Development and validation of a liquid chromatography-tandem mass spectrometry method, using silica column and aqueous-organic mobile phase, for the analysis of clonidine as low as 10 pg/mL in human serum

A bioanalytical method using liquid chromatography-tandem mass spectrometry (LC/MS/MS) was developed and validated for the determination of clonidine in human serum. Clonidine and internal standard clonidine-d(4) were extracted from alkalized human serum using liquid/liquid extraction. The extract was evaporated to dryness, reconstituted, and injected onto a Betasil silica column (50 mm x 3 mm) using an aqueous-organic mobile phase consisting of acetonitrile-water-formic acid (80: 20: 1, v/v/v). The flow rate was 0.7 mL/min. The chromatographic run time was 2.5 min per injection, with retention time of 1.7 min for clonidine. The detection was by monitoring clonidine at m/z 230 --> 213 and IS at m/z 236 --> 219, respectively. The standard curve range was 10-1000 pg/mL. The low limit of quantitation (LLOQ) is 10 pg/mL. The inter-day precision and accuracy of the quality control (QC) samples were <6.4% relative standard deviation (RSD) and <7.9% relative error (RE). The recovery of extraction was 95% for clonidine and 90% for the internal standard.