Original

Chromatography, Vol.28 No.3 (2007)

Original

Direct Analysis of Permethrins in Human Blood by SPE−GC/MS
Hiroko Ogata−Kawata1, Michiko Matsuda1, Nobuhiko Onda1, Jun Ueyama2,
Michihiro Kamijima3, Eiji Shibata4, Masanori Ogawa1 and Yoko Endo1
1

Research Center for Occupational Poisoning, Tokyo Rosai Hospital, Japan Labour Health Organization, Tokyo, Japan２

Department of Medicinal Informatics, Graduate School of Medical Science, Kanazawa University, Kanazawa, Japan ;Department of Medical Technology, Nagoya University School of Health Sciences, Nagoya, Japan３

Department of Occupational and Environmental Health, Nagoya University of Graduate School of Medicine, Nagoya, Japan
４

Department of Health and Psychosocial Medicine, Aichi Medical University School of Medicine, Aichi, Japan

Received for review June 25, 2007, Accepted September 28, 2007.

Abstract
A direct method to determine permethrins in human whole blood with high recovery and high sensitivity was developed using a combinationof solid−phase extraction (SPE) and gas chromatography−mass spectrometry (GC/MS). Whole blood was diluted with pure water and thenloaded on a preconditioned solid−phase Oasis HLB cartridge ; permethrins were eluted by toluene. The extract was mixed with an internal
standard and then concentrated under a N2 gas stream before being analyzed by a capillary GC/MS in electron ionization mode. Compoundsof cis− and trans−permethrin were separated and quantified in selected ion monitoring mode. The SPE recovery efficiency of cis− and trans−
permethrin was 97.7 ± 7.9% and 99.7 ± 7.7%, respectively. The LOD and LOQ found for cis−permethrin and trans−permethrin in whole
blood were 0.21 and 0.67 ng/ml, and 0.20 and 0.64 ng/ml, respectively. Using this method, we measured permethrins in the blood of pest control operators.
Keywords : gas chromatography−mass spectrometry, cis−permethrin, trans−permethrin, solid−phase extraction, whole blood, pest control
operator

metabolites,3−(2,2−dichlorovinyl ) − 2,2 − dimethylcyclopropane −1. INTRODUCTION
Permethrins have been widely utilized worldwide as a syn-carboxylic acid (Cl2CA) and 3−phenoxybenzoic acid (3−PBA).

thetic pyrethroid insecticide in agriculture, forestry, home pest con-

These metabolites are renally eliminated with a half−life of about 6

trol, public health and in similar contexts [1]. It is well known thathr [5]. Many analytical methods for Cl2CA and 3−PBA have been

the toxicity of permethrins is low in mammals, including humans

developed using LC−tandem MS or GC/MS because these com-

[1, 2]. As well all synthetic pyrethroids, there have been reports ofpounds are useful for biological monitoring of permethrin exposure

permethrins−related neurotoxicity and skin paresthesia [2−4].

[6, 7]. In the presence of an esterase inhibitor, permethrin detoxifi-

Biologically, permethrins are rapidly hydrolyzed at the central

cation is blocked or low esterase activity causes delayed detoxifica-

ester bond in the chemical structure and are oxidized to detoxified−tion. In such cases, permethrins accumulate in the human body. Af-

Correspondence : Nobuhiko Onda, Ph. D.
Tel : +81−3−3742−7301
Fax：+81−3−5735−9977
E−mail : ondanobu@tokyoh.rofuku.go.jp

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Chromatography, Vol.28 No.3 (2007)

ter the recent Gulf War (1990), it has been reported that permethrin

2.2. Working standard solutions

has complex effects in the presence of other chemicals and that

One hundred µg/ml of permethrins* in ethanol was prepared as athese enhance the toxicity of permethrin itself [8]. Measurement ofworking standard solution from NANOGENS permethrin mixture,permethrins as well as Cl2CA and 3−PBA present in the blood andand was standardized with the Sigma−Aldrich permethrin solu-

urine of permethrin−exposed humans is required for the biologicaltions. One µg/ml of fenitorothion−d6 was prepared as an internal

study of permethrins.

standard for the GC/MS measurements. Each isomer was identified

There are reports that have analyzed permethrins as pesticides

by the scan spectra obtained with electron ionization (EI) and thein human blood but most of these have been for serum or plasma

retention time. A series of permethrin solutions including a known

samples, and not for whole blood [9−12]. The analytical results foramount of fenitorothion−d6 (IS) were prepared by dilution for theserum or plasma samples may not account for permethrins bound

GC/MS linearity test and for calculation of the limit of determina-

to cell membranes in blood because many pesticides, including per-tion (LOD) and limit of quantitation (LOQ).

methrins, are lipophilic and bind to the cell membrane of erythrocytes [13]. Thus, a method for total assay of permethrins in whole

2.3. Blood samples

blood is necessary. A study has shown that the determination of

For the method development, human blood control samples

synthetic pyrethroid insecticides, including permethrins in whole

were obtained from 2 healthy volunteers who were not exposed tohuman blood, has a high recovery rate using liquid−liquid extrac-

permethrins, and were immediately treated with heparin and stored

tion [14]. Recently, SPE methods have been developed for analyses

at −20°C ; they were used in a week. Blood samples from sevenof environmental, food, and physiological samples to allow simpli-

male pest control operators aged 25−59 years were also immedi-

fication of the elaborate processes of detection required in the labo-

ately treated with heparin, and stored at −80°
C prior to analyses.

ratory. As well known, SPE is simple and effective for cleaning up
biological samples prior to quantitation. The recovery of per-2.4. SPE method for permethrins in blood samples

methrins has been reported to be about 85% for SPE with a C18 car-

The Oasis HLB cartridge used contained a copolymer (m−tridge of human plasma [11, 12]. However, there have been no re-divinylbenzene/N−vinylpyrolidone) packing material (nominal 60

ports of the use of SPE for the determination of permethrins inµm) designed to have hydrophilic−lipophilic properties, in the for-whole blood.

mat of diameter of ca.12 mm x 14 mm long. Preconditioning of theSPE cartridge was performed with elution solvent to be used, ac-This study aimed to develop a method for detecting and quantifying permethrins in whole blood with high rates of recovery andcording to the manufacture’s instruction [15].

high sensitivity using a recent SPE sorbent and GC/MS. The GC/(1) Preconditioning

MS enabled separation and quantification of two isomeric per-Before use, an Oasis HLB cartridge was washed with 5 ml ofmethrins, the cis− and trans−forms ; this is important because cis−

methanol and 5 ml of toluene in this order. Subsequently, 10 ml ofpermethrin toxicity is known to be higher because of its slow de-methanol and 5 ml of Milli−Q water were passed through the car-toxification [1, 2]. Using the method we developed, we then deter-

tridge before the sample solution was loaded.

mined the permethrin concentration in whole blood from pest control operators who were occupationally exposed to insecticides.

*This was a nominal concentration ; the exact values for each level of solution were as follows throughout this study :2. EXPERIMENTAL

1 ng/ml : 0.64 ng cis−permethrin and 0.31 ng trans−permethrin in ml ; to-2.1. Chemicals

tal permethrin 0.95 ng/ml (cis/trans=67.7/32.3),

The permethrin mixture (1 mg/ml in toluene, cis− and trans−

2 ng/ml : 1.28 ng cis−permethrin and 0.61 ng trans−permethrin in ml ; to-permethrin mixture) was purchased from NANOGENS Inc.

tal permethrin 1.89 ng/ml (cis/trans=67.7/32.3),(Watsonville, CA, U.S.A.). The standard solutions, 10 µg/ml of cis

5 ng/ml : 3.2 ng cis−permethrin and 1.5 ng trans−permethrin in ml ; a total−permethrin and trans−permethrin in cyclohexane were purchased

permethrin 4.7 ng/ml (cis/trans=67.7/32.3),
10 ng/ml : 6.4 ng cis−permethrin and 3.1 ng trans−permethrin in ml ; a to-from Sigma−Aldrich Co. (St. Louis, MO, U.S.A.). The 10 µg/ml oftal permethrin 9.5 ng/ml (cis/trans=67.7/32.3),

deuterated−fenitrothion (fenitrothion−d6) in acetone was purchased

20 ng/ml : 12.8 ng cis−permethrin and 6.1 ng trans−permethrin in ml ; to-from Hayashi Pure Chemical Ind. (Osaka, Japan). An Oasis HLB 6

tal permethrin 18.9 ng/ml (cis/trans=67.7/32.3),

cc (500 mg) LP Extraction Cartridge was purchased from Waters

50 ng/ml : 32 ng cis−permethrin and 15 ng trans−permethrin in ml ; total

Co. (Milford, MA, U.S.A.). Ethanol, methanol, acetone and toluene

permethrin 47 ng/ml (cis/trans=67.7/32.3), andof HPLC grade and n−hexane for residual material test purpose

100 ng/ml : 64 ng cis−permethrin and 31 ng trans−permethrin in ml ; a to-were purchased from Wako Pure Chemical (Osaka, Japan).

tal permethrin 95 ng/ml (cis/trans=67.7/32.3).

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Chromatography, Vol.28 No.3 (2007)

Hiroko Ogata−Kawata, Michiko Matsuda, Nobuhiko Onda, Jun Ueyama,
Michihiro Kamijima, Eiji Shibata, Masanori Ogawa and Yoko Endoand of blood permethrin extracted using the SPE were calculated(2) Elution
Five ml of 5−times diluted blood sample was loaded on thebased on 3.14 times the standard deviation (SD) and 10 times thepreconditioned cartridge. The cartridge was washed with 5 ml ofSD, respectively, from the results of the 7 repeated GC/MS runs

Milli−Q water and 5 ml of methanol, and then centrifuged at 3000

using 2.5 µg/ml total permethrin [16].

rpm for 10 min at 4°
C to minimize residual water and methanol. As
elution solvent, 2.5 ml of toluene was loaded on to this cartridge

3. RESULTS AND DISCUSSION

being placed in a test tube. Unless otherwise stated, this cartridge

3.1. Separation and detection of permethrin isomers

was left for 60 min at room temperature, and then the cartridge in

Figure 1 shows SIM chromatograms for permethrin−toluene

the test tube was centrifuged at 3000 rpm for 10 min at 4°
C and thesolution of 10 ng/ml (A) and 2 ng/ml (B), respectively. As de-extract was obtained.

scribed in Experimental , the isomeric identification was made by(3) Concentrationcomparing the retention time ; cis−permethrin was identified at a

The extract was added with a known amount of IS solution,

retention time of 20.86 min (peak 2) and trans−permethrin at 21.03

and was concentrated under a stream of nitrogen gas. Finally, themin (peak 3). The spectra for these were very similar and could notvolume of the extract was made up to 0.5 ml by adding toluene.

have been identified by mass spectrum. Multiple ion detection of
permethrin (m/z 163, 165, 183) was useful for confirming permethrins ; the major fragment ion at m/z 183 was suitable for the

2.5. GC/MS measurements

determination.

All GC/MS measurements were performed with a GCMS−QP
5050A gas chromatograph−mass spectrometer equipped with an

3.2. SPE optimization

AOC−20S autosampler (Shimadzu Co., Kyoto, Japan). Separation
column was DB−1, 100% polydimethylsiloxane capillary column,

To quantitatively recover permethrin from blood sample, SPE

30 m long with an internal diameter of 0.25 mm and film thickness

operations with an Oasis HLB cartridge were examined. The elu-of 0.25 µm (Agilent Technologies Inc., Palo Alto, CA, U.S.A.).

tion solvents tested were methanol, acetonitrile and toluene. Table

Three µl was injected in splitless mode ; split valve was opened 2
min after injection. The injector temperature was set to 250°
C. Helium was used as the carrier gas (inlet pressure : 117 kPa). The column temperature was initially held at 50°
C for 2 min, and programmed to 200°
C at a rate of 20°
C/min, and was held at 200°
C for 1
min, finally programmed to 290°
C at a rate of 7°
C/min and held for
5 min. The MS interface temperature was set at 250°
C. The electron multiplier of MS was set to 1.20 kV. The dwell time for selective ion monitoring (SIM) was 200 ms for each ion. Permethrin
was detected at ionization potential of 70 eV using target ions of
permethrin fragments (m/z 163, 165, 183) and m/z 183, major ionof permethrins was used for the determination. The peak area ratioof permethrin (m/z 183) and fenitrothion−d6 (m/z 283) was used
for quantification.
2.6. Assay validation
2.6.1. Calibration curve
Seven−point calibration plots were made with SPE−processed
control blood samples which were spiked with 1, 2, 5, 10, 20, 50

Figure 1. SIM mass chromatograms of permethrin and fenitrothion−d6 (internal standard) in an ethanol solution.
(A) The peak (1) at 12.75 min is for 100 ng/ml of fenitrothion−d6, peak (2) at retention time 20.85 min is forthe cis isomer of permethrin and peak (3) at 21.03 minis for the trans−isomer of permethrin (10 ng/ml of permethrin in total). (B)Multiple ions for 2 ng/ml total permethrin (m/z 163, 165, 183). (C) Structure of permethrins and the fragmentation.

and 100 ng/ml of permethrin ; three replicates of the SPE was performed at each concentration level. The peak area ratios of permethrin (m/z 183) to IS (m/z 283) were used for the calibration
curves.
2.6.2. Limits of detection and of quantitation
The LOD and LOQ of permethrin standard toluene solution

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Chromatography, Vol.28 No.3 (2007)

Table 2. SPE recoveries of permethrins spiked into diluted bloodand physiological saline

Table 1. “Initial” recovery with solvents used for extraction of
permethrins

Recoveries of spiked permethrins(%, mean ± RSD)

Recoveries of spiked permethrins(%, mean ± RSD)

Methanol (n=5)*Acetonitrile (n=5)
Toluene (n=4)

cis

trans

2±1
16 ± 385 ± 141±1
13 ± 2100 ± 28

Whole blood (n=5)*
Physiological saline
(n=8)

cis

trans

97.7 ± 7.9

99.7 ± 7.7

105.6 ± 12.1

102.7 ± 14.1

* ; number of replicates* ; number of replicates

Figure 2. SIM mass chromatograms (m/z 183) of toluene extract
blanks for the SPE cartridge.
(A) Blank for the SPE cartridge preconditioned without
toluene, and (B) with toluene. Full scale is normalized
to the same signal counts (2,000).
* ; specific blank peaks from Oasis HLB cartridge.
1 shows the “initial” recovery obtained with a standby time of zero

Figure 3. SIM mass chromatograms (m/z 183) of the extract fromthe SPE cartridge loaded with permethrin−spiked blood.
(A) Blank blood and (B) permethrin−spiked blood (50ng/ml of permethrin).
Full scale is normalized to 75,000 signal counts.
* ; specific blank peaks from Oasis HLB cartridge.

before the centrifugation for a 100 ng/ml permethrin solution and 5
ml of each elution solvent. The highest permethrin recovery among
those solvents tested was seen with toluene. Methanol did not help
with permethrin recovery and the recovery was also limited withacetonitrile. Neither methanol nor acetonitrile was strong enough toextract permethrins from the concerned solid phase, whereas toluene efficiently extracts permethrins from it since toluene strongly

chromatograms and the detection of permethrins was more difficultinteracts with poly(divinylbenzene/vinyl pyrolidone) base of theas these could not be easily distinguished from the background.

sorbent and separates permethrins from other lipophilic com-

When the cartridge was preconditioned with toluene, as seen inpounds. Therefore, toluene was of a choice as eluting solvent in

Fig. 2 B, the baseline quality was improved. Some specific peaks

this study. The elution volume was examined using 2.5 or 5.0 ml ofmarked with (*) were identified as homologs of hydrocarbons fromtoluene. Total permethrins recovery by extraction with 5.0 ml of

the GC/MS scan measurements. The chromatograms of the back-toluene was 100 ± 8.7% ; recovery with 2.5 ml of toluene was 110

ground of the cartridge and of the control blood sample spiked with

±7.1% (n=3). The volume of toluene was therefore fixed at 2.5 ml

permethrin are shown in Fig. 3. There was no interference between

throughout the study. Before elution with toluene, methanol was

the specific peaks and both the cis− and trans−permethrin peaks.

passed through the cartridge to minimize residual water which isAs described in Experimental section, the blood samples wereless miscible with toluene and also to remove the unwanted metha-

diluted with water before the SPE. To examine the recovery vari-

nol soluble constituents of blood.

ation with the medium, a physiological saline which is character-Figure 2 shows the elution from Oasis HLB cartridges withized with pH of 7.4 and ionic strength by 0.85% NaCl, was spiked

toluene, when a cartridge was preconditioned without toluene (A)with permethrin. Table 2 shows the recovery rate of permethrin

and with toluene (B). The first shows the higher background on the

spiked in water−diluted blood and in physiological saline. The re-

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Chromatography, Vol.28 No.3 (2007)

Hiroko Ogata−Kawata, Michiko Matsuda, Nobuhiko Onda, Jun Ueyama,
Michihiro Kamijima, Eiji Shibata, Masanori Ogawa and Yoko Endocovery of permethrin in water−diluted blood and the recovery of
permethrin spiked in physiological saline were both close to 100%.
This suggests that permethrins are recovered sufficiently from the
cartridge. In contrast to the present study, the recovery of permethrins in plasma using a Sep−Pak C18 (Waters) cartridge was reported to be 81−93% [11] ; the present SPE with the Oasis HLB
cartridge therefore performs better for permethrin recovery.
A comparative extraction of permethrins from whole blood
was made using liquid−liquid extraction [14] ; we found the same
recovery level as for our SPE method in that the recovery rates ofcis− and trans−permethrin by hexane/acetone=80/20(v/v) were
Figure 4. Linear calibration for cis− and trans−permethrin intoluene and in blood spiked with various levels of permethrin.
The caption, cis−/toluene indicates cis−permethrin intoluene ; similar notation applies for other combination.

105.3 ± 6.9% and 106.6 ± 6.7%, respectively. These results showthat this SPE using an Oasis HLB cartridge is suitable for extracting permethrins from a whole blood sample ; moreover, themethod has a high recovery rate and is ease to use. The present
SPE method requires that blood be diluted only as part of analyticalpreprocessing ; this may potentially be extendable to the analysesof other lipophilic substances in blood.
3.3 Quantitative assay of blood permethrins
3.3.1. Calibration curves for the spiked blood samples
The separate quantification of cis− and trans−permethrin isrequired for biological monitoring because permethrin toxicity isdependent on the isomeric form and also because the metabolic
rates of these isomers are different in body [2]. The proportion of
the two isomers differs among commercial insecticides. However,there are few studies in which each the permethrin isomers wereseparated for quantification. In our study, calibration curves forcis− and trans−permethrin were obtained between 0.64 and 32 ng/
ml, and between 0.31 and 31 ng/ml, respectively, as seen in Fig. 4.
Figure 4 shows good correlation for each isomer dissolved in toluene and in permethrin−spiked control blood.
To evaluate LOD and LOQ, 1.6 ng/ml of cis−permethrin and0.90 ng/ml of trans−permethrin in blood were assayed 7 times. The
LOD of cis− and trans− permethrin in permethrin−spiked whole
blood was calculated as 0.21 ng/ml (1.3 pg) and 0.20 ng/ml (1.2

Figure 5. Permethrins in a pest control operator’s blood.
Cis−permethrin and trans−permethrin were quantified
as 0.78 ng/ml (2.3 pg) and 1.1 ng/ml (3.3 pg), respectively.

pg), respectively. The LOQ of blood permethrin was 0.67 ng/ml(4.0 pg) for cis−permethrin and 0.64 ng/ml (3.8 pg) for the transisomer. The present analysis is superior to previously reported
methods, which have given LOD and LOQ values of 0.5 ng/ml andtrans−permethrin. For other six blood samples, both of cis− and1.0 ng/ml from plasma, respectively [12,17].

trans− permethrin were lower than the LOQ, while the methaboliteof permethrin, 3−PBA were identified and determined from all the

3.3.2. Permethrin detection in whole blood samples from pest control operators

seven blood samples. The operator who reported the highest con-

The developed method was then used for a practical blood

centration level of permethrin in blood was engaged in pest control

sample analysis. From one blood sample, permethrins exceeded theoperation for the longest time (6 hours) against other operators (0.5

LOQ was detected as shown in Fig. 5 with 1.8 ng/ml of total per-to 2 hours). The biological aspects to those results will be dis-

methrin consisting of 0.78 ng/ml cis−permethrin and 1.1 ng/ml

cussed in a separate paper.

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Chromatography, Vol.28 No.3 (2007)[6]

A. O. Olsson, S. E. Baker, J.V. Nguyen, L. C. Romanoff, S.
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4. Conclusion

Chem., 2004, 76, 2453−2461.

The present SPE−GC/MS method for the determination of[7]

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sample and does not require complicated processing. Both the cis−

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