Simultaneous quantitative analysis of sphingoid base 1-phosphates in biological samples by o-phthalaldehyde precolumn derivatization after dephosphorylation with alkaline phosphatasel

This paper describes a simultaneous analytical method for the measurement of sphingoid base I-phosphates and sphingoid bases from a variety of biological samples. This method consists of two steps of sample pretreatment: the enzymatic dephosphorylation of sphingoid base 1-phosphates by alkaline phosphatase (APase) and the subsequent analysis of o-phthalade-hyde (OPA) derivatives of the liberated sphingoid bases by HPLC. By introducing C-17-sphingosine 1-phosphate and C-17-sphingosine as internal standards, not only phytosphingosine 1-phosphate, sphingosine 1-phosphate, and sphinganine 1-phosphate but also phytosphingosine, sphingosine, and sphinganine present in a sample could be quantified in 12 min on a C-18 reversed-phase column with a simple mobile phase of acetonitrile: deionized distilled water (90:10, v/v). With this HPLC method, we could reproducibly analyze the levels of sphingoid base 1-phosphates over a broad range of concentrations from 0.5 to 100.0 pmol from various biological samples including serum, cultured cells, and rat tissue homogenates. The conversion of sphingoid base I-phosphates into sphingoid bases increased the stability of the OPA adducts. Thus, this indirect measurement of sphingoid base I-phosphates increased the sensitivity and reproducibility of the method. This HPLC method was also used to measure the changes in the levels of sphingoid base 1-phosphates in cultured cells after treatment with 1,25-(OH)(2)D-3, a sphingosine kinase activator, or with fumonisin B-1, a sphinganine N-acyltransferase inhibitor. (C) 2002 Elsevier Science (USA).