Chemistry Central Journal

This Provisional PDF corresponds to the article as it appeared upon acceptance. Fully formatted
PDF and full text (HTML) versions will be made available soon.

New Spectrofluorimetric Methods for Determination of Melatonin in the Presence

of
N-{2-[1-({3-[2-(acetylamino)ethyl]-5-methoxy-1H-indol-2-yl}methyl)-5-methoxy-1H-indol-3-yl]
ethyl}acetamide: A Contaminant in Commercial Melatonin Preparations
Chemistry Central Journal 2012, 6:36 doi:10.1186/1752-153X-6-36

Hany W Darwish (hdarwish75@yahoo.com)

Mohamed I Attia (mattia68@hotmail.com)

ISSN 1752-153X

Article type Research article

Submission date 24 January 2012

Acceptance date 2 May 2012

Publication date 2 May 2012

Article URL http://journal.chemistrycentral.com/content/6/1/36

This peer-reviewed article was published immediately upon acceptance. It can be downloaded,
printed and distributed freely for any purposes (see copyright notice below).

Articles in Chemistry Central Journal are listed in PubMed and archived at PubMed Central.

For information about publishing your research in Chemistry Central Journal or any Chemistry
Central journal, go to

http://journal.chemistrycentral.com/info/instructions/

For information about other Chemistry Central publications go to

http://www.chemistrycentral.com

© 2012 Darwish and Attia ; licensee Chemistry Central Ltd.

This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
New spectrofluorimetric methods for determination
of melatonin in the presence of N-{2-[1-({3-[2-
(acetylamino)ethyl]-5-methoxy-1H-indol-2-
yl}methyl)-5-methoxy-1H-indol-3-yl]-
ethyl}acetamide: a contaminant in commercial
melatonin preparations
Hany W Darwish1,2
Email: hdarwish75@yahoo.com

Mohamed I Attia1*,3
*
Corresponding author
Email: mattia68@hotmail.com
1
Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud
University, P.O. Box 2457, Riyadh 11451, Saudi Arabia
2
Department of Analytical Chemistry, Faculty of Pharmacy, Cairo University,
Kasr El-Aini Street, ET 11562 Cairo, Egypt
3
Department of Pharmaceutical Chemistry, Institute of Pharmacy and Food
Chemistry, Würzburg University, Am Hubland, 97074 Würzburg, Germany

Abstract
Background

Melatonin (MLT) has many health implications, therefore it is of valuable importance to

develop specific analytical methods for determination of MLT in the presence of its main
contaminant, N-{2-[1-({3-[2-(acetylamino)ethyl]-5-methoxy-1H-indol-2-yl}methyl)-5-
methoxy-1H-indol-3-yl]ethyl}acetamide (10). For development of these analytical methods,
compound 10 had to be prepared in an adequate amount.

Results

Compound 10 was synthesized in six steps starting from 5-methoxyindole-2-carboxylic acid

(1). Analytical performance of the proposed spectrofluorimetric methods was statistically
validated with respect to linearity, accuracy, precision and specificity. The proposed methods
were successfully applied for the assay of MLT in laboratory prepared mixtures containing
up to 60 % of compound 10 and in commercial MLT tablets with recoveries not less than
99.00 %. No interference was observed from common pharmaceutical additives and the
results were favorably compared with those obtained by a reference method.
Conclusions

This work describes simple, sensitive, and reliable second derivative spectrofluorimetric
method in addition to two multivariate calibration methods, principal component regression
(PCR) and partial least square (PLS), for the determination of MLT in the presence of
compound 10.

Keywords
Melatonin, Contaminants, Synthesis, Spectrofluorimetry, Commercial preparations

Background
Melatonin (N-acetyl-5-methoxytryptamine, MLT, Figure 1) is primarily produced by the
pineal gland in the brain with a marked circadian rhythm normally peaking in the dark to
regulate sleep. MLT acts through activation of two G-protein-coupled receptors, designated
as MT1 and MT2 [1]. In addition, a low-affinity putative MLT binding site called MT3 has
been recently characterized as a melatonin-sensitive form of the human enzyme quinine
reductase 2 [2]. MLT has found widespread use in the treatment of sleep disorders, other
effects described in the literature include its anti- inflammatory, pain modulatory, antitumor,
and antioxidant properties [3-8].

Figure 1 Structures of melatonin (MLT) and compound 10

MLT as well as L-tryptophane (Trp) are naturally occurring indole-based compounds and are
sold over-the-counter as dietary supplement in the United States. Contaminants in Trp
preparations are the etiological agents for the 1989 outbreak of oesinophilia-myalgia
syndrome (EMS), which affected about 1500 people and led to about 30 deaths in the United
States [9]. Administration of 15 mg/day MLT for four weeks to cancer patients has led to
induction of oesinophilia [10,11]. Accordingly, Williamson et al. [12] investigated the
presence of contaminants structurally related to those found in contaminated Trp
preparations. They reported the presence of six structural analogues of Trp contaminants in
three different commercial MLT preparations. Two contaminants were identified to be
hydroxymelatonin isomers with MH+ = 249 whereas, other four contaminants were identified
as melatonin-formaldehyde condensation products with MH+ = 477. Consequently, a tighter
control on nutritional supplements sold and used as drugs was recommended.

Compound 10 (Figure 1) is the most abundant regio-isomer from the four melatonin-
formaldehyde condensation contaminants that were found in the commercial preparations of
MLT [12]. To the best of our knowledge, a preparative methodology for the synthesis of pure
10 has not reported yet. We therefore report herein a preparative method for the synthesis of
compound 10, which is required for the development of spectrofluorimetric methods for
determination of MLT in the presence of compound 10 in commercial MLT preparations. An
evaluation of the literature revealed that only one HPLC/tandem mass spectrometry
(LC/MS/MS) method has been published for the determination of MLT in the presence of
compound 10 [12]. This method offers a high degree of specificity, however its sophisticated
instrumentation and cost factor preclude its use in routine analysis. Therefore, it was
desirable to develop simple and fast procedures that could be applied in quality control
laboratories for the determination of MLT in presence of compound 10. Derivative
spectrofluorimetry and multivariate calibration methods such as PCR and PLS are useful
means of resolving two overlapping spectra and eliminating matrix interference in the assay
of two-component mixtures [13,14].

The principal advantages of these methods lie in the improved sensitivity and selectivity, in
addition to the significant economic advantages over other sophisticated instrumental
techniques such as HPLC/tandem mass spectrometry.

Experimental
Melting points were determined using a capillary melting point apparatus (Gallenkamp,
Sanyo) and are uncorrected. Column chromatography was carried out on silica gel 60 (0.063–
0.200 mm) obtained from Merck. A Bruker AV-400 spectrometer was used to obtain 1 H
NMR (400 MHz) and 13 C NMR (100 MHz) spectra. The NMR resonances were assigned by
means of HH-COSY, HMQC, and HMBC experiments. EI mass spectra were determined on
a Finnigan MAT 8200 spectrometer. IR spectra, recorded as ATR, were obtained by using a
Biorad PharmalyzIR FT-IR instrument. Fluorescence measurements were carried out using a
Shimadzu (Kyoto, Japan) RF-5301 version 3.0 spectrofluorimeter equipped with a 150 W
xenon lamp and 1 cm quartz cells. The slit width of both the excitation and emission
monochromators was set at 5 nm. The calibration and linearity of the instrument were
frequently checked with standard quinine sulphate (0.01 µg ml-1). Wavelength calibration was
performed by measuring λexcitation at 279 nm and λemission at 333 nm; no variation in the
wavelength was observed. Elemental analyses were performed by the microanalytical section
of the Institute of Inorganic Chemistry, University of Würzburg. All reactions were carried
out under an argon atmosphere. MLT was obtained from Merc Inc, New York, USA. Its
purity is certified to be 99.5%. Methanol and ethyl acetate were of analytical-reagent grade.
Commercially available MLT preparation labeled to contain 3 mg MLT was purchased from
the local market.

Synthesis
(5-Methoxy-2,3-dihydro-1  H-indol-1-yl)(5-methoxy-1  H-indol-2-yl)methanone
(3)

A solution of 5-methoxyindoline (2) (0.94 g, 6.28 mmol) in dry CH2Cl2 (5 ml) was added to a
stirred solution of 5-methoxyindole-2-carboxylic acid (1) (1.2 g, 6.28 mmol) and ethyl-3-(3-
dimethylaminopropyl)carbodiimide hydrochloride (EDCI·HCl) (1.80 g, 9.42 mmol) in dry
CH2Cl2 (15 ml). The reaction mixture was stirred for 18 h at room temperature, extracted
with 5 N hydrochloric acid (3 × 5 ml), washed with water (2 × 10 ml), and dried (Na2SO4).
The organic layer was evaporated in vacuo, and the residue was recrystallized from
isopropanol to yield 1.76 g (87%) of 3 (1.76 g, 87%) as a pale yellow powder mp 234–236°C.
FTIR (ATR) ν = 3270, 2935, 1604, 1576, 1406, 797 cm-1. 1 H NMR (DMSO-d6): δ 3.26 (t, 2
H, J = 8.4 Hz, H-3′), 3.79 (s, 3 H, OCH3), 3.81 (s, 3 H, OCH3), 4.52 (t, 2 H, J = 8.4 Hz, H-2´),
6.83 (dd, 1 H, J = 8.8, 2.5 Hz, H-6), 6.93 (d, 1 H, J = 2.5 Hz, H-4′), 6.95-6.96 (m, 1 H, H-6′),
7.04 (s, 1 H, H-3), 7.15 (d, 1 H, J = 2.5 Hz, H-4), 7.43 (d, 1 H, J = 8.8 Hz, H-7), 8.14 (d, 1 H,
J = 8.6 Hz, H-7′), 11.58 (br., 1 H, NH). 13 C NMR (DMSO-d6): δ 28.4 (C-3′), 49.7 (C-2′), 55.3
(OCH3), 55.4 (OCH3), 102.1 (C-4), 104.9 (C-3), 110.7 (C-6′), 111.9 (C-6), 113.1 (C-7), 115.1
(C-4′), 117.6 (C-7′), 127.6, 131.2, 131.4, 133.9, 136.9 (ArC), 153.8, 156.1 (C-5, C-5′), 159.5
(O = C). MS (EI): m/z (%) = 322 (M+, 27), 174 (10), 149 (100), 134 (29). Anal. Calcd for
C19H18N2O3: C, 70.79; H, 5.63; N, 8.69. Found: C, 70.41; H, 5.61; N, 8.75.

(5-Methoxy-1  H-indol-1-yl)(5-methoxy-1  H-indol-2-yl)methanone (4)

A mixture of 3 (0.20 g, 0.62 mmol) and 2,3-dichloro-5,6-dicyanobenzoquinone (DDQ) (0.19

g, 0.68 mmol) was heated at reflux temperature in ethyl acetate (30 ml) for 18 h. The reaction
mixture was evaporated under reduced pressure and the residue was purified by silica gel
chromatography (chloroform/methanol/ammonia, 10:1:0.1) to furnish 0.19 g (96%) of 4 as a
light red powder mp 178–179 °C and. FTIR (ATR) ν = 3290, 2937, 1625, 1517, 1026, 797
cm-1.1 H NMR (CDCl3): δ 3.85 (s, 3 H, OCH3), 3.87 (s, 3 H, OCH3), 6.63 (d, 1 H, J = 3.8 Hz,
H-3′), 6.99 (dd, 1 H, J = 9.1, 2.5 Hz, H-6′), 7.03 (dd, 1 H, J = 8.8, 2.2 Hz, H-6), 7.07 (d, 1 H,
J = 2.2 Hz, H-4), 7.08-7.09 (m, 2 H, H-3, H-4′), 7.37 (d, 1 H, J = 8.8 Hz, H-7), 7.90 (d, 1 H,
J = 3.8 Hz, H-2′), 8.37 (d, 1 H, J = 9.1 Hz, H-7′), 9.70 (br., 1 H, NH). 13 C NMR (CDCl3): δ
55.7 (2 x OCH3), 102.4 (C-4), 103.7 (C-3′), 108.9 (C-3), 109.6 (C-7), 113.0 (C-6′), 113.4 (C-
6), 116.9 (C-7′), 117.7 (C-4′), 127.6 (C-2′), 127.8, 129.2, 130.8, 131.6, 132.5 (ArC), 154.9,
156.7 (C-5, C-5′), 160.8 (O = C). MS (EI): m/z (%) = 320 (M+, 70), 173 (53), 147 (100), 119
(29). Anal. Calcd for C19H16N2O3: C, 71.24; H, 5.03; N, 8.75. Found: C, 70.95; H, 5.08; N,
8.68.

5-Methoxy-1-[(5-methoxy-1  H-indol-2-yl)methyl]-1  H-indole (5)

Compound 4 (0.50 g, 156.03 mmol) was dissolved in dry THF (5 ml) and was added
dropwise to a cooled (0°C) suspension of LiAlH4/AlCl3 in dry diethyl ether (prepared by a
slow addition of AlCl3 (0.32 g, 2.41 mmol) to a suspension LiAlH4 (0.27 g, 7.13 mmol) in
dry diethyl ether (15 ml) at 0°C. The resulting reaction mixture was stirred at 0°C for one
hour and at room temperature for another one hour. The reaction was quenched by a slow
addition of saturated sodium sulphate solution. The solids were removed by filtration, washed
with chloroform (20 ml) and the combined organic phase was dried (Na2SO4) and evaporated
under reduced pressure. The residue was purified by silica gel chromatography
(chloroform/methanol/ammonia, 10:1:0.1) to produce 0.4 g (83%) of 5 as a light red powder
mp 173–174°C. FTIR (ATR) ν = 3384, 2956, 1622, 1485, 795 cm-1.1 H NMR (CDCl3): δ 3.83
(s, 3 H, OCH3), 3.85 (s, 3 H, OCH3), 5.26 (s, 2 H, CH2-N), 6.39 (s, 1 H, H-3) 6.49 (d, 1 H,
J = 3.3 Hz, H-3′), 6.81 (dd, 1 H, J = 8.8, 2.3 Hz, H-6′), 6.86 (dd, 1 H, J = 8.8, 2.5 Hz, H-6),
7.01 (d, 1 H, J = 8.8 Hz, H-7′), 7.04-7.05 (m, 2 H, H-2′, H-4), 7.15 (d, 1 H, J = 2.3 Hz, H-4′),
7.20 (d, 1 H, J = 8.8 Hz, H-7), 7.74 (br., 1 H, NH). 13 C NMR (CDCl3): δ 44.0 (CH2-N), 55.8
(OCH3), 55.9 (OCH3), 101.3 (C-3), 101.6 (C-3′), 102.3 (C-4), 102.8 (C-4′), 110.1 (C-6′),
111.6 (C-6), 112.2, 112.3 (C-7, C-7′), 128.4, 128.5 (ArC), 129.2 (C-2′), 131.4, 131.6, 134.7
(ArC), 154.2 (C-5, C-5′). MS (EI): m/z (%) = 306 (M+, 35), 160 (100), 147 (23). Anal. Calcd
for C19H18N2O2: C, 74.49; H, 5.92; N, 9.14. Found: C, 74.19; H, 5.95; N, 8.89.

2-{[5-Methoxy-3-(2-nitroethyl)-1  H-indol-1-yl]methyl}-5-methoxy-3-(2-
nitroethyl)-1  H-indole (8)

A mixture of 5 (0.250 g, 0.82 mmol), 2-nitroethyl acetate (0.350 g, 2.63 mmol), and tert.
butyl catechol (6 mg) in xylene (20 ml) was heated at reflux temperature for 18 h. The
reaction mixture was evaporated under vacuum and the residue was purified by silica gel
chromatography (chloroform/methanol, 9:0.5) to yield 0.17 g (46%) of 8 as brown viscous
oil. FTIR (ATR) ν = 3421, 2965, 1548, 1212, 795 cm-1. 1 H NMR (CDCl3): δ 3.39 (t, 2 H,
J = 7.1 Hz, CH2-CH2-N), 3.44 (t, 2 H, J = 6.8 Hz, CH2-CH2-N), 3.83 (s, 3 H, OCH3), 3.84 (s,
3 H, OCH3), 4.56 (t, 2 H, J = 6.8 Hz, CH2-CH2-N), 4.61 (t, 2 H, J = 7.1 Hz, CH2-CH2-N), 5.30
(s, 2 H, CH2-N), 6.81 (dd, 1 H, J = 8.8, 2.3 Hz, ArH), 6.86 (dd, 1 H, J = 8.8, 2.5 Hz, ArH),
6.88 (s, 1 H, H-2′), 6.80 (d, 1 H, J = 2.4 Hz, ArH), 6.98 (d, 1 H, J = 2.4 Hz, ArH), 7.09 (d, 1
H, J = 8.8 Hz, ArH), 7.14 (d, 1 H, J = 8.8 Hz, ArH), 7.66 (br., 1 H, NH). 13 C NMR (CDCl3): δ
22.6 (CH2-CH2-N), 23.5 (CH2-CH2-N), 42.0 (CH2-N), 55.9 (OCH3), 56.0 (OCH3), 74.9 (CH2-
CH2-N), 75.7 (CH2-CH2-N), 100.0, 100.6, 110.4, 112.1, 112.6, 112.8 (ArCH), 126.7 (C-2′),
109.5 (C-3, C-3′), 128.9, 130.6, 131.9, 132.0, 133.4 (ArC), 154.5, 154.6 (C-5, C-5′). MS (EI):
m/z (%) = 452 (M+, 17), 233 (58), 186 (100). Anal. Calcd for C23H24N4O6: C, 61.06; H, 5.35;
N, 12.38. Found: C, 60.86; H, 5.24; N, 12.49.

N-{2-[1-({3-[2-(Acetylamino)ethyl]-5-methoxy-1  H-indol-2-yl}methyl)-5-
methoxy-1  H-indol-3-yl]ethyl}acetamide (10)

A mixture of 8 (0.17 g, 0.38 mmol) and 10% Pd/C (70 mg) in absolute ethanol (10 ml) was
hydrogenated under 4 mbar pressure in Parr shaker device at ambient temperature for 18 h.
The reaction mixture was filtered off and the filtrate was evaporated under reduced pressure
to furnish 0.15 g of 9 as pale yellow viscous oil. Crude 9 (0.15 g, 0.38 mmol) was acetylated
using acetic anhydride (0.36 ml, 3.82 mmol) and triethylamine (0.38 ml, 2.66 mmol) in dry
DCM (10 ml) at room temperature for 18 h. The solvent was evaporated under vacuum and
the residue was purified by silica gel chromatography (chloroform/methanol/ammonia,
10:1:0.1) to yield 0.11 g (63%) of 10 as a beige powder mp 88–90°C and was FTIR (ATR)
ν = 3286, 2924, 1635, 1216, 794 cm-1.1 H NMR (CDCl3): δ 1.76 (s, 3 H, CH3), 1.80 (s, 3 H,
CH3), 2.84 (t, 2 H, J = 6.6 Hz, CH2-CH2-N), 2.96 (t, 2 H, J = 6.8 Hz, CH2-CH2-N), 3.39-3.44
(m, 2 H, CH2-CH2-N), 3.48-3.53 (m, 2 H, CH2-CH2-N), 3.81 (s, 3 H, OCH3), 3.82 (s, 3 H,
OCH3), 5.25 (s, 2 H, CH2-N), 5.74 (t, 1 H, J = 5.7 Hz, NH), 5.83 (t, 1 H, J = 5.6 Hz, NH), 6.78
(dd, 1 H, J = 8.8, 2.3 Hz, ArH), 6.81 (dd, 1 H, J = 8.8, 2.5 Hz, ArH), 6.87 (s, 1 H, H-2′), 6.99
(d, 2 H, J = 2.3 Hz, ArH), 7.11 (d, 1 H, J = 8.8 Hz, ArH), 7.14 (d, 1 H, J = 8.8 Hz, ArH), 8.24
(br., 1 H, NH). 13 C NMR (CDCl3): δ 23.1 (CH3), 23.2 (CH3), 24.3 (CH2-CH2-N), 25.3 (CH2-
CH2-N), 39.8 (CH2-CH2-N), 40.2 (CH2-CH2-N), 41.9 (CH2-N), 55.9 (2 x OCH3), 100.5,
100.9, (ArCH), 110.1, 110.3 (C-3, C-3′), 111.9, 112.3, 112.4, 112.5 (ArCH), 126.2 (C-2′),
128.5, 128.8, 130.9, 131.4, 131.9 (ArC), 154.1, 154.2 (C-5, C-5′), 170.3 (O = C), 170.5
(O = C). MS (EI): m/z (%) = 476 (M+, 31), 417 (16), 245 (100), 203 (41), 186 (64). Anal.
Calcd for C27H32N4O4: C, 68.05; H, 6.77; N, 11.76. Found: C, 68.37; H, 6.59; N, 11.66.

Analysis
Preparation of MLT and compound 10 standard solutions

Stock solutions of MLT (100 µg ml-1) and compound 10 (300 µg ml-1) were prepared by
dissolving 10 mg and 30 mg of MLT and compound 10, respectively, in 100 ml methanol.
Appropriate volumes of these stock solutions were diluted to give working solutions of 4 and
3 µg ml-1for MLT and compound 10, respectively. Stock and working solutions were stable
for at least two weeks when stored refrigerated at 4 °C.

Preparation of MLT tablets sample solutions

Ten tablets were weighed and finely powdered. An accurately weighed portion of the powder
equivalent to 3 mg of MLT was extracted with ethyl acetate and the extract was filtered. The
extract was evaporated and reconstituted in methanol to obtain final concentration of 4 µg ml-
1
MLT. Aliquots of tablet extract were diluted with methanol to obtain final concentration of
120 ng ml-1 and the samples were subjected to the analysis according to the Calibration
procedures.

Calibration procedures

Second derivative method

Aliquots equivalent to 20–220 ng ml-1 MLT were accurately transferred from its standard
working solution into separate series of 5-ml volumetric flasks then completed to volume
with methanol. The emission spectra of the prepared standard solutions were scanned from
300 to 450 nm using λexcitation at 279 nm and stored in the computer. The second derivative of
stored emission spectra of MLT were computed with ∆λ = 10 nm. The amplitude of the
second derivative peak of MLT was measured at 324.0 nm. The calibration graph was
constructed by relating the peak amplitudes at 324.0 nm to the corresponding concentrations
of MLT and the regression equation for the data was computed.

PCR and PLS chemometric models

Four level, two factor calibration design [15] was used for construction of 16 samples of the
calibration set by transferring different volumes of MLT and compound 10 from their
standard working solutions into 5-ml volumetric flasks then completed to volume with
methanol (Table 1).

Table 1 The four level two factor experimental design of the calibration set mixtures
shown as concentrations of the mixture components in ng ml-1
Mix. No. MLT Compound 10 Mix. No. MLT Compound 10
1 40 10 9 100 10
2 40 20 10 100 20
3 40 40 11 100 40
4 40 50 12 100 50
5 60 10 13 120 10
6 60 20 14 120 20
7 60 40 15 120 40
8 60 50 16 120 50

The emission spectra of the calibration set were scanned from 300 to 380 nm using λexcitation at
279 nm and stored in the computer. Mean centering of the data proved to be the best
preprocessing method for getting the optimum results.

Constructing the PCR and PLS models

The calibration set fluorescence intensities and their corresponding concentrations were used
to build the PCR and PLS models using PLS-Toolbox 2.0 software for the calculations.

Selection of the optimum number of factors to build the PCR and PLS models
The cross validation method, leaving out one sample at a time, was used to select the
optimum number of factors [16]. Given a set of 16 calibration samples, the PCR and PLS
calibrations were performed on 15 samples. By using this calibration, the concentration of the
sample left out was predicted. This process was repeated a total of 16 times until each sample
had been left out once. The predicted concentrations were then compared with the known
concentrations. The root mean square error of cross validation (RMSECV) was calculated in
the same manner each time a new factor was added to the model. The maximum number of
factors used to calculate the optimum RMSECV was selected to be nine. The method
described by Haland and Thomas [17] was used for selecting the optimum number of factors.

Assay of laboratory prepared mixtures

Aliquots of MLT and compound 10 were transferred from their standard working solutions
into a series of 5-ml measuring flasks, completed to volume with methanol and mixed well.
For determination of MLT, by the proposed methods, the Calibration procedures were
applied.

Results and discussion

MLT dietary supplement tablets are mainly used in treatment of sleep disorders and are sold
over-the-counter. It was reported that commercial MLT preparations contain six
contaminants; four of them (MLT-formaldehyde condensation contaminants) are thought to
be responsible for induction of oesinophilia when MLT was administered in a high dose for
long time [10-12]. Compound 10 is the most abundant regio-isomer from these four
formaldehyde condensation contaminants [12]. Daily value of MLT intake is not established
yet and therefore, it is of sizable importance to develop simple, accurate, and sensitive
methods for the routine analysis of MLT in the presence of its main contaminant, compound
10.

To develop such analytical methods, a considerable amount of pure compound 10 was

required to be prepared. Synthesis of the target compound 10 was carried out according to the
synthetic pathway depicted in Scheme 1. Thus, the commercially available 5-methoxyindole-
2-carboxylic acid (1) was allowed to react with the nucleophile 5-methoxyindoline (2) [18] in
the presence of coupling reagent ethyl-3-(3-dimethylaminopropyl)-carbodiimide
hydrochloride (EDCI.HCl) in DCM at room temperature to furnish the amide 3 in good yield.
Subsequent oxidation of indoline ring of 3 was accomplished using 2,3-dichloro-5,6-
dicyanobenzoquinone (DDQ) in ethyl acetate [19] at reflux temperature to yield the di-indole
derivative 4. Trials to reduce amide bond in 4 using LiAlH4 in THF or in diethyl ether led to
cleavage of the amide bond. Reduction of the amide bond in compound 4 was successfully
achieved using LiAlH4/AlCl3 (3/1) mixture in THF/diethyl ether solvent system at 0°C for
one hour and then at room temperature for another one hour. Introduction of the aminoethyl
side chains into positions 3 and 3′ of compound 5 via adopting our previously reported
procedure [20] was unsuccessful. Briefly, compound 5 was subjected to Mannich reaction
using dimethylamine and formaldehyde in glacial acetic acid produced the Mannich base 6.
Subsequent quaternization of 6 with methyl iodide followed by substitution with potassium
cyanide in the presence of dicyclohexyl[18]-crown[6] did not yield the anticipated compound
7 which might be reduced to its respective diamine derivative that could produce the target
compound 10 upon acetylation. Accordingly, another strategy was adopted to synthesize 10.
Thus, 2-nitroethyl acetate [21] was reacted with 5 in xylene at reflux temperature to yield the
di-nitro derivative 8 which was catalytically hydrogenated in Parr shaker device at 4 mbar
pressure to furnish compound 9. Acetylation of 9 using acetic anhydride and triethylamine in
DCM produced the target compound 10. Assigned structures of the synthesized compounds
were characterized by 1 H NMR, 13 C NMR, and MS spectral data whereas, purity was
determined via microanalyses.

Scheme 1 Synthetic pathway for preparation of compound 10. Reagents and conditions:
i) EDCI.HCl, DCM, rt, 18h; ii) DDQ, ethyl acetate, reflux, 18h; iii) LiAlH4/AlCl3, THF/Et2O,
0°C-rt, 2h; iv) dimethyl amine, HCHO, CH3COOH; v) 1. MeI, CH2CL2, 2. KCN,
dicyclohexyl[18]-crown[6], MeCN; vi) 2-nitroethyl acetate, tert. butyl catechol, xylene,
reflux, 18h; vii) H2, Pd/C, 4 mbar, rt, 18h; viii) acetic anhydride, Et3N, DCM, rt, 18h

Spectrofluorometric technique affords a higher sensitivity when compared with

chromatographic ones. Both MLT and compound 10 exhibited native fluorescence in
methanol with λ emission of 333 after excitation at 279 nm showing great similarity in their
emission spectra. This fact hindered the direct determination of MLT in the presence of
compound 10 (Figure 2).

Figure 2 Zero order excitation and emission spectra of 40 ng ml -1 of MLT and

compound 10 in methanol

In order to ascertain whether determination of MLT in the presence of compound 10 was

feasible, the influence of the variables potentially affecting the fluorescence intensity or the
position of the emission maxima was studied. The stability of the drug solutions was checked
as a function of the preparation time and pH. The fluorescence intensity of MLT and
compound 10 solutions maintained at 4°C was found not to vary within two weeks after
preparation. Fluorescence intensity and fluorescence range of MLT and compound 10 were
stable in pH ranges 2.8-11.2 as they are indole derivatives [22]. Also the effect of diluting
solvent was checked and it was found that methanol and ethanol gave higher sensitivity than
water. Fluorescence intensity for both MLT and compound 10 was stable for at least 2 h.

Second derivative method

By examining first and second-order derivative curves of both MLT and compound 10
emission spectra (∆λ = 10 nm) (Figures 3 and 4), it was clear that MLT can be determined
solely by measuring peak amplitude of the second-order derivative at 324.0 nm where
compound 10 gave zero response (zero- crossing point). The proposed method was validated
according to ICH-guidelines [23] regarding linearity, sensitivity, accuracy, specificity,
repeatability and reproducibility.

Figure 3 First order derivative emission spectra of 40 ng ml -1 of MLT (–) and

compound 10 (…) in methanol using λexcitation at 279 nm

Figure 4 Second order derivative emission spectra of 40 ng ml -1 of MLT (–) and

compound 10 (…) in methanol using λexcitation at 279 nm

Linearity and sensitivity

A linear correlation was obtained between peak amplitude of second derivative spectra at 324
nm and concentrations of MLT in a range of 20–220 ng ml-1 (Figure 5).
Figure 5 Second order derivative emission calibration spectra of MLT in methanol (20-
220 ng ml -1 ) using λexcitation at 279 nm

The regression equation was:

PMLT = 0.01748 C − 0.02736 r = 0.9995

Where P is the peak amplitude of the second derivative spectrum of MLT at 324.0 nm, C is
the concentration of MLT in ng ml-1 and r is the correlation coefficient. LOD and LOQ were
calculated according to the following equations [23]:

LOD = 3.3 σ / S and LOQ = 10 σ / S

Where, σ is the standard deviation of the intercept of regression line and S is the slope of
regression line of the calibration curve. All results are shown in Table 2.

Table 2 Validation report of the proposed second derivative spectrofluorimetric

method for determination of MLT
Parameters MLT
−1
Linear range (ng ml ) 20-220
Intercept ± SD 0.28 ± 0.906
Slope ± SD 1.001 ± 0.008
Correlation Coefficient 0.9995
-1
LOD (ng ml ) 2.988
-1
LOQ (ng ml ) 9.055
a
Accuracy 100.70 ± 1.772
a
Repeatability 99.81.32 ± 1.799
a
Intermediate precision 100.83 ± 2.445
a
corresponding values are average of three determinations ± SD

Accuracy

The accuracy of the proposed method was tested by analyzing triplicate samples of MLT
solutions. The recovery % (mean ± SD) was 100.70 ± 1.772. These results revealed excellent
accuracy (Table 2). The results obtained by applying the proposed method for determination
of MLT in tablets were statistically compared with those results obtained by the reference
spectrophotometric method [24]. It was concluded that with 95% confidence, there is no
significant difference between them, since the calculated t and F values are less than the
theoretical values [25] (Table 3).

Table 3 Analysis of MLT in commercial tablets by the proposed and reference methods
Method Second derivative method PCR method PLS method Reference method24
Mean ± SDa 100.51 ± 2.663 100.99 ± 1.483 100.79 ± 1.479 100.59 ± 3.379
n 5 5 5 5
t test (2.306) 0.041 0.247 0.124 -
 F (6.388) 1.610 5.187 5.220 -
a
Mean ± standard deviation
Values in parentheses are theoretical values for t and F at P = 0.05

Repeatability and reproducibility

Intra-assay precision was assessed by analyzing varying concentrations of MLT (40, 60 and
80 ng ml-1) in triplicate in one assay batch. The inter-assay precision was assessed by
analyzing the same concentrations in triplicate on 3 successive days (Table 2). The average
Recovery % around 100% and low SD indicates high accuracy and high precision of the
proposed method, respectively.

Specificity

MLT was determined in laboratory prepared mixtures containing different percentages of

compound 10. The recovery % (mean ± SD) of 101.09 ± 1.701 proved the high specificity of
the proposed method for quantifying MLT in presence up to 60% of compound 10 (Table 4).
Specificity was also investigated by observing any possible interferences from excepients in
commercial MLT tablets, such as talc, magnesium stearate, dicalcium phosphate, and
microcrystalline cellulose. These excipients did not interfere with the proposed method as
indicated from the obtained good recovery values for the analysis of commercial MLT tablets
(Table 3).

Table 4 Determination of MLT in laboratory prepared mixtures containing different

percentages of compound 10 using the proposed methods
Mix. No. % of Concentration of Second derivative PCR method PLS method
-1
compound MLT (ng ml ) method
10 Recovery % of Recovery % of Recovery % of
MLT MLT MLT
1 7 80 101.11 100.45 100.15
2 20 80 102.69 101.72 101.43
3 40 60 99.75 100.06 99.77
4 50 100 102.84 100.62 100.32
5 60 40 99.05 101.57 101.28
Mean ± SD 101.09 ± 1.701 100.88 ± 0725 100.59 ± 0.728

PCR and PLS chemometric methods

Two chemometric methods – PCR and PLS – were applied for the determination of MLT in
the presence of compound 10. PCR and PLS methods involve the decomposition of the
experimental data, such as spectrofluorimetric data in this case, into systematic variations
(principal components or factors) that explain the observed variance in data. The purpose of
both methods is to build a calibration model between the concentration of the analyte under
study (MLT in our case) and the factors of the data matrix. The main difference between PLS
and PCR methods is in the process of the decomposition of the experimental data. PCR
performs the decomposition of data matrix into principal component without using the
information about the analyte concentration. On the other hand, PLS performs the
decomposition using both spectrum data matrix and analyte concentration [16]. The first step
in the determination of MLT in presence of compound 10 by PCR and PLS methods,
involves constructing the calibration matrix for the binary mixture. In this study calibration
set was optimized with the aid of the multilevel multifacor design method [15]. Table 1 show
the composition of the 16 calibration samples. The emission spectra of these mixtures were
collected and examined, the near zero fluorescence intensity after 380 nm accounted for the
rejection of this part from the spectra. The selection of the optimum number of factors for the
PCR and PLS methods was a very important pre-construction step: if the number of factors
retained was more than required, more noise would be added to the data; if the number
retained was too small, meaningful data that could be necessary for the calibration might be
discarded. In this study, the leave-one out cross-validation method was used and the
RMSECV values of different developed models were compared. Two factors were found
suitable for both PCR and PLS methods. To validate the predictive ability of the suggested
models, PCR and PLS methods were employed to predict the concentration of MLT in five
laboratory-prepared mixtures (validation samples) containing different percentages of
compound 10, where satisfactory results were obtained (Table 4). The predicted
concentrations of the validation samples were plotted against the known concentrations to
determine whether the model accounted for the concentration variation in the validation set.
Plots were expected to fall on a straight line with a slope of 1 and zero intercept. MLT, in all
samples, lay on a straight line and the equations of these lines were y = 0.994 x − 0.205
(r = 0.9995) for PCR and y = 0.997 x − 0.205 (r = 0.9995) for PLS. Both plots had a slope of
almost 1 and an intercept close to zero. The proposed PCR and PLS methods were
successfully used for the determination of MLT in commercial preparations (Table 3). The
results obtained by applying PCR and PLS methods for determination of MLT in tablets were
statistically compared with those results obtained by the reference spectrophotometric method
[24]. It was concluded that with 95% confidence, there is no significant difference between
them, since the calculated t and F values are less than the theoretical values [25] (Table 3).

Conclusion
In this study, simple and sensitive spectrofluorimetric methods were developed for the
analysis of MLT in the presence of its main contaminant, compound 10. Reviewing the
literature exposed that there are no reports for such analysis except a sophisticated
HPLC/Ms/Ms method. Accordingly, compound 10 was synthesized in adequate quantities
starting from 5-methoxyindole-2-carboxylic acid (1) to be used for the development of
appropriate spectrofluorimetric methods for routine analysis of MLT in commercial
preparations. The proposed spectrofluorimetric methods combine the rapidness and simplicity
advantages of traditional spectrometric methods together with other important analytical
merits, such as sensitivity and specificity. Moreover, simplicity was illustrated by the
minimum requirement of chemicals and solvents since methanol was the only organic solvent
used in the procedure. The suggested methods were validated and can be applied for routine
quality control analysis of MLT commercial tablets without prior separation or interference
from impurities/excipients.

Competing interests
The authors declare that they have no competing interest.
Authors’ contributions
HD designed the proposed analytical method, carried out the analytical experimental work,
analyzed the data statistically, participated in the results and discussion, and wrote the
analytical part of the manuscript. MA proposed, planned the work, synthesized compound 10,
participated in the results and discussion, and wrote the synthesis part of the manuscript. All
authors read and approved the final manuscript.

Acknowledgement
The authors thank the Deanship of Scientific Research and the Research Center of the
College of Pharmacy, King Saud University for supporting this study. Authors appreciate the
effort of PD Dr. D. P. Zlotos, Pharmaceuitical Institute, University of Würzburg for offering
the opportunity to conduct synthesis of compound 10.

References
1. Csernus V, Mess B: Biorhythms and pineal gland. Neuroendocrinol Lett 2003, 24:404–
411.

2. Nosjean O, Ferro M, Coge F, Beauverger P, Henlin J-M, Lefoulon F, Fauchere J-L,

Delagrange P, Canet E, Boutin JA: Identification of the Melatonin-binding SiteMT3 as the
Quinone Reductase 2. J Biol Chem 2000, 275:31311–31317.

3. Genovese T, Mazzon E, Muia C, Bramanti P, De Sarro A, Cuzzocrea S: Attenuation in

the evolution of experimental spinal cord trauma by treatment with melatonin. J Pineal
Res 2005, 38:198–208.

4. Peres MFP: Melatonin, the pineal gland and their implications for headache
disorders. Cephalalgia 2005, 25:403–411.

5. Witt-Enderby PA, Radio NM, Doctor JS, Davis VL: Therapeutic treatments potentially
mediated by melatonin receptors: potential clinical uses in the prevention of
osteoporosis, cancer and as an adjuvant therapy. J Pineal Res 2006, 41:297–305.

6. Blask DE, Sauer LA, Dauchy RT: Melatonin as a Chronobiotic/Anticancer Agent:

Cellular, Biochemical, and Molecular Mechanisms of Action and their Implications for
Circadian-Based Cancer Therapy. Curr Top Med Chem 2002, 2:113–132.

7. Mills E, Wu P, Seely D, Guyatt G: Melatonin in the treatment of cancer: a systematic

review of randomized controlled trials and meta-analysis. J Pineal Res 2005, 39:360–366.

8. Sofic E, Rimpapa Z, Kundurovic Z, Sapcanin A, Tahirovic I, Rustembegovic A, Cao G:

Antioxidant capacity of the neurohormone melatonin. J Neural Transm 2005, 112:349–
358.
9. Swygert LA, Back EE, Auerbach SB, Sewell LE, Falk H: Oesinophilia-myalgia
syndrome case-associated contaminants in commercially available 5-
hydroxytryptophane. J Rheumatol 1993, 20:1711–1717.

10. Lissoni P, Ardizzoia A, Tisi E, Rossini F, Barni S, Tancici G, Conti A, Maestroni GJ:
Amplification of eosinophilia by melatonin during the immunotherapy of cancer with
interleukin-2. J Biol Regul Homeost Agents 1993, 7:34–36.

11. Lissoni P, Barni S, Tancini G, Rovelli F, Ardizzoia A, Conti A, Maestroni GJ: A study
of the mechanisms involved in the immunostimulatory action of the pineal hormone in
cancer patients. Oncology 1993, 50:399–402.

12. Williamson BL, Tomlinson AJ, Mishra PK, Gleich GJ, Naylor S: Structural
Characterization of Contaminants Found in Commercial Preparations of
Melatonin:  Similarities to Case-Related Compounds from L-Tryptophan Associated
with Eosinophilia-Myalgia Syndrome. Chem Res Toxicol 1998, 11:234–240.

13. Murillo JA, García LF: Application of First Derivative Fluorescence Spectrometry to
the Simultaneous Determination of Paracetamol and Salicylamide in Pharmaceuticals.
Anal Lett 1996, 29:423–438.

14. Ni Y, Gong X: Simultaneous spectrophotometric determination of mixtures of food

colorants. Anal Chem Acta 1997, 354:163–171.

15. Brereton RG: Multilevel multifactor designs for multivariate calibration. Analyst
1997, 122:1521–1529.

16. Kramer R: Chemometric techniques for quantitative analysis. New York: Marcel Dekker
Inc; 1998.

17. Haland DM, Thomas EV: Partial least-squares methods for spectral analyses. 2.
Application to simulated and glass spectral data. Anal Chem 1988, 60:1202–1208.

18. Gangjee A, Vasudevan A, Queener SF: Synthesis and Biological Evaluation of

Nonclassical 2,4-Diamino-5-methylpyrido[2,3-d]pyrimidines with Novel Side Chain
Substituents as Potential Inhibitors of Dihydrofolate Reductases. J Med Chem 1997,
40:479–485.

19. Lakatosh SA, Luzikov YN, Preobrazhenskaya MN: Synthesis of 6H-pyrrolo[3′,4′:2,3]

[1,4]diazepino[6,7,1-hi]indole-8,10(7H,9H)-diones using 3-bromo-4-(indol-1-
yl)maleimide scaffold. Org Biomol Chem 2003, 1:826–833.

20. Attia MI, Witt-Enderby PA, Julius J: Synthesis and pharmacological evaluation of
pentacyclic 6a,7-dihydrodiindole and 2,3-dihydrodiindole derivatives as novel
melatoninergic ligands. Bioorg Med Chem 2008, 16:7654–7661.

21. Bartoli G, Bosco M, Dalpozzo R, Marcantoni E, Massaccesi M, Sambri L:

Zn(ClO4)2·6H2O as a Powerful Catalyst for a Practical Acylation of Alcohols with Acid
Anhydrides. Eur J Org Chem 2003, 2003:4611–4617.
22. Sorouraddin MH, Rashidi MR, Kalhor EG, Zeynali KA: Simultaneous
spectrofluorimetric and spectrophotometric determination of melatonin and pyridoxine
in pharmaceutical preparations by multivariate calibration methods. Il Farmaco 2005,
60:451–458.

23. ICH, Validation of Analytical procedures: Methodology (Q2AR1), International

Conference on Harmonization, Food and Drug Administration, USA: November 1996 and
November 2005.

24. The Merck Index, An Encyclopedia of Chemicals, Drugs and Biologicals, 12th edn.
1996, 5860.

25. Hinchen JD: Practical statistics for chemical Research, 1st ed. London, 1969.
 CH3
HN
O
MeO
MeO COOH
N
H
N N
H 1
OMe

O
NH
H3C
Graphical abstract 10
 MeO MeO MeO O
COOH COOH i
N + ii
H N N N
H H
OMe
1 2 3

4 3
MeO O MeO 3a
5 2
1 7'
iii 6 1' 7'a 6'
N N 7a N N
H 7 H 2'
OMe 5' OMe
5 3'a
3' 4'
4 iv
CH3
N
CH3 vi
MeO O

N N
H
OMe NO2
H3C
N
H3C MeO
6
N N
H
v OMe

O2N 8
CN
MeO O
vii
N N
H
OMe

NC NH2

7
MeO

N N
H
CH3 OMe
HN
O viii
H2N 9
MeO

N N
H
OMe

O
NH
10
H3C

Scheme 1: Synthetic pathway for preparation of compound 10.

Reagents and conditions: i) EDCI.HCl, DCM, rt, 18h; ii) DDQ, ethyl acetate, reflux, 18h; iii)
LiAlH4/AlCl3, THF/Et2O, 0 °C-rt, 2h; iv) dimethyl amine, HCHO, CH3COOH; v) 1. MeI, CH2CL2, 2.
KCN, dicyclohexyl[18]-crown[6], MeCN; vi) 2-nitroethyl acetate, tert. butyl catechol, xylene, reflux, 18h;
vii) H2, Pd/C,
Scheme 1 4 mbar, rt, 18h; viii) acetic anhydride, Et3N, DCM, rt, 18h.
 CH 3
CH 3
HN
O
HN
O
MeO
MeO
N N
N H
H OMe

MLT
O
NH
H 3C

Figure 1 10
 900

800 compound 10 (ex)

700 MLT (em)

MLT(ex)
600
Intensity

compound 10 (em)
500

400

300

200

100

0
270 290 310 330 350 370 390 410 430 450

Figure 2 Wavelength (nm)

Peak amplitude

Figure 3 Wavelength (nm)

Peak amplitude

324 nm

Figure 4 Wavelength (nm)

Peak amplitude

Figure 5 Wavelength (nm)