Determination of Triazolam and -Hydroxytriazolam in Guinea Pig Hair After a Single Dose

A sensitive liquid chromatography-tandem mass spectrometry method is presented for determination of triazolam and α-hydroxytriazolam in guinea pig hair after a single dose of triazolam. Eighteen guinea pigs were divided into three dosage groups (10, 100, and 500 µg/kg) and administrated a single dose of triazolam intragastrically. Before administration, drug-free hair was shaved from their back. Newly grown hair in shaved area was collected every seven days after administration. About 20 mg of decontaminated hair was cut into small segments and incubated in 2 mL of phosphate buffer (pH 8.4) at 45°C overnight. Triazolam-d4 and α-hydroxytriazolam-d4 were used as internal standards, and liquid-liquid extraction was performed with 3 mL of ethyl ether. The sample was separated on an Allure propyl PFP column with a mobile phase of acetonitrile/20 mM ammonium acetate (7:3, v/v). Detection was implemented with multiple reaction monitoring mode by an API4000 triple-quadrupole tandem mass spectrometer. Limits of detection for triazolam and α-hydroxytriazolam were 1 and 5 pg/mg, respectively. Triazolam and α-hydroxytriazolam could only be detected in the first week, and 100 µg/kg was the minimal dosage detectable in guinea pig hair. The concentration of triazolam in hair was related with administration dosage and hair color. α-Hydroxytriazolam has a higher concentration than triazolam in guinea pig hair.