Analysis of constitutional isomeric phenethylamines and synthetic cathinones by supercritical fluid chromatography and tandem mass spectrometry

Abstract

An ultra high performance supercritical fluid chromatography-tandem mass spectrometry (UHPSFC-MS/MS) method was developed and validated for the determination of a group of basic drugs of abuse in human whole blood. The following compounds were evaluated for the applicability for this technique: 2-, 3-, and 4-fluoroamphetamine, 2-, 3-, and 4-fluoromethamphetamine, 2-, 3-, and 4-methylmethcathinone, 2-, 3-, and 4-methylamphetamine, amphetamine, methamphetamine and 3,4-methylenedioxymethamphetamine. For the validation of the method the following compounds were included: 2-, 3-, and 4-fluoroamphetamine, 2-, 3-, and 4-fluoromethamphetamine, amphetamine, methamphetamine and MDMA. The sample preparation consisted of liquid-liquid extraction using ethyl acetate : heptane (80:20, v/v). The samples were reconstituted in isopropanol before injection. Four 13C6-labelled analogs were used as internal standards. The compounds were separated using an ethylene-bridged hybrid column (3 mm x 100 mm, 1.7 µm) by gradient elution with 40 mM ammonia in methanol and supercritical carbon dioxide.. Quantification was performed by tandem MS using multiple reaction monitoring in positive mode, applying two transitions for the compounds and the internal standards. The run time for the method was 4 min. The calibration curves had r2 above 0.99 for all the compounds.The interday precision was below 15 % for all the 2-, 3-, 4-phenethylamine analytes for the levels above lowest limit of quantification (LOQ), while the classical phenethylamines displayed a precision below 10 % for the levels above LOQ. The intermediate accuracy was below 20 % for all the 2-, 3-, 4-phenethylamine analytes for the levels above LOQ, while the classical phenethylamines demonstrated accuracy below 10 % at all levels. However, the accuracy diverged less than 3 % for most of these levels. LOD varied from 0.007 to 0.02 µM, while LOQ ranged from 0.02 to 0.06 µM for all analytes. Matrix effects were between 67 and 81 % for all the 2-, 3-, 4-isomers, while the classical phenethylamines experienced between 88 and 93 % matrix effect. Extraction recovery was above 80 % for the 2-, 3-, 4-isomers, though the classical phenethylamines had a minimum of 50 % extraction recovery. Carry-over was measured to range between 0.34 and 0.88 % for all analytes. Intraday precision was below 15 % for all analytes, while the intraday accuracy was +/- 20 % for all analytes above LOQ. It was noted that for the conditions of this method chromatographic separation was decreased with repeated injections, proposedly caused by a silyl ether formation or alcohol adsorption on the stationary phase. Additionally, a spray pulsing effect was observed in the UHPSFC-MS interface corresponding to high back pressures.