Investigation and application of liquid chromatography mass spectrometry in the analysis of polar, less volatile and thermal unstable organic pollutants in environmental and biological samples 4

Our focus is firstly on the thermal instability of carbamates in different extraction solvents under MAE conditions, our second focus is to compare the MAE efficiency for relatively more

4 Chapter Four

Microwave-assisted Extraction of Pesticides

4.1 MICROWAVE-ASSISTED EXTRACTION BEHAVIOR OF NON-POLAR AND POLAR POLLUTANTS IN SOIL WITH ANALYSIS BY HPLC

4.1.1 Introduction

As mentioned in the previous chapter, carbamate pesticides are gaining importance in the field of pest control because of their high efficiency as insecticides and nematicides, and their low bioaccumulation potentials However, since they are acetylcholinesterase inhibitors, they are considered hazardous to the environment and to human health [1-3] Evaluation and monitoring of trace levels of these compounds in soil are important because the widespread use of carbamates in agriculture leads to an increase in the presence of their residues in environmental matrices, especially soil

HPLC has been applied routinely in the analysis of carbamates due to their suitability for thermally labile and polar pesticides To obtain the cleanest samples and to avoid pesticide degradation during the extraction from soils, an adequate sample preparation technique with optimum extraction conditions is crucial prior to the final determination

As we discussed before, MAE has become a very attractive sample preparation technique

from solid matrices but also polar and ionic compounds [4] Moreover, simultaneous extraction of multiple samples is possible In the past few years, MAE has been successfully applied to the simultaneous extraction of toxic organic contaminants from different solid matrices, such as PAHs [5-14], PCBs [15-18], organochlorine pesticides [19-21], phenols [22-24], herbicides [25-27], triazines [28-31] and organomercury compounds [32-36] But hitherto, little work has been reported on the MAE of carbamates [37]

In this work, MAE was applied for the first time to study carbamates Our focus is firstly

on the thermal instability of carbamates in different extraction solvents under MAE conditions, our second focus is to compare the MAE efficiency for relatively more polar organic pollutants, such as the carbamates themselves (propoxur, methiocarb, propham, thiuram, chlopropham) and triazines (atrazine, simazine) with that of relatively non-polar substrates, such as PCBs (PCB1242, PCB1248) and PAHs (naphthalene, phenanthrene) Furthermore, we also pay particular attention to the optimization of the parameters that influence MAE efficiency, including the polarities of the solvents, extraction temperature, extraction pressure and heating duration

4.1.2 Experimental

4.1.2.1 Reagents and soil preparation

Analytical-grade PAHs (naphthalene and phenanthrene), PCB (1242 and 1248), triazine (atrazine 98% and simazine 99%), and carbamates (propoxur 99%, methiocarb 99%, propham 99.5%, thiuram 98%, chlorpropham 99.5%) were used in this study (See Chapter 2)

PCB-Three soil samples were collected from local sites (soil-1, soil-2 and soil-3, respectively) The preparation of blank soils, freshly spiked soils and aged spiked soils were described

in Chapter 2

4.1.2.2 MAE procedure and treatment of extracts

In this study, 30 ml extractant was added to the MAE extraction vessel, which contained 2.0 g of spiked soil sample Extraction was performed at 115°C with a heating time of 6 min for PAHs and PCBs, or 4 min for triazines at 80% power After the extraction, the vessels were cooled down to room temperature before they were opened Sample extracts were further clarified by centrifugation at 419 rad/s for 15 min to separate out the fine particulate The supernatant was then evaporated to dryness in a rotary evaporator

Finally, 1 ml of methanol was added to dissolve the residue 10 µl of the solution was directly analyzed by HPLC

In order to examine the thermal degradation behavior of carbamates, the following was performed: 2 ml of standard solution containing 2 ppm of propoxur, thiuram, propham, methiocarb and chlorpropham was evaporated to dryness and, then 30 ml of the appropriate extractant was added to dissolve the residue The solution was transferred to the extraction vessel (no soil inside) and MAE was performed at 95 °C for 6 min at 80% power After the extraction, the vessel was allowed to cool down to room temperature

before it was opened

4.1.2.3 HPLC measurements

For triazines: A Phenomenex (Torrance, CA, USA) ODS 250 × 4.6 mmI.D column was used Detection wavelength was 254 nm The mobile phase was methanol-water (65:35)

at a flow rate of 0.8 ml/min

For carbamates: A Phenomenex ODS 150 × 3.2 mmI.D column was used Detection wavelength was 225nm The mobile phase was acetonitrile-water (40:60) at a flow rate of 0.8 ml/min

PAH analysis was performed on a Waters (Milford, Massachusetts, USA) 600E HPLC

system equipped with a Waters 700 autosampler, a Waters 486 UV-VIS detector and

Millennium version 2.15 control software A Phenomenex ODS 250 × 4.6 mmI.D

column was used Detection wavelength was 254 nm The mobile phase was

acetonitrile-methanol-water (78:7:15) for the first 8 min, then the composition was changed to 100% acetonitrile over 25 min at a flow rate of 0.8 ml/min

4.1.3 Results and Discussion

4.1.3.1 Optimization of the solvents, microwave heating time and temperature

With MAE, temperature, extraction time and suitable solvents appear to be the major parameters affecting the extraction efficiency of the pollutants Employing information that is available from the literature and our own experience on the extraction of some pollutants from soil such as heating for 6 min at 115°C is sufficient to obtain high

considered because of their thermal instability Since the degradation temperature for

most of carbamates studied here is around 110°C, especially for propham with a degradation temperature of 100°C, heating up to only 95°C was applied to the five carbamates

When selecting the solvent, consideration should be given to its microwave-absorbing

properties and analyte solubility in it In order to enhance the absorption of microwave

energy, solvents with a high dielectric constant such as water, methanol and acetone are

preferably applied The temperatures of different solvents achievable with microwave

heating time under MAE conditions are studied and shown in Fig 4-1

ig 4-1 Temperatures of some solvents with heating time under MAE conditions

Hexane-acetone (1:4, v/v) Hexane-acetone (1:1, v/v) Dichloromethane

From Fig 4-1, it is clear that the larger the dielectric constant of the solvent, the more

.1.3.2 Thermal degradation of carbamates under MAE conditions in different

extraction solvent systems

he structures of the carbamates studied are shown in Fig 4-2

t extractants under MAE

rapid the heating is under microwave irradiation In some cases, however, some solvents with high dielectric constants make the concentration step laborious after extraction because of their inherent high boiling points They also have poor extraction selectivity due to polar co-extractives Thus, a mixture of hexane-acetone was selected as the ideal solvent for compounds of environmental significance As Fig 4-1 shows, hexane with a dielectric constant of 1.8 will not be heated whereas by mixing it with acetone, heating will take place within a few seconds Furthermore, due to the good solubility of the carbamates in this solvent mixture the formation of permanent dipoles assures absorption

of microwave radiation, which is considered to be favorable to accelerate the extraction Based on this consideration, three different ratios of hexane-acetone mixtures for MAE were applied in this study

4

T

The extent of thermal degradation of each carbamate in differen

conditions was determined in the absence of soil From Fig 4-3, it can be seen that thermal degradation of all five carbamates occurred in almost all solvents considered, especially thiuram and propham, which underwent degradation in all extractants, except methanol The other three carbamates (propoxur, methiocarb and chlorpropham) degraded more significantly in dichloromethane and hexane-acetone (4:1) than in

ranged from 10% to 100%, depending on the polarity of solvent Based on the results,

some very interesting conclusions can be drawn: (1) The thermal degradation of the five carbamates studied took place in varying degrees under microwave heating 6 min at

95°C (2) The percentage of thermal degradation of carbamates in more polar solvents, such as methanol, acetone and hexane-acetone (1:4, 1:1), was much less than in less polar solvents, such as dichloromethane and hexane-acetone (4:1) Evidence is presented in Fig 4-4, which shows a standard chromatogram and some typical chromatograms of mixtures of the five carbamates in various extractant solvents under the stated MAE conditions The figure shows that thermal degradation occurred seriously and was dependent on the polarity of the extractant The observation may be explained by the principle of “like dissolves like” during MAE Thus, polar analytes are more soluble in polar extractants than in less polar ones This is due to the protective effects of extractants against analytes Therefore, careful selection of extractants to address this issue must be considered in MAE Moreover, although the polarity of water is the strongest among the solvents used, complete degradation of the five carbamates occurred, which indicates that hydrolysis of the carbamates occurred seriously in this extractant under the applied MAE conditions

(CH3)2N C

S

SN(CH3)2

O NHCH 3 OCH(CH3)2

thiuram propoxur

O C NH

Fig 4-3 Plots of percentage of thermal degradation of five carbamates in different extraction solvents:

1) methanol; 2) acetone; 3) hexane-acetone (1:4, v/v); 4) hexane-acetone (1:1); 5) dichloromethane; 6) hexane-acetone (4:1); 7) water

5

4

1

(c) hexane-acetone (1:4)

Fig 4-4 Continued

a

(f) water

a

(e) dichloromethane

Fig 4-4 (a) Chromatography of standard mixture of five carbamates; (b-f) chromatography

obtained after simulated MAE (6 min heating at 95ºC, 80% microwave power) in the

various extractants

Peak identities: 1) propoxur; 2) thiuram; 3) propham; 4) methiocarb; 5) chlorpropham;

a) Unknown

4.1.3.3 The extraction behavior of carbamates spiked in soil

Since propham and thiuram were found to degrade seriously, propoxur, methiocarb and

chlorpropham were selected to investigate the recoveries of carbamates spiked in soil

under the applied MAE conditions The results are shown in Table 4-1 and Fig 4-5 The

recoveries of carbamates (propoxur, methiocarb and chlorpropham) were low in all

extractants used except in methanol Furthermore, the recoveries increased with

increasing extractant polarity For example, the recovery of propoxur increased from 20%

to > 65% when methanol instead of hexane-acetone (4:1) was used as extractant The

principle “like dissolves like” is still applicable to explain these results As carbamates are polar pesticides, their solubility in polar extractants is much higher than in less polar extractants In addition, the dipole-dipole interaction between the analytes and polar exractants is higher than that with the less polar extractants At the same time, polar extractants absorb microwave energy with high efficiency As a result the efficiency of microwave extraction will be higher in polar extractants than less polar extractants What

is surprising is that in less polar extractants, unusually high recoveries of the targets were observed in soil, considering the high thermal degradation in the same solvents without the matrix (previous section) For example, the recovery of propoxur from soil was around 29% when dichloromethane was used as extractant, whereas thermal degradation (no soil involved) was nearly 100% in the same extractant This may be due to the protective effects of the soil matrix afforded to the analytes The recoveries from soil were nearly zero when water was used as extractant, which appears to confirm that carbamates hydrolyzed very significantly in water under the MAE conditions used

Table 4-1 Effects of using different extraction solvents on recoveries of carbamates

spikeda in soil under MAE conditions

Recoveries (%) ± RSD (%) n=6 Extraction solvents

Propoxur Methicarb Chlorpropham

propoxur methicarb chlorpropham

Fig 4-5 Variation of the carbamates from spiked soil with different extractants after heating

6 min at 95 ºC

Extraction solvents: 1) methanol; 2) hexane-acetone (1:4, v/v); 3) hexane-acetone

(1:1); 4) dichloromethane; 5) hexane-acetone (4:1); 6) water

4.1.3.4 MAE of different pollutants from spiked soil

Although there are some studies on the extraction behavior of a certain pollutant extracted from soil by using different extractants under MAE conditions, hitherto, there has been little comparison of extraction behaviors of different pollutants in soil extracted simultaneously in the same extractant by using MAE This was one aspect of our present work The MAE recoveries of some analytes from soil-3 with different solvents are shown in Table 4-2

Table 4-2 The MAE recoveries of different analytes in spiked soila with different solvents

Recovery (%) ± RSD (%) n = 4 Analytes

Methanol Hexane-acetone (1:1) Dichloromethane

Naphthalene 64.4±7.5 79.0±4.1 78.3± 3.3 PAHs

a Soil- 3, collected from campus of National University of Singapore

- No recovery

The data indicate that in each tested solvent high recoveries of PAHs, PCBs and triazines

were obtained and ranged from 70% to 99% with excellent reproducibility, except PAHs

extracted in methanol Although methanol can cause dipole-induced dipole interactions

with the numerous π-electrons on the PAHs because of its permanent dipole, the poor

solubility of PAHs in methanol results in lower recoveries The low recoveries of

carbamates are partially due to their thermal degradation, as determined previously

Fig 4-6 shows the dependence of recoveries of some classes of analytes on different

extractants

0 20 40 60 80 100

Solvent

Fig.4-6 Recoveries of PAHs, PCBs and triazines from spiked soil with different solvents after

microwave heating 6 min at 115 ºC

Extraction solvents: 1) dichloromethane; 2) hexane-acetone (1:1, v/v); 3) methanol

An interesting result observed from Fig 4-6 is that in the same solvent, the recovery increases in going from PAHs to PCBs, and then to triazines (i.e, increasing polarity of analyte) The recovery is probably affected by the interaction between the analyte and the matrix The higher the polarity of the analyte, the larger its dipole Thus it is more easily reoriented and finally desorbed from the soil matrix in the applied microwave field Moreover, when the solvent is rapidly heated in a reproducible way under microwave irradiation, the selective interaction with polar molecules allows local heating and an improvement of extraction efficiency From Fig 4-6, it is also observed that the difference of recoveries is reduced with decreasing polarities of extractants For example, the recovery difference between PCBs and triazines when methanol is used as extractant (14.7%) is larger than that when dichloromethane is used as extractant (2.35%) This suggests that the analyte solubility in the solvent is another important factor on recovery Based on the principle of “like dissolves like”, triazines are more easily dissolved in methanol than in dichloromethane Thus there is a reduction in recovery when dichloromethane is used instead of methanol However, the less polar PCBs are more preferentially dissolved in dichloromethane; therefore, the recovery is slightly better when this solvent is used

4.1.3.5 Influence of matrix, moisture, aging of spiked soil on recovery of tested

pollutants

The effects of matrix, moisture and aged spiked soil on recovery of analytes were investigated and the results are shown in Tables 4-3, Table 4-4 and Table 4-5, respectively

The recovery of propoxur (a representative carbamate) in different spiked soils is shown

in Table 4-3 The table indicates that the effect of different soil matrices on the recovery

of propoxur was not apparent Recoveries were around 60% in all tested soils when methanol was used as the extractant

Table 4-3 Influence of different matrices on recovery of carbamate (Propoxur)

Recovery (%) ± RSD (%) n = 6 Extractant

Soil-1a Soil-2a Soil-3a Soil-4a Soil-5aMethanol 65.4±5.6 64.9±5.6 66.1±6.2 68.1±6.2 65.8±5.2

In order to study the influence of soil moisture on the extraction efficiency, external water was added accurately into the soil in the range of 0% to 20% (w/w) Table 4-4 shows that soil moisture had a positive effect on the recovery of PAHs, PCBs and triazines For example, the recoveries of triazines progressively improved with an increasing degree of moisture in the tested soil This can be explained by the high solubility of triazines in water and the strong ability of water to absorb microwave energy Arising from this observation, water is a safe and environmentally friendly solvent, and should be considered as a viable MAE solvent to extract triazines from soil instead of organic solvents [31] As for PAHs (naphthalene and phenanthrene) and PCBs (PCB1242, PCB1248), suitable soil moisture (i.e., 10%) enhances their recoveries in soil This may be due to the fact that the water present in the matrix can allow important local heating and thus could favour the expansion of the pores and liberate the analytes into the solvent This could accelerate the extraction [9] When the amount of water is significant (i.e., 20%) recovery decreases slightly There are certainly problems of miscibility with the organic solvent used for extraction In this case, the water may act as a barrier and hinder the transfer of the analytes from the matrix to the solvent Although water is helpful for the improvement of the extraction efficiency, as previously suggested, hydrolysis may be responsible for the low recoveries of carbamates (propoxur, chlorpropham) due to the presence of water in the soil

To examine the effect of time-aged soil on the recoveries of carbamates (propoxur, methiocarb and chloropropham) by MAE, freshly spiked soil and spiked soil aged for 60

days were used for investigation As shown in Table 4-5, there was no marked effect of this parameter on carbamate recoveries

Table 4-5 Recoveries of carbamates from aged soil-3

Recovery (%)±RSD (%) Extractant Analyte

Fresh Soil Aged Soil (60 d)

Methiocarb 80.0±5.6 78.1±7.1 Methanol

Chlorpropham 65.7±5.1 62.1±5.9

Methiocarb 29.4±2.9 27.8±4.0 Hexane-acetone (1:4)

Chlorpropham 35.0±3.9 33.7±5.1

Methiocarb 18.4±4.0 20.0±2.9 Hexane-acetone (1:1)

Chlorpropham 24.0±3.1 19.3±5.5

4.1.4 Conclusions

For the first time, the thermal degradation of carbamates (propoxur, thiuram, propham, methiocarb and chlorpropham) was studied under MAE conditions Evidence was presented that the thermal degradation occurred during extraction and was largely determined by the polarity of the extractant The recoveries of carbamates from soil were also dependent on the polarity of the extractant, and the protection afforded by the soil to the analytes was apparent when a less polar solvent was used as extractant Hydrolysis of

Table 4-4 Influence of moisture of soil-3 on recoveries of PAHs, PCBs, Triazines and Carbamates

carbamates occurred in water under the applied MAE conditions and led to low recoveries when soil moisture was present The behavior of breakdown and degradation patterns of carbamates will be studied in a future work In a comparison of extraction behaviour, it was observed that recoveries of the pollutants from soil in the extractants considered (methanol, hexane-acetone (1:1), dichloromethane) were dependent on the relative polarities of the class of analytes: recoveries increase in the order of increasing polarities (Triazines > PCBs > PAHs)

4.2 OPTIMIZATION OF MICROWAVE-ASSISTED EXTRACTION AND SUPERCRITICAL FLUID EXTRACTION OF CARBAMATE PESTICIDES

IN SOIL BY EXPERIMENTAL DESIGN

4.2.1 Introduction

In our previous work, MAE was applied for the first time to study carbamates [38] We found in the study, although MAE has been successfully applied to extract the target PAHs, PCBs and triazine pesticides from soil, significant thermal degradation of the target carbamate pesticides (propoxur, propham, methiocarb and chlorpropham) occurred and subsequently low recoveries were obtained from soil under the applied extraction conditions In order to improve the extraction recovery of carbamate pesticides from soil, the systematic optimization of the MAE procedure is crucial

Besides MAE, SFE is another efficient environmentally friendly technique for the rapid analytical-scale extraction from solid matrices SFE of environmental contaminants has

been reported from various solid matrices The unique properties of supercritical fluids (SCFs) have made SFE a practical alternative to traditional liquid solvent extraction techniques [39, 40] For example, supercritical CO2, which has been used for extraction is

a nontoxic, nonflammable, and generally considered to be a comparatively environmentally-friendly solvent In addition, because CO2 has a very moderate critical temperature (31.3°C) and chemical inertness, SFE is specially recommended for thermally labile compounds such as carbamate pesticides Surprisingly, there have been only a few applications reported using SFE for the analysis of carbamate pesticides Recently, Jeong and Chesney used SFE to extract three N-methylcarbamate pesticides (carbaryl, aldicarb, and carbofuran) from spiked filter paper and silica gel matrices Recoveries from such spiked matrices ranged between 13% and 100%, depending on the analyte and the matrix components [41] The use of statistical techniques on SFE optimization has provided the analyst with a quick, relatively accurate optimization technique that would otherwise have been costly in both time and materials Except for Stuart et al who optimized SFE conditions by using ANOVA techniques to successfully extract three carbamates (carbaryl, aldicarb and pirimicarb) from soil [42], there have been few investigations focusing on optimization of SFE parameters for the extraction of this class of pesticides Considering the above, it is of interest to use SFE for extracting carbamate pesticides from solid matrix and to develop a systematic strategy to optimize the process

In order to obtain optimum MAE and SFE conditions for carbamate extractions, we have