Nano-LC-MS/MS for the quantitation of ceramides in mice cerebrospinal fluid using minimal sample volume

A new nano-liquid chromatography-ESI-MS/MS method has been developed for the sensitive quantitation of C8:0, C16:0, C18:0, C18:1, C20:0, C24:1 and C24:0 ceramide in cerebrospinal fluid of mice using minimal sample volume. Volumes of 2 µL CSF were undertaken a simple, fast extraction procedure involving protein precipitation with methanol and dilution. Ceramides were separated by nano-liquid chromatography with a reversed phase C8 column and detected with a triple quadrupole mass spectrometer. C17:0 ceramide was used as internal standard. The method has been validated in terms of linearity, lower limit of quantitation, precision, accuracy and autosampler stability. Calibration curves covered a range of 2.25-120 pg/µL for most ceramides (7.5-120 pg/µL for C24:0 ceramide). The lower limits of quantitation determined for C8:0, C16:0, C18:1, C18:0, C20:0 and C24:1 ceramide were 0.225 pg on column (2.25 pg/µL) and that for C24:0 ceramide 0.750 pg on column (7.5 pg/µL). Intra- and interday precision and accuracy values, expressed as relative standard deviation and relative error, respectively, were lower than 15% in all cases. Autosampler stability for calibration standards and CSF samples was proven for at least 24 h for all analytes. The suitability of the method has been demonstrated by quantifying the analytes, except the non-endogenous C8:0 ceramide, in cerebrospinal fluid samples of 12 mice. Calculated concentrations ranged from 3 to 120 pg/µL in cerebrospinal fluid for all analytes, except for C24:0 ceramide, which could not be detected in any of the analyzed samples.