Implication médico-légale des toxiques d’origine végétale : approche analytique Jérémy Carlier
To cite this version:
Jérémy Carlier. Implication médico-légale des toxiques d’origine végétale : approche analytique. Chimie analytique. Université Claude Bernard - Lyon I, 2014. Français. NNT : 2014LYO10221. tel-01132579
HAL Id: tel-01132579 https://tel.archives-ouvertes.fr/tel-01132579 Submitted on 17 Mar 2015
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ͻ ǣ ǯ ±ȋȌ ǡ ǯ ǦǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤͳ͵ͷ ǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤͳͶʹ ±ǯ± ǯ Ǧ ȀǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤͳͶ͵ ǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤͳͷͺ ±ǯ± ǯ Ǧ ȀǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤͳͷͻ ǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤͳͲ ǦȀ ǦǦ ±Ã ±ȋ ǤȌǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤͳͳ ǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤͳͺͳ ǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤͳͺʹ ±± ǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤͳͺͺ
ͳͲ
Ǥͳ ǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤʹͶ Ǥʹ ǯ ǡ± ǡǯ ǡ± ǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤʹͷ Ǥ͵ ǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤʹͻ ǤͶ ǯ ǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤ͵Ͳ ǤͷǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤ͵ʹ Ǥ ǯ ǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤ͵Ͷ ǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤ͵ Ǥͺ ǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤ͵ͺ Ǥ ͻ ±ǡ ±ǡ ±ǡ ± ±ǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤͶͲ ǤͳͲ ȋǤȌǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤͶ͵ Ǥͳͳ ǡ ǤǤǤǤǤͶͶ ǤͳʹǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤͶ Ǥͳ͵ ǯ ǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤͶͺ ǤͳͶȋǤȌǤ ǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤͷʹ Ǥͳͷ ±±ǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤͷ͵
ͳ ± ̵ ±± ± ± ǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤǤʹͳ
ͳͳ ±
ǣ ± ǣ ǣ ǣ ǣ ǯ ǣ ǣ ǯ ǣ ǣ ǣ ± Ǧ ǣ Ǧ ǣ ǣ ǣ ǣ ǣ ± ǣ ǣ ǣ ± ǣ ǣ Ã ǤǤǣ ±Ǥǣ ±± ȋȀȌǣ ±ȋȌ ȋȀȌǣ ±±ȋȌ ǣ ǣ ǣ ±
ͳʹ ±
Ǽǽǡǯ±± ± ± ± ±± Ǥ ± ±ǡ ǡǯǯ± ǯ± Ǥ ǡǯ ±± ± ȋǼ ǽȌǤǯ± ± ± ǡ ± ǯ ± ǯ ±Ǥ±±± ° ǡ ±±ȋǤǡ ǤǡǥȌ=?Ǥ ±±ǯȋǤǡǤǡǥȌ =?ǡǯ± ȋ Ǥǡ ǤǡǥȌ =?ǯǯ ±°ȋǤǡ ǤǡǥȌ=?Ǥ °±± ǡ ±± ȋ Ƭ Ǥǡ ȋǤǦ Ȍ ǤǤǤǡǥȌ=?Ǥ ǯ ǡ Ǥ± ± ± ± =?Ǥ ͶͲΨ ± °=? ²=?=?Ǥ ǯ± ± ǡ °±=?Ǥǯ±ǯ ± °=?Ǥǯ ǯ±Ǧ°± ǡ ǡǼǽ± ± =?Ǥ ǡ ǡ ±ȋǡǤǡǥȌ ± ȋ Ǥǡ Ǥǡ ǥȌ =?Ǥ° ǯ ±
ͳ͵ ± ± ±î =?Ǥ
ǯ ± ǯ Ǥ Ǧ ǯ ±± ± ±ǯǤ ǡ ± ° ʹͲͲ ͶǡͺΨʹͲͲǡ± °=?Ǥǡ± ° ȋ Ȍ ǡͷΨ ʹͲͳ͵Ǥ ± ǡȋǤȌǡȋ ǤȌ ǡ ȋ ǤȌ=?Ǥǡ ǯ±±± ° ȋ Ȍ ͵ǡͻΨ ʹͲͳʹȀʹͲͳ͵ =?Ǥ ǡ ± ° ʹͶͻͲ ǡͻΨ ʹͲͳ͵ =?Ǥǡ ǯ ±ͳ͵ͲͲͳ ± ʹͲͲͳʹͲͳͲͷǡͻΨ±ʹͲͳͲǤ ± ͻͳǡΨ ± ± ± ʹǡͻͲǡͻΨ Ǥ± ȋ ǤȌǡǡ ±ȋ ǤȌǡ ȋ ǤȌǡ ȋ ǤȌǡ ǡ ȋ ǤȌ =?Ǥ Ǧǡ ± Ǧ ° Ͷͻ͵Ͷʹǡ͵ΨʹͲͳʹǤ ± ǯ± ȋ ǤȌǡ ǡ ǡ =?Ǥ ͳͻͺ͵ǡ ǯ Ǧ ȋ± °Ȍǡ ȋ± °Ȍǡ òȋ ǤȌ ȋ± °Ȍǯ±ȋ± °Ȍ=?Ǥ
Ǧǡ ± ± Ǥ ǯ±±°Ǥ ǡ ± ±ǡ ǯ°±±Ǥ
ͳͶ ±± ǯÁ ǡ ± ± ±± ǯ Ǥ ²ǡ ǯ°ǯ ǡ Ǥ ǯ ° ǯ± Ǥ ±ǡ ǯ ǯ ° Ǥǡ ± °ǡǯ ǯǡ ± ± ± Ǥǡ °± ± ± ±Ǥ ±ǯ ǯ °°± ǯ ± ±ǡ Ǥ
±ȋ ǡ±Ã±±ǡ ÃȌ ± ± Ǥ ǯ ±±ǡ ǯ ± °±±±ǯ Ǥ ǡ ±ǡ ± ȋǡ ±ǡ Ȍ ± ± ±Ǥ ǯ ǯ ȋ ǤȌ =?ǡ ǯ =?ǡ =? ǯ ȋ ȋ Ƭ Ȍǡ ǡȋǤȌ=?Ȍ± Ǥ
ǯ± ǯ ǯ± Ø ± Ǥ ðǯ ± ± Ǧ±Ǥ±± Ã ǯǡ ǡ ǯÃǡ ǡǦ±ǡ ǯǡ ǡ =?Ǥ
ͳͷ ± ± ǯ Ǥ ± ± Ǥ Ǥ ±ǡ ± ǯ ǯ ǡ± ±±ǡǯ ± ±±Ǥ ǯ ǡ ǡ ± ± ± ±Ǥ ǡ ǡ± ǯ± ± Ǥǯ ǯǡ ǡ ± ² ± ǯ Ǥ ± ±± Ǧ ± ± Ǥ ǯ ǯ ǡǯ Ǥ
ǯ °±ǯ ±± ± Ǧ±Ǥ ǯǯǡ± ± ǯ ± ǯ ±± ǯ ǡ ° ǯ± Ǧ±Ǥ±ǯ ±° ±± ±±Ǥ
± °±±±±Ǥǯ ± ǯ ± ǯ Ǥ ǯ ǡ ±ǯ ±±° ǯ ±±Ǥ
±± ǯ ± Ǧ ±± ±° ±± ±ȋ ǦȀȌǤ
ͳ ±±± ±± ǡ ǡ ± Ǧ±Ǥ ± ±± ± ǡ ±± ± Ǧ±ǡ Ǧ Ǥ ±±± ǯǯ ±ǯ±² ± ± Ǧ Ǥ ±±± ǡ Ǥ ± ± °ǯ ǡǯ ǯ ǯȋǤȌ ǯ± Ǥ ±ǯ ǯǯ ǯǤ
°ǡ±± ǯ ±± Ǥ ǡ ± ±± ȋ Ȍǡ ± ± ± ± ± Ǥ ǦȀ ± ǯ ǯ ǯ Ǥ Ǧ Ȁ ǯ ± Ǧ ± ǯ ǯ±² ±° Ǥ Ǧ ± ± Ǥ ±±± ±Ǥ
±±±± ± ǯ ȋ Ȍ ǡ ǯ ǯ ±ȋ ȌǤ ǯ°±± ǯ ±°±± Ǥ
±±±± ǯ Ǥ ǯ°±± ǯ ± ±Ǥ ± ± ± Ǽ ǯ±±ǽ ȋȌǤ ǯ ǯ ±°ǯ±
ͳ ǯ± ȋ ǤȌǡ ǡǤ
±±°± ǯ ȋȌ ȋȌ ǡ ± Ǧ±Ǥ±±±± ǯ Ǥ ° ±ǯǯ± ȋ ǤȌǤ
ǡ±± ± ȋǤȌǤ±± ±ǯ ǡ ǯ Ǥ
ͳͺ
ͳͻ
± Ǧ±ǡǯ ǯ ǯ ǯ ǯ± °± Ǥ ° ± ±Ǥ ± ± ± ± ± ±± ǯ Ãǡǡ ± ǯ ± ǯ° ±±Ǥ ± ǡ ǡ ± ± ǯ° ȋͳȌ=?Ǥ ± ǯ± Ǥ ± Ǥ ± ± °Ǥ
ǡ ǯ Ǥ ± ± ± ± ǡ ȋ ǤȌ ± °± ò Ǥ ° ± ± ±Ǥ Ǧ ±Ǥ ǯ ǯ ± Ǥ± ǯ ǡ ±ǯ Ǥǯ ±± Ǥǡ± ± ±ǯ à ± Ǥ
ʹͲ ° ± ° ± Ǥ ǡ ǡǤǡÃǡ ±ǡ ȋ±Ȍ ȋͳͺȌ ǡ ͳͻͲͲͳ ǤǤ ȏʹȐ ǡǡ ǡ ȋ͵Ȍ ȋȌ ǡ ǡǡ Ȁ Ǥǡǡ Ȁ Ȁ ǡ ǡ ȀȀ ǡ ǡ ± ǡǡǡ Ȁ Ȁ ǡǡ ǡ Ǥǡ ǡ Ǥǡ ǡ ǡ Ǥǡ ǡǡ ǡ Ãǡ ǡǤǡ ǡ ǡ ǡ ǡ ǡǡǡ ǡ ǡ ǡ ǡǡ ǡǡǡ ǡǡǡ ǡ ͳȋͳȀ͵Ȍ± ̵±±±± Ǥȋǣ ±ǡǣǡǣ ǯ ǡ ǣ ǡ ǣ ǯ ǡ ǣ ǡǣ± Ǧ ǡǣ Ȁǡǣ ǡǣ ǡ ǣǡǣ± ǡǣ ǡ ǣ±ǡ ǣ ǡǤǤǣ ǡ±Ǥǣ±± ǡ ǣ ǡǣ ±ǡǣ ǡ ǣ±ǡ ǣȌ
ʹͳ ° ± ° ± Ǥ ǡ ǡ ǡ±ǡ ǡ ȋ±Ȁ ± ȋ ǤǤ ǦͷͲ ±±ǡ ȏʹȐ ̵± Ǧͷ ͷͷͲȀ ± ǡ ȋͷȌ ȋ± Ȍ ȋȌ ǡǡ Ȍ Ǧͳ Ǧ ȋ± ȌȌ ȋȌ ͳͺ ȽǦ±ǡ ǡǡǡ ȋͳʹ ± ȋͳͺȌ ±±ǡ ȏʹͺȐ ± ǡ ǡǡ ±ǡ Ȍ ±ǡ ǡ ±ǡ ± ǡ ǡ±±ǡ ǡ±ǡ ± ǡǡ ǡǡ ǡǡ ± Ȁ ±ǡ±ǡ ǡ±± ǡ ȋ ±Ȁ Ȍ Ȁ ±ǡ ǡ± ȋ Ȁ Ȍ ǡ±±ǡ ǡ± ǡ ȋͳȌ ȋ ȋ ǡ ±±ǡ ȏʹͻȐ ±±±ǡ±±±ǡ Ȍ ǡȀ ± ±ǡ ±±ǡ±±ǡ Ȍ ̵ ± ǡ ±±± ± ǡǡ ǡǡ± ǡǡ ǡ ǡ ǡ ȋͳȌ ȋ ȋ ǡ ±±ǡ ȏ͵ͲȐ ǡ ǡ Ȍ ͲǦ Ȍ ǡ ± ±ǡ ̵ ͷͲ ± ǡ ͺͲͲȀȀ ǡǡ ǡǡ± ǡ ǡ ǡ±± ȋͲǡʹ ± ȋͳͺȌ ǡ ±±ǡ ȏ͵ͳȐ ȌȋͲǡͷ ± ǡȀ ± ǡǡ Ȍ ̵ ǡǡǡ ȋȌ ±ȋȌ ͳȋʹȀ͵Ȍ± ̵±±±± Ǥȋǣ ±ǡǣǡǣ ǯ ǡ ǣ ǡ ǣ ǯ ǡ ǣ ǡǣ± Ǧ ǡǣ Ȁǡǣ ǡǣ ǡ ǣǡǣ± ǡǣ ǡ ǣ±ǡ ǣ ǡǤǤǣ ǡ±Ǥǣ±± ǡ ǣ ǡǣ ± ǡ ǣ ǡ ǣ ±ǡǣȌ ʹʹ ° ± ° ± Ǥ ǡǡǡ ǡ ǡ ±ȋͳ ȋͳͺȌ ȋͳͺȌ ǡ ͳͻͲͶͲͲ ±±ǡ ȏ͵ʹȐ ǡ ǡǡÃǡ ǡ Ȍ ± ±ǡ ǡ ± ǡǡ ± ǡǦ±ǡǡ±±ǡ ȋͷͲρȌ ȋͳͺȌ ǡ ±±ǡǡ ȏ͵͵Ȑ ǡǡÃǡǡ ǡ ǡ ǡȀ ± ǡǡ ̵ ± ± ǡ ǡ±ǡ ǡ ± ȋ Ȍ ǡ ±±ǡǡ ȏ͵ͶȐ ȋͲǡͷȌ ±± ǡȀ ǡ± ǡ ̵ ǡ ȋȌ Ǧ± ǡ ǡǡǡ ȋͶȌ ȋǦȌ ȋͺȌ ǡ ±±ǡ ȏ͵ͷȐ ǡ ǡ ǡ ǡȀ ± ±ǡǡ ± ǡ ǡ ǡ ̵ ǡǡ± ǡ ǡ ǡ± ± ǡ ± ± ± ǡ ǡǡ ± ǡǡ ǡ ͳȋ͵Ȁ͵Ȍ± ̵±±±± Ǥȋǣ ±ǡǣǡǣ ǯ ǡ ǣ ǡ ǣ ǯ ǡ ǣ ǡǣ± Ǧ ǡǣ Ȁǡǣ ǡǣ ǡ ǣǡǣ± ǡǣ ǡ ǣ±ǡ ǣ ǡǤǤǣ ǡ±Ǥǣ±± ǡ ǣ ǡǣ ± ǡ ǣ ǡ ǣ ±ǡǣȌ
ʹ͵ Ǥ
Ǥͳ Ǥ
ǣ ± ± ǣ ǡ Ǧǡ ǡ ǣ±° °±ȋ Ȍ
±ǡ̵ ȋǤǤȌ ̵ Ǥ ǯǯǯ°ǡ ²ǡ ±± ± Ǥǯ ± ± ±±ÃǦ ± ǡ ǯ Ǥ ± °ǡ ±± ± Ǥ ±± Ǧ ǯ ±±±ȋȋǤȌǣ ȌǤǯ ȋ ǤͳȌǤ
ʹͶ ± ǯ ± ÃǦ ±Ǥ± Ǧ± ± ǡ± Ǥ ǯ ± ± =?Ǥǯ ǤǤȋǤǤȌ± ǯ ± Ǥ ȋǤȌ=?Ǣ ǯ ȋ Ȍ ±° ± ȋǤǤǡǤ ǡǥȌ=?± ± ° ǯ ȋǤ ǤȌ=?Ǣ ǯ ± ±±ǯ° =?ȋ ǤʹȌǤ ǯ ± =?Ǥ
ǡ ° ± ȋͲǡͷͳǡͷΨǯ ÃȌǤ ±± ǯ ± ± ±±=?Ǥ
Ǥʹ ǯ ȋͳα ǡʹα ǡ͵α ͵ǡͶα Ȍǡ±
ȋͳα ǡʹα ǡ͵α ǡͶα Ȍǡǯ ȋͳα ǡʹα ǡ͵α ǡͶα Ȍǡ±
ȋͳα ͵ǡʹα ǡ͵α ͵ǡͶα Ȍ ȋͳα ͵ǡʹα ǡ͵α ͵ǡͶα Ȍ
ʹͷ ǯ Ǧ ±Ǥ ǡ ǯ ° ǡ Ǧǯǡ ± ° ȋ Ǧǣ Ǥ ǤǡǤ ǤȌǤǯ±± ǡ î =?Ǥ ± ǡ ǡǯ ±± ǡ ±±± =?Ǣ ± ±± ±=?Ǥ ǯǡǡ ǡǯȋǤ ȌǡȋǤȌ ǯȋǤ ǤȌ ± ǡ ǡ ± ¢ȋǤȌäᒮᒣᒲȋǤ Ǥ Ȍ =?Ǥ
± ± ǣ ±± ± ± ǡ ǡ ° ± ǡ ǯ ǯ± ±Ǥ± ² ǡ ° ǯǡ±± ±ǡ Ǧǡ ǡ ±ǡ ǡ ±ǡ±Ǥ°² ± ȋ ±Ȍ±± ± ±±± ȋ ±±ǯ± ǣǡǡ ǯǡǯǥȌ=?Ǥ± ± ¢ ±± ±ǡ ±ǡ ² ± =?Ǥ ǡ ǯ ± ǣ ǯ ȋǤ Ȍ ± Ǥ °±±Ǥ
ʹ Ãǯ °=? ± ±± ± ° Ǥ ± ± ǯ ±±±±±± Ǧ°±=?Ǥǯ ǦȀ± =?Ǥ±± ǯ ǦȀ =?ǯ=?±±±± Ǥ±±± ± ǯ Ãǯ Ǥǯ Ǥ±± ±± Ǧ ǯ±±ǯ ǡ Ãǯ ±ǣǯ ǡǯ ǡ± ǡǯ ǡ ǡ ǡ ± ǡ ǡ ± ± ± ± Ǥ ±± ±±
ǦȀ ͺ°̺=?ǤʹͲͳͶǡǤ ±±± ǯ ǡ ± ǡ ǯ ǡ ǡ ± ǦȀ ȋȌ ǯ± ͲǡͲʹͷ Ͳǡͳ ρȀ =?Ǥ±ǯ± ±±ǯ =?Ǥ
ǯ ±
ǯ ± ±Ǥ ± ͳͻͻͲǡ Ǥ ± ± ǯ ǡ ± =?Ǥ ±°ȋǯȌ² ǯ ± ° ǯ ǯ =?Ǥ ±± ͳͻͺͲͳͻͺͶǡ ǡǦ ǯǦ ǡ =?Ǥ ǯ±± ±
ʹ ǯ ǯ ǡ ǯ =?ǡ ȋ ǡȋ ǤȌȌ =? =?Ǥ ǡǯ ±ǡ ± ± ǡ ǣ ±± ± ± ͳͻͷͲ ʹͲͲͲ =? ° ± =?Ǥ ± ° ǯ ° ȋ ǤȌǡ ȋ ǤȌ ȋ ǤȌ ± ±=?ǤǤ± ± ǯǯ± ǯ Ǥ ǯ ǯ±°Ǧ ±±±Ǥǯǯ±±± ±ǯ =?Ǥǯ ±±± ǯǯǯ ±ʹͲͲͲ=?Ǥ ʹͲͳͲ± ǯ =?Ǥ
ͳʹ± ͵ǡ ǯͳͲǤ ͵ǡρȀǯ ²± ±°ǦǡͶ°ǯ ǯ =?Ǥ ǯ ȋǤ Ȍǡ ± ± ±±ͲǡρȀǡͳͻ°ǯ ȋ ±± ±± Ϊ Ȍǡ ǡ ǯ ȋ± ǯ Ȍǡ ± ± ȋ± ± Ȍǯ± ͲǡͳǡͲǡʹǡͲǡ͵ǡͳρȀ=?Ǥ ʹͲͳ͵ǡ Ǥ ± ±± ǯ ǯ Ǥ ǯ±ͳͲǡ͵ǦͳǡͻǡͳͶǡͻǦͺǡͻǡ͵ͳǦͶͺͳǡͳ͵ͻǦʹͶͲǡ ͺǡͶͷͶǡρȀ±±ǡ ǡǯǡ ǡ ǯ ± ± Ǥ ǯ± Ͷͻʹ ρȀ=?Ǥ²±ǡǤ
ʹͺ ǯ Ǥ ± ± ͳʹǡͳ ͻͻ͵ρȀ ǯ ͳͶǡʹȀǤ ° ± ± ͻǡͳ ρȀ ͻͲǡͻ ǡ ͻ Ȁ Ǥ ° ± ± ʹͷͻǡͷ ʹʹͺǡͷ =?Ǥ
Ǥ
Ǥ͵ Ǥ
ǣ± ± ± ǣ ǣ±±±
ǡ ǯ °ǡ ± ± ±± ± ± ǯ± Ǧ Ǥ ± ȋ Ǥ͵ȌǤ
ǤǡǤ ǯ ±ǡ±ǡ
ʹͻ ±ǯǯ± ǯ ǡ ǡ± ǤØ îǡ ǡǡ ǯ° ±ǡ ǯ ±Ȁ±±°=?Ǥ
° ȋȌ±± ±ͶǦ ±ȋȌǡ ± =?ȋ ǤͶȌǤ ±ǯȋ ± Ȍ ± ± ǯ ±± ǡ ǯ=?Ǥ ±ǯ Á ǯǤǡ ±Ǥ ± Ǥ ǯͳͳͲȀ=?Ǥ
ǯ ǯ±±± ±ǡ ǡ Ǧ± ǡ =? ° ǡ ȋǤȌȋ± Ȍ=? ȋǤȌ± Ǧ=?Ǥ
ǤͶ ǯ ȋα Ȍ ȋα Ȍ
͵Ͳ
ǯ±ǡ ± ±± ± ðǦ Ǥ±± ± ±ǣ ± Ǧ± ± Ǧ ° ǡ ǯ ǯ=?Ǥ ± ² ǯ ǯ ǡ ǯ ȋ ǤȌ ±±±±² =?Ǥǡ ±±± ǯ =?Ǥ
ǡǯ ²±ǯ Ǧǡ ± ± ± ±±ǡ± ± ǯ=?Ǥͳͻͻͻǡ ± ±±°± ǯ Ǥ ± ǦȀ±°± ±ǯ± ± Ȁ ±=?Ǥ ʹͲͲͳǡ Ǥ±±± ǯ ǯȋǤȌ Ǥ ± Ǧ ͳ ° ǯ ±ǯ± ±±=?ǤʹͲͲͶ ʹͲͲǡ Ǥ±± ǯ ǯ Ǥ ± Ǧ ±° =?Ǥ ǯ ± =?± ± =?± ±=? ± ± Ǧ Ǥ
ǯ ±
͵ͳ ǡ ǡ ǯ ȋ ǤȌǡ ǯȋ ǤȌǡ Ǥ Ǥǯ ± ± ± ± =?Ǥ Ǥ ͳͻͺͳǡ ǯ Ǥ Ǥ±± ± =?Ǥ ± ° ǯ ±±±ȋͳͲΨ ͳͷͷͶ ͳͷΨ± °͵ʹʹ ± ͳͻͻͳ ʹͲͲͻȌ =?Ǥ ǯ ± ± ǡ ±±± ° =?ǡ=? =?Ǥǡ ±± ± ǯ Ǥ ± ± =?Ǥ
Ǥͷ ǣ ± ± ǣ ±ǡ
͵ʹ ǣǯ ±± Ãͳͺ°° ǡǡǡ ± î±
ǯ ±ǯ°ǯ Ǥǡǡ ±ǡ Ǥǡǡ ǡ ± ͷʹͲ Ǥ± ǡ ±ǡ±ǡǯ± ǯȋ ǤͷȌǤ
ǯǯ ðǡǯ ± ǯ ȋ Ȍȋ ǤȌǯ ðǤ ǯð ͳͳͲȀ =?Ǥ ǡǯ ±ǯî ± ȋɀǦ Ȍȋ ǤȌǡ ± =?Ǥ ±ǯ ± =?Ǥ
ǯ ± ǯ ȋ ʹǦǦͶǡͷǦ ±ǦͷǦ±ÃȌ =?Ǥ ± ±ȋ±°Ǧ Ǧ ±Ǧ ȌǤ ± ± Ǧ ȾǦ ȋ ǡ Ȍǯ Ǧ ±±± ȾǦ Ǥ ± ± ± Á ±° ± ±±°± ±±ǯ±± °±Ǥǯ ǯ Ǽ Ãǽȋ Ȍ ± ±ǡ =?Ǥ ±± °
͵͵ ±ǡ±ǡ ǡǡ ǡ Ǥǯ ± ° Ǥ
ǯ ± ± ° ǯ± ȋ ǤȌ ° ° Ǥ ǤǡǤ ǤȋͻρȀȌǡǤǤ=?Ǥ ±ǯ²ǯǼǯ±±ǽȋȌ Ǧ =?Ǥ ± ± ± ± °ǯͶ͵ͻΨ =?Ǥ ±° ± ǯǯ±Ǥ
Ǥ ǯ ȋ ȌȋȌ
ǯ ±± Ã=?Ǥǯðǡǯǯ ǯ °²±± ǯǡ ± ǯǡǯ Ǽ ±ǽǤ ǯ ± ±± ǯ =?Ǥǯ ± Ãǯǯ=?Ǥ ǯ±±± ǯ ± ñ ± ȋʹͲͲͷȌ=?Ǥ
͵Ͷ ǯ ±± ±±ǡ ± ǡ =?Ǥ ±± ± ǯ± ǡ±Ǥ
ǯ ±±±± ° ±=? ±=?Ǥ
±±±±± ǯ ǯ ±Ǥ ± ǯ ± ǯ ǯǦ± ± =?Ǥ ±± Ǧȋ ʹͷͶ ȌǤ ͳͻǡ Ǥ ± ° ± ± ǯ ȋǣͳǡͶȀȌ=?Ǥ ǯ ±± ± Ǥ ± ° ǯ ǯ ȋ ±ǡ ±±ǡ ǡǥȌ=?Ǥ
ǯ ±
± ǡ ± =?Ǥ ±ǯ ± °Ǥ ǯ ǡ ǡ±±± ÃʹͲͲͲǡǯ± à =?ǤǦ ǯ ǯ ±±±± °± ǯȋØǯ ǡǡ ± =?Ǥǯǯ ±° ر ǡ Ǧî±=?Ǥ
͵ͷ Ǥ
ǤǤ ǣ± ± ± ǣ ǣ
± Ǥ ± ǡ ǡ ² ± ±Ǥ ǯ±ǯ °ǡ ±±Ǥ ±ǡǡ± °ȋ ǤȌǤ
° ȋȌ ȋȌǡ ± =?ȋ ǤͶȌǤȋǤ Ȍ
Ǥ ± Ǧ ǯ°ǣǯ ȋ Ǽ±ǽȌ =?Ǥ °±±±± ǤǦ ±
͵ ±ǯǡ± ±± =?Ǥ ǯ ± ° ǡ ǡ ǯ ±± =?Ǥ Ǽ ǽ ±=?Ǥǯǡǡ± Ǽ±°ǯǽ ǯ =?Ǥ±±±± ǯ ±=?Ǥ ±ͳͻͻǡǤ͵ͲΨ Ǧ ±° =?Ǥ ±ͳͻͺǡǤ ±±ͷͲΨ =?Ǥ
ǯ ± ǯ ± ǡ ± Ǧ±ǤȋǤȌ
ǯ ±
Ǥ ǯ Ǥ ǤǤ Ǥ± ǡǯ°ǯ² ǯ ° =?Ǥ ǯ ± ° ± ² ±ǯî ° ±=?Ǥ± ° ± Ǥ ± ͳͷͲͲ =?Ǥ ǯǯǯ±=?ǤͳͻͻǡǤ ± ± ° ǯ±ͳͻͻͳͻ ǯØ ȋȌǤͷͶΨ ±± =?Ǥ ±͵Ψ ǯ ± ° ʹͶ ° ° ʹͺΨ ± ° ȋ± ͻͳΨȌ =?Ǥ
͵
Ǥ
Ǥͺ Ǥ ǣ ± ± ǣǡ ǡ ǣ ǯ± ±ǡ ǡÁ ǡǯ
ǯǯ°±Ã Ǥǡǯàǯ °ǡ ±ǯ ǯ± Ǥ °± ±±ǯ ±± ǡ Ǥ ǯ° Ǥ±° ȋǤȌ Ǥ Ǥ Ǥ ° ± ǯ ǯǼàǽ ȋ Ǥ ͺȌǤ
͵ͺ ± ǣ ±ǡ ±±=?Ǥ ±±±± ±ǣ ǡͺǦ± ±ǡ ± ǡ ʹǯǦǦ ±Ǧ ±ǡ ± ± =?ȋ ǤͻȌǤ ± ±± ǯ ±± ȋ ± ȌǤ ± ±
±Ã ± ɀǦ ͳǤ ΪȀΪ ± Ϊ ʹΪ ȋ± ΪȀʹΪȌǤ ǯ ΪȀΪ ± ȋ Ȍ ± ± ȋ ±Ȍ=?Ǥǯ ±ǡ ± ǡ ǯǡ Ǧ ǡ ± =?Ǥ ± Ǥ ±ǡ ± ǡͳͲʹͲͲρȀǤ ͳͳͲρȀǡǯ ±=?Ǥ
͵ͻ
Ǥͻ ±ȋǡα ͵Ȍǡ±ȋǡα Ȍǡ
±ȋ Ȍǡȋǡα ͵Ȍ± ±ȋǡα Ȍ
± ǯǡ ° =?Ǥ ǯ ± Ǥ ǯͳͻ° ǡ Ǥ±± ǯǣǯǤ ǯ ± ± ǯ Ǥ±ǡ ± °±±±ȋ°± ° Ȍ=?Ǥ ±ǡ Áǯ ǯǯÁ±ͳͻ° Ǥ± ǡ ǡ ° ± Ǥ± ͳͺʹ͵ ͳͺͶͶǡ
ͶͲ ǯ ǯ Ǥ ǯ ± Ǥǯ ±±±ͳͺͻ ǯ =?Ǥ
ǯ±± ±ǯ± =?Ǥ±ǡǯ± ±± =?Ǥ±±±±±ǡ=?Ǥ
ͳͻͺǡ°± ±° ȋ ȋȌȌ =?Ǥ ʹͲͲͳǡ Ǥ±°±± ± Ǧ ǦȀ ȋ ȌǤ ʹͲͳͲǡ Ǥ ±°± ± ȋδ ͳ ρȀ ° Ȍ ±ǡ ±±±ǤǤȋͲǡͳǡǡͺͲρȀȌ ǦȀǤ°±ǯ ± ǯ =?Ǥ
ǯ ±
±ȋʹͲǦʹͺΨ=?Ȍǯ ± ± ±Ǥǯǯ ±ǯ±=?Ǥ
Ǥǡǯ ǡ ǯ ǯ ǣ ± ǡǦǯÁǡ ǡ ǯǡ ǡ ǡ ǡ± ǡǯǡǥ =?Ǥǯ ǡ±ǯǡ
Ͷͳ ǡ ǯ ° Ǥ ± ° ǯ ǡ ǯǤ ǡ± î °=?Ǥ ǡ± ° ±ͷͲΨ ǡͳͲΨ ȋͷ͵ ± ° ± ͳͻͺͻ ͳͻͻͻȌ =?Ǥ ǯ ±±± ±ǡ± ± ± ð Ǥ ± ǡ Ǥ ± ± ǡ ±± ± ò ±± ǯ° Ǥǯǯ± Ǽ ǽ =?Ǥ ǡ ±±± ± ȋ ǤȌǦ² Ǥ=?Ǥ
ȋǤȌǤ
Ͷʹ ǤͳͲ ȋǤȌǤ ǣ ± ± ǣ ǡ ȋȌǡȋȌǡ ȋȌ ǣ² ǯ ǡǯ
ǯǯ± ǡ ǡ °°Ǥǯ Ǧǯ ǡ±ǡǯȋ ǤͳͲȌǤ
± ǯ ± ǯ±± ° ǯǤ Ǥ ±ǡ ǡ ± ± ±± =? ȋ Ǥ ͳͳȌǤ ǯ ǯ± Á ǯ=?ǤʹͲͲͻǡǤ ± ǡ± ǯ ±=?Ǥ± ± Ǥǯ± ȋȌǤ
ǯ Ǥǯ±ǯ±ǡ
Ͷ͵ °ǡ ǯ ǡ ǯǡ =?Ǥǯ±±±± ± =?±°°Ͷͺ ± ǯ=?Ǥ± °ǯ² Ǧ ǯ ±òǡǯ± òǯ± ±=?Ǥ
Ǥͳͳ ȋǡ Ȍǡ ȋǡȌ ȋȌ
± ǯǯ± ±± ǡ îǯ±
ͶͶ ǯ ± ± =?Ǥ ǯ °±± Ǥ
ǯ ǡǯǯ ° ǯ ǯ ǡ± ǯ =?Ǥ
±± ǯ±±±Ǥ ͳͻͺͶǡǤ±±±± ±=?Ǥ± ǯ±±ͳȀ ͲǡͳȀǤ ͳͻͻʹǡǤ±±±Ǧ ± ͵ρȀ=?Ǥ ʹͲͲͻǡǤ±±± ǯ ± Ǧ ȋ ͳͲͲΨ ± Ȍ =?Ǥ± ǡ ±± ±± ±° ± ǯ ǯǤ±± Ǧ± ǦʹͷͶ ° ±ǯ ±±Ǧ Ǥ ± =?Ǥ ǡ ±° ǯǯ±±±Ǥ
ǯ ±
ǯ ² ±ǯ±ǡ ±± Ǥ ±ǯ ǡ ±=?Ǥ± ǯ ǯ ° ±Ǥǯ±± ± ± ǯ Ǥ ± ͳǡ ͷΨ =?± ° =?Ǥ ǯ ± °
Ͷͷ ǯ =?Ǥ ǡ ǯ ±±± ± ± Ǧ±ͳͻͳͻͺͶ=?Ǥ± ǯ ± ± =?Ǥ
Ǥ
ǤͳʹǤ ǣ ± ± ǣǡǡ ǣǯ± ±±±± ±ǡǡ ±
±± ±± ° Ǥ °ǣǯ ° ±ǯ° Ǣ ǣ ° ± Ǣ ǣ Ǥ
Ͷ ±ȋǤǤȌ ± Ǥ ǯ ǯ ° ǯ ° Ǧ ±±ǡ±òǤ ǡ ° °ǡǤ± ±± ǡ±±±Ǥ ± ǡ ± ±±Ǥ Á ± ǡ °Ǥ Á ° ±±°ȋ ǤͳʹȌǤ
° ± Ã ǡ Ǧ ǡ ȋ± ± ǯ Ȍ Ǧ ȋ Ȍǡ ±=?ȋ Ǥͳ͵ȌǤ ±ǯ ± ǯǯ ǡ ǡ ǡ ± ǡ ǡ ǡ Ǧ Ǥ ° ± =?Ǥ ǯ² ±Ǥ Ã ± ±± ± ǣ Ãǡ ǯ ǡ ǯǡǯ Ǧǡ ǦǦ =?Ǥ
± Ãǣ ǡ ǡ ǡ ǡ ǯ =?Ǥ ± Ã Ͳǡʹ ͲǡͶͷ Ͳǡʹ ͳǡʹΨ =?Ǥ ǡ ǡ Ǥ ±Ͳǡ͵ͲǡͷǡͲǡͷͲǡǡͲǡ͵ΨͲǡ͵Ψ ǡ± =?Ǥ ±ǯȋͺʹͻͲΨ Ã
Ͷ Ȍǡǯ° Ǥ± ǯ ȋͺͶͻͲΨȌ=?Ǥ
Ǥͳ͵ ǯȋȌ ȋȌ
±± ǡ±ǡ ±Ǥ ±± ° =?Ǥ ǯ ± ǯ °ǯ±±Ǥͳͳͺ° ǡ ǯ ±± ±± ±Ǥ ǯ ± ǯ±± ° ǡ Ã =?Ǥ ǡǦȋ ǤȌ ±Ǥ ǯ ǡ± =?Ǥ
Ͷͺ ±ǯ±±±Ǥ ͳ° ǡ ǡ±± ± =?Ǥǡǯ ±± ²±ǡ± =?Ǥ
± ǡ±± ° Ǧǡ ±ǦǤ ±ǯ° =?Ǥ² ± ±ǡ±ǡǦ ǡǦ±ÃǡǦǡǡǡ ² ± =?Ǥ± ± ǯ ǡ ǡ =?Ǥǯ à ± ± =?Ǥ ǯ ± ±± ±ǡ ò ± ± ± ±ǡ ±Ǥ ± ǡ ±ǯ ±±=?Ǥ
ǯ ±° =?ǡ ǯ =?±=? ±± ±± ǦǤʹͲͲʹǡǤ±± ±± ñǤ ±± Ǧ ±Ǧ± ͷΨ±± ± ±° ±Ǥ±ͷρȀ ǯ =?Ǥ ± ± ±=?Ǥͳͻͻͷǡ Ǥ±±±± ǯ Ǧ Ȁȋ ±ȌǤ±
Ͷͻ =?ǤʹͲͲͶǡǤ±±± ± ǯ °Ǥ ±± ǦǦ ±°Ǥ ±ͳȀ ÃͳͲͳͲͲȀ ǯ =?Ǥ ʹͲͲǡ Ǥ ±±±± Ǥ ±± ǦȀ° ± ±ȀȀǦǤ±ʹȀ =?Ǥ ± ± ǯ =? =?±±±±± Ǧǡ Ǧ=?ǡ Ǧ=? ǦȀ=? =?ǯ=? ± ǯ±±Ǥ
ǯ ±
Ǥ± ± ° ±Ǥ ǡ ǯ ±° ± ±±±±± =?Ǥǯ ² =? ȋ ǯ ǯ ǯ =?Ȍǡ ± °=?ǡ ±± ±=? ± =?Ǥ ǡ ¡ ±ʹʹͺ ± °±±ͳͻͷͲͳͻͷ=?Ǥ
±ǯ ° ǯ ǯǤ ± ǯ ±± ͳͲͲ ʹͲͲ ± ͷͲ ² ±ͷ ̵ǡ±±± ǯ =?Ǥǯ ±± ± ± ͷͲͲͳ=?Ǥ± ±ʹǦͶ =?Ǥ Ǥ ±ǡ
ͷͲ ǯǯ± ʹʹͷǡ͵ͲͳͲͲ± ʹͲͲρȀ Ǥ² ± ǯ± ͲǡͳͲǡ͵ρȀ=?Ǥͳͻͺǡ Ǥ± ǯ ǯ ± ±± ± ǯǤ ± ǦʹͲͲρȀ=?Ǥͳͻͻǡ Ǥ± ±±ʹͲͲ ȋʹͲͲͶͲͲǯȌǤ ± ʹͶʹͳρȀǡͳͲ°ǯǤ ° ± ±± =?Ǥͳͻͻͻǡ ±± ǡ ǡ ±± ± Ǥ ǯ±ͶǡͶ͵ʹǡͷρȀ ǡͲǡͺ ǡͷρȀ =?ǤʹͲͲͶǡǤ± ǯ °Ǥ ± Ǧ ͳǡͳ Ͳǡʹ Ȁǡ ͳͶǡʹȀǤ ǯ ± Ã =?Ǥ ʹͲͲǡ Ǥ± ͳͶ Ͷͺ Ȁ ǯ Ǥ±± ° =?Ǥ ʹͲͳʹǡ Ǥ ± ͺǡͶ ͳͷǡͲ Ȁ ͳǡͲͳǡ͵Ȁ ǯ Ǥ=?Ǥ
ȋǤȌǤ Ǥ
ͷͳ ǤͳͶȋǤȌǤ Ǥ
ǣ ± ± ǣǡ±±± ǣ ǯ± ± ǯǯ
Ǥ ȋǤȌ ± ǡǤ ǡ ²ǡ°ǡ± ±± °ǤǦ ǯ Ǥ ǯǡǡ ȋ ǤͳͶȌǤ
± ǡ ±± ǡ±±ǡǡ±±=?ȋ Ǥͻ Ǥ ͳͷȌǤ =?Ǥ ± ±± ± ǯ± Ͳǡ͵ǡ ͲǡͶͷǡͲǡͲͶͺȀ°ǡǡ=?Ǥ Ǥ± ǯ±͵ǦͷʹʹͶρȀ =?Ǥ
ͷʹ ǯ ±± ȋ ǤȌǤ ±Ǧ ±ǡ ±ǡ Ǣ ǡ Ǧ Ȁ Ǣ ±±ǡ ±ǡ²=?ǤØ ± ǯΪȀΪ ȋǤȌ=?Ǥ
Ǥͳͷ ±±ȋα ͵Ȍȋα Ȍ
ǯ±±±± ±͵Ͳ ° ± Ǥ ±±± ± Ǧ =?Ǥ ±± ±±ǡ±=?Ǥ
ͷ͵ °± ǯ Ǥǡ ǯ ± ±ͺͲ ±ǯ =?Ǥ ± ±± ±ǡ ǯ =?Ǥ
±ͻͲǡ±ǯ±±±± ǯ ±± ± Ǧ=?ǤʹͲͳʹǡǤ±±°± ± ± ±±±± ±±± ±Ǥ±± ǦȀ ±Ǧ ° ± °Ǧ°Ǥ ±±±±± ͲǡʹͲǡͷͲρȀ =?Ǥ
ǯ ±
ǯ ± °ǯ=?Ǥ ͷͲͲͲ ǯ ±±±± ǡ °± =?ǤǤ ǯ Ǥ±ǯ± ͵ǡͳ ͳͲǡͻΨ Ψ =?Ǥ ǯ ±±±± Ǥ ±± ǯ ǯ ±=?Ǥǯ =?Ǥ
±ǯ ±ǯ±±ǡ± ±Ǥ ǯ Ø ǦǤ ǯ Ø ±± ° =?Ǥ Ǥ ±±ǯ ± ± ± ǡ
ͷͶ ± ǡ ǯ± ° ǯ ± Ǧ =?Ǥ ǯ =?Ǥǯ± ǯ=?ǤØ Ø±° =?Ǥ°± °ʹͶ =?Ǥ ǯ ǯ± ǡ± ȋ± ± Ȍ ͳͳ ͳͲ ρȀ =?ǤʹͲͳʹǡǤ±±±ǯ ǯ±±±±±±± ʹ͵ͲρȀǤ ǯ± ȋ±±±ǣͲǡʹρȀȌ=?Ǥ
± ±° ± ° Ǥ ± ǯ°± ±Ǥǯ ǯȋǤȌǡ ȋǤȌ ǯ ǯ ȋ ǤȌǡ ǯ °°±Ǥ ǯ ± ȋǤȌǯȋǤ Ȍ ǯ ȋǤȌ ± ± ° Ǥǯ ǯ ± ȋǤ Ȍ ǡ ± ±± °Ǥ ± ± ǡ Ǥ
° ± ° ±ǡ ° ±± ± ±± Ǥ± ± °
ͷͷ ±Ǥ ±°Ǥǡǯ °± ±ǡ ±Ǥ ° ±± Ǥ ± ǯ ±ͳͲǦʹͲΨ ±ǯǯ±°ǯͲǡͷǦͳΨ±=?Ǥ °ǡ± ± ǡ Ǥ ǡ ± ± ͶͲΨ ±±ʹͲͲ ± ȋͻͲ ± ʹͲͲͻǡͳͺΨ Ȍ=?Ǥǡ± ͲͺͷΨ±± =?Ǥ ±± ͳ͵Ͳ ±ǯØ Ǧ ǡ ͳͷǡͺΨ ± ǯ ° Ǥ ±± ° ± ±ǡ ͷͳǡΨ± °=?Ǥ °± Ǥ±± Ǥ± ±± ° ± ±Ǥ ± ǯ± Ǥǯ Ǥ ± °ǡ ± ǯ ° Ǥǯǡ ± °ǡ²± ± Ǥ
Ǥ ȋǤ Ȍǯ±ȋ ǤȌ
± ± ±± ȋͷͲ ͷ ͳͲ ρȀ ȌǤ ± ± ± Ǧ ± ± =?Ǥ ± ±
ͷ ± °Ǥǡǡǯǡ ò ° ±ǯ± ǡ ǯ Ǥǡ ±± ± Ǧ±ǡ ± ȋǯ ± ǯ Ȍ=?Ǥ ± ǡ ± ǯ ±± =?Ǥ
ǯ ± ± Ǥ ± ±ǯ Ǥ ²ǡ ǡ ± ± ǯ =?Ǥ ± ±± ǡǯ ǡ ǯ² ±Ǥ
± ± ǯ ± ± Ǥ ǯ ±± Ã ± ǯ ± ± ± ± Ǥ ǯ ±ǯ ǡǯ ±ǯ ± ± ǡǯǡǯ Ǥ ±ǯ ±±±± ǯ± ±ǯ± ± Ǧ ǦǤ ² ǡ ±± ǡǯ °ǡǯǯ±ǡ ² ± ± ± Ǥ Øǯ ǡǯ ±ǡǯǡǡǯ ± ǯ°ǯ ȋ
ͷ ǯ ǡ ǡǥȌǡ ǯ ǯ Ǥ
± ° ± ǯǯ ǡ ǡǯ ±ǡ ǯǡǡǯǡ Ǥ ǯ ǡ ±ǡ ± ± Ǥ ± ° ±îǯ °± ±îǯ± °±Ǥ
±±° ± ± ǯ ²ǯ ǯ ± ǯǤ± ǯ ± Ǥ ǯ± ± ± Ǧ±Ǥ ± Ǥ
ͷͺ
±
ͷͻ ǦȀǯ Ǧ ǯ±±ǡ ± Ǧ± ± ± ² ±±Ǥ ± Ǧ±ǡ± ± ² ±Ǥ ± ±± ± ±Ǥ± ǯ ±± ± ǯ° ±±Ǥ ± ǯ ǯ ± ǯ ± ± ± ȋǡ ǡ ǡǤǤǤȌǤ
°±ǡ± ±°±±±± =?Ǥ ± ǯ Ã =?ǡǯ ǯ ± =?Ǥǯ ±±±±± ǯ ±± ±° Ǥ±±± ± ±Ǥ ǯ ± Ǧ±±±Ǥ
±ǯǯ ± Ǽ ǽǤ
Ͳ 5ETGGPKPI CRRTQCEJ D[ WNVTCJKIJ RGTHQTOCPEG NKSWKF EJTQOCVQITCRJ[VCPFGO OCUUURGEVTQOGVT[HQTVJGDNQQFSWCPVKHKECVKQPQHVJKTV[HQWTVQZKERTKPEKRNGUQH RNCPVQTKIKP#RRNKECVKQPVQHQTGPUKEVQZKEQNQI[ ,ÃTÃO[%CTNKGTCD ,ÃTÏOG)WKVVQPEF.WFQXKE4QOGWHC(CDKGP$ÃXCNQVG.CWTGPV (CPVQPH;XCP)CKNNCTFC
C .CDQTCVQKTG.#6.7/61:CX/CTKG%WTKG<+,GCP,CWTÂU.C8QWNVGUWT4JÏPG(TCPEG D 'EQNG&QEVQTCNG+PVGTFKUEKRNKPCKTG5EKGPEGU5CPVÃ7PKXGTUKVÃ%NCWFG$GTPCTF*ÏRKVCN.QWKU2TCFGNCXFW &Q[GP.ÃRKPG$TQP(TCPEG E .CDQTCVQKTGFGVQZKEQNQIKG(CEWNVÃFGRJCTOCEKGFG.[QPCX4QEMGHGNNGT.[QP(TCPEG F .CDQTCVQKTGFGRJCTOCEQNQIKGVQZKEQNQIKG%GPVTG*QURKVCNKGT.[QP5WF*QURKEGU%KXKNUFG.[QPEJGOKP FW)TCPF4GXQ[GV2KGTTG$ÃPKVG(TCPEG G .CDQTCVQKTG.#6.7/61:CX4QEMGHGNNGT.[QP(TCPEG H +PUVKVWVOÃFKEQNÃICN(CEWNVÃFGOÃFGEKPGCX4QEMGHGNNGT.[QP(TCPEG
%QTTGURQPFKPICWVJQT6GN 'OCKNCFFTGUULECTNKGT"NCVNWOVQZEQO ,%CTNKGT
#$564#%6 2NCPV RQKUQPKPIU JCXG NGHV VJGKT OCTM QP JKUVQT[ CPF UVKNN ECWUG OCP[ FGCVJU YJGVJGT KPVGPVKQPCN QT CEEKFGPVCN 6JG OGCPU VQ UJQY VQZKEQNQIKECN GXKFGPEG QH UWEJ RQKUQPKPIU UJQWNFDGKORNGOGPVGFYKVJITGCVECTG6JKUCTVKENGRTGUGPVUCVGEJPKSWGHQTOGCUWTKPIVJKTV[ PKPGVQZKERTKPEKRNGUQHRNCPVQTKIKPKPVJGDNQQFEQXGTKPICNCTIGCOQWPVQHVQZKPUHTQONQECN QT GZQVKE RNCPVU ĮNQDGNKPG Į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s*.$ECTVTKFIG6JKTV[ HQWT QH VJG VJKTV[PKPG EQORQWPFU YGTG RWV VJTQWIJC XCNKFCVKQP RTQEGFWTG 6JG CUUC[ YCU NKPGCT KP VJG ECNKDTCVKQP EWTXG TCPIG HTQO QT zI. VQ zI. CEEQTFKPI VQ VJG EQORQWPFU6JGOGVJQFKUUGPUKVKXG .1&HTQOVQzI. 6JGYKVJKPFC[RTGEKUKQPQH VJG CUUC[ YCU NGUU VJCP CV VJG ..13 CPF VJG DGVYGGPFC[ RTGEKUKQP YCU NGUU VJCP HQTzI.HQTCNNVJGEQORQWPFUKPENWFGF6JGCUUC[ CEEWTCE[ YCUKPVJGTCPIGQH VQHQTVJG..136JGGZVTCEVKQPTGEQXGT[CPFOCVTKZGHHGEVTCPIGFHTQOVQ CPFHTQOŌVQTGURGEVKXGN[+VJCURTQXGPWUGHWNCPFGHHGEVKXGKPUGXGTCNFKHHKEWNV HQTGPUKEECUGU -G[YQTFU 6QZKERNCPVU /WNVKTGUKFWGUETGGPKPI (QTGPUKEVQZKEQNQI[ 7NVTCJKIJRGTHQTOCPEGNKSWKFEJTQOCVQITCRJ[VCPFGOOCUUURGEVTQOGVT[ 7*2.%/5/5
ͳ +PVTQFWEVKQP *WOCPMKPFJCUNQPIMPQYPVJGDGPGHKVUCPFFCPIGTUQHRNCPVU6JG[CTGCPGUUGPVKCNHQQF UQWTEG CPF VJG DCUKU QH JGTDCN CPF VTCFKVKQPCN OGFKEKPGU CETQUU VJG YQTNF 6JG WUG QH OGFKEKPCNCPFRQKUQPQWURNCPVUKUTGEQTFGFKPVJGOQUVCPEKGPVYTKVKPIMPQYPVQJWOCPMKPF HTQOVJG%JKPGUG5JGPPQPIDGPECQLKPIVQVJG*KPFW8GFCCPFKPENWFKPIVJG'DGTU2CR[TWU CPFVJGYTKVKPIUQH*KRRQETCVGUCPF6JGQRJTCUVWU2QKUQPKPID[RNCPVUKUCFKTGEVCPFNQIKECN EQPUGSWGPEG QH VJGKT WUG CPF JKUVQT[ KU NKVVGTGF YKVJ YGNNMPQYP ECUGU QH HCVCN RQKUQPKPI YJGVJGT CEEKFGPVCN QT KPVGPVKQPCN 'ZVTCEVU QH RQKUQPQWU RNCPVU UWEJ CU #EQMCPVJGTC 5VTQRJCPVJWU5VT[EJPQUJGNNGDQTG OCPEJKPGGN CPFGWRJQTDKC YGTGWUGFVQVKRCTTQYU CPF URGCTUHQTJWPVKPIQTYCTHCTG=?/QTGTGEGPVN[KVJCUDGGPRQUUKDNGVQKPQEWNCVGVQZKPUUWEJ CU CEQPKVKPG =? QT TKEKP =? YKVJ VJG CKF QH EQORNGZ GSWKROGPV +P CP QEEWNV EQPVGZV VJG OGFKGXCN 'WTQRGCP YKVEJGUŏ JGPDCPGU DGNNCFQPPC CPF OCPFTCMG VJG 5QWVJ #OGTKECP UJCOCPUŏRG[QVGC[CJWCUECVCMKPKCPFOCIKEOKPV#HTKECPKDQICQT5QWVJ2CEKHKEMCXCJCXG NQPI DGGP WUGF Ō CPF CTG UVKNN KP WUG Ō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Ŏ6JG/WLCJKFGGP2QKUQPU*CPFDQQMŏHTGGN[CXCKNCDNGQPVJG KPVGTPGV HQT +UNCOKE VGTTQTKUV ITQWRU UWIIGUVU KP 'PINKUJ RTQEGFWTGU HQT RTGRCTKPI JQOG OCFG RQKUQPU HTQO ECUVQT QKN UGGFU 4KEKPWU EQOOWPKU . QT RQVCVQ VWDGTU 5QNCPWO VWDGTQUWO. =? +PVJG#OGTKECP#UUQEKCVKQPQH2QKUQP%QPVTQN%GPVGTU ##2%% RWDNKUJGFCPPWCN UVCVKUVKEUKPYJKEJRNCPVRQKUQPKPIU YGTG VJG VJNCTIGUV ECWUG QH ECNNU TGEGKXGF D[ RQKUQP EQPVTQNEGPVTGU=?5KPEGVJGOCKPRNCPVURGEKGUTGURQPUKDNGHQTHCVCNRQKUQPKPIUKPVJG 7PKVGF5VCVGUCTG&CVWTC PKPGFGCVJU CPF%KEWVC UGXGPFGCVJU %QPKWOOCEWNCVWO. VYQ FGCVJU CPF 2J[VQNCEEC COGTKECPC . VYQ FGCVJU =? 6JG NCVGUV TGRQTVU RWDNKUJGF D[ 'WTQRGCPRQKUQPEGPVTGUGUVKOCVGVJCVRNCPVRQKUQPKPIUTGRTGUGPVQHGPSWKTKGUKP(TCPEG VJNCTIGUVV[RGQHGPSWKT[ =?KP$GNIKWO KPENWFKPIOWUJTQQOUVJNCTIGUVV[RGQH GPSWKT[ =?KPVJG7- KPENWFKPIOWUJTQQOUVJNCTIGUVV[RGQHGPSWKT[ =? KP5YKV\GTNCPF TFNCTIGUVV[RGQHGPSWKT[ =?+VKUKORQTVCPVVQDGCTKPOKPFVJCVVJKUFCVC KUDCUGFQPECNNUHTQOKPFKXKFWCNUQTJGCNVJRTQHGUUKQPCNUVQRQKUQPEGPVTGUYJQUGOKUUKQPKU VQRTQXKFGCPGOGTIGPE[VQZKEQNQIKECNTGURQPUG6JGOCLQTKV[QHRQKUQPKPIUFQPQVCRRGCTKP VJGUG UVCVKUVKEU UKPEG VJG[ CTG FGCNV YKVJ FKTGEVN[ D[ FQEVQTU QT RJCTOCEKUVU QT DGECWUG VJG RQKUQPGF KPFKXKFWCNU LWFIG KV WPPGEGUUCT[ QT CTG PQV KP C HKV UVCVG VQ ECNN VJG GOGTIGPE[ UGTXKEGU(WTVJGTOQTGVJGENKPKECNGXCNWCVKQPQHRQKUQPKPIKUPQVCNYC[UUVTCKIJVHQTYCTFCPF VJGTGCTGUVKNNOCP[CPCN[VKECNICRUTGICTFKPIUQOGVQZKERTKPEKRNGU VCPIJKPJGONQEMYCVGT FTQRYQTV DKTFNKOG VJKUVNG TKEKP LGSWKTKV[ ŗ 6JG HTGSWGPE[ QH ETKOKPCN RQKUQPKPIU KU WPFQWDVGFN[ WPFGTGUVKOCVGF CU KV KU HQT RQKUQPKPIU FWG VQ VJG CEEKFGPVCN EQPVCOKPCVKQP QH HQQF QT RNCPVDCUGF VTCFKVKQPCN TGOGFKGU CPF PGGFNGUU VQ UC[ PQV GXGT[ ECUG QH RNCPV RQKUQPKPIKUVJGUWDLGEVQHCRWDNKECVKQP
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4GHGTGPEG /CVTKZ 'ZVTCEVKQP #PCN[VKECN %QORQWPFUQTRNCPVURGEKGU 4GEQXGT[ .1& z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Ibogaine and ibogamine were quantified in the powder ingested and the victim’s body fluids by GC-MS/MS after liquid–liquid extraction (Toxi-tubes Aâ). The concentrations of ibogaine measured in the blood samples taken at the scene and in the peripheral blood, urine, and gastric fluid samples taken during the autopsy were 0.65, 1.27, 1.7, and 53.5 lg/mL, while the iboga content in the powder was 7.2%. Moreover, systematic toxicological analyses of biological samples showed the presence of diazepam and methadone in therapeutic concentrations. Death was attributed to the ingestion of a substantial quantity of iboga in the context of simultaneous methadone and diazepam consumption. KEYWORDS: forensic science, forensic toxicology, Tabernanthe iboga, ibogaine, ibogamine, gas chromatography–tandem mass spectrome- try, poisoning Tabernanthe iboga, usually called iboga or eboka,isan Taken in small doses (<50 mg of ibogaine), it has stimulant, angiosperm shrub of the Apocynaceae family that grows wild in anorexigenic, and euphoriant, even aphrodisiac effects (1,3). Its some tropical African forests, particularly in Gabon, but also in use as a stimulant by Congolese hunters has been recorded since the Republic of Congo, Cameroon, and Guinea (1–3). The plant the 19th century (2,3). A medicine based on a weak concentra- grows to a height of 1.5–2 m and grows small yellow, pink, or tion of total extracts of Tabernanthe iboga, LAMBARENEâ, speckled white flowers and orange fruit with sweet-tasting flesh was on the market in France from 1939 to 1968. It was recom- (Fig. 1) (2). Its yellowish roots contain a dozen psychotropic mended for asthenia, physical, intellectual, or psychological indole alkaloids (their bark contains 5–6 percent of these), the depression, convalescence, overwork, age-related or pathological most important being ibogaine, and they are commonly taken in disabilities, although it was widely used as a doping substance traditional magico-religious rituals in some indigenous communi- by mountaineers, cyclists, and long-distance runners until 1966 ties (1–3). Iboga root can be taken fresh, but is generally dried. (4,5). It is ingested in thin strips or grated into a powder, on its own Moderate doses (from 100 mg to 1 g of ibogaine) cause or mixed with palm wine or water (2,3). trance-like visual and auditory hallucinations, altered time perception, and auditory, olfactory, and gustatory synesthesia as well as diarrhea and vomiting (1–3). Iboga is rarely taken as a recreational or psychedelic drug. It is used above all as an 1Laboratoire LAT LUMTOX, 800 av. Marie Curie, Z.I. Jean Jaur es, entheogen in certain African tribes, particularly in the Bwiti 07800 La Voulte-sur-Rhone,^ France. cult in Gabon. In Bwiti, which was inherited from the pyg- 2 Centre d’Evaluation et d’Information sur la Pharmacod ependance de mies, the banzi (initiate) ingests massive doses of iboga during Lyon, 162 av. Lacassagne, 69424 Lyon, France. 3Service de M edecine L egale, Hopital^ Bellevue, 25 bvd. Pasteur, 42055 complex initiation rites in the presence of the tribe and the Saint-Etienne, France. nganga (the community’s chief spiritual healer) and accompa- 4Laboratoire LAT LUMTOX, 71 av. Rockefeller, 69003 Lyon, France. nied by music and dancing. Taking iboga root is in this case a *Presented at the 46th Annual Meeting of The International Association of way of making contact with the ancestors in the “spirit world” – Forensic Toxicologists (TIAFT), June 2 8, 2008, in Schoelcher, Martinique, and of “reconciling oneself with death” (2,3). Although it is an French West Indies; and at the 15th Annual Meeting of the Soci et e Francßaise de Toxicologie Analytique (SFTA), June 6–8, 2007, in Paris, France. ancient religion, the numbers of followers of Bwiti have grown Received 21 Oct. 2011; and in revised form 27 June 2012; accepted 3 in recent years. It forms a bulwark against the modern foreign Sept. 2012. cultures that are establishing themselves in a society undergoing © 2013 American Academy of Forensic Sciences 1 2 JOURNAL OF FORENSIC SCIENCES FIG. 1––Representation of the structures of ibogaine (b) and ibogamine (c), indole alkaloids present in the root bark of Tabernanthe iboga, an angiosperm shrub of the Apocynaceae family (a). major changes and particularly against the Christianity of Eur- in body fluids in the 1990s (12–15), then high-performance ope and Islam of the Middle East (2,3). In the West, ibogaine liquid chromatography combined with mass spectrometry has been used for therapeutic reasons since 1969, when the (HPLC-MS) in blood (16), and more recently HPLC combined Chilean psychotherapist Claudio Naranjo used it as a catalyst with tandem mass spectrometry (HPLC-MS/MS) in body fluids in the therapeutic process. He described the substance as an (10,17). inducer of dreams without loss of consciousness (1,5). From In this article, we report a fatal case of ibogaine poisoning the 1980s onwards, Howard Lotsof maintained that “this sub- through the ingestion of powdered root bark of Tabernanthe stance can be a simple and effective medicinal means to cure iboga. Quantification of ibogaine and ibogamine in the postmor- almost all addictions” and applied for several patents for the tem samples was carried out by an original method of GC com- use of ibogaine to treat chemical dependence on opiates, stimu- bined with a tandem mass spectrometer (GC-MS/MS) after lants (cocaine and amphetamine), alcohol, and nicotine. There liquid–liquid extraction (LLE). were many studies of the anti-addictive effect of ibogaine on animals in the 1990s, but most of the data on humans come Case Report from informal reports by patients’ associations. There are as yet no real clinical trials supported by a proven methodology, The victim, a 27-year-old Caucasian man (1.77 m tall and yet the number of institutions offering ibogaine treatments is weighing 67 kg), was found dead around 11.00 am in 2006 in growing (1,5). Ard eche, France, during a detoxification program organized by a Strong doses of iboga lead to epileptic manifestations, faint- group that specialized in seminars on personal development and ing, paralysis, hypothermia, respiratory failure, and can be lethal. the discovery of iboga. The people in charge of the group, who To date, eighteen deaths linked with ibogaine ingestion have were present at the time of death, claimed that the victim had been recorded (1,5–11). In the late 1960s, the World Health ingested “a teaspoon” of powdered iboga root on the night Assembly classified ibogaine with hallucinogens and stimulants before. The victim had been addicted to various substances for as a substance likely to cause dependency or endanger human roughly 15 years: alcohol, cannabis (resin and herbal), psilocy- health (1). The consumption of iboga, ibogaine, and their bin, benzodiazepines (diazepam, nordiazepam, flunitrazepam), analogs is forbidden in France by the decree of March 12, 2007, cocaine and crack, amphetamine, ecstasy, LSD, morphine, and confirmed by the Council of State, March 20, 2009. heroin by his own admission. He had been undergoing a metha- The most abundant alkaloid present in the roots of the shrub done-based substitution treatment for 4 years (lately 30 mg a is ibogaine or 12-methoxyibogamine (~ 80 percent); there are day) at the time and had taken his last dose two nights prior to weaker concentrations of tabernanthine, ibogaine, and ibogamine his death; he had also been taking a diazepam (VALIUMâ) (~ 5 percent) (Fig. 1) (2,3,6). The effects and toxicity of iboga- treatment for ten years (lately 50 mg a day). ine seem to be related to its simultaneous action on a group of The autopsy was unable to establish any traumatic origin for neurotransmitter systems in the autonomous central nervous sys- the death of the victim, whereas the anatomo-pathological exam- tem and appear not to be attributable to actions at any single ination of the heart–lung block showed the presence of type of receptor, while its mechanism is complex and still only exogenous elements in the pulmonary parenchyma and various partially understood (1,3,9). Ibogaine has been the subject of calibers of bronchial tubes, indicating that regurgitation had several different methods of identification and analysis using taken place that was substantial and extensive enough to have chromatographic techniques combined with mass spectrometry/ caused asphyxiation (Mendelson’s syndrome). No underlying gas chromatography combined with mass spectrometry (GC-MS) cardiac or pulmonary pathology was detected. MAZOYER ET AL. . FATAL POISONING WITH TABERNANTHE IBOGA 3 A sample of blood (subclavian vein) was taken at the scene of The measurements were made in selected reaction monitoring death 7 h after the declaration of death, and samples of periph- mode (SRM). For ibogaine and ibogamine, the pseudomolecular eral blood (femoral artery), urine, and gastric fluid were taken ions were isolated (m/z = 310 and 280, respectively) and quanti- during the autopsy 8 days later. The rest of the powder fied by majority transition (m/z = 225 and 195, respectively). consumed was also analyzed. Specimens were stored at +4°C The identifications were confirmed by the full spectra (full-scan until analyses were carried out 2 weeks later. spectra and full-scan product ion spectra) (Fig. 2). Materials and Methods Systematic Toxicological Analyses (STA) STA procedures include headspace gas chromatography–mass Chemicals and Reagents spectrometry (GC-MS) for the analysis of ethanol and other Ibogaine, ibogamine, and prazepam-d5 standards were pur- volatile substances, GC-MS after acetylation with the use of chased from LGC Standards (Molsheim, France), Toxi-tubes Aâ CARIBOUâ software (personal communication), high-perfor- from Varian (Courtaboeuf, France). Purified water was provided mance liquid chromatography photodiode array detection â by VWR (Fontenay-sous-Bois, France), while methanol and (HPLC-PDA) (18), UPLC-PDA/MS (personal communication) â ethyl acetate for HPLC were provided by Sigma-Aldrich as well as immunoassay techniques, carbon monoxide, and cya- (Saint-Quentin Fallavier, France). nide detection. The stock solution of ibogaine, ibogamine, and their dilutions + ° in methanol were stored at 4 C and protected from light. Praze- Results pam-d5 was used as an internal standard (IS). The calibration curves obtained for ibogaine were linear for concentrations from 10 to 2000 ng/mL in the body fluids and Extraction Procedure from 1 to 100 lg/mg in the powder, with correlation coefficients â Body fluids were extracted by means of Toxi-tubes A . >0.999. The detection limit was calculated from the mathemati- 20 lL of prazepam-d5 (10 lg/mL, 200 ng) and 500 lL of blood cal formula LOD = mbl + 3SDbl (mbl: mean of the blanks; SDbl: or urine, or 50 lL of gastric juice was added to the Toxi-tubes standard deviation of the blanks; n = 30) as 1 ng/mL for the and topped up with 5 mL of deionized water then mixed by gen- two analytes in the blood and urine, while it was 20 ng/mL in tle inversion for 10 min. After centrifugation for 10 min at the gastric fluid. Within- and between-day precision studies gave 3000 g, the organic supernatant was evaporated under a stream relative standard deviations (RSD) always <12% in the whole of air at +50°C. The residue was then reconstituted by 50 lLof calibration range in blood and urine. ethyl acetate and transferred into a vial for injection into the gas The concentrations of ibogaine and ibogamine measured in chromatographic system. the victim’s blood were positive at 650 and 50 ng/mL, respec- Quantification ranges in body fluids were carried out on six tively, in the subclavian blood sample taken at the scene of calibration levels plus a blank at 10, 50, 100, 500, 1000, and death and 1270 and 100 ng/mL, respectively, in the femoral 2000 ng/mL of ibogaine and ibogamine. Drug-free material blood sample taken at the autopsy. The concentrations measured previously tested negative for the targeted molecules was taken in the urine and gastric fluid samples during the autopsy were from different autopsied corpses. also positive for ibogaine (1700 and 53,500 ng/mL, respectively) About 100 mg of the powder was sonicated for 20 min in 1 mL and ibogamine (120 and 4340 ng/mL, respectively) (Table 1, of methanol together with 10 lL of prazepam-d5. After centrifu- Fig. 3). gation for 10 min at 3000 g, 50 lL of the solution was evaporated The results of STA are shown in Table 1. to dryness under a stream of air at +50°C. The residue was then The powder ingested by the victim contained 7.2 percent reconstituted by 50 lL of ethyl acetate and transferred into a vial ibogaine and 0.6 percent ibogamine. A teaspoonful of 1.5–2g for injection into the gas chromatographic system. of the powder contains approximately 108–144 mg of ibogaine Calibration was performed in powdered local dried roots of and 9 to 12 mg of ibogamine. Verbascum thapsus (morphological similarities) previously tested negative for ibogaine and ibogamine by our method. Discussion Toxicological tests identified the presence of significant quan- GC-MS/MS Procedure tities of ibogaine and ibogamine in all the biological samples The gas chromatography used was a Trace GC (Thermo taken from the victim at the scene of death and during the Scientific, Courtaboeuf, France) equipped with an AS3000 autopsy. These results confirm that the individual had consumed autoinjector and a PolarisQ mass spectrometer (electron ionization). iboga root. The analytical column was a FactorFourTM capillary column The ibogaine and ibogamine contents found in the powder 5% phenyl, 95% methyl siloxane, 30 m in length, with an inter- (7.2 and 0.6 percent, respectively) are comparable to those found nal diameter of 0.25 mm (0.50 lm film thickness) from Varian in extracts of powdered Tabernanthe iboga root and to the phar- (Courtaboeuf, France). Helium (6.0 purity) was used as carrier macognostic data reported in the literature (1–3,6). No other gas at a flow rate of 1 mL/min in constant flow mode. The inter- toxic compound, foreign to the plant, was detected by comple- face temperature was +280°C, and ion source temperature was mentary analyses. Bogusz et al. showed that a dose of 20 +200°C. Pulsed injection with surge pressure (1 lL) was carried mg/kg, or the ingestion of 1400 mg of ibogaine, would result in out at +280°C and 200 kPa for 1 min. The initial oven tempera- an ibogaine concentration of 150 ng/mL in a man weighing ture was +50°C for 2 min and was increased to +300°Cat 70 kg (the approximate weight of our victim) 19 h after inges- 25°C/min and held for 8 min. The chromatographic analysis tion (7). In that case, the concentrations measured in the victim lasted 20 min. would be the result of ingesting larger quantities than the 4 JOURNAL OF FORENSIC SCIENCES FIG. 2––Full-scan mass spectra of ibogaine (a) and ibogamine (b). Full-scan production spectra obtained from pseudomolecular ion of ibogaine (m/z = 310) (a’) and ibogamine (m/z = 280) (b’). TABLE 1––Concentrations of the different compounds analyzed, as Netherlands. The 24-year-old victim had ingested a 29 mg/kg measured in the victim’s postmortem samples (ng/mL or ng/g). The quantities dose of ibogaine as part of a heroin addiction treatment and died measured in the gastric contents are given as the total mass recovered from of respiratory failure 19 h later. The concentration of ibogaine the gastric contents (lg in brackets). measured in the femoral blood was 710 ng/mL, and the victim was suspected of secretly taking opiates during the treatment (7). Peripheral _ Blood Blood In 2006, Kontrimaviciut e et al. reported the case of a 48-year- (scene: (autopsy: Gastric old man with a history of drug addiction who died 53 h after subclavian femoral Urine Juice taking iboga. The concentration of ibogaine measured in a sub- Compound vein) artery) (autopsy) (autopsy) clavian blood sample taken at the scene of death, between 6 and Ibogaine 650 1270 1700 53,500 (2675) 12 h after death, was 10,800 ng/mL; the concentrations from Ibogamine 50 100 120 4340 (217) samples of femoral blood, urine, and gastric contents taken dur- Diazepam 413 175 35 786 (39.3) ing the autopsy 48 h later were 5400, 83,300, and 2910 ng/mL, Nordiazepam 350 173 60 650 (32.5) Oxazepam 90 10 40 ND respectively (6). To date, there is no conclusive explanation for Temazepam 40 20 30 ND the cause of these deaths. Some authors implicate that the cere- Methadone 77 74 175 3 (0.15) bellar neurotoxicity produced by ibogaine observed mainly in EDDP 20 21 1300 98 (4.9) rats (1,8), others suspect a deregulation of the autonomic ner- ND, not detected. vous system combined with psychological stress, causing sudden cardiac arrest, the risk of which would be greater if there were a pre-existing cardiac anomaly (9,10,19). In 2009, Hoelen et al. teaspoonful that had been alleged by the group; it could easily reported the case of a woman admitted to hospital as an emer- have been a dose of over 20 g of the iboga powder analyzed. gency after having a heart attack brought about by the consump- To date, at least eighteen cases of fatal ibogaine poisoning tion of a single moderate dose of around 500 mg of ibogaine. have been reported in psychotherapeutic settings or with anti- The patient presented with extreme severe QT interval prolonga- addictive aims, but cases in which the concentrations of the tion (548 ms; QT interval corrected for the heart rate (QTc), active principles have been established in postmortem samples 616 ms) and ventricular tachyarrhythmias. QT interval prolonga- are rare (1,5–11). In 1998, Bogusz et al. reported a fatal case of tion is a risk factor for torsades de pointes and ventricular fibril- iboga poisoning that had occurred in June 1993 in the lation, which can lead to sudden death. The authors proposed a AOE TAL. ET MAZOYER . AA OSNN IHTBRATEIBOGA TABERNANTHE WITH POISONING FATAL FIG. 3––Chromatograms of the victim’s postmortem biological samples (IS: internal standard; RT: retention time). 5 6 JOURNAL OF FORENSIC SCIENCES cause of death associated with the consumption of ibogaine in mainly into the cardiac cavities and large thoracic vessels, the occurrence of QT interval prolongation and ventricular tach- while redistribution into the peripheral system appears to be a yarrhythmias (19). In addition, there are some indications sug- more moderate and slower process. The passive absorption of gesting that taking ibogaine and opiates simultaneously increases xenobiotics from the gastric and digestive contents into the the toxicity of the opiates (1,6,7,11). mesenteric blood can take place after death, as long as there is The STA indicated the presence of methadone and its inac- a concentration gradient, so they can diffuse gradually into the tive metabolite in the victim’s samples. The concentrations of adjacent organs (25). In our case, the concentrations of iboga- methadone measured in the blood were therapeutic for an ine (volume of distribution (Vd) approximately 5 L/kg (26)) inexperienced individual, or infratherapeutic for a long-term and ibogamine measured in the victim’s gastric contents were user like the victim, and consistent with having last been very substantial compared with the other samples. If degrada- taken 48 h before death, as alleged by witnesses (20). Metha- tion of the two molecules had occurred between the two blood done is known to cause QT interval prolongation and fatal samples, the difference may have been offset by diffusion of torsades de pointes, hence the publication of security recom- the gastric and digestive contents into nearby organs, including mendations for doctors in 2009 (21,22). In particular, it is the femoral artery. In addition, the samples were taken from recommended not to exceed a QT interval of 500 ms and not different sites (subclavian vein and femoral artery) and at dif- to prescribe methadone together with other substances that ferent postmortem intervals (7 h and 8 days, respectively). The prolong the interval. subclavian vein is a questionable choice of site for a peripheral The presence in the samples of therapeutic concentrations of blood sample because of its proximity to the heart (27). diazepam and its active metabolites (nordiazepam, oxazepam Indeed, benzodiazepines are often found in higher concentra- and temazepam) confirms that the victim, who was taking a tions in cardiac blood than those in peripheral blood (28) and VALIUMâ treatment, had consumed diazepam. Bearing in mind do not seem to be very prone to postmortem degradation. The his drug-addicted past (dipotassium clorazepate abuse), cocon- concentrations of diazepam (Vd, 0.5–2.5 L/kg (20)), nordiaze- sumption cannot be ruled out. The quantification of these com- pam (Vd, 0.5–2.5 L/kg (20)), oxazepam (Vd, 0.5–2 L/kg (20)), pounds in the urine was not very informative: the relative ratios and temazepam (Vd, approximately 1 L/kg (20)) measured in of the metabolites of diazepam in urine are variable and dose the victim are therefore consistent with early PMR (within 7 h dependent, and their absolute concentration clearly depends on of death). According to this hypothesis, the ibogaine concentra- the individual’s diuresis (20). Reports of methadone-related tions measured by Kontrimavi ciut e_ et al. were more likely to deaths often mention the simultaneous consumption of be the result of PMR than of degradation. Moreover, the con- benzodiazepines (23). A mechanism of action was proposed centrations measured in our victim would have been overesti- recently that explained the potentialization of the harmful cardiac mated. The concentrations of methadone (Vd, 4–5 L/kg (20)) effects of methadone by diazepam (24). and EDDP were same in both samples, which may show that As there is so little information available on postmortem con- they have a slower rate of PMR. centrations of ibogaine after the ingestion of iboga root, it is The legal proceedings against the people in charge of the difficult to come to any firm conclusions about the cause of group who organized the detoxification treatment are still ongo- death. However, our analyses seem to be consistent with the ing. The forensic scientist concluded that the death was a direct results already published. Hoelen et al. reported the case of QT result of taking iboga. Iboga, ibogaine, and their analogs have interval prolongation in a woman who had taken a moderate been classified as controlled substances in France since March dose of iboga. Our victim had taken roughly three times the 2007; the decree came as a direct result of the death of our amount, together with methadone, which is known to prolong victim and another case in France a short time earlier (6). the QT interval, and diazepam, known to potentialize the cardio- toxicity of methadone. The consumption of a substantial amount Conclusion of iboga appears thus to be the cause of death, in the context of simultaneous diazepam and methadone consumption. Moreover, We report a case of fatal poisoning linked to the ingestion of concentrations of diazepam, methadone, and their metabolites powdered Tabernanthe iboga root. The overall analytical picture are insufficient to account for death in a context of regular con- is consistent with a mixed overdose, with ibogaine as the toxic sumption over several years. In this hypothesis, sudden cardiac principle, in association with methadone and diazepam. The death resulted from torsades de pointes caused by QT interval postmortem concentrations are consistent with this hypothesis prolongation. when compared to data already published in the international It is interesting to note that the concentrations of ibogaine scientific literature. Moreover, no cause of death was found at and ibogamine measured in the blood sampled during the the autopsy either by macroscopic or histological analysis. The autopsy are twice as high as those in the blood sampled at the coroner’s report concluded that the cause of death was iboga scene of death. This contradicts the data previously reported by poisoning. Kontrimavi ciut e_ et al., who measured concentrations in the sub- clavian blood sampled at the scene of death that were twice as high as those measured in the femoral blood sampled during References the autopsy 48 h later (6). The hypothesis of postmortem deg- 1. Alper KR. Ibogaine: a review. 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Gallagher CA, Hough LB, Keefner SM, Seyed-Mozaffari A, Archer S, codynamic interactions in cardiac ion channels. J Cardiovasc Pharmacol Glick SD. Identification and quantification of the indole alkaloid iboga- [Research Support, Non-U.S. Gov’t] 2010;56(4):420–30. ine in biological samples by gas chromatography-mass spectrometry. 25. Kintz P. Toxicologie et pharmacologie m edicol egales [Medicolegal toxi- Biochem Pharmacol 1995;49(1):73–9. cology and pharmacology]. Nancy, France: Elsevier France, 1998. 13. Hearn WL, Pablo J, Hime GW, Mash DC. Identification and quantitation 26. Baselt RC. Disposition of toxic drugs and chemicals in man, 6th edn. of ibogaine and an o-demethylated metabolite in brain and biological Foster city, CA: Biomedical Publications, 2002. fluids using gas chromatography-mass spectrometry. J Anal Toxicol 27. Vorpahl TE, Coe JI. Correlation of antemortem and postmortem digoxin 1995;19(6):427–34. levels. J Forensic Sci 1978;23(2):329–34. 14. 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J Chroma- France togr B Analyt Technol Biomed Life Sci 2006;843(2):131–41. E-mail: [email protected] ± ± ± ǯà ǯ ° ±± ±±Ǥ ±± Ǧ Ȁ°±ǯǤ±±±± ǯ=?Ǥ ǯ ǯ ǯà ǯ ±±ʹȀ ǡ ǯ ±± =?Ǥ ± ±° ǡ ǯ ±± ± ±ʹͲǡ±± ±± ± =?Ǥ à ± ± ±ǡ ±±± ±ǡ±± ± ° ± ǯñ =?Ǥ ±± ±ǯ ͳͲͲͲ ± ± Ǥǯ ǯ±±Ǥǯǡǯà ± ± ʹͲͲ ǡ ²±± ° ǯ ± =?Ǥ ± ° ± ± ǦȀǡ ȋ ± ±ȌǤ ± ± ǯ ± ± ±ǯà ±ǡ ± ȋΪͶιǡǯ°ȌǤ ͻͶ ǣ ǯ ǯǡ ǯ ° Ǧ Ȁǡǯ ǦȀ ǯ ñ± =?Ǥ ± ± ǡ ± ±± ±ǡ ± ò ±± ±Ǥ ǯǡ ±ǡ± ±ǡ± =?Ǥ ǯ ± ± ǡ ȋ ǤȌǡ ȋ ǤȌǡȋ ǤȌǡ ȋǤȌȋ ǤȌ =?Ǥ ±ǡ ǡ ǯ ° ± Ǥ ±ǡ ǯ ± ǡ± ± =?Ǥ ǡ ¡°± °±± ± ͳͻͷͲ ͳͻͷ =?Ǥ à ±ǯ ± ±±±± Ǥ ǯ ǯ±±±± Ǧ Ǧ ͷͶ ±Ǧ± ǦͻǤ ± ± ± ± ± =?Ǥ ǯ ±± ± ͳͺ±=?Ǥǯǯ ǯ±±±ǯ±Ǥ ±±±± ǦȀ ͳͺ =?Ǥ ±ǡ ǯ±± ±± ǡ±Ǥ±ǯ Ǧ±±± ±± ±ǯ² ± ǯǯ ǯǤ ͻͷ ± ǯ±± ǡ±±ǯǤ ǯ ±±±± ǡ ° Ǥ ±ǯǯ Ǽ ǽǤ ͻ J Forensic Sci,2014 doi: 10.1111/1556-4029.12412 CASE REPORT Available online at: onlinelibrary.wiley.com TOXICOLOGY J er emy Carlier,1 M.Sc.; Emmanuel Escard,2 M.D.; Michel P eoc’h,3 M.D., Ph.D.; Baptiste Boyer,4 M.D.; Ludovic Romeuf,1 M.Sc.; Thierry Faict,4 M.D., Ph.D.; J erome^ Guitton,5 Pharm.D., Ph.D.; and Yvan Gaillard,1 Pharm.D., Ph.D. Atropine Eye Drops: An Unusual Homicidal Poisoning* ABSTRACT: In March 2009, the body of a 51-year-old man was found in the boot of his car. The body had been frozen before being dis- membered at the abdomen. The autopsy failed to determine the cause of death. Systematic toxicological analyses of the victim’s peripheral blood and urine showed the presence of atropine, a powerful anticholinergic. Atropine was therefore specifically detected and quantified throughout the victim’s biologic samples by HPLC-MS² in the biologic fluids and UHPLC-MS² in the hair. The atropine concentrations were 887 ng/mL in the cardiac blood, 489 ng/mL in the peripheral blood, 6693 ng/mL in the gastric contents (1.1 lg), 6753 ng/mL in the urine, and 2290 pg/mg in the hair. The blood concentrations measured in the decedent were consistent with an overdose of atropine, which was determined as the cause of death. The manner of death was a homicide with criminal intent. KEYWORDS: forensic science, forensic toxicology, case report, homicide, atropine, ultrahigh-performance liquid chromatography/tandem mass spectrometry, hair In March 2009, the body of a 51-year-old man was found in the absence of edema, inflammatory infiltrate, or hemorrhagic the boot of his car, which had been left in a public car park in suffusion. Some hematic lesions, caused by trauma, in the lumbar France. The putrefied body of the victim, who had been missing area on the upper left arm and on the face were confirmed mac- for 29 days, had been cut in half at the abdomen. roscopically by cutaneous incisions in these areas. A diagnosis of The autopsy held the following day established that a profes- peri-mortem dermabrasions on the back or the torso and the but- sional saw had been used, like the ones used in abattoirs for cut- tocks would seem to suggest that the body had been dragged. ting up cattle carcases. The absence of substantial traces of blood The autopsy failed to determine the cause of death, and samples at the edges of the cut section, as well as the relatively good state were collected and submitted for toxicological analysis. of the body and well-preserved intestinal loops and pelvic organs The autopsy samples – peripheral blood, cardiac blood, urine, (given the hypothetical maximum postmortem period of 29 days) gastric fluid, bile, lung, liver, kidney, and hair (vertex posterior; would seem to suggest that the body had been frozen before proximal segment, 1.5 cm in length, cut close to the scalp) – being cut in half. The victim (1.62 m tall and weighing 73.8 kg) were stored protected from light at +4°C(+20°C for the skin had postmortem thermal burn scars on the face, pelvis, and the appendages). Routine toxicological analyses were performed on upper half of the thorax. This was observed macroscopically as the peripheral blood and urine, and in light of these first results, blackish areas on the edges of some of the clothing and the atropine was specifically quantified in all the biologic samples. singed appearance of the hair, and confirmed histologically at the Toxicological analysis of atropine was attempted to be analyzed back of the left shoulder and the area around the left temple by using gas-phase chromatography techniques combined with mass spectrometry (GC-MS) (1,2); however, high-performance liquid- phase chromatography (HPLC) appeared to be more suitable in 1Laboratoire LAT LUMTOX, 800 av. Marie Curie, Z.I. Jean Jaur es, view of the compound’s thermosensitivity. Several sensitive meth- ^ 07800 La Voulte-sur-Rhone, France. ods were validated and published for measuring atropine in bio- 2Unit e interdisciplinaire de m edecine et de pr evention de la violence (UIMPV), Hopitaux^ Universitaires de Gen eve, 4 rue Gabrielle-Perret-Gen- logic fluids and appendages, alone, or combined with scopolamine til1211 Gen eve, 14, Suisse. or other alkaloids (3). Reports of lethal cases (referred to in the 3Service de M edecine L egale, Hopital^ Bellevue, 25 bvd. Pasteur 42055 discussion) are on the other hand infrequent, and postmortem con- Saint-Etienne, France. centrations are rare, or indeed nonexistent in some matrices. 4Institut m edico-l egal, Centre Hospitalier Universitaire, 58 rue Montalem- bert, 63003 Clermont-Ferrand, France. 5Laboratoire de toxicologie, Facult e de pharmacie de Lyon, 8 av. Rocke- feller, 69373 Lyon, France. Materials and Methods *Presented at the 46th Annual Meeting of The International Association of Forensic Scientists (TIAFT), June 2-8, 2008, in La Martinique, French West Chemicals and Reagents Indies. Received 13 Dec. 2012; and in revised form 16 April 2013; accepted 20 Standards of atropine and internal standard cocaine-d3 April 2013. (100 lg/mL) were bought from LGC Standards (Molsheim, © 2014 American Academy of Forensic Sciences 1 2 JOURNAL OF FORENSIC SCIENCES France). The latter was used as internal standard (IS). Subtilisin evaporated to dryness under a stream of air at +45°C, and the A was provided by Sigma-Aldrichâ (type VIII, Bacillus residue was reconstituted with 0.05 mL of acetonitrile/deionized licheniformis; Saint-Quentin Fallavier, France). Acetonitrile for water (50:50, v/v), centrifuged at 14,000 g for 4 minutes for HPLC-MS was obtained from Carlo Erba (Val de Reuil, France), ultimate purification, the supernatant being transferred into a vial methanol for HPLC from Sigma-Aldrichâ, and deionized water for injection into the chromatographic system (HPLC-MS/MS for HPLC from VWRâ (Fontenay-sous-Bois, France). for body fluids and viscera samples, UHPLC-MS/MS for hair Tris buffer (1 mM) was prepared with Trizmaâ hydrochloride sample). This is a standard procedure suggested by the manufac- (≥99%; Sigma-Aldrichâ) diluted in 100 mL of deionized water, turer for extracting nonpolar or weakly polar alkaline com- and the pH was adjusted to 4.7 with sodium hydroxide 1 M pounds, which include atropine. (Sigma-Aldrichâ, Saint-Quentin Fallavier, France). Ammonium formate buffer (2 mM) was prepared with ammonium formate â HPLC-MS/MS Procedure (Body Fluids and Viscera) (≥99%; Sigma-Aldrich ) diluted in 1 L of deionized water, and the pH was adjusted to 3.0 with formic acid (88–91%; Sigma- Separation of the extracted compounds was performed using a â â Aldrich ). C18-ODB Uptisphere column (length: 150 mm; internal diame- ter: 2.1 mm; particle size: 3 lm) (Interchim, Montlucßon, France) at a temperature of +35°C, with a gradient mobile phase com- Systematic Toxicological Analyses posed of acetonitrile and a 2 mM ammonium formate buffer at Systematic toxicological analyses (STA) procedures, per- pH 3.0, for a chromatographic run time of 28 min. The tempera- formed on peripheral blood and urine, include headspace ture of the samples was +10°C and the injection volume was GC-MS for the analysis of cyanide, ethanol, and other volatile 0.01 mL. The separation started with a linear gradient of mobile substances, GC-MS after acetylation with the use of CARIBOUâ phase consisting of 80:20 (v/v) buffer/acetonitrile to 10:90 (v/v) software (B evalot F, Gaillard Y, Lemeur C, personal communi- within 10 min; the conditions were isocratic for 8 min; then, the cation), HPLC photodiode array detection (HPLC-PDA) (4), mobile phase returned to initial conditions within 0.5 min and ultrahigh-performance liquid chromatography-PDA, and MS was equilibrated for 9.5 min. The mobile-phase flow was detection (UHPLC-PDA/MS) (personal communication), 0.19 mL/min for the whole time of the separation. UHPLC-MS/MS (5,6) as well as carbon monoxide detection and Spectrometric detection of atropine, scopolamine, and their IS immunoassay techniques. was performed using an ion trap (LCQ Advantage; Thermo Sci- entific, Courtabœuf, France) after ionization by ESI in positive mode (spray voltage: 5.1 kV; capillary temperature: +220°C; Specific Analysis of Atropine capillary voltage: 21 V; tube lens offset: 35 V). The collision Pretreatment of Viscera and Hair—Viscera (1 g) was crushed gas was high-purity helium (5.6; Linde gas, Molsheim, France), using a homogenizer from PRO Scientific (Oxford, U.S.A.) in and the nebulizing gas was nitrogen (4.5; Linde gas, Molsheim, 1.5 mL of a 1 M Tris buffer at pH 4.7 with 1 mg of subtilisin France). The measurements were made in selected reaction mon- A (protease). After 60 min of incubation at 55°C and centrifuga- itoring mode (SRM). The pseudomolecular ion of atropine was tion (3000 g, 10 min), 1 mL of the supernatant was treated fol- isolated (m/z = 290.1), and the two major transitions (m/z = 124 lowing the same solid-phase extraction (SPE) procedure applied and 260) were used for quantification. The identification was to the body fluids. confirmed by the full-scan product ion spectra (Fig. 1). The hair was decontaminated using the procedure recom- mended by the Society of Hair Testing (7), then desiccated and â UHPLC-MS/MS Procedure (Hair) pulverized using a ball mill from Retsch (Haan, Germany) until a fine gray powder was obtained. A methanolic solution of Separation of the extracted compounds was performed using an â exactly 50 mg of the powdered hair was sonicated for 2 h. After ACQUITY UPLC HSS C18 column (length: 150 mm; internal centrifugation (3000 g, 10 min), the supernatant was evaporated diameter: 2.1 mm; particle size: 1.8 lm) (Waters, Guyancourt, to dryness under a stream of air at +45°C and the residue was reconstituted by 0.15 mL of methanol and diluted in 0.850 mL of deionized water. The sample was then treated following the same SPE procedure applied to the body fluids. Solid-Phase Extraction Procedure Extraction of atropine was performed by means of SPE on Oasisâ HLB cartridge (3 mL, 60 mg) from Waters (Guyancourt, France). The conditioning was carried out with 3 mL of metha- nol followed by 3 mL of deionized water. 1 mL of the biologic fluids, pretreated viscera, or hair was mixed with 0.02 mL of IS (cocaine-d3, 1 lg/mL) and 3 mL of deionized water, vortex mixed for 30 sec and centrifuged at 3000 g for 10 min. The supernatant was then applied to the cartridge and allowed to drain under vacuum at 1 mL/min. Subsequent SPE rinses were carried out with three volumes of 1 mL of an aqueous solution FIG. 1––Structural formula of atropine. Full-scan product ion spectra of methanol (5%). The cartridge was allowed to dry under vac- obtained from pseudo-molecular ion of atropine (m/z = 290.2) (ion trap). uum for 10 min, and the elution was performed by three vol- (m/z = 124 ion corresponds to the tropane nucleus of the molecule; MW: umes of 1 mL of methanol. The eluate was subsequently molecular weight). CARLIER ET AL. . ATROPINE EYE DROPS 3 France) at a temperature of +50°C, with a gradient mobile phase gas flow rate: 0.19 mL/min). The collision gas was high-purity composed of acetonitrile and a 2 mM ammonium formate buffer argon (6.0; Linde gas, Molsheim, France), and the nebulizing at pH = 3.0, for a chromatographic run time of 15 min. The tem- gas was nitrogen (>99%; ZEFIRO 35 LC-MS generator; perature of the samples was +12°C, and the injection volume was F-DGSi, Evry, France). The measurements were made in SRM. 0.01 mL. The separation started with a mobile phase consisting The pseudomolecular ion of atropine was isolated ([M+H]+: of 87:13 (v/v) buffer/acetonitrile for 0.5 min, followed by a linear m/z = 290.2), and the major transition (m/z = 124; collision gradient to 50:50 (v/v) buffer:acetonitrile within 9.5 min, a sec- energy: 20 eV) was used for quantification. The transition ond linear gradient to 95:5 (v/v) buffer:acetonitrile within m/z = 93.1 (collision energy: 35 eV) was selected to confirm the 0.75 min; the conditions were isocratic for 1.5 min; then, the identity of the toxin. mobile phase returned to initial conditions within 0.25 min and was equilibrated for 2.5 min. The mobile phase flow was Results 0.40 lL/min for the whole time of the separation. Spectrometric detection of atropine, scopolamine, and their IS The retention times of atropine and cocaine-d3 were 6.7 and was performed using a triple quadrupole (ACQUITY TQD; 9.1 min, respectively, with the HPLC-MS/MS, and 3.1 and Waters, Guyancourt, France) after ionization by ESI in positive 4.6 min with the UHPLC-MS/MS (Fig. 2). No suppression or mode (desolvation gas flow rate: 450 L/h; cone gas flow rate: enhancement of ionization was observed in the retention times 50 L/h; source temperature: +140°C; desolvation temperature: of the compounds, whichever matrix was tested (suppression/ +350°C; capillary voltage: 3 kV; cone voltage: 50 V; collision enhancement of atropine ionization was qualitatively determined FIG. 2––Chromatographic representation of the hair sample of the victim (1.5 cm from the root). The atropine concentration was 2290 pg/mg. (IS: internal standard). 4 JOURNAL OF FORENSIC SCIENCES by monitoring its signal during the injection of a negative con- plants were mainly consumed for magico-religious, hallucino- trol throughout the simultaneous continuous postcolumn infusion genic, or sometimes aphrodisiac purposes (12). The species can of the standard for the whole time of the chromatographic sepa- be distinguished by their alkaloid composition; while deadly ration). The atropine extraction coefficient was 90.2%. The cali- nightshade and mandrake contain mainly atropine (up to 95% of bration curves obtained were linear for concentrations from 1 to the total alkaloid content), the Datura, Duboisia, Brugmansia, 1000 ng/mL in the whole blood and from 2 to 5000 pg/mg in and Hyoscyamus genera contain a high, sometimes dominant the hair. The limits of detection (LOD) were 0.1 ng/mL and proportion of scopolamine (11). Nowadays, atropine is used as a 1 pg/mg, respectively, (LOD = mbl + 3SDbl;mbl: mean of the medicine, particularly as a preanaesthetic medication and in rean- blanks; SDbl: standard deviation of the blanks, n = 30). The lim- imation, or as a specific antidote to acute poisoning by anticho- its of quantification (LOQ) were 1 ng/mL and 2 pg/mg linesterase or parasympathetic drugs (Veratrum, insecticides, and (LOQ = mbl + 10SDbl). Within- and between-day precision stud- nerve gas). Atropine eye drops, which are more concentrated, ies gave relative standard deviations (RSD) always less than 9% are used in ophthalmological diagnostics and to treat keratitis, in the whole calibration range in the blood and hair, and the uveitis, and iridocyclitis (13). accuracy was from 98.6 to 100.2% and from 100 to 106%, An oral dose of 10 mg of atropine would provoke moderate respectively (8,9). anticholinergic symptoms in most individuals: tachycardia, raised The results of the toxicological investigations are shown in body temperature, reduced saliva production, mydriasis, disorien- Table 1. Quantifications in the viscera and body fluids were car- tation, hallucinations, etc. (14,15). The lethal dose is between ried out from the calibration curve in the blood. It is noteworthy 100 and 200 mg, but quantities as low as 50 mg or even less that no trace of scopolamine was detected (LOD = 0.1 ng/mL in than 5 mg in children have been linked with fatal cases (16,17). the whole blood, LOD = 1 pg/mg in the hair). On the other hand, cases have been reported of people surviving after ingesting doses of 500 mg to 1 g, but these patients often had the benefit of serious hospital care, with the possible admin- Discussion istration of physostigmine or neostigmine as an antidote (14,15,18–20). It is noteworthy that the pharmacokinetics of atro- Cause of Death pine varies widely from one person to another. Two adults, who The analysis revealed that the victim had been undergoing a had each consumed the same quantity, between 200 and 400 mg classic psychotropic treatment comprising mianserin, an antide- of atropine, had blood concentrations of 24 and 217 ng/mL 10 h pressant, alprazolam, an anxiolytic, and zopiclone, a hypnotic. after ingesting 200 g of deadly nightshade berries. The first was The atropine concentrations measured in the peripheral blood delirious, while the second was in a coma with respiratory insuf- were clearly below the therapeutic range, indicating that the last ficiency (19). In another case, a 58-year-old man who acciden- dose had been taken long before death, with no clinical activity tally ingested 500 mg of atropine sulfate had a serum or effect on alertness or behavior (10). More interestingly, the concentration of 244 ng/mL 4 h after ingestion (20). More analysis also showed the presence of atropine in all the victim’s recently, a concentration of 130 ng/mL of atropine was measured samples. in a 48-year-old man 12 h after ingesting between 200 and 300 g Atropine is an anticholinergic and antispasmodic occurring of deadly nightshade berries (21). An 18-year-old man who had naturally in many members of the Solanaceae family: deadly ingested an unknown quantity of 30 mg atropine tablets had a nightshade (Atropa belladonna), the Duboisia, Brugmansia, and postmortem blood concentration of 200 ng/mL (22). Datura genera, the henbanes (Hyoscyamus spp.), and mandrake In the light of this data, the postmortem concentrations mea- (Mandragora officinarum) (11). Their tropane alkaloid content, sured in the victim (489 ng/mL in the peripheral blood) are con- mainly atropine and scopolamine, is the source of their pharma- sistent with a massive overdose of atropine, the cause of death. cotoxicological properties, which have been exploited for several It is worth noting that in the absence of any available compa- centuries. Although sometimes used with criminal intent, to take rable data, the concentrations measured in the victim’s hair are advantage of a victim’s apathetic state or for homicide, these quite uninformative. In 2006, Kintz et al. reported the case of an individual who was poisoned following the consumption of an infusion of Datura inoxia flowers for recreational purposes. TABLE 1––Concentrations of the different compounds analyzed, as mea- Analysis of the patient’s hair identified scopolamine concentra- sured in the victim’s postmortem samples (ng/mL or ng/g). The quantity mea- tions between 14 and 48 pg/mg, while atropine was not detected sured in the gastric contents is given as the total mass recovered from the (23). gastric contents (lg in brackets). Samples Atropine Alprazolam Mianserin Zopiclone Estimation of the Dose of Atropine Administered Peripheral 489 4 N.D. 9 The apparent volume of distribution of atropine in humans is blood Urine 6753 6 Traces 111 reported as between 2 and 3.6 L/kg (10,24). The oral bioavail- Cardiac 887 ability of atropine is estimated to be 50% (25). In these condi- blood tions, a concentration of 489 ng/mL in the peripheral blood of Gastric 6693 an individual weighing 73.8 kg would be consistent with an oral l contents (1.1 g for the dose of between 145 and 260 mg. As we have already noted, whole gastric contents) Bile 6753 the pharmacokinetics of atropine for identical doses varies Liver >123 widely according to interindividual susceptibility, so the dose Kidney 1919 administered is likely to be higher than the estimated oral dose: Lung 412 probably between 200 and 500 mg of atropine. These values do Hair 2290 not, of course, take into account the metabolic elimination of the N.D., not detected. poison (the length of time between consumption and death CARLIER ET AL. . ATROPINE EYE DROPS 5 remains unknown), the postmortem redistribution, and the influ- The victim’s wife and son have been charged with murder. ence of the decomposition and the probable freezing of the Included in the charges against his wife is that of having body. obtained three 10 mL bottles of ATROPINE ALCONâ 1% with- To date, no publication has reported atropine concentration out prescription in the month before his death. Another telling measured in a user’s hair, chronic or not. As a result, the only fact was the decedent was dismembered at abdomen and his son explanation for the concentration of 2290 pg/mg of atropine in was working in an abattoir at the time. the victim’s hair is that he had already had one or more doses before the one that caused his death. This can be explained by Conclusion the ATROPINE ALCONâ 1% (atropine sulfate)-based treat- ment for uveal inflammation that the victim had been taking A case of homicide by atropine poisoning was reported. The (one to two drops on the conjunctiva, two to four times daily victim had ingested a massive dose, probably in the form of according to the ophthalmologist’s prescription). It would have atropine sulfate eye drops, which is very concentrated and easily been interesting to carry out segmental analysis to ascertain accessible. Atropine poisoning is difficult to ascertain at an whether or not there was an increase in the concentration in autopsy, which is why postmortem toxicological investigations, the proximal segment, but the small quantities of the sample and in particular screening methods, are so important. Quantita- were limiting. tion of atropine was conducted in the victim’s peripheral blood, cardiac blood, urine, gastric fluid, bile, lungs, liver, and kidneys with the various methods described. It was not possible to deter- The Manner of the Atropine Overdose mine the influence on the distribution of the poison of the possi- It is unlikely that the overdose was caused by recreational use ble freezing of the body and the conditions of its preservation. It for aphrodisiac purposes in view of the concentrations measured, is noteworthy, however, that the estimated dose matches the data which could only have caused certain death, while recreational from the inquest: the alleged perpetrator had bought three bottles use implies the survival of the consumer. The use of tropane of atropine sulfate in the month before the victim’s death. alkaloids for hallucinogenic purposes essentially involves the Lastly, this is the first time the amount of atropine in a con- consumption of species of the Datura genus, the psychotropic sumer’s hair has been reported. The concentrations measured in action underpinned by the presence of scopolamine. The use of the victim’s hair are probably the result of the chronic dose he atropine alone as a hallucinogen is extremely rare, although it had been taking as a treatment. However, it may be the result of has been described (22). A fatal overdose from the recreational previous unsuccessful murder attempts. use of atropine has never been described. In the same way, the situation in which the body was found would seem to rule out References suicide. The manner of death is thus, based on all the evidence, homicide with criminal intent. 1. Eckert M, Hinderling PH. Atropine: a sensitive gas chromatography-mass spectrometry assay and prepharmacokinetic studies. Agents Actions 1981;11(5):520–31. The Source of Atropine 2. Namera A, Yashiki M, Hirose Y, Yamaji S, Tani T, Kojima T. Quantita- tive analysis of tropane alkaloids in biological materials by gas chroma- Deadly nightshade or mandrake could have been the manner tography-mass spectrometry. Forensic Sci Int 2002;130(1):34–43. of poisoning: a lethal dose would be around 400 g of fresh 3. Beyer J, Drummer OH, Maurer HH. Analysis of toxic alkaloids in body – – deadly nightshade berries or a similar amount of mandrake root samples. 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Ǽ Ãǽȋ Ȍǡ =?Ǥ ǡ ±ȋ ǡ ±± ± ǯ Ȍ=?Ǥ ǯ ±±±± °± =?Ǥ ± ± ǯ ǡ ǯ ǯ ±°ǯ±±±Ǥ±±±±± ͳͻ ǯ ±° Ǧ ± ±± ǯ± ǯ±± ± =?Ǥ ² ±± ǡ ǯ° ǯ± ° ǯ ǯ =?Ǥ ±ǡ± °° ° Ǥ ±± ±ͳͻͻͺʹͲͲͳ ±ǯØ Ǧ ǦǦǤ ǯ ǯ ± ° ǡ ± ǡ ǯ ± ± ±Ǥ ± ǯ ±ǡ ² °ǯ±±±± ǯ±±±Ǥ± ͳ͵ͷ ±°±ͳʹͳͶ ± ° ǯ ǡ ±° Ǥǯ ±±±± ǯ ǯ ±° ǯ ǯ ǯ ± Ǥ ° ±± ±° ǯ± ǯ° ǯ ±ǯ Ǥ ± ǯ ǯ Ǽ ǽǤ ͳ͵ #WVJQT URGTUQPCNEQR[ Forensic Science International 206 (2011) e103–e107 Contents lists available at ScienceDirect Forensic Science International journal homepage: www.elsevier.com/locate/forsciint Rapid communication Fatal intoxication due to ackee (Blighia sapida) in Suriname and French Guyana. GC–MS detection and quantification of hypoglycin-A Yvan Gaillard a,*,Je´re´mie Carlier a,c, Marc Berscht b,Ce´dric Mazoyer a, Fabien Bevalot a, Je´roˆme Guitton c, Laurent Fanton d a LAT-LUMTOX, 800 avenue Marie Curie, 07800 La Voulte sur Rhoˆne, France b Centre hospitalier Franck Joly, Saint Laurent du Maroni, French Guyana, France c Universite´ Claude Bernard, 8 avenue Rockefeller, 69008 Lyon, France d Institut de Me´decine Le´gale, 12 avenue Rockefeller, 69008 Lyon, France ARTICLE INFO ABSTRACT Article history: Between 1998 and 2001 the deaths of 16 Surinamese children were recorded along the Maroni River, Received 24 November 2010 which forms the border between Suriname and French Guyana. After a metabolic origin was eliminated, Received in revised form 20 December 2010 ethnobotanical research in the field led to a hypothesis of intoxication through the ingestion of ackee. Accepted 13 January 2011 Ackee (Blighia sapida) is a large green leafy tree of West African origin. Its unripe fruit contains large Available online 16 February 2011 quantities of two toxic molecules: hypoglycin-A and hypoglycin-B, the former being the more toxic. We have developed a GC–MS procedure allowing us to demonstrate the presence of hypoglycin-A in the Keywords: gastric fluid of one of the deceased children, and to compare the content of hypoglycin-A in fruit collected Ackee on the road to Paramaribo in Suriname (5.1 mg/g) with samples from Burkina Faso (8.1 mg/g) and Hypoglycin-A Jamaican vomiting sickness Jamaica (9.2 mg/g). GC–MS Field research showed the misuse of this little-known plant by Maroon witch doctors. The Death Bushinengue witch doctors were informed about the dangers of ackee, and no new cases have been Forensic science reported to date. Toxicity ß 2011 Elsevier Ireland Ltd. All rights reserved. Plant origin Poisoning 1. Introduction confirmed the presence of ackee on the west bank of the Maroni River in Suriname. Its presence was also confirmed by the LAT 1.1. Background LUMTOX team in the Paramaribo region, where it is grown in plantations established by importing young plants from Jamaica. Between 1998 and 2001 the deaths of 16 Surinamese children were recorded along the River Maroni, which forms the border 1.2. Objectives between Suriname and French Guyana. Some of them were brought by their families to French Guyana and admitted to the pediatric The first aim was to compare the content of hypoglycin-A in department of the Franck Joly Hospital in Saint Laurent du Maroni. unripe fruit collected by the LAT LUMTOX team on the road to These children had similar symptomatic characteristics (hypogly- Paramaribo in Suriname with samples from Burkina Faso and cemia, increased hepatic enzymes) and antecedents (malnutrition, Jamaica which have been supplied by Dr Barennes for Burkina Faso hypovitaminosis) to several reported cases, notably in Burkina Faso, and Dr Jackson for Jamaica. The purpose of this fruit comparison Benin and Haiti. After a metabolic origin was eliminated, the was made to verify that the specie introduced in Suriname was pediatrician considered the possibility of poisoning, and contacted homogeneous on its toxins’ content with those present in Africa the Cayenne Institute of Research and Development (IRD). and Jamaica, the toxicity of which was already proved. Ethnobotanical research carried out by Mr Christian Moretti Five fruits were collected from five different trees at a stage of maturity of the order of 2–3 weeks before the fruit splits into three parts. The flesh of the five arils was removed, desiccated at +25 8C during 30 days then pooled and crushed in a ball mill until the * Corresponding author at: Laboratory of Analytical Toxicology, LAT-LUMTOX, 800 avenue Marie Curie, 07800 La Voulte sur Rhoˆne, France. Tel.: +33 475620524; obtaining of a fine light brown homogeneous powder. fax: +33 475855458. Secondly, the gastric juice of a child who had died in Albina, a E-mail addresses: [email protected], [email protected] (Y. Gaillard). Surinamese border town was analyzed. In order to rule out the 0379-0738/$ – see front matter ß 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.forsciint.2011.01.018 #WVJQT URGTUQPCNEQR[ e104 Y. Gaillard et al. / Forensic Science International[()TD$FIG] 206 (2011) e103–e107 hypothesis of poisoning by any toxin other than hypoglycin-A, a complete toxicological analysis has been conducted with a specific attention to toxic components that were the possible cause of Reye’s syndrome and/or major hypoglycemia. In a second step, a specific technique for identifying hypoglycin-A has been devel- oped. Due to the extremely poor local infrastructure (especially in Suriname) there was no police inquiry, nor was there any autopsy, and no ante-mortem or post-mortem blood or urine samples were taken. Gastric juice collected after a vomiting episode 12–14 h before death was the sole fluid available and subjected to analysis. 2. Ackee 2.1. Botanical and dietary information Fig. 2. Molecular structure of hypoglycin-A and hypoglycin-B. Ackee (Blighia sapida) is a large green leafy tree of West African origin. The fruit is about the size of a small pear and opens insulin secretion; it induces hypoglycemia by inhibiting secondary spontaneously into three parts when ripe. The edible part is the gluconeogenesis, limiting the cofactors (CoA and carnitine) yellow flesh around the three large shiny black seeds (Fig. 1). essential to the oxidation of long-chain fatty acids [4]. Ackee is the national fruit of Jamaica, where it is usually boiled The pathology induced by the consumption of the unripe fruit is in milk or water and served on its own or in meat or fish dishes known in Jamaica as ‘‘Jamaican vomiting sickness’’ or ‘‘ackee such as ‘ackee and salt fish’. In some African countries it is also poisoning’’. The symptoms of ackee intoxication are similar to eaten raw. It tastes a little like hazelnut or avocado [1]. those of Reye’s syndrome. It is an important dietary element, especially in poor popula- Clinical features of the illness include nausea, severe vomiting tions suffering from undernutrition, sometimes exacerbated by leading to loss of consciousness, disturbed mental states, and natural disasters and their consequences on local agriculture. paresthesia. Convulsions can occur, and a coma followed by rapid For example, many cases of ackee poisoning (over 50 of them death (within 12–24 h) in cases where large amounts have been fatal) were recorded in Haiti in November 2000 after flooding had ingested. affected 25,000 families and destroyed maize and sugar cane From a biochemical point of view, there is major hypoglycemia crops [2]. (<0.2 g/l), increased hepatic transaminases and hepatic histology reveals microvesicular steatosis of the liver. 2.2. Toxic compounds In contrast with ackee poisoning, Reye’s syndrome is charac- terized by the release of large amounts of fatty acid as a result of Unripe ackee contains large amounts of two toxic molecules: lipid metabolism anomaly. The causes of the syndrome remain hypoglycin-A (in the seeds and flesh) and hypoglycin-B (in the unknown, although there seems to be a link between the seeds alone), the former being the more toxic (Fig. 2). development of the syndrome and the use of aspirin to treat viral The concentration of hypoglycin-A in the flesh of the unripe fruit infections in children (e.g. chickenpox, flu, gastroenteritis, and is 10–100 times greater than that in the ripe fruit, depending on the upper respiratory tract infection) [5]. season. In ripe fruit, the concentration in the flesh reduces rapidly once exposed to sunlight, but the seeds remain poisonous [3]. 2.4. Cases reported in the literature 2.3. Clinical analysis of intoxication by hypoglycin-A Table 1 is a non-exhaustive list of different cases of ackee poisoning reported around the world. Ackee poisoning is due to hypoglycin-A, a natural hepatic toxin found in large amounts in unripe ackee fruit. Hypoglycin-A is a 3. Analysis of hypoglycin-A [()TD$FIG]powerful hypoglycemic. Its method of action does not stimulate 3.1. Reagents Acetonitrile, methanol, standard D-phenylalanine, N,O-bis- (trimethylsilyl)trifluoroacetamide containing 1% trimethyl chlor- osilane (BSTFA) and N-methyl-N-(trimethylsilyl)trifluoroaceta- mide (MSFTA) were supplied from Sigma–Aldrich (Saint Quentin Fallavier, France). Table 1 List of different cases of ackee poisoning reported around the world. Country of origin Number of deaths Year References Jamaica 271 1980–1991 [10] Ivory Coast >100 1984 [8,11] Togo 132 1988–1989 [1] Jamaica 6 1989–1991 [7] Haiti >10 1991 [2] Jamaica 28 1992 [12] USA 1 1994 [9] Benin 24, 40 1996, 1997 [8] Burkina Faso 29 1998 [7,8] Haiti >50 2008 [2] Fig. 1. Photograph of a ripe ackee fruit held in the hand. #WVJQT URGTUQPCNEQR[ [()TD$FIG] Y. Gaillard et al. / Forensic Science International 206 (2011) e103–e107 e105 Fig. 3. Total ion current chromatogram of an extract of unripe ackee flesh harvested in Suriname: (a) hypoglycin-A; (b) D-phenylalanine. 10 mg of hypoglycin-A synthesized [6] by Dr C.H. Hassall positive mode were acquired from 29 to 400 a.m.u. from 6 to (Roche Products Ltd., Welwyn Garden City, UK) with a purity 25 min. higher than 98% was kindly provided to professor Franc¸oise Be´valot (Faculty of Science, University of Besanc¸on) and generously sent on 3.3. Calibration of the fruit to our laboratory. The range of calibration was determined by spiking some ripe 3.2. Apparatus ackee flesh (which proved to be free of hypoglycin-A when tested using our method beforehand) with hypoglycin-A. The concentra- The gas chromatography used was a Trace GC (Thermo Electron, tion points in the range are 1, 2, 5, 10 and 20 mg/g. The D- Courtaboeuf, France) equipped with an AS3000 autoinjector and a phenylalanine was used as an internal standard. DSQ mass spectrometer. The analytical column was a factor four capillary column 5% phenyl 95% methyl siloxane, 30 m in length, 3.4. Extraction and injection with an internal diameter of 0.25 mm (0.50 mm film thickness) from Varian (Courtaboeuf, France). Helium (6.0 purity) was used as 3.4.1. Unripe fruit the carrier gas at a flow rate of 1 ml/min in constant flow mode. The Ackee flesh was removed from its husk, then desiccated at temperatures were: interface 280 8C and ion source 200 8C. Pulsed +25 8C for 30 days and finally processed for 5 min using a ball mill injection with surge pressure (1 ml) was carried out at 280 8C and (supplied by Retsch, Haan, Germany). 250 kPa for 1 min. The initial oven temperature was 50 8C for 2 min Exactly 100 mg of the resulting fine powder was extracted with and was increased to 300 8Cat158C/min and held for 6.33 min. 5 ml of methanol after the addition of 50 ml of a stock solution of D- The[()TD$FIG] chromatographic run time was 25 min. Repetitive scans in phenylalanine (10 mg/ml in acetonitrile:water, 1:1, v/v) then Fig. 4. Full scan MS spectrum (60–400 a.m.u.) of di-TMS-hypoglycin-A in electronic impact mode after derivation of hypoglycin-A with BSTFA. #WVJQT URGTUQPCNEQR[ e106[()TD$FIG] Y. Gaillard et al. / Forensic Science International 206 (2011) e103–e107 Fig. 5. Full scan MS spectrum (40–400 a.m.u.) of tri-TMS-hypoglycin-A in electronic impact mode after derivation of hypoglycin-A with MSTFA. sonicated for 2 h in an ultrasonic bath (Fisher Scientific, Illkirch, Weber, etc.) refers to hypoglycin-A in a derivative form that France). After centrifugation at 7500 g for 5 min, 100 ml of the would enable analysis in chromatography. superior organic phases was pipetted into a glass vial for GC, dried The data that we are publishing are thus the first data enabling by evaporation under a stream of air at +45 8C, reconstituted with analysts and toxicologists to identify this toxin using gas 20 ml of BSTFA and exposed to silylation at +80 8C for 15 min. chromatography combined with mass spectrometry. 1 ml of this sample was injected into the chromatographic Table 2 shows the retention times and lists the main ions for di- system in electronic impact mode. TMS D-phenylalanine, di-TMS hypoglycin-A and tri-TMS hypogly- Figs. 3 and 4 represent respectively the total ion current cin-A. chromatogram of an extract of unripe ackee flesh harvested in Suriname and the full scan MS spectrum (29–400 a.m.u.) of di- 4.2. In the fruit TMS-hypoglycin-A in electronic impact mode after derivation of hypoglycin-A with BSTFA (Fig. 5). The relative standard deviations (RSDs) of the between-day precision study for each point on the calibration scale established 3.4.2. Gastric juice for the fruit were 5.6, 4.1, 3.0, 2.5 and 2.1% for 1, 2, 5, 10 and 20 mg/ 1 ml of acetonitrile, containing 5 mg/ml of D-phenylalanine, g respectively (n = 10). Linearity was good from 1 to 20 mg/g with was added to 1 ml of gastric juice to enable protein precipitation. r = 0.989. Limit of detection (LoD) was measured after an analysis The sample was centrifuged at 7500 g for 5 min. 1 ml of the of the blanks following the mathematical formula LoD = mean- supernatant was dried by evaporation under a stream of air at blanks + 3SDblanks (n = 30) and was found to be 0.1 mg/g (SD = stan- +45 8C and then derived by 20 ml of BSTFA. dard deviation). Limit of quantification (LoQ) was calculated as 1 ml of this sample was injected into the chromatographic follows: LoD = meanblanks + 10SDblanks (n = 30) and was found to be system in electronic impact mode. 0.3 mg/g. Table 3 shows the concentrations of hypoglycin-A (in mg/g) in 3.4.3. Confirmation method unripe fruit harvested in three different countries. In order to be analytically exhaustive, we sought to obtain the MS spectra of tri-TMS hypoglycin-A in electronic impact mode and 4.3. In the gastric juice in chemical ionization mode after derivation with MSTFA. Analysis of the gastric juice of the deceased child unequivocally 4. Results shows the presence of hypoglycin-A. Systematic toxicological procedures include head-space gas 4.1. Main ions in the mass spectra chromatography–mass spectrometry (GC–MS) for the analysis of ethanol and other volatile substances, GC–MS after acetylation 1 Hypoglycin-A is not available commercially in any form. No with the use of CARIBOU software [13], high performance liquid commercial spectral database (NIST, Wiley, Pfleger-Maurer- chromatography photodiode array detection (HPLC-PDA) [14], Table 2 Retention times and main ions for di-TMS D-phenylalanine and di- and tri-TMS hypoglycin-A. Molecule RT (min) Detection mode Ions (m/z) (abundance %) Di-TMS-phenylalanine 10.39 EI 100(10), 147(21), 192(74), 218(100), 266(10) Di-TMS-hypoglycin-A 8.70 EI 100(28), 147(38), 168(100), 218(60), 242(7) Tri-TMS-hypoglycin-A 9.47 EI 96(34), 146(74), 214(100), 242(20), 254(14) Tri-TMS-hypoglycin-A 9.47 PCI 210(17), 302(56), 312(70), 354(95), 370(100) The underlined ions are used for quantification; RT, retention time; EI, electronic impact; PCI, positive chemical ionization. #WVJQT URGTUQPCNEQR[ Y. Gaillard et al. / Forensic Science International 206 (2011) e103–e107 e107 Table 3 by botanicals is often underestimated in forensic toxicology, the Concentrations of hypoglycin-A (in mg/g) in unripe fruit harvested in three different authors present a useful example of how to track down and sites. identify an unusual botanical toxin. Provenance of fruit analyzed Concentration of hypoglycin-A (mg/g) Suriname 5.1 Acknowledgments Jamaica 9.2 Burkina Faso 8.1 We are indebted to Christian Moretti, Pierre Grenand, Diane Vermont, Franc¸oise Be´valot, Jose Candace Jackson and Hubert Barennes for their precious help. UPLC–PDA/MS [15], inductively coupled plasma spectrometry (ICP-MS) as well as immunoassay techniques (opiates, cocaine, References amphetamine, and methamphetamine), and cyanide detection. These analyses showed no other toxic compound, and in [1] E. Guevart, A. Lawson-Body-Houkportie, G. Tchedre, Toxicite´ du fruit de l’ackee: particular no traces of oral antidiabetics, paracetamol, salicylates, un danger a` connaıˆtre, Afr. Me´d. 29 (1990) 116–118. valproate, quinine or disopyramide. [2] C. Hecdivert, M.S. Sainvil, A.M. Desormeaux, Investigation d’un phe´nome`ne morbide dans les communes de Plaine du Nord et de Milot, Unpublished docu- Gastric juices of people who live in the same area and not ment, Port-au-Prince, Haiti, 2000. suffering poison symptoms have not been analyzed. [3] G.W. Chase, W.O. Landen, A.G. Soliman, Hypoglycine A content in the aril, seeds, and husks of ackee fruit at various stage of ripeness, J. Assoc. Off. Anal. Chem. 73 5. Discussion (1990) 318–319. [4] J. Mills, G.N. Melville, C. Benett, M. West, A. Castro, Effects of hypoglycin A on insulin release, Biochem. Pharmacol. 36 (1987) 495–497. Formal identification of hypoglycin-A carried out enabled to [5] K. Tanaka, E.A. Kean, B. Johnson, Jamaica vomiting sickness. Biochemical investi- confirm the etiology of intoxication by ackee. As they are not gation of two cases, N. Eng. J. Med. 295 (1976) 461–467. [6] C.H. Hassall, K. Reyle, Hypoglycin A, B: biologically active polypeptides from specific, analyses of metabolites of the parent drug have not been Blighia Sapida, Nature 173 (1954) 356–357. targeted for analysis. [7] H.A. Meda, B. Diallo, J.P. Buchet, D. Lison, H. Barennes, A. Ouangre´, M. Sanou, S. Field research showed the misuse of this little-known plant by Cousens, F. Tall, P. Van de Perre, Epidemic of fatal encephalopathy in preschool children in Burkina Faso and consumption of unripe ackee (Blighia sapida) fruit, Maroon witch doctors who used it to ‘‘cure’’ some pathologies, Lancet 353 (1999) 536–540. especially acute forms of diarrhea in children. [8] P. Bouree, P. Frinot Joseph, R.E. Morell-Gil, P. Fernot Joseph, D. Nagiroy, A tree with Dr Rosenblatt (the director of the Albina’s hospital) and the LAT toxic fruits not very known in Africa and in the Island of Hispaniola, Me´d. Afr. Noire 49 (2002) 329–333. LUMTOX team therefore informed the Amerindian shaman, Josef [9] J.A. Mc Tague, R. Forney, Jamaican vomiting sickness in Toledo, Ohio, Ann. Emerg. Hubertus of the dangers of ackee. He in turn passed this Med. 23 (1994) 116–118. information on to the Bushinengue witch doctors who had initially [10] P. Figueroa, O. Nembhard, A. Coleman, M. Betton, Toxic hypoglycemic syndrome – Jamaica, 1989–1991, Morb. Mortal. Wkly Exp. 41 (1992) 53–55. refused to meet the ethnological team led by the Canadian Diane [11] S. Foungbe, Y. Naho, C. Declume, Experimental study of the toxicity of arils from Vermont. Blighia sapida (Sapindaceae) in relation to the poisoning of children of Katiola To date, no new cases have been reported by the pediatric (Ivory Coast), Ann. Pharm. Fr. 44 (1986) 509–515. department of the Franck Joly Hospital in Saint Laurent du Maroni. [12] Y. Gaillard, M. Cheze, G. Pe´pin, Intoxications humaines par les ve´ge´taux supe´ r- ieurs: revue de la litte´ rature, Ann. Biol. Clin. 59 (2001) 764–765. [13] F. Be´ valot, Y. Gaillard, C. Lemeur, Utilisation de macro-commandes pour l’opti- 6. Conclusion misation de l’exploitation des chrommatogrammes de de´pistages syste´matiques obtenus en GC/MS, in: 11th Congress of The Socie´te´ Franc¸aise de Toxicologie Analytique, Dinard, June 10–13, 2003. The work carried out here has enabled us to develop the first [14] Y. Gaillard, G. Pe´ pin, Use of high performance liquid chromatography with method for identifying and measuring the concentration of photodiode-array UV detection for the creation of a 600-compound library. hypoglycin-A in fruit by GC–MS, and the first method for Application to forensic toxicology, J. Chromatogr. A 763 (1997) 149–163. [15] C. Chatenay, F. Be´valot, Y. Gaillard, Use of UPLC–PDA/MS in systematic toxico- identifying hypoglycin-A by GC–MS in the gastric juice of people logical analysis, in: 47th Congress of The International Association of Forensic suffering from poisoning. 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Je´re´my Carlier1,2*, Ludovic Romeuf1,Je´roˆme Guitton3,4,Ce´dric Priez-Barallon1, Fabien Be´valot5, Laurent Fanton6 and Yvan Gaillard1 1Laboratoire LAT LUMTOX, 800 av. Marie Curie Z.I. Jean Jaure` s, La Voulte-sur-Rhoˆne 07800, France, 2Ecole Doctorale Interdisciplinaire Sciences-Sante´, Universite´Claude Bernard, Hoˆpital Louis Pradel, 28 av. du Doyen Le´pine, Bron 69677, France, 3Laboratoire de Toxicologie, Faculte´de Pharmacie de Lyon, 8 av. Rockefeller, Lyon 69373, France, 4Laboratoire de Pharmacologie, Centre Hospitalier Lyon Sud, chemin du Grand Revoyet, Pierre-Be´nite 69495, France, 5Laboratoire LAT LUMTOX, Hoˆpital d’instruction des Arme´es Desgenettes, 71 av. Rockefeller, Lyon 69003, France, and 6Faculte´de Me´decine, Institut Me´dico-le´gal, 12 av. Rockefeller, Lyon 69008, France *Author to whom correspondence should be addressed. Email: [email protected] Atractyloside (ATR) and carboxyatractyloside (CATR) are diterpene toxicity have been documented for over 200 years; its therapeu- glycosides that are responsible for the toxicity of several tic and magico-religious uses have been described throughout Asteraceae plants around the world. Mediterranean gum thistle the ages, and it is still a mainstay of traditional medicine in (Atractylis gummifera L.) and Zulu impila (Callilepis laureola DC.), North Africa, where it is used to treat syphilitic ulcers, whiten in particular, are notoriously poisonous and the cause of many acci- teeth and induce abortions (3, 5). The plant has also been used dental deaths, some suicides and even some murders. There is no in criminal poisonings, on its own or combined with arsenic or current method for measuring the two toxins in biological samples white henbane (Hyoscyamus albus L., containing atropine and that meet the criteria of specificity required in forensic medicine. scopolamine) whose antiemetic properties prevent the rejec- We have endeavored to fill this analytical gap. Analysis was carried tion of any of the poison (6, 7). Criminal poisonings are partic- out using a solid-phase extraction and a high-performance liquid ularly life threatening because of clinicians’ ignorance and the chromatography coupled with high-resolution tandem mass spec- rapid evolution of the symptoms (8). However, it seems that the trometry detection. The method was validated in the whole blood majority of cases of poisoning due to birdlime thistle are acci- with quantification limits of 0.17 and 0.15 mg/mL for ATR and dental, mainly due to its similarity to a wild artichoke, Spanish CATR, respectively. The method was applied to a non-fatal case of golden thistle (Scolymus hispanicus L.) or other edible intoxication with A. gummifera. To the best of the authors’ knowl- Asteraceae (3, 9). Since the nineteenth century, 100 fatal poi- edge, this is the first time that a concentration of ATR and CATR in sonings by A. gummifera have been recorded in the literature. blood (883.1 and 119.0 mg/L, respectively) and urine (230.4 and Birdlime thistle is currently the primary cause of vegetable poi- 140.3 mg/L, respectively) is reported. ATR and CATR were quantified soning in Tunisia (7). in A. gummifera roots by the standard method addition (3.7 and Atractylis gummifera is not the only source of ATR and CATR 5.4 mg/g, respectively). in the genus Atractylis or even in the Asteraceae family. Callilepis laureola DC., a South African herb (8, 9)well known to the Zulu people as impila, is the basis of a very com- mon traditional remedy that has many properties but is also the Introduction cause of many accidents (fatal in 85–90% of cases) (10, 11). The The diterpene glycoside atractyloside (ATR) and its derivative fruit of the cocklebur (Xanthium spp.), a widespread herb in 4-carboxyatractyloside (CATR), or gummiferin, are toxic com- North America (12), causes livestock poisoning in the USA, pounds present in many plants causing fatal poisonings around Argentina and Brazil; and fatal human poisonings have been re- the world (Figures 1 and 2). ported in the People’s Republic of China (4, 13). Other sources of ATR and similar compounds have been identified in European, African, South American, Asian and Australian plants (14) such as Botany and ethnopharmacology Wedelia glauca Ort. (South America) (15)orIphiona aucheri ATR and CATR were first isolated in 1868 (1) and 1964 (2), re- Boiss., used to poison racing camels in the Middle East (16). spectively, from roots of Atractylis gummifera L. from the This list is not exhaustive; there are other species around the Asteraceae family. Known as masticogna in Sicily, musciurida world that are rich in ATR and CATR. in Sardinia, chardon a` glu in France, birdlime or blue thistle in English and el-heddah in Arabic, A. gummifera L. is a thistle that grows wild in all the Mediterranean countries (3). The Biological effects and toxicity of ATR plant has leaves divided into prickly lobes and grouped into and CATR on humans rosettes, pink flowers grouped into inflorescences surrounded The toxicity of ATR and CATR is a direct consequence of their by bracts covered with spikes, which exude a yellowish- specific inhibiting action on oxidative phosphorylation in mito- white latex when the fruit is ripe and a long thick rhizome chondria and their action of opening the mitochondrial perme- where the toxic principles of the plant are concentrated (4) ability transition pores, major actors in apoptosis (11, 17). (Figure 3). Atractylis gummifera is easily available in the Additionally, they inhibit gluconeogenesis, ketogenesis and wild, in traditional markets and North African herbalists as the oxidation of fatty acids and promote anaerobic glycolysis dried fragments of chouˆk el-eulk roots.Itspropertiesand (14, 18). The action of ATR is exerted mainly in cells rich in # The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected] Figure 1. Suggested fragmentation of [M-H]2 of atractyloside. ATR, atractyloside; MM, molecular mass; *, same fragments as carboxyatractyloside (Fig. 2). mitochondria such as hepatocytes and in proximal tubular epi- with metabolic acidosis. Jaundice is a rare symptom inasmuch as, thelial cells, which contain carriers that allow ATR and CATR to in most cases, death follows rapidly, in the hours after ingestion cross the cell membrane (14, 19). after a hypoglycemic coma phase, or in the next few days due to Poisoning by A. gummifera, C. laureola or Xanthium pre- hepatic and/or severe renal insufficiency (5, 11, 13, 14). sents clinically in humans with epigastric pain, diarrhea and vom- For a long time, ATR poisoning could only be estimated by iting and respiratory depression, which causes hypoxemia, postmortem histopathological tissue analysis, when hepatic cen- leading to tissue hypoxia, spasms, stiffness and convulsions. trilobular necrosis and renal tubular necrosis could be observed, Hypoglycemia is preceded by a hyperglycemic phase combined especially in animal experiments (5, 6, 8, 10, 11, 13, 14, 19–21). 2 Carlier et al. Figure 2. Suggested fragmentation of [M-H]2 of carboxyatractyloside. CATR, carboxyatractyloside; MM, molecular mass; *, same fragments as atractyloside (Fig. 1). and Pe´pin (22) finalized the first method of quantifying ATR in whole blood by high-performance liquid chromatography– tandem mass spectrometry (HPLC-MS-MS). In 2001, Laurens et al. (28) developed a method of quantifying ATR in extracts of C. laureola tubers and human gastric lavages by gas chroma- tography coupled with mass spectrometry. In 2004, Steenkamp et al. (23, 24) developed an analytical method of quantifying ATR and CATR in C. laureola tubers and biological fluids by HPLC-MS. These methods are ponderous (28) or lack the nec- essary sensitivity and precision (22) or specificity (23, 24)tobe applied in forensic toxicology. None of these methods has under- gone a complete validation procedure to test its performance. Objective Figure 3. Atractylis gummifera L.: photograph of a flower (author: Fabio Ippolito). This article examines a method of screening and quantifying ATR and CATR in blood and urine by high-performance liquid chro- To date, there are no published data in the international scien- matography coupled with high-resolution tandem mass spec- tific literature on the metabolism and pharmacokinetics of ATR trometry (HPLC-HRMS/MS). The method, validated in whole and CATR, in humans or animals. This can be partly explained blood, is sensitive, specific and fast. A case of human poisoning by the fact that no reliable methods of quantifying ATR or by birdlime thistle is presented. It is the first published identifica- CATR in biological samples had been developed, which has tion of ATR and CATR in blood and urine. The levels of ATR and been pointed out by some authors (22–24). CATR were also measured in extracts of A. gummifera roots bought 7 years earlier. ATR and CATR analysis in plants and body fluids Some limited colorimetric and chromatographic methods of Case Report quantifying ATR or CATR in biological samples have been de- A young woman was hospitalized after accidentally ingesting scribed over the last 30 years (16, 25–27). In 1999, Gaillard macerated birdlime thistle 3 days earlier. She had confused the A Method for Quantifying ATR and CATR in Biofluids 3 plant with an edible species. She was suffering from abdominal 10 min, and the elution was performed by three volumes of pain, vomiting, fatigue and weight loss. Biochemical analysis 0.5 mL of methanol containing 1% of ammonium hydroxide. showed hyperglycemia and high concentrations of ALAT and The eluate was subsequently evaporated to dryness under a ASAT, indicating hepatic cytolysis. The clinician diagnosed bird- stream of air at þ508C, and the residue was reconstituted by lime thistle poisoning after questioning the patient and in light of 50 mL of ACN : acetate buffer (10 : 90, v/v) and transferred into the clinical signs. a vial for injection into the chromatographic system. Blood and urine samples were taken in dry tubes the day the patient was hospitalized (3 days after ingesting the plant) and Validation of the method in whole blood then preserved at 2208C. The method was validated in whole blood according to the inter- national standards (29, 30). The linearity of the method was as- sessed on four calibration ranges (eight calibration points: 0.5, m / Materials and methods 1, 5, 10, 20, 50, 100 and 200 g L) on four different days. The equation of the calibration curve was calculated by the least Chemical and reagents squares method, and the validity of the regression model was Working standard of dipotassium salt of ATR [ 98% by thin-layer confirmed by an ANOVA test. The slope was compared with chromatography (TLC)] and CATR ( 95% by TLC) were bought zero by Student’ t-test [quantification ranges were also carried from Merck (Darmstadt, Germany) and solubilized in a mixture out in urine and bile (blank matrices spiked with the standards), / of acetonitrile : deionized water (50 : 50, v v). Oxazepam-d5 but the methods were not fully validated]. / (100 mg L) was bought from LGC Standards (Molsheim, The limit of detection (LOD) and the lower limit of quantifica- France) and used as internal standard (IS) in the absence of a tion (LLOQ) were calculated by performing the analysis of 10 chemical structure commercially available and comparable to different sources of blank matrices (postmortem blood) and the analytes. The compounds were stored and protected from according to the following formulas: LOD ¼ mbl þ 3SDbl and light at 2208C. LLOQ ¼ mbl þ 10SDbl (mbl: mean of the measured concentration Acetonitrile (ACN) for LC–MS was obtained from Carlo Erba of the blanks; SDbl: standard deviation of the measured concen- (Val de Reuil, France), methanol for HPLC was obtained from ¼ w tration of the blanks; n 10). The upper limit of quantification Sigma-Aldrich (Saint-Quentin Fallavier, France), deionized (ULOQ) was chosen as the concentration of the upper calibra- water and isopropyl alcohol were obtained from Fisher tion standard. Chemical (Optima LC–MS; Illkirch, France) and ammonium hy- The extraction recovery was determined by comparing the droxide (30%) was from Merck. mean peak areas from the samples spiked with compounds be- Ammonium acetate buffer (5 mM) was prepared with ammoni- w fore the complete SPE extraction with those obtained from the um acetate (Sigma-Aldrich ) diluted in water. The pH was subse- m / ¼ w samples spiked after the extraction (20 g L, n 10). quently adjusted to 4.5 with acetic acid (Sigma-Aldrich ). Matrix effect was determined by comparing the mean peak Saline phosphate buffer [hydrochloric acid (HCl)– phosphate- areas from whole blood samples spiked after the extraction buffered saline (PBS)] was prepared with 0.2 g of potassium chlo- and direct injection of the same amount dissolved in the mobile ride (KCl) ( 99.5%; Carlo Erba), 8 g of sodium chloride (NaCl) phase (20 mg/L, n ¼ 10). ( 99.5%; Merck), 0.2 g of monopotassium phosphate (KH2PO4) The within- and between-day precision and accuracy of the ( 99.5%; Merck) and 1.15 g of monosodium phosphate method were calculated on 10 calibration points at 5, 50 and w (NaH2PO4)( 99%; Sigma-Aldrich ) diluted in 1 L of deionized 200 mg/L (blank matrices spiked) on three different days (n ¼ water and the pH adjusted to 4.5 with HCl (37%; Carlo Erba). 90). The within-day precision and accuracy of the method were calculated on 10 calibration points at the LLOQ (n ¼ 10) and at 2,000 mg/L with a dilution factor of 1/20 in another Extraction procedure from body fluids source of postmortem blood (n ¼ 10). Outliers were detected by a Dixon test and removed from the validation data. Extraction The extraction of ATR and CATR was a solid-phase extraction w (SPE) performed on Oasis HLB cartridge (3 mL, 60 mg) from Extraction procedure from A. gummifera L. roots Waters (Guyancourt, France). It is a revised version of the extrac- Dried roots of A. gummifera L. were bought from a herbalist in tion reported by Steenkamp et al. (23, 24). Rabat (Morocco) and processed using a ball mill until a fine light The conditioning step was carried out with 3 mL of methanol, brown powder was obtained. Interestingly, CATR proved unsta- 3 mL of deionized water and then 3 mL of extraction buffer ble in protic solvents such as methanol and was degraded within (HCl–PBS at pH 4.5). The sample (1 mL of blood or urine) was a few days (without transformation into ATR) (esterification), mixed with 10 mL of IS (oxazepam-d5, 1 mg/L) and 2 mL of ace- hence the choice of an acetonitrile extraction, the powder tonitrile, vortex mixed for 30 s and centrifuged at 3,000 g for (50 mg) was dissolved in 5 mL of a solution of ACN : water (50 : 10 min. The supernatant was then evaporated to an approximate 50, v/v), vortex mixed, sonicated for 1 h and centrifuged for volume of 500 mL under a stream of air at þ458C (evaporation of 10 min at 3,000 g. The supernatant was diluted at 1/100. The ex- the acetonitrile) and diluted in 2 mL of HCl–PBS, vortex mixed traction was carried out 7 years after the purchase of the powder. for 30 s and centrifuged at 3,000 g for 10 min. The supernatant The compounds were quantified by the standard method addi- was then applied to the cartridge and allowed to drain under vac- tion: 10, 20, 30, 40, 50 or 60 mL of ATR at 1 mg/L, 50, 100, 150, uum at 1 mL/min. SPE rinse was carried out with 1 mL of extrac- 200, 250 or 300 mLofCATRat1mg/L and 10 mLofISat1mg/L tion buffer. The cartridge was allowed to dry under vacuum for were added to 100 mL of the sample and evaporated to dryness 4 Carlier et al. under a stream of air at þ508C. The residue was reconstituted by (reference standard) in A : B (50 : 50, v/v) with a syringe pump 50 mL of ACN : acetate buffer (10 : 90, v/v) and transferred into a (10 mL/min). vial for injection into the chromatographic system. The equation of the calibration curve was calculated by the method of least squares, and the validity of the regression model was confirmed Results and discussion by an ANOVA test. Mass spectrometry The spectrometric analysis conditions were optimized by post- column infusion of the standards. ATR and CATR are sulfate mol- HPLC-HRMS analysis ecules and produce a better signal in negative ionization mode. The ionization conditions correspond to the values published HPLC conditions w previously (22–24). Separation was performed by HPLC (Ultimate 3000; Thermo 2 2 2 þ 2 w Quantification was performed on the [M H] and [M H 1] Scientific, Courtaboeuf, France) using an XTerra phenyl column (isotopic contribution of 13C) ions of the two molecules in order (length: 150 mm; internal diameter: 2.1 mm; particle size: to maximize the signal-to-noise ratio: the noise signal for the 13C 3.5 mm) (Waters) at þ508C, with a gradient mobile phase com- isotopomer of the two molecules was zero, and adding the inten- posed of acetonitrile (10% isopropyl alcohol) (Solvent A) and a sity of the ions enabled us to artificially increase the signal of the 5 mM ammonium acetate buffer at pH 4.5 (Solvent B) (23, 24). ATR and CATR without increasing the noise. HRMS/MS was per- The chromatographic run time was 12.5 min. The separation formed at high collision energy in order to achieve the loss of a 2 started with a mobile phase consisting of 5 : 95 (v/v) A : B for sulfate group from the [M2H] ion of ATR (725 . 645 amu 1 min; followed by a linear gradient to 98 : 2 (v/v) A : B within 2 2 [M2H2SO3] ) and CATR (769 . 689 amu [M2H2SO3] ) and 2.5 min; the conditions were isocratic for 4 min; then the mobile start the fragmentation of these daughter ions to produce a rich phase returned to the initial conditions within 0.5 min and fragmentation spectrum (Figures 1, 2 and 4). was equilibrated for 4.5 min. The mobile phase flow rate was In an earlier study, we presented a method of measuring ATR 250 mL/min for the whole time of the separation. The temperature and CATR detected by cubed mass spectrometry (MS3). MS3 was of the sampler was þ108C, and the injection volume was 10 mL. chosen because the spectra obtained in MS/MS were not informa- tive enough to enable the identification of the compounds (31). Mass spectrometric conditions On the other hand, the fragmentation lacked repeatability, which Mass spectrometric detection was performed using a quadrupole- invalidated the method for quantification. By turning to HRMS, it orbitrap high-resolution detector (Q Exactive; Thermo Scientific) is possible to overcome the repeatability issue due to the fragmen- after ionization by heated electrospray in negative ion mode. tation, while at the same time maintaining sufficient specificity. Nitrogen was both the collision and nebulizing gas (4.5; Linde gas, Molsheim, France). The mass spectrometer operated in selected ion monitoring mode and targeted MS/MS mode alter- Liquid chromatography nately (Table I). The full-scan product ion spectrum of the com- Given the singular structure of ATR and CATR, it is difficult to pounds was used to confirm the identity of the toxins (Figure 4). achieve and maintain the conditions required for their chromato- The mass spectrometric conditions and the collision energies graphic separation and perfect ionization for mass spectrometry. were optimized by postcolumn infusion of the compounds Those compounds consist of two parts bound via a b-glycosidic linkage: a polar glycoside composed of a glucose and two sulfate groups and the hydrophobic aglycone composed of a nonvolatile Table I diterpene with a perhydrophenanthrenic structure (16)(Figures1 High-Resolution Mass Spectrometry Parameters and 2). Initially, owing to the polar nature of the compounds, we concentrated on hydrophilic interaction chromatography (bare Sheath gas flow rate (psi) 50 silica). Auxiliary gas flow rate (a.u.) 10 Sweep gas flow rate (a.u.) 0 Despite the modifications made to the mobile phase condi- Spray voltage (kV) 24.0 tions, ATR and CATR each formed two peaks, part of which 8 þ Capillary temperature ( C) 250 was eluted with the solvent front. We therefore turned to a Heater temperature (8C) þ250 SIM mode more classic reversed-phase separation. Several columns were Ionization mode Negative w tested: ACQUITY UPLC HSS C18 (length: 150 mm; internal Scan time (min) 3.5–6 m Resolution 140,000 diameter: 2.1 mm and particle size: 1.8 m) (Waters), Synergi Mass tolerance (ppm) 5.0 4u Fusion-RP 80A (length: 150 mm; internal diameter: 2.0 mm w Quantifier ions (scan range) (amu) and particle size: 4 mm) (Phenomenex ; Le Pecq, France) and Oxazepam-d5 290.0750 (288–292) w ATR 725.2154 þ 726.2188 (723–727) XTerra phenyl (length: 150 mm; internal diameter: 2.1 mm CATR 769.2053 þ 770.2086 (767–771) and particle size: 3.5 mm). The latter was chosen as it was the / Targeted MS MS mode only one that enabled separation of the dead volume compounds, Ionization mode Negative Scan time (min) 3.5–6 associated with a satisfactory peak without splitting (anion–p Collision energy (%) 45 and polar–p interactions) in accordance with the observations Resolution 17,500 of Steenkamp et al. (23, 24). ATR, atractyloside; CATR, carboxyatractyloside; SIM, selected ion monitoring; MS/MS, tandem The chromatographic analysis is short (12.5 min) compared mass spectrometry. with methods previously published [25 (22), 72 (28)and A Method for Quantifying ATR and CATR in Biofluids 5 Figure 4. Full-scan product ion spectra of (1) ATR and (2) CATR (mass resolution: 17,500). 35 min (23, 24)]. ATR was eluted at 4.51 min, while CATR was elut- that of Gaillard and Pe´pin (26.2%), which was the only yield ed at 4.66 min. The retention time of oxazepam-d5 (IS) was value published to date (22).Thematrixeffects,ontheother 5.06 min (Figure 5). By using isopropanol in the mobile phase, it hand, are significant (248.1% for ATR and 217.9% for CATR). was possible to avoid the phenomenon of peak splitting and obtain This is a compromise between satisfactory extraction yields better resolution, without affecting the ionization of the com- and chromatographic conditions that enabled rapid analysis pounds, as could be the case with methanol (23, 24). The mobile and satisfactory chromatographic peaks. It is worth noting that phase conditions were optimized to favor the ATR signal over the the matrix effect does not compromise the sensitivity, accuracy CATR signal, which presents less intense background noise. or precision of the method. Extraction recovery and matrix effect Precision, accuracy, LOD and LLOQ The validation parameters of the method in whole blood are The method meets the usual criteria for precision ( 15 or 20% showninTablesII and III. The extraction method was based at the LLOQ) and accuracy (between 85 and 115% or 80 and on the method proposed by Steenkamp et al. (23, 24), adding 120% at the LLOQ) required in forensic toxicology for a working a protein precipitation stage before the SPE and reducing the range from 0.17 and 0.15 to 250 mg/L for ATR and CATR, respec- elution to one stage. tively (LOD ¼ 0.066 and 0.055 mg/L, respectively; Tables II and The extraction yields of ATR and CATR are 71.1 and 48.3%, re- III). This is the first validated method of measuring the two com- spectively. The extraction yield of ATR (71.1%) is greater than pounds to be published. It is also the most sensitive, with an 6 Carlier et al. Figure 5. Chromatogram from the analysis of a sample of blood spiked with 5 mg/L or free of the compounds of interest. ATR, atractyloside; CATR, carboxyatractyloside; IS, internal standard. Table II Validation Data for Quantification of ATR and CATR in Whole Blood: Calibration Ranges Spiked (mg/L) ATR CATR Concentration + SD (mg/L) Accuracy (%) Between-day precision (%) Concentration + SD (mg/L) Accuracy (%) Between-day precision (%) 0.5 0.47 + 0.07 94.4 14.5 0.53 + 0.00 106.9 0.9 1 1.00 + 0.10 99.6 10.2 0.93 + 0.04 92.8 4.0 2 1.86 + 0.20 93.2 10.5 1.68 + 0.09 83.9 5.6 5 4.93 + 0.29 98.7 5.9 5.19 + 0.30 103.9 5.8 10 9.87 + 0.36 98.7 3.6 10.5 + 0.6 105.1 5.5 50 50.5 + 1.2 101.1 2.4 54.1 + 1.5 108.2 2.8 100 99.5 + 1.2 99.5 1.2 104 + 5 104.6 4.5 200 200 + 0.1 100.1 0.1 186 + 3 93.0 1.8 ATR, atractyloside; CATR, carboxyatractyloside; SD, standard deviation. Table III Validation Data for Quantification of ATR and CATR in Whole Blood: QC Spiked (mg/L) ATR (LLOQ ¼ 0.17 mg/L) CATR (LLOQ ¼ 0.15 mg/L) Concentration + SD (mg/L) Accuracy (%) Precision (%) Concentration + SD (mg/L) Accuracy (%) Precision (%) Within day Between day Within day Between day LLOQ 0.176 + 0.032 103.3 18.1 NC 0.141 + 0.011 94.2 7.9 NC 5 4.93 + 0.26 98.6 5.2 1.3 5.24 + 0.36 104.9 6.4 1.6 50 47.9 + 3.4 95.9 5.1 5.8 53.7 + 5.5 107.4 9.8 5.4 200 196 + 9 97.9 3.5 3.7 184 + 12 92.0 8.6 2.2 1,000 (diluted 1/20) 1,010 + 88 101.0 8.7 NC 1,060 + 113 106.0 10.7 NC ATR, atractyloside; CATR, carboxyatractyloside; QC, quality control; SD, standard deviation; LLOQ, lower limit of quantification; NC, not calculated. LLOQ five times lower than that of Steenkamp et al. (1 mg/L), macerated birdlime thistle orally, she presented blood concentra- which was the only LLOQ value published to date (24). The sig- tions of ATR and CATR of 883.1 and 119.0 mg/L, respectively, and nal of the two molecules saturates the detector in weak concen- urine concentrations of 230.4 and 140.3 mg/L (Figure 6). To the tration, leading to a low chosen ULOQ value (250 mg/L). This best of the authors’ knowledge, these are the first blood and weakness is compensated by the possibility of diluting the sam- urine concentrations of ATR and CATR reported in the literature. ple, and the repeatability of this is also validated in this study. Atractylis gummifera roots Case reports The ATR and CATR content in A. gummifera roots is reported in The method enabled the determination of concentrations of ATR Table IV. The concentrations measured are consistent with the and CATR in biological samples of a patient who had consumed values published in the scientific literature, which refer to ATR extracts of A. gummifera roots. Three days after consuming content of between 1.2 and 15.7 mg/gofrhizomedrymass, A Method for Quantifying ATR and CATR in Biofluids 7 Figure 6. Chromatogram from the analysis of blood (top) and urine (bottom) samples from a non-fatal case of intoxication by A. gummifera L. important because the signs of poisoning are nonspecific and Table IV ATR and CATR Content in A. gummifera Roots the clinician’s diagnosis can only be based on anamnesis. The method enabled the quantification of ATR and CATR in n ¼ 5 Concentration + SD (mg/g) Precision (%) the blood and urine of a woman who had consumed extracts ATR 3.9 + 0.2 5.8 of A. gummifera, the cause of many deaths on North Africa. CATR 6.2 + 0.4 6.1 These data provide the first blood concentrations reported of ATR, atractyloside; CATR, carboxyatractyloside; SD, standard deviation. the two toxins in a case of poisoning. according to geographical origin and seasonal variations (3, 11, Conflict of Interest statement 14). It is interesting to note that the concentrations of ATR and None declared. CATR are still high, despite the 7-year gap between drying the roots and analyzing them. CATR is known to be the majority com- pound in fresh birdlime thistle root extracts, but the decarboxy- lation of CATR and ATR during the drying and aging of the plant References does not seem to have reversed this tendency over the years 1. Lefranc, M. (1868) Sur l’acide atractylique et les atractylates, produits (3, 11). This observation is of interest inasmuch as CATR is imme´diats de la racine de l’Atractylis gummifera. Comptes Rendus Hebdomadaires des Se´ances de l’Acade´mie des Sciences, 67, more poisonous than ATR (3). 954–956. 2. Stanislas, E., Vignais, P. (1964) Sur les principes toxiques d’Atractylis gummifera L. Comptes Rendus Hebdomadaires des Se´ances de l’Acade´mie des Sciences, 259, 4872–4875. Conclusion 3. Daniele, C., Dahamna, S., Firuzi, O., Sekfali, N., Saso, L., Mazzanti, G. The article describes the development of a sensitive, rapid and (2005) Atractylis gummifera L. poisoning: an ethnopharmacological simple method of quantifying ATR and CATR in blood and review. Journal of Ethnopharmacology, 97, 175–181. 4. Bruneton, J. Plantes Toxiques: Ve´ge´taux Dangereux Pour l’Homme urine samples by HPLC-HRMS/MS. The validated method meets et les Animaux. 3rd edition. Tec&Doc: Paris, France, 2006. the criteria of specificity required for application in forensic 5.Georgiou,M.,Sianidou,L.,Hatzis,T.,Papadatos,J.,Koutselinis,A. medicine. It makes it possible to provide evidence of ATR and (1988) Hepatotoxicity due to Atractylis gummifera-L. Clinical CATR ingestion that can lead to poisoning. It is particularly Toxicology, 26, 487–493. 8 Carlier et al. 6. Skalli, S., Alaoui, I., Pineau, A., Zaid, A., Soulaymani, R. (2002) 21. Koechel, D.A., Krejci, M.E. (1993) Extrarenal and direct renal actions L’intoxication par le chardon a` glu (Atractylis gummifera L.); a` of atractyloside contribute to its acute nephrotoxicity in pentobarbital- propos d’un cas clinique. Bulletin de la Socie´te´ de Pathologie anesthetized dogs. Toxicology, 79, 45–66. Exotique, 95, 284–286. 22. Gaillard, Y., Pe´pin, G. (1999) Poisoning by plant material: review of 7.Hamouda,C.,Amamou,M.,Thabet,H.,Yacoub,M.,Hedhili,A., human cases and analytical determination of main toxins by high- Bescharnia, F. et al. (2000) Plant poisonings from herbal medication performance liquid chromatography–(tandem) mass spectrometry. admitted to a Tunisian toxicologic intensive care unit, 1983–1998. Journal of Chromatography B: Biomedical Sciences and Veterinary and Human Toxicology, 42, 137–141. Applications, 733, 181–229. 8. Candy, H.A., Pegel, K.H., Brookes, B., Rodwell, M. (1977) The occur- 23. Steenkamp, P.A., Harding, N.M., van Heerden, F.R., van Wyk, B.E. rence of atractyloside in Callilepis laureola. Phytochemistry, 16, (2004) Determination of atractyloside in Callilepis laureola using 1308–1309. solid-phase extraction and liquid chromatography-atmospheric pres- 9. Brookes, K., Candy, H.A., Pegel, K.H. (1983) Atractylosides in sure ionisation mass spectrometry. Journal of Chromatography A, Callilepis laureola (Asteraceae). South African Journal of 1058, 153–162. Chemistry, 36, 65–68. 24. Steenkamp, P.A., Harding, N.M., van Heerden, F.R., van Wyk, B.E. 10. Wainwright, J., Schonland, M.M., Candy, H.A. (1977) Toxicity of (2006) Identification of atractyloside by LC–ESI–MS in alleged herb- Callilepis laureola. South African Medical Journal, 52, 313–315. al poisonings. Forensic Sciences International, 163, 81–92. 11. Stewart, M.J., Steenkamp, V. (2000) The biochemistry and toxicity of 25. Boulay, F., Brandolin, G., Lauquin, G.J., Vignais, P.V. (1983) Synthesis atractyloside: a review. Therapeutic Drug Monitoring, 22, 641–649. and properties of fluorescent derivatives of atractyloside as potential 12. Kupiecki, F.P., Ogzewalla, C.D., Schell, F.M. (1974) Isolation and char- probes of the mitochondrial ADP/ATP carrier protein. Analytical acterization of a hypoglycemic agent from Xanthium strumarium. Biochemistry, 128, 323–330. Journal of Pharmaceutical Sciences, 63, 1166–1167. 26. Bye, S.N., Coetzer, T.H., Dutton, M.F. (1990) An enzyme immunoassay 13. Zhang, Y.G., Huang, G.Z. (1988) Poisoning by toxic plants in China. for atractyloside, the nephrotoxin of Callilepis laureola (Impila). Report of 19 autopsy cases. American Journal of Forensic Toxicon, 28, 997–1000. Medicine and Pathology, 9, 313–319. 27. Steenkamp, V., Stewart, M.J., Zuckerman, M. (1999) Detection of 14. Obatomi, D.K., Bach, P.H. (1998) Biochemistry and toxicology of the poisoning by Impila (Callilepis laureola)inamotherandchild. diterpenoid glycoside atractyloside. Food and Chemical Toxicology, Human & Experimental Toxicology, 18, 594–597. 36, 335–346. 28. Laurens, J.B., Bekker, L.C., Steenkamp, V., Stewart, M.J. (2001) Gas 15. Schteingart, C.D., Pomilio, A.B. (1984) Atractyloside, toxic compound chromatographic–mass spectrometric confirmation of atractyloside from Wedelia glauca. Journal of Natural Products, 47, 1046–1047. in a patient poisoned with Callilepis laureola. Journal of 16. Roeder, E., Bourauel, T., Meier, U., Wiedenfeld, H. (1994) Diterpene Chromatography B: Biomedical Sciences and Applications, 765, glycosides from Iphiona aucheri. Phytochemistry, 37, 353–355. 127–133. 17. Sherer, B. (1973) The new atractyloside type compound as a high 29. Shah, V.P., Midha, K.K., Dighe, S., McGilveray, I.J., Skelly, J.P., Yacobi, A. affinity ligand to the adenine nucleotide carrier. FEBS Letters, et al. (1991) Analytical methods validation: bioavailability, bioequiva- 31, 15–19. lence and pharmacokinetic studies. Conference report. European 18. Andreyev, A.Y., Bondareva, T.O., Dedukhova, V.I., Mokhova, E.N., Journal of Drug Metabolism and Pharmacokinetics, 16, 249–255. Skulachev, V.P., Tsofina, L.M. et al. (1989) The ATP/ADP-antiporter 30. Polettini, A. Applications of LC-MS in Toxicology.1stedition. is involved in the uncoupling effect of fatty acids on mitochondria. Pharmaceutical Press: London, UK, 2006. European Journal of Biochemistry, 182, 585–592. 31. Romeuf, L., Mazoyer, C., Ahid, C., Cherrah, Y., Ollagnier, M., Basset, T. 19. Stuart, B.P., Cole, R.J., Gosser, H.S. (1981) Cocklebur (Xanthium stru- et al. (2008) Identification and quantification of atractyloside (ATR) marium, L. var. strumarium) intoxication in swine: review and re- and carboxyatractyloside (CATR) by HPLC-MS2 and MS3 in the body definition of the toxic principle. Veterinary Pathology, 18, 368–383. fluids of two rabbits poisoned by oral feeding and two actual cases of 20. Obatomi, D.K., Brant, S., Anthonypillai, V., Bach, P.H. (1998) Toxicity human poisoning by Atractylis gummifera. In the 46th annual meet- of atractyloside in precision-cut rat and porcine renal and hepatic ing of The International Association of Forensic Toxicologists, tissue slices. Toxicology and Applied Pharmacology, 148, 35–45. Martinique, France. A Method for Quantifying ATR and CATR in Biofluids 9 ±±±± ǯ ǯ Ǧ Ȁ°±ǯǤ± ±± ± ° ± Ǧ±Ǥ ǯ° ± ± Ǧ ±=?Ǥ ± ± ͺͺ͵ǡͳ ͳͳͻǡͲρȀ ǡʹ͵ͲǡͶͳͶͲǡ͵ǯǤ ǯ ° ± ± Ǥ° ǯ ±±±Ǥ ±°±±±±°ǯǯ ±ȋǯ ±ÃȌǤ ±± Ǥ ǯ± ͵ǡͶ ͺǡͻ ρȀ ǡ ǯ ǯ±±Ǥ ±±° ° ±± ±Ǥ ° ±° ±±±±°ǯ ± Ǥǯ ǯ²± ±°±± ǯ Ǥ±² ± ± ǯ ± Ǥ ± ǯ± ± ± ±±Ǥ ǯ± ǯ ± ± ±ǯ± ±Ǥ ͳͲ ǦȀ ǦǦ ±Ã ± ȋǤȌ ± ǯ ± ±ǡÁ ǡǯ Ǥ ±Ã ± ± =?Ǥ ǯ ±ǡǡ ǯ=?Ǥ ǯ ǯ ± ° =?Ǥ ±±° ǯ ȋǯ±Ȍǡî ǯ =?Ǥ ǯ ǡ °ǯ ǡ ± °ǯ ± ± =?Ǥ± î ± Ǥ ǯ ±± ± ͳͻͺͻ ͳͻͻͻ =?Ǥ ǡ ȋǤȌǡǦ²ǤǤ=?Ǥ ǯ ǯ± ǡ ǯ ±ǡ ± Ǥ ǡ± ± ±±±=?Ǥ ± ±±ǡ ±± Ǥ± ± Ǥǯ ± ±ǡ±± Ǥ ͳͳ ǯ ±±ǯ ±Ã ǤǤ ±ǯǯ Ǽ ǽǤ ͳʹ #WVJQT URGTUQPCNEQR[ Journal of Chromatography B, 962 (2014) 1–8 Contents lists available at ScienceDirect Journal of Chromatography B jou rnal homepage: www.elsevier.com/locate/chromb The principal toxic glycosidic steroids in Cerbera manghas L. seeds: Identification of cerberin, neriifolin, tanghinin and deacetyltanghinin by UHPLC–HRMS/MS, quantification by UHPLC–PDA-MS a,∗ b c d a Jérémy Carlier , Jérôme Guitton , Fabien Bévalot , Laurent Fanton , Yvan Gaillard a Laboratoire LAT LUMTOX, 800 av. Marie Curie Z.I. Jean Jaurès, 07800 La Voulte-sur-Rhône, France b Laboratoire de toxicologie, Faculté de pharmacie de Lyon, 8 av. Rockefeller, 69373 Lyon, France c Laboratoire LAT LUMTOX, Hôpital Desgenettes, 71 av. Rockefeller, 69003 Lyon, France d Institut médico-légal, Faculté de médecine, 12 av. Rockefeller, 69008 Lyon, France a r t i c l e i n f o a b s t r a c t Article history: The toxicity of the sea mango (Cerbera manghas L.) is well known. The plant is ranked as one of the deadliest Received 22 October 2013 of the southern Asian coastline. Cardenolidic heterosides are responsible for the cardiotoxicity of trees Accepted 11 May 2014 of the Cerbera genus. We have identified and determined the concentration of the principal glycosidic Available online 17 May 2014 steroids present in the seeds of sea mangos (Thailand). Drug screening of an extract of the seeds was carried out using ultra-high performance liquid chromatography coupled to photodiode array detection Keywords: and mass spectrometry (UHPLC–PDA-MS) with quantification at 219 nm. Identification was confirmed by Cerbera manghas L. UHPLC–HRMS. Deacetyltanghinin (m/z 549.3055 ± 2 ppm), neriifolin (m/z 535.3259 ± 2 ppm), tanghinin Cardenolides (m/z 591.3169 ± 2 ppm) and cerberin (577.3375 ± 2 ppm) were the most abundant glycosidic steroids Forensic toxicology present in the sea mango seeds. A seed of the dried ripe fruit had concentrations of 1209.1, 804.2, 621.4 Ultra-high performance liquid chromatography–photodiode array-mass and 285.9 g/g, respectively. A seed of the fresh unripe fruit had concentrations of 49.4, 47.0, 3.5 and spectrometry (UHPLC–PDA-MS) 2.3 g/g. Ultra-high performance liquid © 2014 Elsevier B.V. All rights reserved. chromatography–high resolution tandem mass spectrometry (UHPLC–HRMS/MS) 1. Introduction for 50% of plant poisonings, or 10% of total poisonings (537 deaths recorded between 1989 and 1999) [5]. Suicide is the principal cause Trees and shrubs of the Cerbera genus grow beside muddy of these poisonings in a part of the world where suicidal poison- waterways and in mangrove swamps in South Asian coastal areas, ing is a major problem [6]. However, it has also been established tropical Pacific islands and as far afield as Australia and Madagas- that there are at least as many deaths by homicide. For various car. Their drupes, which look similar to the mango (Mangifera indica social and political reasons, these murders go largely unpunished L.), comprise a fibrous envelope containing one or two extremely in India. Some Western cities are home to large populations with an poisonous kernels. The seeds contain cardiotoxic heterosides, ancestral knowledge of the poisonous properties of Cerbera, but the including cerberin, cerberoside and neriifolin (deacetylcerberin) active toxic principles of Cerbera are not generally covered by rou- [1–3]. tine chromatographic testing techniques in analytical toxicology Cerbera seeds were used as an ordeal poison on the high plateaus laboratories: so a case of acute poisoning brought about by Cerbera of Madagascar until the 19th century. A person accused of theft or seeds could very easily go unnoticed at the analytical level, which is witchcraft would be forced to swallow a tanghin seed and could why the New Scientist labelled Cerbera the ‘toxin of the perfect mur- prove their innocence by surviving. In its time, the tanghin seed der’ [7]. In eastern Sri Lanka Cerbera accounts for no more than 20% was responsible for tens of thousands of fatal poisonings [4]. Nowa- of plant poisonings, behind yellow oleander (Thevetia peruviana days, poisonings appear to be localized, despite the widespread K. Schum.) (7 deaths 2000–2001) [8]. Accidental poisonings occur distribution of the plant in the world. In the Indian states of Ker- through ignorance of the plant, or indirectly through consumption ala and Tamil Nadu, deaths caused by Cerbera poisoning account of the coconut crab (Birgus latro L.) [9]. People who have been poisoned present the clinical picture of acute digitalis poisoning common to cardiotoxic cardenolides. The + + ∗ symptoms, caused by inhibition of the Na /K ATPase pump, are Corresponding author. Tel.: +33 475 62 05 24; fax: +33 475 85 54 58. E-mail address: [email protected] (J. Carlier). hyperkalaemia, diarrhoea and vomiting, hypotension, bradycardia http://dx.doi.org/10.1016/j.jchromb.2014.05.014 1570-0232/© 2014 Elsevier B.V. All rights reserved. #WVJQT URGTUQPCNEQR[ 2 J. Carlier et al. / J. Chromatogr. B 962 (2014) 1–8 and dysrhythmia, leading to death after a few hours [8–11]. The Saint-Quentin Fallavier, France) diluted in 1 L of deionized water mortality rate is high (20–28% [12]) but there is no correlation with (V.W.R., Fontenay-sous-Bois, France) and the pH was adjusted to 3.8 ® the quantity of seeds consumed; the ingestion of a single seed can with acetic acid (Sigma–Aldrich , Saint-Quentin Fallavier, France). prove fatal [11]. Despite the dangerous nature of the plant and the incidence 2.2. Sample preparation of poisonings, there are few methods of measurement of the toxic principles of Cerbera. In 1978 Malathy and Krishnamoorthy The seeds were removed from the fruit, then desiccated and ® succeeded for the first time in detecting Cerbera poisoning by com- pulverized using a ball mill from Retsch (Haan, Germany) until a paring autopsy samples with extracts of tanghin seeds (thin-layer fine powder was obtained. A methanolic solution of the powdered chromatography) [13]. In 2001, in an internal memo, the authors seeds (100 g/L) was sonicated for 1 hour then centrifuged (3,000 × g, proposed the first method for detecting neriifolin (among other 10 min). The supernatant was diluted in methanol to 0.5 g/L of seeds cardenolides) in post-mortem fluids. In 2009, Deveaux et al. mea- from the dried fruit and 5 g/L of seeds from the fresh fruit. sured neriifolin in the plasma of two victims of fatal poisoning (<1 and 7 g/L) and cerberin, cerberigenin and neriifolin in the seeds 2.3. Screening procedures of dried C. manghas fruit (0.71, 6.8 and 70 g/g) [9,14]. There are at least two simple explanations as to why laboratories A sample of the solution of powdered seeds from the dried fruit do not routinely detect Cerbera toxins: on the one hand, toxicol- (100 L at 0.5 g/L) was evaporated to dryness under a stream of ◦ ogists’ lack of knowledge of this type of poisoning; and on the air at +45 C. The residue was reconstituted with 50 L of 5 mM other hand, the distinct lack of commercially available analytical ammonium acetate buffer at pH = 3.8:acetonitrile (85:15, v/v), cen- standards for these compounds, with the exception, fortunately, of trifuged at 14,000 × g for 4 min, the supernatant being transferred neriifolin. Rare as it might be, homicide by poisoning using natural into a vial for injection into the chromatographic system. toxins is greatly underestimated, which is why this work has been Separation was performed by ultra-high performance liquid ® carried out: making it possible to know more about the active toxic chromatography (UHPLC) (ACQUITY UPLC ; Waters, Guyancourt, ® ® principles in Cerbera that can be detected in autopsy samples in France) using an ACQUITY UPLC BEH C18 column (length: cases of human poisoning. In this study we propose to identify and 150 mm; internal diameter: 2.1 mm; particle size: 1.7 m) (Waters, ◦ measure the principal cardenolide heterosides present in the seeds Guyancourt, France) at +50 C, for a gradient mobile phase com- of fresh and dried C. manghas fruit for the first time. posed of acetonitrile (Solvent A) and a 5 mM ammonium acetate buffer at pH = 3.8 (Solvent B). The chromatographic run time was 15 min. The separation started with a mobile phase consisting of 2. Experimental 15:85 (v/v) A:B for 1.4 min; followed by a linear gradient to 80:20 (v/v) A:B within 9.6 min; the conditions were isocratic for 0.2 min; 2.1. Chemical and reagents then the mobile phase returned to initial conditions within 0.8 min and was equilibrated for 3 min. The mobile phase flow rate was Fresh and dried C. manghas fruit was kindly provided by 450 L/min for the whole time of the separation. The temperature Botanique Co. (Bangkok, Thailand) (Fig. 1). ◦ ® of the samples was +12 C and the injection volume was 5 L. Standard of neriifolin was bought from Sigma–Aldrich (Saint- Spectrophotometric detection was performed using a photo- Quentin Fallavier, France). Internal standard (IS) digoxin-d3 was ® diode array detector (ACQUITY PDA; Waters, Guyancourt, France). bought from LGC Standards (Molsheim, France). Both were then ® Mass spectrometric detection was performed using a quadrupole diluted in methanol (Sigma–Aldrich ; Saint-Quentin Fallavier, ◦ ® detector (ACQUITY SQD; Waters, Guyancourt, France) after ion- France) and stored at a temperature of −20 C. ization by electrospray in positive and negative-ion mode. The Acetonitrile for LC–MS (liquid chromatography–mass spec- nebulizing gas was nitrogen (4.5; Linde gas, Molsheim, France). The trometry) was obtained from Carlo Erba (Val de Reuil, France) mass spectrometer operated in full-scan mode from 80 to 900 amu. and methylene chloride and hexane were obtained from The analytical conditions were optimized by post-column infusion V.W.R. (Fontenay-sous-Bois, France). Ammonium acetate buffer ® of a neat solution of neriifolin in 50:50 (v/v) A:B with a syringe (5 mM) was prepared with ammonium acetate (Sigma–Aldrich , pump (10 L/min) (Table 1). The data were processed by the anal- ysis software Empower Pro 2 (Waters, Guyancourt, France). 2.4. Identification procedure The solution of powdered seeds from the dried fruit (0.5 g/L) was diluted in methanol to a concentration of 1 mg/L. One millil- itre of the solution was then evaporated to dryness under a stream ◦ of air at +45 C. The residue was reconstituted with 50 L of 5 mM ammonium acetate buffer at pH = 3.8:acetonitrile (85:15, v/v), cen- trifuged at 14,000 × g for 4 min, the supernatant being transferred into a vial for injection into the chromatographic system. The chromatographic conditions were strictly the same as described in the screening procedures in order to achieve the same chromatographic profile. Mass spectrometric detection was performed using a quadrupole-Orbitrap high-resolution detector (Q Exactive; Thermo Scientific, Courtabœuf, France) after ionization by heated electrospray in positive-ion mode. Nitrogen was both the collision and nebulizing gas (4.5; Linde gas, Molsheim, France). The mass spectrometer operated in full-scan mode and targeted-MS/MS Fig. 1. Cerbera manghas L.: a dried ripe fruit and its seed. alternately. The mass spectrometric conditions and the collision #WVJQT URGTUQPCNEQR[ J. Carlier et al. / J. Chromatogr. B 962 (2014) 1–8 3 Table 1 Spectrophotometry and mass spectrometry parameters. Screening procedure Identification procedure Quantification procedure Spectrophotometry parameters HRMS parameters: full-scan mode Spectrophotometry parameters Scan time (min) 0.0–15.0 Sheath gas flow rate (psi) 60 Scan time (min) 5.0–9.0 Scan range (nm) 200–400 Auxiliary gas flow rate (a.u.) 20 Scan range (nm) 200–400 Resolution (nm) 1.2 Sweep gas flow rate (a.u.) 0 Resolution (nm) 1.2 Sampling rate (points/s) 20 Spray voltage (kV) +4.0 Sampling rate (points/s) 20 ◦ Filter time constant (s) 0.100 Capillary temperature ( C) +330 Filter time constant (s) 0.100 ◦ Integration algorithm Traditional Heater temperature ( C) +250 Integration algorithm Traditional First injection: full-scan mode MS parameters: full-scan mode Ionization mode Positive Ionization mode Positive/negative Scan time (min) 5.0–9.0 Scan time (min) 0.0–15.0 Scan range (amu) 533–595 Scan range (amu) 80–900 Resolution 140,000 Desolvation gas (L/h) 750 Mass tolerance (ppm) 2.0 Cone gas (L/h) 50 Reference mass Polysiloxane (536.1654 amu) Spray voltage (kV) +3.5 Second injection: targeted-MS/MS mode ◦ Desolvation temperature ( C) +400 Ionization mode Positive ◦ Source temperature ( C) +150 Scan time (min) 5.0–9.0 Cone voltage (V) +20/−20 Inclusion list (amu) Compound (1) 549.3055 Compound (2) 535.3259 Compound (3) 591.3169 Compound (4) 577.3375 Collision energy (%) 10 Resolution 17,500 MS, mass spectrometry; MS/MS, tandem mass spectrometry; HRMS, high resolution mass spectrometry. energy were optimized by post-column infusion of a neat solution The chromatographic conditions were the same as described in of neriifolin in 50:50 A:B (v/v) with a syringe pump (10 L/min) the screening procedures. The spectrophotometric conditions are (Table 1). Once their exact mass was determined, the compounds reported in Table 1. of interest were fragmented and their full-scan product ion spectra The quantification of the compounds of interest was performed were recorded (Table 1). at 219 nm (maximum of absorbance of cardenolide compounds). The identified compound neriifolin was the only commercially available benchmark and was quantified by the standard addi- tion method (+50, 100, 150, 200, 250 and 300 L of neriifolin at 2.5. Quantification procedure 10 mg/L). The equation of the calibration curve was calculated by the method of least squares and the validity of the regression model A sample of the solution of powdered seeds (1 mL) from the was confirmed by an ANOVA test. The other compounds were thus dried fruit (0.5 g/L) or fresh fruit (5 g/L) and 50 L of a methanolic quantified using the calibration curve of neriifolin. solution of digoxin-d3 at 10 mg/L (IS) were evaporated to dryness ◦ The limit of detection (LOD) and the lower limit of quantifica- under a stream of air at +45 C. The residue was reconstituted with tion (LLOQ) were calculated by performing the analysis of ten blank 50 L of 5 mM ammonium acetate buffer at pH = 3.8:acetonitrile matrices (seeds of dried fruits of Acokanthera oblongifolia (Hochst.) (85:15, v/v), centrifuged at 14,000 × g for 4 min, the supernatant Codd, a cardiotoxic plant from the Apocynaceae family, historically being transferred into a vial for injection into the chromatographic system. used as a poison arrow in East Africa [15], kindly provided by Dr. Fig. 2. Chromatograms obtained from screening of an extract of dried C. manghas fruit seeds (5 g of seeds on column). (solid line: total ion current in positive mode, dotted line: total ion current in negative mode, dashed line: UV absorption at 219 nm). #WVJQT URGTUQPCNEQR[ 4 J. Carlier et al. / J. Chromatogr. B 962 (2014) 1–8 Gilbert Gault from VetAgroSup, France) and according to the for- was composed of ions with m/z 549 (relative intensity: 47%), 566 + mulas: LOD = mbl + 3SDbl and LLOQ = mbl + 10SDbl (mbl: mean of the (100%) and 587 (9%), representing hydrogen [M1+H] , ammonium + + measured concentration of the blanks; SDbl: standard deviation of [M1+NH4] and potassium [M1+K] adducts, respectively, of com- − the measured concentration of the blanks; n = 10). pound (1) of mass M1. Only the chlorine adduct [M1+Cl] (583 amu) was observable in negative-ion mode. The second peak, eluted at 3. Results and discussion 6.7 min, was composed of ions with m/z 535 (63%), 552 (100%) and 573 (34%), representing hydrogen, ammonium and potassium 3.1. Screening adducts, respectively, of compound (2) of mass M2. Only the chlo- − rine adduct [M2+Cl] (569 amu) was observable in negative-ion In mass spectrometry, four main chromatographic peaks are mode. The third peak, eluted at 7.6 min, was composed of ions shown in the seeds from dried C. manghas fruit (methanolic extract with m/z 591 (100%), 608 (91%) and 629 (29%), representing hydro- of 5 g of seeds on column) (Fig. 2): the first peak, eluted at 6.2 min, gen, ammonium and potassium adducts, respectively, of compound Fig. 3. Comparison of the UV absorption spectrums of compounds (1), (3) and (4) shown by screening, and the spectrum from an analysis of pure standard neriifolin (left). Full-scan product ion spectra of the compounds (1), (3) and (4) (right). #WVJQT URGTUQPCNEQR[ J. Carlier et al. / J. Chromatogr. B 962 (2014) 1–8 5 Fig. 4. Chromatograms from MS and spectrophotometric analysis of pure standard neriifolin (100 ng on column) and of an extract of dried C. manghas fruit seeds (5 g on column) (left). Comparison of the UV absorption spectrum of compound (1) and the spectrum from an analysis of pure standard neriifolin (right). (solid line = sum of ions 535, 552 et 573 amu in positive-ion mode, dashed line = UV absorption at 219 nm). − (3) of mass M3. Only the chlorine adduct [M3+Cl] (625 amu) was 2.2; Thermo Scientific, Courtabœuf, France), of which only two observable in negative-ion mode. The last peak, eluted at 8.0 min, match a known structure (ChemSpider website) and only one was composed of ions with m/z 577 (100%), 594 (91%) and 615 matches a steroid structure (C30H46O8): the empirical formula of (29%), representing hydrogen, ammonium and potassium adducts, neriifolin. The difference between the experimental m/z ratio and + respectively, of compound (4) of mass M4. Only the chlorine adduct the exact theoretical mass of neriifolin ([M+H] = 535.3265 amu) − [M4+Cl] (611 amu) was observable in negative-ion mode. The was ppm = −1.205. The fragmentation spectrum of the compound reduction in the proportion of ammonium and potassium adducts matches that of neriifolin (Fig. 5): the fragments 339.23, 357.24 + is logically correlated with the reduction in the proportion of the and 375.25 amu belong to the genin ([(digitoxigenin − 2H2O) + H] , + + ammonium acetate buffer in the mobile phase at the moment of [(digitoxigenin − H2O) + H] and [digitoxigenin + H] respectively) the elution of the compounds. and the fragment 129.06 amu belongs to the sugar part of the + These four compounds presented a similar UV absorption spec- molecule ([(thevetose − CH3OH) + H] ). trum, matching that of steroids with a cardenolide structure, with Through HRMS analysis of compound (4) it was possible to mea- maximum absorption max = 219 nm (Fig. 2). The spectrum for com- sure an m/z ratio of 577.3375 amu. This experimental m/z ratio pounds (2) and (4) was identical. The spectrum for compounds (1) corresponds with nine possible chemical formulas to ± 2 ppm, of and (3) was also identical, and differed from the other two by a which only two match a known structure and only one matches a low-intensity absorption peak of max = 270 nm (Figs. 3 and 4). steroid structure (C32H48O9). Fragments 339.23, 357.24 and 375.25 observed in the fragmentation spectrum of neriifolin and match- 3.2. Identification ing the digitoxigenin, are found in the spectrum of compound (4) (Fig. 3). By comparing the molecular structures listed on the From the UV spectrum, mass and retention time of compound ChemSpider website it was possible to identify cerberin. The differ- (2) it was possible to identify neriifolin by comparison with the ence between the experimental m/z ratio and the exact theoretical +