability of the developed workflow for NTA and suspect screening was tested to further expand the coverage of the targeted biomonitoring assay. SPE Sorbent Selection. SPE cartridges with two different sorbent types, HLB and PSA+C18, were considered and the stability and robustness of the material were tested. For this purpose, loading, washing, and elution procedures were individually evaluated. In this first evaluation, analytical standards in LC-MS grade water (H2O) were used as samples to minimize influences of the sample matrix. Analyte trapping efficiency (analyte loss, %) for both SPE sorbents was tested using H2O or 2% methanol (MeOH/H2O, 2/98, v/v) for sample loading and washing. Subsequently, RE for diverse elution solvents were compared including 3% formic acid in methanol (FA/MeOH, 3/97, v/v), 3% ammonia in MeOH (NH3/MeOH, 3/97, v/v), and pure MeOH. Details including conditioning of SPE sorbents, analyte spiking procedures and spiking levels, as well as calculations of the trapping efficiency and RE, are reported in the SI. Sample Buffer Selection Before Loading. Urine and plasma are commonly used sample matrices for human biomonitoring and, especially in the case of urine, varying concentrations of matrix components can influence analyte extraction. To ensure method reliability, commonly utilized buffers, PBS (pH 7.4, 200 mM) and NH4AC (pH 6.0, 2.5 M), and pure H2O were tested for sample dilution before the SPE cleanup. Based on results of our previously published work, a final dilution ratio of 2 was used . Optimization of Washing, Elution, and Reconstitution Steps. Water was selected as the washing solvent and the required volume was optimized by comparing the SSE of either washing one or two times with 1 mL. MeOH was selected as the primary component of the elution solvent due to its widespread application in SPE processes . Considering the strong anion-exchange interactions of PSA with commonly encountered acidic compounds in exposomics , a basic MeOH solution (3% NH3) was used as the elution solvent to compare the RE of the mixed-mode sorbent (PSA+C18). For HLB, both acidic (3% FA) and basic MeOH (3% NH3) were evaluated due to both anion and cation exchange properties of HLB. Furthermore, MeOH and two commonly used solvents, acetonitrile (ACN) and isopropanol (ISO), and a mixture of MeOH/ACN/ISO (1/1/1, v/v/v), were evaluated as elution solvents. The solvent mixture was selected based on results obtained from urine samples. Elution volumes of 200 µL, 400 µL, and 800 µL were subsequently compared using pooled urine samples as a matrix. A typical workflow to improve detection limits is to evaporate raw extracts and reconstitute them in a defined volume. Despite its clear benefits for enhancing the detection of low-abundant analytes, this step is rather time-consuming, especially when dealing with a large number of samples. To further enhance sample throughput and assess the effect of evaporation and reconstitution, two workflows were tested and compared. One set of SPE extracts was dried using a CentriVap Vacuum concentrator (Labconco) and reconstituted with 50% MeOH (MEOH/H2O, 1/1, v/v). In contrast, a second set of extracts was diluted with H2O, resulting in a final mixture of 50% MeOH for comparison. The final concentrations in the extracts of both sets were the same. Method Transfer to 96-well Plates. After the optimization, pooled urine and plasma were used to test extraction and cleanup using the developed workflow for SPE cartridges. Then, the method was transferred to a 96-well plate SPE packed with HLB (Waters Corporation, USA) and the following parameters: SPE plates were conditioned with 1 mL of MeOH, followed by 1 mL of H2O. Then, 400 μL of urine or plasma sample were mixed with 400 μL of PBS. The mixture was loaded on the SPE plate and washed twice with 1mL of H2O each. Gravity flow was selected for sample loading onto SPE and washing to minimize analyte loss. Analytes were eluted by gravity using 200 µL of MeOH twice, for a total of 400 μL, with negative vacuum by manifold before and after the elution to control the flow rate no more than 3 drops per second. RE and SSE were calculated and compared to results determined for SPE cartridges to prove applicability for high-throughput applications. Assessing the Potential of SPE for NTA and Suspect Screening. SPE is a promising technology to enhance method performance for targeted applications, but systematic testing about chemical coverage is a necessity before any potential application in non-targeted analysis. The coverage of other typically detected analytes in human samples and the effect on signal intensities were evaluated by comparing results of the novel 96-well-based SPE approach to results from a PPT method as the reference method for non-targeted applications. SRM 1950 (plasma) and SRM 3672 (urine) were extracted with both workflows and were analyzed using LC-HRMS for NTA. Unknown compounds were annotated using publicly available spectral libraries and a set of 234 authentic analytical standards was spiked into samples for compound identification and to test the method for targeted screening. Results were reported based on well-established reporting schemes, with identified compounds reported with the highest confidence level ("level 1": identified based on authentic chemical standards). Therefore, standards were only spiked into extracts from the SPE process. Spiking levels of these standards are given in Table . After annotation, the effect of the sample preparation method on relative feature intensities was compared. Results were further investigated as a function of retention times and classifications of the annotated features. Details are outlined in the SI. Data Acquisition. Targeted LC-MS/MS. Front-end LC separation was performed using our previously published method . Identical separation gradients were used for LC-MS/MS and LC-HRMS(/MS) using an Agilent 1290 Infinity II and a Thermo Scientific Vanquish Horizon, respectively. An Acquity HSST3 column (1.8 μm, 2.1× 100 mm, Waters) was used with gradient elution using a flow rate of 0.4 mL/min and 0.3 mM ammonium fluoride in H2O as eluent A and ACN as eluent B. Details are reported in Table .