O31: Comprehensive non-MS drug screening by UPLC-PDA-CAD with durable quantitative calibration

Abstract

Introduction Mass spectrometric (MS) methods are today widely used in analytical toxicology, but in comprehensive quantitative drug screening it is laborious to maintain quantitative calibration and use of historic calibration is not feasible. In non-MS gas chromatographic (GC) and liquid chromatographic (LC) methods, historic calibration is widely used. However, GC is not amenable to analysis of current polar drugs and traditional LC-UV methods do not have high enough identification power to meet the requirements of screening. Ultra-performance LC (UPLC) with two consecutive detectors appears to possess high potential for simultaneous multi-component screening and quantification. In this study, a comprehensive quantitative screening method for 170 basic drugs in blood samples was developed and validated using UPLC coupled with photodiode array (PDA) and corona charged aerosol detector (CAD). Methods Blood samples were extracted with organic solvent (ethyl acetate: butyl acetate, 25:75) in basic conditions. After extraction, the organic solvent was evaporated to dryness and samples were reconstituted with the UPLC mobile phase. Dibenzepine was used as an internal standard. Chromatographic separation was performed at 60 °C using a HSS C18 column (150 mm × 2,1 mm, particle size 1,8 μm), and the mobile phase consisted of 0.1% trifluoroacetic acid and methanol. After a three minute isocratic phase, a linear gradient from 5% to 95% methanol in 15 minutes followed. The flow rate was 0.4 mL/min. UV spectra were collected in the range of 210–400 nm, and wavelength of 230 nm was used for quantification. Substance identification was based on the UV spectrum, retention times on both detectors, and the response ratio of CAD and PDA at wavelength 230 nm. Calibration was carried out using a single calibration point at the vicinity of the upper limit of therapeutic range of each drug. Individual calibration curves were created for both detectors and the average result was used for quantification. Results The retention times were found be very stable, the relative standard deviation of retention times being Conclusion The developed method allows the detection and quantification of 170 basic drugs in therapeutic and toxic concentrations in a single run. Due to the stability of calibration and good linearity, historic one-point calibration can be utilized. Adding the CAD detector with universal response after the PDA detector increases the reliability of both identification and quantification. In addition, drugs with poor UV absorption can be detected and quantitated. This method provides a tool for comprehensive quantitative screening for ordinary basic drugs in blood and leaves LC-MS target analysis to be applied to low-dose compounds.