Matrix solid-phase dispersion extraction versus solid-phase extraction in the analysis of combined residues of hexachlorocyclohexane isomers in plant matrices

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Journal of Chromatography A, 1176 (2007) 43–47

Matrix solid-phase dispersion extraction versus solid-phase extraction in the analysis of combined residues of hexachlorocyclohexane isomers in plant matrices P.C. Abhilash, Sarah Jamil, Nandita Singh ∗ Eco-Auditing Group, National Botanical Research Institute, Council of Scientific and Industrial Research, Rana Pratap Marg, Lucknow 226 001, India Received 2 October 2007; received in revised form 31 October 2007; accepted 2 November 2007

Abstract This paper describes a method based on matrix solid-phase dispersion (MSPD) to determine the presence of combined residues of hexachlorocyclohexane (HCH) isomers (␣-, ␤-, ␥- and ␦-) in various plant matrices including vegetables, fruits, leaves, grains and roots, by gas chromatography with 63 Ni electron-capture detection. The MSPD method consists of sample homogenization, cellular disruption, exhaustive extraction, fractionation and clean up by simple processes in which a small amount of sample (5 g) was blended with Florisil and the mixture passed into a small chromatographic column and eluted with 10 ml of n-hexane–ethyl acetate solvent mixture (70:30; v/v) and repeated with another 10 ml of the same solvent mixture. A comparison with classical solid-phase extraction (SPE) showed MSPD to be efficient, fast, simple and easy to perform. The detection limit of various HCH isomers was found to be in the range of 2.15–5.68 ng and method detection limit varied from 0.465 to 1.136 ng g−1 . Mean recoveries were found in the range of 91–98%. Till date, there are no official methods or standards by Central Pollution Control Board or Bureau of Indian standards that take into account India’s real life conditions in the analysis of pesticide residues in plant matrices and the MSPD method described herein has proved to be a feasible one for the analysis of combined residues of HCH isomers in various plant materials. © 2007 Elsevier B.V. All rights reserved. Keywords: Matrix solid-phase dispersion; Solid-phase extraction; Hexachlorocycloheaxane; Plant matrices

1. Introduction Organochlorine pesticide residue is an important component of the chemical pollutants found in all environmental media [1]. They are potentially hazardous to living systems because of their inclination to bioaccumulate in the lipid component of biological species and their resistance to degradation [2]. One of the avenues for human exposure to these compounds is through consumption of agricultural products contaminated with pesticides [3]. Hexachlorocyclohexane (HCH) is one of the highly toxic and persistent organochlorine pesticides that have caused serious environmental problems since it began to be produced at the beginning of the 1940s [4]. Eight isomers of ␣, ␤, ␥, ␦, ␧, ␨, ␩, and ␪ exist, all with same molecular formula, but which are differentiated by variations in the axial equatorial positions of

∗

Corresponding author. Tel.: +91 522 2205831/35x302; fax: +91 522 2205847. E-mail address: [email protected] (N. Singh). 0021-9673/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2007.11.005

the molecules of chlorine around a ring of six carbons [5]. Of these isomers, only ␥-HCH (also known as lindane) possesses insecticidal activity [5,6]. It is refined from technical grade HCH (13–26% of ␥-HCH) resulting in a significant quantity of waste HCH consisting of primarily ␣- and ␤-isomers, which in addition to lindane becomes an environmental liability due to its highly persistent nature and toxicity [5]. Concern over pesticide residues in plant materials (i.e., fruit, vegetables, cereals, medicinal plants, leafy vegetables, oils, etc.) has led to the development of many methods for monitoring these compounds [7]. Modern trends in analytical chemistry are towards the simplification and miniaturization of sample preparation, as well as the minimization of organic solvents used. In view of this aspect, several novel extraction techniques are being developed in order to reduce the analysis step, increase the sample throughput and to improve the quality and sensitivity of analytical methods [8]. Analysis of pesticide residues in plant samples are commonly carried out by gas chromatography (GC) with electron-capture detection (ECD), nitrogen–phosphorus detection (NPD), or

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coupled with mass spectrometry (MS) [9–11]. In these methods pesticides are mainly extracted from plant matrices using conventional techniques such as liquid–liquid extraction or soxhelt extraction [9–13]. However, these conventional methodologies for pesticide extraction of plant matrices are time consuming and require larger samples and greater volumes of hazardous solvents [14]. Solid-phase extraction (SPE) using ready-made cartridges under vacuum or pressurized condition has proven very effective for cleaning, extracting and concentrating pesticides in plant samples [15]. Supercritical fluid extraction (SFE), microwave-assisted extraction (MAE), solid-phase microextraction (SPME), and stir-bar sorption extraction (SBSE) are other techniques employed very recently with successful results [16–20]. However, the use of most of these latest techniques requires special system. Not all Indian laboratories are equipped for these latest procedures and certainly these costly techniques are not affordable for routine analysis of large number of samples. To overcome analytical cost and ensure quality control in analytical procedures, multi residue techniques such as MSPD (matrix solid-phase dispersion) have been successfully employed [21–23]. The MSPD techniques include sample homogenization, cellular disruption, exhaustive extraction, fractionation and clean up by a simple process in which a small amount of sample (0.1–5 g) is blended with the selected solidphase (such as C18, C8, C2, silica, Florisil or alumina) and the mixture packed in to a small chromatographic column [22–25]. Gravitational elution with an appropriate solvent in this MSPD column usually provides clean extracts. When necessary, further purification can be performed using a ‘co-column’ for clean up, coupled to the first one containing MSPD material. Matrix solidphase dispersion has been widely used in the last years for the isolation of a wide range of drugs, pesticides, naturally occurring constituents and other compounds from different complex plant and animal tissues providing, in many cases, equivalent or superior results to older official methods conducted by more classical extraction and/or SPE techniques [26–30]. However, MSPD in pesticides analysis has been reported almost exclusively to food analysis [9,31–34]. The aim of this work was to study the suitability of MSPD as an alternative method to conventional SPE for the analysis of combined residues of HCH isomers (␣, ␤, ␥, ␦) in multiple plant matrices (root, stem, leaf, fruit, bulb, underground stem, etc.). The MSPD method was optimized and compared to the classical SPE method for the determination of HCH isomers in plant samples. The analytical results confirm that the described MSPD method is simple and gives good recoveries for the fortified plant matrices, giving sensitivity limits that are well below those set by the international regulations (WHO and Codex alimanetarious) for pesticide residues. 2. Experimental 2.1. Solid-phases for MSPD Graphitized carbon bulk material ENVI-Carb 120–400 mesh (surface area 100 m2 g−1 ), C18, C8, C2 and Florisil (60–100

mesh) were obtained from Supelco (Bellefonte, PA, USA) and neutral alumina (60–230 mesh, activity I), anhydrous Na2 SO4 , NaCl and MgSO4 from Merck (Darmstadt, Germany). 2.2. Reagents Certified analytical standards of pesticides, purity >99%, were obtained from Supelco. Acetone, ethyl acetate, n-hexane and cyclohexane (gas chromatography grade) were purchased from Merck. Individual stock solutions (1.0 mg ml−1 ) of each isomer were prepared in cyclohexane and stored in a freezer at −20 ◦ C. 2.3. Plant matrices Leaf, stem and root samples of Withania somnifera (L.) and Ocimum sanctum Linn. were obtained from Botanic garden of National Botanical Research Institute (NBRI). Vegetable and fruit samples were purchased from Narahi vegetable market, Lucknow and wheat and legume samples were purchased from near by grocery store. Extracts from all the matrices were pre-checked to confirm the absence of any pesticide before fortification and sample processing. Table 1 shows the details of matrices tested for this method validation. For the recovery studies, blank samples were fortified before the extraction by addition of mixed standard solution of HCHs isomers to give 0.001, 0.05 and 0.5 mg kg−1 of each compound. They were then subjected to the procedures described in Sections 2.5 and 2.6. 2.4. Chromatographic procedure Analyses were carried out in a Perkin-Elmer (Norwalk, CT, USA) Clarus 500 series gas chromatograph fitted with a 63 Ni electron-capture detector. A split/splitless injector was used in a splitless mode, applying an injection volume of 1 ␮l. For separation, a 35% diphenyl and 65% dimethyl polysiloxane capillary column (30 m × 0.32 mm i.d., 0.5 ␮m) Elite 35 (Perkin-Elmer) was employed. The operational conditions were: injector and detector temperature, 250 and 300 ◦ C, respectively, the oven Table 1 Matrices selected in the method validation Commodity group

Plant species/matrices/parts tested

Citrus fruit Pome fruit Berries and small fruits Various fruits Root/tuber vegetables Bulb vegetables Fruiting vegetable Brassica vegetables Leafy vegetables Medicinal leaf Stem Grains/cereals Pulses

Lemon, orange Apple Grapes Bananas, mangoes Carrot, potato Onion Cucumber, tomato Cabbage, cauliflower Spinach Withania leaf, ocimum leaf Withania stem, ocimum stem Wheat Green gram

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temperature was: 100 ◦ C, held for 2 min; 15 ◦ C min−1 to 300 ◦ C, held for 5 min. Nitrogen (N2 ) was used as a carrier gas at a programmed flow of 1.5 ml min−1 . 2.5. SPE method The SPE method was adapted from US Environmental Protection Agency (EPA) Method 8081A [35]. 2.5.1. Extraction and clean up procedure 2.5.1.1. Leaf, stem and root samples. Plant samples of 10 g were crushed with a 20-ml of a suspension of light petroleum:acetone (4:1, v/v) in a pestle and mortar. The extract was then homogenized in a vortex mixer with 3 g anhydrous Na2 SO4 for 5 min. The extraction process was followed by a clean up by SPE with Florisil. Glass column (30 cm × 1.5 cm i.d.) were packed from the bottom with glass wool plug/cotton and 5 g of Sigma–Aldrich branded Florisil (60–100 mesh size) with a top layer of 2 g anhydrous Na2 SO4 . Samples were eluted with 35 ml of same solvent mixture (light petroleum:acetone 4:1, v/v), concentrated by using a rotary evaporator and then reconstituted in 1 ml hexane and kept in 4 ◦ C for final analysis. 2.5.1.2. Bulb, vegetables and fruit sample. Ten grams of chopped samples were homogenized with 50 ml of acetonitrile for 30 min at a higher speed of 5000 rpm. 2 g of NaCl and Na2 SO4 was added to this and homogenized for five more minutes. The extract was eluted through a Florisil column (same as above) with 50 ml of acetonitrile:toluene (3:1, v/v). The extract was then concentrated by a rotary evaporator and finally reconstituted in 2 ml toluene. 2.6. Matrix solid-phase dispersion (MSPD) method 2.6.1. Extraction of plant matrices Leaf, stem, root, grain and legume samples were dried at 35 ◦ C for 24 h, powdered, sieved (1–2 mm) and stored at 4 ◦ C for posterior analysis. Fruits and vegetables were chopped and blended in a Polytron mixer PT 6100 (Kinematica, Luzern, Switzerland) for 3 min at 3000 rpm in order to achieve representative samples. Five grams of the homogenized samples were weighed directly and used for the extraction purposes. 2.6.2. Dispersion method Five grams of plant matrices were gently ground with 0.5 g Florisil (deactivated with 3% acetone) in a glass mortar for 5 min and 1 g MgSO4 and 0.5 g NaCl was added to this mixture and ground firmly for five more minutes. This mixture was transferred into a glass column filled with neutral alumina deactivated with 3% acetone (2 g) and anhydrous sodium sulfate (0.5 g). A mixture of n-hexane–ethyl acetate solvent 70:30 (v/v, 10 ml) was utilized for elution in the column and repeated with another 10 ml of same solvent mixture. The resulting extract was concentrated, resuspended with n-hexane (1 ml) and kept in 4 ◦ C for posterior analysis.

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2.6.3. Sample clean up If necessary, sample clean up was performed using an additional column filled with neutral alumina deactivated with 3% acetone (2 g) and anhydrous sodium sulfate (0.5 g). The column was then eluted with a mixture of n-hexane–ethyl acetate solvent 70:30 (v/v, 10 ml). 2.7. Matrix effect Matrix effect expressed as the signal from the pesticides in different plant matrix compared to the signal in solvent was tested in all matrices. To an aliquot of blank extract in cyclohexane a HCH mixture, 5–10% of the extract volume was added to a final concentration of 0.001, 0.5 and 0.5 mg kg−1 of selected crops. The GC-ECD signal of the standard additions was compared to the signal of standard in cyclohexane. 2.8. Method validation To confirm that the MSPD method is suitable for its intended use, a validation process was carried out by establishing the basic analytical requirements of the performance to be appropriate for quantitative determination of HCH isomers in various plant samples. Precision, linear dynamic range and both instrumental and method detection limits were evaluated for the analytical approach developed. Individual stock solutions (1.0 mg ml−1 ) of each isomer were prepared in cyclohexane and stored in a freezer at −20 ◦ C. Mixed working standard solutions in cyclohexane at concentration of 0.001–1 mg ml−1 were prepared by diluting the stock solutions with cyclohexane and stored at 4 ◦ C in the dark. Matrix matched standard solutions were prepared at the same concentrations as that of the calibration solutions by adding appropriate amounts of standard solutions to blank matrix extracts. The instrumental limit of detection (LOD) was calculated based on the signal response of the lowest concentration entering the column that is 3σ above the mean blank signal (where σ is the standard deviation of the blank signal). Precision was evaluated in terms of repeatability at different days using same matrix and fortification levels. The method detection limit (MDL) was estimated from the LOD multiplied by the final volume and divided by the sample mass and injected volume (MDL = (LOD × final volume)/(sample mass × injected volume)). 3. Results and discussion The proposed method is meant to be a multi-residue method for HCH isomers in many different matrices. In multiresidue monitoring of multiple matrices the most important issues are the selection and optimization of the method, selectivity and sensitivity of the method, confirmation of positives, accuracy of quantitation, timely analysis and cost in resources. The SPE was adapted and employed as the referential methodology through out this study. The alternative MSPD-based method was optimized and compared with SPE method.

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3.1. Optimization of MSPD method The efficiency of MSPD depends on the type and quantity of solid-phases and sorbent, sample volume elutropic strength and the volume of the elution solvent. In the initial experiments several solid-phases were tested for extraction of different HCH isomers from plant matrices. Among the solid-phases tested were graphitized carbon black (Envi-carb), C18, C8, C2, silica, Florisil or alumina. The best results, representing a compromise between good recoveries for different isomers, were obtained for Florisil. The use of Florisil for microdispersion of plant matrices produced higher recoveries than those obtained with non-polar solid phases. Moreover, Florisil produced the cleanest chromatographic profiles with lower baselines than other solidphases, a fact that was attributed to the preferential adsorption of polar sample components, interfering with compounds such as pigments and chlorophylls on the Florisil surface. For further optimization the sample volume (5 g), sorbent mass (0.5, 1 and 2 g), elution solvent strength (n-hexane–ethyl acetate; 70:30, v/v), and elution method (gravity or vacuum) were varied to assess optimal conditions. The use of a suitable solvent mixture led to the successful and selective elution of the analytes from the extraction column, retaining a large number of matrix co-extracts. In this case, 10 ml of the nhexane–ethyl acetate mixture (70:30, v/v) proved to be the most suitable eluting condition to reach the highest recoveries. All the MSPD elution was conducted by gravity flow. With this MSPD extraction procedure, the extracts were generally clear enough to allow for direct chromatographic determination, but higher reproducabilites were achieved when an additional clean up was performed. The use of neutral alumina for extract clean up was optimized (2 g) and this material was utilized in the same column with the MSPD material for clean up (co-column). 3.2. Method validation 3.2.1. Precision, linear ranges, instrumental and method detection limit, matrix effect In order to evaluate not only the extraction efficiency of the proposed method but also the precision, 12 extractions of different matrices (lemon, carrot, spinach, green gram) were carried out on 12 consecutive days. The precision for different isomers,

Table 2 Comparative precision (RSD, %), instrumental limit of detection (LOD) and method detection limits (MDL) of MSPD and SPE methods Isomer

␣-HCH ␤-HCH ␥-HCH ␦-HCH

MDL (ng g−1 )

Precision RSD (%)

LOD (ng)

MSPD

SPE

MSPD

SPE

MSPD

SPE

7.05 6.95 5.40 9.85

8.11 7.25 6.73 10.45

2.15 2.85 3.45 5.68

2.91 2.54 3.86 6.15

0.465 0.570 0.690 1.136

0.498 0.504 0.770 1.230

expressed as relative standard deviation, is shown in Table 2. It ranged from 5.40% to 9.85% for various HCH isomers using MSPD method. The calibration curves were prepared using standard solutions of the HCH isomers in chromatographic grade cyclohexane. The response of the detector for HCHs was linear in the studied range of 0.001–1 mg kg−1 with correlation coefficients of 0.9978–0.9999. The detection limit of various HCH isomers was found in the range of 2.15–5.68 ng and method detection limit was varied from 0.465 to 1.136 ng g−1 . Matrix effect, expressed as the signal from the isomers in matrix compound to the signal in solvent was tested in all matrices. In general, the measured matrix effect is quite small, with a mean value of 94% and a relative standard deviation of 8%. 3.3. Comparison of the proposed MSPD method with the SPE method The extraction efficiency of MSPD was also compared with that of the SPE method (modified from EPA method 6081A). Student’s t-test was used to statistically compare the recovery and repeatability data of the two methods. This analysis revealed no significant difference between the mean values at the confidence level of 95%. The overall range of recovery for various HCH isomers from 20 different commodities at 3 different fortification level (0.001, 0.5 and 0.5 mg kg−1 ) was 91–98% for MSPD and 90–95% for SPE method, respectively. The efficiency provided by MSPD was similar or even better than that obtained by the SPE method. Beside its efficiency, the MSPD method also requires the use of smaller amounts of reagents and less time. The total solvent volume utilized by the MSPD method for each assay was approximately 20 ml, the solid-phase materials

Table 3 Mean HCH isomers (±S.E.) in real plant samples (mg kg−1a ) Plant

Matrix

␣-HCH

␤-HCH

Lantana camara L.

Root Leaf

1.85 ± 0.015 c 0.47 ± 0.045 b

6.38 ± 0.370 g 3.39 ± 0.095 de

4.20 ± 0.300 f 1.93 ± 0.045 c

1.00 ± 0.010 b
Erianthus munja

Root Stem Leaf

1.30 ± 0.045 bc 1.96 ± 0.120 c
2.91 ± 0.040 d 2.74 ± 0.045 d 2.10 ± 0.110 d

1.10 ± 0.055 b 0.55 ± 0.030 b 0.38 ± 0.055 ab

0.075 ± 0.451 a 0.11 ± 0.046 a
Withania somnifera (L.) Dunal

Root Stem Leaf Fruit

0.87 ± 0.020 b 3.24 ± 0.015 e 1.42 ± 0.030 bc
3.47 ± 0.020 e 8.39 ± 0.020 j 4.54 ± 0.030 f
a

␥-HCH

1.87 3.00 4.08 0.084

± ± ± ±

␦-HCH

0.015 c 0.010 de 0.020 f 0.025 a

0.99 ± 0.005 b 1.28 ± 0.041 bc 0.33 ± 0.020 ab
Means (n = 3) with different letters indicate significant differences at p < 0.05 level (analysis of variance (ANOVA)). S.E.: standard error; LOD: limit of detection.

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4.5 g and the time 40 min, i.e., respectively 1/3 to 1/5, 1/2 to 1/3 and 1/2 of those consumed in the SPE procedure. Furthermore, the MSPD procedure does not require sophisticated instruments or materials, and is an interesting analytical alternative for the majority of pesticide monitoring laboratories in India. 3.4. Real sample analysis The proposed MSPD method was applied to the analysis of real plant matrices collected from the campus and near by areas of a lindane producing factory located at Chinhat Small Scale industrial area, Lucknow. The lindane plant was established in 1992 with an installed capacity of lindane production of 25 metric tonnes per month (300 metric tonnes per annum). The industry has also in house formulation facility for producing various lindane formulations; lindane EC, carbaryl lindane 4.4 granules, lindane 6% granules, lindane 6.5 WDP, lindane 1.3%. It can be observed that HCH isomers were present in almost all samples and the concentration of various HCH isomers in the plant matrices analyzed varied between 0.075 and 8.39 mg kg−1 (Table 3). 4. Conclusions The results of the study point out that the proposed method of extraction by MSPD provides a rapid and sensitive procedure for the simultaneous determination of HCH isomers in plant matrices. The method is simple and with a low solvent consumption, reducing the risk for human health and environment. Considering some aspects of the Indian condition (i.e. HCH and lindane commonly used and possibly higher residue levels found in “real samples”, laboratory infrastructure, etc.) the MSPD-based method described herein was developed as an alternative to the SPE method. This method provided analytical results equivalent to the classical SPE method adopted from the EPA, with the advantages of being cheaper, simpler and faster. Satisfactory results were obtained in the routine analysis of real samples, confirming the reliability and efficacy of this method for the analysis of HCH residues in vegetation samples. Therefore, this proposed procedure may be useful as a screening protocol to identify pesticides in various plant matrices by industrial, pharmaceutical and official regulatory laboratories. Acknowledgments The authors wish to thank Director, NBRI for providing necessary facilities and his continuous encouragements. The authors gratefully acknowledge that the present work is a partial output of CSIR—Net Work Project SMM-005. One of the authors (PCA) thankfully acknowledges the University Grants Commission, Government of India for Doctoral Fellowship (UGC-JRF-SRF).

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