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7.

4
II.7.4 Carbamate pesticides
by Kiyoshi Ameno

Introduction

Among many carbamate pesticides commercially available in Japan, those with relatively high
toxicities are shown in > Table 4.1 [1]. Carbamate pesticides are generally classified into
N-methylcarbamate insecticides and N-allylcarbamate herbicides in view of their chemical
structures and biological actions. The number of fatalities due to poisoning by carbamate pes-
ticides is 50–100 every year in Japan; many of them are poisoned by methomyl [2]. According
to statics reported by National Research Institute of Police Science of Japan, the number of
fatalities is highest with paraquat plus diquat, followed by organophosphates and then carba-
mates among pesticides. The toxicity of carbamate pesticides is due to inhibition of acetylcho-
linesterase (AchE) by their binding with the active site of the enzyme; the inhibition of the
hydrolysis reaction of acetylcholine (Ach) results in the accumulation of Ach, provoking
poisoning symptoms, such as miosis, lacrimation, sweating, hypersalivation and convulsion of
extremities. The binding of carbamate pesticides to AchE is much weaker than that of organo-
phosphorus pesticides, and the former pesticides are easily decomposeda in mammalian bodies.
Therefore, the damages of organs by carbamate pesticides have not been reported.
For analysis of carbamate pesticides, methods by GC [3–7], GC/MS [5, 7, 8–10], HPLC
[11] and LC/MS [10] were reported. In this chapter, the extraction procedures and analytical
methods using the above 4 instruments are described for this group of pesticides.

Reagents and their preparation

• The authentic standards of compounds listed in > Table 4.1 and ethion can be purchased
from Wako Pure Chemical Industries, Ltd., Osaka, Japan. A standard mixture of seven
N-methylcarbamate pesticides can be obtained from Kanto Chemicals (Tokyo, Japan)b.
Carbofuran, carbaryl and ethion are separately dissolved in acetone to prepare 100 µg/mL
solutions as stock solutions for internal standards (ISs)c. Carbendazole (Aldrich, Milwau-
kee, WI, USA) is dissolved in distilled water to prepare 100 µg/mL solution. o-Methoxyl-
phenol can be also obtained from Aldrich.
• Sep-Pak C18 and Oasis MCX cartridges are purchased from Waters (Milford, MA, USA).
• The solutions to be prepared are: n-hexane/ethyl acetate (1:1, v/v); acetonitrile/distilled water
(1:1, v/v); methanol/distilled water (5:59, v/v); 0.1 M HCl solution; and 2 % NaCl solution.
• 0.1 M Phosphate buffer solution (pH 7.0): 6.81 g of KH2PO4 is dissolved in 400 mL distilled
water, and the pH of the solution is adjusted to 7.0 with 1.0 M NaOH solution; the final
volume is adjusted to 500 mL with distilled water.
• 5 % Ammonia/methanol solution: 2.5 mL of 3 M NH4OH solution is mixed with 47.5 mL
methanol.
• HPLC mobile phases: methanol/distilled water (65:35, v/v); acetonitrile/2 mM ammonium
formate (30:70 and 80:20, v/v, pH 3.0).

© Springer-Verlag Berlin Heidelberg 2005


560 Carbamate pesticides

⊡ Table 4.1
Structures, properties and toxicities of carbamate pesticides

Name, MF, MW, property and structure Use Acute oral LD50
(rat, mg/kg)
bendiocarb insecticide 40 ~ 156
C11H13NO4
MW: 223.2
MP: 125 ~ 129

benfuracarb insecticide 223 205


C20H30N2O5S
MW: 410.5
MP: 110

carbaryl insecticide 850 500


C12H15NO2
MW: 201.2
MP: 142
carbofuran insecticide 8
C12H15NO3
MW: 221.3
MP: 153 ~ 154
carbosulfan insecticide 623 657
C20H32N3O3S
MW: 380.5
BP: 124 ~ 128

ethiofencarb insecticide 200 approx.


C11H15NO2S
MW:: 225.3
MP: 33.4

fenobucarb insecticide 250 185


C12H17NO2
MW: 207.3
MP: 31 ~ 32

furathiocarb insecticide 53
C18H26N2O5S
MW: 382.5
BP: 250<
Carbamate pesticides 561

⊡ Table 4.1
(continued)

isoprocarb insecticide 450


C11H15NO2
MW: 225.3
MP: 93 ~ 96
metolcarb insecticide 580 498
C9H11NO2
MW: 162.2
MP: 76 ~ 77

methomyl insecticide 17 24
C5H13N2O2S
MW: 162.2
MP: 78 ~ 79
oxamyl insecticide 5.4
C7H13N3O3S
MW: 219.3
MP: 100 ~ 102
pirimicarb insecticide 147
C11H18N4O2
MW: 238.3
MP: 90.5

propoxur insecticide 50
C11H15NO3
MW: 209.2
MP: 90
thiodicarb insecticide 66
C10H18N4O4S3
MW: 354.5
MP: 173 ~ 174

XMC insecticide 542


C10H13NO2
MW: 179.2
MP: 99
xylylcarb insecticide 375 325
C10H13NO2
MW: 179.2
MP: 79 ~ 80

MF: molecular formula; MW: molecular weight; MP (°C): melting point; BP (°C): boiling point.
562 Carbamate pesticides

Extraction methods

It is absolutely necessary to extract carbamate pesticides from crude biomedical specimens


containing many impurity compounds as pretreatments before instrumental analysis. As spec-
imens, body fluids (whole blood, serum and urine), tissues and stomach contents are objects
for analyses. The whole blood should be completely hemolyzed before extraction. Homogeni-
zation is needed for tissues (organs) specimens; for stomach contents, the supernatant fraction
after their centrifugation should be used.

Liquid-liquid extraction

i. A 2-g aliquot of specimens (blood, tissues and stomach contents d) is mixed with 10 mL
acetonitrile (containing an appropriate IS), homogenized with a Polytron homogenizer
and centrifuged at 3,000 rpm for 5 min to obtain clear supernatant solution.
ii. For the sediment, the above i) step of extraction is repeated two times.
iii. The three acetonitrile supernatant solutions thus obtained are combined and mixed with
80 mL of 2 % NaCl solution and 25 mL of n-hexane/ethyl acetate (1:1, v/v) in a 250-mL
volume separating funnele.
iv. The funnel is shaken for 10 min (with a shaking machine).
v. The n-hexane/ethyl acetate layer is obtained.
vi. The layer is evaporated to dryness under reduced pressure in a rotary evaporator at room
temperature.
vii. The residue is dissolved in 100 µL methanol.
viii. A 2-µL aliquot of it is injected into GC or GC/MS; a 20-µL aliquot into HPLC.
ix. For quantitation, each calibration curve is constructed using peak area ratios of a target
compound to IS. A ratio obtained from a specimen is applied to the curve to calculate its
concentration.

Solid-phase extraction-1 [4]

i. A Sep-Pak C18 cartridge f is washed and activated by passing chloroform, acetonitrile,


acetonitrile/distilled water (1:1) and distilled water, 10 mL each, successively.
ii. A 1-mL volume of a specimen (blood, serum or urine) is mixed with 9 mL distilled water,
stirred well g and poured into the Sep-Pak C18 cartridge.
iii. It is washed with 10 mL distilled water.
iv. A target compound and IS are eluted with 3 mL chloroform.
v. A small amount of aqueous layer (upper) of the eluate is carefully removed with a Pasteur
pipette h.
vi. The above organic extract is dehydrated with anhydrous Na2SO4i.
vii. It is evaporated to dryness under a stream of nitrogen at room temperature.
viii. The residue is dissolved in 100 µL methanol j.
ix. A 2-µL aliquot of it is injected into GC or GC/MS; a 20-µL aliquot into HPLC or LC/MS.
GC and GC/MS analysis 563

Solid-phase extraction-2 [10]

i. An Oasis MCX cartridge k is activated by passing 1 mL methanol and 1 mL of 0.1 M phos-


phate buffer solution (pH 7) through it.
ii. A 1-mL volume of serum is mixed well with 100 µL of IS (for example carbendazole) so-
lution and poured into the cartridge.
iii. The cartridge is washed with 1 mL distilled water.
iv. The first elution is made by passing 1 mL methanol through it.
v. The cartridge is washed with 1 mL of 0.1 M HCl solution.
vi. The second elution is made by passing 1 mL methanol again.
vii. The third elution is made by passing 1 mL of 5 % ammonia/methanol.
viii. The three eluates obtained at the steps iv, vi and vii are combined l, and evaporated to dry-
ness under a stream of nitrogen.
ix. The residue is dissolved in 100 µL methanol for GC or GC/MS analysis; an appropriate
amount is injected into GC (/MS). For HPLC (/MS), the residue is dissolved in 100 µL of
a mobile phase to be used and injected into HPLC (/MS).

GC and GC/MS analysis

GC conditions

GC columnsm: DB-5 and DB-1 fused silica capillary columns (30 m × 0.25 mm i.d., film thick-
ness 0.25 µm, J&W Scientific, Folsom, CA, USA).
GC conditions; instrument: Shimadzu GC14B (Shimadzu Corp., Kyoto, Japan); detector:
flame thermionic ionization detector (FTD)n; column temperature: 50 °C → 20 °C/min →
120 °C → 5 °C/min → 260 °C(10 min); injection and detector temperature o: 230 °C; carrier gas:
He (13 kPa).

GC/MS conditions

GC column: a DB-5MS fused silica capillary column (30 m × 0.25 mm i.d., film thickness
0.25 µm, J&W Scientific).
GC/MS; instrument: Shimadzu GC-MS5000 (Shimadzu Corp.); column temperature:
120 °C (1 min) → 20 °C/min → 240 °C (8 min); injection and separator temperature: 230 °C;
carrier gas: He (2.0 mL/min); ionization mode: EI (70 eV).

Assessment of the method

For GC analysis of carbamate pesticides, an FTD detector is recommendable, because it is


specific and sensitive, and is not influenced by impurities and organic solvents appreciably.
Qualitative analysis only with a single column is insufficient; multiple columns of different
properties should be used for testing an identity of a compound. > Table 4.2 shows retention
564 Carbamate pesticides

⊡ Table 4.2
Retention times of carbamate pesticides obtained by GC*

Carbamate Retention time Carbamate Retention time


(min) (min)
oxamyl 5.9 bendiocarb 15.1
methomyl-oxime 7.1 carbofuran 16.5
propamocarb 9.3 ethiofencarb 18.8
metolcarb (MTMC) 11.2 pirimicarb 18.8
isoprocarb 12.2 carbaryl 19.7
methomyl 12.2 methiocarb 20.7
XMC 12.6 benthiocarb 21.1
xylylcarb (MPMC) 13.3 diethofencarb 21.4
propoxur (PHC) 13.6 furathiocarb 31.5
fenobucarb 13.7 benfuracarb 33.6
* Detection limits were 0.5–1.0 ng on-column.
Data were obtained in cooperation with Dr. S. Hatta (environmental chemist). Column: a DB-5 fused silica capillary
column (30 m × 0.25 mm i. d., film thickness 0.25 µm).

⊡ Table 4.3
EI mass spectra of carbamate pesticides

Carbamate Mass spectral ions (m/z)


M+ Base Others
oxamyl 162 72 98 145 115
propamocarb 188 58 129 143 70
metolcarb (MTMC) 165 108 77 58 107
isoprocarb 193* 121 136 91 103
methomyl-oxime 105 88 58
methomyl 162* 105 58 88 103
XMC 179 122 107 91 77
xylylcarb (MPMC) 179 122 107 91 77
propoxur (PHC) 209* 110 152 81 58
fenobucarb 207* 121 150 91 77
bendiocarb 223 151 166 126 108
carbofuran 221 164 149 122 131
ethiofencarb 225 107 168 77 57
pirimicarb 238 166 72 132 152
carbaryl 201 144 115 116 145
methiocarb 225 168 153 109 91
benthiocarb 257 100 82 125 89
diethofencarb 267 151 225 207 124
furathiocarb 382* 163 135 194 325
benfuracarb 410* 190 163 135 102
APC 274 160 217 176 190
M+: molecular ion; Base: base peak; *: very small peak or not detectable.
HPLC and LC/MS analysis 565

times of carbamate pesticides, when a DB-5 fused silica capillary column was used for GC
analysis.
> Table 4.3 shows principal ions (5 peaks each including a molecular ion) observed in EI
mass spectra for carbamate pesticides obtained by GC/MS [4, 9]. The intensities of molecular
ions are generally low; there are compounds without appearance of molecular ionsp. Sufficient
qualitative analysis is achieved by measuring retention times and mass spectra of compounds
tested. The sensitive quantitation analysis can be made using the selected ion monitoring
(SIM).
Methomyl can be analyzed by GC (/MS) in its underivatized from; but a method using the
conversion of methomyl into methomyl-oxime by alkali, followed by silylation (TMS) for GC/
MS, was reported [8]. This method is aimed to overcome the thermolability of underivatized
methomyl, and a good mass spectrum including the molecular ion can be obtained for the
methomyl-oxime-TMS (M.W. 177); the mass spectrum shows ions at m/z 177 (molecular ion),
162, 130, 121 and 105. Chemical ionization (CI) mode is preferably used for quantitation,
because it gives the base peaks at m/z 178 and 197 of protonated ions of methomyl-oxime-TMS
and o-methoxyphenol-TMS (IS). The quantitation can be made with peak area ratios of the ion
at m/z 178 to that at m/z 197. The quantitative range is reported to be 0.1–6 µg/mL; the details
of the method can be obtained from the reference [8].

HPLC and LC/MS analysis

HPLC conditions [11]

HPLC columnq: Finepak SIL C18 (25 cm × 4.6 mm i.d., particle size 5 µm, Nihonbunko, Tokyo,
Japan).
HPLC conditions; instrument: Shimadzu HPLC10A (Shimadzu Corp.); detector: UV
(wavelength, 254 nm); mobile phase: methanol/distilled water (65:35, v/v); its flow rate: 1 mL/
min.

LC/MS conditions [10]

LC column: NucleosilC18 5 µm (15 cm × 1.0 mm i.d., Polymer Laboratories, Marseille,


France).
LC/MS conditions; MS instrument: PE-Biosystems AP100 (PE Biosystems, Marseille,
France); HPLC pump: Shimadzu LC10AD (Shimadzu Corp.); autosampler: Series 200 Perkin-
Elmer (Perkin-Elmer, Courtaboeuf, France); mobile phase: gradient 30–80 % acetonitrile/2 mM
ammonium formate (pH 3); its flow rate: 50 µL/min; ionization mode: ion spray (+ 5 keV).

Assessment of the method

The analysis of carbamate pesticides by HPLC is much inferior to that by capillary GC in reso-
lution ability, but does not suffer from decomposition of analytes by heat. In HPLC analysis of
566 Carbamate pesticides

⊡ Table 4.4
Retention times and detection limits (on-column) of carbamate pesticides obtained by HPLC

Carbamate Retention time (min) Detection limit (ng)


methomyl-oxime 3.5 35
methomyl 4.0 54
metolcarb (MTMC) 5.4 10
propoxur (PHC) 5.5 16
carbaryl (NAC) 6.3 45
ethiofencarb 6.4 12
xylylcarb (MPMC) 6.5 11
XMC (macball) 6.8 6
isoprocarb (MIPC) 6.9 11
EMPC 7.1 35
fenobucarb 8.6 9
terbucarb 23.0 2
allyxycarb (APC) 25.0 7

methomyl and methomyl-oxime, the peaks are frequently interfered with by a solvent peak; to
avoid the problem, the ratio of water content of the mobile phase is increased to about 50 %.
There are various detectors commercially available for HPLC; they are useful for improving
specificityr and sensitivitys.
> Table 4.4 shows retention times and detection limits for carbamate pesticides obtained
under the above HPLC-UV conditions.
The analysis by LC/MS has been established recently; it requires no concern about the
decomposition by heat and also give mass spectra. The method is very useful for qualitative
analysis and enables sensitive quantitation in the SIM mode. > Table 4.5 shows ions selected

⊡ Table 4.5
Selected ions for qualitative and quantitative LC/MS analysis of carbamate pesticides

Carbamate Selected ion (m/z)


Quantitative Qualitative
aldicarb 89 116
ethiofencarb 107 –
methomyl 88 106
carbaryl 145 155
carbofuran 123 165
chlorpropham 172 126
methiocarb 121 169
pirimicarb 239 182
formetanate 110 93
fenoxycarb 302 88
propham 138 120
Toxic and fatal concentrations 567

for qualitative and quantitative analyses. The sensitivity obtained by SIM of LC/MS is as high
as that of GC/MS.

Toxic and fatal concentrations

Although the number of fatalities due to carbamate pesticide poisoning is not small in the
world, the reports on their concentrations in blood and organs are not many. The concen-
trations reported in literature and measured in the author’s laboratories are summarized
in > Table 4.6.
The blood methomyl concentrations in fatalities by its poisoning is 0.6–57 µg/mL (mean
concentration in 13 cases, 20 µg/mL) [3, 8, 12–14]. When the blood methomyl concentrations
of survived subjects, after ingestion followed by treatments, are also taken into consideration,
the fatal blood concentration of methomyl is estimated not lower than 1.0 µg/mL.
The respective blood concentrations of carbofuran, benfuracarb and furathiocarb after
their ingestion for suicidal purposes are as follows. The blood carbofuran concentrations
in 7 fatality cases after ingestion of the pesticide only were 0.32–29.3 µg/mL (n = 7, average
9.2 µg/mL) [7, 10, 15, 16]. The blood benfuracarb concentrations after its ingestion were 0.30–
2.32 µg/mL (n = 3, average 0.98 µg/mL) and the blood concentrations of carbofuran, the
metabolite of benfuracarb, were not lower than 1.45 µg/mL [6], suggesting that benfuracarb is
rapidly metabolized into carbofuran in human bodies. After furathiocarb ingestion, its blood
concentrations were 0.1–21.6 µg/mL (n = 7, average 3.6 µg/mL); but carbofuran, also the meta-
bolite of furathiocarb, has never been analyzed simultaneouslyt [5]. As shown in > Table 4.1,
the acute toxicities of furathiocarb and benfuracarb in rats are 8–30 times less than that of
carbofuran; the both compounds are metabolized into a highly toxic metabolite carbofuran in
mammals. In furathiocarb and benfuracarb poisoning cases, carbofuran should be analyzed
simultaneously.
In fatal cases of propoxur poisoning, its blood concentrations were 0.3–41.1 µg/mL (n = 6,
average 10.7 µg/mL) [17] and ethanol was also detected from blood in many cases. This sug-
gests that ethanol enhances the toxicity of propoxur.

Notes

a) Many of carbamate pesticides are generally decomposed by alkali and occasionally by


strong acids. The contact of carbamate pesticides with strongly alkaline and acidic com-
pounds should be avoided. Carbosulfan is decomposed even by weak acids. The metabo-
lism of carbamate pesticides is rapid and gives unstable intermediate metabolites [1]. In
this chapter, the methods for analysis of the metabolites are not dealt with.
b) The standard metabolic solution contains aldicarb, ethiofencarb, oxamyl, carbaryl, fenobu-
carb, bendiocarb and methiocarb (10 µg/mL each). It is useful for the testing of retention
times and as the standards for quantitative analysis.
c) In GC and GC/MS analysis, one or two of carbofuran, carbaryl and ethion (an organophos-
phorus compound) are used as IS(s) according to retention time(s) of target compound(s).
The peak of ethion appears at about 27 min by GC analysis (DB-5). The solutions of the
above ISs should be prepared at the final concentration of 5 or 20 µg/mL.
568 Carbamate pesticides

⊡ Table 4.6
Blood concentrations of carbamate pesticides in their fatal poisoning cases

Carbamate Subject No. Blood conc. (µg/mL) Ref.


methomyl 1 0.7 [3]
2 1.4
3 44.0 (1 h) [8]
4 0.6 [12]
5 35.0 4.0 (Al) [13]
6 57.0 3.2 (Al)
7 8.0 0.5 (Al)
8 5.6
9 28.0
10 4.8
11 19.1
12 12.8 [14]
13 43.2
average 20.0
carbofuran 1 8.0 [15]
2 29.3 [16]
3 11.6 [7]
4 10.0
5 4.0
6 0.32
7 1.5 [10]
average 9.2
benfuracarb 1 2.32 (NA) [6]
2 0.31 1.47 (CA)
3 0.30 1.45 (CA)
average 0.98 1.46 (CA)
furathiocarb 1 21.6 [5]
2 0.7
3 0.1
4 1.0
5 0.4
6 0.32
7 0.8
average 3.6
propoxur 1 1.3 0.10 (Al) [17]
2 13.6 2.26 (Al)
3 0.6 1.67 (Al)
4 7.5 0.04 (Al)
5 41.1 1.38 (Al)
6 0.3 0.07 (Al)
average 10.7
NA: not analyzed; CA: carbofuran (µg/mL); Al: ethanol (mg/mL); 1 h: 1 h after ingestion.
Toxic and fatal concentrations 569

d) When a high concentration of a carbamate pesticide is present in stomach contents, 10 vol-


umes of acetone is added to the specimen, mixed and centrifuged; the supernatant solution
directly or after condensation can be analyzed by GC or GC/MS.
e) For urine or other relatively clean specimens, 1 mL of it is mixed with 5 mL ethyl acetate,
shaken and centrifuged to separate the ethyl acetate layer without the use of the separating
funnel. The same extraction procedure is repeated two times, and the organic layers are
combined, and then subjected to the step vi.
f) In the solid-phase extraction with a Sep-Pak C18 cartridge, the flow rate should not be
higher than 3 mL/min (the speed at drop by drop). To prevent the lower recovery rates, the
Sep-Pak C18 cartridge should not be dried through the whole procedure. Under the wet
conditions, the recovery rates are about 100 %.
An Oasis HLB cartridge (Waters) with a polymer-resin base does not give low recoveries
due to drying up. According to a report by Waters, an Oasis HLB cartridge is activated by
passing t-butyl methyl ether, methanol and distilled water, 3 mL each, through it. A speci-
men solution diluted with buffer solution at pH 3 containing a carbamate pesticide is
poured into the cartridge, followed by washing with distilled water and 5 % methanol
solution, 3 mL each, and by elution with 6 mL of 5 % methanol/t-butyl methyl ether. The
recovery is not lower than 90 %.
g) If necessary, the mixture is centrifuged to obtain clear supernatant solution.
h) It is possible to pass it through a water-repellant filter paper (Advantec, 2S) to remove
water.
i) For GC and GC/MS analysis, this procedure is necessary; for HPLC analysis, the dehydra-
tion is not necessary, because the mobile phase contains water.
j) Acetonitrile acts to shorten the life span of the FTD and should not be used.
k) There is no lowering of recovery rates due to drying up of the Oasis MCX cartridge.
l) For GC and GC/MS analysis, anhydrous Na2SO4 should be added for dehydration of the
combined eluates.
m) Fused silica wide-bore capillary columns with the same stationary phases can be used.
n) An FID can be also used, though many impurity and solvent peaks may appear, resulting
in much lower sensitivity.
o) When the injection temperature is high, the carbamate pesticides are decomposed; rela-
tively low injection temperature should be used.
p) Even for compounds exhibiting almost no molecular peaks in the EI mode, relatively
intense (not lower than 40 %) protonated molecular peaks can be obtained in the CI mode
[4, 9].
q) Even with the similar ODS columns, the retention times are greatly different according to
manufacturers and lots, and should be confirmed using the authentic standard for each
column.
r) Using a photodiode array detector (DAD), it is possible to make tentative identification of
a compound by comparing the absorption spectrum of a test compound with that of the
authentic one.
s) When N-methylcarbamate pesticides are heated in NaOH solution, methylamine is pro-
duced. The methylamine can react with o-phthalaldehyde and 2-mercaptoethanol to form
a highly fluorescent compound. Various HPLC systems equipped with the post-column
labelling method are commercially available from various manufacturers. Using this
method, aldicarb-sulfoxide, aldicarb- sulfone, oxamyl, methomyl, 3-hydroxycarbofuran,
570 Carbamate pesticides

aldicarb, propoxur, carbofuran, carbaryl and methiocarb can be analyzed at ppb levels
simultaneously.
t) Carbofuran as a metabolite of furathiocarb has never been analyzed. This does not mean
“not detectable”, but means “not determined”. If it is analyzed, an appreciable amount of
carbofuran seems to be detected.

References

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