Chemicals and materials
Azacyclonol and fexofenadine-d6 were purchased from MedChemExpress (Monmouth Junction, NJ, USA). Fexofenadine was purchased from Combi-Blocks (San Diego, CA, USA). Cyclizine was purchased from Toronto Research Chemical (Toronto, ON, Canada). Cyclizine and fexofenadine-d6 were used as internal standards. Acetic acid (HPLC grade), acetonitrile, and methanol were purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan). Pipradrol was kindly donated by Dr. Ruri Kikura-Hanajiri of the National Institute of Health Sciences in Tokyo, Japan. Oasis® HLB cartridges (WAT094225, Waters, Milford, USA) were used for the extraction from the urine samples. Blank urine verified in-house with a full toxicological screening for drugs was collected from a healthy volunteer who gave informed consent.
Sample collection
Urine samples were routinely collected from patients at the Department of Emergency and Critical Care in Tokai University Hospital who were diagnosed with poisoning due to overdose. Urine samples collected for toxicological analysis were stored in a freezer (−30°C) until analysis.
Sample preparation
Five microliters each of fexofenadine-d6 and cyclizine (1 μg mL-1) was added to 0.2 mL of the urine sample. Subsequently, 0.8 mL of milli-Q water was added to each sample. The samples were mixed well and were applied to the HLB cartridges.
The HLB cartridges were conditioned with 1 mL each of methanol and milli-Q water. The samples were applied to the conditioned cartridges and were passed through them at a flow rate of 5 mL min-1 using a vacuum pump. The cartridges were then washed with 1 mL of milli-Q water and 1 mL of 5% methanol and were dried under a vacuum for 1 min. The residual compounds were eluted with 1 mL chloroform-methanol (9:1). The resultant solution was dried under a stream of nitrogen at room temperature. The dry residue was dissolved in 80 μL acetonitrile and 20 μL acetic acid and was filtered through a 0.45-μm membrane filter prior to injection into the LC system.
LC–MS conditions
Chromatographic separation was evaluated on an Eclipse Plus C18 analytical column (Agilent, Santa Clara, CA, USA; 50 mm × 2.1 mm ID, 1.8 μm particles), on a Poroshell 120 EC-C18 analytical column (Agilent, Santa Clara, CA, USA; 100 mm × 3.0 mm ID, 2.7 μm particles), and on a Xterra MS C18 (Waters, Milford, MA, USA; 150 mm × 2.1 mm ID, 3.5 μm particles) at 40 °C using the Agilent 1200 LC system (Agilent, Santa Clara, CA, USA). Separation was performed with the gradient elution of 0.1% acetic acid–acetonitrile at a flow rate of 0.3 mL/min. The injection volume was 5 μL.
Electrospray ionization tandem MS (ESI-MS) detection was performed on an Agilent 6410 triple quad tandem mass spectrometer (Agilent, Santa Clara, CA, USA). The positive ionization mode was used, and the ions were monitored in the multiple reaction monitoring (MRM) mode. The ESI source parameters were as follows: high-purity drying gas (N2) flow rate, 6 L min-1; temperature, 300°C; capillary voltage, 4000 V; and nebulizer, 15 psi.
Validation
The method was validated for three compounds. Linearity was evaluated through the analysis of the azacyclonol, pipradrol and fexofenadine working solutions with human blank urine in final concentrations of 1, 5, 10, 25, 50, 100, 250, 500, and 1000 ng mL-1. The coefficient of determination should have a R2 ≥ 0.990. The lower limit of detection and quantification (LLOQ) for all analytes were 0.5 ng mL-1 and 1 ng mL-1, respectively. The precisions and accuracies of the method were estimated by replicating analysis of the QC samples at four concentration levels: 1 (LOQ) ng mL-1, 20 (low QC) ng mL-1, 200 (medium QC) ng mL-1, and 900 (high QC) ng mL-1. The precision and accuracy were expressed as relative standard deviation (RSD%) and RE related error (RE%), respectively. Intra-assay impression (n = 5; assays = 4) was 1.6% – 12.8% for all analytes, while inter-assay impression (n = 20) was less than 10.4%.