10.1.

02
AOAC Official Method 985.22
Organochlorine
and Organophosphorus Pesticide Residues
Gas Chromatographic Method
First Action 1985
Final Action 1986
(Applicable to residues of acephate, -BHC, chlorpyrifos, dieldrin,
monocrotophos, and omethoate in lettuce, strawberries, and toma-
toes.)
A. Principle
Nonfatty sample is blended with acetone and filtered; pesticides
are transferred from aqueous filtrate to organic phase by shaking
with petroleum ether and CH
2
Cl
2
; after drying, organic phase is
concentrated in presence of petroleum ether and then acetone to
remove CH
2
Cl
2
; aliquot of concentrated organic phase is injected
into various GC systems for determination of wide variety of pesti-
cide residues.
Absence of cleanup steps permits examination for residues of
many chemical types, including many that would not be recovered
through methods requiring Florisil or charcoal column chroma-
tographic step.
B. Reagents and Apparatus
(Solvents must be purified and final distillation must be conducted
in all-glass apparatus. Caution: See Appendix B, safety notes on
distillation, flammable solvents, acetone, and petroleum ether.)
(a) Solvents.Acetone, CH
2
Cl
2
, petroleum ether, distilled in
glass.
(b) Sodium sulfate.Anhydrous, granular.
(c) Glass wool.Rinse with acetone and alcohol several times
and dry. Washed glass wool will be somewhat brittle.
(d) High-speed blender.Waring Blendor, or equivalent.
(e) Kuderna-Danish concentrator.500 mL with Snyder col-
umn and fitted with volumetric flask or graduated receiving tube.
Calibrate receiving tube with acetone delivered from a buret. Use
buret-corrected volume for sample weight calculation.
(f) Separatory funnels.1 L, with Teflon stopcocks.
(g) Gas chromatograph.(1) For organochlorine residues.
Instrument containing any suitable methyl silicone column, such as
2% OV-101, on 80100 mesh Chromosorb W (HP), 6 ft 2 mm id
glass, and Hall 700A HECD halogen-specific detector. Column,
200; He carrier gas, 60 mL/min; detector 900, H
2
reaction gas
60100 mL/min; n-propanol solvent 0.35 mL/min; electrometer
range 10 in OPR/FLT mode; attenuation 5. (2) For organophospho-
rus residues.(If not available, see Sections E and F.) Instrument
with column containing 2% stabilized DEGS on 80100 mesh
Chromosorb W (HP), 4 ft 2 mm id silanized glass, and P-specific
flame photometric detector (526 nm filter). Column 180; detector
200; He carrier gas, 60 mL/min. Condition column (disconnect
detector) by passing carrier gas through column 0.5 h at 80.
Program temperature at 12/min to 230 and hold overnight. Es-
tablish stable flame at electrometer setting that will produce 50% full
scale deflection for 1.5 ng chlorpyrifos and 6 ng monocrotofos. If
necessary, increase air/O
2
until 50% response. Baseline noise
should be <2%.
(h) Reference standard materials.Acephate, BHC, chlorpyri-
fos, p,p-DDT, dieldrin, methamidophos, monocrotophos, and
omethoate (U.S. Environmental Protection Agency, Pesticides and
Industrial Chemical Respository [MD-8], Research Triangle Park,
NC 27709). Prepare all standards in acetone. Mixed standards.For
Hall system, standard solution should contain at least chlorpyrifos,
dieldrin, and p,p-DDT. For flame photometric detector, standard
solution should contain at least methamidophos and chlorpyrifos.
Do not use mixed standard solutions for quantitation of unknowns.
(i) Standard solutions.Prepare all stock solutions and dilutions
in glass-distilled acetone. Prepare GC standard solutions so 4 L
injection causes 3070% full scale deflection in properly functioning
system. Suggested concentrations are given in Table 985.22. Check
responses before beginning analysis. Store all standard solutions in
tightly stoppered containers in refrigerator. Let equilibrate 1 h at
room temperature before using.
C. Preparation of Sample
Chop or blend fruits and vegetables and mix thoroughly. Weigh
100 g chopped or blended sample into high-speed blender jar, add
200 mL acetone, and blend 2 min at high speed. Do not add Celite.
Filter with suction through 12 cm Bchner funnel fitted with shark-
skin paper. (Note: Rinse filter paper with acetone before filtration of
sample to remove artifacts that can interfere with analysis.) Collect
extract in 500 mL suction flask. Filtration is normally complete in
<1 min. Continuation of vacuum for excessive period can reduce
volume of extract and cause error in calculation.
Place 80 mL sample extract in 1 L separatory funnel, and add 100
mL petroleum ether and 100 mL CH
2
Cl
2
. Shake vigorously 1 min.
Transfer lower aqueous layer to second 1 L separatory funnel. Dry
upper organic layer in first separatory funnel by passing through ca
1
1

2
in. Na
2
SO
4
supported on washed glass wool in 4 in. funnel,
collecting in 500 mL Kuderna-Danish concentrator fitted with volu-
metric flask or calibrated receiving tube. To separate funnel with
aqueous phase, add 7 g NaCl and shake vigorously 30 s until most
NaCl is dissolved. Add 100 mL CH
2
Cl
2
, shake 1 min, and dry lower
organic phase through same Na
2
SO
4
. Extract aqueous phase with
additional 100 mL CH
2
Cl
2
, and dry as above. Rinse Na
2
SO
4
with ca
50 mL CH
2
Cl
2
. Attach Snyder column on Kuderna-Danish concen-
trator (boiling chips may be added) and start evaporation slowly by
placing only receiver tube into steam. After 100150 mL has evapo-
rated, concentrator may be exposed to more steam. When liquid level
in hot concentrator tube is ca 2 mL, add 100 mL petroleum ether
through Snyder column and reconcentrate to ca 2 mL. Add 50 mL
petroleum ether and repeat concentration step. Add 20 mL acetone
and reconcentrate to ca 2 mL. Do not let solution go to dryness during
any concentration step. Adjust volume of extract to suitable definite
volume with acetone.
Calculation of equivalent sample weight.Calculate equivalent
sample weight in final solution as follows:
mg sample equivalent
L final extract
=
100
80
200 + W 10

1
mL final volume
where 200 = mL acetone blended with 100 g sample; W = amount
(mL) H
2
O present in sample; and 10 = adjustment for water-acetone
volume contraction. Thus, when sample contains 85% H
2
O (85
mL/100 g) and final extract volume is 7 mL, each L contains:
1996 AOAC INTERNATIONAL
100
80
200 + 85 10

1
7
=
4.15 mg sample equivalent/ L final extract
D. Determination
Check that both GC systems are working properly by injecting
mixed standard solution into each. Inject ca 12 mg sample equivalent
into each system. Tentatively identify any GC responses on basis of
retention times. Quantitate residue peak(s) by area comparison
withthat obtained from known amount of reference material(s). To
ensure valid measurement of residue amount, area of peaks from
residue and reference standard should be within 25%. Caution:
Repeated injection of sample extracts which have had minimum
cleanup can be detrimental to GC columns. Replace packing material
at front of GC columns as needed to maintain chromatographic
quality and prolong column life.
E. Reagents and Apparatus
(a) Fused silica capillary columns.With bonded methyl sili-
cone and methyl 50% phenyl silicone stationary phases. Column
length: 30 m. Inside diameter: 530 m. Film thickness:
(b) Guard column.Deactivated fused silica tubing, 530 m id,
5.0 m length.
(c) Capillary column connectors.Low or zero dead volume,
suitable for connecting analytical column to retention gap. Various
styles, ferrule, adhesive, and press in types are available from
chromatographic supply companies.
(d) Silanized direct injection adapter.4 mm id.
(e) Column inlet liner.For 4 mm id columns.
(f) Glass wool.Pesticide grade or silanized. Silanized glass
wool free of nitrogen, chlorine, phosphorus, or sulfur contaminates.
F. Instrumetnt Setup
(a) Connecting column and guard column.Cut off column and
retention gap ends with a capillary cleaving tool. Examine cuts with
a 20 magnifier to assure that ends are square and smooth. Clean ends
of column/retention gap with a wiper wetted with methanol. Attach
the retention gap to one end of the column using an appropriate
capillary column connector and secure retention gap to column cage.
(b) Inlet adapter (see Figure 985.22).Rinse adapter with a few
mL of methanol. Attach capillary column/inlet adapter reducing
union to column over end of the adapter, exercising care not to
fracture the end of the adapter when tightening the nut and ferrule.
Place a small, loose plug of glass wool in the inlet end and push to
the bottom of the adapter at the restrictor. Use the minimum amount
necessary to cover top of restrictor. Place column liner for 4 mm id
columns in the open end of the adapter and carefully insert the as-
Table 985.22 Concentrations of Standard Solutions
a
Compound
Instrument Used
B(g)(2)
E
acephate 0.5 0.4
-BHC 0.1 0.1
chlorpyrifos 0.5 0.5
dieldrin 0.2 0.2
p,p-DDT 0.5 0.5
methamidophos 0.2 0.4
monocrotophos 2.0 0.4
omethoate 2.0 0.4
a
in ng/L

sembled adapter into the injection port and secure. Place reducing
union nut and ferrule on guard column. Cut off and clean end of
guard column as described above.
(Caution: A square, clean cut is essential to properly attach reten-
tion gap to adapter.)
Insert the end of the guard column through the column/adapter
reducing union. Push the guard column firmly into the flared portion
of the restrictor to form a seal between the polyamide coating of the
guard column and the adapter restrictor. A proper seal is evidenced
by formation of a ring at point of contact between the tubing and
restrictor walls. Tighten column nut on reducing union.
(1) Detector connections.Install the column into the detector
following the detector manufacturers instructions for positioning
the column end in the detector.
(Caution: The position of the column end in the detector is criti-
cal to proper operation.)
(2) Carrier gas.Use He carrier gas to get the best column
efficiency and compatibility with the flame photometric detector.
Adjust flow so that chlorpyrifos elutes in 4 0.5 min. For 30 m
530 id columns, flows of 2030 mL/min He are typical.
(3) Makeup gas.Use of makeup gas is recommended for all
systems regardless of carrier flow. The makeup gas can be N
2
or He.
Adjust makeup flow so that total flow of carrier and makeup gas
equals the optimum gas flow recommended by the manufacturer for
Figure 985.22Modified direct injection inlet assem-
bly for wide-bore capillary gas chromatography
1996 AOAC INTERNATIONAL
the particular detector. Makeup gas flows of 540 mL/min are
typical.
(4) GC system operation.Operate P-specific flam photometric
detector (526 nm filter) per manufacturers intructions. Column 200
C; detector 225C; injector 225C. Establish stable flame that will
produce approximately 50% full scale deflection for 1.5 ng chlor-
pyrifos and 1 ng acephate. (Note: Injection of a sample extract on
systems with new or cleaned inlet adapters may be necessary to
condition system and achieve specified sensitivity for acephate.)
References: JAOAC 68, 64(1985); 70, 329(1987).
J. AOAC Int. 77, 92(1994).
Revised: March 1996
CAS-30560-19-1 (acephate)
CAS-319-84-6 (-BHC)
CAS-2921-88-2 (chlorpyrifos)
CAS-60-57-1 (dieldrin)
CAS-50-29-3 (DDT)
CAS-1113-02-6 (omethoate)
CAS-10265-92-6 (methamidophos)
CAS-6923-22-4 (monocrotophos)
1996 AOAC INTERNATIONAL