Multiresidue method for high-performance liquid chromatography determination of carbamate pesticides residues in tea samples

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Multiresidue method for high-performance liquid chromatography

determination of carbamate pesticides residues in tea samples

Chia-Chang Wu a; Chun Chu b; Yei-Shung Wang c; Huu-Sheng Lur b

a Tea Research Extension Station, Nantou, Taiwan b Department of Agronomy, National Taiwan University, Taipei, Taiwan c Department of Agricultural Chemistry, National Taiwan University, Taipei, Taiwan

To cite this Article Wu, Chia-Chang, Chu, Chun, Wang, Yei-Shung and Lur, Huu-Sheng'Multiresidue method for high-performance liquid chromatography determination of carbamate pesticides residues in tea samples', Journal of Environmental Science and Health, Part B, 44: 1, 58 — 68

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ISSN: 0360-1234 (Print); 1532-4109 (Online) DOI: 10.1080/03601230802519744

Multiresidue method for high-performance liquid

chromatography determination of carbamate pesticides

residues in tea samples

CHIA-CHANG WU1_{, CHUN CHU}2_{, YEI-SHUNG WANG}3_{and HUU-SHENG LUR}2

1_{Tea Research Extension Station, Nantou, Taiwan}

2_{Department of Agronomy, National Taiwan University, Taipei, Taiwan}

3_{Department of Agricultural Chemistry, National Taiwan University, Taipei, Taiwan}

A multiresidue method was developed to determine 19 carbamate pesticides in tea samples. Optimizations of different parameters, such as the type of extraction solvents, clean-up cartridges, and elution solvents were carried out. The developed method used acetonitrile as extraction solvent, amino cartridge for adsorbents and acetone-n-hexane as the eluting solution. Nineteen carbamate residues were then analyzed by high-pressure liquid chromatography (HPLC) with fluorescence detector. The present results showed good linearity by correlation coefficients of more than 0.9999 for all analyses. Limits of detection and quantification varied from 0.0005–0.023 mg L−1, 0.008–0.077 mg L−1, respectively. Recoveries of sixteen carbamate pesticides ranged from 65% to 135% at the spiked level of 0.5, 1.0 and 2.0 mg L−1. The relative standard deviations were lower than 20% and coefficient of variations were lower than 15%. The results indicate that the proposed method provides an effective multi and trace level screening determination of carbamate pesticides residues for tea samples.

Keywords: Carbamate pesticides; tea; multiresidue analysis. Introduction

Carbamate pesticides are widely used on a large number of crop protections, and are increasingly used instead of organochlorine and organophosphorus pesticides. Carba-mate pesticides have been available commercially since the 1950s; they have become increasingly important in recent years, due to their broad spectrum of biological activity, high insect toxicity but a generally low toxicity toward warm-blooded species. Therefore, they are widely applied to control the insects, mites, and nematodes in agricultural

products.[1]

The action of carbamate pesticides are binding to the enzyme acetylcholinesterase, disrupting nerve function, re-sulting in paralysis and death. As a result, they are con-sidered toxic for the environment and for human beings. A large number of carbamate pesticides are presently used in crop plantations, and pose a potential risk to human

Address correspondence to Huu-Sheng Lur, Department of Agronomy, National Taiwan University,Taipei, Taiwan 106; E-mail; [email protected]

Received July 20, 2008.

health.[2]_{Therefore, in recent years continuous monitoring}

of these compounds on crops has become very important for public health.

Multi-residue methods (MRMs) are known to be cost-effective techniques for pesticide residues analysis. Government agencies of most countries have recently es-tablished MRMs and set up monitoring protocols for pesti-cide residues on agriculture products. Nevertheless, MRMs analysis generally consists of a serious of laborious steps, such as the pesticide extraction with organic solvents, clean up by cartridges, concentration, and chromatographic sepa-ration and identification. There has been an increasing need of analytical methods for identification and quantification of pesticide residues in tea. Almost all the common ana-lytical methods to measure organophosphorus, pyrethroid and organochlorine residues in tea are based on the use of chromatographic techniques, mainly gas chromatography

(GC) and have been employed extensively.[3−5] Carbamate

pesticides and their degradation products or derivatives, however, are polar, no-volatile and thermally unstable, that

can not be directly analyzed by GC.[6]_{Since the 1970s,}

high-performance liquid chromatography (HPLC) has become a preferred choice for the determination of carbamate pesti-cides in crops, fruits, vegetables, foodstuffs and environment

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bodies.[4,6−8] _{So far to our knowledge, there has been no}

literature reporting the MRM with high-pressure liquid chromatography (HPLC) to detect carbamate pesticides residues for tea samples. The aim of this paper was to de-velop an effective MRM for simultaneous determination of carbamate pesticides in tea samples using HPLC. In addi-tion, the optimization of solid-phase extraction (SPE) was also assessed in the MRM.

Materials and methods

Reagents

All pesticide standards were divided to two groups, group A (aldicarb sulfoxide, oxamyl, 3-OH carbofuran, aldicarb, metolcarb, thiodicarb, carbofuran, carbaryl, iso-procarb, and fenobucarb) and group B (aldicarb sulfone, methomyl, butocarboxin, 3-keto carbofuran, propoxur, meobal, macbal, methiocarb and promecarb). The chemi-cals were obtained from Riedel de-Ha¨en (Hannover, Ger-many) with purity of 98.6–99.9%. All the solvents and chemicals used in this study were of analytical grade from Merck (Germany).

Calibration, limits of detection and quantiﬁcation

A standard stock solution (1000 mg L−1) was prepared in

acetonitrile and stored at−20◦C. The working solutions

re-quired for preparing a standard curve (0.05, 0.1, 1.0, 2.5,

5.0 and 10 mg L−1) were prepared daily from the standard

stock solution by serial dilutions. All measurements were performed in three replicates for each level was analyzed; calibration curves were calculated by linear least-squares regression using peak areas.

The limits of detection (LODs) and quantiﬁcation (LOQs) with this procedure were deﬁned as the concen-trations of each of the pesticides in tea samples (expressed

as mg L−1) that gave signals of 3 and 10 times the noise,

respectively.[9,10]

Recovery of pesticides from fortiﬁed samples

Tea samples spiked with 0.5, 1.0 and 2.0 mg L−1carbamate

pesticides were used for quantiﬁcation analysis to evalu-ate the recovery (%), and for determining the coefﬁcient of variation (CV) of each carbamate pesticide. The recovery assays were replicated three times.

Extraction

The extraction and cleaning procedure was modiﬁed from

(1999).[11]_{Samples of 10 grams of tea powder were}

homog-enized with 40 mL distilled water, and were extracted with 50 mL or 100 mL of different extraction solvents (acetoni-trile, dichloromethane, acetone, and n-hexane) by

homoge-nization for 1 min with the polytron. Extracts were filtered through a Whatman GF/C 110 mm filter paper with vac-uum pump, transferred to flasks, and then added tested extraction solvent to 100 mL. Immediately after addition of 15 g NaCl and shaking for 1 min, the upper 10 mL extraction solvent layer were collected, and evaporated to dryness with nitrogen gas. The residue was then dissolved in 5 mL acetone-dichloromethane (1/9 (v/v)). The follow-ing clean-up was accomplished with an amino cartridge (Agilent Technologies, USA) rinsed with 5 mL acetone-dichloromethane (1/9 (v/v)). The concentrated extract (in acetone-dichloromethane, 1/9 (v/v)) was transferred to the top of the cartridge, and the elution was collected carefully. Four 8 mL of the acetone-dichloromethane was continu-ously added twice to the cartridge and collected together. Collected eluent was dried by nitrogen gas, dissolved again

in 1 mL acetonitrile, and ﬁltered through a 0.2µm

mem-brane before HPLC injection.

Cartridge studies

The extracted dried residue was dissolved in a 5 mL mix-ture (acetone-dichloromethane, 1/9 (v/v)). Recovery anal-ysis during the clean-up step was evaluated with ﬁve types of cartridges including octyl (Agilent Technologies, USA), octadecyl (Agilent Technologies, USA), ﬂorisil (J. T. Baker, USA), silica gel (J. T. Baker, USA), and amino (Agilent Technologies, USA). The cartridges were rinsed with 5 mL of elution solution (acetone-dichloromethane, 1/9 (v/v)). Concentrated extract residue was loaded onto the top of the cartridge, and the elution was collected carefully. And 4 mL of the elution solution was added twice (8 mL in total) to the cartridge and the eluent was collected together. Col-lected residue eluent was dried by nitrogen gas, dissolved in

1 mL acetonitrile, and passed through 0.2µm membrane

ﬁlters before HPLC injection.

Elution studies

Evaluation of elution solution was performed with differ-ent types of solvdiffer-ent mixtures (acetone-dichloromethane,

methonal-dichloromethane, n-hexane-dichloromethane,

and acetone-n-hexane) and an amino cartridge. The ratio of each elution mixtures were 1/9 (v/v). Concentrated extract was loaded to the top of the cartridge rinsed with 5 mL of mixture. The elution was collected. For elution volume analysis, different volumes (1 mL, 2 mL, 4 mL) of the elution mixtures were continuously added twice to the cartridge (in total 2 mL, 4 mL, and 8 mL respectively), and the eluted solutions were collected together. The collected eluent was again dried by nitrogen gas, dissolved in 1

mL acetonitrile and ﬁltered through a 0.2µm membrane

before HPLC injection.

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Sample analysis

The present developed MRM were practically used for mar-ket tea samples analysis. Six thousand and forty eight tea samples were collected by the Agriculture and Food Agency (AFA), Council of Agriculture from 12 tea plantation ar-eas of Taiwan (Yilan, Taipei, Tayoyuan, Hsinchu, Miaoli, Taichung, Nantou, Yunlin, Chiayi, Kaohsiung, Taitung and Hwalien) from 2004 to 2005.

The samples were ground to a powder in a mortar with

a pestle, then homogenized and stored at −20◦C before

analysis. Samples were analyzed within four weeks after collection.

Sample extraction and clean up procedure were con-ducted as the previously optimized and described by us-ing an amino cartridge (Agilent Technologies, USA). The puriﬁed residues were reconstituted with 1 mL acetonitrile

and ﬁltered through a 0.2µm membrane prior to HPLC

analysis.

Apparatus and experimental conditions

Analysis of carbamate pesticides was performed on an Ag-ilent Liquid Chromatograph (Model 1100, AgAg-ilent Tech-nologies, USA), equipped with a quaternary pump, an auto sampler, a ﬂuorescence detector, column thermostat and a post-column deliver reactor (Pickering 5100X). Separation

was carried out with a reversed phase column (C18, 5µM

spherical, 4.6× 250 mm, Astec, USA). Column oven

tem-perature was kept at 40◦C, and the sample injection volume

was 10µL. Mobile phases for gradient elution were distilled

water (mobile phase A) and acetonitrile (mobile phase B)

delivered at a constant ﬂow rate of 1.0 mL min−1. The

gradi-ent program of the mobile phase was as follows: 70% A and 30% B at initial, 60% A and 40% B at 5 min, 35% A and 65% B at 6 min, 40% A and 60% B at 20 min, 30% A and 65% B at 23 min and than hold 2 minutes. The post-column reactor utilized sodium hydroxide and o-phthalaldehyde/thioﬂuor

reagents and delivered at 100◦C by post-column deliver

re-actor. Detection of pesticides as ﬂuorescent derivatives was carried out using 330 and 460 nm as wavelengths for exci-tation and emission, respectively.

Results and discussion

Chromatogram of carbamate pesticides

Figure 1 showed chromatograms of the separation for 19 anilities in standard solution of carbamate pesticides. The chromatograms revealed a sharp and symmetrical peak for each standard molecule, as well as with tea sample. The retention times for all compounds investigated are listed in Table 1. A good separation was obtained in a relatively short running time, the ﬁrst retention time was 3.91 min for aldicarb sulfoxide and the ﬁnal retention time was 22.98 min for promecarb. The chromatograms result suggested

that detection of all nineteen carbamate pesticides can be done within 25 min by the HPLC.

Linearity

Condition of uniform variance over the linear range was checked. All measurements were performed over ﬁve

dif-ferent levels ranging between 0.05 and 10.00 mg L−1(three

replicates for each level were analyzed). Calibration curves were calculated by linear least-squares regression using peak areas. The results are summarized in Table 1, the cal-ibration curves were found to have good linearity by

cor-relation coefﬁcients (r2) of more than 0.999 in all analyses,

indicating that the linear regression method can answer the concentrations of the analyses under study, within the con-centration range investigated.

LODs and LOQs

LODs for 19 carbamate pesticides investigated were at low-nanogram per milliliter levels, with the range of 0.0005 mg

L−1and 0.023 mg L−1 (Table 1). The highest LODs were

0.023 mg L−1for aldicarb and carbofuran, and the lowest

LODs for were 0.0005 mg L−1for isoprocarb and

fenobu-carb. In LOQs the highest was 0.077 mg L−1for carbofuran

and the lowest were 0.008 mg L−1for meobal and macbal

(Table 1).

The LODs and LOQs data obtained in the present exper-imental conditions were all satisfactory, being 10–100 times lower than maximum residue limits (MRLs) of the carba-mate pesticides required by the regulation of Taiwan, the

European Union (EU), and Japan (Table 2).[12−15] These

data conﬁrm the usefulness of our method in detecting the carbamate pesticide residues in tea samples.

Recovery test and coefﬁcient of variations (CV) of tea samples

The recovery, relative standard deviations (RSD) and CV of nineteen carbamate pesticides from tea samples spiked

with 0.5–2 mg L−1 were listed in Table 3. In tea spiked

with 2 mg L−1, 17 carbamate pesticides were detected and

the recoveries were within the range of 65% to 135%. In the same recoveries ranges we also detected sixteen and seventeen carbamate pesticides spiked with levels of 1 mg

L−1 and 0.5 mg L−1, respectively. It might be due to the

high volatility during the nitrogen drying processed under

35◦C, aldicarb sulfoxide was not detected within the spiking

range.

Good results were obtained with the mean recoveries of the carbamate pesticides that fell within the recommended

range of 65% to 135% by EPA method 531.2,[16]_suggesting

that this method is suitable for the residue analysis. Table 3 also showed levels of RSD for the carbamate pesticides. Almost all the carbamate pesticides RSD were lower than

20%, expected in a tea sample spiked with 0.5–2 mg L−1.

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Fig. 1. Chromatograms for: A: standard mixture of pesticides group A containing of 1 mg kg−1of: 1. aldicarb sulfoxide, 2. oxamyl, 3. 3-OH carbofuran, 4. aldicarb, 5. metolcarb, 6. thiodicarb, 7. carbofuran, 8. carbaryl, 9. isoprocarb, 10. fenobucarb; B: standard mixture of pesticides group B with 1 mg kg−1of: 1. aldicarb sulfone, 2. methomyl, 3. butocarboxin, 4. 3-keto carbofuran, 5. propoxur, 6. meobal, 7. macbal, 8. methiocarb, 9. promecarb; C: tea sample spiked with standard mixture group A; D: tea sample spiked with standard mixture group B; E: blank tea sample. Analysis condition is shown in “Apparatus and conditions” section.

The RSD values were in agreement with that recently

re-ported in the EU guidelines.[9]

The CV values were calculated in Table 3, almost all the carbamate pesticides CV were lower than 15%, as was

ex-pected in a tea sample spiked with 0.5 mg L−1. We found

that butocarboxin and 3-OH carbofuran with the CV were higher than 15%, a similar result was also found in a tea

sample spiked with 1 mg L−1and 2 mg L−1of butocarboxin

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Table 1. Linearity, calibration curves, limits of detection (LODs), and limits of quantiﬁcation (LOQs) for the carbamate pesticides under study.

Parameters of linearity

Pesticide RT (min) Linear range (mg L−1) r2 _Slope _Intercept _{LOD mg L}−1 _{LOQ mg L}−1

Group A Aldicarb sulfoxide 3.912 0.05–10 0.9999 9.794× 101 _0.101 _0.009 _0.028 Oxamyl 4.962 0.05–10 0.9999 9.941× 101 _−0.497 _0.008 _0.026 3-OH Carbofuran 7.018 0.05–10 0.9999 9.222× 101 0.059 0.008 0.026 Aldicarb 10.971 0.05–10 0.9999 1.104× 102 _0.572 _0.023 _0.076 Metolcarb 13.113 0.05–10 0.9999 1.355× 102 _2.042 _0.017 _0.057 Thiodicarb 14.204 0.05–10 0.9999 1.078× 102 _1.373 _0.021 _0.069 Carbofuran 15.244 0.05–10 0.9999 1.030× 102 _0.632 _0.023 _0.077 Carbaryl 18.981 0.05–10 0.9999 1.587× 102 _0.699 _0.015 _0.050 Isoprocarb 18.865 0.05–10 0.9999 1.108× 102 _0.521 _0.0005 _0.016 Fenobucarb 21.981 0.05–10 0.9999 1.023× 102 _−6.463 _0.0005 _0.018 Group B Aldicarb sulfone 5.062 0.05–10 0.9994 9.498× 101 _0.726 _0.014 _0.046 Methomyl 5.814 0.05–10 0.9997 1.267× 102 _2.187 _0.010 _0.034 Butocarboxin 9.798 0.05–10 0.9997 5.097× 101 _0.481 _0.022 _0.074 3-Keto Carbofuran 10.844 0.05–10 0.9998 6.682× 101 _2.777 _0.014 _0.048 Propoxur 14.819 0.05–10 0.9995 1.080× 102 _3.937 _0.011 _0.035 Meobal 16.296 0.05–10 0.9995 1.197× 102 _3.728 _0.003 _0.008 Macbal 16.906 0.05–10 0.9996 1.245× 102 _3.430 _0.002 _0.008 Methiocarb 22.147 0.05–10 0.9996 9.166× 101 _4.830 _0.003 _0.010 Promecarb 22.984 0.05–10 0.9997 1.043× 102 3.139 0.003 0.010

and 3-OH carbofuran, respectively. According to GARP

(Group of Analysts of Residues of Pesticides),[17] _the

ac-ceptable values of variation coefﬁcient are lower than 15%. However, the US-EPA can accept values of variation

co-efﬁcient up to 30%.[18]_{Thus, the obtained values from}

ex-traction and cleanup procedure of this study are reliable for routine analysis of carbamate pesticides in tea.

Effects of extraction solvents

In trace analysis, when residue levels are close to the limit of sensitivity of the instrument, the trace matrix compo-nents can interfere with determination processes, so the

Table 2. The maximum residue levels of carbamate pesticides (mg kg−1) regulated in different areas.

Pesticide Taiwan European Union Japan

Aldicarb NE 0.05 NE Carbaryl 2.0 NE 1.0 Carbofuran 1.0 0.2 0.2 Carbosulfan NE 0.1 NE Macbal NE NE 10.0 Methomyl 1.0 0.1 20.0 Thiabendazole NE 0.1 NE Thiodicarb NE NE 25.0 Propoxur NE 0.1 NE

NE: Not established.

extraction and the elution solvents should be adequately selected. A number of studies indicate that liquid-liquid extraction (LLE) methods have been used to extract car-bamate pesticides from several vegetable matrices, and the most frequently employed solvents are dichloromethane

and n-hexane.[19]

Because of the different structural characteristics and po-larity of the carbamate pesticides, it is important to opti-mize the extraction conditions. Several extraction solvents for analysis recoveries were further evaluated in the present study (Table 4). There were signiﬁcant differences in ex-tracting efﬁciency among the extraction solvents for most of carbamate pesticides in tea samples. Comparing four solvents n-hexane had the lowest recoveries with all the recovery values below 65%. It might be due to that car-bamate pesticides were less soluble in n-hexane than in other three kinds’ solvents. There were fourteen and thir-teen carbamate pesticides on a range of recoveries between 65% and 135% when acetonitrile and acetone were used as an extraction solvent respectively. On the other hand, if dichloromethane was used as extraction solvent only four carbamate pesticides showed the recovery range of 65% to 135%.

Thus, acetonitrile or acetone revealed to be an optimal extraction solvent for the carbamate pesticides extraction in tea samples. Considering the whole average recovery values of all tested carbamate pesticides and the nitrogen dryness process, acetonitrile was selected as the extraction solvent in the following experiments.

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Table 3. Recoveries of carbamate pesticides from the tea samples spiked at different concentration levels. Spiked level (mg L−1) Pesticide 0.5 1.0 2.0 Group Aa Oxamyl 78.5b_{± 6.7}c_(8.5)d _75.9_{± 6.5 (8.6)} _65.5_{± 5.7 (8.7)} 3-OH Carbofuran 41.9± 12.3 (29.3) 36.2± 7.3(6.8) 47.5± 14.7(30.9) Aldicarb 116.5± 11.8 (10.2) 93.2± 3.2 (3.4) 94.8± 3.6 (3.8) Metolcarb 140.4± 16.4 (11.7) 91.0± 5.7(6.2) 100.0± 4.2(4.2) Thiodicarb 102.2± 14.0 (13.7) 69.0± 3.1 (4.5) 82.0± 3.8 (4.7) Carbofuran 119.7± 13.4 (11.2) 115.5± 1.2(1.0) 96.9± 3.5 (3.6) Carbaryl 187.8± 9.7 (5.2) 125.7± 4.6(3.6) 111.1± 3.2(2.9) Isoprocarb 84.0± 8.5 (10.1) 98.5± 5.0 (5.1) 81.4± 3.3 (4.0) Fenobucarb 115.5± 7.6(6.6) 89.4± 3.8(4.2) 89.4± 4.3(4.8) Group B Aldicarb sulfone 86.7± 3.4(3.9) 88.6± 6.9 (7.8) 67.1± 3.8(5.7) Methomyl 95.9± 8.1(8.4) 88.8± 6.3 (7.1) 74.6± 3.8(5.1) Butocarboxin 94.8± 19.8 (20.9) 62.9± 11.7(18.5) 76.1± 6.1 (8.0) 3-keto Carbofuran 118.1± 15.2 (12.8) 102.2± 4.5(4.4) 87.5± 4.4 (5.0) Propoxur 88.5± 12.1 (13.7) 115.3± 2.2(1.9) 85.2± 5.2 (6.1) Meobal 85.1± 8.7 (10.2) 98.0± 5.4 (5.6) 82.4± 4.7 (5.7) Macbal 103.1± 2.5(6.2) 137.8± 7.4(5.4) 103.5± 5.4(5.2) Methiocarb 92.8± 6.4(6.9) 94.4± 4.2 (4.4) 95.1± 5.1 (5.3) Promecarb 64.1± 4.3(6.7) 87.4± 2.8 (3.2) 103.5±6.4(6.2)

a_{Aldicarb sulfoxide was not detected.}

b_{Recovery (%)}_{= 100% × (Level of carbamate pesticides in spiked sample level of carbamate pesticides in blank sample) / Level of carbamate}

pesticides in spiked tea sample.

c_{Relative standard deviation (RSD).}

d_{Coefﬁcient of variations (CV, %) are shown in parenthesis.}

All values represent the average for three determinations.

Table 4. Recoveries of carbamate pesticides from the tea samples with different extraction solvents.

Extraction Solvent

Pesticide Acetonitrile Dichloromethane Acetone n-Hexane

Group Aa Oxamyl 95.9b_{± 0.3}c_(0.3)d _74.1_{± 0.5 (0.7)} _77.1_{± 0.2 (0.2)} _ND 3-OH Carbofuran 55.6± 0.4(0.6) 50.8± 0.6 (1.1) 79.7± 0.4 (0.4) ND Aldicarb 60.7± 0.5 (0.9) 34.2± 0.1 (0.3) 37.3± 0.1 (0.2) 4.0± 0.0 (0.8) Metolcarb 84.6± 0.6 (0.8) 52.1± 0.4 (0.7) 55.4± 0.1 (0.1) 9.8± 0.1 (0.9) Thiodicarb 85.1± 0.3 (0.3) 57.9± 0.5 (0.8) 60.8± 0.3 (0.4) 3.1± 0.3 (10.2) Carbofuran 103.9± 0.4 (0.4) 59.6± 0.3 (0.6) 65.7± 0.2 (0.3) 11.8± 0.3 (2.5) Carbaryl 112.8± 0.8(0.7) 69.7± 0.3 (0.4) 79.5± 0.3 (0.3) 12.4± 0.0 (0.3) Isoprocarb 102.0± 0.6(0.6) 61.0± 0.1 (0.2) 75.8± 0.4 (0.5) 20.0± 0.1 (0.4) Fenobucarb 99.9± 1.0 (1.0) 63.1± 0.4 (0.6) 63.7± 0.2 (0.3) 36.9± 1.7 (4.7) Group B Aldicarb sulfone 91.6± 0.3 (0.3) 85.7± 1.8 (2.0) 106.6± 0.2 (0.2) ND Methomyl 85.4± 0.2 (0.2) 63.7± 0.1 (0.2) 82.0± 0.2 (0.2) ND Butocarboxin 68.7± 0.1 (0.2) 55.4± 0.1 (0.3) 69.8± 0.1 (0.1) 5.0± 0.0 (0.5) 3-keto Carbofuran 55.7± 0.3 (0.5) 43.6± 0.3 (0.6) 65.9± 0.2 (0.2) 2.8± 0.1 (5.2) Propoxur 95.9± 0.3 (0.3) 67.9± 0.3 (0.4) 97.6± 0.1 (0.1) 10.1± 0.1 (1.4) Meobal 55.2± 0.1 (0.2) 41.5± 0.1 (0.3) 62.0± 0.7 (1.1) 8.1± 0.1 (0.6) Macbal 71.3± 0.3 (0.4) 51.9± 0.1 (0.1) 80.5± 0.8 (1.0) 10.2± 0.1 (0.9) Methiocarb 73.3± 0.3 (0.4) 49.6± 0.3 (0.6) 67.6± 0.1 (0.2) 14.3± 0.4 (2.9) Promecarb 79.5± 0.3 (0.3) 50.6± 0.3 (0.6) 66.8± 0.3 (0.5) 15.5± 0.4 (2.9)

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Table 5. Recoveries of carbamate pesticides from the tea samples with different extraction volumes.

Extraction Volume Pesticide 50 mL 100 mL Group Aa Oxamyl 129.7b_{± 0.7}b_(0.5)d _72.8_{± 0.9 (1.3)} 3-OH Carbofuran 81.7± 1.8(2.1) 49.0± 0.4 (0.8) Aldicarb 107.9± 1.0 (0.9) 51.7± 0.3 (0.7) Metolcarb 135.0± 1.1 (0.8) 66.3± 0.3 (0.5) Thiodicarb 116.0± 0.6 (0.5) 63.3± 0.4 (0.7) Carbofuran 165.2± 1.3 (0.8) 84.1± 0.5 (0.6) Carbaryl 171.1± 1.3 (0.7) 88.1± 0.3 (0.3) Isoprocarb 161.1± 1.4 (0.8) 75.7± 0.4 (0.5) Fenobucarb 168.2± 1.4 (0.9) 80.0± 0.5 (0.6) Group B Aldicarb sulfone 166.7± 1.5 (0.9) 93.3± 0.8 (0.8) Methomyl 140.2± 1.0 (0.7) 76.4± 0.6 (0.8) Butocarboxin 136.4± 1.0 (0.8) 62.0± 0.9 (1.4) 3-keto Carbofuran 123.8± 1.1 (0.9) 67.3± 0.5 (0.8) Propoxur 185.1± 2.0 (1.1) 85.1± 0.7 (0.8) Meobal 171.6± 1.4 (0.8) 78.5± 0.7 (0.9) Macbal 187.5± 1.4 (0.7) 93.3± 0.8 (0.8) Methiocarb 167.8± 1.5 (0.9) 76.4± 0.6 (0.8) Promecarb 166.5± 1.4 (0.9) 62.0± 0.9 (1.4)

b_{Recovery (%)}_{= 100% × (Level of carbamate pesticides in spiked}

sam-ple level of carbamate pesticides in blank samsam-ple) / Level of carbamate pesticides in spiked tea sample.

Effects of etraction solvent volumes

The second parameter studied was the different volumes of extraction solvent for the extraction of the pesticides. Com-parison with different extraction solvent volumes (50 mL and 100 mL) in carbamate pesticides analysis of tea samples is described in Table 5. The results showed that the recover-ies of these two extraction volumes were above 65%, except that aldicarb sulfoxide was not detected.

When used 50 mL acetonitrile to extract the tea samples, there were eight carbamate pesticides on a range of recov-eries between 65% and 135%, and 10 carbamate pesticides on a range of recoveries between 165.2% and 187.5%. On the other hand, under 100 mL acetonitrile extraction there were 13 carbamate pesticides on a range of recoveries be-tween 65% and 135%, and ﬁve carbamate pesticides on a range of recoveries between 49.0% and 63.3%.

In this study, the extraction solvent volumes of 100 mL acetonitrile were efﬁcient in extracting carbamate pesticide residues in tea samples.

Solid phase extraction (SPE)

SPE has being increasingly used in pesticides analysis, mainly for sample clean-up. Several papers have described

the determination of pesticides in fruit, vegetables,[18]_and

water[20,21]by use of solvent extraction and solid-phase

ex-traction. SPE columns not only offer the potential of sim-plifying the puriﬁcation of the initial extract and reducing the amount of solvent consumed, but also decrease matri-ces effects that inﬂuence the analysis and the detection of studied compounds.

The physical and chemical properties of pesticides and adsorbents used in SPE devices vary widely. The SPE ex-traction procedure compared in this work was based on non-polar cartridge (octyl, octadecyl), moderately polar cartridge (florisil, silica gel), and polar cartridge (amino). Table 6 illustrates the recoveries obtained with the five types of cartridges. By using amino cartridge there were sixteen carbamate pesticides that could be detected with recoveries ranged from 65% to 135%. The other adsorbents showed relative low recoveries. For florisil or octyl cartridges there were 14 and 13 carbamate pesticides that could be detected within the range of 65% to 135%, and particularly for sil-ica gel cartridge there were only 12 carbamate pesticides that could be detected within the range of 65% to 135%. Aldicarb sulfoxide was not detected by all five cartridges tested, perhaps due to its polarity and volatility. It was also found that the recovery of 3-OH carbofuran was very low (36.2%) by using an amino cartridge. In other four type cartridges 3-OH carbofuran was even not detected.

To our knowledge, no comprehensive data on the SPE procedure for carbamate pesticides analysis in tea sam-ples has been documented. The present study demonstrated amino cartridge to be an appropriate adsorbent for a rou-tine residues analysis of carbamate pesticides in tea samples.

Effects of elution solvents

After the sorbent type of SPE was determined, efﬁciency of different elution solvents for the amino cartridge was further evaluated. The recovery values of different elution mixture solvents are shown in Table 7. Using the N-hexane dichloromethane and acetone-dichloromethane mixture as elution solvents, the recoveries of sixteen and ﬁfteen carba-mate pesticides were in the range above 65%, respectively. On the other hand, only four carbamate pesticides had re-coveries above 65% when using methanol-dichloromethane as elution solvents. None of recoveries of carbamate pesti-cides by acetone-n-hexane elution exceeded 30%, suggesting that acetone-n-hexane was not a suitable elution solvent for the amino cartridge SPE clean up of tea sample extractions.

Effects of elution volumes

The other parameter studied was the elution volume for the amino cartridge SPE clean up process. Resultant recovery values for elution volumes of 2 mL, 4 mL and 8 mL are shown in Table 8. Better recoveries were obtained with 4 mL and 8 mL of n-hexane-dichloromethane, with sixteen

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Table 6. Recoveries of carbamate pesticides from the tea samples with different packing materials of cartridges. Type of cartridge

Pesticide Florisil Amino Octyl Octadecyl Silica gel

Group Aa Oxamyl 23.4b_{± 1.2}c_(4.9)d _75.9_{± 6.5 (8.6)} _ND _ND _67.9_{± 8.2 (12.1)} 3-OH Carbofuran ND 36.2± 7.3 (6.8) ND ND ND Aldicarb 94.7± 6.7 (7.1) 93.2± 3.2 (3.4) 81.2± 2.3 (2.8) 82.5± 4.4 (5.4) 95.8± 23.8(24.9) Metolcarb 97.4± 1.5 (1.5) 91.0± 5.7 (6.2) 81.9± 3.4 (4.1) 81.2± 3.5 (4.4) 71.2± 16.9(23.8) Thiodicarb 43.2± 3.8 (8.9) 69.0± 3.1 (4.5) 55.9± 0.4 (0.7) 58.3± 2.6 (4.4) 70.6± 5.1 (7.2) Carbofuran 78.2± 3.5 (4.5) 115.5± 1.2(1.0) 70.9± 2.1 (3.0) 70.0± 5.0 (7.1) ND Carbaryl 123.6± 8.5(6.8) 125.7± 4.6(3.6) 123.5± 2.5 (2.0) 121.8± 7.3 (6.0) ND Isoprocarb 84.4± 6.8 (8.1) 98.5± 5.0 (5.1) 85.0± 1.4 (1.6) 83.1± 4.7 (5.7) 68.6± 19.6(28.6) Fenobucarb 85.7± 5.6 (6.6) 89.4± 3.8 (4.2) 90.7± 1.9 (2.1) 89.7± 3.0 (3.3) 80.0± 10.7(13.4) Group B Aldicarb sulfone 51.3± 6.9 (13.4) 88.6± 6.9 (7.8) ND ND 66.6±22.7 (34.1) Methomyl 66.2± 4.4 (6.6) 88.8± 6.3 (7.1) ND ND 70.2± 2.6 (3.8) Butocarboxin 90.6± 3.0 (3.3) 62.9± 11.7 (18.5) 78.3± 7.9 (10.1) 78.1± 3.9 (5.0) 98.5± 7.4 (7.5) 3-keto Carbofuran 98.4± 13.8 (14.1) 102.2± 4.5 (4.4) 79.0± 6.7 (8.5) 80.2± 4.8 (18.4) 125.0± 15.3 (12.2) Propoxur 85.7± 6.2 (7.3) 115.3± 2.2 (1.9) 104.3± 4.1 (3.9) 103.8± 6.8 (6.6) ND Meobal 88.2± 7.1 (8.1) 98.0± 5.4 (5.6) 87.7± 4.4 (5.0) 88.2± 4.6 (5.2) ND Macbal 133.4± 9.6(7.2) 137.8± 7.4 (5.4) 134.3± 5.3 (4.0) 131.0± 3.1 (2.4) ND Methiocarb 86.3± 2.7 (3.1) 94.4± 4.2 (4.4) 88.3± 4.6 (5.2) 88.4± 3.6 (4.1) 88.5± 5.8 (6.5) Promecarb 82.0± 3.4 (4.1) 87.4± 2.8 (3.2) 87.2± 3.8 (4.4) 87.7±7.1 (8.1) 84.1± 3.6 (4.2)

Table 7. Recoveries of carbamate pesticides from the tea sample with different cartridge elution solvents. Elution solvent

Acetone- Methonal- N-hexane-

Acetone-Pesticide dichloromethane (1/9) dichloromethane (1/9) dichloromethane (1/9) hexane (1/9)

Group Aa Oxamyl 86.6b_{± 0.5}c_(0.6)d _76.1_{± 0.4 (0.6)} _84.6_{± 0.5 (0.6)} _ND 3-OH Carbofuran 51.1± 0.3 (0.7) 46.7± 0.4 (0.9) 92.6± 0.6 (0.7) 0.9± 0.1 (9.3) Aldicarb 54.8± 0.4 (0.7) 37.4± 0.3 (0.5) 51.2± 0.6(1.2) 6.1± 0.2 (3.1) Metolcarb 75.5± 0.6 (0.7) 53.3± 0.2 (0.4) 63.6± 0.4 (0.6) 18.5± 0.4 (2.2) Thiodicarb 75.2± 0.5 (0.7) 55.8± 0.3 (0.5) 72.3± 0.6 (0.8) ND Carbofuran 88.6± 0.7 (0.8) 61.2± 0.3 (0.5) 78.2± 0.6 (0.7) 16.7± 0.3 (2.0) Carbaryl 97.6± 0.8 (0.8) 67.4± 0.2 (0.3) 84.6± 0.4 (0.5) 17.2± 0.2 (1.4) Isoprocarb 87.8± 0.7 (0.8) 58.2± 0.2 (0.3) 74.3± 0.3 (0.3) 29.4± 0.5 (1.6) Fenobucarb 83.8± 0.9 (1.1) 56.5± 0.3 (0.6) 70.1± 0.3 (0.5) 30.4± 0.5 (1.6) Group B Aldicarb sulfone 79.1± 1.8 (2.3) 77.6± 1.0 (1.3) 95.8± 0.6 (0.6) ND Methomyl 84.7± 0.3 (0.4) 66.8± 0.3 (0.5) 84.8± 0.3 (0.4) 1.0± 0.1 (5.9) Butocarboxin 76.7± 0.4 (0.6) 60.1± 0.4 (0.7) 73.1± 0.3 (0.4) 7.7± 0.1 (1.7) 3-keto Carbofuran 61.7± 0.4 (0.7) 52.3± 0.6 (1.2) 68.9± 0.3 (0.5) 7.4± 0.3 (3.8) Propoxur 87.3± 0.5 (0.5) 64.9± 0.2 (0.4) 81.5± 0.7 (0.9) 16.8± 0.3 (2.0) Meobal 69.7± 0.7 (1.0) 54.6± 0.6 (1.1) 76.6± 0.8 (1.0) 19.9± 0.5 (2.7) Macbal 79.9± 0.5 (0.6) 61.8± 0.5 (0.8) 90.6± 0.7 (0.8) 23.6± 0.6 (2.6) Methiocarb 79.8± 0.5 (0.6) 58.6± 0.5 (0.9) 77.3± 0.7 (0.8) 24.3± 0.5 (2.0) Promecarb 81.1± 0.4 (0.5) 59.2± 0.4 (0.8) 74.9± 0.9 (1.2) 27.6± 0.6 (2.2)

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Table 8. Recoveries of carbamate pesticides from the tea samples with different elution volumes. Elution volume Pesticide 2 mL 4 mL 8 mL Group Aa Oxamyl 66.1b_{± 0.6}c_(0.9)d _84.4_{± 0.5 (0.6)} _104.3_{± 0.5 (0.5)} 3-OH Carbofuran 31.7± 1.6 (5.2) 31.3± 1.1 (3.4) 70.4± 1.5 (2.2) Aldicarb 59.2± 0.3 (0.5) 57.1± 0.5 (0.8) 61.8± 0.3 (0.5) Metolcarb 67.8± 0.8 (1.2) 67.3± 0.3 (0.5) 76.7± 0.3 (0.4) Thiodicarb 66.6± 0.3 (0.5) 65.4± 0.4 (0.7) 74.6± 0.4 (0.5) Carbofuran 86.7± 0.6 (0.7) 86.1± 0.5 (0.6) 95.8± 0.6 (0.6) Carbaryl 90.3± 0.6 (0.6) 88.3± 0.4 (0.4) 99.9± 0.4 (0.4) Isoprocarb 77.1± 0.7 (0.9) 78.7± 0.3 (0.4) 87.0± 0.4 (0.4) Fenobucarb 81.1± 0.6 (0.8) 81.4± 0.3 (0.4) 92.2± 0.4 (0.5) Group B Aldicarb sulfone 63.3± 0.7 (1.0) 84.2± 0.5 (0.6) 95.9± 0.6 (0.7) Methomyl 67.8± 0.2 (0.3) 73.1± 0.4 (0.6) 75.1± 0.4 (0.5) Butocarboxin 66.7± 0.2 (0.4) 68.7± 0.5 (0.7) 64.9± 0.7 (1.1) 3-keto Carbofuran 52.6± 0.9 (1.7) 65.9± 0.3 (0.5) 67.4± 0.9 (1.4) Propoxur 80.9± 0.5 (0.6) 83.3± 0.6 (0.8) 85.0± 0.5 (0.6) Meobal 80.4± 0.7 (0.8) 80.4± 0.8 (1.0) 85.0± 0.9 (1.1) Macbal 90.5± 0.7 (0.8) 91.6± 0.8 (0.9) 94.7± 0.9 (1.0) Methiocarb 74.4± 0.4 (0.5) 80.3± 0.5 (0.6) 76.8± 0.4 (0.5) Promecarb 72.9± 0.3 (0.4) 76.6± 0.4 (0.6) 77.4± 0.4 (0.5)

b_{Recovery (%)}_{= 100% × (Level of carbamate pesticides in spiked sample level of carbamate. pesticides in blank sample) / Level of carbamate}

carbamate pesticides in a range higher than 65%. When 8 mL of acetone-dichloromethane was used as the elution solvent, only the recoveries of aldicarb and butocarboxin were less than 65%, which were 61.8% and 64.9%, respec-tively. Using 4 mL of acetone-dichloromethane as the elu-tion solvent, the recoveries under 65% were 31.3% and 57.1% for 3-OH carbofuran and aldicarb, respectively. But using 2 mL of acetone-dichloromethane the recoveries of four analytes were above 65%.

The results suggest that 8 mL of

n-hexane-dichloromethane was sufﬁcient for the amino cartridge clean up of tea samples in carbamate pesticides analysis. Evaluation the carbamate pesticides in tea sample

The developed protocol was practically applied for a dietary exposure assessment of carbamate pesticides in made-tea samples by our laboratory. This study was undertaken be-tween 2004 and 2005 as part of the tea pesticide residue monitoring program. We collected 4,133 made-tea sam-ples from tea processors and markets around almost all Taiwan area. Tea samples were analyzed by the present de-veloped multiresidue pesticides analysis method with op-timum conditions as described above. In total 19 carba-mate pesticides residues could be monitored for tea

sam-ples by the developed method. The results are expressed in Table 9, and the percentages of positive samples were rela-tively low. Only two kinds of carbamate pesticides residues, carbaryl and carbofuran, were detected in tea samples. Car-baryl was the most frequently detected carbamate pesti-cides in the tea samples, from 2004 to 2005, and the fre-quency was 0.086% to 0.384%, respectively. Carbofuran was only detected in 2005, the frequency was 0.043%. The detected residues level of carbaryl ranged from 0.01 mg

kg−1to 1.39 mg kg−1for samples between 2004 and 2005,

and the level of carbofuran was 0.01 mg kg−1 in 2005.

In Taiwan these two kinds of carbamate pesticides have been legally recommended to control insects on tea bushes. The present detected residue levels of carbaryl and car-bofuran were lower than their correspondent maximum residues levels (MRLs) regulated by the Taiwan govern-ment. The other carbamate pesticides residues were not de-tected from these samples. That may be due to the dissipa-tion of carbamate pesticides during the growth, processing

and roasting.[22] The surveyed result is similar to that of

our previous reports for tea fresh-leaf samples.[23]The data

also suggest that the residues of carbamate pesticides on ﬁeld-sprayed tea plants can be dissipated almost completely when good agriculture practice (GAP) is followed by tea farmers.

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Table 9. Levels of carbamate pesticides residues detected in made-tea samples from Taiwan during 2004 to 2005.

2004a ₂₀₀₅

No. of positive Concentration range No. of positive Concentration range

Pesticide samples (mg kg−1) samples (mg kg−1)

Aldicarb NDb ₀ _ND ₀ Carbaryl 7c_(0.384%)d _0.17–1.39 _{2 (0.086%)} _0.01–0.02 Carbendazim ND 0 ND 0 Carbofuran ND 0 1 (0.043%) 0.01 Carbosulfan ND 0 ND 0 Isoprocarb ND 0 ND 0 Macbal(XMC) ND 0 ND 0 Methiocarb ND 0 ND 0 Methomyl ND 0 ND 0 Metolcarb ND 0 ND 0 Thiodicarb ND 0 ND 0 Thiabendazole ND 0 ND 0 Propoxur ND 0 ND 0

a_{Surveyed samples in 2004 and 2005 year were 1820 and 2313, respectively.} b_{Not detected.}

c_{Number of detected.}

d_{Detection rate (%) in parenthesis.}

Conclusion

The work shows an efﬁcient HPLC-based multiresidue method with good extraction and SPE cartridge clean-up step for carbamate pesticide residues in tea samples. The amino cartridge for adsorbent was superior to other car-tridges in terms of the high extraction efﬁciency, and re-producibility. The method allows determination of 0.0005–

0.023 mg L−1levels of carbamate pesticide residues in tea

samples. Overall results indicate that the presented method has satisfactory reproducibility, recovery, and accuracy for nineteen carbamate pesticides analysis in tea samples. Re-sults of practical survey of carbamate pesticides residues in tea samples of Taiwan suggest that residue levels were rel-atively low, and posed low risk to consumer health as well.

Acknowledgments

We are especially grateful to the Council of Agriculture (Taiwan) providing ﬁnancing this project. The authors also wish to thank Yu Chu Liang, Hsiu Jung Hsieh, Pao Hua Chen, Li Hua Lin, Li Chen Lin, Su Chuan Hsieh, Zhen Wei Lin, Song Hui Chen and Chen Chang Chen for their skill-ful technical assistance. The authors also thank the Taiwan Agricultural Chemicals and Toxic Substances Research In-stitute of Dr. Hong Ping Li for supplying the analytical methods.

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