We report an HPLC assay for melatonin that incorporates automated inje
ction, methanol/water mobile phase, and fluorescence detection. Plasma
samples were extracted by solid and liquid phases. Recovery was > 70%
for 1-10 mL of plasma extracted, approximately 40 pg-250 ng of melato
nin. Samples were dried and reconstituted in 100 mL/L methanol. Inject
ions were 25 muL or 150 muL, depending on sample concentration, and th
e melatonin peak was eluted in 380 mL/L methanol. The detection limit
of the assay was 6 pg on the column, allowing a practical sensitivity
in plasma of 11 pmol/L for 8-mL samples and 34 pmol/L for 2-mL samples
. More than 100 plasma samples from volunteers and patients were assay
ed and the results compared with an established RIA. The mean daytime
concentration of melatonin was 20.7 pmol/L (SEM = 1.2) and 18.5 pmol/L
(SEM = 1.6) for HPLC and RIA, respectively, and the mean nighttime co
ncentration was 82.4 pmol/L (SEM = 6.5) and 82.2 (SEM = 7.3), respecti
vely.