7. 4
7. 4
© Springer-Verlag Berlin Heidelberg 2005
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 classi ed into
N-methylcarbamate insecticides and N-allylcarbamate herbicides in view of their chemical
structures and biological actions.  e 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.  e 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.  e binding of carbamate pesticides to AchE is much weaker than that of organo-
phosphorus pesticides, and the former pesticides are easily decomposed
a
in mammalian bodies.
 erefore, 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
•  e 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

(rat, mg/kg)
bendiocarb
C
11
H
13
NO
4
MW: 223.2
MP: 125 ~ 129
insecticide 40 ~ 156
benfuracarb
C
20
H
30
N
2
O
5
S
MW: 410.5
MP: 110
insecticide 223 205
carbaryl
C
12
H
15
NO

H
15
NO
2
S
MW:: 225.3
MP: 33.4
insecticide 200 approx.
fenobucarb
C
12
H
17
NO
2
MW: 207.3
MP: 31 ~ 32
insecticide 250 185
furathiocarb
C
18
H
26
N
2
O
5
S
MW: 382.5
BP: 250<

2
O
2
S
MW: 162.2
MP: 78 ~ 79
insecticide 17 24
oxamyl
C
7
H
13
N
3
O
3
S
MW: 219.3
MP: 100 ~ 102
insecticide 5.4
pirimicarb
C
11
H
18
N
4
O
2
MW: 238.3

H
13
NO
2
MW: 179.2
MP: 79 ~ 80
insecticide 375 325
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  uids (whole blood, serum and urine), tissues and stomach contents are objects
for analyses.  e whole blood should be completely hemolyzed before extraction. Homogeni-
zation is needed for tissues (organs) specimens; for stomach contents, the supernatant fraction
a er 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.  e 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 funnel
e
.
iv.  e funnel is shaken for 10 min (with a shaking machine).
v.  e n-hexane/ethyl acetate layer is obtained.

i
.
vii. It is evaporated to dryness under a stream of nitrogen at room temperature.
viii.  e 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.
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 bu er 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.  e cartridge is washed with 1 mL distilled water.
iv.  e  rst elution is made by passing 1 mL methanol through it.
v.  e cartridge is washed with 1 mL of 0.1 M HCl solution.
vi.  e second elution is made by passing 1 mL methanol again.
vii.  e third elution is made by passing 1 mL of 5 % ammonia/methanol.
viii.  e three eluates obtained at the steps iv, vi and vii are combined
l
, and evaporated to dry-
ness under a stream of nitrogen.
ix.  e 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 columns

(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

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).  e quantitation can be made with peak area ratios of the ion
at m/z 178 to that at m/z 197.  e 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 column
q
: Finepak SIL C
18
(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  ow 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  ow rate: 50 µL/min; ionization mode: ion spray (+ 5 keV).
Assessment of the method
 e analysis of carbamate pesticides by HPLC is much inferior to that by capillary GC in reso-
lution ability, but does not su er from decomposition of analytes by heat. In HPLC analysis of
HPLC and LC/MS analysis
566 Carbamate pesticides
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 %.

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
567
for qualitative and quantitative analyses.  e 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.  e concen-
trations reported in literature and measured in the author’s laboratories are summarized
in
> Table 4.6.
 e 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, a er ingestion followed by treatments, are also taken into consideration,
the fatal blood concentration of methomyl is estimated not lower than 1.0 µg/mL.
 e respective blood concentrations of carbofuran, benfuracarb and furathiocarb a er
their ingestion for suicidal purposes are as follows.  e blood carbofuran concentrations
in 7 fatality cases a er ingestion of the pesticide only were 0.32–29.3 µg/mL (n = 7, average
9.2 µg/mL) [7, 10, 15, 16].  e blood benfuracarb concentrations a er its ingestion were 0.30–

Blood concentrations of carbamate pesticides in their fatal poisoning cases
Carbamate Subject No. Blood conc. (µg/mL) Ref.
methomyl 1
2
3
4
5
6
7
8
9
10
11
12
13
average
0.7
1.4
44.0 (1 h)
0.6
35.0 4.0 (Al)
57.0 3.2 (Al)
8.0 0.5 (Al)
5.6
28.0
4.8
19.1
12.8
43.2
20.0

0.31 1.47 (CA)
0.30 1.45 (CA)
0.98 1.46 (CA)
[6]
furathiocarb 1
2
3
4
5
6
7
average
21.6
0.7
0.1
1.0
0.4
0.32
0.8
3.6
[5]
propoxur 1
2
3
4
5
6
average
1.3 0.10 (Al)
13.6 2.26 (Al)

g) If necessary, the mixture is centrifuged to obtain clear supernatant solution.
h) It is possible to pass it through a water-repellant  lter 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)  ere is no lowering of recovery rates due to drying up of the Oasis MCX cartridge.
l) For GC and GC/MS analysis, anhydrous Na
2
SO
4
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 di erent according to
manufacturers and lots, and should be con rmed using the authentic standard for each
column.
r) Using a photodiode array detector (DAD), it is possible to make tentative identi cation 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.  e methylamine can react with o-phthalaldehyde and 2-mercaptoethanol to form
a highly  uorescent compound. Various HPLC systems equipped with the post-column

tion of pesticides of various classes in clinical and forensic toxicology. Forensic Sci Int 121:116–123
11) Tsunoda N (1985) Analysis of N-methylcarbamate pesticides by high-performance liquid chromatography.
Forensic Toxicol News 3 (3):33–34 (in Japanese)
12) Driskell WJ, Groce DF, Hill RH Jr et al. (1991) Methomyl in the blood of a pilot who crashed during aerial spray-
ing. J Anal Toxicol 5:339–340
13) Tsatsakis AM, Tsakalof AK, Siatitsas Y et al. (1996) Acute poisoning with carbamate pesticides: the Cretan expe-
rience. Sci Justice 36:35–39
14) Tsatsakis AM (1998) More fatal methomyl poisoning in Creta. Sci Justice 38:282–283
15) Picotte P, Perreault P (1991) Suicide with carbofuran. Bull Int Assoc Forensic Toxicol 21 (2):38–40
16) Ferslew KE, Hagardorn AN, McCormick WF (1992) Poisoning from oral ingestion of carbofuran (Furadan 4F), a
cholinesterase-inhibiting carbamate insecticide and its effects on cholinesterase activity in various biological
fluids. J Forensic Sci 37:337–344
17) Pfordt J, Magerl H, Vock R (1987) Tödliche Vergiftungen mit Propoxur
®
. Z Rechtsmed 98:43–48