Multiresidue determination of chlorophenoxy acid herbicides in human urine samples by use of solid-phase extraction and capillary LC–UV detection

Chlorophenoxy acid herbicides are intensively applied to get rid of unwanted plants because of their low cost and selectivity. Due to their toxicity, which depends on their chemical form, the European Community has established legal directives to restrict their use and to control their maximum residue levels in several matrices. Determination of chlorophenoxy acids—2,4-dichlorophenoxyacetic acid (2,4-D), 4-chloro-2-methylphenoxyacetic acid (MCPA), 2-(2,4-dichlorophenoxy)propanoic acid (2,4-DP), 2-(4-chloro-2-methylphenoxy)propanoic acid (MCPP), 4-(4-chloro-2-methylphenoxy)butanoic acid (MCPB) and 2-(2,4,5-trichlorophenoxy)propanoic acid (2,4,5-TP) in spiked human urine samples has been carried out by capillary LC, after solid-phase extraction on a column packed with silica C₁₈ restricted-access material. Chromatographic analysis was performed in gradient-elution mode at 25 °C, with injection of 20 μL low-organic-solvent composition herbicide solutions for focusing purposes on the head of the capillary column, and diode array detection at 232 nm. Urine samples collected during 24 h from healthy and unexposed volunteers were spiked in the concentration range 25–150 μg L⁻¹; recoveries obtained were between 66 and 100% (n = 6 for each spiked level) and RSDs (relative standard deviations) were between 1 and 5%. Detection limits in the urine samples from volunteers were between 3.5 and 6.0 μg L⁻¹. The developed methodology has allowed the clean-up and preconcentration of low volumes of untreated human urine without previous treatment, showing the effectiveness of the employed SPE sorbent for extracting the target analytes and ultimately resulting in the reduction of the sample-preparation time.     

Chiral Determination of Salbutamol, Salmeterol and Atenolol by Two-Dimensional LC–LC: Application to Urine Samples

A heart-cut two-dimensional high-performance liquid chromatography method for enantiomeric determination of salbutamol, salmeterol and atenolol in urine is presented. It involves the use of two separations in a liquid chromatography?Cliquid chromatography achiral?Cchiral coupling. Target compounds were previously separated in a primary column (Kinetex? HILIC, 2.6???m, 150?×?2.1?mm I.D.) with a mixture of MeOH:ACN:ammonium acetate buffer (5?mM, pH 6) 90:5:5 (v/v/v) as mobile phase at a flow rate of 0.40?mL?min^?1. Enantiomeric separation was carried out by transferring peak of each compound through a switching valve to a vancomycin chiral column (Chirobiotic? V, 2.6???m, 150?×?2.1?mm I.D.) using MeOH:ammonium acetate buffer (2?mM, pH 4) 97:3 (v/v) as mobile phase at a flow rate of 0.50?mL?min^?1. Ultraviolet detection was done at 227?nm. The method was applied to determine target analytes in urine samples after enzymatic hydrolysis with ??-glucuronidase from Helix pomatia, followed by a solid-phase extraction procedure using Isolute^? HCX mixed-mode cartridges. Extraction recoveries ranged from 82 to 90?% in urine samples. Detection limits were 0.091?C0.095???g for each enantiomer of atenolol and between 0.058 and 0.076 and 0.18?C0.14???g for enantiomers of salbutamol and salmeterol, respectively (3?mL of urine). Linearity ranges were between 0.5 and 10???g?mL^?1. Intraday and interday reproducibilities of enantiomeric ratio and enantiomeric fraction, expressed as relative standard deviation, were between 1.9 and 9.0?%. The optimized method was successfully applied to the analysis of urine samples obtained from excretion studies in volunteers and in freeze-dried urine samples, containing urinary components with MW?<?10,000 and components with MW?>?10,000, spiked with different amounts of studied drugs.     

Effect of temperature on the separation of chlorophenoxy acids and carbamates by capillary high-performance liquid chromatography and UV (or diode array) detection

In this work, the effect of temperature in isothermal and programmed modes on several chromatographic parameters such as retention factor, selectivity, resolution and plate number has been discussed. A critical comparison of isocratic/isothermal, gradient/isothermal and isocratic/program temperature modes has been made. Two representative families of pesticides have been selected for this study. One includes ionisable chlorophenoxy acids and two of their esters, some of which show similar polarities. The other one contains several weakly polar carbamates. Analysis was carried out using a reversed-phase capillary high-performance liquid chromatography (HPLC) system and focusing technique with UV or diode array detection (DAD).     

Chiral Determination of Salbutamol,Salmeterol and Atenolol by Two-Dimensional LC–LC: Application to Urine Samples

A heart-cut two-dimensional high-performance liquid chromatography method for enantiomeric determination of salbutamol, salmeterol and atenolol in urine is presented. It involves the use of two separations in a liquid chromatography–liquid chromatography achiral–chiral coupling. Target compounds were previously separated in a primary column (Kinetex™ HILIC, 2.6 μm, 150 × 2.1 mm I.D.) with a mixture of MeOH:ACN:ammonium acetate buffer (5 mM, pH 6) 90:5:5 (v/v/v) as mobile phase at a flow rate of 0.40 mL min⁻¹. Enantiomeric separation was carried out by transferring peak of each compound through a switching valve to a vancomycin chiral column (Chirobiotic™ V, 2.6 μm, 150 × 2.1 mm I.D.) using MeOH:ammonium acetate buffer (2 mM, pH 4) 97:3 (v/v) as mobile phase at a flow rate of 0.50 mL min⁻¹. Ultraviolet detection was done at 227 nm. The method was applied to determine target analytes in urine samples after enzymatic hydrolysis with β-glucuronidase from Helix pomatia, followed by a solid-phase extraction procedure using Isolute^® HCX mixed-mode cartridges. Extraction recoveries ranged from 82 to 90 % in urine samples. Detection limits were 0.091–0.095 μg for each enantiomer of atenolol and between 0.058 and 0.076 and 0.18–0.14 μg for enantiomers of salbutamol and salmeterol, respectively (3 mL of urine). Linearity ranges were between 0.5 and 10 μg mL⁻¹. Intraday and interday reproducibilities of enantiomeric ratio and enantiomeric fraction, expressed as relative standard deviation, were between 1.9 and 9.0 %. The optimized method was successfully applied to the analysis of urine samples obtained from excretion studies in volunteers and in freeze-dried urine samples, containing urinary components with MW < 10,000 and components with MW > 10,000, spiked with different amounts of studied drugs.

     

Determination of chlorophenoxy acid herbicides and their esters in soil by capillary high performance liquid chromatography with ultraviolet detection, using large volume injection and temperature gradient

A simple method for the simultaneous determination of chlorophenoxy acid herbicides and their esters in soil is presented. Compounds studied are: 2,4-dichlorophenoxyacetic acid (2,4-D), 4-(2,4-dichlorophenoxy)butanoic acid (2,4-DB), 2,4-dichlorophenoxyacetic-1-butyl ester (2,4-D-1-butyl ester), and 2,4-dichlorophenoxyacetic-1-methyl ester (2,4-D-1-methyl ester).

The chromatographic analysis was carried out by HPLC, after ultrasonic extraction, on a C₁₈ packed capillary column with temperature gradient, large injection volumes and UV detection at 232 nm. Samples were spiked with amounts between 2.5 and 6.0 μg g⁻¹ of each herbicide; recoveries obtained were between 72 and 97% (n=3 for each spiked level) and detection limits were between 0.3 and 0.5 μg g⁻¹.

Application of this procedure to the analysis of herbicides in ester and acid forms showed the effectiveness of the methodology proposed.     

Capillary liquid chromatography of chlorophenoxy acid herbicides and their esters in apple juice samples after preconcentration on a cation exchanger based on polydivinylbenzene-N-vinylpyrrolidone

A capillary liquid chromatography (cLC) method with gradient elution has been used to determine chlorophenoxy acid herbicides: 2,4-dichlorophenoxyacetic acid, 4-chloro-2-methylphenoxyacetic acid, 2-(2,4-dichlorophenoxy)propanoic acid, 2-(4-chloro-2-methylphenoxy)propanoic acid, 4-(2,4-dichlorophenoxy)butanoic acid, 4-(4-chloro-2-methylphenoxy)butanoic acid, 2-(2,4,5-trichlorophenoxy)propanoic acid, 2,4-dichlorophenoxyacetic-1-methyl ester and 2,4-dichlorophenoxyacetic-1-butyl ester in spiked apple juice samples with amounts between 0.025 and 0.150 mg kg(-1) of each herbicide. Clean-up and preconcentration of acid and esters were carried out in an Oasis MCX polymer. Detection limits obtained by cLC, between 0.005 and 0.018 mg kg(-1), allowed the determination of chlorophenoxy acids and their esters in apple juice samples around the levels permitted by the European Regulations, with recoveries in the range 84-99% and RSDs between 1 and 4%.     

Determination of heterocyclic aromatic amines by capillary high-performance liquid chromatography with diode array detection in ready-to-eat cooked ham treated with electron-beam irradiation

Heterocyclic aromatic amines (HAs) are a group of mutagenic and carcinogenic substances present in significant amounts in cooked meat and fish that can potentially be formed during food processing operations. This paper proposes a capillary liquid chromatography method with diode array detection for the trace-level determination of three HAs, namely, MeIQx (2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline), norharman (9H-pyrido[3,4-b]indole) and harman (1-methyl-9H-pyrido[3,4-b]indole), in ready-to-eat (RTE) cooked ham processed by electron-beam (accelerated electrons) irradiation to eliminate pathogenic microorganisms and to extend its shelf-life. The HAs selected have frequently been detected and quantified in a wide range of food and could be potential markers to indicate the presence of these toxic compounds. The method is based on the separation in an Inertsil C(8) capillary column (150 mm x 0.3-mm internal diameter, 3 microm) by gradient elution mode using a mixture of acetonitrile and 30 mM ammonium acetate pH 4.5 buffer as the mobile phase. Detection was at 250 and 265 nm and, to improve sensitivity, large injection volumes (20 microL) and on-column focusing techniques based on the injection of HA samples in low organic solvent strength solutions were employed. A simple and short solid-phase extraction and purification procedure was also optimized for sample preparation. Nonirradiated and irradiated RTE cooked ham samples at doses between 1 and 8 kGy were analyzed. HAs were not detected in any of the samples analyzed; so both types of samples were spiked at concentration levels in the range 5-25 ng g(-1), which may be found in meat products. The quality parameters of the method developed in the food matrix were established, and detection limits around 0.3 ng g(-1) were obtained. Spiked recoveries between 70 and 79% (n = 3 for each spiked level) relative standard deviations between 1 and 5% were also obtained, showing the effectiveness of the proposed method.     

Large injection volumes in capillary liquid chromatography: Study of the effect of focusing on chromatographic performance

This paper describes a multivariate approach to study the effect on chromatographic conditions and to optimize such conditions in capillary liquid chromatography when high injection volumes are required. Several separations have been evaluated by using isocratic and gradient solvent elution, as well as isocratic elution combined with temperature programming. In this study, easily ionisable organic compounds with low logP have been used as representative analytes. Injection volume and nature of the injection solution have been evaluated in order to increase the sensitivity (peak area) and column performance (N values). The equations obtained by multiple linear regressions and response surfaces allow achieving the optimum on-column focusing conditions for chlorophenoxy acids, carbamates and heterocyclic amines.     

Enantiomeric separation of chlorophenoxy acid herbicides by nano liquid chromatography-UV detection on a vancomycin-based chiral stationary phase

Enantiomeric separation of mecoprop, dichlorprop, and fenoprop herbicides in their acid form, commonly used to control the growth of broad-leaved weeds, was carried out by nano-liquid chromatography (nano-LC) at a flow rate of 60 nL/min, using a packed capillary column with vancomycin-modified silica particles of 5 microm. The length of chiral stationary phase was 21 cm, while the total and effective lengths were 43 and 33cm, respectively. Inner diameter was 0.075 mm. Separated peaks were detected at 195 nm. Several mixtures of methanol, water, and 500 mM ammonium acetate buffer at different pH's were tested as mobile phase, and experimental parameters such as resolution (Rs), capacity factor (k), efficiency (N/m), and enantioselectivity factor (alpha) were measured under all the test conditions. Baseline enantiomeric separation was obtained for the three studied herbicides with alpha in the range 1.6-1.9, using as the mobile phase aqueous solutions containing 85% methanol, 5% of 500mM ammonium acetate pH4.5 buffer, and 10% water. Experimental results show that the vancomycin stationary phase presents a great enantiorecognition capability towards chlorophenoxy acid herbicides on using nano-LC.     

Direct chiral liquid chromatography determination of aryloxyphenoxypropionic herbicides in soil: deconvolution tools for peak processing

In this paper, the enantiomeric separation of two aryloxyphenoxypropionic esters (fluazifop-butyl and quizalofop-ethyl) and a safener herbicide (mefenpyr-diethyl), which is widely used for protecting crop plants, has been studied by direct liquid chromatography (LC) with UV detection on an α₁-acid glycoprotein as chiral stationary phase. Optimization of separation conditions was done by factorial experimental design. Experimental factors and ranges selected were propanol (5–10%), phosphate buffer pH (6.5–7.0), and column temperature (15–25 °C). Responses were expressed in terms of enantioresolution (R _s) and adjusted retention time of the second eluted enantiomer (t _r2′). The chemometric method used to explore data was response surface analysis. Multiple response analyses were carried out to determine the combination of experimental factors which simultaneously optimize experimental responses. Under optimum conditions for enantioseparation of each herbicide, partially overlapped or fully resolved enantiomers were obtained. Deconvolution tools were employed as an integration method to fit chromatographic data and to achieve a more precise enantiomeric ratio (ER) and enantiomeric fraction (EF) values. Applicability of both direct chiral LC and peak deconvolution methods was evaluated in spiked soil samples at different R/S enantiomeric ratios. Acceptable and reproducible recoveries between 71% and 96% with precision in the range 1–6% were achieved for herbicide-spiked levels from 0.50 to 9.0 μg g^–1. In addition, parameters such as R _s, ER, and EF were calculated and compared with values obtained using the common valley drop integration method.