PESTICIDE RESIDUE IN THE

CRUDE DRUG
Analytical Pharmacognosy
Graptophyllum pictum with and without pesticide
 Section 9
Pesticide residues in the Source of contamination,
crude drug and their maximal residue level and
10 analysis the determination of the
residue
 PLANTS
SOIL
FRUIT
STORAGE
PESTICIDE

Chemically:
1. Chlorinated Hydrocarbon (aldrin,
benzen hexachloride)
RODENTICIDE
2. Chlorinated Phenoxy alkanoate
INSECTICIDE
3. Organo Phospor
HERBICIDE
4. Carbamate
FUNGICIDE
5. Dithiocarbamate
MOLUSCIDE
6. Anorganic Pesticide: Al phosphide,
Ca arsenate, Pb arsenate
7. Natural pesticide
Retain long residual
action 8. Mixed Pesticide
Maximum Residue Level

ARL = ADI x 60 kg (bw) x extraction factor

MDI x safety factor 100
ARL = Acceptable residue level
MDI = Mean Daily Intake

Theoretical
ADI (mg/kg) x 60 x extraction factor
maximally
tolerable level = Average dailiy intake (kg) x 100
(mg/kg)
 ADI of SOME PESTICIDE
(mg/kg bw)

HCB 0,0005 MALATHION 0,0200

LINDAN 0,0080 PARATHION 0,0050
ALDRIN 0,0001 PARAQUAT 0,0040
DIELDRIN 0,0001 PROPOXUR 0,0200
ENDRIN 0,0002 DIAZINON 0,0020
DDT 0,0200 PIRETRIN 0,0400
Example
ADI aldrine dan dieldrine = 0,0001 mg/kg
= 0,0060 mg/60kg
MDD of Tongkat Ali = 10 g
MRL aldrine and dieldrine in Tongkat Ali =
0,0060 mg =
0,010 kg x 100
0,006 mg/kg
 WHO/PHARM/80.502
I. ADI aldrine dan endrine = 0,0001 mg/kg bw
= 0,006 mg/60 kg
MRL 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) Untuk simplisia, MRL
MRL = 0,06 mg/0,003 kg = 20 mg/kg
tidak perlu sama
III. ADI DDT = 0,05 mg/kg bw = 3 mg/60 kg bw dengan batas
MDD Valeriana = 20 g maksimal pada
MRL = 3 mg/20 g
= 150 mg/kg
sayuran

MRL of vegetables according to FAOP/WHO

Aldrine and dieldrine= 0,02 – 0,2 mg/kg
Chlordane = 0,02 – 0,5 mg/kg
DDT = 0,50 – 7,0 mg/kg
For Unknown Pesticide

Calculated toward the most toxic, ex:

aldrine C12H8Cl6 Æ Cl = 58,3%
dieldrine C12H8Cl6OÆ Cl = 55,85%
Approximately 50 %
Ex. For Valerian = 50% x 0,3 mg/kg
= 0,15 mg/kg
Method of Determination
SAMPLE

Extraction
POOR RECOVERY
Lost during process

Partition/Adsorption DECOMPOSED/ME
TABOLIZED

Mixed Non Pesticide

Pesticide components
UNKNOWN
SAMPLE NOT
ALWAYS OK
Determination
PESTICIDE of UNKNOWN
HISTORY
TEST for Pesticide Group

Chlorinated Organo Phospor Heavy Metal,

Hydrocarbon Arsen

TOTAL P
TOTAL of Cl TOTAL of
ORGANIC
ORGANIC Heavy Metal,
Arsen
DITHIOKARBAMATE

TOTAL CARBON
DISULFIDE
Sample Handling
1. 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 Filtrate LCC with PAE 1-2 min +
10 mL NaCl sat + 600 mL
water, shake
Grind and sieve on
710 or 840

Organic phase Water phase

20-50 g + 350 ml (dispose)
acetonitrile-water Na2SO4
(65:35) (15 g)

Fill with 40-50 mL PAE

Shake for 5 min 22 mm
and filter Elution:
1. Ether-PAE (6%) 200 mL
(Aldrin, BHC, DDT,PCB)
Florisil
10 cm
Residue 2. Ether-PAE (15%) 200
mL (endrin, dieldrin,
Fat, protein, aa,
phospate organic,
starch, other
parathion
compounds
3. Ether-PAE (50%) 200
mL (malathion)
Destruction of organic compound
with burning
Chlorine Æ chlorida
Phosphor Æ ortophosphate

Principle of determination:
Extract concentrated Ædried on sample holder Æ burnt under
atmospheric oxygen Æ gas absorbed in solutionÆ Cl-
/orthophophateÆ colorimetry.

Equipments: combustion flask

1. Erlenmeyer 1 L borosilicate with stopper
2. 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.
Method
1. 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 mL
flask and measure as that of the sample.
Combustion of Phosphor

• Dip sample holder made from filter paper into NaOH

Methanol, 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 Phosphate
Principle:
Phosphate + molibdate Æ complex phosphomolibdate
Æ reduced
Æ molibdenum
Æ blue 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 mL
ammonium molibdate, mix, + 0,4 mL ANSA (aminonaphtosulfonic
acid) mix and heat at 100oC for 12 + 2 min. cool and measure at 820
nm
Determination of Arsen
Molibdenun blue <-- 1,5 – 15 μg
Gutzeit:
• 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 Æ 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 Æ 250 mL
The Scheme of the equipment:
Arsenate Æ As + HgBr2 Æ colour

Sensitivity 10 – 30 mg (~0,1 ppm)

HgBr2 paper
Hole 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,
70 ml
leave for 40 menit
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 mL
ditizone-benzen, organic phase + 40 mL KCN,
mix for 30 min.
Benzene layer is measured at 525 nm.
Gas Chromatography
Capilary column
Detection system ECD
Gas: 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 240oC
Detector 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 Reading
Beltran, 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
SPME–GC–MS. Batas
methoxychlor Æ
deteksi: < 1 ng/g sampel.
• solid-phase microextraction (SPME) Æ is
frequently used in sample preparationÆ
GC, GC-MS, GLC MS.