Dispersive Liquid-liquid Microextraction Technique Research Articles

Development of cost-effective dispersive liquid-liquid micro-extraction technique for preconcentration of multi-residue herbicides in environmental waters prior to Chromatographic analysis

Development of cost-effective dispersive liquid-liquid micro-extraction technique for preconcentration of multi-residue herbicides in environmental waters prior to Chromatographic analysis

A sustainable and innovative method to determine parabens in body creams for exposure and risk assessment

Methylparaben (MeP), ethylparaben (EtP), propylparaben (PrP), and butylparaben (BuP) are among the most widely used preservatives in cosmetics, drugs, and foods. These compounds have been associated with toxic effects due to the overuse of products with parabens in their formulation. The toxicity of parabens may be correlated to endocrine disruption, owing to their ability to mimic the actions of estradiol. In this paper, a simple, sustainable, robust, and innovative dispersive liquid-liquid microextraction (DLLME) technique was developed and employed to extract these xenobiotics from body cream samples, aiming to calculate the margin of safety (MoS) to assess the risk of exposure. The validated method presented suitable linearity (r > 0.99), lower limits of detection (ranging from 0.01 to 0.04 % w/w), and satisfactory precision and accuracy (ranging from 4.33 to 10.47, and from −14.25 to 13.85, respectively). Seven of the ten analysed samples presented paraben contents within the acceptable concentration according to European legislation. The MoS value obtained for PrP (37.58) suggested its reduced safety, indicating that PrP may significantly contribute to systemic exposure resulting from the use of personal care products.

Determination of insecticide residues in beverages based on MIL-100(Fe) dispersive solid-phase microextraction in combination with dispersive liquid-liquid microextraction followed by HPLC-MS/MS

A novel dispersive solid-phase microextraction method based on a metal-organic framework (MIL-100(Fe)) combined with a dispersive liquid-liquid microextraction technique was proposed for the extraction and enrichment of four insecticides in beverages. The qualitative and quantitative analysis of these insecticides was conducted using HPLC-MS/MS. To optimize the extraction process, several parameters were investigated, and the main variables were optimized using CCD-based RSM. The developed method displayed a wide linear range of 1.000–1000 ng/L and R2 values >0.993 for all four calibration curves. The method demonstrated high sensitivity, with LODs and LOQs of 0.3–0.6 ng/L and 0.8–1.0 ng/L, respectively. In addition, the greenness of the proposed method was assessed using the Complex GAPI tool, and the results showed that the proposed method exhibits benefits, such as minimal usage of organic solvents and negligible matrix influence, making it a suitable method for the detection of insecticide residues in beverages.

A selective dispersive liquid-liquid micro-extraction technique for trace level pollutants enrichment of pharmaceutical residues from hospital wastewaters followed by liquid chromatographic analysis

In this study a dispersive liquid-liquid micro-extraction (DLLME) method was utilized for extraction of trace pharmaceuticals in wastewater samples. Factors influencing the extraction performances were tested and accordingly 900 µL dichloroethane as the extraction solvent, 1400 µL acetonitirle as dispersive solvent, 10 min extraction time at 4000 rpm centrifugation, and pH 5 were found optimum. Acetonitrile and 0.2% formic acid in water were used as eluent. The column temperature was maintained at 25 °C and the optimum detection wavelengths of 273 nm for sulfamethoxazole and 280 nm for ciprofloxacin were used. For both the analytes, the coefficients of determinations (r2) were found to vary from 0.9989 to 0.9997, confirming good linearity in the concentration range of 0.005-100 µg mL-1 for sulfamethoxazole and 0.01-100 µg mL-1 for ciprofloxacin. The LOD and LOQ were in the range of 0.78-1.58 ng mL-1 and 2.24-5.28 ng mL-1, respectively; the RSDs were 0.41-3.21% for intra-day precision and 0.37-6.44% for inter-day precision. The concentrations of the three pharmaceuticals determined ranged from 0.76-1.53 μg mL-1 in the wastewater samples collected from Black Lion Hospital. However, ciprofloxacin and doxycycline were not detected and only low concentration of sulfamethoxazole was detected in the wastewater samples collected from Menelik II Hospital.
 KEY WORDS: Antibiotics, DLLME, Dispersive solvent, Hospital wastewater
 Bull. Chem. Soc. Ethiop. 2024, 38(1), 27-42. DOI: https://dx.doi.org/10.4314/bcse.v38i1.3

Dispersive liquid-liquid microextraction coupled with gas chromatography-mass spectrometry (GC-MS) for the determination of soy sauce aroma compounds

In the present work, a dispersive liquid-liquid microextraction (DLLME) method combined with gas chromatography-mass spectrometry (GC-MS) was successfully developed for accurate determination of aroma-active components in soy sauce. Several key experimental parameters were optimized, including the type of dispersant, vortex time and the volume ratio of extractant and dispersant. The optimal conditions were 1200 μL acetone as dispersant, 800 μL dichloromethane as extractant and 3 min of vortex mixing. The proposed method was validated, achieving good linearities with R2 ≥ 0.9941, recoveries between 82 and 129% with 4-hydroxy-2,5-dimethyl-3(2H)-furanone (HDMF) and 4-hydroxy-2 (or 5)-ethly-5 (or 2)-methyl-3(2H)-furanone (HEMF) within 84–103%, relative standard deviations ranging from 0.02 to 8.60% with HDMF and HEMF between 0.26 and 1.44%, and limits of detection and limits of quantification in the range of 0.08–35.95 μg/L and 0.25–119.83 μg/L, respectively. The entire sample processing with DLLME was completed in roughly 15 min with a total consumption of 2 mL of organic solvent, and 93 volatiles identified in the obtained extracts, with 87.1% (81 volatiles) showing relative standard deviations lower than 10%. Compared to conventional liquid-liquid extraction (LLE), DLLME exhibited obvious advantages of simplicity, rapidity, stability and low solvent consumption, favouring the extraction of acids and ketones. This is the first application of the DLLME technique for the analysis of aroma compounds in soy sauce and is suitable for rapid determination of key aroma compounds like HDMF and HEMF, especially for routine detection and large batch analysis of soy sauce, which can provide reliable information for soy sauce flavour analysis.

A green ternary polymeric deep eutectic solvent used in dispersive liquid-liquid microextraction technique for isolation of multiclass pesticides in fruit juice samples

A green dispersive liquid-liquid microextraction procedure based on a ternary polymeric hydrophobic deep eutectic solvent (DES) for monitoring multi-residue pesticides in eleven various fruit juices was developed before Gas Chromatography coupled with micro-Electron Capture Detector (GC-µECD). Six ternary polyethylene glycol (PEG)-based DESs were synthesized, and their extraction efficiency was evaluated. Among these DESs, [thymol]:[polyethylene glycol]:[acetic acid] (1:2:2) showed the highest signal intensity. Moreover, three other binary PEG-based DESs and a non-polymeric DES were also synthesized and compared to the previous DES. The extraction yield obtained by polymeric one was much higher than non-polymeric. Some significant parameters influencing the extraction efficiency including the type and volume of DES (extraction solvent), disperser solvent, pH, and ionic strength were optimized as follows: [Thy]:[PEG]:[HOAc] (1:2:2), 400 µL of DES, ACN as disperser solvent (700 µL), no pH adjustment and no salt addition. The DES was characterized by ATR-FTIR and (1H and 13C) NMR spectroscopy. Under the optimized conditions, the limits of quantification, the linear ranges and extraction recovery were 0.009–0.182 µgL−1 0.003–50.0 µgL−1, 67.9–108.4%, respectively. The enrichment factors were 11–170. Intra-day and inter-day RSDs% were 1.4–5.1% and 2.0–12.6%, respectively. This method was applied for analyzing pesticide residues in eleven fruit juices, and the pre-concentration factor and relative extraction recoveries were in the range of 12–169 and 71–108%, respectively.

Trace determination of triazine herbicides in fruit and vegetables using novel hydrophobic deep eutectic solvent-based dispersive liquid-liquid microextraction followed by high-performance liquid chromatography-ultraviolet.

In the present research, a novel hydrophobic deep eutectic solvent-based dispersive liquid-liquid microextraction technique was established and combined with high-performance liquid chromatography-ultraviolet for the determination of triazine herbicides in fruit and vegetable samples. A deep eutectic solvent was synthesized using l-menthol as a hydrogen bond acceptor and ethylene glycol as a hydrogen bond donor and used as a green extractant. The characterization of deep eutectic solvent was investigated by Fourier-transform infrared, nuclear magnetic resonance, and thermogravimetric analysis. Under the optimum conditions, relative standard deviation values for intra-day and inter-day of the method based on seven replicate measurements of 50.0μg/kg of triazines were in the range of 2.8%-5.5% and 3.7%-7.2%, respectively. The calibration graphs were linear in the range of 3.0-500μg/kg and the limits of detection were in the range of 1.0-2.0μg/kg. The relative recoveries of different fruit and vegetable samples that have been spiked with two levels of target compounds were 91.5%-109.8%. The method has good linearity, sensitivity, accuracy, and precision. It is also environmentally friendly and was successfully used to determine the concentrations of triazines in fruit and vegetable samples.

Investigation of 33 types of honey samples: application of an efficient dispersive liquid-liquid microextraction technique coupled with gas chromatography-mass spectrometry to determine 16 polycyclic aromatic hydrocarbons

In this research, for the first time, 16 polycyclic aromatic hydrocarbons (PAHs) in 33 types of honey samples have been analyzed using an efficient, sensitive and rapid high-density solvent-microextraction (HDS-ME) technique coupled to gas chromatography-mass spectrometry (GC-MS). Important parameters in the extraction process were optimized using central composite design (CCD). Good linear relationships were chosen for 16 PAHs in the range of 1–200 ng g−1, with a correlation coefficient (R2) higher than 0.94. The limits of detection and limits of quantification were 0.3–0.8 ng g−1 and 0.9–2.4 ng g−1, respectively. The relative standard deviations for six similar analyses at the optimized point were less than 9%. The recoveries of the proposed method were obtained between 86% and 102%. High recovery, low consumption of the solvent, speed, ease of use and good elimination of matrix interference compared to other methods are the advantages of the proposed method. The performance of the present work was confirmed by the standard addition method and satisfactory results were obtained. The concentrations of PAHs in honey samples were found lower than 11 ng g−1.

New microextraction methods for the evaluation of bromhexine HCl in pure and pharmacological formulations.

In this study, ion-pair reactions were used to investigate bromhexine HCl. The development of simple, accurate, sensitive, low-cost, and efficient extraction methods for bromohexine HCl separation, such as the DLLME and cloud point extraction techniques, are described as bromhexine HCl estimation methodologies. These methods employed the interaction of bromohexine HCl with alizarin yellow reagent to produce a yellow complex in an acidic medium (pH = 5). The complex's maximum absorbance intensity was 480 nm, and the stoichiometry for both continuous variation and molar ratio methods was determined to be 1:1. The dispersive liquid liquid microextraction (DLLME) method's concentration range (1-23µg.mL-1), the Beers law was obeyed with correlation coefficient (R2=0.998), limit of detection as (0.055µg.mL-1), limit of quantification as (0.183µg.mL-1) and molar absorptivity as (23930.2L.mol-1.cm-1).In the second technique, the cloud pointextraction method, The linearity of calibration curve above was the range between (1-40 µg.mL-1), the correlation coefficient (R2=0.998) and molar absorptivity was (13202.88L.mol-1.cm-1), (LOD) and (LOQ) were (0.141µg.mL-1) and (1.4641µg.mL-1), respectively. The proposed techniques can be used for the determination of bromohexine HCl in both pure and pharmaceutical formulations with very good success and pharmaceutical formulations with very good success.

A fast-screening dispersive liquid-liquid microextraction-gas chromatography-mass spectrometry method applied to the determination of efavirenz in human plasma samples.

We demonstrate the suitability of a fast, green, easy-to-perform, and modified sample extraction procedure, i.e., dispersive liquid-liquid microextraction (DLLME) for the determination of efavirenz (EFV) in human plasma. Data acquisition was done by gas chromatography-mass spectrometry (GC-MS) in the selected ion monitoring (SIM) mode. The simplicity of the method lies in, among others, the avoidance of the use of large organic solvent volumes as mobile phases and non-volatile buffers that tend to block the plumbing in high-performance liquid chromatography (HPLC). Chromatographic and mass spectral parameters were optimized using bovine whole blood for matrix matching due to insufficient human plasma. Method validation was accomplished using the United States Food and Drug Administration (USFDA) 2018 guidelines. The calibration curve was linear with a dynamic range of 0.10-2.0μg/mL and an R2 value of 0.9998. The within-run accuracy and precision were both less than 20% at the lower limit of quantification (LLOQ) spike level. The LLOQ was 0.027μg/mL which compared well with some values but was also orders of magnitude better than others reported in the literature. The percent recovery was 91.5% at the LLOQ spike level. The DLLME technique was applied in human plasma samples from patients who were on treatment with EFV. The human plasma samples gave concentrations of EFV ranging between 0.14-1.00μg/mL with three samples out of seven showing concentrations that fell within or close to the recommended therapeutic range.

Bioavailability of Aflatoxins in Cultured Fish and Animal Livers Using an In Vitro Dialyzability Approach.

The objective of the present study was to investigate the bioavailability of aflatoxins (AFs) from fish, and chicken and rabbit livers using an in vitro dialyzability approach. Ultrahigh-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) was used to assess the aflatoxin content in samples, as well as in dialyzate and residue fractions after the in vitro procedure. A vortex-assisted dispersive liquid-liquid microextraction (VALLME) technique was used for preconcentrating AFs before determination. Raw samples showed bioavailability ratios of 41-45% for aflatoxin B1 (AFB1), 28-38% for aflatoxin B2 (AFB2), and 42% for aflatoxin G2 (AFG2). Aflatoxin G1 (AFG1) was not detected. The culinary process (steaming or grilling) was found to change AFs' bioavailability (higher bioavailability ratios were found in cooked samples). AFB2 was found to be transformed into other compounds during the in vitro process, and the presence of AFB2 and AFB2 transformation/degradation products was investigated and confirmed by high-resolution mass spectrometry (HRMS).

Simultaneous preconcentration and determination of zirconium in environmental samples using ultrasound-assisted ionic liquid based dispersive liquid-liquid microextraction combined with spectrophotometry

ABSTRACT The Green, delicate and rapid ultrasound-assisted ionic liquid-based dispersive liquid-liquid microextraction technique was developed to preconcentrate trace quantities of zirconium Zr(IV) ion in real samples prior to spectrophotometric detection. The proposed method uses (1-hexyl-3-methylimidazolium tris(pentafluoroethyl)trifluorophosphate) ionic liquid (IL) as an extraction solvent for Zr(IV) ions after the complexation with quinalizarin at pH 5.0. The effect of various analytical parameters has been investigated. The calibration graph was linear in the range of 2.0–400 µg L − 1 and the limit of detection was 0.6 µg L-1. The preconcentration factor was 100. Relative standard deviation (RSD%) at 200 µg L-1 of Zr(IV)for intra-day and inter-day precisionwere 2.0 and 2.7%, respectively (n = 10). Certified reference materialswere used to verify the accuracy.The proposed method was applied to preconcentrate and determine the content of Zr(IV) in real environmental (water, soil and plants) samples with satisfactory results.

Determination of Methadone and Tramadol in Vitreous Humor Specimens Using Dispersive LiquidLiquidMicroextractionandUltraHighPerformance Liquid Chromatography

Background: Drug abuse is spreading rapidly all over the world. Methadone and tramadol are among not only the most abused opioids but also important from the forensic point of view. Therefore, we need to devise a simple and sensitive method for the sample preparation and identification of abused drugs in postmortem specimens. Methods: A simple and rapid Dispersive Liquid-Liquid Microextraction (DLLME) technique coupled with Ultrahigh Performance Liquid Chromatography (UHPLC) was developed for the extraction and analysis of methadone and tramadol from postmortem vitreous humor samples. Different parameters affecting the extraction recovery, such as the type and volume of extraction and dispersion solvents, pH value, sensitivity, and specificity, were optimized and studied. Results: Under optimized conditions, the recovery ranges were 82.3%-89.6% and 85.4%-87.1% for methadone and tramadol, respectively. The linear range was 25-100 ng/mL for both methadone and tramadol with a correlation coefficient (R2) of more than 0.98. Limit of Detection (LoD) and Limit of Quantification (LoQ) were 3 and 8 ng/mL for methadone and 6 and 16 ng/mL for tramadol. The accuracy level of the methods for methadone and tramadol detection were 99.4%-100% and 99.7%-99.9%, respectively. The method was specific enough for the qualitative and quantitative determination of methadone and tramadol. Conclusion: The obtained results showed that DLLME combined with UHPLC is a fast and straightforward method for determining methadone and tramadol in postmortem vitreous humor specimens.

Supramolecular solvent-based microextraction for the preconcentration of Pb2+ and Cd2+ prior to spectrophotometric detection.

Supramolecular solvent-based dispersive liquid-liquid microextraction technique has been developed as a preconcentration tool for the determination of trace level of Pb2+ and Cd2+. Dodecanol dispersed in tetrahydrofuran has been utilized as a supramolecular-solvent system for the extraction of analytes prior to their quantitative determination with graphite furnace atomic absorption spectrophotometer. Both Pb2+ and Cd2+, which were efficiently extracted by supramolecular solvent system, were complexed with dithizone followed by the addition of supramolecular solvent. The experimental variables that could possibly influence the extraction efficiency, i.e. pH value, temperature, sample volume, centrifugation time, rate of centrifugation, ionic strength, etc. were subjected to the optimization step. An interference study was also conducted to check the selectivity of developed method. Limit of detection calculated for Pb2+ and Cd2+ was 0.015 and 0.061 mg L-1, respectively. The limit of quantification was 0.05 and 0.2 mg L-1 for Pb2+ and Cd2+, respectively. The analytical signal was enhanced to 30 times in case of Pb2+ and 27 times in case of Cd2+. The results obtained revealed that the developed method is rapid, simple, sensitive, and efficient for the determination of both analytes in real water samples.

A simple preconcentration method for highly sensitive determination of Pb in bee products by magnetic ionic liquid dispersive liquid-liquid microextraction and electrothermal atomic absorption spectrometry

A highly efficient preconcentration method based on the dispersive liquid-liquid microextraction technique (DLLME), that incorporated a magnetic ionic liquid (MIL) as extractant and electrothermal atomic absorption spectrometry (ETAAS) detection, was developed to determine Pb at ultra-trace levels in different bee products. Initially, the analyte was chelated with 1,5-diphenylcarbazide in 2 mol L-1 HCl and extracted by 150 μL of trihexyl(tetradecyl)phosphonium tetrachloromanganate (II) ([P6,6,6,14]2MnCl4). Subsequently, the extractant phase was separated by a magnet and an aliquot was injected into the graphite furnace of ETAAS. Phase separation was possible without a centrifugation step by using a MIL, thus reducing the analysis time and simplifying the proposed method. Under optimal experimental conditions, an extraction efficiency of 97% and a sensitivity enhancement factor of 107 were obtained. A detection limit (LOD) of 3 ng L-1 with a relative standard deviation of 3.6% (at 100 ng L-1 Pb, n = 10) and a linear range of 10-3000 ng L-1 were obtained. This work reports the first application of the MIL [P6,6,6,14]2MnCl4 along with the DLLME technique for the determination of Pb at trace levels in different bee products, such as honey, mead, honey vinegar and honey beer.

Salting-out strategy for speciation of selenium in aqueous samples using centrifuge-less dispersive liquid-liquid microextraction.

The centrifuge-less dispersive liquid-liquid microextraction (DLLME) technique was used to separate selenium species in aqueous samples. According to the salting-out effect, a simple approach was used to eliminate the centrifugation step. The optimization of the independent variables was performed using chemometric methods. Under optimal conditions, this methodology was statistically validated. The linearity was between 20 and 300 μg L-1. The limit of detection and quantification were calculated 3.4 μg L-1 and 10.4 μg L-1, respectively. The values of reproducibility and repeatability were determined ≤ 9.5% and ≤ 6.4, respectively. The possibility of the method was successfully assessed by analyzing the analytes in real samples clarified satisfactory recoveries (98.1-101.4% for Se (IV) and 98.4-101.5% for Se (VI)).

Proposal of a new, fast, cheap, and easy method using DLLME for extraction and preconcentration of diazepam and its transformation products generated by a solar photo-Fenton process

This work evaluated the formation of transformation products (TPs) during the degradation of diazepam (DZP) by a solar photo-Fenton process. Six TPs were identified, three of them for the first time. After elucidation of the TPs, a new, cheap, fast, and easy method was employed to extract and preconcentrate DZP and its TPs, using dispersive liquid-liquid microextraction (DLLME). The method was optimized using factorial and Doehlert designs, with the best results obtained using acetonitrile as disperser solvent and chloroform as extraction solvent, with volumes of 1000 and 650 µL, respectively. When DZP degradation was performed in ultrapure water, the extraction/preconcentration of DZP and its TPs by DLLME was very similar to the results obtained using a traditional SPE method. However, when hospital wastewater was used as the matrix, more limited extraction efficiency was obtained using DLLME, compared to SPE. Meanwhile, all the TPs extracted by SPE were also extracted by the DLLME technique. Furthermore, DLLME was much less expensive than SPE, besides being faster, easier, and requiring only small amounts of organic solvents. This work reports a new and very important tool for the extraction and preconcentration of TPs formed during degradation using techniques such as advanced oxidation processes (AOPs), since without this step it would not be possible to identify all the TPs formed in some complex wastewater matrices.

Determination of Gemfibrozil in Drug Matrix and Human Biological Fluid by Dispersive Liquid-Liquid Microextraction with High Performance Liquid Chromatography

A simple and rapid dispersive liquid-liquid microextraction technique coupled with high performance liquid chromatography (HPLC) was developed for the extraction and analysis of gemfibrozil in drug matrix and biological fluids like human plasma and urine samples. In this method, carbon tetrachloride at microliter volume level and methanol were used as extraction and disperser solvents, respectively. The effects of factors including the type and volume of disperser and extraction solvents, pH and extraction time on the performance of the sample preparation were carefully evaluated. Under optimum conditions, the obtained enrichment factors were between 9.2 - 9.3 and extraction recoveries were high and ranged between 91.2 - 93.2%. The calibration curve was linear in the range of 0.1 - 100 mg/L with a detection limit of 12.3 µg/L. The relative standard deviation (RSD) for ten replicate measurements of gemfibrozil was 1.3%. The performance of the method was checked by the analysis of the gemfibrozil in presence of other drugs in water samples.

Application of an agitation-assisted dispersed solvent microextraction for analysis of naphthalene and its derivatives from aqueous matrices.

Agitation-assisted dispersive liquid-liquid extraction without a dispersing solvent is lately receiving considerable attention owing to the low to no solvent loss relative to its predecessor, which suffers severe extracting solvent loss. Herein, we report the application of a simple agitation-assisted dispersive liquid-liquid microextraction method, without a disperser solvent, for the extraction of naphthalene and its derivatives from aqueous solutions. Under the optimised conditions, namely, 25 μL 3:1 mixture of dichloroethane and ethylacetate with 20 s agitation, in 2-mL aqueous solutions containing 10% NaCl, the method demonstrated acceptable figures of merit: linearity-R2 ≥ 0.9914 in the concentration range 0.5-50 ng/mL, repeatability (%RSD ≤ 12.9 for n = 15) and limits of detection (0.034-0.081 ng/mL). The recoveries obtained from the spiked dam water sample were also satisfactory (94-103%). These parameters are comparable with those reported in literature, especially for dispersive liquid-liquid microextraction techniques albeit for different analytes. Despite only naphthol being detected in one of the three sampled sites, the method shows considerable promise for routine monitoring of river and dam water quality subject to accuracy validation using certified reference materials.

Development of Dispersive Liquid-Liquid Microextraction Procedure for Trace Determination of Malathion Pesticide in Urine Samples

Measurement of pesticides in biological matrices is become a serious challenge for researches because of their very low concentration in different matrices. The aim of this study was to develop a new sample preparation method with high accuracy and validity, simplicity and short retention time for determination of malathion. Dispersive liquid-liquid micro-extraction (DLLME) technique coupled with high-performance liquid chromatography equipped with ultraviolet detector (HPLC-UV) developed for trace extraction and determination of malathion pesticide in human urine samples. This study was done in 2017 at Tehran University of Medical Sciences, Tehran, Iran. One variable at a time (OVAT) method was used to optimize parameters affecting the malathion extraction. Different parameters such as extraction solvent, disperser solvent, and volume of the extraction solvent, volume of the disperser solvent, centrifugation time and speed, salt addition, and sample pH were studied and optimized. Under the optimized conditions, the limit of detection and enrichment factor of the method were 0.5 μg L-1 and 200, respectively. The calibration curve was linear in the concentration range of 2-250 μg L-1. The relative standard deviation for six replicate experiments at 200 μg L-1 concentration was less than 3%. The relative recoveries of spiked urine samples were 96.3%, 101.7% and 97.3% at three different concentration levels of 50, 200 and 1000 μg L-1, respectively. DLLME procedure was successfully developed for the extraction of malathion from human urine samples. Compared to other extraction techniques, the proposed procedure had some advantages such as shorter extraction time, better reproducibility, and higher enrichment factor.