Drugs and Poisons in Humans - A Handbook of Practical Analysis (Part 57)

Introduction:Oleander (Nerium oleander and Nerium indicum) is a relatively small evergreen tree of an Indian origin, and growing in Honshu, Shikoku, Kyushu and Okinawa islands in Japan. The plant contains cardiac glycosides in its leaves, stems and flowers and is known as one of poisonous plants; poisoning and fatal cases for domestic animals and humans due to ingestion of this plant were reported [1–6]. The main toxin of oleander is oleandrin. Oleandrin can be measured using cross-reaction of an immunoassay kit for digoxin [1], TLC [2], HPLC [7, 8] and LC/MS [3, 6]. Oleandrin is thermolabile; it is difficult...
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Drugs and Poisons in Humans - A Handbook of Practical Analysis (Part 57) 6.7II.6.7 Oleander toxins by Chiaki Fuke and Tomonori AraoIntroductionOleander (Nerium oleander and Nerium indicum) is a relatively small evergreen tree of anIndian origin, and growing in Honshu, Shikoku, Kyushu and Okinawa islands in Japan. Theplant contains cardiac glycosides in its leaves, stems and flowers and is known as one of poison-ous plants; poisoning and fatal cases for domestic animals and humans due to ingestion of thisplant were reported [1–6]. The main toxin of oleander is oleandrin. Oleandrin can be measured using cross-reaction of an immunoassay kit for digoxin [1],TLC [2], HPLC [7, 8] and LC/MS [3, 6]. Oleandrin is thermolabile; it is difficult to analyze it byGC or GC/MS, because it gives 4 peaks due to decomposition. In this chapter, a method for LC/MS analysis of oleandrin and its metabolite desacetylole-andrin [9] together with their related compounds, such as oleandrigenin and gitoxigenin, con-tained in human specimens, is presented. The structures and their molecular weights of oleandrin and its related compounds areshown in > Figure 7.1.⊡ Figure 7.1Structures and molecular weights of oleandrin and its related compounds.Reagents and their preparation• A 1-mg aliquot each of oleandrin, oleandrigenin, desacetyloleandrina, gitoxigenin and digi- toxigenin (Sigma, St. Louis, MO, USA) is dissolved in 10 mL acetonitrile (100 µg/mL) separately.• A 0.1-mL volume of the above digitoxigenin solution is diluted with acetonitrile to 10 mL (1 µg/mL; internal standard, IS).© Springer-Verlag Berlin Heidelberg 2005520 Oleander toxins HPLC conditions Instrument: a Hitachi M-8000 type LC/3DQMS system; column: GH-C18 (III) (150 × 2.1 mm i.d., particle size 5 µm, Hitachi Ltd., Tokyo, Japan); column temperature: 40 °C; mobile phase: methanol/water (6:4, v/v); its flow rate: 0.2 mL/min. MS conditions Ionization: sonic spray ionization (SSI)b; shield temperature: 250 °C; aperture-1 temperature: 150 °C; aperture-2 temperature: 120 °C; drift voltage: 70 V; ion detection mode: positive; microscan: 5 s; mass defect: 55/100 amu; scan range: m/z 350–650; low mass cutoff: m/z 120; accumulation time: 500 ms. MS/MS conditionsc Ion accumulation step: Ion accumulation mass range: m/z 350–650; low mass cutoff: m/z 120; ion accumulation time: 300 ms; ion accumulation voltage: 0 V. Ion isolation step (MS-1): Isolation mass range: m/z 595.48–602.77; low mass cutoff: m/z 569.06; isolation time: 10 ms; isolation voltage: 0.175 V. CID step (MS-2): CID mass range: m/z 584.15–614.64; low mass cutoff: m/z 190; CID time: 50 ms; CID voltage: 0.188 V. Procedure i. A 1-mL volume of a specimend is mixed with 4 mL distilled water and 100 µL IS solution. ii. The above mixture is extracted with 2 mL of 1-chlorobutane by shaking for 15 min. iii. It is centrifuged at 2,000 g for 5 min; the organic phase is transferred to a test tube. iv. The steps ii and iii are repeated twice. v. The organic phases are combined and evaporated to dryness under a stream of nitrogen with warming at 40 °C. vi. The residue is dissolved in 0.5 mL of 80 % methanol aqueous solution, and washed with 1 mL hexane twicee. vii. The 80 % methanol layer is evaporated to dryness under a stream of nitrogen with warm- ing at 40 °C in a water bath. viii. The residue is dissolved in 100 µL mobile phase and centrifuged at 12,000 g for 5 min; a 5-µL aliquot of the supernatant solution is injected into LC/MS. ix. Each calibration curve is constructed using spiked specimens with digitoxigenin as IS. The concentration of an oleander toxin in a specimen is calculated using the calibration curve. Oleander toxins 521Assessment of the methodOleandrin is one of cardiac glycosides and exerts its effect at low concentrations. To detect itstherapeutic concentrations, the detection limit by an analytical method should be in the nano-grams/mL order. When the present method was used in an oleander poisoning case, oleandrincould be detected from blood and cerebrospined fluid (CSF), showing the applicability of themethod. > Figure 7.2 shows a TIC and mass spectra for the authentic standards of the five com-pounds. The spectra showed intense [M + Na]+ adduct ions at m/z 599 for oleandrin, m/z 557for desacetyloleandrin, m/z 455 for oleandrigenin and m/z 397 for digitoxigenin used as IS; forgitoxigenin, both [M + Na]+ and [M + Na – H2O]+ ions appeared ...