Liquid Liquid-liquid Microextraction Research Articles

Determination of Five Sulfonamides in Water by High Performance Liquid Chromatography Combined with Ionic Liquid Liquid-liquid Microextraction

A novel method for the determination of sulfamethazine (SM2), sulfamonomethoxinc (SMM), sulfamethoxazole (SMZ), sulfadiazine (SD), sulfaquinoxaline (SQX) from the water has been developed using ionic liquid dispersive 1iquid-liquid microextraction coupled with high performance liquid chromatography. The influence of extraction parameters including types of extraction solvent, volume of extraction solvent, types of dispersant solvent, volume of dispersant solvent, extraction time, and pH were investigated. Using 80 μL 1-Hexyl-3-methylimidazolium hexafluorophosphate ([C6MIM]PF6) as extraction solvent, 800 μL acetonitrile as dispersant solvent, pH=4.0, extraction 3min, centrifugal after storage 2min. Under the optima1 conditions, good linear relationships were obtained in the five sulfonamides residues concentrations of 5~150 μg•L-2 with the correlation coefficients of 0.9988-0.9994, limits of detection (LOD) were 0.74~1.21 μg•L-2 (S/N=3), limits of quantification (LOQ) were 2.51~4.06 μg•L-2 (S/N=10). The proposed method was applied to the determination of five sulfonamides residues in water, the recoveries were from 88.5% to 98.8%, with the corresponding intra-day RSDs less than 5.1%, inter-day RSDs less than 9.5%. The method was considered to be simple, fast, and precise to satisfy the requirements of the residual analysis of sulfonamides in water.

Vortex-assisted liquid-liquid–liquid microextraction followed by high performance liquid chromatography for the simultaneous determination of fourteen phenolic acids in honey, iced tea and canned coffee drinks

A vortex-assisted liquid-liquid–liquid microextraction method followed by high performance liquid chromatography-diode array detection for the determination of fourteen phenolic acids (cinnamic, m-coumaric, chlorogenic, syringic, ferulic, o-coumaric, p-coumaric, vanillic, p-hydroxybenzoic, caffeic, 2, 4-dihydroxybenzoic, sinapic, gentisic and gallic acids) in honey, iced tea and canned coffee drink samples has been developed. The separation was achieved using a Poroshell 120-EC-C18 column under a gradient elution at a flow rate of 0.6mLmin−1 and mobile phase composed of methanol and acetic acid (1%, v/v). Under the optimum chromatographic conditions, the fourteen phenolic acids were separated in less than 32min. The extraction was performed using a small volume (400µL) of ternary organic solvents (1-pentanol, propyl acetate and 1-hexanol) dispersed into the aqueous sample (10mL) and assisted by vortex agitation (2500rpm for 45s), the analytes were next back-extracted from the organic solvent using 0.02M KOH (40µL) with vortex speed and time of 2500rpm and 60s, respectively. Under these conditions, enrichment factors of 30–193-fold were achieved. The limits of detection (LODs) were 0.05–0.68µgL−1. Recoveries in honey, iced tea and canned coffee drinks were in the range 72.2–112%. The method was successfully applied for the determination of the phenolic acids in honey, iced tea and canned coffee drinks.

Hollow Fiber Supported Ionic Liquid Liquid-Liquid Micro-Extraction for Determination of Triclosan in Environmental Water Samples

Hollow Fiber Supported Ionic Liquid Liquid-Liquid Micro-Extraction for Determination of Triclosan in Environmental Water Samples

Determination of Azithromycin in Biological Samples by LLLME Combined with LC

A simple liquid–liquid–liquid microextraction (LLLME) technique combined with liquid chromatography (LC) was developed for the extraction and quantitative determination of azithromycin (AZI) in biological fluids. In this technique, AZI was extracted from 2.5 mL basic sample solution (donor phase) at pH 10.5 into an organic phase (90 μL), for 30 min. Then back-extracted into a 5 μL microdrop acidic aqueous solution (acceptor phase) at pH 2.5 immersed in the organic phase from the tip of a microsyringe for 20 min with a stirring rate of 800 rpm and donor phase temperature of 35 °C. After a prescribed time, the acceptor microdrop was returned into the microsyringe and injected into the LC. Optimization of experimental parameters on LLLME efficiency was investigated. Under optimized conditions, a preconcentration factor of 85 and limit of detection of 0.03 μg mL−1 were obtained. The calibration curve was linear (r 2 = 0.998) in the concentration range of 0.1–15 μg mL−1. Within-day relative standard deviation (RSD) (S/N = 3) and between-day RSD were 5.1 and 6.8%, respectively. Finally, the feasibility of the proposed method was evaluated by extraction and determination of AZI in plasma and urine samples and satisfactory results were obtained.

A new concept of hollow fiber liquid–liquid–liquid microextraction compatible with gas chromatography based on two immiscible organic solvents

A new concept of liquid–liquid–liquid microextraction (LLLME) was introduced based on applying two immiscible organic solvents in lumen and wall pores of hollow fiber (HF). With this methodology, analytes of interest can be extracted from aqueous sample, into a thin layer of organic solvent (dodecane) sustained in the pores of a porous hollow fiber, and further into a μL volume of organic acceptor (acetonitrile or methanol) located inside the lumen of the hollow fiber. Some chlorophenols (CPs) were selected as model compounds for developing and evaluating of the method performance. The analysis was performed by gas chromatography–electron capture detection (GC–ECD) without derivatization. The factors affecting the HF-LLLME of target compounds were investigated and the optimal extraction conditions were established. Under the optimum conditions, preconcentration factors in a range of 208–895 were obtained. The performance of the proposed method was studied in terms of linear dynamic ranges (LDRs from 0.02 to 100 ng mL −1), linearity ( R 2 ≥ 0.995), precision (RSD % ≤ 8.1) and limits of detection (LODs in the range of 0.006–0.2 ng mL −1). In addition to preconcentration, HF-LLLME also served as a technique for sample clean-up.

Determination of hippuric acid in biological fluids using single drop liquid–liquid-liquid microextraction

A simple, sensitive, and inexpensive single drop liquid–liquid-liquid microextraction (LLLME) followed by isocratic reverse phase high performance liquid chromatography (RP-HPLC) and UV detection was developed for determination of hippuric acid in human urine and serum samples. The analyte was extracted from an acidic aqueous sample solution (pH 3) through a thin layer of organic solvent membrane and back-extracted to a basic acceptor drop (pH 11) suspended on the tip of a 10-μL HPLC syringe in the organic layer. The influence of several important parameters on extraction efficiency of hippuric acid was evaluated. Under optimized experimental conditions, the calibration graph was linear in the concentration range of 1–400 μg L−1 with coefficient of determination 0.998. The limits of detection and quantification were 0.3 and 1.0 μg L−1, respectively. Intra-day and inter-day precision were in the range of 1.1–2.7% and 1.1–3.1%, respectively. This procedure was successfully applied to the determination of hippuric acid in spiked urine and serum samples with satisfactory results. The relative recoveries of urine and serum samples ranged from 91.4 to 99.3%, with relative standard deviations varying from 1.6 to 4.2%.

Preconcentration and Determination of Carbamate Pesticide Residues in Vegetable Samples by Electrokinetic Flow Analysis with On-Line Hollow Fiber Liquid–Liquid–Liquid Microextraction and Spectrophotometry

ABSTRACT A simple, convenient, sensitive, and environmentally friendly analysis method for carbamate pesticide residues in vegetable samples was proposed by using electrokinetic flow analysis (EFA) with on-line hollow fiber liquid–liquid–liquid microextraction (LLLME) and ultraviolet spectrophotometry (UV). On-line LLLME conditions were investigated, including organic solvents, flow rate of sample loading, extraction times and sample volume, and so on. With dodecanol phase immobilized on the hollow fiber, the on-line LLLME unit could be operated for 150 hr. In each regeneration of dodecanol phase on the hollow fiber, the consumption of dodecanol was 50 µL, namely., the consumption rate was 0.33 µL h−1. By introducing 5.0 mL sample solution, the analytical time was 22 min and the enrichment factor of carbaryl achieved 300. The linear calibration range was from 0.0033 to 1.0 µg mL−1 carbaryl, which was used as a converted concentration for total carbamate pesticides. The relative standard deviation (R.S.D.) of peak height was 2.7% (n = 5) and the limit of detection (LOD) was 1 ng mL−1 carbaryl (K = 3, n = 11).

Quantitation of atorvastatin in human plasma using directly suspended acceptor droplet in liquid–liquid–liquid microextraction and high-performance liquid chromatography-ultraviolet detection

A simple and sensitive methodology based on liquid–liquid–liquid microextraction (LLLME) followed by high-performance liquid chromatography-ultraviolet detection (HPLC-UV) has been successfully developed for the determination of atorvastatin (AT) in human plasma. AT was first extracted from 4.5 mL acidic aqueous sample (diluted plasma, donor phase, pH 1) at temperature 45 °C through 400 μL 1-octanol for 4.5 min, while being agitated by a stirring bar at 1250 rpm. Then, a 5.5 μL free suspended basic aqueous droplet (acceptor phase, pH 10) was delivered to the top-center position of the organic membrane. The mixture was stirred at 650 rpm for 7.5 min and the analyte was back-extracted into the droplet. Finally, the acceptor phase was taken into a microsyringe and injected directly into the HPLC. An enrichment factor of 187 along with substantial sample clean up was obtained under the optimized conditions. The calibration curve showed linearity in the range of 1–500 ng mL −1 with regression coefficient corresponding to 0.996. Limits of detection (S/N = 3) and quantification (S/N = 10) were 0.4 and 1 ng mL −1, respectively. A reasonable relative recovery (91%) and satisfactory intra-assay (4.4–7.0%, n = 6) and inter-assay (4.9–7.7%, n = 8) precision illustrated good performance of the analytical procedure. This technique was eventually applied for the determination of AT in human plasma after oral administration of 40 mg single dose of drug. The protocol proved to be highly cost-effective and reliable for the screening purpose.

Liquid–Liquid–Liquid Micro Extraction Combined with CE for the Determination of Rare Earth Elements in Water Samples

A simple method for determination of rare earth elements (REEs) by liquid–liquid–liquid microextraction (LLLME) coupled with capillary electrophoresis and ultraviolet technique was developed. In the LLLME system, 40 mmol L−1 4-benzoyl-3-methy-1-phenyl-5-pyrazolinone (PMBP) acted as extractant and 4% (v/v) formic acid was used as back-extraction solution. The parameters influencing the LLLME, including the type of the organic solvent, sample pH, formic acid concentration, PMBP concentration, extraction time, volume of organic solvent, stirring rate and phase volume ratio, were investigated. Under the optimized conditions, the detection limits (S/N = 3) of REEs were in the range of 0.19–0.70 ng mL−1. The developed method was successfully applied to the determination of trace amounts of REEs in water samples.

Determination of 3-nitroaniline in water samples by directly suspended droplet three-phase liquid-phase microextraction using 18-crown-6 ether and high-performance liquid chromatography

Liquid–liquid–liquid microextraction (LLLME) with directly suspended droplet in high-performance liquid chromatography (HPLC) has been applied as a new, rapid and easy method for the determination of 3-nitroaniline in environmental water samples. The target compound was extracted from the aqueous sample solution (donor phase, pH 13) into an organic phase and then was back-extracted into a directly suspended droplet of an acidic aqueous solution (acceptor phase, pH 0.3). In this method, without using a microsyringe as supporting device, an aqueous large droplet is freely suspended at the top-center position of an immiscible organic solvent, which is laid over the aqueous sample solution while being agitated. Then, the droplet was withdrawn into the microsyringe and directly was injected into the HPLC system with UV detection at 227 nm. Up to 148-fold enrichment of the analyte could be obtained under the optimal conditions [i.e. donor phase: 0.1 M sodium hydroxide solution (4.5 mL); organic phase: o-xylene/1-octanol (90:10, v/v; 250 μL); acceptor phase: 0.5 M hydrochloric acid and 500 mM 18-crown-6 ether (6 μL); extraction time: 60 s; back-extraction time: 6 min and stirring rate: 600 rpm]. The limit of detection was 1 μg/L ( n = 7) and the relative standard deviation (RSD, n = 5) was 4.9 at S/N = 3. The calibration graph was linear in the range of 5–1500 μg/L with r = 0.9983. All experiments were carried out at room temperature (22 ± 0.5 °C).

On-column liquid–liquid–liquid microextraction coupled with base stacking as a dual preconcentration method for capillary zone electrophoresis

A simple and efficient dual preconcentration method of on-column liquid–liquid–liquid microextraction (LLLME) coupled with base stacking was developed for capillary zone electrophoresis (CZE) in this paper. Four N-methyl carbamates were used as target compounds to evaluate the enrichment means. The carbamates in sample solutions (donor phase) were extracted into a dodecanol phase immobilized on a porous hollow fiber, hydrolyzed and back extracted into 0.20 μL running buffer (acceptor phase) of 30 mmol/L methylamine hydrochloride (pH 11.6) containing 0.5 mmol/L tetradecyltrimethylammonium bromide inside the hollow fiber, stacked further with 0.5 mol/L NaOH injected at −10 kV for 60 s, and separated by CZE. Analytical parameters affecting the LLLME, base stacking and CZE were investigated, including sample solution volume, pH and temperature, extraction time, stirring rate, buffer component, buffer pH, NaOH concentration, stacking time, etc. The enrichment factors of the carbamates were higher than 1100. The relative standard deviation (RSD) of peak height and limits of detection (LODs) were 4.5–5.5% ( n = 6) and 2–4 ng/mL ( S/ N = 3) for standard solutions, respectively. The proposed method was applied to the analysis of vegetable and fruit samples with the RSD less than 6.0% ( n = 3) and LODs of 6–10 ng/g ( S/ N = 3). The calibration solutions were prepared by diluting the stock solutions with blank sample solutions, and the calibration concentrations ranged from 0.012 to 1.0 μg/mL ( r > 0.9951). The analytical results demonstrated that the LLLME coupled with base stacking was a simple, convenient and reliable on-column sample pretreatment method for the analysis of anionic analytes in CZE.

Determination of fentanyl in biological and water samples using single-drop liquid–liquid–liquid microextraction coupled with high-performance liquid chromatography

A single-drop liquid–liquid–liquid microextraction (LLLME) method coupled with high-performance liquid chromatography (HPLC) was developed for the determination of fentanyl in biological (plasma and urine) and wastewater samples. Fentanyl is a potent synthetic narcotic analgesic administered in the form of a transdermal patch for the management of chronic pain. Fentanyl was extracted from 0.01 M NaOH solution (donor phase) into a thin layer of organic phase (100 μL), then back-extracted into 5 μL of the acidic acceptor microdrop (1 × 10 −3 M HClO 4) immersed in the organic membrane from the tip of a 25-μL HPLC syringe. After the extraction, the microdrop was withdrawn into the syringe and injected directly into a HPLC system for analysis. The parameters influencing the extraction efficiency including the organic solvent and its volume, acceptor microdrop volume, composition of the donor and acceptor phases, stirring rate, temperature, salt addition and pre- and back-extraction times were investigated and optimized. At the most appropriate conditions (100 μL of n-octane, 3.6 mL of the donor phase maintained at 0.01 M NaOH, 5 μL of 1 × 10 −3 M HClO 4 as the acceptor microdrop, stirring rate of 1000 rpm for pre-extraction and 700 rpm for back-extraction, 30 °C, no salt addition, 30 min for pre-extraction and 20 min for back-extraction), an enrichment factor (EF) of 355 was obtained. The limit of detection (LOD) was 0.1 ng mL −1 (based on S/N = 3) and intra- and inter-day relative standard deviations less than 9% were obtained. The calibration graph was linear within the range of 0.5–1000 ng mL −1 with the correlation coefficient ( r) of 0.9999. Finally, the feasibility of the proposed method was evaluated by extraction and determination of fentanyl in plasma, urine and wastewater samples and satisfactory results were obtained.

Improved liquid–liquid–liquid microextraction method and its application to analysis of four phenolic compounds in water samples

An improved liquid–liquid–liquid microextraction (LLLME) technique has been put forward based on the principle of single drop LLLME. In the technique, a vial insert was firstly utilized as acceptor phase container. Because the diameter of the bottom of the vial insert was small, the contact area between the acceptor phase and the vial insert was bigger than that between microsyringe and microdrop of acceptor phase in single drop LLLME, and the stability of microdrop was increased markedly. More acceptor phase could be held in the improved method than that in single drop LLLME, and the sensitivity of the method was increased. The sample vial and vial insert were horizontally placed so that the density of organic solvent has little effect on the selection of organic solvents. Aqueous ammonia and toluene were selected as the acceptor phase and the organic phase, respectively. The improved method was successfully applied to determine four phenolic compounds in real aqueous samples. Good recoveries that ranged from 82.2% to 117.2% were obtained. The intra-day and inter-day reproducibilities (RSD) were under 4.8% and 6.8%, respectively. The extraction efficiency of the improved method was 11–47 times higher than that of single drop LLLME method. The improved LLLME method is economical, rapid, simple, efficient, low organic solvent consumption and no cross-containment. This method is very suitable for the extraction of ionizable and chargeable analyte in complex environmental or biological samples.

Application of liquid–liquid–liquid microextraction and high-performance liquid-chromatography for the determination of sulfonamides in water

This work presents a novel liquid–liquid–liquid microextraction (LLLME) technique for the extraction of sulfonamides from aqueous systems; it combines with high-performance liquid-chromatography-ultraviolet absorbance detection (HPLC/UV). In this experiment the sulfonamides were successively extracted from a donor phase (i.e., a water sample) into several microliters of an organic phase and then from the organic phase into an acceptor phase (i.e., an aqueous extract) by LLLME. The following separation and quantitative analyses were performed using HPLC/UV with 265 nm detection. Extraction condition such as solvent identity, agitation, extraction time, acceptor phase NaOH concentration, donor phase pH, and salt addition were optimized. Relative standard deviation (RSD, 2.6–5.3%), coefficient of estimation ( R 2, 0.9972–0.9999), and method detection limit (MDL, 0.11–0.77 ng mL −1) were achieved under the selected conditions. The proposed method was successfully applied to the analyses of three practical water samples and the relative recoveries of sulfonamides from the spiked water samples were in the range of 86.2–108.7%. The proposed method also confirms microextraction to be robust to monitoring trace levels of sulfacetamide, sulfadiazine, sulfathiazole, sulfamerazine, sulfadimidine, sulfamonomethoxine, sulfamethoxazole, and sulfaquinoxaline in aqueous samples.

Determination of methylmercury and phenylmercury in water samples by liquid–liquid–liquid microextraction coupled with capillary electrophoresis

A novel method for determination of methylmercury (MeHg) and phenylmercury (PhHg) by liquid–liquid–liquid microextraction (LLLME) coupled with capillary electrophoresis (CE) with ultraviolet (UV) technique was developed. The method based on MeHg and PhHg was complexed with 1-(2-pyridylazo)-2-naphthol (PAN) to form hydrophobic complexes. When the sample solution was stirred, analytes were extracted into the organic layer (200 μL toluene) and back-extracted simultaneously into the 4.0 μL 0.1% (w/v) l-cysteine microdrop. The factors affecting on the LLLME of two mercury species, including sample pH, complex reagent concentration, extraction time, volume of organic solvent, stirring rate and phase volume ratio, were investigated. Under the optimized conditions, the detection limits (S/N = 3) of MeHg and PhHg were 0.94 and 0.43 ng mL −1 (as Hg), respectively. The precisions (RSDs, c = 10 ng mL −1, n = 7) were in the range of 3.3–3.4% for migration time, 6.1–7.2% for peak area response, and 6.7–7.5% for peak height response for the two mercury species. The enrichment factors of 324 for MeHg and 210 for PhHg were obtained with 40 min LLLME. The developed method was successfully applied to the determination of trace amounts of MeHg and PhHg in water samples.

Directly Suspended Droplet Three Liquid Phase Microextraction of Diclofenac Prior to LC

Directly suspended droplet liquid–liquid–liquid microextraction (LLLME) has been used to determine residues of diclofenac (2-[2-(2,6-dichlorophenyl) aminophenyl] ethanoic acid), in environmental water samples. In this technique a free suspended droplet of an aqueous solvent is delivered to the top-center position of an immiscible organic solvent floating on the top of an aqueous sample while being agitated by a stirring bar placed on the bottom of the sample cell. Recently, diclofenac was found as an environmental contaminant in sewage, surface, ground and drinking water samples. In the present work, diclofenac was extracted from water samples by LLLME and analysed by HPLC with UV detection at 281 nm. Factors such as organic solvent, extraction and back extraction times, stirring rate and the pH of acceptor and donor phases were optimized. Enrichment factor and detection limit (LOD, n = 7) were 102 and 0.1 μg L−1, respectively. The linearity ranged from 0.5 to 2,000 μg−1 with a %RSD (n = 5) of 7.2 at S/N = 3. All experiments were carried out at room temperature (22 ± 0.5 °C).

Hollow fiber-based liquid–liquid–liquid microextraction combined with high-performance liquid chromatography for the speciation of organomercury

A novel and extremely highly sensitive method for the speciation of organomercury in sea food and environmental water samples was developed by hollow fiber liquid–liquid–liquid microextraction (LLLME) coupled to high-performance liquid chromatography (HPLC) with ultraviolet (UV) detection at 254 nm. By the use of toluene filled the pores of the hollow fiber wall as the organic phase and Na 2S 2O 3 aqueous solution in the lumen of hollow fiber as acceptor phase, the simultaneous preconcentration of methylmercury (MeHg), ethylmercury (EtHg), and phenylmercury (PhHg) could be realized, and the target mercury species in the post-extraction acceptor phase were directly injected into HPLC for their separation. The factors affecting on the liquid–liquid–liquid microextraction of three organomercury species, including organic solvent, the pH of donor phase, the concentration of complexing agent, extraction time and stirring rate were investigated and the optimal extraction conditions were established. With the consumption of 3.8 mL of sample solution, the enrichment factors were about 120, 215 and 350 for MeHg, EtHg, and PhHg, respectively. The limits of detection were in the range of 0.3–3.8 ng mL −1 (as Hg) with precisions (RSDs (%), c = 30 ng mL −1 (as Hg), n = 7) ranging from 6.4 to 8.9%. The developed technique was validated by analyzing a certified reference material (DORM-2, dogfish-muscle, NRCC) and an environmental water sample, and the analytical results were satisfactory.

Single drop liquid–liquid–liquid microextraction of methamphetamine and amphetamine in urine

Single drop liquid–liquid–liquid microextraction (LLLME) combined with high performance liquid chromatography (HPLC)–UV detection was investigated for the determination of a popular drug of abuse, methamphetamine (MAP), and its major metabolite, amphetamine (AP), in urine samples. The target compounds were extracted from NaOH modified sample solution to a thin layer of organic solvent membrane, and back-extracted to an acidic acceptor drop suspended on the tip of a 50-μL HPLC syringe in the aforementioned organic layer. This syringe was also used for direct injection after extraction. Factors affecting extraction efficiency were studied. At optimal conditions, the overall enrichment factor (EF) was 500-fold for AP and 730-fold for MAP, respectively. The method exhibited a wide linear range (1.0–1500 μg/L), low detection limit (0.5 μg/L), and good repeatability (RSD < 5.0%) for both analytes. The feasibility of the method was demonstrated by the analysis of human urine samples.

Orthogonal array designs for the optimization of liquid–liquid–liquid microextraction of nonsteroidal anti-inflammatory drugs combined with high-performance liquid chromatography-ultraviolet detection

Orthogonal array designs (OADs) were applied for the first time to optimize liquid–liquid–liquid microextraction (LLLME) conditions for the analysis of three nonsteroidal anti-inflammatory drug residues (2-(4-chlorophenoxy)-2-methylpropionic acid, ketoprofen, and naproxen) in wastewater samples. Six relevant factors were investigated: type of organic solvent, composition of donor phase and acceptor phase, stirring speed, extraction time and salt concentration. In the first stage, mixed-level orthogonal array design, an OA 16 (4 1 × 2 12) matrix was employed to study the effect of six factors, by which the effect of each factor was estimated using individual contributions as response functions. Based on the results of the first stage, 1-octanol was chosen as organic solvent for extraction. The other five factors were selected for further optimization using an OA 16 (4 5) matrix and a 4 × 4 table to locate more exact levels for each variable. The relative standard deviations for the reproducibility of optimized LLLME varied from 6.2 to 7.1%. The coefficients of determination for calibration curves were higher than 0.9950. The method detection limits for drugs spiked in ultrapure water were in the range of 0.03–0.3 ng/mL. The final optimized conditions were applied to the analysis of drug residues in three wastewater samples in Singapore.

Liquid–liquid–liquid microextraction for the enrichment of polycyclic aromatic hydrocarbon metabolites investigated with fluorescence spectroscopy and capillary electrophoresis

The extraction and pre-concentration of phenol, 2-naphthol, and several hydroxyl polycyclic aromatic hydrocarbon (PAH) metabolites were investigated, using liquid–liquid–liquid microextraction (LLLME). The PAH metabolites are a very important class of compounds, and they have not been investigated previously by LLLME. For several of the hydroxyl PAH metabolites, the enrichment factors were small when using LLLME with an alkaline acceptor phase. Changing the acceptor phase to 1-octanol, which gave a two-phase system, improved the enrichment factors significantly for several of the hydroxyl PAH metabolites. For example, the enrichment factor was improved by a factor of 68.5 for 3-hydroxybenzo[a]pyrene. Enrichment factors were investigated as a function of time and stirring rate. At about 55 min the enrichment factor reached a maximum for the two-phase system and at approximately 75 min for the three-phase microextraction system. However, a 30 min extraction time was used for most of the experiments. Also, fluorescence spectroscopy was used to determine the enrichment factors and the mass distribution of the solute between the phases. Fluorescence spectroscopy was very effective in determining the very small concentrations of the solute in the various phases. In addition, capillary electrophoresis and LLLME were combined to demonstrate the substantial enrichment of 2-naphthol by combining these two approaches.