1 SCIENTIFIC REPORTS | (2018) 8:1167 | DOI:10.1038/s41598-018-19313-1 www.nature.com/scientificreports High-throughput analysis using non-depletive SPME: challenges and applications to the determination of free and total concentrations in small sample volumes Ezel Boyacı 1,3 , Barbara Bojko 1,4 , Nathaly Reyes-Garcés 1 , Justen J. Poole 1 , Germán Augusto Gómez-Ríos 1 , Alexandre Teixeira 2 , Beate Nicol 2 & Janusz Pawliszyn 1 In vitro high-throughput non-depletive quantitation of chemicals in biofuids is of growing interest in many areas. Some of the challenges facing researchers include the limited volume of biofuids, rapid and high-throughput sampling requirements, and the lack of reliable methods. Coupled to the above, growing interest in the monitoring of kinetics and dynamics of miniaturized biosystems has spurred the demand for development of novel and revolutionary methodologies for analysis of biofuids. The applicability of solid-phase microextraction (SPME) is investigated as a potential technology to fulfll the aforementioned requirements. As analytes with sufcient diversity in their physicochemical features, nicotine, N,N-Diethyl-meta-toluamide, and diclofenac were selected as test compounds for the study. The objective was to develop methodologies that would allow repeated non-depletive sampling from 96-well plates, using 100 μL of sample. Initially, thin flm-SPME was investigated. Results revealed substantial depletion and consequent disruption in the system. Therefore, new ultra-thin coated fbers were developed. The applicability of this device to the described sampling scenario was tested by determining the protein binding of the analytes. Results showed good agreement with rapid equilibrium dialysis. The presented method allows high-throughput analysis using small volumes, enabling fast reliable free and total concentration determinations without disruption of system equilibrium. Non-depletive determinations of compounds in biological matrices play a signifcant role in many bioanalyti- cal studies (e.g., pharmacokinetic, toxicokinetic, protein binding determinations, etc.) 1,2 . Barring a few excep- tions, most current bioanalytical assays seek protocols that are applicable to relatively small sample volumes (e.g. 100 μL) 3 . For instance, methods that enable high-throughput analysis, while only requiring one hundred micro- liters of biofuid or less, would be highly desirable in a hospital environment where many samples are generated, fast determination and diagnosis are imperative, and only small samples can be taken from certain patients, such as newborns. Further, recent paradigm shifs 4–9 in the toxicological risk assessment arena have spurred the demand for characterization of the exposure in in vitro assay systems to enable non animal-based risk assess- ments 10,11 , a challenge currently being faced by many industries. While nominal concentrations of chemicals added to test systems have long served as the basis of potency calculations due to the accessibility of the nominal 1 Department of Chemistry, University of Waterloo, 200 University Avenue West, Waterloo, Ontario, N2L 3G1, Canada. 2 Unilever U.K., Safety and Environmental Assurance Centre, Colworth Science Park, Sharnbrook Bedford, MK441LQ, United Kingdom. 3 Present address: Department of Chemistry, Middle East Technical University, Ankara, 06800, Turkey. 4 Present address: Department of Pharmacodynamics and Molecular Pharmacology, Faculty of Pharmacy, Collegium Medicum in Bydgoszcz, Nicolaus Copernicus University in Torun, 85-067, Bydgoszcz, Poland. Correspondence and requests for materials should be addressed to J.P. (email: Janusz@uwaterloo.ca) Received: 23 August 2017 Accepted: 27 December 2017 Published: xx xx xxxx OPEN