Humans are consistently exposed to a myriad of chemicals during their daily lives through various sources, including dietary intake and environmental pollutants . Exposomics represents the comprehensive analysis of all environmental and food-related exposures and associated influences on various health outcomes . To allow a full assessment of the totality of chemical exposures in human samples, sensitive, specific, and robust analytical techniques, such as liquid chromatography-tandem mass spectrometry (LC-MS/MS) are required. Multi-class or next-generation human biomonitoring (HBM) methods have recently been developed to monitor a wide range of environmental chemicals and are needed to address the full complexity of chemical exposure . In contrast, non-targeted analysis (NTA) is an emerging technique for exposomics using high-resolution mass spectrometry (HRMS) and allows the exploration of unknown chemicals in human samples . Furthermore, suspect screening methods use large chemical databases and spectral libraries to prioritize detected features and annotate compound identities . Both targeted and non-targeted exposomic methods face significant challenges related to complex biological sample matrices , and ensuring repeatability and consistency across large sample sets is mandatory, especially when dealing with low-abundance analytes (i.e. pg/mL levels or lower) . Addressing these challenges in exposomics requires sophisticated optimization of the entire analytical workflow, including sample preparation protocols. Efficient sample pretreatment before instrumental analysis can reduce interferences, separate, and concentrate analytes in diverse matrices and various sample preparation techniques have been optimized to analyze the exposome. Liquid extraction (LE) and protein precipitation (PPT) are widely used extraction methods for exposomics due to their comparably wide chemical coverage . However, these techniques are prone to preserve matrix interferences which can hamper overall method performance due to limited sample clean-up. Recently, solid phase extraction (SPE) got more attention as an option for sample preparation in exposomics and is discussed as a promising approach for future large-scale applications, owing to its capability to reduce matrix effects (ME), enhance sensitivity, and improve consistency in highthroughput studies . SPE has been frequently used for the purification and extraction of various analytes from diverse sample matrices for HBM including urine , blood , and various other biological matrices . However, a systematic development of an exposome-scale SPE method compatible with both, targeted and non-targeted data acquisition strategies has rarely been performed. This challenge originates from the complexity of designing a balanced method with suitable method robustness and broad chemical space coverage. Most existing SPE methods focused on a limited selection of targeted analytes to obtain reliable protocols without assessing the potential coverage for NTA due to the lack of certified reference standards, which restricts their utility in NTA. In this study, a targeted high-throughput sample pretreatment method using SPE cartridges was optimized to assure the applicability of the workflow for urine and plasma samples. The predicted octanol-water partition coefficient (logP) from PubChem (pubchem.ncbi.nlm.nih.gov) was utilized to evaluate the chemical coverage in terms of analyte lipophilicity of compounds in this study. Target analytes with a wide range of physicochemical properties, including polarity, presents a central challenge for SPE methods in exposomics, due to varying analyte-sorbent interactions. This issue is a key factor that needs to be addressed during method development aiming for a comprehensive chemical coverage. Here, 94 highly diverse analytes from multiple toxicant classes including perfluorinated substances (PFAS), plasticizers, personal care products, food processing by-products, pesticides, industrial chemicals, air pollutants, disinfecting agents, mycotoxins, phytoestrogens and phytotoxins were used to optimize an SPE protocol for high-throughput applications. Optimization steps included selecting SPE sorbents and sample buffers, optimizing washing and elution steps, and improving efficiency. Subsequently, the protocol was transferred to 96-well plates, and analytical performance and throughput capacity were rigorously tested. Finally, the potential for NTA was explored using NIST standard reference materials (SRMs) for urine and plasma. The results indicate the feasibility of the approach for scaling and large-scale exposome-wide association studies. Experimental Section Biological samples and chemicals. Human pooled plasma was purchased from Innovative Research (Novi, MI, USA). Pooled urine was obtained from a female volunteer who abstained from consuming food and beverages stored in plastic containers, phytoestrogens rich food, and cosmetics containing parabens for two days before sample collection . Standard reference materials, including urine (SRM 3672) and plasma (SRM 1950) were obtained from the National Institute of Standards and Technology (NIST) (Gaithersburg, USA) for NTA. Detailed information on standards, including selection and spiking levels can be found in Tables . The selection of target analytes and analyte classes was intended to be as broad as technically feasible with differing structures, real-life exposure levels, and toxicological modes of action. It was based on our previous studies on next-generation biomonitoring (Jamnik et al. 2022) . Method development and optimization. The sample pretreatment approach was developed and optimized in several steps. Initially, two universal SPE sorbents, a commercial hydrophilic-lipophilic balanced sorbent (HLB) from Waters and an in-house prepared mixed-mode SPE sorbent, were compared. The mixed-mode sorbent was prepared from two SPE sorbents, primary second amine (PSA) and C18 (PSA+C18), by mixing equal weights before packing. Details of the preparation are described in the Supplementary Information (SI). Packed SPE cartridges (HLB or PSA+C18) were then used to optimize the full processes including sample loading, washing, and elution. A mixture of 94 reference standards was spiked into urine, plasma, and H2O samples both before ("prespiked") and after ("postspiked") the SPE process. Analyte loss (%), extraction recovery (RE, %), and signal suppression and enhancement (SSE, %) were calculated based on the peak areas from these "prespiked" and "postspiked" samples. Analyte loss was defined as the fraction of an analyte that was not retained by the SPE sorbent. It was calculated as the ratio of peak areas of the effluents collected during sample loading and washing from "prespiked" samples compared to peak areas of "postspiked" effluents. Analyte loss of 0% was achieved for complete analyte retention, whereas completely unretained analytes are characterized by a loss of 100%. The term extraction recovery (RE) was used to describe the recovery of the full SPE process including loading, washing and elution. Thus, RE was calculated as the ratio of peak areas of a "prespiked" and "postspiked" sample. Full analyte recovery results in RE of 100%, whereas 0% indicates no analyte recovery of the SPE procedure. RE greater than 100% indicate contamination or carryover introduced during sample preparation or sample injection. Matrix effects in terms of signal suppression and enhancement (SSE) depend on the influence of the sample matrix on the analyte's signal intensity. This was calculated as the ratio of peak areas for "postspiked" samples to a standard in pure solvent . SSE values close to 100% indicate negligible effects of the sample matrix on the detector response, which is required for accurate analyte quantification. To evaluate the method performance and provide a rationale for the individual optimization steps, the metrics described above were classified into three categories: "good," "acceptable," and "poor," as shown in Table . Noteworthy, they do not represent method validation criteria but are used for method optimization. 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 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 HBM 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 the 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 (Table ) 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 reported 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 . A QTrap 6500+ mass spectrometer equipped with an electrospray ionization (ESI) source (Sciex) was operated using one of our previously published multiple reaction monitoring (MRM) methods with fast polarity switching . Full method parameters including MRM transitions and ESI parameters are provided in the SI. Non-targeted HRMS(/MS). An Orbitrap Exploris 480 mass spectrometer (Thermo Scientific) was used for HRMS(/MS) measurements. Samples were analyzed using full scan mode and AcquireX was used to generate fragment spectra using a mass resolution of 120,000 at m/z 200, with full method parameters reported in the SI. Data Analysis Quality Control. Several types of QC samples were used to ensure reliable results. A systems suitability test sample (SST) was measured before and after each analytical sequence to ensure favorable instrument performance (see SI and Table ). In addition, a multi-analyte reference standard mixture consisting of 94 highly diverse chemicals in pure solvent was injected. Solvent blanks were used to monitor instrumental background and carry-over and process blanks were used to monitor background concentrations/contaminations. Targeted Data Analysis. RE and SSE values were evaluated for the set of 94 analytes 6 using standards in neat solvent, pooled urine, and pooled plasma. Peak integration was carried out using Multiquant (v3.0.2, Sciex) and Skyline (v21.2.2.536, McCoss Lab) . Calculation of RE and SSE was performed using Microsoft Office 365. Graphics were created by Origin 2021b (v9.8; OriginLab Corporation). Non-targeted Data Analysis. Raw data acquired in polarity switching mode was split based on the polarity using Freestyle (Thermo). Then, raw data files were analyzed using MSDIAL (v5.2) . Full data processing settings including data extraction, peak picking, compound identification, and alignment are reported in the SI. To evaluate the qualitative analyte coverage for NTA and suspect screening, MassBank of North America (MoNA, ) was used as a spectral library. Annotations were then classified based on the level of confidence scheme according to Schymanski et al. using confidence levels 1 (confirmed by authentic standards), level 2 (confirmed by spectral library match), and level 3 (confirmed by spectral library match, but with isomeric structures). Additional data analysis and visualization was performed using Microsoft Office 365, Origin 2021b, MetaboAnalyst (v6.0), and Inkscape (version 1.2.2; Inkscape).