Development of an HPLC method for determination of pentachloronitrobenzene, hexachlorobenzene and their possible metabolites
© Khan et al; licensee BioMed Central Ltd. 2011
Received: 24 December 2010
Accepted: 23 November 2011
Published: 23 November 2011
Pentachloronitrobenzene (PCNB) and hexachlorobenzene (HCB) are highly toxic and widespread in every environmental compartment. Some of metabolic products such as amino/nitro containing chlorinated aromatic compounds can be determined by gas chromatography coupled with electron capture detector (GC-ECD). However, it is difficult to identify some of chlorophenolic and chloroquinolic intermediates produced from PCNB and HCB by the above mentioned technique. Therefore, for analysis of these compounds and their metabolites, we have developed a high performance liquid chromatography (HPLC) based method.
The extraction of PCNB and HCB from soil and minimal salt medium was carried out with ethyl acetate and hexane respectively with good recoveries (98% for PCNB and 97% for HCB). The validation of the proposed extraction and HPLC method was done by analysis of PCNB and HCB biodegradation and their metabolites identification from anaerobic enriched soil samples.
A rapid, sensitive and simple HPLC based analytical method was developed for the analysis of PCNB, HCB and their possible intermediates.
Farmers use pesticide (herbicides, insecticides, fungicides, molluscicides, rodenticides, acaricides and nematicides) for the protection of crop . Chlorinated aromatic compounds (CACs) have been widely used in industrial, agricultural and domestic products such as pesticides, soil fumigants, disinfectants, toilet deodorants, solvents, and also used as precursors for the production of dyes [2, 3].
As a result of worldwide extensive use, CACs are widespread and released as environmental pollutants in coastal marine sediments, freshwater lake sediments, sewage sludge, waste water, groundwater, rivers and estuaries, and soils [4–6]. Due to their persistence, toxicity, and bioaccumulation potential, CACs are subject to regulation in most of the developed countries . The main environmental impact of pesticide is caused by their mobilization and transport from agriculture fields to pollute soils, sediment, water and also percolating through the soil and reaching the ground water [8–11].
Pentachloronitrobenzene (PCNB) is an important group of organochlorine fungicide which has been widely used either for seed dressing or for soil treatment to control a wide range of phytopathogenic fungi in crops [12, 13]. Hexachlorobenzene (HCB) is also used as a fungicide and is a side product of industrial emission related to the manufacture of chlorinated solvents and pesticides [14–16]. Both organochlorine fungicide (PCNB and HCB) are widespread and found in every environmental compartment i.e. soil, water, and air [17, 18]. Lipophilic nature of these compounds results in their bioaccumulation at different trophic levels via the food chain, thereby increasing risk to different living beings [19, 20]. The accumulation of organochlorine pesticide is due to their resistance to both biotic and abiotic degradation in environmental condition . Public concern has prompted the need for removal of these contaminants from the environment. Therefore, there has been an increase in effort to remove PCNB, HCB and their possible intermediates from the environment either by abiological or biological means during the past decades. The biotransformation products of PCNB include pentachloroaniline (PeCA), tetrachloroaniline (TeCA), trichloroaniline (TCA), dichloroaniline (DCA), chloroaniline (CA), pentachlorophenol (PCP), pentachlorothiophenol, pentachlorothioanisole and pentachloromethyl-phenyl sulfoxide [22–25]. The biotransformation products of HCB are pentachlorobenzene (PeCB), tetrachlorobenzene (TeCB), trichlorobenzene (TCB), dichlorobenzene (DCB), pentachlorophenol (PCP), tetrachlorohydroquinone (TeCH) and dichlorohydroquinone (DiCH) [14–16]. Some of these polychlorinated and their nitro or amino derivatives metabolites such as PeCB, TeCB, TCB, DCB, PeCA, TeCA, TCA, DCA and CA can be determined by gas chromatography coupled with electron capture detector (GC-ECD) [26–29]. However, it is difficult to identify some of chlorophenolics and chloroquinolics intermediates such as PCP, tetrachlorophenol (TeCP), trichlorophenol (TCP), dichlorophenol (DCP) and TeCH, DiCH by GC due to their relative polarity, low vapour pressure, chemical reactivity, causing adsorption and tailing of the chromatographic peaks [30, 31]. Therefore, for determination of these CACs and chemically different nature (polar/ionic) of the putative intermediates, there is a need to develop simple, quick extraction and analytical method. In the present study we report a high performance liquid chromatography (HPLC) method for determination of PCNB, HCB and their possible metabolites.
Solvents selection for extraction
The solvents were selected for extraction of PCNB, HCB and their possible metabolites by dissolving increasing concentrations of compounds up to their maximum solubility into different organic solvents. Solubility tests revealed that all compounds were best soluble in ethyl acetate whereas HCB was only soluble in hexane. The selected solvents were used for the determination of extraction efficiency of PCNB, HCB and their possible metabolites.
Extraction efficiency and quantification
Recoveries (%) of PCNB and HCB from spiked soil calculated by HPLC
Spiked concentration of PCNB (gm kg-1)
Concentration (Mean ± SD) (gm kg-1)
Spiked concentration of HCB (gm kg-1)
Concentration (Mean ± SD) (gm kg-1)
0.0489 ± 0.0016
0.0488 ± 0.0023
0.0979 ± 0.0025
0.0978 ± 0.0025
0.1968 ± 0.0016
0.1957 ± 0.0028
0.2976 ± 0.0018
0.2949 ± 0.0053
0.3938 ± 0.0013
0.3937 ± 0.0019
To determine any interference caused by the endogenous and soil component, a blank soil sample was analyzed. No interference was identified from the soil sample. The limit of detection (LOD) were also calculated by applying the 3s criterion equation: y-yb = 3sb, where as 'y' is lowest concentration signal and 'yb' is a blank signal, i.e. LOD corresponds to a signal equal to 3 times of standard deviation of the background noise. The detection limit for PCNB was found to be 0.0001 μg ml - 1 and 0.0003 μg ml - 1 for HCB. Similar exercise was carried out for both PCNB and HCB from MSM. Result indicated that extraction efficiency of PCNB and HCB from MSM was very close to the extraction efficiency as determined for the soil sample (data not shown).
UV-vis absorption spectra and retention time of PCNB, HCB and their possible intermediates
Details of retention times and corresponding UV-vis absorption spectra of PCNB, HCB and their possible intermediates determined by HPLC
Name of the compounds
Retention time (min)
Determination of PCNB, HCB depletion rate and identification of metabolites
Due to high electronegative properties of PCNB, HCB and their intermediates, the identification is possible by gas chromatography coupled with electron capture detector (GC-ECD) and mass-spectroscopy (GC-MS) [30, 32, 33]. Although, the detection efficiency by these methods is good, yet there are several drawbacks like the need to derivatize such as methylation, acetylation and pentafluorobenzylation and the loss of compounds by thermal decomposition [24, 26, 27, 34]. Similarly, there are a few reports describing extraction procedures for PCNB and HCB but they are cumbersome as they required additional steps, of sample cleanup or sample enrichment followed by quantitative determination by liquid chromatographic analysis [24, 35]. In the present work, solubility analysis was carried out which indicated that all compounds were highly soluble in ethyl acetate, except HCB which was soluble in hexane. Based on their solubility, ethyl acetate and hexane were used for extraction of PCNB and HCB respectively from the soil sample as well as from the MSM. The proposed liquid-liquid extraction and HPLC analysis procedure showed good recoveries of 98% with 0.0001 μg ml - 1 LOD for PCNB and 97% with 0.0003 μg ml - 1 LOD for HCB without the need to cleanup the extracted samples. In the proposed HPLC method there is no need for the derivatization such as methylation, acetylation or pentafluorobenzylation for the detection of chlorophenolic and chloroquinolic intermediates. The proposed liquid-liquid extraction and analytical method is one of the most suitable for the application to any toxic organochlorine residue determination from different environmental compartment such as soil, sediment and sludge.
A rapid, sensitive and simple analytical method was developed for the determination of polychlorinated fungicides such as PCNB, HCB and their possible intermediates including chlorophenolic as well as chloroquinolic intermediates using HPLC techniques. The developed method gave good recoveries, good peaks resolution and sensitive detection levels without internal interference.
Chemicals, reagents and standard stocks
The standards of PCNB, HCB including their possible intermediates such as PeCA, PCP, TeCA, TCA, DCA, PeCB, TeCB, TeCH, TCB, TeCNB, TeCP), TCP, TCNB, DCP, DCNB, DCA, DiCH and DCB were purchased from Sigma Aldrich (St, Louis, MO, USA). Ethyl acetate, methanol, and hexane (HPLC grade) were obtained from Merck (Darmstadt, Germany). The stock solution (100 μg ml-1) of the above compounds was prepared in dimethyl sulphooxide (DMSO) of PCNB and in hexane of HCB. Stock solutions were diluted to obtain 10 μg ml-1 working solutions. All chemicals, aqueous solutions and standards were filtered through 0.22 μm filter (Millipore, Milford, Mass., USA) and stored at 4°C. The composition of minimal salt medium (MSM) used for determining the extraction efficiency of PCNB and HCB compounds was the same as described by Fazlurrahman et al. .
Sample preparation and fortification
Soil samples (collected locally) were air dried, homogenized, grounded, sieved and stored in stoppered glass flasks at 18°C. Fifteen gram soil sample was fortified by adding five working concentration of PCNB and HCB equivalent to 0.05, 0.1, 0.2, 0.3 and 0.4 mg kg - 1 respectively. The standard solution(s) was mixed in 20 ml MSM and added to the soil verifying that the solution covered the soil particles completely. After fortification, the samples were mixed with the help of glass rod followed by vortexing and kept in dark at room temperature for 24 h for equilibration. Since most of the microbial degradation of pesticide were also carried out in MSM, therefore the extraction efficiency of PCNB and HCB was also estimated from 10 ml MSM containing 0.5, 1.0, 2.0, 3.0 and 4.0 μg ml - 1 respective compounds.
Extraction efficiency of PCNB and HCB compounds from different sources
Chromatographic apparatus and conditions
The liquid chromatographic system consisted of Water-HPLC 600 and multi-solvent delivery system pump with Waters 996 photodiode array detector (Waters, Corporation, Milford, MA, USA). A reverse phase Waters Spherisorb 5 μm C18 (150 × 4.6 mm) column (Waters, Corporation, Milford, MA, USA) was used as the stationary phase. The separation of analytes on HPLC was conducted by using RPC18 column with mobile phase methanol: water (96:4, v/v) at a column temperature of 25°C and a flow rate of 1.0 ml min - 1. The injection volume was 5 μl. The detection of the analytes was realized by measuring the UV-absorption with PDA detector at 300 nm.
Chlorinated aromatic compounds
- and (DiCH):
high performance liquid chromatography
- and (DCA):
- and (GC-ECD):
gas chromatography coupled with electron capture detector
- and (GC-MS):
minimal salt medium.
We are thankful to Council of scientific and industrial research for financial support. This is IMTECH communication number 05/2010.
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