Capillary liquid chromatography and tandem mass spectrometry for the quantification of enkephalins in cerebrospinal fluid

A capillary LC-MS/MS system was evaluated for the absolute quantification of enkephalins in cerebrospinal fluid (CSF). On column focusing on a C18 trapping column, in-line with the analytical column, was used for preconcentration. Quantification was performed with a triple quadrupole instrument in the multiple reaction monitoring mode. Weighted linear regression analysis proves to be a good linearity in a dynamic range of two orders of magnitude. The method was validated, yielding calibration curves with correlation coefficients greater than 0.9914. Assay precision and accuracy were evaluated by direct injection of enkephalin fortified artificial CSF (aCSF) samples at three concentration levels. Mean accuracy of analysed concentrations was between 97.63 and 107.6%. LOD and LOQ were assessed at, respectively, 0.5 and 1 pmol/mL. Validation results show that it is feasible, with a capillary LC-MS/MS system, to quantify neuropeptides in the low femtomole range in aCSF. The obtained coefficients of variation, however, indicate that the use of appropriate isotopically labelled internal standards in neuropeptide quantification using narrow bore LC, combined with ESI-MS, may be highly beneficial.