NERIUM OLEANDER  Chemical Safety Poisons Information Monograph  

searcher & illustrated by : Karint NymPh : 10 Dec 2006 

for more detail : Click     http://www.LunarCot.co.th 

1.   NAME 

        1.1  Scientific name     Nerium oleander L.

        1.2  Family                  Apocynaceae

        1.3  Common name(s) and synonyms

Adelfa (Puerto Rico, Spain); Alheli Extranjero (Puerto Rico);

Baladre (Catalunya-Spain); Espirradeira (Brazil);

Flor de Sao Jose (Brazil); Laurel de jardín (Argentina Uruguay);

Laurel rosa (Argentina, Uruguay); Laurier rose (France);

Loandro (Brazil); Loureiro rosa (Brazil); Oleana (Hawaii);

Oleander (Brazil, UK, USA); Oliwa (Hawaii); Rosa Francesa (Cuba);

Rosa Laurel (Mexico); Rose bay (UK).  

2.  SUMMARY

2.1     Main risks and target organs oleander ingestion causes both cardiac and gastrointestinal signs and symptoms.  The main toxic principles are the glycosides, which induce cardiac arrhythmia and eventually death.

2.2     Summary of clinical effects The clinical picture usually starts with gastrointestinal signs:  nausea, vomiting, abdominal pain and diarrhoea.  Later, cardiovascular and neurological symptoms can occur.  Sinus bradycardia or different degrees of atrioventricular (AV) block are the most frequent cardiac features.  In severe cases, ectopic beats occur which may be followed by ventricular tachycardia and fibrillation.  The main neurological symptoms are: tremor, drowsiness and ataxia.  Hypotension and unconsciousness may also occur. Seizures have been described.

2.3         Diagnosis is based on the clinical features (malaise, vomiting, abdominal pain, bradycardia, dizziness), electrocardiographic changes and glycosides investigated by digoxin immunoassay.  The triad of gastrointestinal distress, circumoral erythema and cardiac dysrhythmia should alert to the possibility of N. oleander poisoning. A plant specimen or parts obtained from gastric lavage or vomiting should be collected for botanical identification. N. oleander can be recognized by microscopic examination of its epidermis, where stoma cells are typical.

2.4     First-aid measures and management principles.  Treatment should aim at:

- gut decontamination by means of emesis or lavage;

- correction of electrolyte imbalance;

- correction of severe bradycardia with atropine or electrical pacing

- corrections of ventricular dysrhythmias;

- administration of digoxin-specific Fab antibodies could be considered if available.

2.5     Poisonous parts : All parts, including the sap, either fresh, dried or boiled, are toxic.

A single leaf intensively chewed has been reported to be lethal.

2.6     Main Toxins Poisonous Principles are cardiac glycosides.

The one most studied is oleandrin, but there are more than ten other glycosides whose chemical structure is well known:        5 ß-cardenolides, such as oleandrine;

                                          5 a-cardenolides, such as uzangenin-type;

                                          oleandrigenin (16-Acetylgitoxigenin) (ol). 

The "ol" being: oleandroside (oleandrin); glucose (glucosyloleandrin); gentiobioside (gentiobiosyl oleandrin); diginoside (nerigoside); digitoxigenin (digitalose/diginose); oleagenine - oleasides (A,E) (diginose, gentiobiose-diginose).

Adyregenin is a compound with no cardiac effect.

3.  CHARACTERISTICS

3.1  Description of the plant

3.1.1    Special identification features oleander is an evergreen shrub reaching four metres in height. Leaves are 10 to 22 cm long, narrow, untoothed and short-stalked, dark or grey-green in colour.  Some cultivars have leaves variegated with white or yellow.  All leaves have a prominent mid rib, are "leathery" in texture and usually arise in groups of three from the stem.  The plant produces terminal flower heads, usually pink or white, however, 400 cultivars have been bred and these display a wide variety of different flower colour: deep to pale pink, lilac, carmine, purple, salmon, apricot, copper, orange and white (Huxley, 1992).  Each flower is about 5 cm in diameter and five-petalled.  The throat of each flower is fringed with long petal-like projections.  Occasionally double flowers are encountered amongst cultivars.  The fruit consists of a long narrow capsule 10 to 12 cm long and 6 to 8 mm in diameter; they open to disperse fluffy seeds.  Fruiting is uncommon in cultivated plants.  The plant exudes a thick white sap when a twig or branch is broken or cut (Font-Quer, 1974; Schvartsman, 1979; Lampe & McCann, 1985; Pearn, 1987).

3.1.2  Habitat  Where the species grows in the wild (i.e. in the Mediterranean), it occurs along watercourses, gravely places and damp ravines.  It is widely cultivated particularly in warm temperate and subtropical regions where it grows outdoors in parks, gardens and along road sides.  Elsewhere, where the plant is not frost-tolerant (e.g. in central and western Europe), it may be grown as a conservatory or patio plant.

3.1.3  Distribution  Oleander is cultivated worldwide as an ornamental plant; it is native only in the Mediterranean region (Kingsbury, 1964; Hardin & Arena, 1974).

3.2  Poisonous parts of the plant - The whole plant, including the sap, is toxic.

3.3  The toxin(s)

3.3.1  Name(s) Oleandrin, a glycoside, is the main toxin.  Its chemical name is

16b-acetoxy-3b-[(2,6 dideoxy-3-0-methyl-a2-L-arabino-hexopyranosyl) oxy]-14-hydroxy-5ß, 14ß-card-20(22)-enolide (Reynolds, 1989).

3.3.2  Description, chemical structure, stability  Oleandrin:

CAS number:  465-16-7

Structural formula:  C32H48O9

Molecular weight:  576.7

Structural name:  oleandrin

3.3.3  Other physico-chemical characteristics Oleandrin forms colourless, odourless, acicular crystals which are very bitter (Shaw & Pearn, 1979).  The concentration of oleandrin in the plant tissues is approximately 0.08% (Schvartsman, 1979).

Solubility:  insoluble in water; it has little resistance to light but it is heat-stable (Pearn, 1987; Reynolds, 1989).

3.4         Other chemical contents of the plant : Oleander contains at least 2% cardiac glycosides.

Rosagenin may be extracted from the bark and has a strychnine-like action.  

Several flavones (0.5%) and volatile oils (unimportant amount), as well as rubber, fats, sugars and hydrocyanic acid, can be isolated from its leaves (Schvartsman, 1979; Shaw & Pearn, 1979; Pearn, 1987).

    4.  USES/CIRCUMSTANCES OF POISONING

4.1  Uses

4.1.1  Uses

         Miscellaneous pharmaceutical product.  Other therapeutic preparation

4.1.2  Description

        Preparations containing the active principles were used formerly as rodenticides, insecticides, and as remedies for indigestion, fever, ringworm, malaria, leprosy, venereal diseases and as abortifacients.  Therapeutic use of oleander glycosides as cardiac drugs were assessed and documented in the 1930s (Shaw & Pearn, 1979; Osterloh et al., 1982).  The USSR pharmacopoeia contains an oleandrin solution (solution Neriolini) and oleandrin tablets, and oleandrin solution (oleandrin 22 mg, alcohol 74 mL, distilled water to 100 mL).  This contains 7 to 9 'frog units' per mL (1 mg oleandrin = 34 to 44 frog units).  Oleandrin tablets each contain 100 mg of the active principle (Reynolds, 1989).

4.2     High risk circumstances

The high risk circumstances of exposure are: children playing with the ornamental shrub (tasting, chewing, ingesting portions of the plant), and inappropriate medicinal use of plant infusion.  Intentional poisoning by ingestion of plant infusions has been reported.

4.3     High risk geographical areas

Although the plant is native to the Mediterranean basin,

it has been introduced as an ornamental shrub in many tropical and subtropical countries (Mahin et al., 1984).  The plant is common in Australia and southern Africa and ingestion of plant parts is one of the major causes of childhood admission to hospital (Shaw & Pearn, 1979).

    5.  ROUTES OF EXPOSURE

         5.1     Oral

Parts of the plant can be ingested accidentally or in suicide attempts (Jouglard et al., 1973; Pronczuk & Laborde, 1988).  Extracts or herbal teas made of N. oleander have              been ingested for suicidal or medicinal purposes (Haynes et al., 1985; Blum & Rieders, 1987).  Ingestion of water contaminated with N. oleander leaves (from a flower bowl),              meat cooked or stirred with the stems, and honey made by bees visiting the flowers have produced toxic effects (Hardin & Arena, 1974).

        5.2      Inhalation

Smoke from burning N. oleander may be toxic (Hardin & Arena, 1974;  Shaw & Pearn, 1979).

        5.3      Dermal

                  No data available.

        5.4      Eye

                  No data available.

        5.5      Parenteral

                  No data available.

        5.6      Others

                  A fatal case, following rectal and oral administration of N. oleander extract, has been described (Blum & Rieders, 1987).

    6.  KINETICS

6.1     Absorption by route of exposure.  Oleandrin is well absorbed orally (Schvartsman, 1979).

6.2     Distribution by route of exposure. 

         Wide body distribution is expected: high concentrations of oleandrin have been measured in blood, liver, heart, lung, brain, spleen and kidney in a fatal case of N. oleander extract poisoning (Blum & Rieders, 1987).

6.3         Biological half-life by route of exposure

         No data available.

6.4     Metabolism

         No data available.

6.4         Elimination and excretion. 

         Oleandrin is eliminated very slowly from the body (one to two weeks)

         (Shaw & Pearn, 1979).

    7.  TOXINOLOGY

7.1     Mode of Action

       The cardiac effects of the glycosides are due to direct cardiotoxicity and an indirect effect via the vagal nerve.  The direct effect is due to the inhibition of the Na-K ATP-ase pump sodium-potassium adenosine triphosphatase enzyme system).  This specific action increases intracellular sodium ion and serum potassium concentrations.  The sodium influx lowers the membrane potential threshold, increasing excitability.  The chronotropic effect is primarily central, mediated by an increase of vagal tone which decreases the rate of sinoatrial node depolarization (Osterloh et al., 1982).

        7.2  Toxicity

               7.2.1  Human data

                        7.2.1.1    Adults  :  Between 7 to 20, or a "handful", of the leaves have been ingested by adult patients who were intoxicated but recovered completely;  the dose of oleandrin in this sample is not known (Pronczuk & Laborde, 1988; Shumaik et al., 1988).

                        7.2.1.2  Children  :  A single leaf may be lethal to a child (Shaw & Pearn, 1979).

7.2.2  Relevant animal data

         One leaf of N. oleander may be sufficient to kill a sheep (Shaw & Pearn, 1979).  The lethal dose of N. oleander leaves reported for several animal species is about 0.5 mg/kg.  Animals poisoned by eating the plant often develop bloody diarrhoea, due to a direct effect on the gastrointestinal tract.  The bitter and astringent taste of the plant's leaves means that all but starving creatures are likely to be exposed. (Szabuniewicz et al., 1971; Pearn, 1987).

             7.2.3  Relevant in vitro data

                        No data available.

        7.3  Carcinogenicity

               No data available.

        7.4  Teratogenicity

               No data available.

        7.5  Mutagenicity

               No data available.

7.6  Interactions : Pre-medication with digoxin or other cardiac glycosides may increase the severity of poisonings.  The well-known interaction between digoxin and quinidine (increasing digoxin levels) should be considered.

8.  TOXICOLOGICAL ANALYSES AND BIOMEDICAL INVESTIGATIONS

8.1  Material sampling plan

      8.1.1  Sampling and specimen collection 

               8.1.1.1  Toxicological analyses

Suspect material, scene residues, vomited or washed out gastric contents should be collected for analysis, as well as preparations containing N. oleander.  Plasma samples should be taken for the digoxin immunoassay. 

Morphology and anatomy:  pecimens of the leaves, flowers and branches should be collected for botanical and pharmacognostical identification. 

Suspect material and plant portions found in vomit or scene residues should be taken for the macroscopic and microscopic analysis. 

               8.1.1.2  Biomedical analyses

Blood and urine should be collected for routine biochemical analysis.  Serum samples should be taken for measurements of the serum potassium concentration. 

               8.1.1.3  Arterial blood gas analysis

Blood should be collected for arterial blood gas analysis in unconscious                             patients. 

               8.1.1.4  Haematological analyses

Blood should be collected for routine haematological analyses. 

               8.1.1.5  Other (unspecified) analyses

                          No data available.

         8.1.2  Storage of laboratory samples and specimens 

                    8.1.2.1  Toxicological analyses

Biological samples should be stored at 4°C in a refrigerator.  For macroscopic and microscopic analysis fresh plant material should be kept between sheets of newspaper.  Dried plant material should be protected from moisture.  Plant portions found in vomit or scene residues have to be stored in clean bottles or sealed plastic bags. 

                    8.1.2.2  Biomedical analyses

                          Biological samples should be stores at 4°C in a refrigerator. 

                    8.1.2.3  Arterial blood gas analysis

                          Storage is not possible. 

                    8.1.2.4  Haematological analyses

                           Samples should be stored at 4°C in a refrigerator. 

                    8.1.2.5  Other (unspecified) analyses

                           No data available.

             8.1.3  Transport of laboratory samples and specimens 

                    8.1.3.1  Toxicological analyses

No special requirements.

For macroscopic and microscopic analysis: no special requirements. 

                    8.1.3.2  Biochemical analyses

                          No special requirements. 

                    8.1.3.3  Arterial blood gas analysis

                          Transport to the laboratory immediately. 

                    8.1.3.4  Haematological analyses

                           No special requirements. 

                    8.1.3.5  Other (unspecified) analyses

                           No data available.

        8.2  Toxicological Analyses and their Interpretation

             8.2.1  Tests on toxic ingredient(s) of material 

                    8.2.1.1  Simple Qualitative Test(s) 

A.  Identification of the plant parts (additional to 3.1.1) The powdered leaves are characteristically a light yellow green in colour and taste bitter.

      Anatomical:  The lower surface of the leaf has characteristic abaxial grooves containing (single-cell hair) and vimicelled trichomes. The cells of the mesophyllum contain calcium oxalate crystals (35 µm).   

                             B.   Identification of toxic, active ingredients in plant or preparation   

(i) Specific reaction:

- Principle of test (or method): In general cardenolides form with aromatic nitro compounds in alkaline solution (e.g. Kedde-reagent) typical and very specific re, violet-red or blue-violet products                             (Meisenheimer adducts).

- Sampling: plant parts, (preferably leaves, flowers, fruit).

- Chemicals and reagents   

                          Chemicals:

                             *  dioxane

                             *  dichloromethane (methylene chloride)

                             *  3,5-dinitro benzoic (C2H4N2O6)

                             *  ethanol

                             *  methanol

                             *  2 N NaOH (8,4 g NaOH dissolved in 100 mL water)

                             *  oleandrine

                             *  oleander glycoside mixture -1 for TLC- comparison

                        Reagents:

*  Keddle-reagent: dissolve 3g of 3,5-dinitro benzoic acid in ethanol.  Take 5 mL and mix with 5 mL of a 2 N NaOH take directly before use.

Equipment:

*  round bottle flask, 50 mL

*  pipettes 0.1 mL and 0.5 mL

*  test tubes

*  filter-paper or silica gel plate (5 × 5 cm)

*  Extrelut R- column (20 mL, Nr. 11737 Merck AG, D-6100

                             *  Darmstadt

            - Test sample (or specimen) preparation

*  Extract 1 g of material (suspect material, scene residues) with 20 mL methanol 50% for 15 minutes under reflux on a water bath.

                             *  Filtrate and evaporate the extract to a volume of 10 mL.

                             *  Apply the extract on an ExtrelutR- column

                                 and wait for 20 minutes.

*  Extract the retain material with 60 mL of dioxan-methanol-dichloromethane [8+1+1].

                             *  Collect the extract and evaporate to 10 mL.

Reference compound

Dissolve 10 mg of oleandrine in 10 mL ethanol

Dissolve 10 mg of the "Oleander glycoside mixture" in 10 mL ethanol.

            - Procedure:

                            *  Spot 100 µL of the extract on a filter-paper

                                 or on a silica gel plate [5 × 5 cm]

                            *  Add 5 drops of the Keddle-reagent

                            *  An immediate blue, red or violet-blue colour indicates the

                                presence of cardenolides (Colour vanish). 

                                Analysis calibration procedure and calculation of results not necessary. 

  Quality control

Carry out a blank test by taking 100 µL of water instead of the sample.

   Carry out a positive control test by taking 100 µL of a cardenolide solution such as oleandrin in ethanol.   

- Specificity :  A specific reaction with the lactone ring of the cardenolide -molecule.  Cardenolides respond with a blue, red or violet- blue colour reaction.   

                          - Analytical performance: detection limit 3 µg of a cardenolide.   

- Analytical assessment of the result: a positive result indicates the presence of a cardenolide.   

   - Medical interpretation of the result: consider the possibility of ingestion of cardenolides.

                             (ii) Thin Layer Chromatography   

                             - Principle of test:

                             TLC separation of the Oleander cardenolide mixture, (Wagner & Bladt, 1995), detection in

                             UV-254 and detection of the cardenolide zones with Kedde-reagent.   

                             - Sampling: 1 to 2 g of the suspect material or residue.   

                             - Chemicals and reagents

                             Chemicals:   

                             *  chloroform

                             *  dioxane

                             *  dichloromethane (methylene chloride)

                             *  3,5-dinitro benzoic acid

                             *  thyl acetate

                             *  methanol

                             *  2 M sodium hydroxide (8,4 g NaOH dissolved in 100 mL water)

                             *  sulfuric acid (conc.)

                             *  water

                             *  TLC-Silicagel 60 F 254 plates (with fluorescence marker) (e.g. Merck

                                Darmstadt, Roth D 7500 Karlsruhe Nr. 2-8867 and 2-8864)

                             *  oleandrine

                             *  oleander glycoside mixture -1 for TLC-

                                comparison

                             Reagents:  

                             *  Keddle-reagent: dissolve 3 g of 3,5-dinitro benzoic acid

                             *  in ethanol.

                             *  Take 5 mL and mix with 5 mL of a 2 M NaOH directly

                             *  before spraying.   

                             *  Solvent system for TLC: ethylacetate-methanol-water

                                [81+11+8]

                             *  Reference compounds:

                                Dissolve 10 mg of oleandrine in 10 mL ethanol

                                Dissolve 10 mg of lanatoside C in 10 mL ethanol

                                Dissolve 10 mg of the "Oleander glycoside mixture" in 10 mL ethanol.

                             - Equipment:   

                             *  round bottle flask 50 mL

                             *  TLC tank

                             *  UV lamp (254 nm)

                             *  fume cupboard

                             *  spray apparatus

                             - Test sample (or specimen) preparation   

                             *  Extract 1 g of material (suspect material, scene residues) with 20 mL methanol (50%)

                                for 15 minutes under reflux on a water bath.

                             *  Filtrate and evaporate the extract to a volume of 10 mL.

                             *  Apply the extract on an Extrelut20 -column and wait for minutes.

                             *  Extract the retained material with 60 mL of dioxanmethanol- dichloro methane

                                [8+1+1].

                             *  Collect the extract and evaporate to 10 mL. 

                             *  Reference compounds

                                Dissolve 10 mg of oleandrine in 10 mL ethanol

                                Dissolve 10 mg of lanatoside C in 10 mL ethanol

                                Dissolve 10 mg of the "Oleander glycoside

                                mixture" in 10 mL ethanol.

                             - Procedure:

                             *  Apply 20 µL of each cardenolide reference

                                solution to the TLC plate along the

                                baseline.

                             *  Apply 20 µL and 40 µL of the test sample

                                preparation to the TLC plate along the

                                baseline.

                             *  Prepare the TLC tank with the solvent

                                mixture.

                             *  Place the prepared TLC plate in the tank

                                and allow to run for 12 cm

                             *  Take the TLC plate out and allow to dry.

                             *  Detection

Place the plate under UV lamp (UV-254 nm) to visualize the spots and mark with a pencil.  Cardenolides show weak fluorescence quenching.  Spray the TLC with Kedde-reagent and immediately blue, violet-blue and red-violet spots indicate the presence of cardenolides.  Plant extracts show 8-10 Kedde-positive zones in the R-range 0.1 - 0.9.

Another re-agent used may be conc. Sulfuric acid (unspecified reaction).  Mostly red to red-brown spots indicate the presence of cardenolides.  Plant extracts show at least 8-10 red zones in the R-range 0.1-0.9.  Calculate the Rf-values of the zones and compare the Rf-values with the reference compound and the Rf-values of the cardenolid compounds given in table 2.  Analytical calibration procedure and calculation or results is not applicable.

                                - Quality control:  Carry out positive control test by taking

                                  20 µL of a cardenolide solution such as oleandrine in ethanol.

                                Compare the TLC-fingerprint of an authentic N. oleander cardiac

                                glycosides-extract.

                                - Specificity:

                                A specific reaction with the lactone ring of the cardenolide-structure.

                                Cardenolides respond with blue, red or violet-blue colour.

                                - Analytical performance:  detection limit 3 µg of a cardenolide.   

- Analytical assessment of the result:  a positive result indicates the presence of a cardenolide.   

- Medical interpretation of the result: consider the possibility of ingestion of cardenolides.

8.2.1.2          Advanced Qualitative Confirmation Test(s)

              With the HPLC-method a cardenolide profile of oleander glycosides in plant extracts or oleander-preparations can be achieved.  The analysis is performed on a RP-18 column by gradient elution (acetonitrile -               water) and detection in UV-220 nm. (Tittel & Wagner, 1981).

               Depending on habitat, distribution and time of harvest, the cardenolide content in leaves of different origin shows marked differences in the distribution of the major glycosides.

                    8.2.1.3  Simple Quantitative Method(s)

-  Principle of test:  The purified cardenolide fraction of suspect material or scene residue, is measured after addition of Kedde-reagent at its absorption maximum (540 nm). 

- Sampling: 2 g plant material, suspect material, scene residue, stomach content

- Chemicals and reagents: chemicals (analytical grade)

*  3,5-dinitro benzoic acid (C2H4N2O6)

*  ethanol

*  methanol

2 N NaOH (8,4 g of a NaOH are dissolved in 100 mL of water)

*  water (distilled)

*  Aluminium oxide (Al2O3) acidic

Reagents:

*  Kedde-reagent:  Dissolve 3 g of 3,5-dinitro benzoic acid in ethanol

    Take 5 mL and mix with 5 mL of a 2 N NaOH directly before spraying.

                             Equipment:

                             flasks: 5 mL and 20 mL

                             volumetric flasks: 25 mL and 50 mL

                             pipettes: 1,2,3,4 mL

                             chromatographic column [15 mm diameter,

                             200 mm length]

                             with tap and glass filter (medium porosity

                             grade)

                             timer: 2 minutes

                             spectrophotometer visible

                             glass cuvettes (1 cm path length)

                             Test sample (or specimen) preparation:

                                       - Weigh a precise amount of powdered plant material (2 g).

                                       - add 35 mL of mixture of 24 mL ethanol + 36 mL water.

                                       - heat for 20 minutes under reflux.

                                       - filtrate after cooling through filter-paper.

                                       - wash with residue twice with 5 mL portions of the ethanol

- water mixture (24+36).

                                       - combine the filtrates and add the ethanol

- water mixture to mark in a volumetric flask 50 mL.

                             Preparation of the column:

- 4.2 g Al2O3 and 20 mL water are shaken in a flask for approximately 5 minutes.

- after standing for 5 minutes the supernatant water is discarded.

- wash four times the Al2O3-material with 50 mL portions of ethanol.

- discard the ethanol. add 50 mL of ethanol, shake well and transfer  the Al2O3 material into the column.

-  wait for 15 minutes, then add 10 mL ethanol.

-  open the tap and discard the ethanol till approximately 1/2 cm above the column material (Al3O2).  Close the tap.

Procedure:

-  apply exactly 2 mL of the filtrate to the column.

-  eluate 4 times with O.5 mL ethanol and finally with 16 mL ethanol.

-  collect the cardenolide containing fractions in a volumetric flask and make up to 25 mL volume with ethanol and 4 mL Kedde-reagent in a flask as blank (solution).

-  take exactly 4 mL of the cardenolide fraction and add 4 mL Kedde-reagent, mix thoroughly.

-  transfer into the glass cuvette.

-  read the absorbence of the test solution exactly 2 minutes (with timer) after addition of the reagent against the blank at 540 nm as the coloration is time-dependent!

-  Analytical calibration procedure and calculation or results:

-  calibrate with four standards of a solution of 10 mg oleandrine 100 mL ethanol.

-  take 1/2/3/4 mL and add each time 4 mL of the Kedde-reagent.

-  measure each solution exactly after 2 minutes at 540 nm against the blank.

-  construct a calibration function.

-  compare the absorbence of the test solution with the calibration function.

-  alculate the oleandrin amount as:

     a × 1562.5

          % =      ----------

     e × (100-t)

a = mg oleandrin found in the calibration function

e = sample material in analytical solution

t = loss of liquid (%)

   (t = weigh a precise amount of powdered plant material (1 g) and heat: at 110°C till the material has a constant weight:  the difference in weight indicates the loss of liquid of the material).

-  Quality control: It is important that test and reference solution are measured exactly after 2 minutes after adding the Kedde-reagent (coloration is time dependent!).   

-  Specificity: the method is generally applicable for cardenolides.  The total cardenolides are calculated as oleandrine.   

-  Analytical performance : Mean coefficient of variation 6%. Detection limit is 1 µg of cardenolide.   

-  Analytical assessment of the result

   Consider the presence of cardenolides.   

-  Medical interpretation of the result

   Consider the possibility of ingestion of cardenolides.

8.2.1.4  Advanced Quantitative Method(s)

           Methods for toxic, active ingredients.  With the HPLC-method of external standard a quantitative estimation of the oleander glycosides can be achieved.  The analysis is performed on a RP-18 column isocratic and by gradient elution (acetonitrile-water) and detection in UV-220 nm. (Tittel & Wagner, 1981).

column      µ-Bondapak .. C-18 (300 × 4 mm ID)

                  flow        2 mL/minutes

detection   220 nm

solvent     acetonitrile-water (40+60) isocratic

                  oleandrin   Rt 12.5 minutes

                  adynerin    Rt 14 minutes

            Quantitative estimation of glycosides other than oleandrine and adynerine is possible by linear gradient elution (20 to 50%                              acetonitrile in 45 minutes).

8.2.2     Tests for biological specimens

                    8.2.2.1  Simple Qualitative Test(s)

                    8.2.2.2  Advanced Qualitative Confirmation Test(s)

                    8.2.2.3  Simple Quantitative Method(s)

                    8.2.2.4  Advanced Quantitative Method(s)

                    8.2.2.5  Other Dedicated Method(s)

8.2.3     Interpretation of toxicological analyses

8.3     Biomedical investigations and their interpretation

8.3.1  Biochemical analysis

8.3.1.1  Blood, plasma or serum

                             "Basic analyses"

                             "Dedicated analyses"

                             "Optional analyses"

           8.3.1.2  Urine

                             "Basic analyses"

                             "Dedicated analyses"

                             "Optional analyses"

          8.3.1.3  Other fluids

8.3.2  Arterial blood gas analyses

8.3.3  Haematological analyses

                    "Basic analyses"

                    "Dedicated analyses"

                    "Optional analyses"

             8.3.4  Interpretation of biomedical investigations

8.4     Other biomedical (diagnostic) investigations and their interpretation

8.5     Overall interpretation of all toxicological analyses and toxicological investigations

Sample collection : Plant specimens should be collected for botanical            identification if possible.  They should be transported between two newspaper sheets (preferably the leaves and flowers).  Plant portions found in vomit should be stored in a plastic bag.

Biomedical analysis : ECG is valuable for diagnosis, prognosis and treatment since cardiac effects similar to digitalis glycosides may occur (Goldfrank et al., 1994).  The serum potassium concentration should be measured because of its prognostic importance.

Toxicological/toxinological analysis :

Plasma samples should be taken for digoxin immunoassay. In a mild case of N. oleander poisoning the apparent digoxin level was 1.5 ng/mL (Shumaik et al., 1988).  In a fatal case of N. oleander poisoning "digoxin" level was 5.8 mcg/l.  A cross reactivity between oleander glycosides was demonstrated in a radioimmunoassay. Using this method, 5.8 mcg/l of digoxin corresponds to 580 mcg/l of total cardiac glycoside concentration (Osterloh et al., 1982).  Cheung et al. (1984) found radioimmunoassay to be a rapid and convenient method to confirm N. oleander poisoning, but the apparent "digoxin" concentration was not proportional to the              cardiac glycoside concentration.

               Test for active ingredient

Immunoassay is a rapid and convenient method for confirming ingestion of N. oleander.  The competitive immunoassay method allows rapid screening of suspected specimens.  This method is also useful for confirming the presence of cardiac glycosides in serum (Radford et al., 1986).  Cross reactivity has been demonstrated between the cardiac glycosides in N. oleander and the digoxin radioimmunoassay (Osterloh et al., 1982; Haynes et al., 1985).  Oleandrin can be detected by thin layer chromatography, with location by fluorescence and chromogenically by means of p-anisaldehyde.

Quantification can be performed by fluorescence spectrophotometry with excitation at 355 nm and fluorescence scanning from 340 to 580 nm (Blum & Rieders, 1987).

Test for biological sample Parts of the plant can be confirmed by microscopic examination of the epidermis, where stoma cells