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

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

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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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  1. 6.7 II.6.7 Oleander toxins by Chiaki Fuke and Tomonori Arao 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 poison- ous 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 to analyze it by GC 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 are shown in > Figure 7.1. ⊡ Figure 7.1 Structures 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 2005
  2. 520 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.
  3. Oleander toxins 521 Assessment of the method Oleandrin is one of cardiac glycosides and exerts its effect at low concentrations. To detect its therapeutic 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, oleandrin could be detected from blood and cerebrospined fluid (CSF), showing the applicability of the method. > 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 557 for desacetyloleandrin, m/z 455 for oleandrigenin and m/z 397 for digitoxigenin used as IS; for gitoxigenin, both [M + Na]+ and [M + Na – H2O]+ ions appeared at m/z 413 and 395, respec- tively. ⊡ Figure 7.2 TIC and mass spectra for each peak obtained by LC/MS for the authentic compounds (1 µg/mL each) of oleandrin and its related compounds.
  4. 522 Oleander toxins > Figure 7.3 shows a TIC and mass chromatograms (MCs) for the above 5 compounds, which had been spiked into blood (0.1 µg/mL) and extracted from it. There were no interfering impurity peaks for each test compound in the chromatogram of blank blood. The recovery rates for oleandrin, desacetyloleandrin and oleandrigenin were not lower than 70 %; but that for gitoxigenin was as low as about 20 %. There was good linearity in the ⊡ Figure 7.3 TIC and MCs obtained by LC/MS for an extract of blood, into which oleandrin and its related compounds had been spiked (0.1 µg/mL each).
  5. Oleander toxins 523 range of 5–100 ng/mL for oleandrin, desacetyloleandrin and oleandrigenin. The detection limits from blood were 3 ng/mL for oleandrin, 2 ng/mL for desacetyloleandrin and oleandri- genin, and 30 ng/mL for gitoxigenin. Poisoning case, and toxic and fatal concentrations A 49-year-old female boiled an oleander branch with leaves in water, and took a large amount of the extract solution; she underwent therapy, but died one day later. The blood and CSF specimens obtained at the postmortem inspection of the above victim were analyzed by the present method. The TIC and MCs obtained for the victim by LC/MS are shown in > Fig- ure 7.4. Oleandrin could be detected; but desacetyloleandrin, oleandrigenin and gitoxigenin could not. The peak at m/z 599 observed in the MC was confirmed to be due to oleandrin by MS/MS analysis as shown in > Figure 7.5. The concentration of oleandrin was 10 ng/mL for both blood and CSF. The blood or plasma concentrations in cases of poisoning by oleandrin, digoxin and digi- toxin are shown in > Table 7.1. There is another report dealing with LC/MS detection of oleandrin in an oleander poisoning case [6] except our case; there are also 2 reports dealing with the immunoassay detection of oleandrin using its cross-reaction [1, 4]; the immunoassay kit had been developed for measurements of digoxin, and thus the values of oleandrin in blood were expressed as the concentrations of digoxin (5.8 and 4.2 ng/mL). However, the digoxin immunoassay method does not give quantitative results for oleandrin; it seems useful only for tentative qualitative analysis, but is not reliable for its quantitation. ⊡ Table 7.1 Concentrations of oleandrin, digoxin and digitoxin in blood or plasma of cardiac glycoside poisoning cases Compound Concentration Specimen Outcome Ref. (ng/mL) oleandrin 1.1 blood survived [6] 10 blood dead the present case digoxin 7–24 plasma dead [10] 22 blood dead [10] 30 blood dead [10] digitoxin 260 plasma survived [10] 320 plasma dead [10]
  6. 524 Oleander toxins ⊡ Figure 7.4 TIC and MCs obtained by LC/MS for an extract of blood in a case of oleander poisoning.
  7. Oleander toxins 525 ⊡ Figure 7.5 Authentic oleandrin blood extract MS/MS mass spectra of an extract of blood in a case of oleander poisoning and of the authentic oleandrin. Product ions were obtained from peaks detected by mass chromatography at m/z 599. Notes a) Desacetyloleandrin was synthesized by deacetylation in anhydrous methanol with sodium methoxide as catalyst. b) Sonic spray ionization (SSI) is relatively similar to atmospheric pressure chemical ionization (APCI). The mobile phase is electrically neutral; but in a small region, especially around the surface layer of the solution, charge separation can occur. In SSI, nebulization is done so that the surface layer of the solution, in the region of charge separation, is stripped by fast nitrogen gas flow and electrically charged airborne droplets are created. The diameters of these electrically charged droplets shrinks by vaporization of solvent molecules from the surface, and protonated molecular ions are formed in the gas phase. The interface does not require heating upon nebulizing; thus it is suitable for sensitive analysis of thermolabile compounds. c) The MS/MS conditions with 3-dimensional QMS for oleandrin are described here; the con- ditions are highly dependent on a compound to be analyzed. It is essential to optimize conditions for each compound. d) As specimens, blood, plasma and urine can be used. e) Such washing to remove compounds of low polarity is useful, especially when repeated analyses are required. References 1) Osterloh J, Herold S, Pond S (1982) Oleander interference in the digoxin radioimmunoassay in a fatal ingestion. JAMA 247:1596–1597 2) Blum LM, Rieders F (1987) Oleandrin distribution in a fatality from rectal and oral Nerium oleander extract administration. J Anal Toxicol 11:219–221 3) Rule G, McLaughlin LG, Henion J (1993) A quest for oleandrin in decayed human tissue. Anal Chem 65:857–863 4) Nakata M, Miyata S, Endo K et al. (1995) A fatal case of oleander poisoning. J Okinawa Med Assoc 34:97 (in Japanese)
  8. 526 Oleander toxins 5) Gupta A, Joshi P, Jortani SA et al. (1997) A case of nondigitalis cardiac glycoside toxicity. Ther Drug Monit 19:711–714 6) Tracqui A, Kintz P, Branche F et al. (1998) Confirmation of oleander poisoning by HPLC/ MS. Int J Legal Med 111:32–34 7) Tor ER, Holstege DM, Galey FD (1996) Determination of oleander glycosides in biological matrices by high-per- formance liquid chromatography. J Agric Food Chem 44:2716–2719 8) Namera A, Yashiki M, Okada K et al. (1997) Rapid quantitative analysis of oleandrin in human blood by high- performance liquid chromatography. Jpn J Legal Med 51:315–318 9) Takaesu H, Fuke C, Arao T et al. (1998) A study on the methods of verifying oleander poisoning through the analysis of biological materials. Jpn J Forensic Toxicol 16:136–137 (in Japanese with an English abstract) 10) Moffat AC, Jackson JV, Moss MS et al. (eds) (1986) Clarke’s Isolation and Identification of Drugs, 2nd edn. The pharmaceutical Press, London, pp 541–544

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