Liquid chromatography/electrospray ionisation-mass spectrometry method for the quantification of sphingosine and sphinganine in cell cultures exposed to fumonisins.
Fumonisins, mycotoxins produced by Fusarium verticillioides, are potent inhibitors of the de novo sphingolipid biosynthesis via inhibition of the key enzyme ceramide synthase. The cellular response to a fumonisin exposure is obvious as an alteration of the ratio of the sphingoid bases sphingosine (SO) and sphinganine (SA). We developed a new column liquid chromatography/electrospray ionisation-mass spectrometry (LC-ESI-MS) method for the rapid, simultaneous and quantitative determination of these bases in cell cultures of immortalised human kidney epithelial cells (IHKE cells). For sample preparation, cell lysates were only diluted, centrifuged and directly used for LC-MS measurements. Quantification was carried out using phytosphingosine (PSO) as an internal standard. Detecting the protonated molecule [M+H](+) signals of SO (m/z 300) and SA (m/z 302) in the selected ion monitoring (SIM) mode, detection limits of 10 pg for SO (signal-to-noise ratio S/N=3:1) and 25 pg for SA (S/N=3:1) were established. The average recovery for SO and SA was higher than 90% for control IHKE-cells, respectively. The developed LC-ESI-MS method allows the sensitive, selective and rapid monitoring of sphingosine and sphinganine in cell matrices with a drastically reduced time for sample preparation.
Seefelder
W
W
Lehrstuhl für Lebensmittelchemie, Universität Würzburg, Am Hubland, 97074 Würzburg, Germany.
Schwerdt
G
G
Freudinger
R
R
Gekle
M
M
Humpf
H-U
HU
D002138:Calibration; D002461:Cell Line, Transformed; D002853:Chromatography, Liquid / Q000379:methods*; D037341:Fumonisins / Q000494:pharmacology*; D006801:Humans; D012015:Reference Standards; D012680:Sensitivity and Specificity; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D013110:Sphingosine / Q000031:analogs & derivatives* / Q000032:analysis*
D037341:Fumonisins; D013110:Sphingosine; C005682:safingol