Widiastuti Setyaningsih

European Master in Quality Analytical in Laboratory
Gdansk University of Technology
E­mail widiastuti_setyaningsih@yahoo.com
Mobile +48889664465 or +6287878121767

AM0309-FUNDAMENTAL OF BIOCHEMICAL ANALYSIS

Date : May 28, 2010

DETERMINATION OF MELATONIN FROM CHITOSAN NANOPARTICLES

Author : Firat Yerlikaya, Yesim Aktas, Yilmaz Capan

Pubisher : Springer Fachmedien Wiesbaden GmbH. Page : 1-4

INTRODUCTION
Melatonin, N-acetyl-5-methoxytryptamine, is a neurohormone produced in the
brain by the pineal gland from the amino acid tryptophan that varies with the
body's sleep cycles (Paredes S, et al., 2009). The studies of Melatonin is
considerably important since being a promising bio-material which potentially
offers many advantages i.e.
Regulate other hormones and maintains body's circadian rhythm; Blocks the growth of prostate
and breast cancer cells; Protect against sunburn and other skin damage (Rohr, U., et al., 2002).
Help strengthen the immune system (Tututi, M., et all., 2009).
As a chronobiotic which deal with abnormal timing of the circadian system: jetlag, shiftwork,
delayed sleep phase syndrome, some sleep problems of the elderly (Arendt, J., et al., 2005) and
help promote sleep in children in neurodevelopmental disabilities (Wassmer, E., et al., 2006)
As antioxidant (Sainz, R., et al., 2003), anti-inflammatory agent (Cuzzocrea, S., et al., 1999)
and anti-hypertensive effect (Xia, C., et al., 2008)
Melatonin is naturally found in animals, plants, and microbes (Paredes S, et all., 2009). Foods may
contain trace amounts of melatonin (Coates, Paul. 2005) such as chitosan which is commonly produced
from crustaceans (e.g. shrimp, crab and lobster) by treating shells with NaOH and HCl (Nair, R., et al.,
2009).
Chitosan is a linear poly-saccharides composed of randomly distributed
deacetylated and acetylated unit (http://en.wikipedia.org/wiki/Chitosan)
which are β(1-4) 2-acetamido-2-deoxy-β-D- glucopyranose (N-acetylglu-
cosamine) and 2-amino-2-deoxy-β-D-glucopyranose (D-glucosamine).
Possesses biocompatible and biodegradable properties (Tiyaboonchai,
W,. 2003), it has received much attention as functional biopolymer which is applied in pharmaceuticals
as prospective drug delivery carriers and higher drug entrapment and faster in vitro drug release
(Hemant, K,. et all. 2010).

Schaffazick, S., et al. (2005) prepare melatonin-loaded nanoparticles to protect the compound from
oxidation cited with Gibaly, I., et al. (2003) experiment of melatonin-loaded chitosan microcapsules. In
the following year, nanocapsule suspensions and nanocapsule spray-dried powders containing
melatonin was developed by Schaffazick, S., et al. (2006). Recently, Melatonin-loaded chitosan
nanoparticles is being interesting to study (Hafner, A., et al., 2009) and promising in pharmaceutical
market.
This bioactive compound with a potential influence of dietetic and/or drug intake to human health pose
challenges to the currently available analytical methods. There was a gap between knowledge and
insufficiently standardized analysis method for melatonin determination.

Analysis melatonin in chitosan present some challenges i.e. The content of melatonin in sample is very
low (Parilla, M., et al., 2009) ; The amphiphilic makes difficult to choose a solvent yielding a complete
recovery and accurate results (Hardeland, R., et al., 2006); Potent antioxidant (Sainz, R., et al., 2003)
and react quickly during sample preparation and/or analysis.

From the review paper written by Parrilla, M., et al., (2009), mentioned many analysis technique for
melatonin such as immunological technique (radioimmunoassay, enzyme linked sorbent assay and
enzyme-immunoassay), chromatographic technique (GC-MS, LC-FD, LC-CD, LC-MS, etc)* and other
techniques (chemiluminescene and spectrophotometrically).

Simonin et al., (1999) has proved that chromatographic techniques to be a good alternative to
immunoassay techniques due to the detection limit of GC was lower than that obtained with
immunoassay technique. GC is very sensitive with element-specific detectors that permit considerably
lower detection limits. The extra estimation of melatonin determined by immunoassay technique to that
determined by GC-MS related with the cross-reactivity process with immunoassay antiserum.

Gas Chromatographic (GC) techniques require samples that are volatile below 300◦C, this technique is
not applicable for very-high-boiling or nonvolatile materials (Snyder, L., et al., 2010). Considering the
physical properties of melatonin which is non-volatile material (MSDS of Melatonin from
ScienceLab.com), this technique is not suit to applied. Also with such high sensitivity of melatonin to
temperature it is likely that the high analysis temperature would denature melatonin hence a likely error
for the test results

Liquid chromatography can be used for good alternative to determine melatonin in chitosan. It is
characterized by the use of high-pressure pumps for faster separation, more effective columns for
enhanced separation, and better control of the overall process for more precise and reproducible results
(Snyder, L., et al., 2010). And the most important to apply in chitosan sample, it has the advantage of
being run at ambient temperature, which helps prevent breakdown of the melatonin in chitosan.

In this research, Firat Yerlikaya and team work with LC/UV** to determine melatonin from chitosan
nanoparticles. LC/UV is separation of the sample on a reverse-phase column, which provides good
selectivity without risk of thermal breakdown of the analyte. The method is simple, quick, and
reproducible. Besides that, this method is refer to the criteria of European Pharmacopoeia (Ph. Eur.)
and International Conference on Harmonization (ICH) which present a specific, accurate, precise and
sensitive method for determination of melatonin from chitosan nanoparticles. The sensitivity is in the
low to mid-ppb range, with very good recovery and excellent precision.

Note for abbreviation

* GC-MS, Gas Chromatography coupled with Mas Spectroscopy

LC-FD, Liquid Chromatography coupled with Fluorometric Detection
LC-CD, Liquid Chromatography coupled with Coulometric Detection
LC-MS, Liquid Chromatography coupled with Mas Spectroscopy

** LC-UV, Liquid Chromatography coupled with Ultra Violet Detection

EXPERIMENTAL STRATEGY

Sample Preparing and Solution

Stock Solution Standard Solution Chitosan Nanoparticle

Melatonin Stock Solution Chitosan Suspensions

(Protasan UP CL 113) (Aktas et al., 2005)
Dilution Mobile Phase
Weighing concentration (mg/mL) Supernatant Centrifugation
10 mg 0.05; 0.25; 0.5; 1; 2; 4; 8
Natant
Mobile Phase Dissolving Melatonin Standard
100 mL Volumetric Flask Spiking

Stock Solution Standard Curve Homogenization

Vortex 1 minute

Filtration
0.20 µm polytetrafluoroethylene

Sample Analysis (Melatonin Determination)

Attribute Required Condition
Apparatus Agilent Technologies 1200 Series
Data Processing ChemStation (Agilent Technologies, USA)
Column Clipeus C18 (5 µm, 150 9 4.6 mm i.d.)
Mobile Phase 0.1 M Triethylammonium Acetate : Acetonotrile (70:30, V/V), Flow rate 10 mL min -1
VWD 223 nm (UV-1800)
System Suitability 1 mg mL-1 melatonin standard solusion (n = 6)

Analytical Method Validation

Attribute Description
Reference European Pharmacopoeia (Ph. Eur.) and International Conference on Harmonization
(ICH) Guideline Q2 (R1) with suggestions of EMEA (CPMP/ICH/381/95)
Specificity Comparing blank CS nanoparticles dispersed in mobile phase and blank CS
nanoparticles spiked with 1.00 lg mL-1 of melatonin standard solution
Range The method must be calibrated and validated in range 0.05 – 8.00 µg mL-1
Linearity Regression analysis using least square method with n = 3. The significance between y-
intercept and slope evaluate using Student t test.
Accuracy Melatonin standard solution (0.05, 1.00 and 8.00 µg mL-1) were replicated three times
and spiked to the chitosan nanoparticles to evaluate the accuracy and repeatability (intra-
day). Result expressed as recovery %.
Precision The method for accuracy evaluation was repeated for two consecutive days to evaluate
the intermediate precision (inter-day). Result expressed as RSD% (Residual Standard
Deviation)
Detection and Evaluate using the Signal : Noise (S/N) method.
Quantification Limit
RESULTS AND COMMENTS

This method was acceptable according to European Pharmacopoeia (Ph. Eur.) and International
Conference on Harmonization (ICH). There are three main results in this paper, i.e. (1). Specificity; (2).
Linearity and Range; (3). Accuracy and Precision; (4). Detection and Quantification Limit.

SPECIFICITY

Results
Specificity is defined as the degree to which the method can quantify the analyte accurately in the
presence of interference. The authors confirm that their method is specific by the presence of
interfering peaks with melatonin peaks in the chromatogram which is shown bellow.

The experimental results show that the method developed was specific for melatonin analyte as
justified by the findings when blank chitosan nanoparticles solution composing of mobile phase
constituent was injected and assessed the chromatogram pattern at retention time of melatonin at 4.074
minutes (retention time) and total run time was 5 minutes.

Comments
The chromatogram shown negative peak at around 1.5 minutes. The negative peak might formed
because of the absorption of the melatonin analyte lower than absorption of the mobile phase. This can
be solved by changing to a mobile phase with lower UV absorption. Besides that, when a non-
absorbing anion elutes, it dilutes the UV absorbing background and causes a negative peak. So that, the
detector output leads are usually reversed to make the chromatogram look normal.

Since the interferences in the matrix of chitosan nanoparticles which are have low absorption or non-
absorbing properties can caused abnormal chromatogram (negative peak), pre-treatment with cleaning
steps is necessary to apply.

LINEARITY AND RANGE

Results
The method managed to come up with calibration curve for melatonin determination in chitosan
nanoparticles. This method gave a linear fit equation signal Y = 119.39 X + 0.264 with linearity of
99.97% in range 0.005 until 8.00 µg mL-1.
Comments
The regression analysis which is designed using least square method and a student t test to evaluate the
significance of y-intercept and the slope were not discussed and shown in the data of the analysis
results in this paper. But since the linearity came to close to 100% (p<0.05), the relation between
concentration and the peak area found to be linear.

The working range agrees with levels of melatonin reported by other researchers where their results for
melatonin in biological sample falls within the these range, e.g. 0.1 µg g-1in wolf berry reported by
Manchester et al., (2000) and 7.11 µg g-1 in huang qin plant reported by Reiter et al., (2000).

ACCURACY AND PRECISION

Results
The accuracy and precision results are shown in table bellow.

Comments
The accuracy can be determined by comparing the response of the method to a reference material with
a known value assigned to the material. Certified Reference Materials (CRMs) are suggested as
reference value for trueness experiments because they are traceable to the international standards with a
known uncertainty (Eurachem guide, 1998).

In all reviews of the melatonin determination there is no available certified reference material for
melatonin (Parrilla, M., et al., 2009). So that, in this case, chitosan nanoparticles is analysed by the
method its original state and spiking of a known amount of the melatonin analyte.

The accuracy which is expressed by Recovery (%) between value 99 until 101% for three concentration
shows that the method has good accuracy and the pre-tereatment did not significantly removed the
melatonin analyte with the interference during the cleaning steps.

The presision of the method which is expressed as relative standard deviation (RSD) for intra-day
(repeatability) and inter-day (intermediate precision) of three replicates at three concentration levels
within the working range of the method was found to be less than 2%. This performance of the method
compares better with Mwaikono, K., (2010) research where the repeatability was less than 3% and the
intermediate precision was less than 7% for melatonin determination using LC-UV in biological
samples.

DETECTION AND QUANTIFICATION LIMIT

Results
The detection limit for the method was 9 ng mL -1 (S/N = 3:1) and the quantification limit was 31 ng
mL-1 (S/N = 10:1).
Comments
The detection and quantification limit were evaluated using Signal-to-Noise (S/N) approach. The
detection limit in this paper is defined as injected amount of melatonin that results in a peak with a
height at least three times as high as the baseline noise level. The detection limit obtained for the
method was 9 ng mL-1. This value is relatively higher than previous researchers (Chen, H., 2000) for
determining melatonin using LC-UV which was obtained 4 ng mL-1 at a signal-to-noise ratio of 1: 4.
The detection limit can be improved by sample pre-treatment where melatonin wil be concentrated
before injection.

S/N approach define the limit of quantification as the concentration of the melatonin (analyte)
substance in the chitosan nanoparticles (sample) that will give signal-to-noise (S/N) ratio of 10 : 1. The
result for quantification limit was 31 ng mL -1 which is affected by both the detector sensitivity and the
accuracy of sample preparation at such a low concentration. To investigate the effect of both factors;
solution of different concentration are prepared by spiking known amounts of analyte substances and
repeatedly analyzed to determine the S/N ratio.

CONCLUSIONS AND COMMENTS

It was possible to bring confidence to the purity of melatonin peak when UV detector was used in-line
for melatonin determination since the developed method of an RP-LC UV was acceptable according to
European Pharmacopoeia (Ph. Eur.) and International Conference on Harmonization (ICH). This
method was found to be specific, accurate, precise and sensitive. But, some doubts in this research
especially in the experimental design needs to be evaluated to improve the method. i.e.
The wavelenght was set at 223 nm in this work is out of range which previous works have been
agreed. Chung, et al., (2004) defined the the range of 280 ± 2 nm working range condition of the
UV detector, this excitation wavelength was found to be in agreement with several other authors
works such as Chen, H., et al., (2000), Arnao and Hernandez-Ruiz (2009), Iriti, et al., (2006) when
they worked on melatonin in biological samples.
Regarding to the determination of melatonin concentration in chitosan nanoparticles in which using
standard curve in defined working range, the research has not been achieved in the sense since the
working range of the method does not cover the levels of melatonin found in chitosan
nanoparticles.
Biological matrix such as chitosan nanoparticles have several compounds which can interfere with
melatonin peak. So that, cleaning step in pre-treatment of the sample is needs to be done prior to
sample injection. This treatment also help to avoid the abnormality of chromatogram (negative
peak formation) by removing the interference with low absorption or non-absorbing properties
Furher research on improvement of the variables is highly recommended and validation of the
parameters such as robustness for more variable such as different instruments, analyst, materials,
etc., and establishment of the measurement uncertainty for the method if necessary.
With improvement on the method lower values of melatonin found in chitosan nanoparticles can be
achieved.
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