This study aimed to investigate the applicability of cork in a solid phase-based extraction method coupled to a 96-deepwell plate for LC-MS/MS determination of new psychoactive substances and other drugs of abuse in blood. Nowadays, there is a concern in developing new analytical methods that follows the principles of “green analytical chemistry”. These include the miniaturization of methods, utilization of minimal sample and solvent volumes, development of greener solvents and sorbents, among others. Regarding sorbents, natural compounds such as cork can be a great choice to be employed as extraction phase due to its large availability and low cost. Nevertheless, these new materials can help overcome analytical challenges related to postmortem analysis. Regarding sample preparation, blood samples were primarily submitted to protein precipitation by mixing 200 μL of blood, 250 μL of water, 250 μL of zinc sulfate and 50 μL of NaOH, followed by agitation and centrifugation. An aliquot of 500 μL of the supernatant was added to a 96-deepwell plate containing 30 mg of cork powder with particle size ≤ 250 μm. Extraction was performed by agitation for 15 minutes in an orbital shaker at 300 rpm, with consecutive removal of the aqueous phase. Desorption was performed by the addition of 1150 μL of acetonitrile with further agitation at 300 rpm for 2 minutes. The organic layers were recovered and dried under nitrogen stream, reconstituted in 30 μL of methanol and 2 μL was injected in the analytical system. Sample preparation parameters were optimized through univariate and multivariate strategies with the software Statistica 8.0 (Statsoft, Tulsa, USA). A Nexera UFLC system coupled to a LCMS-8045 triple quadrupole mass spectrometer (Shimadzu, Japan) was used for the analysis. Separation was performed on an Acquity UPLC® BEH C18 (1.7 μm, 2.1 × 50 mm) column (Waters, USA) using water (A) and acetonitrile (B) both containing 0.1% formic acid as mobile phases. Total run time was 7.5 minutes. The method was validated according to ANSI/ASB Standard 036 for the following parameters: lower limit of quantification (LLOQ), linearity, carryover, selectivity, precision, bias and dilution integrity. To verify the applicability of the method, three real postmortem blood samples were analyzed. The optimization of extraction parameters was carried out for the following items: choice of cork conformation (powder or sheet), cork particle size, cork mass, extraction time, desorption time, solvent type and solvent volume. The best parameters values are described in “Methods” session. LLOQs ranged between 0.5 and 5 ng/mL. Calibration curves were linear through the ranges of 0.5–85 ng/mL for ethylone, MDA, MDMA, N-ethylpentylone, 25B-NBOMe, 25C-NBOMe, 25I-NBOMe, fentanyl, pentylone and LSD; 1–85 ng/mL for cathinone, mephedrone and PCP; and 5–100 ng/mL for 25-NBOH, with determination coefficient (r2) > 0.99 and absence of carryover effects. Bias, within-run and between-run precision and dilution integrity results were within the acceptable values. The three samples were positive for the presence of MDA and MDMA with concentrations ranging between 23.11–26.59 ng/mL and 79.21–198.13 ng/mL, respectively. Sample preparation optimization was found to be relevant for the achievement of better analytical responses. Additionally, the use of the 96-deepwell plate allows simultaneous processing of multiple samples, with further possibility of automation. This study demonstrate that cork can be employed as a biosorbent for the determination of NPS and other drugs of abuse in postmortem blood through proper optimization, validation and application of the methodology. Therefore, it can be an alternative extraction phase to be considered in method development for toxicological purposes.
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