Microcolumn liquid chromatography and analysis of single cells

Low nanoliter (nL) injection volume and high separation efficiencies allow microcolumns (below 50 micron ({dollar}mu{dollar}m) inner diameter (i.d.)) to be used for single cell analysis. A packed microcolumn liquid chromatography system with electrochemical detection was used for quantitation of histamine, 5-hydroxytryptamine (5-HT) and biogenic amines in single mast cells. On average, a single mast cell contains 150 femtomoles of histamine and 3.8 femtomoles of 5-HT (n = 12). Extracellular amines affected endogenous cell content due to uptake and metabolism characteristics of mast cells.; The effect of i.d. on microcolumn efficiencies was examined by packing columns with 5 {dollar}mu{dollar}m octadecylsilane-modified silica particles in columns ranging in i.d. from 33 to 12 {dollar}mu{dollar}m. By collecting reduced plate height data of test analytes, norepinephrine (NE), 3,4-dihydroxybenzylamine (DHBA) and dopamine (DA) versus flow velocity, van Deemter curves were plotted. From evaluation of the magnitudes of the A, B and C terms, a linear decrease of the A term as column diameter decreased was the most significant contributor to a lower overall plate height at the optimum velocity.; Improvement in microcolumn efficiencies was achieved by using smaller i.d. microcolumns packed with smaller particles. Columns with 25 {dollar}mu{dollar}m i.d. ranging in length from 110 to 125 cm packed with 3 {dollar}mu{dollar}m octadecylsilane-modified silica particles were used to baseline-separate deuterated NE (D{dollar}sb3{dollar}-NE) and deuterated E (D{dollar}sb3{dollar}-E) from protiated NE and E in a single chromatographic run. This method was applied to determine the activity of phenylethanolamine N-methyltransferase (PNMT) and amounts of endogenous NE and E in single bovine adrenal medullary cells.; Neuropeptides in single VD1 neurons from the visceral ganglion of Lymnaea stagnalis were analyzed by reversed-phase packed microcolumn with matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The microcolumn effluent mixed with matrix was deposited onto a target every 60 seconds producing nL sample spot sizes of approximately 1 mm in diameter which were analyzed by MALDI-TOF-MS. The structure of a native peptide was elucidated (partially) by post source decay analysis.