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Finally, to verify the results, paired aliquots of 27 real hair samples were analyzed with both PLE and a protocol optimized by design-of-experiment strategies planned to maximize the extraction yield; in this case a comparable efficiency was observed, suggesting that exhaustive EtG extraction was obtained with both approaches. This finding opens new perspectives in the eligible protocols devoted to hair EtG analysis, in terms of speed, automation, and reproducibility.Cannabis is the most commonly used drug of abuse in pregnancy and afte