Drugs and Poisons in Humans - A Handbook of Practical Analysis (Part 56)

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Drugs and Poisons in Humans - A Handbook of Practical Analysis (Part 56)

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Introduction: Datura metel a (jimsonweed) is a plant belonging to the Solanacea family; and contains tropane alkaloids (belladonna alkaloids), such as atropineb (dl-hyoscyamine) and hyoscine (dl-scopolamine), in its seeds and every part of the plant. Its seeds and leaves have long been being used as a folk medicine in Japan. Seishu Hanaoka, a Japanese surgeon, first used this plant for general anaesthesia in 1804. Many other species of solanaceous plants, such as Atropa, Scopolia and Duboisia, contain tropane alkaloids, and are distributed worldwide; many of them have been being used as folk medicines and also sometimes causing poisoning cases. The...

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  1. 6.6 II.6.6 Tropane alkaloids by Akira Namera Introduction Datura metel a (jimsonweed) is a plant belonging to the Solanacea family; and contains tropane alkaloids (belladonna alkaloids), such as atropineb (dl-hyoscyamine) and hyoscine (dl-scopo- lamine), in its seeds and every part of the plant. Its seeds and leaves have long been being used as a folk medicine in Japan. Seishu Hanaoka, a Japanese surgeon, first used this plant for gen- eral anaesthesia in 1804. Many other species of solanaceous plants, such as Atropa, Scopolia and Duboisia, contain tropane alkaloids, and are distributed worldwide; many of them have been being used as folk medicines and also sometimes causing poisoning cases. The seeds of Datura metel are being sold in gardening stores; people are growing this plant in their own gardens. There were poisoning cases in which Datura metel was eaten by mistaking it for Jew’s mallow; its root eaten for that of burdock and its seeds eaten for sesame [1]. More enlightenment is required for recognition of Datura and Atropa plants as being poisonous. GC/MS analysis Reagent and their preparation • A 1-mg aliquot of l-hyoscyamine (Sigma, St. Louis, MO, USA) is dissolved in 1 mL aceto- nitrile. • A 1-mg aliquot of l-scopolamine (Sigma) is dissolved in 1 mL acetonitrile. • A 1-mg aliquot of atropine-d3 (Sigma) is dissolved in 10 mL acetonitrile (0.1 mg/mL) to serve as an internal standard (IS). • A 990-µL volume of N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA, Pierce, Rockford, IL, USA and other manufacturers) is mixed with 10 µL of trimethylchlorosilane (TMCS, Pierce and other manufacturers) (99:1, v/v) and used as a derivatization reagent. • A 38.1-g amount of sodium tetraborate (Na2B4O7 · 10 H2O) is dissolved in distilled water to prepare 1,000 mL solution (0.1 M, pH 9.3). • Extrelut NT-20 c (granule for repacking, Merck, Darmstadt, Germany) is washed with 3 volumes of diethyl ether and dried under a stream of air in a draft until the disappearance of the ether smell. Moreover, the granule is dried by warming at 40 °C for 1 h. A 2-g aliquot each of the granule is packed in a glass tube (15 cm × 10 mm i.d.) to be used as an Extrelut columnd. © Springer-Verlag Berlin Heidelberg 2005
  2. 510 Tropane alkaloids GC/MS conditions GC column: an HP-5MS fused silica capillary column (30 m × 0.25 mm i.d., film thickness 0.25 µm, Agilent Technologies, Palo Alto, CA, USA). GC/MS conditions; instrument: a GC5890 Series II gas chromatograph/an MS 5971A mass spectrometer (Agilent Technologies); column (oven) temperature: 50 °C (1 min) → 20 °C/min → 300 °C (5 min); injection temperature: 250 °C; detector temperature: 280 °C; carrier gas: He (50 kPa); monitoring ions: m/z 124,140 and 361 for l-hyoscyamine-TMS; m/z 138, 154 and 375 for l-scopolamine-TMS; m/z 127, 143 and 364 for atropine-d3-TMS. Procedure i. A 0.5 mL volume of a specimen, 5 µL IS solution and 1.0 mL borate solution (0.1 M, pH 9.3) are placed in a test tube, mixed well and poured into an Extrelut column. ii. After leaving for 15 min, target compounds are eluted with 4 mL dichloromethane. iii. The eluate is evaporated to dryness under a stream of nitrogen. iv. The residue is mixed with 50 µL of BSTFA/TMCS (99:1) and warmed at 45 °C for 20 min for TMS derivatization in a glass vial with a Teflon cap. v. The resulting solution is mixed with 100 µL dichloromethane; a 1-µL aliquot of it is in- jected into GC/MS for analysis. Assessment of the method > Figure 6.1 shows SIM chromatograms for l-hyoscyamine and l-scopolamine, which had been extracted from human serum (the concentration of each compound, 1 µg/mL). These chromatograms were obtained using a slightly polar capillary column; similar chromatograms can be also obtained with a nonpolar capillary column. Even with the same type of columns, there is variation in their property among their lots; it is essential to confirm the retention times using the authentic standards for each column. There is a method for GC(/MS) analysis of the tropane alkaloids without any derivatization; but the derivatization of the alkaloids is recommended, because the underivatized alkaloids are thermolabile and thus cause decompo- sition by heat [2] and low reproducibility. By this method, good linearity can be obtained in the range of 10–5,000 ng/mL and the detection limit is about 5 ng/mL for each compound [3]. After intravenous injection of 3 mg atropine sulfate, a peak blood concentration at 11 µg/mL could be obtained [4]. Therefore, therapeutic concentrations of atropine can be measured by this method.
  3. GC/MS analysis 511 ⊡ Figure 6.1 SIM chromatograms for tropane alkaloids obtained by GC/MS. The amount of each alkaloid spiked into 1 mL serum was 1 µg.
  4. 512 Tropane alkaloids HPLC analysis Reagents and their preparation • The acetonitrile solutions of l-hyoscyamine and l-scopolamine are prepared as described in the GC/MS section. • A 2-g aliquot of phosphoric acid is dissolved in Milli Q watere to prepare 1,000 mL solution, followed by adjustment of its pH to 3.0 with NaOH solution (phosphate buffer solution). • Mobile phase: 100 mL acetonitrile (HPLC grade) is mixed with 900 mL of the above phos- phate buffer solution, passed through a filter (0.45 µm) and degassed using an ultrasonic cleanerf. • A 5-mL volume of methanol is mixed with 95 mL of Milli Q water (5 %, v/v). • Each Oasis® HLB cartridge (1 cc/30 mg, Waters, Milford, MA, USA) is washed with 1 mL methanol and 1 mL water to activate itg. HPLC conditions HPLC column: a Discovery C18 octadecyl chemical-bonded silica columnh (150 × 4.6 mm i.d., particle size 5 µm, Supelco, Bellefonte, PA, USA). HPLC conditions; instrument: an LC-10A high-performance liquid chromatograph (Shimadzu Corp., Kyoto, Japan); mobile phase: acetonitrile/phosphate buffer solution (1:9, v/v); column (oven) temperature: 40°C; flow rate: 1.0 mL/min; detection wavelength: 215 nm. Procedure i. A 0.5-mL specimen is poured into an activated Oasis® HLB cartridge. ii. The cartridge is washed with 1 mL of 5 % methanoli. iii. The target compounds are eluted with 1 mL methanol. iv. The eluate is evaporated almost to dryness under reduced pressure. v. The residue is dissolved in 100 µL of the mobile phase; a 10-µL aliquot of it is injected into HPLC. Assessment of the method > Figure 6.2 shows HPLC chromatograms for the authentic standards of l-hyoscyamine and l-scopolamine, for blank serum and for serum, into which both compounds had been spiked. Under the present conditions, l-hyoscyamine could be completely separated from l-scopol- amine. However optical isomers of each compound cannot be separated. Tropic acid, one of the metabolites for both hyoscyamine and scopolamine, can be detected simultaneously. How- ever, for confirmation of drug identity, the UV spectral patterns of the test peak should be compared with those of the corresponding authentic standard together with the coincidence of
  5. HPLC analysis 513 ⊡ Figure 6.2 HPLC-UV chromatograms for tropane alkaloids. The amount of each alkaloid spiked into 1 mL serum was 1 µg.
  6. 514 Tropane alkaloids their retention times. The detection limit of this method is about 0.01 µg/mL, and thus toxic and fatal concentrations of the compounds can be detected. Chiral analysis of atropine by CE/MS [5] Reagents and their preparation • The acetonitrile solutions of l-hyoscyamine and l-scopolamine are prepared as described in the GC/MS section. • A 1-mg aliquot of dl-hyoscyamine (atropine, Sigma) is also dissolved in 1 mL acetonitrile. • A 0.92-g amount of formic acid and 286 g of 2,3,6-tri-O-methyl-β-cyclodextrin (Sigma and other manufacturers) are dissolved in Milli Q water to prepare 1,000 mL solution; after degassing, an appropriate volume of the solution is used as an electrophoretic medium. CE/MS conditions CE column: a fused-silica capillary column (900 mm × 50 µm i.d., 360 µm o.d., Agilent Tech- nologies). CE conditions: an HP3D capillary electrophoresis system (Agilent Technologies); detector: an Agilent 1100 mass spectrometer; electrophoretic medium: solution containing 200 mM tri- methyl-β-cyclodextrin and 50 mM formic acid; impressed voltage: 30 kV; column (oven) tem- perature: 20 °C; sheath solution: 50 % methanol containing 5 mM ammonium acetate (4 µL/ min); nebulizer gas: nitrogen (10 psi); drying gas: nitrogen (6 L/min, 300 °C); monitoring ions: m/z 290.2 (l-hyoscyamine), m/z 304.1 (l-scopolamine). Procedure i. A specimen for analysis is seeds of a plant (Datura metel and others). ii. A fixed amount of seeds is crushed using a mortar. iii. The resulting powder and 1 mL methanol containing 1 % ammonia are placed in a test tube. iv. The mixture is sonicated for 3 min for extraction. v. The supernatant solution is passed through a membrane filter (0.22 µm); 200 µL of the fil- trate is evaporated to dryness under a stream of nitrogen. vi. The residue is dissolved in 100 µL Milli Q water and injected into CE/MS. Assessment of the method In the upper panels of > Figure 6.3, electropherograms for the mixture of the authentic l-hy- oscyamine, atropine (dl-hyoscyamine) and l-scopolamine. l-Hyoscyamine could be separated from d-hyoscyamine, enabling the optical resolution. However, since d-hyoscyamine is not
  7. Poisoning cases, and toxic and fatal concentrations 515 ⊡ Figure 6.3 Electropherograms for tropane alkaloids obtained by CE/MS. The upper panels show those for the authentic standard solution; the lower panels those using the extract of seeds of Datura metel. available, the optical resolution ability for scopolamine is not clear. Since small shifts appear in migration times of compounds for each electrophoresis in CE analysis, it is essential to confirm migration times using the authentic standard compounds. By this method, excellent linearity could be obtained in the range of 1–100 µg/mL for aqueous solution of l-hyoscyamine, d-hyoscyamine and l-scopolamine; their detection limit was about 0.5 µg/mL. Poisoning cases, and toxic and fatal concentrations There are many tropane alkaloid poisoning cases, in which solanaceous plants are ingested by mistake. However, in many cases, atropine or hyoscyamine was only detected from the plant or
  8. 516 Tropane alkaloids foods, which the poisoned victim had eaten; the number of reports dealing with the determi- nation of the alkaloids in blood and/or urine of the poisoned patients is very limited. Poisoning cases, in which large amounts of atropine sulfate are ingested for suicidal purpose, were also reported. The therapeutic blood atropine concentrations are 5–70 ng/mL; but its half-life is as short as 2–3 h. Case 1 [6]: a 71-year-old female parboiled and ate a plant in the evening, which had been grown in her garden; she had believed it to be the Jew’s mallow. About 20 min after ingestion, paresthesia appeared in the hand and then extended to her whole body; she fell into the cloud- ing of consciousness. She was brought to a nearby clinic by an ambulance car at about 10:00 p. m. A doctor at the clinic suspected atropine poisoning, because of her clouded consciousness, mydriasis, dry mouth and palpitations. From about 1:00 p. m. on the next day, gastrolavage was performed; her consciousness was recovered and general conditions improved. The same plant as that eaten by her was examined by a specialist and proved to be Datura tatula; the causative food for the poisoning could be identified. Case 2 [7]: a pharmacy college student (male) ingested about 1 g of atropine together with al- cohol; he was sent to a hospital and survived. His blood atropine concentration was 130 ng/mL. Case 3 [7]: a 18-year-old male ingested atropine tablets (30 mg per tablet, but the number of the tablets is not known), and died. The atropine concentration in his whole blood was 200 ng/mL. Notes a) There are many toxic solanaceous plants in the world. Some examples except Datura metel are: Scopolia japonica native to Japan, Hyoscyamine niger (henbane) and Atropa belladonna (deadly nightshade) both native to Europe, Duboisia myoporoides (pituri) native to Australia, Datura stramonium (jimsonweed) native to India, Datura (Brugmansia) suaveolens (angel’s trumpet), Datura ferox and Datura stramonium. b) Atropine means the racemic form of hyoscyamine. The tropane alkaloids being contained in Datura metel (jimsonweed) are mainly l-hyoscyamine and l-scopolamine together with trace amounts of d-hyoscyamine ( > Fig. 6.3). c) The Extrelut granule might be contaminated by impurities upon packing. It is, therefore, desirable to well wash the granule with a solvent to be used for elution. When various elution solvents are to be used, the granule can be appreciably cleaned by washing with diethyl ether. d) Packed Extrelut columns are commercially available. e) The Milli Q water means the one, which had been passed through the Millipore filter (Mil- lipore, Bedford, MA, USA) and widely used in laboratories. For a mobile phase for HPLC, the Mill Q water is used in place of distilled water. f) The mobile phase for HPLC should be prepared by degassing air dissolved in the liquid using aspiration under reduced pressure together with sonication. Since the pressure is reduced inside the container, a pressure-resistant glassware should be used. When the re- duced pressure state is left for a long time, acetonitrile is evaporated from the mobile phase, resulting in the change of composition ratio of acetonitrile and phosphate buffer solution. g) The flow rate for the solid-phase extraction cartridge should equally be 1–2 mL/min for adsorption, washing and elution. When the flow rate is too slow upon washing, a part of the
  9. Poisoning cases, and toxic and fatal concentrations 517 target compound may be lost due to elution with the washing solution. Just after activation of the cartridge, it should not be dried. However, just before the final elution, the cartridge should be dried under reduced pressure. These manipulations should not be confused. Here, the most typical procedure is presented, but there are other procedures using differ- ent solvent systems. h) According to the kinds of columns to be used (manufacturers, type No., column diameter and column length), the turn of the drugs to be eluted and their retention times become different. When more residual silanol groups exist in a column, longer retention times of target compounds and more broadening of their peaks take place. To overcome these prob- lems, the increase in the ratio of methanol or acetonitrile in a mobile phase or addition of 10–20 mM triethylamine as a counter ion may be useful to some extent. i) The washing of the cartridge at this step should be performed perfectly. If not, the final elu- ate becomes contaminated by protein impurities, which appear as insoluble particles upon its condensation; in such a case the filtration or centrifugation of the final extracts is re- quired before injection into HPLC. References 1) Goto K, Endo Y, Mori C et al. (1996) A case of poisoning by Datura metel as a result of its mistaken ingestion for Jew’s mallow. Jpn J Toxicol 9:355 (in Japanese) 2) Eckert M, Hinderling PH (1981) Atropine; a sensitive gas chromatography-mass spectrometry assay and pre- pharmacokinetic studies. Agents Actions 11:520–531 3) Unpublished result 4) Metcalfe RF (1981) A sensitive radioreceptor assay for atropine in plasma. Biochem Pharmacol 30:209–212 5) Namera A, Minoura K, Yashiki M et al. (2000) Chiral analysis of tropane alkaloids using CE/MS. Jpn J Legal Med 54:148 (in Japanese) 6) Kimura S (1995) Food poisoning by Datura tatura. Jpn J Food Hyg 36:663–664 (in Japanese) 7) Baselt RC, Cravey RH (1995) Disposition of Toxic Drugs and Chemicals in Man, 4th edn. Chemical Toxicology Institute, Foster City, CA, pp 63–65

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