Figure 2. Detection of N-acetyltryptamine, N-acetylserotonin and melatonin by liquid chromatography/tandem mass spectroscopy.

The SPE extract (10 µL of 50 µL total) from 1 mL of human plasma was injected onto the UPLC RP column and eluted as described. N-Acetyltryptamine, N-acetylserotonin, melatonin, and the internal deuterated standards were analyzed by MRM mass spectrometry, monitoring the indicated transitions. (A.) Calibration curves for N-acetyltryptamine, N-acetylserotonin and melatonin. The indicated concentrations of N-acetyltryptamine, N-acetylsertonin and melatonin (from 0.01 to 6 nM) were added to a constant concentration of deuterated internal standards (d₃-N-acetyltryptamine and, d₇-N-acetylsertonin and d₄-melatonin). The integrated peak of MRM signal intensity was quantified and the ratio of analyte to internal standard was calculated. Each point indicates the mean ± SD (n=3), and a linear fit to the data points was calculated for each compound. (B.) N-Acetyltryptamine and melatonin in human plasma: Top: MRM signal for internal standard (98 fmol) d₃-N-acetyltryptamine. Second from top: MRM signal for plasma N-acetyltryptamine (arrow, 12.64 min). Second from bottom: MRM signal for the internal standard (85 fmol) d₄-melatonin. Bottom; MRM signal for plasma melatonin (peak at 12.13 min). (C.) N-Acetylserotonin in rhesus pineal tissue: Top: MRM signal for internal standard (198 fmol) d₇-N-acetylserotonin. Bottom; MRM signal for N-acetylserotonin (peak at 7.68 min) in extracts from rhesus pineal gland (1.5 mg tissue) collected at dawn. Further details appear in the Materials and Methods section.