Micro-QuEChERS Extraction for Broad-Spectrum LC-QQQ-MS Quantification of Nitazene Analogs in Plasma: A Validated Method With Casework Application.

Nitazenes are highly potent synthetic opioids of increasing concern in clinical and forensic toxicology, thus underscoring the need for sensitive and selective analytical methods for biological specimens. This study describes the development and validation of an LC-MS/MS method for the quantification of nitazene analogs and related metabolites in plasma using micro-QuEChERS sample preparation. The validated concentration range was 0.05-20 ng/mL, with coefficients of determination R2 > 0.99. The limit of detection (LOD) was 0.025 ng/mL, and the lower limit of quantification (LLOQ) was 0.05 ng/mL. Bias ranged from -2.6% to 15.7%, within-run precision from 3.4% to 12.7%, and between-run precision from 4.4% to 14.9%, as evaluated at three quality control levels. Matrix effects and recovery were analyte-dependent but compatible with satisfactory overall validation performance. No interferences or carryover were observed. Method applicability was demonstrated in an authentic intoxication case, in which N-pyrrolidino protonitazene was quantified at 1.4 ng/mL, representing, to our knowledge, the first clinical case reported in Brazil. The case was clinically characterized by severe opioid toxicity requiring repeated naloxone administration. The proposed method combined low sample volume, simple sample preparation, and suitable analytical performance for targeted analysis of emerging nitazene compounds. These findings support the use of micro-QuEChERS and LC-MS/MS as a practical approach for targeted nitazene quantification in clinical and forensic toxicology.