An efficient enrichment method using immobilized metal affinity chromatography (IMAC) was developed for selective extraction of bioactive sphingoid base-1-phosphates (SB1Ps) from adventitious roots of Hypericum perforatum cultured in bioreactor. The phosphate-selective IMAC enrichment coupled with LC-MS/MS enabled sensitive analysis of low-abundance SB1Ps present in the root biomass, which would not be feasible otherwise due to severe interferences from complex biological matrices. The time-dependent growth rate and production of SB1Ps from adventitious roots were investigated. The level of phytosphingosine-1-phosphate, which was found to be the major SB1P5, reached a maximum amount of 635.6 pmol per gram of dry weight after 3 weeks of culture and decreased between 3 and 5 weeks of culture subsequently. On the other hand, sphingosine-1-phosphate and sphinganine-1-phosphate were present at levels of 18.91 and 73.15 pmol per gram of dry weight, respectively, after a week of culture and their level decreased thereafter. (C) 2012 Elsevier Ltd. All rights reserved.