Quantitation of cardioexcitatory Asn-D-Trp-Phe-NH2 diastereomers in Aplysia's central nervous system by nanoscale liquid chromatography-tandem mass spectrometry.

A tripeptide, Asn-D-Trp-Phe-NH(2) (NdWFa) that contains a D-amino acid residue (i.e. D-tryptophan) was previously identified in Aplysia's central nervous system (CNS) and found to be cardioexcitatory. However, the occurrence of its diastereomers including NWFa, theoretically the precursor of NdWFa, remains largely unknown. In this work, a nanoscale liquid chromatographic/tandem mass spectrometric (nano-LC-MS/MS) method was developed for a sensitive determination of the diastereomers of NWFa. Resolution of the diastereomers including NWFa, NdWFa, NWdFa, and dNWFa was achieved on capillary columns packed with C(18) silica particles with an MS detection-friendly mobile phase consisting of water, acetonitrile, and formic acid. Columns of different internal diameters (IDs) ranging from 75 to 250 microm were evaluated to achieve the best sensitivity. With the use of a 75 microm ID column integrated with a nanoelectrospray emitter, the method had limits of detection (LOD) of 0.21 nM (or 0.49 pg on column, 5 microl injected) NdWFa in tissue homogenate (S/N=3). The five major ganglia in Aplysia californica's CNS (i.e. buccal, cerebral, abdominal, plural, and pedal) were analyzed. NdWFa was detected only in abdominal ganglion at the ng/g tissue level. Further, its diastereomer, NWFa, was also detected for the first time and also only in abdominal ganglion at a significantly lower level. The levels of both NWFa and NdWFa varied from animal to animal in the range from 0 to 81 ng/g tissue.