PESTICIDE RESIDUE IN THE CRUDE DRUG

Analytical Pharmacognosy

Graptophyllum pictum with and without pesticide

Section 9

10

Pesticide residues in the crude drug and their analysis

Source of contamination, maximal residue level and the determination of the residue

PESTICIDE

PLANTSSOILFRUITSTORAGE

RODENTICIDE

INSECTICIDE

HERBICIDE

FUNGICIDE

MOLUSCIDE

Chemically:

1. Chlorinated Hydrocarbon (aldrin, benzen hexachloride)

2. Chlorinated Phenoxy alkanoate

3. Organo Phospor

4. Carbamate

5. Dithiocarbamate

6. Anorganic Pesticide: Al phosphide, Ca arsenate, Pb arsenate

7. Natural pesticide

8. Mixed PesticideRetain long residual action

Maximum Residue Level

ARL = ADI x 60 kg (bw) x extraction factorMDI x safety factor 100

ARL = Acceptable residue levelMDI = Mean Daily Intake

Theoretical maximally tolerable level = (mg/kg)

ADI (mg/kg) x 60 x extraction factor

Average dailiy intake (kg) x 100

ADI of SOME PESTICIDE(mg/kg bw)

HCBLINDANALDRINDIELDRINENDRINDDT

0,00050,00800,00010,00010,00020,0200

MALATHIONPARATHIONPARAQUATPROPOXURDIAZINONPIRETRIN

0,02000,00500,00400,02000,00200,0400

Example

ADI aldrine dan dieldrine = 0,0001 mg/kg = 0,0060 mg/60kg

MDD of Tongkat Ali = 10 gMRL aldrine and dieldrine in Tongkat Ali =

0,0060 mg =0,010 kg x 1000,006 mg/kg

WHO/PHARM/80.502I. ADI aldrine dan endrine = 0,0001 mg/kg bw

= 0,006 mg/60 kgMRL aldrine dalam Valeriana = 0,006 mg/0,02kg

= 0,3 mg/kg

II. ADI chlordane = 0,001 mg/kg bw= 0,06 mg/60 kg bw

MDD Digitalis = 3 g (BP 1,5 g)MRL = 0,06 mg/0,003 kg = 20 mg/kg

III. ADI DDT = 0,05 mg/kg bw = 3 mg/60 kg bwMDD Valeriana = 20 gMRL = 3 mg/20 g

= 150 mg/kg

MRL of vegetables according to FAOP/WHOAldrine and dieldrine= 0,02 – 0,2 mg/kgChlordane = 0,02 – 0,5 mg/kgDDT = 0,50 – 7,0 mg/kg

Untuk simplisia, MRL tidak perlu sama

dengan batasmaksimal pada

sayuran

For Unknown Pesticide

Calculated toward the most toxic, ex:aldrine C12H8Cl6 Cl = 58,3%dieldrine C12H8Cl6O Cl = 55,85%Approximately 50 %Ex. For Valerian = 50% x 0,3 mg/kg

= 0,15 mg/kg

Method of Determination

SAMPLE

Extraction

Partition/Adsorption

Mixed Pesticide

Non Pesticide components

Determination

POOR RECOVERY

Lost during process

DECOMPOSED/METABOLIZED

UNKNOWN SAMPLE NOT ALWAYS OK

PESTICIDE of UNKNOWN HISTORY

TEST for Pesticide Group

Chlorinated Hydrocarbon

Organo Phospor Heavy Metal, Arsen

DITHIOKARBAMATE

TOTAL CARBON DISULFIDE

TOTAL of ClORGANIC

TOTAL P ORGANIC TOTAL of

Heavy Metal, Arsen

Sample Handling1. Directly analysed,

– Keep in closed container, cooled.– Extracted and keep the extract.

2. Light may decompose the pesticide.3. Direct handling should not deteriorate the sample or create

analytical error. 4. Solvents and reagent must not contain material that disturb

reaction, alter the result, causing degradation of pesticide. 5. The most rapid and simplest method as the priority. 6. Change of specification need to be justified with supporting data.7. Concentrating of the extract must be careful, especially evaporation

to avoid the lost of pesticide residue. Adding oil into the volatile pesticide that does not affect the colorimetric measurement may be conducted to prevent the lost of the pesticide.

Sample

Grind and sieve on 710 or 840

20-50 g + 350 ml acetonitrile-water (65:35)

Shake for 5 min and filter

Residue

Fat, protein, aa, starch, other compounds

Filtrate LCC with PAE 1-2 min + 10 mL NaCl sat + 600 mLwater, shake

Organic phase Water phase

(dispose)Na2SO4 (15 g)

Florisil

22 mm

10 cm

Fill with 40-50 mL PAE

Elution:

1. Ether-PAE (6%) 200 mL(Aldrin, BHC, DDT,PCB)

2. Ether-PAE (15%) 200 mL (endrin, dieldrin, phospate organic, parathion

3. Ether-PAE (50%) 200 mL (malathion)

Destruction of organic compound with burningChlorine chloridaPhosphor ortophosphate

Principle of determination:Extract concentrated dried on sample holder burnt under

atmospheric oxygen gas absorbed in solution Cl-/orthophophate colorimetry.

Equipments: combustion flask1. Erlenmeyer 1 L borosilicate with stopper2. Fuse a platinum wire with 1 mm diameter on the tip of the stopper.3. At the end of the wire stick platinum gauze 36 mesh, 1,5 x 2 cm to

hold compound on the sample holder clear from the absorbing liquid during combustion.

Sample holder• For small amount is made from halide free filter

paper with 5 cm length and 3 cm width• For few mL, is made conical from cellose

acetate with 4 cm diameter. Both side is sealed with hair drier (seam + 5 mm). The seam is immersed with acetone, dried, washed 10 min. with NaOH (240 g/L), wash with water then put in a funnel wit 6.5 cm diameter.

• For P, use halide free filter holder with 4 cm2

size.

Method1. Sample is transferred to conical sample holder ( the

solven must not dissolve the sample holder, such as: acetone).

2. Let it dry.3. With glove, fold sample holder into 1 cm2 and then put in

the center of platinum gauze.4. Insert filter paper 1 x 3 cm as a fuse on top of the holder,

between the folds.5. Wet the flask with water.6. Fill the flask with oxygen through a tube by which the

end of the tube on the surface of the liquid.

7. Ignite the end of the fuse and soon insert the stopper. 8. Hold tightly the stopper. 9. When combustion start, incline the flask to avoid

incompletely burnt material from falling to the liquid.10. Soon after combustion complete, shake for 10’ to

dissolve chlorine. 11. Wash the wall with water. 12. Withdraw the stopper, wash the stopper and the

platinum with water. 13. Transfer into 50 mL flask and adjust the volume.

Determination of Cl-

Principle:• Displacement of thiocyanate by Cl- in Hg(CNS)2. • Sensitivity 0,5 μg Cl-.• With Fe3+ form Fe(CNS)3 stable colour ~ Cl-,

measurement at 460 nm.

Procedure• 15 mL aliquot + 1 mL Fe amonium sulphate (0,25 mL) +

3 mL Hg(CNS)2 mix for 10’ measure at 460 nm.• Calibration curve is prepared using NaCl containing 5

μg Cl per mL, transfer 0, 2, 4, 6, 8, 10 mL into 50 mLflask and measure as that of the sample.

Combustion of Phosphor

• Dip sample holder made from filter paper into NaOHMethanol, suspend in hot air stream.

• Transfer aliquot (+ 0,20 mL) into sample holder, wash the flask with 0,20 mL CHCl3 to complete the transfer.

• Let the solution evaporate and fold into 1 cm2 and insert into the platinum sample holder.

• Insert fuse 1 x 3 cm on top of holder between the folds.• Fill in 10 mL solfuric acid = 37 g/L to the flask.• Ignite as that of Cl- combustion.• Transfer the combustion products into 25 mL flask.

Determination of PhosphatePrinciple:Phosphate + molibdate complex phosphomolibdate

reducedmolibdenumblue intensive at 820 nm

Uncontaminated extract will yield 0,05 - 0,1 ppm.Test result 0,1 ppm does not mean contaminated

Procedure:7 mL in calibrated 10 mL tube + 2,2 mL H2SO4 3 M mix + 0,4 mLammonium molibdate, mix, + 0,4 mL ANSA (aminonaphtosulfonicacid) mix and heat at 100oC for 12 + 2 min. cool and measure at 820 nm

Determination of ArsenMolibdenun blue <-- 1,5 – 15 μgGutzeit:• Less quantitative• For limit test• Simpler than Gutzeit.

Sample preparation:a. 25-70 g in 800-1000 mL Kjeldahl flask, + 25 mL water, 25-50 mL

HNO3 + 20 mL H2SO4 heat + a little of HNO3 untill all organic matter is destroyed.

b. Cool + 75 mL water + 25 mL saturated ammonium molibdate to expel N in the solution, reheated.

c. Cool + water 250 mL

The Scheme of the equipment:Arsenate As + HgBr2 colour

Sensitivity 10 – 30 mg (~0,1 ppm)

HgBr2 paperHole diameter l 6,5 mm

Pb acetate cotton

120 mL

Hole diameter 2 mm

Tip of the hole 1 mm

25-50 mL aliquot, 1 g KI, 10 g Zn, leave for 40 menit70 ml

Determination of Pb

Ash is dissolved in 20 mL HCl (7g/L). Filter and wash with 5 mL water. Filtrate is adjusted to 100 mL.

Solution + 2 mL ammonium citrate, 2 drops methyl red, ammonia, form yellow colour mix and heat for 10-15 min. + 1,5 mL ammonia, + 10 mLditizone-benzen, organic phase + 40 mL KCN, mix for 30 min.

Benzene layer is measured at 525 nm.

Gas Chromatography

Capilary columnDetection system ECDGas: argon-methane (95:5)I. Silica column 30 m (id 0,25

mm), chemically bonded 5% fenil/95% methylpolysyloxane. at 60o (0,5 min.), increase by 30o/min. up to 180oC (2 min.), increase 2o/min. to 250o (5 min.).Injection temperature 240oCDetector temp. 300oC

II. Silica column 15 m (id 0,25 mm), chemically bonded 7% cyanopropyl, 7% phenyl, 86% methyl-polysyloxane. Temp. 60o (0,2 min), increase 30o/min. to 180oC (1 min.), increase 2o/min. to 250o (5 min.).Injection: on column injection with 1 mL volume. Detector temp. 300oC

Further ReadingBeltran, J. , F. J. López and F. Hernández (2000) Solid-

phase microextraction in pesticide residue analysis, J. Chromatography 885, 389-404.

Bao-Huey Hwang and Maw-Rong Lee (2000) Solid-phase microextraction for organochlorine pesticide residues analysis in Chinese herbal formulations, J. Chromatography (2000): 898, 245-256.

Y. Picó, , G. Font, J. C. Moltó and J. Mañes (2000) Pesticide residue determination in fruit and vegetables by liquid chromatography –mass spectrometry , J. Chromatography : 882, 153-173

Steve Schachterle and Carl Feigel (1996)Pesticide residue analysis in fresh produce by gas chromatography-tandem mass spectrometry, J. Chromatography : 754, 411-422

• 19 pesticide (fungiside, herbiside, organic phosphor and chlor pesticides) were added tp 7 kind of fruits and vegetables. Analysis by GC-MS-MS. Spectrum is used for confirmation, detection limit 1-5 ppb.

• Pestisida β-, γ- and δ-hexachlorocyclohexane, p,p′-DDD, p,p′-DDE, p,p′-DDT, o,p′-DDT, aldrin, dieldrin, endrin, endrin aldehyde, endrin ketone, endosulfan (I, II and sulfate), heptachlor, heptachlor epoxide, and methoxychlor SPME–GC–MS. Batas deteksi: < 1 ng/g sampel.

• solid-phase microextraction (SPME) is frequently used in sample preparationGC, GC-MS, GLC MS.