SOFT 2004 Meeting Abstracts - Society of Forensic Toxicologists

M3
AMPHETAMINE BINDING TO SYNTHETIC MELANIN AND SCATCHARD ANALYSIS OF BINDING
DATA
Lata Gautam*, Karen S Scott, and Michael Cole: Department of Forensic Science and Chemistry, APU, Cambridge,
United Kingdom, CBI IPT
The determination of drug use and drug history has historically been carried out through the analysis of biological
fluids (blood and urine). In the past decade, hair analysis has been exceedingly used in order to determine recent past
drug use as well as long-term drug histories through the use of segmental analysis. In order to fully understand the
implications of a positive hair test result, it is important to understand how the drugs are incorporated into hair and how
they bind. Keratin, melanin and lipids are possible drug binding sites in hair. Keratin and melanin have many polar
groups which serve as attachment points for the drugs. Previous research into drug-hair binding has shown that
different amounts of drug bind in hair of different colours. There are no structural differences in hair of different
colours other than in the type and content of melanin present. For this reason, this investigation focuses on
amphetamine binding to synthetic melanin in order to help to explore the possible binding mechanisms.
The binding study was carried out at room temperature in the presence of light and oxygen using 0.1 M phosphate
buffer at pH 7.4. Both compounds were found to be stable under these conditions. Amphetamine-d-sulphate and
melanin, synthesized by the oxidation of tyrosine, were purchased from Sigma Chemical Company. As melanin is only
sparingly soluble in buffer, it was solubilised in dimethyl sulphoxide (DMSO) prior to dilution with buffer. An indirect
spectroscopic method was used by measuring the decrease in absorbance of amphetamine as the concentration of
melanin was increased. Aliquots of amphetamine were titrated against the melanin solution. Samples were incubated
for 45 minutes with constant shaking. Background correction was done at each step using the same amount of melanin
as present in the sample for matrix matching. The interaction was monitored using a Genesys 6v, UVNis
spectrophotometer in scanning mode from 250-264 nm focussing on changes in the amphetamine spectral fingerprint at
257.2 nm. Positive controls were prepared using the same amount of amphetamine as in the sample with the addition of
buffer instead of melanin at each step. Buffer was used for the background correction to run positive controls.
To determine the classes of binding sites, association/dissociation constants and the binding capacity of melanin, the
data were analysed by Scatchards' method (Scatchard et al., 1949). As the molecular weight and the structure of
melanin is still not clear, the number of binding sites can not be calculated directly as an integer. Instead, the ratio of
the number of pmoles of drug bound and the dry weight of melanin in pg is considered. This study has proved that
amphetamine binds to synthetic melanin in-vitro. Data analysis from the Scatchard method has yielded a curvilinear
plot with upward concavity. The curvilinear plot indicates multiple binding sites and the upward concavity indicates
negative co-operativity.
Scatchard Analysis of Melanin Binding
Capacity for Amphetamine
0.60,.......-----------------------,
•
0.10
•
•
• •• • • •
0.00
0.20 0.25 0.30 0.35 0.40 0.45 0.50 0.55 0.60 !
Reference: Scatchard G., (1949) The attractions of proteins for small molecules and ions, Annals 0/ the New York
Academy o/Science. 51:660-672
Keywords: Hair, Melanin, Scatchard Analysis
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