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The use of HPLC for determining amphetamines has not received much attention because of the poor response of UV spectrometric detectors to these compounds when present at low concentration levels. However, HPLC coupled with mass spectrometry can provide the sensitivity required for the analysis of analytes at low concentrations in biological samples. In comparison to GC–MS and LC-UV analyses, LC–MS–MS offers superior sensitivity, selectivity and robustness for a wide range of analytes without any need for derivatization. LC–MS–MS technology also makes possible the development of highly selective, sensitive and precise methods for the simultaneous analysis of amphetamines and their metabolic derivatives. In combination with LC–MS–MS, limits of quantitation (LOQ) of 0.54 ng/mL are achieved for MAMPH, HMMA, and MDMA and LOQ of 1.0 ng/mL are achieved for AMPH and MDA on Gemini C18 HPLC columns.