SOFT 2004 Meeting Abstracts - Society of Forensic Toxicologists

A41
DETERMINATION OF STRYCHNINE IN HUMAN BLOOD USING SOLID PHASE
EXTRACTION AND GCIEI-MS
M. Barroso'· I, E. Gallard0 2 , S. Avila l , C. Margalho l , E. Marques I ,M. Lopez-RivaduIla 2 , D.N. Vieira l
INational Instutute of Legal Medicine, Delegation ofCoimbra, Portugal
2Institute of Legal Medicine, University of Santiago de Compostela, Spain
Introduction: Strychnine is the main alkaloid in Strychnos nux vomica, an Indian tree. This plant was first
introduced in Germany in the XVI century, as a poison to rats and other pests. Although widely used in the
past, even with pharmaceutical purposes, its use is now limited in many countries. Strychnine is not
commercialized in Portugal since 1974, but sometimes it is still associated with forensic intoxications,
because small amounts may remain in storage, particularly in rural areas. The objective of this work was
the development and validation of a simple and rapid method for the determination of strychnine in human
blood, employing solid phase extraction (SPE) and GC/EI-MS.
Materials and Methods: Oasis HLB (30 mg) extraction cartridges were obtained from Waters (Milford,
MA, USA). Stock solutions of strychnine and papaverine (internal standard) were prepared in methanol,
protected from light and stored at 4°C until use.
The sample was submitted to the following procedure: to 500 J.1L of blood were added 2 mL of distilled
water, and the mixture was centrifuged at 3000 rpm for 5 minutes. The supernatant was then applied to
previously conditioned SPE columns. After elution of the sample the columns were washed with 1 mL of
5% methanolic solution, and then dried under full vacuum for 15 minutes. The elution was performed with
1 mL of chloroform, which was afterwards evaporated to dryness under a gentle stream of nitrogen. The
dry residue was reconstituted in 50 J.1L of methanol, and an aliquot of 1 J.1L was injected in the GC.
The GC oven temperature program started at 150°C for 1 minute, then raised by 35 °C/min to 200°C, held
for 1 minute and finally elevated by 40 °C/min to 270°C, where it was kept for 7 minutes.
The injector port was set to 200 "C. The mass spectrometer temperature was 280 "C, and it was operated in
the SIM (Selective Ion Monitoring) mode. The selected ions were 334, 120 and 162 for strychnine; and
338, 324 and 308 for papaverine.
Results: The calibration curve was established in spiked blood within a range of 0.10 to 2.50 J.1g/mL, and
the correlation coefficient was 0.9994. The limits of detection and quantification were respectively 60.27
and lOO nglmL. The precision (coefficient of variation), calculated at both low and high concentrations,
was inferior to lO%. Accuracy was superior to 90% for all calibrators. Mean recovery for strychnine was
90.66%. The method was applied to authentic samples obtained from the Laboratories of Forensic
Toxicology of the National Institute ofLegal Medicine, Coimbra and Lisbon, Portugal.
Conclusion: One may conclude that the proposed technique is analytically suitable for the extraction and
determination of strychnine in blood, since it is linear within the studied range, and presents adequate
precision and accuracy. Therefore, it can be applied in forensic cases where the compound is involved.
Keywords: Strychnine, SPE, GClMS
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