> Table 4.2 shows retention GC and GC/MS analysis... ⊡ Table 4.2Retention times of carbamate pesticides obtained by GC* Carbamate Retention time min Carbamate Retention time min * Det
Trang 1© Springer-Verlag Berlin Heidelberg 2005
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 classifi ed into N-methylcarbamate insecticides and N-allylcarbamate herbicides in view of their chemical
structures and biological actions Th 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 Th 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 Th e 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
Th 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
• Th 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 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)
• Th e 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 buff er 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 fi nal 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)
Trang 2⊡ Table 4.1
Structures, properties and toxicities of carbamate pesticides
Name, MF, MW, property and structure Use Acute oral LD 50
(rat, mg/kg)
bendiocarb
C11H13NO4
MW: 223.2
MP: 125 ~ 129
benfuracarb
C20H30N2O5S
MW: 410.5
MP: 110
carbaryl
C12H15NO2
MW: 201.2
MP: 142
carbofuran
C12H15NO3
MW: 221.3
MP: 153 ~ 154
carbosulfan
C20H32N3O3S
MW: 380.5
BP: 124 ~ 128
ethiofencarb
C11H15NO2S
MW:: 225.3
MP: 33.4
insecticide 200 approx.
fenobucarb
C12H17NO2
MW: 207.3
MP: 31 ~ 32
furathiocarb
C18H26N2O5S
MW: 382.5
BP: 250<
Trang 3561 Carbamate pesticides
⊡ Table 4.1
(continued)
isoprocarb
C11H15NO2
MW: 225.3
MP: 93 ~ 96
metolcarb
C9H11NO2
MW: 162.2
MP: 76 ~ 77
methomyl
C5H13N2O2S
MW: 162.2
MP: 78 ~ 79
oxamyl
C7H13N3O3S
MW: 219.3
MP: 100 ~ 102
pirimicarb
C11H18N4O2
MW: 238.3
MP: 90.5
propoxur
C 11 H 15 NO 3
MW: 209.2
MP: 90
thiodicarb
C10H18N4O4S3
MW: 354.5
MP: 173 ~ 174
XMC
C10H13NO2
MW: 179.2
MP: 99
xylylcarb
C10H13NO2
MW: 179.2
MP: 79 ~ 80
MF: molecular formula; MW: molecular weight; MP (°C): melting point; BP (°C): boiling point.
Trang 4Extraction 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 fl uids (whole blood, serum and urine), tissues and stomach contents are objects for analyses Th e whole blood should be completely hemolyzed before extraction Homogeni-zation is needed for tissues (organs) specimens; for stomach contents, the supernatant fraction aft 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 Th 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 funnele
iv Th e funnel is shaken for 10 min (with a shaking machine)
v Th e n-hexane/ethyl acetate layer is obtained.
vi Th e layer is evaporated to dryness under reduced pressure in a rotary evaporator at room temperature
vii Th e 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 Th e above organic extract is dehydrated with anhydrous Na2SO4i
vii It is evaporated to dryness under a stream of nitrogen at room temperature
viii Th 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
Trang 5Solid-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 buff 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 Th e cartridge is washed with 1 mL distilled water
iv Th e fi rst elution is made by passing 1 mL methanol through it
v Th e cartridge is washed with 1 mL of 0.1 M HCl solution
vi Th e second elution is made by passing 1 mL methanol again
vii Th e third elution is made by passing 1 mL of 5 % ammonia/methanol
viii Th 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 Th 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 columnsm: DB-5 and DB-1 fused silica capillary columns (30 m × 0.25 mm i.d., fi lm thick-ness 0.25 µm, J&W Scientifi c, Folsom, CA, USA)
GC conditions; instrument: Shimadzu GC14B (Shimadzu Corp., Kyoto, Japan); detector:
fl ame 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., fi lm thickness 0.25 µm, J&W Scientifi c)
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 specifi c and sensitive, and is not infl uenced by impurities and organic solvents appreciably Qualitative analysis only with a single column is insuffi cient; multiple columns of diff erent properties should be used for testing an identity of a compound > Table 4.2 shows retention
GC and GC/MS analysis
Trang 6⊡ Table 4.2
Retention times of carbamate pesticides obtained by GC*
Carbamate Retention time
(min)
Carbamate Retention time
(min)
* 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
+
Trang 7times 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] Th e intensities of molecular ions are generally low; there are compounds without appearance of molecular ionsp Suffi cient qualitative analysis is achieved by measuring retention times and mass spectra of compounds tested Th e 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] Th is 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) Th e quantitation can be made with peak area ratios of the ion
at m/z 178 to that at m/z 197 Th 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 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 fl 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-Perkin-Elmer, Courtaboeuf, France); mobile phase: gradient 30–80 % acetonitrile/2 mM ammonium formate (pH 3); its fl ow rate: 50 µL/min; ionization mode: ion spray (+ 5 keV)
Assessment of the method
Th e analysis of carbamate pesticides by HPLC is much inferior to that by capillary GC in reso-lution ability, but does not suff er from decomposition of analytes by heat In HPLC analysis of
HPLC and LC/MS analysis
Trang 8methomyl 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 %
Th ere are various detectors commercially available for HPLC; they are useful for improving specifi cityr and sensitivitys
> Table 4.4 shows retention times and detection limits for carbamate pesticides obtained
under the above HPLC-UV conditions
Th e analysis by LC/MS has been established recently; it requires no concern about the decomposition by heat and also give mass spectra Th e method is very useful for qualitative analysis and enables sensitive quantitation in the SIM mode > Table 4.5 shows ions selected
⊡ Table 4.4
Retention times and detection limits (on-column) of carbamate pesticides obtained by HPLC
Carbamate Retention time (min) Detection limit (ng)
⊡ Table 4.5
Selected ions for qualitative and quantitative LC/MS analysis of carbamate pesticides
Carbamate Selected ion (m/z)
Quantitative Qualitative
Trang 9for qualitative and quantitative analyses Th 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 Th e concen-trations reported in literature and measured in the author’s laboratories are summarized
in > Table 4.6.
Th 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, aft 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
Th e respective blood concentrations of carbofuran, benfuracarb and furathiocarb aft er their ingestion for suicidal purposes are as follows Th e blood carbofuran concentrations
in 7 fatality cases aft er ingestion of the pesticide only were 0.32–29.3 µg/mL (n = 7, average
9.2 µg/mL) [7, 10, 15, 16] Th e blood benfuracarb concentrations aft er 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 Aft er 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 Th is 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 Th e contact of carbamate pesticides with strongly alkaline and acidic com-pounds should be avoided Carbosulfan is decomposed even by weak acids Th e 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) Th e 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)
Th e peak of ethion appears at about 27 min by GC analysis (DB-5) Th e solutions of the above ISs should be prepared at the fi nal concentration of 5 or 20 µg/mL
Toxic and fatal concentrations
Trang 10⊡ Table 4.6
Blood concentrations of carbamate pesticides in their fatal poisoning cases
Carbamate Subject No Blood conc (µg/mL) Ref.
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
[3] [8] [12] [13]
[14]
2 3 4 5 6 7 average
8.0 29.3 11.6 10.0 4.0 0.32 1.5 9.2
[15] [16] [7]
[10]
2 3 average
2.32 (NA) 0.31 1.47 (CA) 0.30 1.45 (CA) 0.98 1.46 (CA)
[6]
2 3 4 5 6 7 average
21.6 0.7 0.1 1.0 0.4 0.32 0.8 3.6
[5]
2 3 4 5 6 average
1.3 0.10 (Al) 13.6 2.26 (Al) 0.6 1.67 (Al) 7.5 0.04 (Al) 41.1 1.38 (Al) 0.3 0.07 (Al) 10.7
[17]
NA: not analyzed; CA: carbofuran ( µg/mL); Al: ethanol (mg/mL); 1 h: 1 h after ingestion.