1H NMR assignment 

                    The chemical shifts and coupling constants of melatonin using CDCl3 as the standard are shown in Table 1 below;

Chemical Shift/ppm
Coupling constant
3H, 2-CH3
3H, Acetone
2H and  b-CH2
6.5 Hz, 
2H and l-CH3
6.5 Hz
3H, OCH3
Broad and Singlet
1H and 6-H
2 and 8.5 Hz
1H and 4H
2 Hz
1H and 7H
8.5 Hz
Broad and Singlet
IR Spectrum
 Wavenumber / cm-1
C=O , amide I
C=O , amide II
Aromatic C=C
1620, 1587, 1492
1217, 1180
Aromatic Substitution
828, 810, 800

Method of measuring melatonin

                   A variety of methods now exist for the assay of melatonin in body fluids. Melatonin has the advantage of being non-species specific and hence facilitates comparison of pineal activity in species with different breeding habits. Its formation, except in unusual circumstances, appears to be highly localised in the pineal. Furthermore, as a great deal of information on pineal activity has been derived from melatonin synthesizing enzymes, it is important to substantiate this information by measurement of the end product.
                    Melatonin can be measured by different techniques whose major advantages and disadvantages are list in Table 1 as shown below; 

Table 1 : Methods of measuring melatonin
Methods Descriptions
Bioassay Rana pipiens, Xenopus laevis, Nannostomus beckfordi anomalous, specific, intensitive (> 100 pg), tedious
Fluorometry Not specific (lengthy separation procedures), insensitive (>10 ng)
Gas chromatography,   
mass spectrometry
Highly specific, Highly sensitive (<0.01 pg)   


                    The quantitative bioassay for melatonin is based on the dermal melanophore response (nucleocentric aggregation of melatonin granuals) of Larval Rana pipiens exposed to melatonin  in their bathing medium. Groups of light adapted (melatonin-dispersed) tadpoles are exposed for 30 minutes to media containing either known concentrations of authetic melatonin or aqueous extracts of biological samples. They are then killed and fixed by the addition of formaldehyde to the media. A calebration curve is plotted relating the melanophore response evaluated microscopically according to the Hogben index (Waring,1963) to melatonin concentration. From such a curve the melatonin content of extracts can be accurately estimated.

 Melatonin Radioimmunoassays

                    Various methods have been used to produced anti-melatonin antisera for radioimmunoassays (RIA) as shown in Table 2. As the molecule is not itself antigenic, it must be coupled to a carrier molecule. Methods of coupling melatonin itself or its analoques are shown in Table 2. All these structurally defferent antigens have produced relatively specific, high titred antisera of sufficient sensitivity to be used in serum determinations of melatonin. The type of protein carrier and mode of injection do not appear to influence greatly the formation of antibodies. Further more, integrity of the N-acetyl group does not appear to be necessary for specificity. Rabits were used as experimental animals. Iodinated hapten, iodinated antigen and tritiated melatonin (3H-MT) have all been used as tracers. A notable advantage of the iodinated hapten assay system is the high sensitivity of the standard curve. All RIA methods require prior extraction of melatonin at neutral of alkaline pH into a non-polar solvent. Further purification by column or thin-layer chromatography is employed according to the specificity of the antiserum and the tissue or the fluid to be examined.

Table 2 : Methods of producing anti-melatonin serum in rabbits
Indol derivative Protein carrier Conjugation reation Hapten/protein molar ratio Tracer Titer
melatonin BSA formaldehyde condensation (Mannich reation) 3-50 moles/mole 3H-melatonin
N-acetyl - 5 - methoxy-tryptophan TG,BSA carbodiimide coupling 10-47 moles/mole 3H-melatonin 1:20,000
Indomethacin HSA carbodiimide coupling
3H-melatonin 1:13,000
N-succinyl- 5- methoxy- tryptamine BSA mixed anhydride 30 moles/mole 125I- N-3- (4 - hydroxy-phenyl)-  
propionyl- 5-methoxy- tryptamine
Abbreviations : BSA = bovine serum albumin, TG = thyroglobulin, HSA = Human serum albumin.

                     Validation of melatonin RIA

                    Even after extraction and further prification, the major problem in RIA of melatonin is that of specificity. Two antibodies, R8 and K244 are used in such system. Classical criteria for the validation of RIA are shown in Table 3. 

Table 3 : Specificity of melatonin RIA-criteria
1. Low cross-reactivity of structural analogues
2. Parallelism between sample aliquots and the standard curve
3. Chromatographic identity of immunoreactivity with melatonin in several solvent systems
4. Good correlation with bioassay and/or gas-chromatogrpahy-mass-spctrometry
Further validation of RIA :
1. Consistent high recovery of added melatonin
2. Acceptable intra- and inter-assay variability
3. Low extraction blank

 Chromatographic identity of melatonin and selected analoques

                    The establishment of chromatographic identity of extracted material with melatonin has been accomplished using primarily the four solvent systems shown in Fig. 1. The Rf values of selected melatonin analoques are shown in order to illustrate the usefulness of the silica gel G-butanol system, and the difficulty of separating N-acetyltrptamine from melatonin.

Fig. 1: Migration of melatonin and selected analoques in different thin-layer chromatographic systems.
Abbreviations: MT = melatonin, NAT = N-acetyl-tryptamine, O-Ac5MTol = O-acetyl-5-methoxytryptophol,
6HOMT = 6-hydroxy-melatonin, NAS = N-acetylserotonin, 5MT = 5-methoxytryptamine,
N-Ac5MTP = N-acetyl-5-methoxytryptophan.

                    Using K244 antibody,  immunoreactive materials other than melatonin were not found. R8 antibody shows a little interference by two other unidentify products as well as possibly N-acetyltryptamine, as shown in Fig. 2. Melatonin in urine poses considerable problems compared with other fluids. Extracted urine in the K244 system shows several immunoreactive peaks on chromatography. It is possible to measure melatonin in urine with a reasonable degree of specificity by RIA after prior chromatographic separation but the procedure is tedious.  High pressure liquid chromatography (HPLC) can be used to improve the tecnique.

Fig.2 : Chromatographic localization of immunoreactivity of extracted plasma in two different thin-layer chromatographic systems
assayed by K244 and R8. The major peak in each case corresponds to the Rf value of authetic melatonin.
Gas Chromatography - Mass Spectrometry (GCMS) for melatonin 

                    GCMS is probably one of the most effective methods in the detection and identification of unknown compounds of for the assay of compounds which are difficult or impossible to estimate by other methods. It is a tecnique when the abundance of one or more ions is monitored continuously as the compounds elute from the gas chromatograph into the mass spectrometer ion source. A concurrent increase of two or more ions at the proper retention time and in the proper ion ratioo can be taken as a strong evidence for the pressence of a given compound. In the study of pineal chemistry, mass spectrometer was applied as early as 1959 for the identifecation of 5-hydroxyindole acetic acid which had been isolated from bovine pineal (Lerner et al., 1958).
                    Basic function of MS

                    In the ion source of the mass spectrometry, sample material is ionized, conventionally by an electron beam. The positively charged ions thus formed are accelerated through a potential field, after which they enter the mass analyser, which can either be a magnetic sector of quadrupole field. In either case, the fragment ions are separated according to their mass-to-charge ratio (m/e) and detected by a secondary electron multiplier. Since the charge on an ion in this case is normally equal to one, the m/e usually corresponds to the mass of the ion in atomic mass units (amu). Scanning the field produced a mass spectrum which in almost all cases is unique for a given compound.

                    GCMS assay for melatonin

                    The melatonin assay in this case is based upon selected ion monitoring of the m/e 232 and /or 245 ions from trimethylsilyl (TMS) derivatives (Fig. 3). An internal standard is simply an analoque of melatonin having a considerably longer retention time, with intensed ions at m/e 232 and m/e 245. These ions, which have exactly the same structure as those arising from melatonin, can be used as standards for high resolution measurements on melatonin as well as other methoxy indoles. This type of standard is preferred to deuterium labeled analoques because of problems with deuterium exchange, and difficulty in preparing such compounds with high isotopic purity.

Fig.3 : Partial mass spectrum of the TMS melatonin (b) and its derivatives (a) and (c).
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