Fluorometric detection of serotonin and serotonin-O.P.T.
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Specifications and blank evaluations for the spectrophotofluorometric detection of serotonin and serotonin after reaction with ortho-phthaldialdhyde (O.P.T.) are presented. Sensitivity (detection limit) and precision for the serotonin-direct method were 55.9 ng./ml. and 2.7 ng./fluorescence intensity unit, respectively. There was a linear relationship between serotonin concentration and fluorescence produced in the range 2,797 ng./ml. to the detection limit.
Blank fluorescence was composed of scattering, reagent, reagent and glassware contaminants and tissue components. The largest and most variable of these exhibited a peak of maximal fluorescence at ca. 400 nm. wavelength. It arose primarily from glassware and reagent contaminants and was not easily controllable. The blank altered the height and configuration of the serotonin spectrum producing atypicallity and resulting in inability to obtain an accurate reading for the serotonin component. No single common blank could be produced or described for correction of all specimens. The tissue blank for mid-jejunum was less than 0.2% of gross serotonin fluorescence at 545 nm. wavelength.
Computer analyses indicated that the serotonin-O.P.T. blank was composed of peaks at the following wavelengths: 389, 407, 426, 448, 474 and 507 nm. A single chloroform wash was found to remove blanks peaking at 389, 426 and 474 nm. wavelengths eliminating atypicallity and permitting a more accurate reading of serotonin-O.P.T. at 473 nm. wavelength. A common reagent blank was also achieved. The chloroform wash produced a 17% loss of serotonin-O.P.T. Computer analyses further indicated that the characteristic serotonin-O.P.T. peak at 473 nm. wavelength was a composite of peaks at 459 and 490 nm.
The use of O.P.T. permitted the fluorometric detection of serotonin down to a concentration of 12.3 ng./ml. with a precision of 0.1 ng./fluorescence intensity unit, and with standard linearity from 313 ng./ml. to the detection limit. These specifications represented 4.5- and 27.3-fold increases in sensitivity and precision, respectively, over the serotonin-direct method. The use of O.P.T. with a chloroform wash increased detection sensitivity to 75 ng./ml.; precision remained unaffected.
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