FUNDAMENTAL-STUDY ON THE QUANTIFICATION OF ENKEPHALIN PEPTIDES BY LC/MS

A liquid chromatographic (LC) and mass spectrometric (MS) method was studied for the quantitative determinations of the enkephalin peptides, leucine-enkephalin (LE; Tyr-Gly-Gly-Phe-Leu) and methionine-enkephalin (ME; Tyr-Gly-Gly-Phe-Met), which have opioid-like activity in the mammalian central nervous system. The protonated molecular [M+H](+) ions and deprotonated molecular [M-H](-) ions from LE and ME were detected as major pseudo-molecular ions by positive and negative MS detection, respectively. The production of these ions at the atmospheric pressure chemical ionization interface was affected by the composition of the mobile phase for the LC. The use of an aqueous mobile phase containing acetonitrile and volatile organic bases gave a good production of the pseudo-molecular ions for the peptides. In this case, [M-H](-) ions of LE and ME were more sensitively detected than their [M+H](+) ions. The linear calibration graphs of the peptides were obtained by negative ion and selected-ion monitoring MS when the aqueous mobile phase containing acetonitrile and 20 mM ammonium formate (pH 9.0) was used. The detection limits of LE and ME were 270 pmol and 690 pmol injected, respectively, with a signal-to-noise ratio of 5.