SOFT 2004 Meeting Abstracts - Society of Forensic Toxicologists

M40
EV ALUA TION OF THE IDS ONE-STEP ELISA KIT FOR THE SCREENING OF KETAMINE IN
HAIR
C.Y. Yong*, H.S. Leong, and C.P. Lui: Narcotics II Laboratory, Centre for Forensic Science, Health
Sciences Authority, 11 Outram Road, Singapore 16907S.
An evaluation study was conducted with the commercially available ketamine microtiter plate enzymelinked
immunoassay (ELISA) ofInternational Diagnostic System (IDS), USA, in the screening ofketamine
in hair. The assay principle is based on the competitive binding between the ketamine-enzyme conjugate
and the free ketamine extracted from the hair for a limited number of antibody-binding sites. Aliquots of20
III of the sample or calibrator was added to the antibody-coated microwell followed by 100 III of diluted
enzyme conjugate. After an incubation period of 30 min at room temperature, any unbound material was
removed with washing. An enzyme substrate was then added and further incubated for 15 min for the
colour development. An acid stop solution was added to stop the reaction. The absorbance is measured at
450 nm with 650 nm as the reference filter. The absorbance obtained is inversely proportional to the
amount ofketamine present in the sample.
During the sample preparation, the hair specimens were washed and decontaminated before being
pulverized in a Retch ball mill. About 25 mg of the pulverized hair was digested overnight at 45°C in 1 ml
of 0.5 M hydrochloric acid. The samples were allowed to cool before the addition of 1 ml of 0.5 M sodium
hydroxide and 1 ml of phosphate buffer solution (pHS). 20 fll of the clear supernatant were used for the
immunoassay.
The objective of this study is to determine if a commercially available ELISA system was sufficiently
sensitive for the routine screening ofketamine in hair. We evaluate the immunoassay performance in terms
of precision, accuracy, efficiency, sensitivity and specificity. The limit of detection (LOD) of the kit was
found to be 0.6 ng/mg. The linearity range for ketamine was found to be up to 4.3 ng/mg using spiked hair
samples. The intra-day % CV (within-day precision) for ketamine at concentrations of 0.6, O.S and 1 ng/mg
was found to be from 3.73 % to 6.12 %. Interference study was evaluated by measuring the extent of cross
reactivity of various cOmmon drugs of abuse such as opiates, 11-nor-b. 9 -THC-9-carboxylic acid,
amphetamines and their ring analogues, cocaine and its metabolites, benzodiazepines, buprenorphine and
its metabolites, LSD and its metabolites with ketamine. All the drugs above do not interfere with the
detection of ketamine using ELISA. Only a low cross-reactivity was observed for low concentrations of
norketamine.
A total of 62 hair segments from suspected ketamine abusers were screened using the ELISA test kit. These
hair specimens were obtained from suspected ketamine abusers. The effciency, sensitivity and specificity of
the immunoassay were evaluated in comparison with gas chromatography/ mass spectrometry (GCIMS)
using these hair specimens. Confirmatory cut-off for ketamine was 0.6 ng/mg. True positives, true
negatives, false positives, and false negatives were determined using immunoassay cut-off at 0.8 ng/mg and
1.0 ng/mg. An optimum immunoassay cut-off concentration of 1.0 ng/mg was determined for the kit. At
this cut-off, a total of 55 hair samples were screened positive and 7 screened negative. Comparing with
GCIMS results, 2 false negatives and no false positives were determined. The immunoassay exhibited an
efficiency of 96.7 %, a sensitivity of96.4 % and a specificity of 100 %.
The results collected support that the IDS One-StepTM ELISA test kit provides a suitable preliminary
screening procedure for the detection of ketamine in hair specimens. It offers a rapid, sensitive, and
effective method to determine the presence ofketamine in hair in suspected ketamine abusers.
Keywords: ELISA, Ketamine, Hair Analysis
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