Detection and application of 40 piperazine compounds in hair suitable for rapid separation of isomers

A simple and sensitive procedure, using benzylpiperazine-D₇ (BZP-D₇) as an internal standard, has been developed and validated for the qualitative and quantitative analysis of 40 piperazines in hair. Drugs were extracted from 20 mg of hair with 0.5 mL of methanol containing 1 ng/mL BZP-D₇. After ultrasonication, centrifugation and filtration, the supernatant was analyzed by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) operating in the multiple reaction monitoring mode. Piperazine-type substances were separated in 10 min on a T₃ column using a mobile phase gradient composed of A (water, formic acid 0.1 %, acetonitrile 5 %, and 20 mmol/L ammonium acetate) and B (acetonitrile). The developed and validated method showed good selectivity, sensitivity (limit of detection: 0.5-20 pg/mg and lower limit of quantitation: 5-20 pg/mg), linearity (R² > 0.99), accuracy, precision, and dilution integrity. The method also showed good recovery and acceptable matrix effects for most of the targeted compounds. This analytical approach was successfully applied for the identification and quantification of piperazine-type substances in hair from rat and guinea pig.