ANALYSIS OF METHIONINE ENKEPHALIN IN HUMAN PITUITARY BY MULTIDIMENSIONAL REVERSED-PHASE HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY, RADIORECEPTOR ASSAY, RADIOIMMUNOASSAY, FAST-ATOM-BOMBARDMENT MASS-SPECTROMETRY, AND MASS-SPECTROMETRY MASS-SPECTROMETRY

Methionine enkephalin (ME = YGGFM) was measured in five individual human post-mortem pituitaries using four different analytical methods, with the objective of comparing the molecular specificities of the methods. Radioreceptor assay (RRA) used a receptor-rich preparation from brain and [H-3]etorphine as radioligand to determine ME-like receptoractivity (ME-LR). Radioimmunoassay (RIA) measured ME-like immunoreactivity (ME-LI). Pituitary samples analyzed by RRA and RIA were purified first with a high-performance liquid chromatography (HPLC) gradient on a polymer analytical column. Fast atom bombardment mass spectrometry (FAB-MS) in two different detection modes quantified ME using the protonated molecular ion NH+ of ME at 574 a.m.u. and B/E linked-field selected reaction monitoring (SRM) to monitor the specific unimolecular metastable transition that produced the unique amino acid sequence-determining tetrapeptide fragment ion YGGF+ from the MH+ precursor ion. Both FAB-MS methods used the deuterated internal standard YGG[H-2(5)-F]M. Samples analysed with FAB-MS were purified first with multi-dimensional reversed-phase HPLC. The first dimension was an ODS gradient, and the second dimension was a polymer isocratic elution. The following ME amounts were measured (mean +/- standard error of the mean): ME-LR, 7.0 +/- 1.0-mu-g g-1 tissue; ME-LI, 1.8 +/- 0.7-mu-g g-1 tissue; MH+, 2.7 +/- 0.6-mu-g g-1 tissue; SRM, 3.0 +/- 0.8-mu-g g-1 tissue. The FAB SRM method provided the highest level of molecular specificity amount these four analytical methods used to measure picomole amounts of endogenous ME in a human pituitary.