Validation of a SPE-LC–MS/MS method for the determination of ketamine and norketamine in micropulverized hair after a single IV dose

• Extensively pulverized hair of 60 μm particle size was used for extraction.
• Extraction was performed in 10 min instead of overnight.
• Ketamine was detected in real samples after a single 1 mg kg−1 IV dose.

A SPE-LC–MS/MS method for the determination of ketamine (KET) and norketamine (NKET) was developed and validated. Extensive pulverization (25 min at 25 Hz) of previously cooled samples (5 min in liquid nitrogen) allowed for extraction in a phosphate buffer (pH 6) solution after 10 min vortex agitation at room temperature, simplifying the coupling of the extraction to an effective mixed-mode SPE (solid phase extraction) clean-up procedure. The extraction optimization was performed with samples fortified by drug incorporation according to a published procedure involving incubation of blank matrices for 16 days. The method was validated for selectivity, matrix effect, linearity, LLQ (lower limit of quantification), precision, accuracy, recovery, carryover and stability after preparation and has proven to be accurate and reliable within a range of 0.02–10 ng/mg for KET and 0.04–4 ng/mg for NKET, meeting proposed KET cutoffs for discrimination from chronic use. In addition, the method was sensitive enough to detect the drugs after unique small (1 mg/kg) intravenous doses received by patients submitted to general anesthesia before surgical procedures. Ketamine levels varied from 0.060 to 0.111 ng/mg and norketamine was positive (