SOFT 2004 Meeting Abstracts - Society of Forensic Toxicologists

A72
COMPARISON OF CHEMICAL DERIVATIVES FOR SYSTEMATIC TOXICOLOGICAL
ANALYSIS OF AUTOPSY BLOOD USING GAS CHROMATOGRAPHY MASS
SPECTROMETRY
Tareq AL-Ahmadi', R.A. Anderson
Forensic Medicine and Science, University of Glasgow, Glasgow G12 8QQ, UK.
Aims: Different derivatisation techniques have been developed for drug analysis in whole blood using GC­
MS. The method was validated for six drugs of abuse and their metabolites.
Method: A mixed standard stock solution was prepared to give a final concentration of I Ilg/ml for each
analyte and this was used to spike whole blood. Solid phase extraction (SPE) was carried out using the
polymeric phase Phenomenex Strat-X (60 mg/3ml cartridges containing 331lm particles). Blank blood
(lml) was mixed with internal standard solution and different concentrations of mixed standard stock
solution, and phosphate buffer (pH 6, 3.5ml). This was centrifuged for to minutes and transferred to SPE
columns. The extracts, in methanol, were evaporated to dryness and derivatised using three different
methods: (a) acylation·esterification with PFPAI PFP-OH (2:1 vlv, 150 Ill); (b) acylation-methylation with
PFPAI trimethylsilyldiazomethane (TMS) (5:1 v/v, 120 Ill); (c) silylation with MTBSTFA containing 1%
TBDMSCI (30 Ill). Reaction vials were heated with a microwave oven for one minute. After
derivatisation, vials for methods (a) and (b) were cooled to room temperature and evaporated to dryness
under a stream of nitrogen. The derivatised extracts were reconstituted in 50111 of ethyl acetate prior to
analysis by GC-MS. Extracts derivatised by method (c) were analysed directly. A Thermo-Finnigan Trace
GC·MS instrument was used in selected ion monitoring (S1M) mode except method (c) which used full
scan mode. The GC was equipped with an HP5 column (30m x 0.32 mm x 0.25Ilm) from J&W scientific
and split/splitless injection port at 280°C. The oven temperature was at tOO°C for 2 min, programmed at
12°C/min to 300°C. Ions monitored for quantitative analysis were as follows:
Derivative Amp MA MOR BZE THC
THC­
COOH
PFPAIPFP·OH 118 204 414 82 417 459
PFPAITMS 118 204 414 82 417 489
MTBSTFA 158 172 341 282 371 515
The method was subsequently applied to 35 forensic autopsy case samples.
Results: Recoveries for all drugs of interest were found to be over 70%. Limits of detection (LOD) were
calculated as 3 times the standard error of the regression line plus the intercept. LOD's in blood ranged
from 0.4 ng/ml to 3.7 ng/ml with PFPA/PFP-OH, O.3ng/ml to 1.4ng/ml with PFPA/TMS and with
MTBSTFA were from 1.9 ng/mJ to 7.3 ng/mt. Limits of quantification (LOQ) were calculated as to times
the standard error. They were ::::::12.4 ng /ml with PFPAIPFP·OH, ::::::4.7 ng/ml with PFPAlTMS and ::::::19.8
ng/ml with MTBSTF A of blood. The case samples analysed were found to contain various drugs of abuse
and prescription drugs. The different derivatisation techniques gave varying results; however acylation·
methylation gave the best sensitivity and chromatography.
Conclusion: A validated, sensitive and specific method for the extraction and quantification of drugs abuse
in blood is presented. An alternative derivatisation method (acylation-methylation) is proposed which
gives better sensitivity and improved chromatography for detection and quantification of drugs of abuse
compared to silylation or acylation alone.
Keywords: Drugs of abuse, Derivatisation, GC-MS
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