© Springer-Verlag Berlin Heidelberg 2005 Introduction Datura metel a jimsonweed is a plant belonging to the Solanacea family; and contains tropane alkaloids belladonna alkaloids, such
Trang 1© Springer-Verlag Berlin Heidelberg 2005
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, fi rst 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
Th e seeds of Datura metel are being sold in gardening stores; people are growing this plant
in their own gardens Th ere 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)trifl uoroacetamide (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
Trang 2510 Tropane alkaloids
GC/MS conditions
GC column: an HP-5MS fused silica capillary column (30 m × 0.25 mm i.d., fi lm 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 Aft er leaving for 15 min, target compounds are eluted with 4 mL dichloromethane iii Th e eluate is evaporated to dryness under a stream of nitrogen
iv Th e 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 Tefl on cap
v Th e 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) Th ese 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 confi rm the retention times using the authentic standards for each column Th ere 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] Aft er intravenous injection of 3 mg atropine sulfate, a peak blood concentration at 11 µg/mL could be obtained [4] Th erefore, therapeutic concentrations of atropine can be measured by this method
Trang 3SIM chromatograms for tropane alkaloids obtained by GC/MS The amount of each alkaloid
spiked into 1 mL serum was 1 µg.
⊡ Figure 6.1
Trang 4512 Tropane alkaloids
HPLC analysis
Reagents and their preparation
• Th e 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 buff er solution)
• Mobile phase: 100 mL acetonitrile (HPLC grade) is mixed with 900 mL of the above phos-phate buff er solution, passed through a fi lter (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 buff er solution (1:9, v/v); column (oven) temperature: 40°C; fl ow 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 Th e cartridge is washed with 1 mL of 5 % methanoli
iii Th e target compounds are eluted with 1 mL methanol
iv Th e eluate is evaporated almost to dryness under reduced pressure
v Th e 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 confi rmation 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
Trang 5HPLC-UV chromatograms for tropane alkaloids The amount of each alkaloid spiked into 1 mL
serum was 1 µg.
⊡ Figure 6.2
Trang 6514 Tropane alkaloids
their retention times Th e 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
• Th e 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; aft er 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 fi xed amount of seeds is crushed using a mortar
iii Th e resulting powder and 1 mL methanol containing 1 % ammonia are placed in a test tube
iv Th e mixture is sonicated for 3 min for extraction
v Th e supernatant solution is passed through a membrane fi lter (0.22 µm); 200 µL of the fi l-trate is evaporated to dryness under a stream of nitrogen
vi Th e 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
Trang 7available, the optical resolution ability for scopolamine is not clear Since small shift s appear in migration times of compounds for each electrophoresis in CE analysis, it is essential to confi rm 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
Th ere 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
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.
⊡ Figure 6.3
Trang 8516 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 Th e 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 aft er 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 Th e 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 identifi ed
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 Th e atropine concentration in his whole blood was
200 ng/mL
Notes
a) Th ere 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 Th e 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) Th e 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) Th e Milli Q water means the one, which had been passed through the Millipore fi lter (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) Th e 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 pressuresistant 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 buff er solution g) Th e fl ow rate for the solid-phase extraction cartridge should equally be 1–2 mL/min for adsorption, washing and elution When the fl ow rate is too slow upon washing, a part of the
Trang 9target compound may be lost due to elution with the washing solution Just aft er activation
of the cartridge, it should not be dried However, just before the fi nal elution, the cartridge should be dried under reduced pressure Th ese manipulations should not be confused Here, the most typical procedure is presented, but there are other procedures using diff er-ent solver-ent 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 diff erent 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) Th e washing of the cartridge at this step should be performed perfectly If not, the fi nal elu-ate becomes contaminelu-ated by protein impurities, which appear as insoluble particles upon its condensation; in such a case the fi ltration or centrifugation of the fi nal 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