SOFT 2004 Meeting Abstracts - Society of Forensic Toxicologists

A59
DETERMINATION OF ACONITINE ALKALOIDS BY HPLCITOF-MS: COMPARISON OF
ELECTRO-SPRAY AND LASER-SPRAY IONIZATIONS
Rina Kaneko·'], Satoshi Hattori], Shiho Furuta l , Yukari Hirata l , Kanako Watanabe 2 , Hiroshi Seno\ Makoto
Hamajima l , Akira Ishiil; IDepartment of Legal Medicine, Fujita Health University School of Medicine,
Japan, 2Department of Legal Medicine, Hamamatsu University School of Medicine, Japan, 3Department of
Legal Medicine, Aichi Medical University, Japan
Background: Aconitum species (Ranunculaceae) are widely distributed in Northern Asia and North America.
Their roots are popularly used in herbal medicines in China and Japan; however, their diesterditerpene-type
alkaloids are extremely toxic. For example, the LD50 values ofaconitine in mice are 1.8 mg/kg (orally) and
0.12 mg/kg (intravenously); the estimated minimal lethal dose in man is about 2 mg. Accidental poisoning
cases by aconite usually happen in Japan. Also, it is sometimes used in suicidal and homicidal cases,
because of its high toxicity. It is thus important to determine aconitine alkaloids in body fluids with high
sensitivity. We have succeeded in determining aconitine and its related compounds by HPLC/time-of-flight
(TOF)-mass spectrometry (MS). Also, we have tried to detect aconitine alkaloids by laser-spray ionization
and compared its sensitivity with that by conventional electro-spray ionization.
Methods: Aconitine alkaloids (aconitine, mesaconitine, hypaconitine, and jesaconitine) and
methyllycaconitine (internal standard; IS) spiked in plasma samples were purified using a BondElut Certify
HF column. Extracted and dried aconitine alkaloids are reconstituted with 50/lL of mobile phase, which
consists of70 % of20mM ammonium acetate (solvent A) and 30 % of acetonitrile (solvent B). HPLC was
employed in the isocratic mode (A:B 70:30) at flow rate of 0.2mLlmin. MS was performed on a JEOL
(Tokyo, Japan) AccuTOF HPLC-TOF mass spectrometer; the ionization used was electro-spray ionization
(ESI) in positive ion mode. The MS conditions were as follows: spray voltage; 2.2kV, orifice voltage; 75V
(for quantitation) or 135V (for identification), nebulizing gas (nitrogen); lOLimin. For the laser-spray
ionization, a 10 W infrared laser was used.
Results: All aconitine alka:Ioids and IS are completely separated on chromatogram. In human plasma samples,
the calibration curves gave good linearity in the range of 5 and 300 ng/mL in all the compounds; their
detection limits were about 0.3 to 1 ng/mL. Their limits for identification were about 0.7 to 2 ng/mL. The
intraday CV values were less than 21 % for 200 ng/mL. In the laser-spray ionization, the ion current
intensity was about 5 times higher compared to that by electron-spray ionization.
Conclusion: The calibration curves gave good linearity in the range of 5 and 300 ng/mL; aconitine alkaloids
can be sufficiently detectable at toxic concentrations. Thus this method is useful for quantitation and
identification of aconitine alkaloids. Laser spray ionization could be also applied to identify trace amounts
of aconitine alkaloids.
Keywords: Aconitine, Mass spectrometry, Laser spray
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