Pesticide Analytical Manual Vol. I
400–1
Transmittal No. 96-E1 (9/96)
Form FDA 2905a (6/92)
Table of Contents
page date
401: Method for N-Methylcarbamates
Basic References 401-1 1/94
General Principles 401-1 1/94
Applicability 401-1 1/94
Reference Standards 401-1 1/94
Steps of the method 401-1 1/94
Validation 401-2 1/94
E1 Extraction with methanol 401-3 1/94
E2 Extraction with methanol, reduced sample size 401-4 1/94
C1 Liquid-Liquid Partitioning and Charcoal/Celite 401-5 1/94
Column Cleanup
DL1 HPLC, Post-Column Derivatization, 401-9 1/94
Fluorescence Detection
Chapter 1
Regulatory Operations
Chapter 2
General Analytical
Operations and Information
Chapter 3
Multiclass
MRMs
Chapter 5
GLC
Chapter 4
Selective
MRMs
Chapter 6
HPLC
Pesticide Analytical Manual Vol. I
SECTION 400
400–2
Transmittal No. 96-E1 (9/96)
Form FDA 2905a (6/92)
page date
DL2 HPLC, Fluorescence Detection 401-13 1/94
Confirmation 401-13 1/94
402: Method for Acids and Phenols
Basic Reference 402-1 1/94
General Principles 402-1 1/94
Applicability 402-1 1/94
Reference Standards 402-1 1/94
Steps of the method 402-1 1/94
Validation 402-2 1/94
E1 Extraction with solvents from acidified, 402-3 1/94
denatured products
E2 Extraction with acidified methylene chloride 402-7 1/94
E3 Extraction with acidified methanol 402-9 1/94
E4 Extraction with acidified water/methanol 402-10 1/94
E5 Extraction with acidified methanol 402-11 1/94
E6 Dissolution in methylene chloride/hexane 402-13 1/94
E7 Extraction with acidified methylene chloride 402-15 1/94
C1 GPC Cleanup, methylation, and Florisil 402-17 1/94
column cleanup
C1a GPC Cleanup 402-17 1/94
C1b Methylation 402-21 1/94
C1c Florisil column cleanup 402-22 1/94
Determination 402-25 1/94
Confirmation 402-25 1/94
403: Method for Phenylurea Herbicides
Basic Reference 403-1 1/94
General Principles 403-1 1/94
Applicability 403-1 1/94
Reference Standards 403-1 1/94
Steps of the method 403-1 1/94
Validation 403-2 1/94
E1 Extraction with Methanol 403-3 1/94
C1 Liquid-Liquid Partitioning and Florisil 403-5 1/94
Column Cleanup
DL3 HPLC, Post-Column Photolysis and 403-7 1/94
Derivatization, Fluorescence Detection
DL4 HPLC, Different mobile phase 403-10 1/94
Confirmation 403-11 1/94
404: Method for Benzimidazoles
Basic Reference 404-1 1/94
General Principles 404-1 1/94
Pesticide Analytical Manual Vol. I
400–3
Transmittal No. 96-E1 (9/96)
Form FDA 2905a (6/92)
page date
Applicability 404-1 1/94
Reference Standards 404-1 1/94
Steps of the Method 404-1 1/94
Validation 404-3 1/94
E1 Extraction with methanol, transfer to 404-5 1/94
methylene chloride
E2 Extraction with methanol, removal of oil with 404-7 1/94
hexane, transfer to methylene chloride
E3 Extraction with methanol, removal of oil with 404-8 1/94
methylene chloride, transfer to methylene chloride
DL5 HPLC, Ion Pair Mobile Phase, UV and 404-9 1/94
Fluorescence Detector
DL6 HPLC, Concentrated Ion Pair Mobile Phase, 404-12 1/94
UV and Fluorescence Detector
DL7 HPLC, Ion Pair Mobile Phase, Changes in 404-14 1/94
Detector Settings
Confirmation 404-14 1/94
Figures
401-a Method for N-Methylcarbamates 401-2 1/94
401-b Vacuum Rotary Evaporator 401-3 1/94
401-c HPLC System for Determination of 401-9 1/94
N-Methylcarbamates
401-d HPLC Chromatogram of Carbamates 401-12 1/94
and Metabolites
402-a Method for Acids and Phenols 402-2 1/94
402-b Delivery Tube Apparatus 402-3 1/94
403-a Method for Phenylureas 403-2 1/94
403-b HPLC System for Determination of 403-7 1/94
Phenylurea Herbicides
403-c HPLC Chromatograms of Carrot Extract 403-10 1/94
403-d Chromatographic Pattern Comparison 403-11 1/94
404-a Method for Benzimidazoles 404-2 1/94
404-b Chromatograms of Benzimidazole Compounds 404-12 9/96
404-c Effect of Ion Pairing Solution Concentration 404-13 1/94
on Chromatography of MBC
Tables
401-a: Recovery of Chemicals Through Method 401 401-a–1 9/96
(E1-E2 + C1 + DL1)
401-b: Recovery of Chemicals Through Method 401 401-b–1 9/96
(E1-E2 + C1 + DL2)
402-a: Recovery of Chemicals Through Method 402 402-a–1 9/96
(E1-E7 + C1 + DG1 or DG3 or DG4)
403-a: Recovery of Chemicals Through Method 403 403-a–1 9/96
(E1 + C1 + DL3 and DL4)
Pesticide Analytical Manual Vol. I
SECTION 400
400–4
Transmittal No. 96-E1 (9/96)
Form FDA 2905a (6/92)
page date
404-a: Recovery of Chemicals Through Method 404 404-a–1 9/96
(E1-E3 + DL5)
Transmittal No. 94-1 (1/94)
Form FDA 2905a (6/92) 401–1
SECTION 401Pesticide Analytical Manual Vol. I
401: METHOD FOR N-METHYLCARBAMATES
BASIC REFERENCES
Krause, R.T. (1979) J. Chromatogr. 185, 615-624
Krause, R.T. (1980) J. Assoc. Off. Anal. Chem. 63, 1114-1124
Krause, R.T. (1985) J. Assoc. Off. Anal. Chem. 68, 734-741
GENERAL PRINCIPLES
N-methylcarbamate insecticide residues, including carbamate metabolites, are ex-
tracted with methanol. The extract is cleaned up by partitioning and column
chromatography on a charcoal/Celite column. Residues are selectively determined
with an HPLC system consisting of a reverse phase (RP) column, post-column
hydrolysis and derivatization, and detection of the resultant derivative with a fluo-
rescence detector.
Variations in the determinative step may be used for additional residues not of the
N-methylcarbamate structure.
APPLICABILITY
Consult Guide to PAM I for additional information pertinent to the appropriate
application of multiresidue methodology.
Method is applicable to residues with an N-methylcarbamate structure in either
nonfatty or fatty foods when determinative step includes post-column hydrolysis and
derivatization. See Table 401-a, following method description, for chemicals tested
through the method.
Method is applicable to naturally fluorescent residues when post-column hydrolysis
and derivatization are not performed. See Table 401-b, following Table 401-a.
Certain commodities, e.g., oranges, contain naturally fluorescent co-extractives that
interfere with analysis.
Limit of quantitation is 0.01 ppm carbofuran in high moisture products (fresh fruits
and vegetables) and about 0.02 ppm in dry products.
REFERENCE STANDARDS
Dissolve reference standards of N-methylcarbamates in methanol to produce con-
centrations of 1 μg/mL. Store solutions in actinic glassware, and keep in refrigera-
tor when not in use. Most carbamate standards stored in this manner are stable for
several months. However, methiocarb sulfone and sulfoxide degrade within hours
and days, respectively, even with stated storage precautions.
STEPS OF THE METHOD
Extraction (E) Recommended Use
E1 (p. 401-3) Extraction with methanol high moisture products
E2 (p. 401-4) Extraction with methanol, reduced low moisture products
sample size
Cleanup (C)
C1 (p. 401-5) Two stage liquid-liquid partitioning all products
and charcoal/Celite column cleanup
SECTION 401
401–2
Transmittal No. 94-1 (1/94)
Form FDA 2905a (6/92)
Pesticide Analytical Manual Vol. I
Determination (D) Recommended Use
DL1 (p. 401-9) HPLC, post-column hydrolysis and N-methylcarbamate
derivatization, fluorescence detection residues
DL2 (p. 401-13) HPLC, fluorescence detection naturally fluorescent
residues
Figure 401-a
Method for N-Methylcarbamates
VALIDATION
The following combination has undergone interlaboratory validation and is recom-
mended for use:
E1 + C1 + DL1
Validation report:
Krause, R.T. (1985) J. Assoc. Off. Anal. Chem. 68, 726-733. Collaborative study
leading to AOAC official final action status for aldicarb, aldicarb sulfone,
bufencarb, carbaryl, carbofuran, 3-hydroxycarbofuran, methiocarb, methomyl,
and oxamyl in grapes and potatoes.
AOAC official method reference: Official Methods of Analysis of the AOAC (1990) 15th
ed., 985.23.
>75% water
E1
<75% water
E2
C1
DL2
naturally
fluorescent
chemicals
DL1
N-methylcarbamates
Transmittal No. 94-1 (1/94)
Form FDA 2905a (6/92) 401–3
SECTION 401Pesticide Analytical Manual Vol. I
E1 EXTRACTION WITH METHANOL
Reference
Krause, R.T. (1980) J. Assoc. Off. Anal. Chem. 63, 1114-1124
Principles
Residues are extracted from high moisture products (>75% water) with methanol,
found to be the most effective extractant for N-methylcarbamates in tests using
radiolabeled materials. The filtered extract is concentrated with a system that permits
evaporation of the relatively high boiling point methanol without destroying heat-
labile residues.
Apparatus
Buchner funnel (Buchner), porcelain, 12 cm diameter
evaporator, vacuum rotary with circulating chilled liquid (see Figure 401-b).
Maintain 1+1 water/ethylene glycol solution in condensing coils and around
receiving flask at –15° C with 1/2 horsepower cooling unit. Insulate con-
denser with Styrofoam or other material. Control evaporator vacuum with
vacuum pump and gauge; manometer may be used but is not preferred.
filter paper, Sharkskin, or 597
S&S, to fit Buchner
flask, round-bottom (r-b), 2 L, Ts
24/40
homogenizer, Polytron Model
PT 10-35, with PT 35K genera-
tor containing knives, head
equipped with metal (not
Teflon) bushing
homogenizer jar, four-side,
glass, 1 qt
magnetic stirrer, star, 10 mm di-
ameter × 8 mm height
vacuum filtration flask, 500 mL
Reagents
methanol, distilled from all-glass
apparatus
Directions
? Add 150 g chopped high mois-
ture product and 300 mL
methanol to homogenizer jar.
? Homogenize mixture 30 sec at about half speed (setting of 7) and then 60
sec at full speed.
? Vacuum filter homogenate through Buchner fitted with filter paper and
collect filtrate in 500 mL vacuum filtration flask. Reduce vacuum during
filtration if filtrate begins to boil.
? Transfer portion of filtrate equivalent to 100 g sample to 2 L r-b flask.
Figure 401-b
Vacuum Rotary Evaporator
To remove higher boiling solvents from solutions
containing heat-labile residues. Water/ethylene
glycol at –15° C cools receiving flask of rotary
evaporator and (insulated) evaporator con-
denser.
Condenser
(insulated)
Motor
Cooling
unit
Water bath
35 C
Constant
temperature
bath circulator
-15 C
Needle
valve
Pump
Manometer
To
vacuum
pump
SECTION 401
401–4
Transmittal No. 94-1 (1/94)
Form FDA 2905a (6/92)
Pesticide Analytical Manual Vol. I
mL water
100 g sample
volume 100 g sample = + 200 mL methanol – 10 mL contraction factor
? Add enough water to r-b flask to total 100 mL water.
? Add star magnetic stirrer to r-b flask. Place 250 mL trap on 2 L r-b flask
and attach to vacuum rotary evaporator.
? Circulate refrigerated (–15° C) (1+1) water/ethylene glycol through evapo-
rator condensing coils; maintain receiving flask at –15° C by immersion in
refrigerated bath.
? Apply vacuum slowly to minimize frothing by regulating with needle valve.
After full vacuum is applied, slowly place flask in 35° C water bath. Con-
centrate extract to 75 mL.
ALTERNATIVE:
E2 EXTRACTION WITH METHANOL, REDUCED SAMPLE SIZE
Principle
Reduced sample size permits same amount of solvent to extract residues effectively
from low moisture products (<75% water).
Directions
? Proceed as in E1, except extract 75 g ground low moisture product with 300
mL methanol.
? Transfer portion of filtrate equivalent to 50 g sample to 2 L r-b flask.
mL water
50 g sample
volume 50 g sample = + 200 mL methanol
? Continue as in E1, "Add enough water to r-b flask to total 100 mL water."
Transmittal No. 94-1 (1/94)
Form FDA 2905a (6/92) 401–5
SECTION 401Pesticide Analytical Manual Vol. I
C1 LIQUID-LIQUID PARTITIONING AND CHARCOAL/CELITE COLUMN
CLEANUP
Reference
Krause, R.T. (1980) J. Assoc. Off. Anal. Chem. 63, 1114-1124
Principles
Residues in aqueous extract are transferred to acetonitrile by liquid-liquid partition-
ing in the presence of sodium chloride. Co-extractives are removed from the
acetonitrile solution by partitioning them into petroleum ether, which is discarded.
Residues are partitioned from acetonitrile into methylene chloride. Methylene
chloride solution is cleaned up on a charcoal/Celite column, and residues are eluted
with toluene/acetonitrile.
Apparatus
chromatographic column, 22 mm id × 300 mm, Teflon stopcock, coarse
porosity fritted disc
chromatographic column, 25 mm id, plain
evaporator, vacuum rotary, as described in E-1
flasks, round-bottom (r-b), 250 and 500 mL, 1 L, Ts 24/40
magnetic stirrer, star, 10 mm diameter × 8 mm height
separatory funnel (separator), 250 and 500 mL
vacuum adapter, side arm, with Ts 24/40 joints
Reagents
acetonitrile, distilled from all-glass apparatus; see Section 204 for distillation
directions
Celite 545
charcoal (Nuchar S-N), produced by Westvaco Corp. and available from
Eastman Kodak, Cat. No. 118 0454
1+4 (w/w) charcoal/Celite, combined after each is prepared as directed
below; mix thoroughly and store in sealed container
dichlorodimethylsilane
glass wool, Pyrex
hydrochloric acid, concentrated, reagent grade
isopropanol, distilled from all-glass apparatus
methanol, distilled from all-glass apparatus
methyl red
methylene chloride, distilled from all-glass apparatus
petroleum ether, distilled from all-glass apparatus
sodium chloride, reagent grade
2% (w/v) sodium chloride/water
20% (w/v) sodium chloride/water
SECTION 401
401–6
Transmittal No. 94-1 (1/94)
Form FDA 2905a (6/92)
Pesticide Analytical Manual Vol. I
sodium sulfate, anhydrous, granular, reagent grade; see Section 204 for han-
dling directions
toluene, distilled from all-glass apparatus
eluant: 25% (v/v) toluene/acetonitrile
Preparation of Silanized Celite 545
? Slurry 150 g Celite 545 with 1 L (1+1) hydrochloric acid/water in 2 L beaker,
cover with watch glass, and stir magnetically while boiling 10 min.
? Cool slurry, filter, and wash with distilled or HPLC grade water until filtrate
is neutral.
? Wash Celite with 500 mL methanol followed by 500 mL methylene chloride,
and then air dry Celite in hood on watch glass to remove solvent.
? Transfer Celite to 1 L glass-stoppered (g-s) Erlenmeyer flask. Heat unstop-
pered flask in 120° C oven overnight and then cool flask in desiccator.
? Place flask in hood and carefully pipet 3 mL dichlorodimethylsilane onto
Celite. Stopper flask, mix well, and let flask remain at room temperature 4
hr.
? Add 500 mL methanol to flask, mix, and let stand 15 min.
? Filter silanized Celite and wash with isopropanol until neutral.
? Air dry silanized Celite in hood to remove isopropanol.
? Dry silanized Celite in 105° C oven 2 hr and cool in desiccator. Store sila-
nized Celite in g-s container.
? Test Celite for total silanization with two tests. Place about 1 g Celite in 50 mL
water; silanized Celite will float. Place second 1 g Celite in 20 mL toluene
saturated with methyl red; silanized Celite will appear yellow. If particles of
Celite are dispersed in water and/or appear pink with methyl red/toluene
solution, active sites still exist on Celite; repeat silanization.
Purification of Charcoal
? Slurry 100 g Nuchar S-N with 700 mL hydrochloric acid, cover with watch
glass, and stir magnetically while boiling 1 hr.
? Add 700 mL water, stir, and boil additional 30 min.
? Cool slurry, filter, and wash with water until neutral.
? Wash Nuchar S-N with 500 mL methanol followed by 500 mL methylene
chloride, and air dry Nuchar S-N in hood to remove solvent.
? Dry Nuchar S-N in 120° C oven 4 hr. Cool in desiccator. Store Nuchar S-N in
g-s container.
Testing of Charcoal/Celite
? Prepare cleanup column of (1+4) (w/w) charcoal/Celite as described be-
low.
? Prepare methanol solution of 5 μg/mL each carbaryl, methiocarb, methio-
carb sulfoxide, and methomyl. Use freshly prepared mixed standard solu-
tion; methiocarb sulfoxide degrades in solution.
Transmittal No. 94-1 (1/94)
Form FDA 2905a (6/92) 401–7
SECTION 401Pesticide Analytical Manual Vol. I
? Pipet 5 mL solution into 250 mL r-b flask and 5 mL into 25 mL actinic
volumetric flask.
? Dilute solution in volumetric flask to 25 mL with methanol; use as HPLC
reference standard.
? Evaporate standard solution in r-b flask just to dryness with vacuum rotary
evaporator as described below. After last trace of methanol has evaporated,
remove r-b flask from evaporator and dissolve carbamate residue in 10 mL
methylene chloride.
? Transfer methylene chloride solution in r-b flask to prepared adsorbent
column and elute as described below.
? After evaporation of eluate in r-b flask, dissolve residue in 25 mL methanol,
filter aliquot through filtration device, and quantitate recovery of carba-
mates as in DL1. Nuchar S-N is considered satisfactory if recovery is ≥95%.
Directions
Partitioning
? Transfer concentrated extract from E1 or E2 to 500 mL separator con-
taining 15 g sodium chloride. Shake separator until sodium chloride is
dissolved.
? Wash r-b flask with three 25 mL portions acetonitrile, transferring each to
500 mL separator; shake separator 30 sec, and let layers separate 5 min.
? Drain aqueous phase into 250 mL separator containing 50 mL acetonitrile,
shake 20 sec, let layers separate, and discard aqueous layer.
? Add 25 mL 20% aqueous sodium chloride solution to acetonitrile in 500
mL separator, shake 20 sec, let layers separate, and transfer aqueous
solution to 250 mL separator.
? Shake 250 mL separator 20 sec, let layers separate, and discard aqueous
layer.
? Add 100 mL petroleum ether to 500 mL separator, shake 20 sec, let layers
separate, and drain acetonitrile layer into second 500 mL separator.
? Transfer acetonitrile in 250 mL separator to first 500 mL separator which
contains petroleum ether, shake 20 sec, let layers separate, and transfer
acetonitrile to second 500 mL separator.
? Add 10 mL acetonitrile to first 500 mL separator, shake, let layers separate,
and transfer acetonitrile to second 500 mL separator. Discard petroleum
ether layer.
? Add 50 mL 2% aqueous sodium chloride solution to acetonitrile in second
500 mL separator. Extract mixture successively with 100, 25, and 25 mL
methylene chloride, shaking each 20 sec (shake 25 mL portions gently).
? Drain lower methylene chloride/acetonitrile layers through 22 mm id
column containing about 5 cm sodium sulfate. Collect eluate in 1 L r-b flask.
? Add star magnetic stirrer to r-b flask. Place 250 mL trap on 1 L r-b flask and
attach to vacuum rotary evaporator.
? Circulate refrigerated (–15° C) (1+1) water/ethylene glycol through evapo-
rator condensing coils; maintain receiving flask at –15° C by immersion in
refrigerated bath.
SECTION 401
401–8
Transmittal No. 94-1 (1/94)
Form FDA 2905a (6/92)
Pesticide Analytical Manual Vol. I
? Apply vacuum slowly to minimize frothing by regulating with needle valve.
After full vacuum is applied, slowly place flask in 35° C water bath.
? Remove r-b flask from evaporator immediately after last traces of solution
have evaporated and add 10 mL methylene chloride to r-b flask.
Charcoal/Celite Cleanup
? Fit one-hole No. 5 rubber stopper onto tip of chromatographic column
with stopcock, add side-arm vacuum adapter and 500 mL r-b flask, open
stopcock, and connect apparatus to vacuum line.
? Place 0.5 g silanized Celite in chromatographic column, tamp, add 5 g
charcoal/Celite mixture, and tamp again. Add 1-2 cm glass wool plug on
top of adsorbent.
? Prewash column with 50 mL 25% toluene/acetonitrile eluant. Close stop-
cock when prewash solution is about 0.5 cm from top of glass wool.
? Disconnect vacuum, discard solution in r-b flask, and reconnect flask to
apparatus.
? Transfer 10 mL methylene chloride extract from partitioning steps to
column and elute column at 5 mL/min.
? Wash 1 L r-b flask with 10 mL methylene chloride and then with 25 mL
eluant. Transfer each separately to column and elute each to top of glass
wool before adding next solution.
? Add 100 mL eluant and elute column at 5 mL/min. Turn off stopcock
when top of eluant reaches top of glass wool.
? Evaporate solution in 500 mL r-b flask just to dryness using vacuum evap-
orator as above. Remove flask from evaporator immediately after all solu-
tion has evaporated.
? Immediately pipet 5 mL methanol into 500 mL r-b flask to dissolve resi-
due.
? Cleaned up extract contains 20 g sample equivalent/mL solution for high
moisture products and 10 g sample equivalent/mL solution for low mois-
ture products.
Transmittal No. 94-1 (1/94)
Form FDA 2905a (6/92) 401–9
SECTION 401Pesticide Analytical Manual Vol. I
DL1 HPLC, POST-COLUMN DERIVATIZATION, FLUORESCENCE DETECTION
Reference
Krause, R.T. (1978) J. Chromatogr. Sci. 16, 281-288
Principles
Residues in methanol solution are separated on a C-8 reverse phase HPLC column
using acetonitrile/water gradient mobile phase. Residues eluting from the column
are hydrolyzed in-line to methylamine under alkaline conditions. Methylamine is
reacted, also in-line, with o-phthalaldehyde and 2-mercaptoethanol to form a
fluorophore that is measured by a fluorescence detector. This post-column
derivatization-fluorescence detection determinative step is very selective for residues
containing the N-methylcarbamate structure.
Apparatus
filtration device for solutions, 10 mL syringe with Luer-Lok tip, fitted with
either (a) 13 mm diameter Swinny stainless steel filter holder and 13 mm
diameter filters, 5.0 μm LS-type, or (b) disposable membrane filters, 13 mm
diameter, 0.22 μm nylon membrane, encased in polypropylene.
(Preassembled devices that do not require a syringe are also available.)
HPLC system (Figure 401-c) must meet system suitability test below. Complete
system consists of:
1) mobile phase delivery system, programmable HPLC gradient system
2) injector, automatic sampler with 10 μL injection loop
3) guard column, stainless steel, containing 25-37 μm pellicular C-8 or C-18
packing
4) column oven or heater
Figure 401-c
HPLC System for Determination of N-Methylcarbamates
AcetonitrileWater
Fluorescence
detector
Recorder/data
handling
Post-column
reaction
Hydrolysis
chamber
NaOH OPA-MERC
100 C
Valve
injector
Guard
column
C-8
analytical
column
25 cm
SECTION 401
401–10
Transmittal No. 94-1 (1/94)
Form FDA 2905a (6/92)
Pesticide Analytical Manual Vol. I
5) analytical column, 25 cm × 4.6 mm id, containing 6 μm Zorbax C-8 spherical
particles. Column packing should consist of 5 or 6 μm spherical silica
particles bonded with monofunctional octyl silane reagent to form mono-
molecular bond.
6) connecting tubing, No. 304 stainless steel (1.6 mm od × 0.18 mm id) to
connect injector, column, and first tee
7) post-column derivatization unit, as shown in telescoped portion of Figure
401-b. Units used during collaborative study of method were assembled
from the following parts:
a) reservoirs for sodium hydroxide and OPA-MERC reaction solutions, 60
cm × 25 mm id glass columns with Teflon fittings; pressurize reservoirs
with nitrogen gas, adjusted to create appropriate reagent flow. (Pumps
can be substituted for nitrogen gas pressure.)
b) 6 m × 0.5 mm id Teflon restriction coil, to connect each reservoir to 15
cm × 0.18 mm id stainless steel tubing, which in turn is connected to
0.74 mm id stainless steel mixing tee (Valco Instruments Co., Cat. No.
ZVT-062) for connection to flow of mobile phase
c) carbamate hydrolysis chamber, stainless steel tubing, 3 m × 0.48 mm id
No. 321, coiled to fit in small oven capable of maintaining constant,
uniform 100° C
d) reaction tube, 25 cm stainless steel tubing between tee that delivers
OPA-MERC solution and 1.5 cm × 0.3 mm id detector cell tubing
Commercial post-column derivatization units that replace these compo-
nents are now available from several manufacturers (ABI, Pickering Instru-
ments, Waters). Systems with dual piston pumps are preferred.
8) fluorescence detector, dual monochromator, equipped with ≤20 μL cell
9) recorder, strip chart recorder or computing integrator compatible with
detector
Reagents
acetonitrile, UV grade distilled from all-glass apparatus. Before use, degas
acetonitrile in glass bottles by applying vacuum and slowly stirring with mag-
netic stirrer 5 min. Acetonitrile other than HPLC grade may cause broad,
nonreproducible peaks in chromatograms.
2-mercaptoethanol (MERC), 98+%
methanol, distilled from all-glass apparatus
o-phthalaldehyde (OPA), chromatographic grade
sodium borate buffer solution, 0.05 M. Add 19.1 g ACS grade sodium tetrabor-
ate decahydrate (Na
2
B
4
O
7
?10 H
2
O) and about 500 mL degassed HPLC grade
water to 1 L volumetric flask. Heat flask in steam bath to dissolve sodium
tetraborate, cool to room temperature, and dilute to volume with degassed
HPLC grade water. Mix well, but gently, to minimize re-incorporation of air into
solution.
sodium hydroxide solution, 0.05 N. Prepare clear supernate sodium hydroxide
as follows: to one part sodium hydroxide (reagent quality containing <5%
sodium carbonate), add one part water and swirl until solution is complete.
Stopper and set aside until sodium carbonate has settled, leaving clear liquid
Transmittal No. 94-1 (1/94)
Form FDA 2905a (6/92) 401–11
SECTION 401Pesticide Analytical Manual Vol. I
(about 10 days). Pipet 27 mL clear supernate sodium hydroxide into 100 mL
volumetric flask. Dilute to volume with water and mix (5 N sodium hydroxide).
Pipet 10 mL 5 N sodium hydroxide into 1 L volumetric flask. Dilute to volume
with degassed HPLC grade water and mix well, but gently, to minimize re-
incorporation of air into solution.
water, HPLC grade, commercial product or prepared from water purification
equipment that produces distilled, deionized water. For HPLC, degas water
as described for acetonitrile. Water must be adequately purified to prevent
plugging HPLC column and extraneous peaks in chromatograms. All water
used in HPLC procedure must be HPLC grade. (“Water” that does not specify
HPLC grade means distilled water.)
OPA-MERC reaction solution. Weigh 500 mg OPA, transfer to 1 L volumetric
flask, add 10 mL methanol, and swirl to dissolve OPA. Add about 500 mL 0.05
M sodium borate buffer solution and 1 mL 2-mercaptoethanol. Dilute to vol-
ume with sodium borate buffer solution. Mix well, but gently, to minimize re-
incorporation of air into solution. (Borate solution purchased in plastic
bottles or low purity grades of OPA may cause excessively high background
fluorescence.) Solution is acceptable for about 2 days when stored at room
temperature, about 1 week when stored in refrigerator or under helium.
System Operation
? Adjust mobile phase flow rate to 1.50±0.02 mL/min with 50% acetonitrile/
HPLC grade water.
? Adjust flow rates of 0.05 N sodium hydroxide and OPA-MERC reaction
solution to 0.50±0.02 mL/min each. Operate column oven at 35° C and
hydrolysis chamber at 100° C.
? Set fluorescence detector excitation and emission wavelengths to 340 and
455 nm, respectively, and slit widths to 15 and 12 nm, respectively. Set
detector photomultiplier tube gain to low and time constant to 1 sec.
? Equilibrate system 10 min with 12% acetonitrile/HPLC grade water, inject
sample, and begin 30 min linear gradient to 70% acetonitrile/HPLC grade
water.
? Adjust sensitivity so that 10 ng carbofuran produces 50±5% full scale
deflection on printer-plotter. Baseline noise should be <2%, and carbam-
ates should elute as shown in Figure 401-c.
? If system will not be used for several days, replace aqueous mobile phase
with methanol and pump through system. Drain sodium hydroxide and
OPA-MERC reaction solutions from their reservoirs, and wash reservoirs
and associated tubing with water, then methanol. When starting system,
change mobile phase to HPLC grade water, and wash reservoirs and
associated tubing with water before adding reaction solutions.
System Suitability Test
See Chapter 6, HPLC, for further information about evaluating HPLC systems.
? Prepare mixed standard solution containing 1 μg/mL each aldicarb sul-
foxide, aldicarb sulfone, carbofuran, and carbaryl.
? Chromatograph solution, using HPLC system operation described above.
Retention times will be about 6.5, 8, 20, and 21 min, respectively.
SECTION 401
401–12
Transmittal No. 94-1 (1/94)
Form FDA 2905a (6/92)
Pesticide Analytical Manual Vol. I
? Baseline resolution should be achieved between aldicarb sulfoxide and
aldicarb sulfone and between carbaryl and carbofuran.
Directions
See Figure 401-d for typical chromatogram of carbamate pesticides and metabolites.
? Filter methanol extract from C1 r-b flask through filtration device.
? Collect filtrate in 10 mL centrifuge tube or other suitable container. About
4.5 mL filtrate will be collected. Exact volume of filtrate collected is not
critical because sample concentration (g sample/mL methanol) is known.
? If solution requires dilution, pipet aliquot into another container and dilute
to volume, as needed.
? Inject 10 μL methanol solution into HPLC system.
? Tentatively identify residue peaks on basis of retention times. Measure
peak area or height and determine residue amount by comparison to
peak area or height obtained from known amount of appropriate refer-
ence standard(s). To ensure valid measurement of residue amount, sizes
of peaks from sample residue and reference standard should match within
±25%. Chromatograph reference standard(s) immediately after sample.
Chromatographed at conditions described in DL1, with post-column derivatization. 1) aldicarb
sulfoxide; 2) aldicarb sulfone; 3) oxamyl; 4) methomyl; 5) 3-hydroxycarbofuran; 6) methiocarb
sulfoxide; 7) aldicarb; 8) carbofuran; 9) carbaryl; 10) methiocarb; 11) bufencarb.
Figure 401-d
HPLC Chromatogram of Carbamates and Metabolites
1
2
3
810
11
11
9
7
6
5
4
0 5 10 15 20 25 30
Retention (min)
10% FSD
Transmittal No. 94-1 (1/94)
Form FDA 2905a (6/92) 401–13
SECTION 401Pesticide Analytical Manual Vol. I
ALTERNATIVE:
DL2 HPLC, FLUORESCENCE DETECTION
Reference
Krause, R.T. (1983) J. Chromatogr. 255, 497-510
Principles
Residues in methanol solution are separated on a C-8 reverse phase HPLC column
using an acetonitrile/water gradient mobile phase. Naturally fluorescent residues
eluting from the column are detected and measured by a fluorescence detector.
Directions
? Set up and operate an HPLC system in the same manner as DL1, except:
operate hydrolysis chamber at ambient temperature.
set detector excitation and emission wavelengths at 288 and 330 nm,
respectively.
turn off pumps or nitrogen flow that add sodium hydroxide and OPA-
MERC reaction solutions to mobile phase.
? Perform determination as in DL1.
CONFIRMATION
Confirm tentative identification of naturally fluorescent residues by using DL2.
See Table 401-b for list of chemicals for which this confirmatory step is appropri-
ate. Use excitation and emission wavelengths that are optimum for residue being
confirmed.
Confirm N-methylcarbamates that include phenolic structure by injecting final
extract into HPLC with post-column hydrolysis-electrochemical detection method
described in Krause, R.T. (1988) J. Chromatogr. 442, 333-343. This method is based
on selective detection of phenolic group of insecticides, rather than carbamate
moiety. Intact carbamates are separated by reverse phase HPLC using gradient
acetonitrile/water mobile phase as described above. Eluted carbamates are hydro-
lyzed in-line with dilute sodium hydroxide at 100° C, and resulting phenols are
detected with coulometric electrochemical detector. Technique has been tested
with six carbamates (bufencarb, carbaryl, carbofuran, 3-hydroxycarbofuran,
isoprocarb, and methiocarb) and four crops (apples, cabbage, grapes, and toma-
toes).
SECTION 401
401–14
Transmittal No. 94-1 (1/94)
Form FDA 2905a (6/92)
Pesticide Analytical Manual Vol. I
Transmittal No. 94-1 (1/94)
Form FDA 2905a (6/92) 402–1
Pesticide Analytical Manual Vol. I SECTION 402
402: METHOD FOR ACIDS AND PHENOLS
BASIC REFERENCE
Hopper, M.L. et al. (1992) J. AOAC Int. 75, 707-713
GENERAL PRINCIPLES
Acidic and phenolic residues are extracted from commodity acidified with sulfuric
acid by various techniques dictated by the type of commodity. The extract is cleaned
up by gel permeation chromatography (GPC). Residues in the concentrated extract
are methylated by ion pair alkylation and further cleaned up by Florisil column
chromatography. The resulting methyl esters are determined by GLC. Certain
residues can be determined only by element-selective GLC detectors.
APPLICABILITY
Consult Guide to PAM I for additional information pertinent to the appropriate
application of multiresidue methodology.
Method is applicable to a wide variety of fatty and nonfatty foods; several different
extraction steps are available for the different food types. Cleanup by GPC is most
practical when a large number of analyses are being performed. Method was originally
designed for chlorophenoxy acids, but has been found applicable to a variety of
acidic and phenolic residues. See Table 402-a, following method description, for
pesticides and metabolites tested through the steps of this method.
REFERENCE STANDARDS
Use reference standards of methyl esters/ethers of acids or phenols, if available.
Otherwise, use standards of the acids/phenols methylated through C1a and cleaned
up through C1b. Prepare stock solutions in acetone.
STEPS OF THE METHOD
Extraction (E) Recommended Use
E1 (p. 402-3) Extraction with solvents from animal tissues, dairy prod-
acidified, denatured products ucts, fats, and shortenings
E2 (p. 402-7) Extraction with acidified methylene fruits, vegetables other
chloride than legumes, and
beverages
E3 (p. 402-9) Extraction with acidified methanol legumes
E4 (p. 402-10) Extraction with acidified water/ grains and cereals
methanol
E5 (p. 402-11) Extraction with acidified methanol sugar and high sugar pro-
cessed foods
E6 (p. 402-13) Dissolution in methylene chloride/ vegetable oils
hexane
E7 (p. 402-15) Extraction with acidified methylene water
chloride
Cleanup and methylation (C)
C1 (p. 402-17) GPC cleanup, methylation, and all products
Florisil column cleanup
Transmittal No. 93-1 (1/94)
Form FDA 2905a (6/92)402–2
SECTION 402 Pesticide Analytical Manual Vol. I
Determination (D) Recommended Use
DG1 (p. 302-25) GLC, 100% methyl siloxane column, halogenated acids and
200°, EC detector phenols
DG3 (p. 302-29) GLC, 100% methyl siloxane column, halogenated acids and
200°, ElCD-X phenols
DG4 (p. 302-31) GLC, 100% methyl siloxane column, acids and phenols con-
200°, ElCD-N taining nitrogen
VALIDATION
The following combination has undergone validation in a single laboratory, over
many years, with repeated recoveries performed in conjunction with FDA’s Total
Diet Study; these are recommended for use:
E1 + C1 + DG1
Validation report:
Hopper, M.L. et al. (1992) J. AOAC Int. 75, 707-713. Where slight differences
occur between the method description in Section 402 and in the Hopper et
al. 1992 publication, this section reflects standard operating procedure in the
Total Diet Study.
Figure 402-a
Method for Acids and Phenols
animal tissues,
dairy products,
fats and
shortenings
E1
fruits,
vegetables
other than
legumes, and
beverages
E2
legumes
E3
grains
and
cereals
E4
sugar and
high sugar
processed
foods
E5
C1
DG1/3
residues
with
halogen
DG4
residues
with
nitrogen
vegetable
oils
E6
water
E7
Transmittal No. 94-1 (1/94)
Form FDA 2905a (6/92) 402–3
Pesticide Analytical Manual Vol. I SECTION 402
E1 EXTRACTION WITH SOLVENTS FROM ACIDIFIED, DENATURED PRODUCTS
Reference
Hopper, M.L. et al. (1992) J. AOAC Int. 75, 707-713
Principles
Fat and residues are dissolved in ethyl ether and petroleum ether after the fatty
product has been denatured with oxalate and alcohol and acidified with sulfuric acid.
Ether extract is washed with large quantities of water to remove co-extractives.
Apparatus
blender, high speed; explosion-proof Waring Blendor, 1 qt jar
centrifuge, explosion-proof, to hold 500 mL bottles
centrifuge bottle, glass, 500 mL. Use glass stopper or cover rubber stopper
with aluminum foil to avoid contamination.
delivery tube apparatus (Figure 402-b), fabricated in laboratory
Kuderna-Danish concentrator (K-D),
500 mL, with Snyder column, plain
receiving flask
separatory funnel (separator), 250
mL and 1 L
Reagents
boiling chips, 20-30 mesh carbo-
rundum
ethyl ether, distilled from all-glass
apparatus, with 2% ethanol as pre-
servative, see Section 204 for perox-
ide test
methanol, distilled from all-glass ap-
paratus
methylene chloride, distilled from
all-glass apparatus
petroleum ether, distilled from all-
glass apparatus
sodium (or potassium) oxalate, re-
agent grade
sodium chloride aqueous solution,
saturated
10% sulfuric acid, reagent grade
1+1 (v/v) ethyl ether/petroleum ether
50% (v/v) methylene chloride/hexane
Directions
For each batch of samples analyzed, fortify commodity with chlorophenoxy acid
herbicide(s) and pentachlorophenol and analyze it with others to verify adequacy of
recoveries.
Figure 402-b
Delivery Tube Apparatus
Vacuum
Rubber
stopper
with two
holes
Glass tubing
Inlet
Delivery
Glass tube, inserted in one hole of two-hole
rubber stopper, is used to draw upper solvent
layer from centrifuge bottle into separatory
funnel. Siphon tube is straight or bent in U-
shape and inlet end placed at interface of two
phases in centrifuge bottle. Second hole in
stopper is fitted with another glass tube.
Vacuum drawn through second tube causes
upper phase from centrifuge bottle to transfer
into separator.
(Corrigan, E. (Nov. 1963) (FDA) Bureau By-Lines 5, 20;
Sawyer, L.D., and Baca, J.R. (May 1978) LIB 2188, FDA,
Rockville, MD.)
Transmittal No. 93-1 (1/94)
Form FDA 2905a (6/92)402–4
SECTION 402 Pesticide Analytical Manual Vol. I
Animal Tissues
? Weigh 50 g sample into blender jar.
? Add 50 mL distilled water, 100 mL methanol, 10 mL 10% sulfuric acid,
and about 2 g sodium or potassium oxalate.
? Blend at high speed 2 min and transfer to centrifuge bottle with aid of
powder funnel. Rinse blender jar and funnel with 50 mL ethyl ether and
add to bottle.
Dairy Products
? Grind cheese and other solid products before analysis. Weigh appropriate
amount into centrifuge bottle: 100 g milk or other relatively low fat com-
modity, 25-50 g cheese or other high fat commodity.
? Add 100 mL methanol, 10 mL 10% sulfuric acid, and about 2 g sodium or
potassium oxalate; mix. Add 50 mL ethyl ether.
Fats and Shortenings
? Weigh 25 g sample into centrifuge bottle.
? Add 50 mL distilled water, 100 mL methanol, 10 mL 10% sulfuric acid,
and about 2 g sodium or potassium oxalate; mix. Add 50 mL ethyl ether.
All
? Shake centrifuge bottle vigorously 1 min; then add 50 mL petroleum
ether and shake vigorously 1 min.
? Centrifuge about 5 min at 1500 rpm. Transfer top (solvent) layer, with
delivery tube apparatus, into 1 L separator containing 500-600 mL water,
30 mL saturated sodium chloride solution, and 10 mL 10% sulfuric acid.
? Re-extract aqueous residue in centrifuge bottle twice, shaking vigorously
1 min with 50 mL (1+1) ethyl ether/petroleum ether. After each extrac-
tion, centrifuge and transfer solvent layer into same 1 L separator.
? Mix combined extracts and water in separator thoroughly but cautiously
to prevent emulsion formation. Drain and discard water.
? Rewash (gently) solvent layer twice with 100 mL water, 10 mL 10% sulfu-
ric acid and 30 mL saturated sodium chloride solution; discard wash solu-
tion each time. If emulsions form, add additional 5 mL saturated sodium
chloride solution to wash.
? After final wash is discarded, transfer ether layer to 250 mL separator. Let
stand ≥30 min. Drain and discard any water and emulsion from separator.
? Transfer ether to K-D with plain receiving flask, add boiling chips, and
evaporate solvent.
? Cool, add 50 mL methylene chloride to extracted fat, and mix. Add boiling
chips and evaporate on steam bath until level in receiving flask does not
change and there is still methylene chloride in Snyder column traps. Cool.
? Use approximate fat content of commodity, as listed in Section 201, to
determine what dilution is required to achieve concentration of ≤0.16 g
fat/mL. Quantitatively transfer fat from receiving flask to glass-stoppered
(g-s) graduate of appropriate volume. Use 50% methylene chloride/hex-
ane for rinsing during transfer and then dilute solution to predetermined
volume with that solvent mixture.
Transmittal No. 94-1 (1/94)
Form FDA 2905a (6/92) 402–5
Pesticide Analytical Manual Vol. I SECTION 402
? Transfer aliquot of solution to tared vessel; evaporate to dryness at steam
bath temperature under current of dry air. Weigh and record weight of
fat extracted. If necessary, adjust remaining solution volume so that
solution contains ≤0.16 g fat/mL.
? Clean up extract by C1; equivalent weight of whole product cleaned up
is:
original sample weight
mL final extract
· mL loaded onto GPC
Transmittal No. 93-1 (1/94)
Form FDA 2905a (6/92)402–6
SECTION 402 Pesticide Analytical Manual Vol. I
Transmittal No. 94-1 (1/94)
Form FDA 2905a (6/92) 402–7
Pesticide Analytical Manual Vol. I SECTION 402
E2 EXTRACTION WITH ACIDIFIED METHYLENE CHLORIDE
Reference
Hopper, M.L. et al. (1992) J. AOAC Int. 75, 707-713
Principle
Residues are extracted from fruits and vegetables with methylene chloride after
acidification.
Apparatus
blender, high speed; explosion-proof Waring Blendor, 1 qt jar
centrifuge, explosion-proof, to hold 500 mL bottles
centrifuge bottle, glass, 500 mL. Use glass stopper or cover rubber stopper
with aluminum foil to avoid contamination.
delivery tube apparatus, see Figure 402-a in E1; apparatus with straight tube
inlet (rather than U-shape) is preferred
graduated cylinder (graduate), 250 mL
Kuderna-Danish concentrator (K-D), 500 mL, with Snyder column, 10 mL
graduated receiving flask
separatory funnel (separator), 250 mL
Reagents
boiling chips, 20-30 mesh carborundum
glass wool, Pyrex
hexane, distilled from all-glass apparatus
methylene chloride, distilled from all-glass apparatus
10% sulfuric acid, reagent grade
Directions
For each batch of samples analyzed, fortify commodity with chlorophenoxy acid
herbicide(s) and pentachlorophenol and analyze it with others to verify adequacy
of recoveries.
? Weigh 100 g sample (chopped fruits or vegetables other than legumes or
beverages) into blender jar. Add 10 mL 10% sulfuric acid and 250 mL
methylene chloride.
? Blend 2 min at high speed and pour into centrifuge bottle with aid of
powder funnel.
? Centrifuge 5 min at 1500 rpm. Transfer top (water) layer, with delivery
tube apparatus, into 1 L separator, then discard.
? Carefully decant methylene chloride (leaving cake in centrifuge bottle)
through funnel containing glass wool plug into separator. Let stand
≥30 min. Drain and discard any water and emulsion from separator.
? Transfer methylene chloride to graduate and record volume.
Transmittal No. 93-1 (1/94)
Form FDA 2905a (6/92)402–8
SECTION 402 Pesticide Analytical Manual Vol. I
mL methylene chloride recovered
250 mL
· 100 g ·
mL loaded onto GPC
10 mL
? Transfer measured volume of methylene chloride to K-D with 10 mL
receiving flask. Add boiling chips and concentrate to 5 mL on steam bath.
Dilute extract to 10 mL with hexane and mix.
? Clean up extract by C1; equivalent weight of product cleaned up is:
Transmittal No. 94-1 (1/94)
Form FDA 2905a (6/92) 402–9
Pesticide Analytical Manual Vol. I SECTION 402
E3 EXTRACTION WITH ACIDIFIED METHANOL
Reference
Hopper, M.L. et al. (1992) J. AOAC Int. 75, 707-713
Principle
Residues are extracted from legumes with methanol after acidification and parti-
tioned into methylene chloride.
Apparatus
blender, high speed; explosion-proof Waring Blendor, 1 qt jar
centrifuge, explosion-proof, to hold 500 mL bottles
centrifuge bottle, glass, 500 mL. Use glass stopper or cover rubber stopper
with aluminum foil to avoid contamination.
graduated cylinder (graduate), 100 and 250 mL
Kuderna-Danish concentrator (K-D), 500 mL, with Snyder column, 10 mL
graduated receiving flask
separatory funnel (separator), 2 L
Reagents
boiling chips, 20-30 mesh carborundum
glass wool, Pyrex
hexane, distilled from all-glass apparatus
methanol, distilled from all-glass apparatus
methylene chloride, distilled from all-glass apparatus
sodium chloride aqueous solution, saturated
10% sulfuric acid, reagent grade
Directions
For each batch of samples analyzed, fortify commodity with chlorophenoxy acid
herbicide(s) and pentachlorophenol and analyze it with others to verify adequacy
of recoveries.
? Weigh 100 g chopped legume vegetable into blender jar. Add 10 mL
10% sulfuric acid and 200 mL methanol.
? Blend mixture in blender jar 2 min at high speed and pour into centri-
fuge bottle with aid of powder funnel.
? Centrifuge 5 min at 1500 rpm and carefully pour top layer through
funnel containing glass wool plug into 250 mL graduate. Measure vol-
ume re-covered if <250 mL or take 250 mL.
? Pour measured extract into 2 L separator and add 100 mL methylene
chloride. Shake separator 30 sec, then add 30 mL saturated sodium
chloride solution, 10 mL 10% sulfuric acid, and 650 mL water.
? Shake 30 sec and allow emulsion to settle ≥30 min. Drain and discard
any water and emulsion from separator.
Transmittal No. 93-1 (1/94)
Form FDA 2905a (6/92)402–10
SECTION 402 Pesticide Analytical Manual Vol. I
? (If emulsion does not break, drain emulsion into centrifuge bottle and
centrifuge at 1500 rpm 5 min. Siphon off and discard water layer. Pour
methylene chloride through funnel containing glass wool plug into 250
mL separator. Let stand ≥30 min to ensure complete separation of any
remaining water. Drain and discard any water and emulsion from separa-
tor.)
? Transfer methylene chloride layer to 100 mL graduate and record vol-
ume.
? Transfer measured volume of methylene chloride to K-D with 10 mL
receiving flask. Add boiling chips and concentrate extract on steam bath
to about 5 mL. Dilute to 10 mL with hexane and mix.
? Clean up extract by C1; equivalent weight of legumes cleaned up is:
mL extract recovered after centrifugation
200 + 10 + (100 g · % moisture)
· 100 g ·
mL loaded onto GPC
10 mL
mL methylene chloride recovered
100 mL
·
ALTERNATIVE:
E4 EXTRACTION WITH ACIDIFIED WATER/METHANOL
Principle
Water/methanol replaces methanol for extraction from grains and cereal pro-
ducts to accommodate their low moisture.
Additional Reagents
30% (v/v) water/methanol
Directions
? Weigh 50 g ground grain or cereal product into blender jar. Add 10 mL 10%
sulfuric acid and 340 mL 30% water/methanol.
? Continue as in E3, “Blend mixture in blender jar 2 min. . .”
? Equivalent weight of grains or cereal products cleaned up is:
250 mL
340 mL + 10 mL
· 50 g ·
mL loaded onto GPC
10 mL
mL methylene chloride recovered
100 mL
·
Transmittal No. 94-1 (1/94)
Form FDA 2905a (6/92) 402–11
Pesticide Analytical Manual Vol. I SECTION 402
E5 EXTRACTION WITH ACIDIFIED METHANOL
Reference
Hopper, M.L. et al. (1992) J. AOAC Int. 75, 707-713
Principles
Residues are extracted from sugar or high sugar processed foods with aqueous
methanol after acidification; residues are then transferred to methylene chloride
by partitioning.
Apparatus
blender, high speed; explosion-proof Waring Blendor, 1 qt jar
centrifuge, explosion-proof, to hold 500 mL bottles
centrifuge bottle, glass, 500 mL. Use glass stopper or cover rubber stopper
with aluminum foil to avoid contamination.
graduated cylinder (graduate), 250 mL
Kuderna-Danish concentrator (K-D), 500 mL, with Snyder column, 10 mL
graduated receiving flask
separatory funnel (separator), 250 mL and 2 L
Reagents
boiling chips, 20-30 mesh carborundum
glass wool, Pyrex
methanol, distilled from all-glass apparatus
methylene chloride, distilled from all-glass apparatus
sodium chloride aqueous solution, saturated
10% sulfuric acid, reagent grade
Directions
For each batch of samples analyzed, fortify commodity with chlorophenoxy acid
herbicide(s) and pentachlorophenol and analyze it with others to verify adequacy
of recoveries.
? Weigh 50 g sugar or high sugar processed food into blender jar. Add 10
mL 10% sulfuric acid, 100 mL water, and 200 mL methanol.
? Blend 2 min at high speed. Pour mixture through powder funnel con-
taining glass wool plug into 2 L separator.
? Add 250 mL methylene chloride and shake 30 sec. Add 700 mL water,
10 mL 10% sulfuric acid, and 35 mL saturated sodium chloride solution.
Shake separator 1 min and let emulsion settle.
? Drain remaining emulsion and methylene chloride into 500 mL centri-
fuge bottle and centrifuge 5 min at 1500 rpm. Siphon off and discard
water layer.
? Pour methylene chloride layer through funnel containing glass wool
plug into 250 mL separator. Let stand ≥30 min to ensure complete
separation of any remaining water. Drain and discard any water and
emulsion from separator.
Transmittal No. 93-1 (1/94)
Form FDA 2905a (6/92)402–12
SECTION 402 Pesticide Analytical Manual Vol. I
? Transfer methylene chloride layer to 250 mL graduate and record volume.
? Transfer measured volume of methylene chloride to K-D fitted with 10 mL
receiving flask. Add boiling chips and concentrate extract on steam bath to
about 5 mL. Dilute to 10 mL with hexane and mix.
? Clean up extract by C1; equivalent weight of product cleaned up is:
50 g ·
mL loaded onto GPC
10 mL
mL methylene chloride recovered
250 mL
·
Transmittal No. 94-1 (1/94)
Form FDA 2905a (6/92) 402–13
Pesticide Analytical Manual Vol. I SECTION 402
· mL loaded onto GPC
16 g
100 mL
E6 DISSOLUTION IN METHYLENE CHLORIDE/HEXANE
Reference
Hopper, M.L. et al. (1992) J. AOAC Int. 75, 707-713
Principle
No actual extraction of residues is done for vegetable oils; instead, they are dissolved
in solvent for subsequent cleanup on GPC.
Reagents
hexane, distilled from all-glass apparatus
methylene chloride, distilled from all-glass apparatus
50% (v/v) methylene chloride/hexane
Directions
For each batch of samples analyzed, fortify commodity with chlorophenoxy acid
herbicide(s) and pentachlorophenol and analyze it with others to verify adequacy
of recoveries.
? Weigh 16 g pure vegetable oil into 100 mL volumetric flask. Dilute to
volume with 50% methylene chloride/hexane (0.16 g/mL).
? Clean up oil solution by C1; equivalent weight of product cleaned up is:
Transmittal No. 93-1 (1/94)
Form FDA 2905a (6/92)402–14
SECTION 402 Pesticide Analytical Manual Vol. I
Transmittal No. 94-1 (1/94)
Form FDA 2905a (6/92) 402–15
Pesticide Analytical Manual Vol. I SECTION 402
E7 EXTRACTION WITH ACIDIFIED METHYLENE CHLORIDE
Reference
Hopper, M.L. et al. (1992) J. AOAC Int. 75, 707-713
Principle
Residues in water are extracted with methylene chloride after acidification.
Apparatus
Kuderna-Danish concentrator (K-D), 500 mL, with Snyder column, two-ball
micro Snyder column, 10 mL graduated receiving flask
separatory funnel (separator), 1 L
Reagents
acetone, distilled from all-glass apparatus
methylene chloride, distilled from all-glass apparatus
10% sulfuric acid, reagent grade
Directions
For each batch of samples analyzed, fortify commodity with chlorophenoxy acid
herbicide(s) and pentachlorophenol and analyze it with others to verify adequacy
of recoveries.
? Weigh 500 g water, transfer to 1 L separator. Add 10 mL 10% sulfuric acid
and 60 mL methylene chloride. Shake vigorously 1 min.
? Allow layers to separate and drain methylene chloride layer into K-D
with 10 mL receiving flask. Repeat extraction with two 60 mL portions
methylene chloride. Combine all extracts.
? Add boiling chips and evaporate to near dryness on steam bath. Add 50
mL acetone and evaporate to about 3 mL.
? No GPC cleanup is necessary. Proceed to methylation, C1b. Entire solu-
tion (therefore, entire weight of original product) is methylated.
Transmittal No. 93-1 (1/94)
Form FDA 2905a (6/92)402–16
SECTION 402 Pesticide Analytical Manual Vol. I
Transmittal No. 94-1 (1/94)
Form FDA 2905a (6/92) 402–17
Pesticide Analytical Manual Vol. I SECTION 402
C1 GPC CLEANUP, METHYLATION, AND FLORISIL COLUMN CLEANUP
C1a GPC CLEANUP
Reference
Hopper, M.L. (1982) J. Agric. Food Chem. 30, 1038-1041
Principles
Co-extractives in the extract are separated from residues on GPC by molecular size
exclusion; larger molecules (fats, etc.) elute first and are discarded. Advance calibra-
tion of the GPC column dictates the optimum amount of eluate to discard in order
to remove most large molecule co-extractives and recover as much residue as possible.
Once calibrated, the column can be used repeatedly.
Apparatus
filtration device for solutions, 10 mL syringe with Luer-Lok tip, fitted with
either (a) 13 mm diameter Swinny stainless steel filter holder and 13 mm
diameter filters, 5.0 μm LS-type, or (b) disposable membrane filters, 25 mm
diameter, 5 μm Teflon membrane, encased in polypropylene. (Pre-assembled
devices that do not require a syringe are also available.)
GPC apparatus; automated equipment optional but recommended. GPC
apparatus must include:
1) sample introduction valve
2) pump, low pressure, suitable for use with organic solvents, capable of 5
mL/min flow
3) sample loading loop, 1/16" Teflon tubing coiled in cylindrical form,
about 13 mL capacity
4) pulse dampener, about 6' of 1/8" copper tubing coiled and closed at one
end, installed between pump and sample introduction valve with a con-
necting tee. Pulse dampener is needed only when pump is not pulseless.
GPC column, glass, 25 mm id × 300 or 500 mm with organic solvent plunger
kit
GPC syringe, 10 mL syringe with Luer-Lok tip, with Millipore Swinny stain-
less steel adapter, Millipore 5.0 μm LS-type filter
Kuderna-Danish concentrator (K-D), 500 mL, with Snyder column, two-ball
micro Snyder column, 5 and 10 mL volumetric or graduated receiving
flasks
Reagents
Bio-Beads SX-3 resin, 200-400 mesh (Bio-Rad Laboratories, Richmond, CA;
pretested resin is available from ABC Laboratories)
hexane, distilled from all-glass apparatus
methylene chloride, distilled from all-glass apparatus
eluant: 1+1 (v/v) methylene chloride/hexane
Transmittal No. 93-1 (1/94)
Form FDA 2905a (6/92)402–18
SECTION 402 Pesticide Analytical Manual Vol. I
Preparation of GPC column
? Weigh 35 g Bio-Beads SX-3 into 400 mL beaker.
? Add 150 mL 50% methylene chloride/hexane.
? Stir beads with glass or steel rod until all beads have swelled and lumps are no
longer present.
? Pour slurry into GPC column with aid of stirring rod.
? Hold column in upright position with plunger tightened about 25 mm
from bottom end of usable length of column, ignoring threaded ends.
? Add slurry to column continuously so beads never become completely
settled until all beads have been added.
? Place other plunger in column after beads have settled and liquid has
drained completely.
? Compress each plunger an equal distance from its respective end until bed
length is about 200 mm.
? Connect column to GPC solvent delivery system, and pump solvent from
bottom to top of column until all air is expelled.
? Adjust flow rate of system to 5 mL/min and check column pressure. Adjust
operating pressure for column to 8-11 psig by moving plunger(s).
? Allow GPC system to equilibrate by pumping solvent through it.
? Re-adjust flow rate to 5 mL/min if it has changed.
Calibration of GPC column
Elution of Fat
? Melt butter and decant butterfat and solids through fluted filter paper into
suitable container; do not include water layer. Heat funnel if necessary to
facilitate filtration.
? Weigh 4 g warm, filtered butterfat into 25 mL g-s graduate; dilute to 25 mL
with 50% methylene chloride/hexane; mix until fat is dissolved (0.16 g fat/
mL).
? Filter fat solution through filtration device and load 5 mL onto GPC column.
? Elute with 50% methylene chloride/hexane.
? Collect column effluent in tared beakers in 10 mL increments from 0 to
100 mL.
? Evaporate solvent from each beaker, cool, and weigh to calculate amount
of fat eluted in each 10 mL increment. (For manual GPC, collect 10 mL
fractions in separate graduates and transfer to tared beakers for evapora-
tion and calculation of fat.)
? 95% of fat should elute in first 60 mL. If >5% of fat appears in 60-70 mL
fraction or later, reject column and prepare new one by repacking with
original batch of beads. Visual evaluation of yellow band of fat as it passes
through column usually shows tailing or streaking when column is inad-
equate. Use new batch of beads if second column is still inadequate.
Transmittal No. 94-1 (1/94)
Form FDA 2905a (6/92) 402–19
Pesticide Analytical Manual Vol. I SECTION 402
Elution of Pesticides
? Prepare mixed standard solution containing 1.2 μg ethion/mL, 0.4 μg
diazinon/mL, 0.4 μg heptachlor epoxide/mL, 0.2 μg dicloran/mL, and
0.6 μg dieldrin/mL in 50% methylene chloride/hexane.
? Place 5 mL aliquot standard solution in K-D with 10 mL receiving flask;
add 50 mL hexane and 2-3 boiling chips; concentrate to 10 mL. This
removes methylene chloride prior to determinative step.
? Filter mixed standard solution through filtration device and load 5 mL
onto GPC column.
? Elute with 50% methylene chloride/hexane.
? Collect 10 mL fractions from 0 through 160 mL.
? Transfer each fraction to K-D with 10 mL receiving flask and add 50 mL
hexane and 2-3 boiling chips; concentrate to 10 mL.
? Calculate recoveries by comparison to mixed standard solution that has
also undergone concentration to 10 mL. Use determinative steps DG1 and
DG2.
? Column is normal if diazinon and ethion start to elute in either 50-60 mL
or 60-70 mL fraction, and dicloran starts to elute in 90-100 mL fraction.
? Determine what volume should be discarded (usually first 60 mL) and what
should be collected (usually 60-160 mL fraction) by examining fat and
mixed standard elution profiles developed above. Use these calibrated
fraction volumes in subsequent calibration steps and in sample cleanup.
Elution of Pesticides from Fat
? Weigh 1.6 g warm, filtered butterfat into tared 10 mL g-s graduate, add 5
mL mixed standard solution from pesticide elution test above, dilute
to volume with 50% methylene chloride/hexane, and mix until fat is
dissolved.
? Filter fortified fat solution through filtration device and load 5 mL onto
GPC column.
? Elute with 160 mL 50% methylene chloride/hexane.
? Discard and collect respective volumes determined during calibration.
? Transfer collected fraction to K-D with 10 mL receiving flask and concen-
trate as in pesticide elution test.
? Calculate recoveries as in pesticide elution test.
? For normal column, ≥80% diazinon, parathion, and ethion, and ≥95% of
organochlorine pesticides are recovered.
Elution of Herbicides
? Prepare mixed standard solution of 0.1 μg 2,4,5-T/mL and 0.05 μg
pentachlorophenol/mL in 50% methylene chloride/hexane by diluting
acetone stock solutions.
? Load 5 mL onto GPC column and elute with 160 mL 50% methylene
chloride/hexane.
? Discard and collect respective volumes determined above.
Transmittal No. 93-1 (1/94)
Form FDA 2905a (6/92)402–20
SECTION 402 Pesticide Analytical Manual Vol. I
? Transfer collected fraction to K-D with 5 mL receiving flask.
? Add 2-3 boiling chips and concentrate.
? Cool, add 50 mL acetone and fresh boiling chips, and reconcentrate to
about 3 mL.
? Cool solution and methylate as in C1b.
? Clean up methylated solution on Florisil as in C1c and concentrate to 5 mL.
? Calculate amount of herbicide in concentrated extract using determinative
step DG1 and reference standards of pentachlorophenol methyl ether
and 2,4,5-T methyl ester. Convert amount of methylated chemicals to
amount of corresponding acid/phenol by multiplying respective values
by 0.95, which represents ratio of molecular weights of acid/phenol to
methylated chemical for both 2,4,5-T and pentachlorophenol (255.49/
269.52 and 266.35/280.37, respectively). Calculate percentage recovered by
comparing converted values to amounts added (0.5 μg 2,4,5-T and 0.25 μg
pentachlorophenol).
? Gel column is acceptable if ≥80% of added 2,4,5-T and pentachlorophenol
are recovered.
Directions
? Use GPC column prepared and calibrated as described above. Column can
be used repeatedly.
? Centrifuge cloudy solutions of extract from E1-E6 before loading them
onto GPC. Filter all solutions through filtration device before GPC.
? Load filtered sample extract onto GPC column in 5 mL loops. About 0.8 g
fat or 15-50 g equivalent weight of nonfatty product can be loaded in each
loop. Use original sample weight, aliquots taken during extraction, volume
of final extract, and loading loop size to calculate amount being loaded.
? If targeted limit of quantitation is lower than can be achieved with this size
aliquot, load additional aliquots of extract in separate 5 mL loops and
combine concentrated eluates from each after GPC cleanup.
? Elute column with 160 mL 50% methylene chloride/hexane. Discard vol-
ume previously calibrated and collect remainder in beaker or graduate.
Transfer collected eluate to K-D fitted with 10 mL receiving flask. Rinse
collection vessel with several mL acetone and add to concentrator.
? Add 2-3 boiling chips and concentrate to about 3 mL. Cool, add 50 mL
acetone and fresh boiling chips, and reconcentrate to about 1 mL. Use
micro-Snyder column to reach final volume if necessary. Methylate
cleaned up extract by procedure in C1b.
Transmittal No. 94-1 (1/94)
Form FDA 2905a (6/92) 402–21
Pesticide Analytical Manual Vol. I SECTION 402
C1b METHYLATION
Reference
Hopper, M.L. (1987) J. Agric. Food Chem. 35, 265-269
Principles
Acids and phenols in cleaned up extract solution are ionized by an alkali
(tetrabutylammonium hydroxide) and methylated with methyl iodide to their re-
spective esters and ethers.
Apparatus
graduated cylinder (graduate), 10 mL, g-s
microliter syringes: 25, 50, or 100 μL, for adding reagents
water bath, capable of maintaining 40° C
Reagents
acetone, distilled from all-glass apparatus
hexane, distilled from all-glass apparatus
methyl iodide, certified grade
tetrabutylammonium hydroxide (TBAH) titrant, 1.0 M in methanol
Cautions
Note the following cautions that must be observed during methylation:
Use well ventilated hood and protective gloves when adding reagents for
methylation.
With each batch of samples, also methylate aliquot of same mixed stan-
dard solution of chlorophenoxy acids and pentachlorophenol used for forti-
fication in above recovery tests. Clean up methylated standard on Florisil
column.
Determine completeness of methylation by calculations using primary stan-
dards of chlorophenoxy acid methyl esters and pentachlorophenyl methyl
ether (i.e., not esters/ether generated by this methylation step). If methy-
lation reaction appears incomplete by test, prepare fresh mixed standard
solution of acids and pentachlorophenol (these solutions have been found
susceptible to degradation).
Methylate fresh mixed standard and old mixed standard, and clean up both
by Florisil chromatography. Determine degree of methylation in both old
and new standards as above.
The comparison between degrees of methylation will indicate whether prob-
lem is caused by standard or by methylation step. If low recoveries are not
repeated with new standards, problem can be assumed to be with old stan-
dards. If recoveries of old and new standards are both poor (i.e., consistently
<40% or >120%), methylation step is at fault.
Presence of water may prevent complete methylation.
Transmittal No. 93-1 (1/94)
Form FDA 2905a (6/92)402–22
SECTION 402 Pesticide Analytical Manual Vol. I
Directions
? Dilute concentrated extract from GPC, C1a, to 3 mL with acetone.
? Add 80 μL 1.0 M TBAH/methanol and 40 μL methyl iodide. Immediately
stopper tube.
? Mix, then place stoppered tube in 40° C water bath for 1.5 hr, with water
level of bath above fluid level in tube.
? Remove tube from water bath and attach to 250 mL K-D. Add 50 mL hexane
and boiling chips. Evaporate to about 1 mL (avoiding dryness).
? Dilute to appropriate volume with hexane, add 2 mL distilled water, and
shake stoppered tube. Discard water.
? Clean up methylated extract on Florisil, C1c.
C1c FLORISIL COLUMN CLEANUP
Reference
Griffitt, K.R. et al. (Feb. 1983) “Miniaturized Florisil Cleanup of Chlorophenoxy
Acid Herbicides and Pentachlorophenol in Total Diet Samples,” LIB 2695, FDA,
Rockville, MD
Principle
Solution of methylated residues is cleaned up by adsorption chromatography on
Florisil column.
Apparatus
chromatographic column, 10 mm id × 300 mm, Teflon stopcock, coarse por-
osity fritted disc
Kuderna-Danish concentrator (K-D), 125 or 250 mL, with Snyder column,
graduated or volumetric receiving flask
Reagents
acetonitrile, distilled from all-glass apparatus; see Section 204 for distillation
directions
boiling chips, carborundum, 20 mesh, or other suitable boiling chips
ethyl ether, distilled from all-glass apparatus, with 2% ethanol as preservative;
see Section 204 for peroxide test
Florisil, PR grade; see Section 204 for handling and testing directions and
calculation of lauric acid (LA) value
hexane, distilled from all-glass apparatus
methylene chloride, distilled from all-glass apparatus
sodium sulfate, anhydrous, granular, reagent grade; see Section 204 for han-
dling directions
eluants: 1—20% methylene chloride/hexane (v/v). Dilute 200 mL methyl-
ene chloride with hexane. Allow mixture to reach room tempera-
ture, and adjust volume to 1 L with hexane.
Transmittal No. 94-1 (1/94)
Form FDA 2905a (6/92) 402–23
Pesticide Analytical Manual Vol. I SECTION 402
2—50% methylene chloride/0.35% acetonitrile/49.65% hex-
ane (v/v/v). Pipet 3.5 mL acetonitrile into 500 mL methylene
chloride and dilute with hexane. Allow mixture to reach room
temperature and adjust to 1 L with hexane.
Florisil Check
For each batch of Florisil, verify that weight calculated from LA value is suitable by
this test.
? Place activated Florisil (weight = 110/LA value × 4 g) in chromato-
graphic column; add about 2 cm sodium sulfate.
? Add 5 mL mixed standard solution of 0.1 μg 2,4,5-T methyl ester/mL,
0.05 μg pentachlorophenyl methyl ether/mL, and 0.2 μg picloram me-
thyl ester/mL.
? Elute as directed below, concentrate eluates to 5 mL, and examine by
DG1.
? Calculate recoveries by comparison to original mixed standard solution.
? Elute column further with about 100 mL ethyl ether and collect in 10
mL fractions.
? Concentrate each fraction to 5 mL, and examine each by DG1.
? Determine volume of ethyl ether needed to elute picloram methyl ester
from column and record for each batch of Florisil.
? Pentachlorophenyl methyl ether should elute in eluate 1; 2,4,5-T methyl
ester (and methyl esters of other chlorophenoxy acids) should elute in
eluate 2. Picloram methyl ester should elute in about 100 mL ethyl
ether.
? If test chemicals do not elute as expected, test adjusted weights of Florisil
(similar to tests described in Section 204) or use different batch of
Florisil.
Directions
? Add appropriate weight of Florisil (determined above) to 10 mm id ×
300 mm column; add about 2 cm sodium sulfate.
? Prewash column with 15 mL hexane. Do not allow column to go to
dryness.
? Place K-D fitted with appropriate receiving flask under column.
? Quantitatively transfer methylated extract from C1b to column. Rinse
flask with hexane and add to column. Extract and rinse volume together
should be ≤15 mL.
? With stopcock completely open, elute column with 35 mL eluant 1.
? Change K-Ds and elute column with 60 mL eluant 2.
? To analyze for picloram, change K-Ds and elute column with appropri-
ate volume of ethyl ether determined above.
? Add boiling chips to K-Ds and concentrate each eluate to suitable defi-
nite volume. When volume <5 mL is needed, use two-ball micro-Snyder
or micro-Vigreaux column during evaporation.
Transmittal No. 93-1 (1/94)
Form FDA 2905a (6/92)402–24
SECTION 402 Pesticide Analytical Manual Vol. I
? Add 50 mL hexane to K-D containing Eluate 2 and reconcentrate to
remove final traces of acetonitrile. Add 50 mL hexane to K-D containing
ethyl ether eluate and reconcentrate.
Transmittal No. 94-1 (1/94)
Form FDA 2905a (6/92) 402–25
Pesticide Analytical Manual Vol. I SECTION 402
DETERMINATION
Determine methylated residues in concentrated solution from C1 with determinative
steps DG1, DG3, and DG4 (see Section 302).
Chlorinated residues are determined using DG1 or DG3; residues containing nitro-
gen may be determined using DG4.
Inject volume of concentrated extract equivalent to the following weights, based on
whole product:
Products ≥20% fat Products <20% fat
Eluate 1 ≤5 mg ≤10 mg
Eluate 2 ≤10 mg ≤20 mg
Ethyl ether ≤10 mg ≤20 mg
CONFIRMATION
Confirm tentatively identified residues according to the principles discussed in
Section 103. Review PESTDATA (Appendix I) to find GLC systems applicable to
residue; rechromatograph on other systems if available.
Transmittal No. 93-1 (1/94)
Form FDA 2905a (6/92)402–26
SECTION 402 Pesticide Analytical Manual Vol. I
Pesticide Analytical Manual Vol. I SECTION 403
403–1
Transmittal No. 94-1 (1/94)
Form FDA 2905a (6/92)
403: METHOD FOR PHENYLUREA HERBICIDES
BASIC REFERENCE
Luchtefeld, R.G. (1987) J. Assoc. Off. Anal. Chem. 70, 740-745
GENERAL PRINCIPLES
Phenylurea herbicide residues are extracted with methanol. The extract is cleaned
up by partitioning and column chromatography on a Florisil column. Residues are
selectively determined with an HPLC system consisting of a reverse phase (RP)
column, post-column photodegradation and derivatization, and detection of the
resultant derivative by fluorescence detection.
APPLICABILITY
Consult Guide to PAM I for additional information pertinent to the appropriate
application of multiresidue methodology.
Method is applicable to residues of phenylurea herbicides in nonfatty foods. See
Table 403-a, following method description, for pesticides tested through the method.
Limits of detection and quantitation for selected compounds are also included in
Table 403-a; method is applicable to determination of at least 14 phenylurea
herbicides at concentrations of 0.05, 0.5, and 1.0 ppm in 14 nonfatty food prod-
ucts.
REFERENCE STANDARDS
Prepare stock solutions (1 mg/mL) of each standard in HPLC grade isopropyl
alcohol. Combine appropriate amounts of stock standard solutions and further
dilute with (1+1) acetonitrile/HPLC grade water for working standards. Use stan-
dards at concentration of 1.25 μg/mL for HPLC determination. Keep stock solu-
tions refrigerated prior to use and prepare working standards weekly.
STEPS OF THE METHOD
Extraction (E) Recommended Use
E1 (p. 403-3) Extraction with methanol nonfatty foods
Cleanup (C)
C1 (p. 403-5) Liquid-liquid partitioning and nonfatty foods
Florisil column cleanup
Determination (D)
DL3 (p. 403-7) HPLC, post-column photolysis and phenylureas
derivatization, fluorescence detection
DL4 (p. 403-10) HPLC, different mobile phase alternative to DL3,
confirmation of
identity
SECTION 403 Pesticide Analytical Manual Vol. I
Transmittal No. 94-1 (1/94)
Form FDA 2905a (6/92)403–2
Figure 403-a
Method for Phenylureas
VALIDATION
The following combination has undergone interlaboratory validation and is rec-
ommended for use:
E1 + C1 + DL3
Validation report:
Luchtefeld, R.G. (May 1989) “Validation of a Multiresidue Procedure for
Detemining Phenylurea Herbicides,” LIB 3309, FDA, Rockville, MD
nonfatty foods
E1
C1
DL3 and DL4
phenylurea
residues
Pesticide Analytical Manual Vol. I SECTION 403
403–3
Transmittal No. 94-1 (1/94)
Form FDA 2905a (6/92)
E1 EXTRACTION WITH METHANOL
Reference
Luchtefeld, R.G. (1987) J. Assoc. Off. Anal. Chem. 70, 740-745
Principles
Residues of phenylurea herbicides are extracted from nonfatty foods with metha-
nol, and the methanol solution is separated from food solids by centrifugation.
Apparatus
centrifuge, explosion-proof, to hold 500 mL bottles
centrifuge bottle, glass, 500 mL. Use glass stopper or cover rubber stopper
with aluminum foil to avoid contamination.
funnel, glass
graduated cylinder (graduate), 250 mL
homogenizer, Polytron Model PT 10-35, with PT 35K generator containing
knives, head equipped with metal (not Teflon) bushing
Reagents
glass wool, Pyrex
methanol, distilled from all-glass apparatus
Directions
? Weigh 50 g sample into centrifuge bottle. Add 100 mL methanol and
homogenize mixture 1.5 min using Polytron with speed set at 8.
? Centrifuge homogenate 5 min at 1500 rpm and decant 80-100 mL
through funnel fitted with glass wool plug into 250 mL graduate.
? Add 100 mL methanol to material in centrifuge bottle and homogenize
1.0 min with speed set at 8.
? Centrifuge homogenate and decant as much liquid as possible through
funnel into same 250 mL graduate.
? Record volume of combined methanol extract.
SECTION 403 Pesticide Analytical Manual Vol. I
Transmittal No. 94-1 (1/94)
Form FDA 2905a (6/92)403–4
Pesticide Analytical Manual Vol. I SECTION 403
403–5
Transmittal No. 94-1 (1/94)
Form FDA 2905a (6/92)
C1 LIQUID-LIQUID PARTITIONING AND FLORISIL COLUMN CLEANUP
Reference
Luchtefeld, R.G. (1987) J. Assoc. Off. Anal. Chem. 70, 740-745
Principles
Co-extractives are removed by adding sodium chloride to the methanol extract
and partitioning with hexane, which is discarded. Residues are partitioned from
methanol into methylene chloride. Concentrated methylene chloride extract is
cleaned up on a Florisil column, and residues are eluted with acetone/methylene
chloride.
Apparatus
chromatographic column, 10 mm id × 300 mm, Teflon stopcock, coarse
porosity fritted disc
chromatographic column, 25 mm id, plain
Kuderna-Danish concentrators (K-D), 250 and 500 mL, with Snyder col-
umn and 5 mL graduated receiving flasks
separatory funnel (separator), 500 mL and 1 L
Reagents
acetone, distilled from all-glass apparatus
acetonitrile, HPLC grade
boiling chips, 20-30 mesh carborundum
Florisil, PR grade; see Section 204 for handling and testing directions
hexane, distilled from all-glass apparatus
methylene chloride, distilled from all-glass apparatus
sodium chloride, reagent grade
sodium chloride aqueous solution, saturated
sodium sulfate, anhydrous, granular, reagent grade; see Section 204 for
handling directions
eluant: 20% (v/v) acetone/methylene chloride
1+1 (v/v) acetonitrile/HPLC grade water
Directions
Partitioning
? Transfer entire methanol extract (E1) to 500 mL separator.
? Add 30 mL saturated sodium chloride solution and 50 mL hexane, and
shake mixture 30 sec. Let layers separate.
? Transfer lower aqueous layer to 1 L separator containing 500 mL water
and 30 mL saturated sodium chloride solution. Discard hexane.
? Add 200 mL methylene chloride to the 1 L separator and shake 1 min.
? Drain methylene chloride through 25 mm × 50 mm column of sodium
sulfate into 500 mL K-D fitted with 5 mL receiving flask.
SECTION 403 Pesticide Analytical Manual Vol. I
Transmittal No. 94-1 (1/94)
Form FDA 2905a (6/92)403–6
? Re-extract aqueous phase with two 100 mL portions methylene chloride.
Drain each extract through sodium sulfate into K-D.
? Rinse sodium sulfate with 50 mL methylene chloride; add rinse to K-D.
? Add boiling chip to K-D and evaporate combined extracts and rinse to
about 3 mL for transfer to Florisil column.
Florisil Cleanup
? Place 4 g activated Florisil in 10 mm id chromatographic column; add 1
cm sodium sulfate. Wash column with 30 mL methylene chloride and
discard wash.
? When methylene chloride reaches top of sodium sulfate, place 250 mL
K-D with 5 mL graduated receiving flask under column. Transfer concen-
trated extract to column. Rinse container with about 3 mL methylene
chloride and add rinse to column.
? When extract reaches top of sodium sulfate, rinse column with two 3 mL
portions methylene chloride; allow each rinse to reach top of sodium
sulfate.
? When final rinse reaches top of sodium sulfate, elute column with 50 mL
20% acetone/methylene chloride.
? Concentrate solution on steam bath to about 3 mL. Remove receiving
flask and evaporate to dryness, using stream of nitrogen and water bath at
40° C.
? Pipet 2 mL (1+1) acetonitrile/HPLC grade water into receiving flask.
? Calculate sample equivalent in final cleaned up extract according to fol-
lowing formula:
=
·
g sample
mL extract
mL methanol filtrate collected
200 mL + (50 g · % water in sample) – 5
50 g
2 mL final extract
Pesticide Analytical Manual Vol. I SECTION 403
403–7
Transmittal No. 94-1 (1/94)
Form FDA 2905a (6/92)
DL3 HPLC, POST-COLUMN PHOTOLYSIS AND DERIVATIZATION,
FLUORESCENCE DETECTION
Reference
Luchtefeld, R.G. (1985) J. Chromatogr. Sci. 23, 516-520
Principles
Residues in acetonitrile/water solution are separated on a C-18 RP HPLC column
using methanol/water gradient mobile phase. Residues eluting from the column
are photolyzed in-line to primary amines by exposure to UV light; the flow’s
passage through a sleeve of Teflon tubing increases exposure to UV light. The
amines are reacted in-line with o-phthalaldehyde and 2-mercaptoethanol to form
fluorophores that are measured by a fluorescence detector. The determinative
step is very selective for phenylureas.
Apparatus
filtration device for solutions, 10 mL syringe with Luer-Lok tip, fitted
with either (a) 13 mm diameter Swinny stainless steel filter holder and
13 mm diameter filters, 5.0 μm LS-type, or (b) disposable membrane filters,
13 mm diameter, 0.22 μm nylon membrane, encased in polypropylene.
(Preassembled devices that do not require a syringe are also available.)
HPLC apparatus (Figure 403-b) must meet system suitability test below.
Complete system consists of:
1) mobile phase delivery system: dual pump gradient system
2) injector with 40 μL valve loop injector
3) guard column, direct connect cartridge system containing prepacked
C-18 cartridge (Alltech Associates, Deerfield, IL 60015, Cat. No. 28013)
4) column oven or heater, to maintain analytical column at constant tem-
perature
Figure 403-b
HPLC System for Determination of Phenylurea Herbicides
MethanolWater
Valve
injector
Guard
column
C-18 (ODS)
analytical column
Fluorescence
detector
Recorder/data
handling
Post-column
reaction
OPA-MERC
Teflon
reaction coil
UV lamp, sleeve of
Teflon tubing
SECTION 403 Pesticide Analytical Manual Vol. I
Transmittal No. 94-1 (1/94)
Form FDA 2905a (6/92)403–8
5) analytical column, 25 cm × 4.6 mm id, containing spherical particles
with monomeric bonded layer of octadecylsilane (ODS, C-18); e.g.,
Econosphere C-18, Alltech Associates
6) post-column photolysis and derivatization unit as shown in telescoped
portion of Figure 403-a. Unit is assembled from:
a) UV lamp for photodegradation, 17 cm × 9 mm od (Model 80-1178-
01) with power supply (Model 90-0001-01), BHK Inc., Pomona, CA
b) Teflon sleeve for photodegradation lamp, 10′ × 0.5 mm id delay
coil (Cat. No. 5-9206, Supelco, Inc., Supelco Park, Bellefonte, PA).
Place coiled Teflon tubing over lamp and connect one end to mix-
ing tee and other end to column.
c) low flow-rate pump for OPA-MERC solution (Model 396-31, LDC/
Milton Roy, Riviera Beach, FL). Connect cone-shaped coil of 13′ ×
1/8" od stainless steel tubing as pulse dampener between pump and
0.5 mm id Teflon tubing, which is connected to tee.
d) mixing tee, stainless steel, 0.25 mm bore, 1/16" standard fittings
(No. ZT1C, Valco Instruments, Inc., Houston, TX)
e) reaction coil, 10′ × 0.5 mm id delay coil (Supelco; Cat. No. 5-9206),
with one end connected to mixing tee and other end to detector
7) fluorescence detector
Reagents
acetic acid, glacial, reagent grade
2-mercaptoethanol (MERC), reagent grade. Prepare stock solution by dilut-
ing 10 mL MERC to 100 mL with methanol.
methanol, HPLC grade. Before use, degas in glass bottles by helium sparging
or other suitable method.
monobasic potassium phosphate, certified ACS grade. Prepare 0.002 M solu-
tion by dissolving 0.27 g in 1 L HPLC grade water.
o-phthalaldehyde (OPA), reagent grade. Prepare stock solution by dissolving
300 mg OPA in 100 mL methanol.
sodium borate buffer solution. Dissolve 19.1 g ACS grade sodium tetraborate
decahydrate (Na
2
B
4
O
7
?10 H
2
O) in approximately 500 mL HPLC grade wa-
ter. Dilute to 1 L with HPLC grade water and adjust pH to 10.5 with sodium
hydroxide, using pH meter. Filter through filtration device.
water, HPLC grade, commercial product or prepared from water purification
equipment that produces distilled, de-ionized water. For HPLC, degas water
as described for acetonitrile. Water must be adequately purified to prevent
plugging HPLC column and extraneous peaks in chromatograms. All water
used in HPLC procedure must be HPLC grade. (“Water” that does not specify
HPLC grade means distilled water.)
OPA-MERC solution. Transfer about 250 mL degassed sodium borate buffer
solution to 500 mL volumetric flask. Add 25 mL OPA stock solution and
5 mL MERC stock solution. Dilute to volume with borate buffer solution with
mixing.
Pesticide Analytical Manual Vol. I SECTION 403
403–9
Transmittal No. 94-1 (1/94)
Form FDA 2905a (6/92)
System Operation
? Operate column overnight with 0.3 mL/min (90+10) 0.002 M monoba-
sic potassium phosphate/methanol mobile phase, using single or dual
pump system. Change mobile phase to 10% methanol/HPLC grade wa-
ter and operate 30 min at 1.0 mL/min. If chromatogram baselines be-
gin to drift after several days of use, repeat this procedure.
? Maintain analytical column at 35° C. Equilibrate system with 40% metha-
nol/HPLC grade water at 1.0 mL/min, with 0.2 mL/min OPA-MERC
solution added through mixing tee. Allow detector to stabilize after
starting OPA-MERC solution flow and turning on UV photodegradation
lamp.
? After injecting sample or standard, begin 30 min linear gradient from
40% methanol/HPLC grade water to 80% methanol/HPLC grade wa-
ter. Operate detector at excitation wavelength of 340 nm and emission
wavelength of 455 nm. Adjust detector sensitivity to obtain 50% recorder
or integrator deflection when 40 μL 1.0 μg/mL diuron solution is in-
jected. Diuron elutes in approximately 24 min under these conditions.
? Flush pump and tubing used for addition of OPA-MERC solution daily
after use by pumping 3% glacial acetic acid in water through system at
1.0 mL/min for about 20 min.
? After each day’s use, rinse HPLC column 20 min with 80% methanol/
HPLC grade water. Column may be stored in this mobile phase.
System Suitability Test
See Chapter 6, HPLC, for further information about evaluating HPLC systems.
? Prepare solution containing 1 μg/mL each of metobromuron and diu-
ron.
? Chromatograph solution three times. Retention times are approximately
22 and 24 min for metobromuron and diuron, respectively.
? Determine relative standard deviation (RSD) (standard deviation/mean)
of peak heights measured in three chromatograms; also determine reso-
lution between the two peaks, according to formulas in Figure 602-a.
? RSD is <3% and resolution will be ≥1.5 on adequate system.
Directions
Figure 403-c compares chromatogram from this system to one from system using
UV detection at 245 nm.
? Filter both acetonitrile/water sample extract, C1, and reference stan-
dard solution(s) through filtration device; inject 40 μL filtrate into HPLC
system.
? Compare detector responses (peak height is preferred) to sample ex-
tract and to reference standard using same chromatographic conditions.
? Use (1+1) acetonitrile/HPLC grade water to make further dilutions of
sample extract, as necessary to make peak heights of sample and stan-
dard match closely.
SECTION 403 Pesticide Analytical Manual Vol. I
Transmittal No. 94-1 (1/94)
Form FDA 2905a (6/92)403–10
Figure 403-c
HPLC Chromatograms of Carrot Extract
0 5 10 15 20 25
B
A
(A) HPLC determinative step, Section 403 DL3; (B)
Detection by UV detector at 245 nm. Large peak in
chromatogram A is caused by interference from car-
rot sample.
[Reprinted with permission of Association of Official Analytical Chem-
ists, from J. Assoc. Off. Anal. Chem. (1987) 70, 740-745, Figure 1,
page 741.]
ALTERNATIVE:
DL4 HPLC, DIFFERENT MOBILE PHASE
Reference
Luchtefeld, R.G. (1985) J. Assoc. Off. Anal. Chem. 70, 740-745
Principles
HPLC chromatographic pattern for phenylureas is changed by using a gradient
mobile phase of acetonitrile/water instead of methanol/water. Differences pro-
vide useful confirmatory evidence of residue identity.
Additional Reagent
acetonitrile, HPLC grade. Before use, degas in glass bottles by helium sparging
or other suitable method.
Directions
Figure 403-d demonstrates different chromatographic patterns produced by DL3
and DL4. Table 403-a lists retention times (relative to diuron) for 14 phenylurea
herbicides on the two systems.
? Set up and operate HPLC system as in DL3, except use 30 min linear
gradient from 30% acetonitrile/HPLC grade water to 80% acetonitrile/
HPLC grade water as mobile phase. All other operations remain the same.
Pesticide Analytical Manual Vol. I SECTION 403
403–11
Transmittal No. 94-1 (1/94)
Form FDA 2905a (6/92)
Figure 403-d
Chromatographic Pattern Comparison
1 fluometuron
2 metobromuron
3 diuron
4 linuron
5 chlorbromuron
6 chloroxuron
1
2
3
4
5
6
1
3
2
4
5, 6
035
B
A
Minutes
Chromatography of six phenylurea herbicides on Econosphere ODS
column, 25 cm × 4.6 mm id with two different mobile phases. [A]
linear gradient from 40-80% methanol/water in 30 min; 1 mL/
min flow rate. [B] linear gradient from 30-80% acetonitrile/water
in 30 min; 1 mL/min flow rate.
[Reprinted with permission of Association of Official Analytical Chemists, from J.
Assoc. Off. Anal. Chem. (1987) 70, 740-745, Figure 5, page 743.]
CONFIRMATION
Confirm tentatively identified residues according to the principles discussed in
Section 103. Rechromatograph on whichever HPLC system, DL3 or DL4, was not
used in original determination.
SECTION 403 Pesticide Analytical Manual Vol. I
Transmittal No. 94-1 (1/94)
Form FDA 2905a (6/92)403–12
Transmittal No. 94-1 (1/94)
Form FDA 2905a (6/92)
SECTION 404Pesticide Analytical Manual Vol. I
404–1
404: METHOD FOR BENZIMIDAZOLES
BASIC REFERENCE
Gilvydis, D.M., and Walters, S.M. (1990) J. Assoc. Off. Anal. Chem. 73, 753-761
GENERAL PRINCIPLES
Residues are extracted with methanol and partitioned into methylene chloride
after initial acidification and again after subsequent alkalinization of the extract.
Residues are separated and quantitated by reverse phase (RP) ion pair HPLC with
both UV and fluorescence detectors.
APPLICABILITY
Consult Guide to PAM I to find additional information pertinent to appropriate
application of multiresidue methodology.
Method is applicable to fruits and vegetables and determines residues of allophanate,
MBC (resulting from use of benomyl, carbendazim, or thiophanate-methyl), thia-
bendazole, and thiophanate-methyl. UV detector responds to all residues to which
the method is applicable, but fluorescence detector responds only to MBC and
thiabendazole. Alternative determinative step permits examination for residues in
commodities from which interfering materials are co-extracted. Method variations
applicable to coffee beans are designed for determination of MBC only.
Method limit of quantitation is 0.1 ppm for each residue. UV detection is nonse-
lective and prone to crop interferences, so limit of quantitation may be affected by
the particular commodity. Limit of quantitation for thiabendazole can be increased
to 0.01 ppm by use of a fluorescence detector at conditions of maximum absor-
bance for the compound.
See Table 404-a, following method description, for results of recovery tests.
REFERENCE STANDARDS
Obtain reference standards from repository or commercial sources.
Prepare stock solutions (25 μg/mL) in acetone of each of following: allophanate,
MBC, thiabendazole, and thiophanate-methyl. (Do not use benomyl as reference
standard, because overnight standing is required to ensure complete decomposition
to MBC.) Solutions are stable in acetone and in HPLC mobile phase.
Prepare mixed standard solution by combining 1 mL of each stock standard solution,
evaporate to dryness with gentle heat (30-40° C) under stream of nitrogen, dissolve
residue in 4.0 mL methanol, add 6.0 mL HPLC ion pairing solution, and mix to give
final concentration of 2.5 μg/mL of each standard.
STEPS OF THE METHOD
Choose from these method options:
Extraction (E) Recommended Use
E1 (p. 404-5) extraction with methanol, transfer fruits and vegetables
to methylene chloride
E2 (p. 404-7) extraction with methanol, removal green coffee beans
of oil with hexane, transfer to
methylene chloride
Pesticide Analytical Manual Vol. ISECTION 404
404–2
Transmittal No. 94-1 (1/94)
Form FDA 2905a (6/92)
E3 (p. 404-8) extraction with methanol, removal roasted coffee beans
of oil with methylene chloride,
transfer to methylene chloride
Cleanup: There are no traditional cleanup steps in this method.
Determination (D) Recommended Use
DL5 (p. 404-9) HPLC, 4.1 mM ion pairing fruits and vegetables
reagent in mobile phase, UV
and fluorescence detection
DL6 (p. 404-12) HPLC, 32.7 mM ion pairing citrus, coffee beans
reagent in mobile phase, (green and roasted)
UV and fluorescence detection
DL7 (p. 404-14) HPLC, 4.1 mM ion pairing reagent thiabendazole
in mobile phase, changes in
detector settings
Figure 404-a
Method for Benzimidazoles
fruits other than
citrus, vegetables green coffee beans
DL5
MBC
thiophanate-methyl
thiabendazole
allophanate
roasted coffee beans
MBC
(coffee beans)
DL7
thiabendazole
citrus
E1 E2
DL6
MBC
thiophanate-methyl
thiabendazole
allophanate
E3
Transmittal No. 94-1 (1/94)
Form FDA 2905a (6/92)
SECTION 404Pesticide Analytical Manual Vol. I
404–3
VALIDATION
Several combinations of method options are possible. The following combinations
have undergone interlaboratory validation and are recommended for use:
E1 + DL5
Validation report:
Gilvydis, D.M. (July 1990) Quarterly Report on methods research, FDA internal
communication
E2 + DL6
Validation report:
Jacobs, R.M., and Yess, N.J. (1993) Food Addit. and Contam. 10, 575-577
E3 + DL5
Validation report:
Roy, R.R. (1993) FDA private communication of results (applied to many
different commodities) from Interagency Agreement No. FDA 224-90-2479,
work performed by USDA National Monitoring and Residue Analysis Labora-
tory, Gulfport, MS
Pesticide Analytical Manual Vol. ISECTION 404
404–4
Transmittal No. 94-1 (1/94)
Form FDA 2905a (6/92)
Transmittal No. 94-1 (1/94)
Form FDA 2905a (6/92)
SECTION 404Pesticide Analytical Manual Vol. I
404–5
E1 EXTRACTION WITH METHANOL, TRANSFER TO METHYLENE CHLORIDE
Reference
Gilvydis, D.M., and Walters, S.M. (1990) J. Assoc. Off. Anal. Chem. 73, 753-761
Principle
Residues are extracted with methanol and partitioned into methylene chloride after
initial acidification and again after subsequent alkalinization of the extract.
Apparatus
Buchner funnel (Buchner), porcelain, 12 cm diameter
evaporator, vacuum rotary
filter paper, sharkskin, to fit Buchner funnel
flask, round-bottom (r-b), 1 L
separatory funnel (separator), 500 mL
shaker, mechanical, Burrell wrist action
vacuum filtration flask, 1 L
Reagents
All solvents must be suitable for liquid chromatography and spectrophotom-
etry and must be filtered through 0.2 μm filter.
hydrochloric acid, reagent grade, 1 M
methanol, distilled from all-glass apparatus
methylene chloride, distilled from all-glass apparatus
sodium chloride, 1% aqueous solution
sodium hydroxide, 5 M and 1 M solutions
sodium sulfate, anhydrous, granular, reagent grade; see Section 204 for
handling directions
Directions
? Weigh 50 g finely chopped sample into 500 mL glass-stoppered (g-s)
Erlenmeyer flask. Add 100 mL methanol and shake 10 min on mechani-
cal shaker. To analyze bananas (whole or pulp only), shake vigorously by
hand prior to mechanical shaking to disperse mass.
? Filter contents with suction through Buchner fitted with filter paper into
vacuum filtration flask. Rinse Erlenmeyer and filter cake with additional
50 mL methanol.
? Transfer filtrate to 500 mL separator, and add 10 mL 1 M hydrochloric
acid and 100 mL 1% sodium chloride solution; for low moisture crops,
e.g., wheat grain, use 150 mL 1% sodium chloride solution. Mix and
allow to cool.
? Extract aqueous filtrate with two 100 mL portions methylene chloride,
shaking vigorously 1 min each time.
Pesticide Analytical Manual Vol. ISECTION 404
404–6
Transmittal No. 94-1 (1/94)
Form FDA 2905a (6/92)
? Dry methylene chloride layers through column of about 70 g sodium sulfate,
collecting in r-b flask. Save aqueous methanol layer in separator for later
extractions.
? Rinse sodium sulfate with about 50 mL methylene chloride and collect in
r-b flask.
? Evaporate methylene chloride in r-b flask just to dryness using vacuum
rotary evaporator with ≤30° C water bath.
? Dissolve material in r-b flask in 4 mL methanol. This extract contains
residues of allophanate and thiophanate-methyl and some MBC and thia-
bendazole.
? Determine residues in this extract with DL5, except use DL6 for citrus.
Avoid delay in determination to minimize loss of thiophanate-methyl by
degradation.
? Drain aqueous methanol phase from separator into beaker. Rinse separa-
tor with two 10 mL portions water, and add rinsings to beaker.
? Adjust pH of solution in beaker to 7.5-8 with 5 M and 1 M sodium hydrox-
ide solutions and 1 M hydrochloric acid, as necessary. Do not allow solu-
tion to become strongly alkaline during adjustment.
? Return solution to separator and extract with two 100 mL portions methy-
lene chloride, shaking vigorously 1 min each time.
? Dry methylene chloride layers through column of about 70 g sodium
sulfate, collecting in r-b flask. (Sodium sulfate column used to dry previ-
ous methylene chloride layer, above, may be reused.) Rinse sodium sul-
fate with about 50 mL methylene chloride and collect in r-b flask.
? Evaporate combined methylene chloride extracts to dryness in vacuum
rotary evaporator. Dissolve material in r-b flask with 4 mL methanol. Most
MBC and thiabendazole are recovered in this extract.
? Determine residues with DL5, except use DL6 for citrus.
Transmittal No. 94-1 (1/94)
Form FDA 2905a (6/92)
SECTION 404Pesticide Analytical Manual Vol. I
404–7
E2 EXTRACTION WITH METHANOL, REMOVAL OF OIL WITH HEXANE,
TRANSFER TO METHYLENE CHLORIDE
Reference
Gilvydis, D.M., and Walters, S.M. (Aug. 1989) “Modification of LIB 3217 for
Carbendazim (MBC) in Green and Roasted Coffee Beans,” LIB 3353, FDA, Rockville,
MD
Principles
Residues are extracted from green coffee beans with methanol and the filtrate is
acidified. Oils are removed from acidified extract by partitioning into hexane,
which is discarded. Aqueous methanol extract is then made alkaline, and residues
are extracted into methylene chloride by partitioning. Only MBC is targeted by
this analysis, so extraction from acidified filtrate (done in E1) is not performed.
Apparatus
Buchner funnel (Buchner), porcelain, 12 cm diameter
evaporator, vacuum rotary
filter paper, sharkskin, to fit Buchner funnel
flask, round-bottom (r-b), 1 L
separatory funnel (separator), 500 mL
shaker, mechanical, Burrell wrist action
vacuum filtration flask, 1 L
Reagents
All solvents must be suitable for liquid chromatography and spectrophotom-
etry and must be filtered through 0.2 μm filter.
hexane, distilled from all-glass apparatus
hexane, methanol-saturated
hydrochloric acid, reagent grade, 1 M
methanol, distilled from all-glass apparatus
methylene chloride, distilled from all-glass apparatus
sodium chloride, 1% aqueous solution
sodium hydroxide, 5 M and 1 M solutions
sodium sulfate, anhydrous, granular, reagent grade; see Section 204 for
handling directions
Directions
? Grind coffee beans, using centrifugal mill, to pass 20-mesh screen.
? Weigh 50 g prepared sample into 500 mL g-s Erlenmeyer flask. Add
100 mL methanol and shake 10 min on mechanical shaker.
? Filter contents with suction through filter paper in Buchner and rinse
with additional 50 mL methanol. Transfer filtrate to 500 mL separator.
Pesticide Analytical Manual Vol. ISECTION 404
404–8
Transmittal No. 94-1 (1/94)
Form FDA 2905a (6/92)
? Add to filtered extract in separator 10 mL 1 M hydrochloric acid and 150
mL 1% sodium chloride solution. Mix and allow to cool.
? Extract acidified methanol once with 100 mL methanol-saturated hexane.
Drain lower aqueous layer into beaker and discard hexane.
? Adjust pH of solution in beaker to 7.5-8 with 5 M and 1 M sodium hydrox-
ide solutions and 1 M hydrochloric acid, as necessary. Do not allow solu-
tion to become strongly alkaline during adjustment.
? Return solution to separator and extract with two 100 mL portions methyl-
ene chloride, shaking vigorously 1 min each time.
? Dry methylene chloride layers through column of about 70 g sodium
sulfate, collecting in r-b flask. Rinse sodium sulfate with about 50 mL
methylene chloride and collect in r-b flask.
? Evaporate combined methylene chloride extracts to dryness in vacuum
rotary evaporator. Dissolve material in r-b flask with 4 mL methanol. Resi-
dues of MBC will be in solution. Determine with DL6.
ALTERNATIVE:
E3 EXTRACTION WITH METHANOL, REMOVAL OF OIL WITH
METHYLENE CHLORIDE, TRANSFER TO METHYLENE
CHLORIDE
Reference
Gilvydis, D.M., and Walters, S.M. (Aug. 1989) “Modification of LIB 3217 for
Carbendazim (MBC) in Green and Roasted Coffee Beans,” LIB 3353, FDA, Rock-
ville, MD
Principle
Roasted coffee beans contain more oil than green coffee beans, so methylene
chloride, rather than hexane, is used to remove oil.
Directions
? Extract as in E2, except after acidification of filtered extract and cooling of
mixture:
? Extract acidified methanol once with 100 mL methylene chloride, shaking
vigorously 1 min. Drain lower aqueous layer into beaker and discard meth-
ylene chloride.
? Continue, as in E2, by making solution alkaline.
Transmittal No. 94-1 (1/94)
Form FDA 2905a (6/92)
SECTION 404Pesticide Analytical Manual Vol. I
404–9
DL5 HPLC, ION PAIR MOBILE PHASE, UV AND FLUORESCENCE DETECTOR
Reference
Gilvydis, D.M., and Walters, S.M. (1990) J. Assoc. Off. Anal. Chem. 73, 753-761
Principles
Benzimidazole residues are chromatographed on RP HPLC system. MBC and thia-
bendazole are ionized in acidic mobile phase and paired with negatively charged
counter-ions from sodium decanesulfonate to control system selectivity and sepa-
rate analytes from interfering sample co-extractives. Thiophanate-methyl and
allophonate, neutral at the conditions in the column, are unaffected by mobile
phase modifications and chromatograph according to RP principles.
UV and fluorescence detectors each respond to MBC and thiabendazole; UV de-
tector also responds to thiophanate-methyl and allophanate.
Apparatus
Filtration device for ion pairing solution: stainless steel glass filter holder,
300 mL capacity, fitted with hydrophilic membrane of pH range 2-10, 0.45
μm pore size, 47 mm diameter. Use with vacuum filtration flask, 1 L.
Filtration device for sample solutions, 10 mL syringe with Luer-Lok tip,
fitted with nylon 13 mm diameter disposable filter unit, 0.45 μm pore size.
HPLC system must meet system suitability test below. Complete system
consists of:
1) mobile phase delivery system, constant volume isocratic pump
2) injector, automatic sample injection module, preferably with loop vol-
ume ≥25 μL
3) analytical column, 25 cm × 4.6 mm id, containing highly end-capped
5 μm C-18 bonded silica suitable for chromatography of basic com-
pounds
4) guard column, compatible with analytical column, packed with same or
comparable C-18 bonded silica
5) column oven, with configuration that accommodates guard and analyti-
cal columns
6) variable wavelength UV detector or photodiode array detector, equipped
with flow cell of about 8 μL
7) fluorescence detector, dual monochromator, equipped with 5 μL flow
cell
8) recorder, strip chart recorder or computing integrator compatible with
each detector
Reagents
1-decanesulfonate, sodium salt, 98% pure
ion pairing solution, 4.1 mM 1-decanesulfonate, sodium salt. Pipet 7.0 mL
phosphoric acid into 200 mL HPLC grade water; dissolve 1.0 g 1-
decanesulfonate, sodium salt in this mixture. Pipet 10.0 mL triethylamine
into solution and dilute to 1 L with HPLC grade water. Filter through
<1 μm porosity membrane. (pH of solution should be about 2.4.)
Pesticide Analytical Manual Vol. ISECTION 404
404–10
Transmittal No. 94-1 (1/94)
Form FDA 2905a (6/92)
methanol, distilled from all-glass apparatus
mobile phase, prepared by mixing manually 65 parts ion pairing solution with
35 parts methanol. (Do not mix mobile phase using pump, because exother-
mic reaction caused by mixing generates bubbles that prevent stable pump
operation.) Before use, degas by sparging with helium while stirring solvent
with magnetic stirrer.
phosphoric acid, 85%
triethylamine, 99% pure
water, HPLC grade, commercial product or prepared from water purification
system that produces distilled, de-ionized water. Resistivity of water must be
>12 megohms-cm.
System Operation
? Connect fluorescence detector in tandem with (following) UV detector.
? Set column oven temperature at 40° C and equilibrate system with mobile
phase at flow rate of 1.5 mL/min for ≥30 min or until constant retention
times are achieved for all four analytes.
? Rinse system with 50% methanol/water when not using for extended pe-
riod (e.g., ≥24 hr). When not in use for shorter periods, maintain slow
flow (0.1-0.2 mL/min) of mobile phase to prevent salt deposition.
? To rid column of highly retained sample components as necessary, rinse
column with methanol after rinsing with 50% methanol/water; then rinse
with 50% methanol/water again before introducing mobile phase. Mobile
phase must be rinsed from column with aqueous solvent before adding
methanol and vice versa to avoid salt precipitation and possible clogging of
system.
? Operate UV detector initially at absorbance wavelength of 250 nm; imme-
diately following elution of allophanate, reset wavelength to 280 nm and
rezero baseline for determination of thiophanate-methyl, MBC, and thia-
bendazole. Adjust detector and/or recorder sensitivity so that 40-70% full
scale deflection (FSD) is obtained for 62.5 ng of each standard (25 μL
mixed standard solution).
? Operate fluorescence detector at excitation wavelength of 280 nm (20 nm
slit width) and emission wavelength of 310 nm (10 nm slit width). Adjust
detector, attenuator, and/or recorder sensitivity so that 60-80% FSD is
obtained for 62.5 ng MBC (25 μL mixed standard solution); at these
conditions, 62.5 ng thiabendazole will cause 40-50% FSD.
? Alternatively, use wavelength-programmable fluorescence detector and
wavelength-programmable or diode array UV detector for concurrent de-
terminations of residues at optimum wavelength settings for each.
System Suitability Test
See Chapter 6, HPLC, for further information about evaluating HPLC systems.
? Prepare mixed standard solution (2.5 ng/μL for each compound) as di-
rected above. Prepare additional dilutions to produce mixed standard
solutions of 4 ng/μL and 0.4 ng/μL.
? Allow HPLC system to equilibrate at conditions described in System Opera-
tion.
Transmittal No. 96-1 (9/96)
Form FDA 2905a (6/92)
SECTION 404Pesticide Analytical Manual Vol. I
404–11
? Inject at least three 25 μL portions 2.5 ng/μL mixed standard solution.
Determine following parameters:
1) retention time and peak height for each peak; relative standard de-
viations (RSD) for repetitive retention times and peak height mea-
surements
2) column efficiency (N) for thiabendazole peak
3) asymmetry factor (As) for thiabendazole peak
? Inject each of three different concentrations of mixed standard solu-
tions (10-100 ng/25 μL injection). Plot peak height vs. amount injected.
? HPLC systems adequate for analysis for benzimidazoles will meet follow-
ing minimum criteria: retention times of about 8, 15, 18, and 23 min for
allophanate, thiophanate-methyl, MBC, and thiabendazole, respectively
(retention times may vary among columns but should remain constant
for particular column); RSD <0.5% for retention times and <3% for
peak heights of individual peaks in three consecutive chromatograms;
N>12,000 and As <1.3 for thiabendazole peak.
? Examine systems not meeting these criteria for problems, using various
troubleshooting sections of Chapter 6. Correct problems uncovered by
troubleshooting until system meets criteria defined above.
? System will typically respond linearly to 10-100 ng of each compound,
but linear range may vary among systems. Perform quantitative analyses
only within calculated linear range of system as determined above. Di-
lute sample extracts as needed to permit injection of analyte level within
linear range. Adjust amounts injected so that peak heights of analyte
and reference standard do not differ >25% from one another.
Directions
See Figure 404-b for typical chromatograms produced by HPLC system.
? To extract from E1-E3 (dissolved in 4.0 mL methanol), add 6.0 mL ion
pairing solution; mix. Residue must be dissolved in methanol prior to
adding ion pairing solution.
? Filter through 0.45 μm porosity membrane; filter will plug as solution is
applied, so filter only volume needed for HPLC determination, about 1
mL.
? Inject 25 μL sample solution and chromatograph as described in System
Operation.
? Compare chromatographic response (peak retention times, heights, and/
or areas) with that of standard solution and calculate residue amount.
? If further dilutions are necessary, use mixture of 4:6 methanol:ion pair-
ing solution as diluent.
? To convert calculated MBC (MW 191.2) to equivalent benomyl (MW
290.4), multiply by 1.52.
? To convert calculated MBC to equivalent thiophanate-methyl (MW 342.4),
multiply by 1.79.
? Peaks of 50% FSD at conditions established for screening analysis are
equivalent to about 0.5 ppm each of thiophanate-methyl, allophanate,
and thiabendazole; MBC peak of 50% FSD at these conditions repre-
sents about 0.3 ppm.
Pesticide Analytical Manual Vol. ISECTION 404
404–12
Transmittal No. 96-1 (9/96)
Form FDA 2905a (6/92)
Figure 404-b
Chromatograms of Benzimidazole Compounds
Chromatograms of: (A) peach extract partitioned from the acidic phase of 404 E1, (B)
standard solution, (C) peach extract partitioned from basic phase of 404 E1. HPLC operation
as directed in DL5. Sample contains 0.14 ppm field-incurred MBC.
ALTERNATIVES:
DL6 HPLC, CONCENTRATED ION PAIR MOBILE PHASE, UV AND
FLUORESCENCE DETECTOR
Reference
Gilvydis, D.M., and Walters, S.M. (Aug. 1989) “Modification of LIB 3217 for
Carbendazim (MBC) in Green and Roasted Coffee Beans,” LIB 3353, FDA, Rockville,
MD
Principles
Concentration of ion pairing reagent is increased eight times to increase k′ values
of analytes and improve separation from early eluting co-extractives.
Additional Reagents
ion pairing solution, 32.7 mM 1-decanesulfonate, sodium salt. Pipet 7.0 mL
phosphoric acid into 200 mL HPLC grade water; dissolve 8.0 g 1-
decanesulfonate, sodium salt in this mixture. Pipet 10.0 mL triethylamine
into solution and dilute to 1 L with HPLC grade water. Filter through <1 μm
porosity membrane. (pH of solution should be about 2.4.)
UV Detector
Fluorescence
Detector
A
B
C
A
B
C
TIME (min) TIME (min)
515255 15
250 nm 280 nm
allophanate
thiophanate-methyl
MBC
thiabendazole
MBC
thiabendazole
Transmittal No. 94-1 (1/94)
Form FDA 2905a (6/92)
SECTION 404Pesticide Analytical Manual Vol. I
404–13
UV Detector
MBC
minutes
O27
mobile phase, prepared by mixing manually 63 parts ion pairing solution
with 37 parts methanol. Do not mix using pump. Before use, degas by
sparging with helium while stirring solvent with magnetic stirrer.
Directions
See Figure 404-c for depiction of improvements provided by higher concentration
of ion pairing solution in mobile phase.
? Perform HPLC determination as in DL5, except use ion pairing solution
and mobile phase described here. At these conditions, MBC will elute in
about 26 min.
Figure 404-c
Effect of Ion Pairing Solution Concentration on Chromatography of MBC
minutes
O27
Fluorescence Detector
MBC
HPLC operated as directed in DL6, 32.7 mM 1-decanesulfonate, sodium salt
Chromatograms of extract from green coffee beans, extracted according to 404 E2, and
chromatographed at conditions directed in DL5 and DL6. Superimposed chromatograms of
MBC represent 0.3 ppm.
UV Detector
MBC
minutes
O26
minutes
O
26
Fluorescence Detector
MBC
HPLC operated as directed in DL5, 4.1 mM 1-decanesulfonate, sodium salt
Pesticide Analytical Manual Vol. ISECTION 404
404–14
Transmittal No. 94-1 (1/94)
Form FDA 2905a (6/92)
DL7 HPLC, ION PAIR MOBILE PHASE, CHANGES IN DETECTOR
SETTINGS
Reference
Gilvydis, D.M., and Walters, S.M. (1990) J. Assoc. Off. Anal. Chem. 73, 753-761
Principle
Changes in detector settings provide increased sensitivity to thiabendazole.
Directions
? Perform HPLC determination as in DL5, except adjust wavelength of UV
detector absorbance to 305 nm and adjust excitation and emission wave-
lengths of fluorescence detector to 305 and 345 nm, respectively.
? Dilute sample extract containing thiabendazole and rechromatograph.
UV detector response to thiabendazole at this setting will be about twice
that obtained at 280 nm, and fluorescence detector response to thia-
bendazole will be about 10 times that obtained at 280 and 310 nm.
CONFIRMATION
See Section 103 for additional information about confirmation.
Confirm initial findings of residues by rechromatographing with alternative systems
described here, as appropriate to residue(s) found.
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- 3. 美国FDA农残分析手册（第一巻英文）：ch3tabs
- 4. 美国FDA农残分析手册（第一巻英文）：ch4tabs
- 5. 美国FDA农残分析手册（第一巻英文）：chapter1
- 6. 美国FDA农残分析手册（第一巻英文）：chapter2
- 7. 美国FDA农残分析手册（第一巻英文）：chapter3
- 8. 美国FDA农残分析手册（第一巻英文）：chapter4
- 9. 美国FDA农残分析手册（第一巻英文）：chapter5
- 10. 美国FDA农残分析手册（第一巻英文）：chapter6
- 11. 美国FDA农残分析手册（第一巻英文）：indices
- 12. 美国FDA农残分析手册（第一巻英文）：Introduction