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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.