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A novel approach has been developed for the illicit drugs quantitative determination using dried blood spots (DBS) on filter paper. The illicit drugs tested were opiates (morphine and its 3- and 6-glucuronide metabolites, codeine, 6-monoacetylmorphine), cocainics (ecgonine methylester, benzoylecgonine. cocaine, cocaethylene) and amphetamines (amphetamine, methamphetamine, MDA, MDMA, MDEA). The described method, requiring a small blood volume, is based on high performance liquid chromatography coupled to tandem mass spectrometry using on-line extraction. A Whatman card 903 was spotted with 30 pi of whole blood and left overnight to dry at room temperature. A 3-mm diameter disk was removed using a manual punch, suspended in 150 mu L of water for 10 min with ultra-sonication, and then 100 mu L. was injected in the on-line LC-MS/MS system. An Oasis HLB was used as an extraction column and a C18 Atlantis as an analytical column. The chromatographic cycle was performed with 20 mM ammonium formate buffer (pH 2.8) (solvent A) and acetonitrile/solvent A (90:10, v/v) gradient in 16 min. Detection was performed in positive electrospray ionization mode (ESI+) with a Quattro Micro (Waters). Recoveries of all analytes were up to 80%. DBS were stored in duplicate at 4 degrees C and -20 degrees C for up to 6 months. Illicit drugs seemed to be much more stabled at -20 degrees C. Furthermore, it was tested whether analysis of DBS may be as reliable as that of whole blood investigating authentic samples; significant correlations were obtained. This DBS assay has potential as rapid, sensitive and inexpensive option for the illicit drugs determination in small blood volumes, which seems of great interest in suspected cases of driving under the influence of drugs. (C) 2011 Elsevier B.V. All rights reserved.