HPLC assay of melatonin in plasma with fluorescence detection.
We report an HPLC assay for melatonin that incorporates automated injection, 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 melatonin. Samples were dried and reconstituted in 100 mL/L methanol. Injections were 25 microL or 150 microL, depending on sample concentration, and the 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 assayed 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 concentration was 82.4 pmol/L (SEM = 6.5) and 82.2 (SEM = 7.3), respectively.
Peniston-Bird
J F
JF
Joint Academic Unit of Obstetrics, Gynaecology and Reproductive Physiology, London Hospital Medical College, Whitechapel, UK.
Di
W L
WL
Street
C A
CA
Kadva
A
A
Stalteri
M A
MA
Silman
R E
RE
D000293:Adolescent; D000328:Adult; D000368:Aged; D002648:Child; D002851:Chromatography, High Pressure Liquid / Q000379:methods* / Q000706:statistics & numerical data; D002940:Circadian Rhythm; D005260:Female; D005453:Fluorescence; D006801:Humans; D008550:Melatonin / Q000097:blood*; D008875:Middle Aged; D011247:Pregnancy; D011786:Quality Control; D011863:Radioimmunoassay; D012016:Reference Values
D008550:Melatonin