Measurement of melatonin and 6-sulphatoxymelatonin.

Melatonin and its major metabolite, 6-sulphatoxymelatonin, can routinely be measured by RIA or ELISA. Plasma, serum or saliva samples maybe used for melatonin measurement and urine for the metabolite. Melatonin is a hormone produced by the pineal gland witha clear circadian rhythm giving low levels by day and a peak during the night in normally entrained individuals. The timing of sample collection for measurement is therefore crucial. Melatonin levels are primarily assessed to determine the timing of an individual's internal body clock and therefore indicate any misalignment to the 24 h day.

Determination of melatonin in saliva using automated solid-phase extraction, high-performance liquid chromatography and fluorescence detection.

A sensitive bioanalytical method for the determination of melatonin in saliva by solid-phase extraction (SPE), high-performance liquid chromatography (HPLC) and fluorescence detection has been developed and validated. Saliva was collected with a Salivette sampling device (Sarstedt) and a mixed-mode SPE column was used for the extraction of melatonin and internal standard (N-acetyl-6-methoxytryptamine) from the saliva. Chromatographic separation was performed using a HyPurity C18 LC column (150 x 2.1 mm) with mobile phase acetonitrile-ammonium hydrogen carbonate buffer, 0.015 M, pH 6.8 (23:77, v/v). Excitation and emission wavelengths were set to 285 nm and 345 nm, respectively. The within-day precision for the method at 50 pmol/L was 7.9 % and the between-day precision was 10.5 %. The limit of quantification was 50 pmol/L.

Potassium hexacyanoferrate(III)-mediated fluorometric determination of melatonin.

This paper describes the development of a novel, simple and sensitive high-performance liquid chromatographic method for the determination of melatonin using precolumn fluorometric derivatization. A strong fluorescence was induced when alkaline solution of melatonin was heated in the presence of potassium hexacyanoferrate (III). This fluorescent reaction was used for the precolumn derivatization of melatonin. Solid phase extraction was used for concentrating melatonin below a level of detection limit.

Comparison of melatonin products against USP's nutritional supplements standards and other criteria.

OBJECTIVE: To evaluate and compare the quality of a sample of melatonin products, as measured by the United States Pharmacopeial Convention (USP) General Tests and Assays for Nutritional Supplements (other than Microbial Limits) and certain other tests. DESIGN: Five immediate-release, two sublingual, and two controlled-release products were randomly gathered from a health food store, groceries, and pharmacies in the Baltimore-Washington metropolitan area. MAIN OUTCOME MEASURES: Weight variation, disintegration (not applicable for controlled-release products), and drug dissolution, based on USP standards. Twelve-hour dissolution profiles were obtained from the controlled-release products. All tablets were also evaluated for friability following the USP procedure and for hardness following unofficial procedures. RESULTS: All products passed the weight variation test. Two products showed excessive friability. Three immediate-release products failed both the disintegration and the dissolution tests. One of the three products demonstrated a threefold difference in hardness. One controlled-release product released 90% of melatonin in four hours in the dissolution test; the other released 90% of its content in 12 hours. CONCLUSION: Some products showed evidence of poor formulation and/or poor quality control as indicated by excessive friability, failure to disintegrate and dissolve, and excessive variation in hardness. In vitro release profiles of the two controlled-release products were substantially different. The poor quality of some supplements should be a concern to consumers and health care providers.

Structural characterization of contaminants in commercial preparations of melatonin by on-line HPLC-electrospray ionization-tandem mass spectrometry.

Three different commercially available melatonin preparations were analyzed by on-line HPLC-electrospray ionization-tandem mass spectrometry. All three samples contained the same impurities at the approximately 0.1-0.5% level of parent melatonin. Based on accurate mass-HPLC-MS and tandem mass spectrometric analyses, two contaminants (both MH+ = 249) were identified as hydroxylation products of melatonin. One compound (MH+ = 265) was determined to be a C-2 oxidation product of hydroxymelatonin and a group of four regioisomers (MH+ = 477) were identified as melatonin-formaldehyde condensation products. These latter contaminants are structural analogues of the case-associated peak "E" found in L-tryptophan implicated in onset of eosinophilia-myalgia syndrome. The significance of these findings is discussed.