Analysis of methionine enkephalin in human pituitary by multi-dimensional 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 ³H]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 MH⁺ 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 YGG²H₅-F]M. Samples analyzed 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 ± 1.9 μg g⁻¹ tissue; ME-LI, 1.8 ± 0.7 μg g⁻¹ tissue; MH⁺, 2.7 ± 0.6 μg g⁻¹ tissue; SRM, 3.0 ± 0.8 μg 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.