Ceramide-1-phosphate (C1P) is a bioactive sphingolipid with roles in several biological processes. Currently, high performance liquid chromatography-electrospray ionization-tandem mass spectrometry (HPLC ESI-MS/MS) offers the most efficient method of quantifying C1P. However, the published protocols have several drawbacks causing overestimations and carryovers. Here, the reported overestimation of C1P was shown to be due to incomplete neutralization of base hydrolyzed lipid extracts leading to the hydrolysis of SM to C1P. Actual quantity of C1P in cells (6 pmols/10(6) cells) was much lower than previously reported. Also, the major species of C1P produced by ceramide kinase (CERK) was found to be d(18:1/16:0) with a minority of d(18:1/24:1) and d(18:1/24:0). The artifactual production of C1P from SM was used for generating C1Ps as retention time markers. Elimination of carryovers between samples and a 2-fold enhancement in the signal strength was achieved by heating the chromatographic column to 60 (degrees) C. The role of ceramide transport protein (CERT) in supplying substrate to CERK was also revalidated using this new assay. Finally, our results demonstrate the presence of additional pathway(s) for generation of the C1P subspecies, d(18:1/18:0) C1P, as well as a significant portion of d(18:1/16:0), d(18:1/24:1), and d(18:1/24:0). In conclusion, this study introduces a much improved and validated method for detection of C1P by mass spectrometry and demonstrates specific changes in the C1P subspecies profiles upon downregulation of CERK and CERT.