Trace analysis of benzophenone-derived compounds in surface waters and sediments using solid-phase extraction and microwave-assisted extraction followed by gas chromatography–mass spectrometry

This study describes a procedure for determining eight benzophenone-derived compounds in surface waters and sediments. These include the pharmaceutical ketoprofen, its phototransformation products 3-ethylbenzophenone and 3-acetylbenzophenone, and five benzophenone-type ultraviolet (UV) filters. The proposed analytical method involves the pre-concentration of water samples by solid-phase extraction (SPE) and microwave-assisted extraction (MAE) of sediment samples followed by derivatization and analysis by gas chromatography–mass spectrometry. Different parameters were investigated to achieve optimal method performance. Recoveries of 91 to 96 % from water samples were obtained using HLB Oasis SPE cartridges, whereas MAE of sediments (30 min at 150 °C) gave recoveries of 80 to 99 %. Limits of detection were between 0.1 and 1.9 ng L^?1 for water samples and from 0.1 to 1.4 ng g^?1 for sediment samples. The developed method was applied to environmental samples and revealed the presence of UV filters in the majority of the surface waters with up to 690 ng L^?1 of 2-hydroxy-4-methoxybenzophenone. By contrast, ketoprofen (≤2,900 ng L^?1) and its degradation products (≤320 ng L^?1) were found in only two rivers, both receiving wastewater treatment plant effluents. Sediment analysis revealed benzophenone to be present in concentrations up to 650 ng g^?1, whereas concentrations of other compounds were considerably lower (≤32 ng L^?1). For the first time, quantifiable amounts of two ketoprofen transformation products in the aqueous environment are reported.     

Surfactant-assisted dispersive liquid–liquid micro-extraction combined with magnetic solid-phase extraction for analysis of polyphenols in tobacco samples

A novel and simple two-step micro-extraction technique combining surfactant-assisted dispersive liquid–liquid micro-extraction and magnetic solid-phase extraction prior to high-performance liquid chromatography was established for analysis of polyphenols including chlorogenic acid, caffeic acid, and scopoletin in tobacco samples. In the developed system, Fe₃O₄ nanoparticles were synthesized by a one-step chemical co-precipitation method and used to remove hydrophobic substances in tobacco samples by physical adsorption. Low-density solvent (1-heptanol) and cationic surfactant cethyltrimethyl ammonium bromide were employed as extraction solvent and disperser agent, respectively. Under the optimized experimental conditions, a good linearity of the method was obtained over the concentration range from 0.1 to 1000 ng mL^?1 for target analytes. The limits of detection (S/N?=?3) were 0.05 ng mL^?1 for CGA, 0.10 ng mL^?1 for CFA, and 0.12 ng mL^?1 for SP, respectively. Finally, the applicability of the developed method was evaluated by extraction and determination of these three phenolic compounds in tobacco samples and satisfactory average recoveries of spiked samples were between 96.6 and 102.7%.     

Determination of migrated phthalic acid residues into edible oils using a green mode of air-assisted liquid–liquid microextraction followed by high-performance liquid chromatography–diode array detector

In this study, for the first time, an organic solvent-free air-assisted liquid–liquid microextraction method has been reported for the extraction and preconcentration of phthalic acids (o-phthalic acid, m-phthalic acid, and p-phthalic acid) from edible oil samples. The method is based on the repeated aspirating/injection of an alkaline aqueous solution and the oil sample mixture in a conical bottom centrifuge tube to form a cloudy solution. After phase separation by centrifuging, the sedimented phase is directly analyzed by high-performance liquid chromatography–diode array detection. Under the optimum extraction conditions, the method showed low limits of detection and quantification between 0.11–0.29 and 0.28–0.91 ng mL^?1, respectively. Extraction recoveries and enrichment factors were from 81 to 97% and 406 to 489, respectively. The relative standard deviations for the analysis of 5 ng mL^?1 of each analyte were less than 5.9% for intraday (n = 6) and interday (n = 5) precisions. Finally, different oil samples were successfully analyzed using the proposed method and m-phthalic acid, and p-phthalic acid were determined in some of them at ng mL^?1 level.     

Sensitive quantification of uranium using cloud point extraction coupled with inductively coupled plasma-optical emission spectrometry

This article reports the utilization of cloud point extraction as a preconcentration strategy prior to U(VI) determination by inductively coupled plasma-optical emission spectrometry. Complexes of U(VI) with Cyanex-301 were preconcentrated into mixed-micellar medium using Triton X-100 and Cetylpyridinium bromide at ambient temperature. Optimal values of parameters impacting the extraction efficiency were determined. The proposed technique has linearity range of 5–200 ng mL^?1 with r = 0.99 and detection and quantification limits of 0.57 and 0.85 ng mL^?1, respectively. The method has good selectivity for U(VI) over various cations and was successfully applied to U(VI) determination in water samples with satisfactory results.     

Development of novel magnetic solid-phase extraction sorbent based on Fe<Subscript>3</Subscript>O<Subscript>4</Subscript>/carbon nanosphere/polypyrrole composite and their application to the enrichment of polycyclic aromatic hydrocarbons from water samples prior to GC–FID analysis

We describe a magnetic nanocomposite that consists of Fe₃O₄/carbon nanosphere/polypyrrole (Fe₃O₄/CNS/PPy). The synthesized nanocomposites were characterized by scanning electron microscopy, transmission electron microscopy, and Fourier transform infrared spectroscopy. The nanocomposite was successfully applied to extract of the polycyclic aromatic hydrocarbons (PAHs) from water samples. Compared to Fe₃O₄/PPy, the Fe₃O₄/CNS/PPy nanocomposite exhibits improved properties in terms of extraction. The amount of adsorbent, salt effect, extraction time, desorption time, type, and the volume of desorption solvent were optimized. Following the desorption of the extracted analytes, the PAHs (i.e., naphthalene, 2-methylnaphthalene, 2-bromonaphthalene, fluorene, and anthracene) were quantified by gas chromatography–flame ionization detector. The PAHs can be determined in 0.05–100.00 ng mL^?1 concentration range, with limits of detection (at an S/N ratio of 3) ranging from 0.01 to 0.05 ng mL^?1. The repeatability of the method was investigated with relative standard deviations of lower than 9.9% (n = 5). Also, the recoveries from spiked real water samples were in the range of 88.9–99.0%. The results indicate that the novel material can be successfully applied for the extraction and analysis of PAHs from water samples.     

Determination of endocrine disrupting chemicals in environmental solid matrices by extraction with a non-ionic surfactant (Tween 80)

A readily applicable method based on extraction by aqueous non-ionic surfactant solutions (Tween 80) and RP-HPLC coupled to fluorescence detection, has been developed for the simultaneous determination of the phenolic endocrine disrupting chemicals (EDCs) nonylphenol (NP), nonylphenol monoethoxylate (NP1EO) and nonylphenol diethoxylate (NP2EO) and bisphenol A (BPA) in environmental solid matrices. Clean up of sample extracts was performed on Si-C18 solid phase extraction (SPE) cartridges. The overall Tween 80 extraction-SPE-RP-HPLC procedure was validated for accuracy and precision by analyzing sediment samples spiked with known amounts of EDCs. Recoveries for NP, NP1EO, NP2EO and BPA and limits of detection are in agreement with conventional extraction methods. The developed methodology was successfully applied to the analysis of target compounds in Italian river sediments, river suspended matter and benthonic macroinvertebrate organisms (oligochaetes Lumbriculus variegatus). Results confirmed that this relatively simple procedure performed satisfactorily in the determination of phenolic EDCs in environmental solid matrices of different complexity and that it can be a suitable alternative method to conventional systems even for routine analyses.     

Comparative solid phase extraction study on the U(VI) preconcentration by using immobilized thermotolerant <Emphasis Type="Italic">Bacillus vallismortis</Emphasis> and <Emphasis Type="Italic">Bacillus mojavensis</Emphasis>

Bacillus vallismortis and Bacillus mojavensis were loaded onto Amberlite XAD-4 resin and used for solid phase extraction (SPE) of uranium(VI). A quick and simple UV–Vis spectrophotometric method was used to determine U(VI) ion. The best experimental conditions were determined as being a pH of 5.0; a sample flow rate of 2.0 mL min^?1; 200.0 mg of biosorbent; 800 mg of Amberlite XAD-4, and 5.0 mL of 1 mol L^?1 HCl as desorption solution for both immobilized bacteria. The preconcentration factors were achieved as 80 for both solid phase extractor. The developed methods were validated by applying to reference water and tea samples.     

Magnetic titanium oxide nanoparticles for hemimicelle extraction and HPLC determination of organophosphorus pesticides in environmental water

We report on a method for the extraction of organophosphorus pesticides (OPPs) from water samples using mixed hemimicelles and magnetic titanium dioxide nanoparticles (Fe₃O₄@TiO₂) modified by cetyltrimethylammonium. Fe₃O₄@TiO₂ nanoparticles were synthesized by a hydrothermal process and then characterized by scanning electron microscopy and Fourier transform IR spectrometry. The effects of the quantity of surfactant, extraction time, desorption solvent, pH value, extraction volume and reuse of the sorbent were optimized with respect to the extraction of OPPs including chlorpyrifos, dimethoate, and trichlorfon. The extraction method was applied to analyze OPPs in environmental water using HPLC along with UV detection. The method has a wide linear range (100–15,000 ng L^?1), good linearity (r?>?0.999), and low detection limits (26–30 ng L^?1). The enrichment factor is ~1,000. The recoveries (at spiked levels of 100, 1,000 and 10,000 ng L^?1) are in the range of 88.5–96.7 %, and the relative standard deviations range from 2.4 % to 8.7 %.

Quantification of Thiram in Honeybees: Development of a Chemiluminescent ELISA

Abstract

A Chemiluminescence Enzyme‐Linked Immuno‐Sorbent Assay (CL‐ELISA) for determination and quantification of the fungicide thiram in honeybees was developed in an indirect competitive format. The assay was optimized by determining: the optimal coating conjugate concentration and anti‐thiram antiserum dilution, the effect of the incubation time on the competitive step, the tolerance to organic solvents. The IC₅₀ and the limit of detection (LOD) values were 60 ng mL^?1 and 9 ng mL^?1, respectively, similar to those of colorimetric ELISA with a calibration range of 9–15,000 ng mL^?1. Cross reactivity of some related compounds such as some dithiocarbamates, a thiocarbamate, the ethylenethiourea and the tetramethylthiourea were tested. The assay was then applied to honeybees sample extracts obtained by using the liquid‐liquid extraction or the graphitized carbon‐based solid phase extraction.

The calibration curves in honeybee extracts from liquid‐liquid procedure gave an IC₅₀ of 141 ng mL^?1 and a LOD of 17 ng mL^?1. In case of extracts obtained by SPE these values were 139 ng mL^?1 and 15 ng mL^?1, respectively. The average recovery value from honeybee extracts spiked with 75 ng mL^?1 of thiram was 72% for SPE, higher than for liquid‐liquid extraction (60%). On the opposite, when the honeybees were directly spiked with 2 and 10 ppm the average recovery was higher for liquid‐liquid extraction (54%), than for SPE (31%). Finally, the assay was applied to honeybee samples collected during monitoring activities in Italy and Russia.     

GC–MS Determination of Antioxidants in Ground Water Contaminated with JP-8

Accidental spills and leaks of kerosene-based fuel require the differentiation of the exact fuel type between kerosene and JP-8. The detection of the antioxidants, 2,6-Di-tert-butylphenol (DTBP) and 2,4-dimethyl-6-tert-butylphenol (DMTBP) in ground water can be an important clue to distinguish between the two. We have developed a method to determine trace phenolic antioxidants in ground water without derivatization by a gas chromatography–mass spectrometry, and then applied it to distinguish JP-8 from kerosene. 25 ground water samples were collected from 25 monitoring wells in an area contaminated with kerosene-based fuel. The antioxidants from ground water were extracted with methylene chloride. Solid phase extraction (SPE) was compared to liquid extraction (LLE), but LLE was selected due to poorer reproducibility and recovery of SPE. Extraction of the compounds from ground water gave recoveries of about 90% and a detection limit of 0.02 μg L^?1. The method was used to analyze groundwater samples contaminated with fuel. DMTBP was detected in concentrations of 0.05–4.65 μg L^?1 in 12 of the samples. Since DMTBP is the only antioxidant used in JP-8 in Korea, this suggests that the fuel in the contaminated samples is JP-8.     

Magnetic nanofibrous polyaniline nanocomposite for solid-phase extraction of naproxen from biological samples prior to its spectrofluorimetric determination

Nanofibrous polyaniline–magnetite (PANI/Fe₃O₄) nanocomposite was in situ prepared through adsorption of magnetite nanoparticles onto PANI nanofibers surface and utilized as an efficient sorbent for magnetic solid-phase extraction of naproxen from water and biological samples, followed a desorption step and spectrofluorimetric determination. Field-emission scanning electron microscopy, transmission electron microscopy, Fourier-transform infrared spectroscopy, thermal gravimetric analysis and X-ray diffraction techniques were employed for characterization of the prepared nanocomposite. The important parameters influencing the extraction efficiency including PANI/Fe₃O₄ mass ratio, adsorbent dose, extraction time, sample solution pH, ionic strength, type and volume of desorption solvent and the elution time were studied and optimized. The investigated nanocomposite was successfully applied to the extraction of naproxen in spiked tap water, urine and plasma samples, with a relative recovery in the range of 90–98%. The reusability of PANI/Fe₃O₄ was examined for ten successive cycles, and the results confirm that the efficiency did not change significantly. A linear calibration plot was obtained in the range of 40–1000 ng mL^?1 with a limit of detection about 17 ng mL^?1 under the optimum conditions. The relative standard deviation (RSD) was found to be 2.34% (n = 8, concentration level of 100 ng mL^?1). The kinetics and thermodynamics of the extraction process were also studied.     

Determination of four phenolic endocrine disrupting chemicals in Dianchi Lake,China

An efficient derivatization method using phenyltrimethylammonium (PTA-OH) has been developed to determine simultaneously four phenolic endocrine disrupting chemicals, 4-n-nonylphenol (4-n-NP), 4-tert-octylphenol (4-t-OP), bisphenol A (BPA) and 4-cumylphenol (4-CP) in surface water of Dianchi Lake (China) by solid-phase extraction (SPE) and gas chromatography-mass spectrometry (GC-MS). Compared with silylation of target phenols using N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA)?+?1% trimethylchlorosilane (TMCS), methylation by PTA-OH displayed a higher response and stability based on the investigations of various derivatization conditions, including derivatization solvent, amount of derivatization reagent, reaction temperature and time. Experiments were carried out to examine the performance of the proposed method based on the correlation coefficient, the method quantification limit (MQL), mean recovery rate and relative standard deviation (RSD). Under optimum derivatization conditions, MQLs of the methylated target compounds were all below 1?ng?L^?1. Results revealed that the proposed method exhibited a satisfactory precision and reproducibility for the separation and determination of target phenols. The proposed method had been applied to determine four phenols in surface water of Dianchi Lake located in southwest of China. The concentrations of 4-n-NP, 4-t-OP, BPA and 4-CP were determined to be 13.6-141.6?ng?L^?1, N.D.-56.5?ng?L^?1, N.D.- 4713.6?ng?L^?1 and 23.3-48.5?ng?L^?1, respectively.     

Dissolved carbon dioxide flotation-assisted in-syringe dispersive liquid–liquid microextraction coupled with microsampling flame atomic absorption spectrometry for selective determination of palladium in water samples

Dissolved carbon dioxide flotation-assisted in-syringe dispersive liquid–liquid microextraction (DCF-IS-DLLME) followed by microsampling flame atomic absorption spectrometry was developed as a simple, inexpensive and fast method for extraction and determination of Pd(II). In the proposed approach, N,N′-bis (naphthylideneimino) diethylenetriamine (NAPdien) was utilized as a selective complexing reagent for Pd(II) ion. Several influential factors on the extraction efficiency including types and volumes of extraction and disperser solvents, pH of the sample solution, concentration of NAPdien and interfering ions were studied. By applying the optimal conditions, a preconcentration factor of 28.7 and limit of detection of 2.5 ng mL^?1 were provided by the proposed method. Linearity was in the range of 10–400 ng mL^?1 with a correlation coefficient (R ²) of 0.9968. Intra-day RSD% values for five repetitive measurements of the spiked solutions at the concentrations of 20 and 100 ng mL^?1 were 5.2 and 2.4%, respectively, whereas it was obtained within the range of 3.6–18.6% for the real samples. Inter-day RSD% values of the spiked solutions were found to be 9.6 and 8.7%, respectively. The results demonstrated that except for Fe²⁺ and Fe³⁺, no remarkable interfering effect was created by the other studied ions for determination of Pd(II) so that the tolerance limits (W _Ion/W _Pd(II)) of the major cations and anions were in the range of 1000–10,000. Finally, DCF-IS-DLLME was successfully applied for determination of Pd(II) in different water samples and the obtained relative recoveries in the range of 94.5–105% illustrated favorable accuracies for the proposed method.     

Determination of Low-Molecular-Mass Aldehydes in Water Samples by Liquid Chromatography and Chemiluminescence Detection Using Continuous in situ Derivatization/Preconcentration with Dansylhydrazine

Abstract

A simple, expeditious, and sensitive method has been developed for the determination of low-molecular-mass aldehydes in water samples by liquid chromatography and peroxyoxalate–chemiluminescence detection. The method is based on continuous solid-phase extraction with in situ derivatization/preconcentration of the aldehydes using dansylhydrazine, which was first adsorbed on an RP–C₁₈ mini-column. For 10 mL of aqueous sample, the limits of detection (LOD) for C₁ to C₄ aldehydes were 20–30 ng L^?1, except for formaldehyde, which had an LOD of 400 ng L^?1. Application was illustrated by the determination of these aldehydes in water samples; the interday precision was always less than ca. 7%, and relative recoveries were more than 96%.     

LC–MS/MS Determination of Catecholamines in Urine Using FMOC-Cl Derivatization on Solid-Phase Extraction Cartridge

A method for the determination of catecholamine derivatives in human urine is proposed that includes the derivatization of target compounds on a solid-phase extraction cartridge and determination of the analytes by a UHPLC method with tandem mass-spectrometric detection. 9-Fluorenyl-methoxycarbonyl chloride was used as the derivatization agent. The limits of detection for the analytes were 2.5 ng mL^?1 for 9-fluorenyl-methoxycarbonyl-adrenaline, 5 ng mL^?1 for 9-fluorenyl-methoxycarbonyl-octopamine, and 25 ng mL^?1 for 9-fluorenyl-methoxycarbonyl-dopamine. The proposed procedure was tested on real samples obtained from volunteers.     

Fast Analysis of Synthetic Pyrethroid Metabolites in Water Samples Using In-Syringe Derivatization Coupled Hollow Fiber Mediated Liquid Phase Microextraction with GC-ECD

A fast and efficient method has been demonstrated for the trace determination of six important metabolites of synthetic pyrethroids including cis- and trans-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropane-1-carboxylic acid (cis- and trans-Cl₂CA), cis-3-(2,2-dibromovinyl)-2,2-dimethylcyclopropane-1-carboxylic acid (cis-Br₂CA), 4-fluoro-3-phenoxybenzoic acid (4-F-3-PBA), 3-phenoxybenzoic acid (3-PBA), and 2-phenoxybenzoic acid (2-PBA) in environmental water samples using hollow fiber (HF)-mediated liquid-phase microextraction (LPME) coupled with in-syringe derivatization (ISD) followed by gas chromatography (GC) with electron capture detector (ECD) analysis. This method utilizes a HF membrane segment impregnated with extraction solvent as the LPME sampling probe, which was connected to a microsyringe pre-filled with derivatizing agents, and it was immersed into sample solution for extraction. After extraction, the extracting solution was subjected to derivatization reaction that was performed inside the syringe barrel followed by GC-ECD analysis. Under optimal conditions, the best extraction efficiency was obtained using sampling probe (2.0 cm hollow fiber) impregnated with 1-octanol immersed into water sample (5.0 mL, adjusted pH below 1.0) and stirring (1,250 rpm) for 10 min at 70 °C and diisopropylcarbodiimide (2 μL) and 1,1,1,3,3,3-hexafluoro-2-propanol (1 μL) were the derivatizing agents used. The detection limits of 3 ng mL^?1 for cis- and trans-Cl₂CA, 2 ng mL^?1 for cis-Br₂CA, 6 ng mL^?1 for 4-F-3-PBA, and 0.6 ng mL^?1 for 3-PBA and 2-PBA. The method showed good linearity (R ² = 0.973?0.998), repeatability from 4.0 to 13 % (n = 5), recovery from 79.2 to 95.7 %, and enrichment factors ranged between 109 and 159 for target analytes spiked in water samples. The proposed method and conventional methods were compared. Results suggested that the proposed HF-LPME-ISD/GC-ECD method was a rapid, simple, inexpensive, and eco-friendly technique for the analysis of metabolites of pyrethroids.     

1-Butyl-3-methylimidazolium hexafluorophosphate ionic liquid-based liquid–liquid microextraction for the determination of 4-nonylphenol and 4- tert -octylphenol in environmental waters

In the present study, room-temperature ionic liquid (RTIL) 1-butyl-3-methylimidazolium hexafluorophosphate was used as extraction solvent in a liquid–liquid microextraction (LLME) procedure followed by liquid chromatography for determining 4-nonylphenol (4-NP) and 4-tert-octylphenol (4-t-OP) in environmental water samples. RTIL-based LLME was a simple, inexpensive, and fast sample preparation method, and its parameters such as extraction time, addition of salt, selection of phase ratio, and pH value were optimized. The optimized method had acceptable limits of detection (LOD) and a precision of 2?µg?L^?1 and 8.1% for 4-NP and 0.6?µg?L^?1 and 3.7% for 4-t-OP, respectively. The proposed method was successfully applied in river water and effluent from a sewage-treatment plant, and the recoveries spiked at 6?µg?L^?1 and 25?µg?L^?1 levels were in the range of 82–113%.     

Diphenyl diselenide grafted onto a Fe<Subscript>3</Subscript>O<Subscript>4</Subscript>-chitosan composite as a new nanosorbent for separation of metal ions by effervescent salt-assisted dispersive magnetic micro solid-phase extraction

Diphenyl diselenide was immobilized on chitosan loaded with magnetite (Fe₃O₄) nanoparticles to give an efficient and cost-effective nanosorbent for the preconcentration of Pb(II), Cd(II), Ni(II) and Cu(II) ions by using effervescent salt-assisted dispersive magnetic micro solid-phase extraction (EA-DM-μSPE). The metal ions were desorbed from the sorbent with 3M nitric acid and then quantified via microflame AAS. The main parameters affecting the extraction were optimized using a one-at-a-time method. Under optimum condition, the limits of detection, linear dynamic ranges, and relative standard deviations (for n?=?3) are as following: Pb(II): 2.0 ng·mL^?1; 6.3–900 ng·mL^?1; 1.5%. Cd(II): 0.15 ng·mL^?1; 0.7–85 ng·mL^?1, 3.2%; Ni(II): 1.6 ng·mL^?1,.6.0–600. ng·mL^?1, 4.1%; Cu(II): 1.2 ng·mL^?1, 3.0–300 ng·mL^?1, 2.2%. The nanosorbent can be reused at least 4 times.

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Graphical abstract Fe₃O₄-chitosan composite was modified with diphenyl diselenide as a sorbent for separation of metal ions by effervescent salt-assisted dispersive magnetic micro solid-phase extraction.

     

Simultaneous derivatization and extraction of iodine from milk samples by hollow fiber liquid-phase microextraction followed by gas chromatography-electron capture detection

A simple, low-cost and sensitive method is demonstrated for derivatization and extraction of iodine from milk samples using hollow fiber liquid-phase microextraction (HF-LPME) and gas chromatography-electron capture detection. Iodide ions are converted to iodine under acidic medium and in the presence of an oxidant. The generated iodine reacted with 3-pentanone in extraction vial to give 2-iodo-3-pentanone and was extracted into 4 μL of 1-octanol located in the lumen of a hollow fiber. Organic solvent was selected using one variable at a time optimization method and the other main factors affecting derivatization and HF-LPME procedures were evaluated using a Taguchi’s L₁₆ (4⁵) orthogonal array. Under optimal conditions, the method showed low limit of detection (0.5 ng mL^?1), wide linear range (1–2,000 ng mL^?1) with good correlation coefficient (0.9997) and acceptable relative standard deviation (4.6 %, n = 5). Finally, the developed method was successfully applied for determination of iodide in real samples including infant milk formulas and cow milk with reasonable relative recoveries (99.8–110.5 %).     

Evaluation of Two Techniques for Extraction of Cortisol from Human Saliva

The aim of the study was to compare two kinds of extraction of cortisol from human saliva. The first was made with dichloromethane and the second by solid-phase extraction. Saliva was sampled in the evening into plastic tubes then cortisol was extracted and analyzed by LC with detection at 240 nm. The limit of detection was 1 ng mL^?1 for extraction with dichloromethane and 3 ng mL^?1 for SPE extraction. Both methods of extraction were applied to the analysis of saliva samples without any interference peaks.