Matrix effect on mass spectrometry response was investigated with commercially available electrospray ionisation (ESI) and atmospheric pressure chemical ionisation (APCI) sources coupled with a single quadruple mass spectrometer. A post-column infusion system was used to observe the MS signal alterations of methadone, selected as the model compound, in plasma. For this purpose, two sample preparation procedures were tested: (1) conventional off-line sample preparations with liquid-liquid extraction (LLE), solid-phase extraction (SPE) and protein precipitation (PP) with perchloric acid (PA) and acetonitrile (ACN) and (2) on-line SPE with two different extraction columns packed with a large particle support (LPS) and with restricted access material (RAM), respectively. Whatever the sample preparation procedures, APCI source appeared to be less liable to matrix effect than ESI source. Among the different off-line sample preparations, LLE was the most efficient extraction procedure. With other techniques, MS signal was affected not only by endogenous material but also by procedure introduced compounds. Moreover, on-line SPE-LC-MS configuration exhibited matrix effects, which depend on the API source and the extraction support.