Identification of bacterial <Emphasis Type="Italic">N</Emphasis>-acylhomoserine lactones (AHLs) with a combination of ultra-performance liquid chromatography (UPLC), ultra-high-resolution mass spectrometry,and in-situ biosensors

N-Acylated homoserine lactones (AHLs) are produced by Gram-negative bacteria as communication signals and are frequently studied as mediators of the “quorum sensing” response of bacterial communities. Several reports have recently been published on the identification of AHLs from different species and attempts have been made to study their role in natural habitats, for example the surface of plant roots in the rhizosphere. In this article, different analytical methods, including bacterial biosensors and chromatographic techniques, are reviewed. A concept for assignment of the structures of AHLs is also presented. The retention behaviour of derivatives of AHLs containing β-keto or hydroxyl groups and/or double bonds has been evaluated in relation to the separation behaviour of AHLs with saturated and unsubstituted alkanoyl chains. Samples have also been analysed by high resolution mass spectrometry (Fourier-transform ion-cyclotron-resonance mass spectrometry, FTICR-MS), nano liquid chromatography–electrospray ionization ion trap mass spectrometry (nano-LC–MS) and by the aid of a biosensor. The results obtained from ultra performance liquid chromatography (UPLC), FTICR-MS, nano-LC–MS, and bioassays have been compared to attempt structural characterisation of AHL without chemical synthesis of analytical standards. The method was used to identify the major AHL compound produced by the rhizosphere bacterium Acidovorax sp. N35 as N-(3-hydroxydecanoyl)homoserine lactone.     

Detection of quorum sensing molecules in Burkholderia cepacia culture supernatants with enzyme-linked immunosorbent assays

The Burkholderia cepacia complex (Bcc) employs a quorum sensing (QS) mechanism which is a cell density-dependent bacterial communication system to regulate certain gene expressions. As with many other Gram-negative bacteria, Burkholderia cepacia species use (N-acyl-)homoserine lactones (AHLs or HSLs) as signalling molecules. Because of the essential role of QS in bacterial behavior, the aim of this study was to demonstrate the applicability of our in-house-developed enzyme-linked immunosorbent assays (ELISAs) for the detection of bacterial activities via HSLs in B. cepacia strain LA3 culture supernatants. For this purpose the previously developed monoclonal antibodies (mAbs) HSL1/2-2C10 and HSL1/2-4H5 were exploited. N-3-Oxo-decanoyl-L-homoserine lactone (3-oxo-C10-HSL) was used as main analyte throughout all experiments. With the bacterial culture medium (named ABC medium) a matrix effect in both ELISAs was visible (slight increase in optical density, shift in test midpoints (IC₅₀) and working ranges). For example, ELISA with mAb HSL1/2-2C10 and enzyme tracer HSL3-HRP (HSL derivative conjugated to horseradish peroxidase) had an IC₅₀ of 120 μg L⁻¹ for 3-oxo-C10-HSL in phosphate-buffered saline versus 372 μg L⁻¹ in ABC medium. A significant increase of HSLs in B. cepacia strain LA3 culture supernatants after 12 h to 48 h of growth was observed. Although the analytical result of these immunoassays cannot distinguish HSLs from homoserines (HSs), the appearance of these compounds can be easily followed. Hydrolysis and spiking experiments were carried out with these biological samples. According to our knowledge, these are the first immunoassays for the detection of quorum sensing molecules in biological culture supernatants. This study provides a cost-effective, fast, and sensitive analytical method for detection of HSLs/HSs in biological samples without complex sample preparation and will offer a quick idea about B. cepacia activities. The low sample amount requirement (less than 1 mL) constitutes a tremendous advantage for many analytical questions with biological samples.     

Development and characterization of rat monoclonal antibodies for N-acylated homoserine lactones

Quorum sensing (QS) is a communication mechanism between bacteria using diffusible chemical signaling molecules, which are called autoinducers (AI). By detecting the concentration of quorum sensing molecules through binding to a specific receptor protein, bacteria regulate their gene expressions when the concentration of autoinducers and thus the cell density reaches a threshold level. Many Gram-negative bacteria use acylated homoserine lactones (HSLs) as autoinducers. Because of the broad biological functions of HSLs, interest in detection and analysis of HSLs is increasing with a view to their medical, biotechnological, and agricultural applications. In this study, an anti-HSL antibody-based immunochemical detection method has been developed. Four structurally distinct HSL haptens, named HSL1, HSL2, HSL3, and HSL4, have been designed for antibody and assay development. New rat anti-HSL monoclonal antibodies (mAbs) have been produced in-house and characterized with enzyme-linked immunosorbent assays (ELISA), both in the coating antigen and in the enzyme tracer format. Eight mAbs (HSL1-1A5, HSL1-8E1, HSL1/2-2C10, HSL1/2-4H5, HSL4-4C9, HSL4-5E12, HSL4-5H3, and HSL4-6D3) will be presented in this paper. We demonstrate that the anti-HSL mAbs have distinguished sensitivity and selectivity toward HSLs depending upon their chemical structures. The optimized assays are capable of detecting HSLs in the microgram per liter (low micromolar to nanomolar) range. The best IC₅₀ (test midpoint) was 134 ± 30 μg L⁻¹ (n = 54) for N-(3-oxodecanoyl)-l-homoserine lactone (3-oxo-C10-HSL) using mAb HSL1/2-2C10 and HSL1–HRP in the enzyme tracer format. In the coating antigen format, the most selective mAb for N-octanoyl-l-homoserine lactone (C8-HSL) was mAb HSL4-4C9. Additionally, anti-HSL mAbs showed higher sensitivity against hydrolyzed HSLs, namely homoserines. These compounds might also occur under certain biological conditions. This study marks the beginning of new ways for quick and cost-effective HSL detection, requiring small sample amounts (less than 1 mL) and little to no sample preparation.     

Specific assay for homoserine and its lactone in Pisum sativum. Preparation of homoserine hydroxamic acid

The existence of homoserine lactone in Pisum sativum seedlings is demonstrated. L-Homoserine lactone reacts with hydroxylamine, at neutral or alkaline pH, to form homoserine hydroxamic acid. Procedures are described for preparing L-homoserine lactone and L-homoserine hydroxamic acid. The hydroxamic acid yields a color with maximum absorbance at 492 nm with Fe³⁺ in 0.25 N HCl. This reaction permitted assay for total homoserine and homoserine lactone. Six-day old Pisum sativum seedlings, with cotyledons removed, were extracted with 90% ethanol. Evaporation of the ethanol and addition of Na₂SO₄ solution and toluene and centrifugation removed protein lipids and esters. After clarification with activated charcoal, homoserine lactone content was estimated by reaction with NH₂OH and Fe³⁺ reagents. For total homoserine, protein precipitation was with 2 N HCl and toluene. Evaporation to dryness at 60 °C under vacuum converted all homoserine to the lactone. The values found for total homoserine (μmols/g, wet weight) and preformed lactone (%) with the various growth media used were as follows: nitrate 87.4 (14.7%), NH₂OH 75.2 (6.3%), water 70.5 (7.9%), urea 56.4 (18.9%). Acetic anhydride added to homoserine hydroxamic acid forms acetohydroxamic acid, which yields a color with maximum absorbance at 505 nm with Fe³⁺. This color reaction is seven times as sensitive as the reaction of Fe³⁺ with homoserine hydroxamic acid itself.     

Identification of unsaturated N-acylhomoserine lactones in bacterial isolates of Rhodobacter sphaeroides by liquid chromatography coupled to electrospray ionization-hybrid linear ion trap-Fourier transform ion cyclotron resonance mass spectrometry

The identification of two unsaturated N‐acylhomoserine lactones (AHLs) produced by Rhodobacter sphaeroides bacteria, based on liquid chromatography (LC) coupled to a hybrid quadrupole linear ion trap (LTQ)‐Fourier transform ion cyclotron resonance (FTICR) mass spectrometer upon electrospray ionization (ESI), is presented. Besides the confirmation of the signaling molecule already described in the literature, i.e. (Z)‐N‐tetradec‐7‐enoyl‐homoserine lactone (C_14:1‐HSL), we have discovered the occurrence, at low, yet significant levels, of another monounsaturated compound, C_12:1‐HSL, which may extend the number of small diffusible chemical signals known for R. sphaeroides. Both unsaturated AHLs were identified by high‐resolution FTICR mass spectrometry in extracts of bacterial culture media and the occurrence of a C=C bond was assessed upon their conversion into bromohydrins. Collision‐induced dissociation (CID) spectra were then collected on the LTQ mass analyzer. A careful comparison of tandem MS spectra of monounsaturated (i.e., C_12:1‐HSL and C_14:1‐HSL) and saturated AHLs (i.e. C₁₂‐HSL and C₁₄‐HSL) led to the emphasis of two series of product ions, exhibiting 14 Da spaced m/z ratios. Both series were referred to progressive fragmentations at the aliphatic end of the AHL acyl chains, followed by neutral losses of terminal alkenes (i.e. CH₂=CH(CH₂)_nH). In particular, the series located at the higher end of the explored m/z range (>200 Da), observed only for monounsaturated species, enabled the location of the C=C bond between carbons 7 and 8 of the acyl chain. Copyright © 2011 John Wiley & Sons, Ltd.     

Single drop microextraction of homoserine lactones based quorum sensing signal molecules, and the separation of their enantiomers using gas chromatography mass spectrometry in the presence of biological matrices

N-acylated homoserine lactones (AHLs) are produced by Gram-negative bacteria as communication signals and are frequently studied as mediators of the “quorum sensing” response of bacterial communities. AHLs are optically active components and the L-form is known to have activity in the quorum sensing. However, the knowledge regarding the stereospecific production of the AHLs in bacterial cultures is limited; therefore, there is a need for a fast and easy method for their chiral analysis. A method was developed for the preconcentration of the AHLs using single drop microextraction or liquid-liquid microphase extraction in toluene for their analysis using GC-MS. The performance of the method were determined and discussed for the chiral separation of these autoinducers using a capillary column coated with heptakis-(2,3-di-O-acetyl-6-O-t-butyldimethyl-silyl)-β-cyclodextrin. The salient feature of this study is the demonstration, that Burkholderia cepacia LA3 produced D-decanoyl-homoserine lactone beside L-decanoyl- and L-octaonyl- enantiomers.     

Comparison of off- and in-line solid-phase extraction for enhancing sensitivity in capillary electrophoresis using ochratoxin as a model compound

This paper proposes and compares two approaches based on off- and in-line solid-phase extraction (SPE), intended to enhance sensitivity in capillary electrophoresis with ultraviolet detection (CE-UV) using as a model the determination of ochratoxin A (OA) in river water samples. In the off-line SPE mode, the reversed-phase sorbent (octadecilsylane, C₁₈) selectively retains the target analyte (OA) and allows large volumes of the sample (70 mL) to be introduced and subsequently eluted in a small volume (0.1 mL) of an appropriate solution. In the in-line SPE mode, a custom-made microcartridge is inserted near the inlet of the capillary, which is filled with the same C₁₈ sorbent. This solid phase selectively retains OA present in a sample volume as low as approximately 640 μL for subsequent elution with ca. 135 nL of an appropriate eluent. The limit of detection (LOD) obtained with the in-line SPE method was 1 ng L^-1, which is 3 orders of magnitude lower than that obtained with CE-UV and roughly 1 order lower than that provided by the off-line SPE-CE-UV method.     

Determination of sulfonylurea herbicides in soil extracts by solid-phase extraction and capillary zone electrophoresis

Summary Sulfonylurea herbicides in soil extracts were concentrated using off-line solid-phase extraction (SPE), and determined by capillary zone electrophoresis (CZE) and UV detection. The method involves extraction of soils with 0.1 M NaHCO₃ solution and subsequent preconcentration by using C₁₈ cartridges prior to separation of the pesticide using CZE. The results show that a C₁₈ cartridge is suitable for the purification of sulfonylurea herbicides in soil extracts with the recoveries ranging from 65–103%. The separation conditions affecting the resolution and detection sensitivity was systematically investigated. The sulfonylureas were resolved well using 30 mM sodium acetate (NaAc)/acetic acid (HAc)+10% acetonitrile (ACN) buffer at pH 4.80. The calibration plots for the test solutes in the concentration of 0.2–50 mg L⁻¹ were linear with detection limits in the range of 0.05–0.10 mgL⁻¹. The proposed method has been successfully demonstrated for the determination of sulfonylurea herbicides in soil samples.     

Response of aerobic sludge to AHL-mediated QS: Granulation,simultaneous nitrogen and phosphorus removal performance

The effects of different species and concentrations’ signal molecules on aerobic activated sludge system were investigated through batch experiments. Results showed that the fastest NH₄⁺-N oxidization rate and the most extracellular polymeric substances (EPS) secretion were obtained by adding 5 nmol/L N-hexanoyl-l-homoserine lactone (C₆-HSL) into the aerobic activated sludge. Further study investigated the correlation among N-acyl-homoserine lactones-mediated quorum sensing (AHLs-mediated QS), nutrient removal performances and microbial communities with the long-term addition of 5 nmol/L C₆-HSL. It was found that C₆-HSL-manipulation could enhance the stability and optimize the decontamination performance of aerobic granular sludge (AGS) system. Microbial compositions considerably shifted with long-term C₆-HSL-manipulation. Exogenous C₆-HSL-manipulation inhibited quorum quenching-related (QQ-related) activities and enhanced QS-related activities during the stable period. The proposed C₆-HSL-manipulation might be a potential technology to inhibit the growth of harmful bacteria in AGS, which could provide a theoretical foundation for the realization of more stable biological wastewater treatments.     

Comparison of adsorbents for on-line solid-phase extraction of polycyclic aromatic hydrocarbons before liquid chromatography with UV detection

Summary On-line solid-phase extraction (SPE) coupled with reversed-phase liquid chromatography and UV detection at 254 nm has been used for the determination of trace-level polycyclic aromatic hydrocarbons (PAH) in soil extracts. Five commercially available adsorbents (C₈, C₁₈, PLRP-S, PRP-1, and Bond-Elut Env) were evaluated. Results showed that recovery of the PAH decreased with increasing molecular weight, because of their poorer solubility. Recovery of high-molecular-weight PAH was significantly improved by addition of 10% (v/v) acetonitrile to the sample before loading of the SPE adsorbent. PAH recovery ranged from 64.0 to 108% when a 50 mL sample spiked with 1 μg L⁻¹ was applied to these adsorbents. Determination of PAH was possible with detection limits below 0.05 μg L⁻¹, which corresponds to 0.2 μg kg⁻¹ soil. The method was successfully used to determine PAH in soil extracts.     

Improved HPLC determination of fluoxetine and norfluoxetine in human plasma

Summary An HPLC method with fluorescence detection has been developed for the determination of fluoxetine and its main metabolite norfluoxetine in human plasma. Pretreatment of the biological samples by liquid-liquid extraction was used to improve the sensitivity of a previously published SPE procedure. The method uses 200 μL plasma and recovery is good for both analytes. On a C₈ column with a mixture of perchlorate buffer and acetonitrile as mobile phase fluoxetine, norfluoxetine and the internal standard (paroxetine) were eluted in less than 9 min, without interference from the biological matrix. Response for both analytes was linearly dependent on concentration over the range 2.5–500 ng mL⁻¹, and repeatability (RSD%) was <4%. The limit of detection was 1 ng mL⁻¹ for both fluoxetines. Application to plasma samples from depressed patients treated with fluoxetine gave good results. There was no interference from other common CNS drugs. This method seems to be a useful tool for clinical monitoring, because it requires small plasma samples and is highly sensitive and highly selective.     

Heterocyclic Chemistry Applied to the Design of N-Acyl Homoserine Lactone Analogues as Bacterial Quorum Sensing Signals Mimics

N-acyl homoserine lactones (AHLs) are small signaling molecules used by many Gram-negative bacteria for coordinating their behavior as a function of their population density. This process, based on the biosynthesis and the sensing of such molecular signals, and referred to as Quorum Sensing (QS), regulates various gene expressions, including growth, virulence, biofilms formation, and toxin production. Considering the role of QS in bacterial pathogenicity, its modulation appears as a possible complementary approach in antibacterial strategies. Analogues and mimics of AHLs are therefore biologically relevant targets, including several families in which heterocyclic chemistry provides a strategic contribution in the molecular design and the synthetic approach. AHLs consist of three main sections, the homoserine lactone ring, the central amide group, and the side chain, which can vary in length and level of oxygenation. The purpose of this review is to summarize the contribution of heterocyclic chemistry in the design of AHLs analogues, insisting on the way heterocyclic building blocks can serve as replacements of the lactone moiety, as a bioisostere for the amide group, or as an additional pattern appended to the side chain. A few non-AHL-related heterocyclic compounds with AHL-like QS activity are also mentioned.     

Determination of five nitrobenzoic acids in groundwater by solid-phase extraction and liquid chromatography–mass spectrometry

A method involving solid-phase extraction (SPE) and reversed-phase liquid chromatography–mass spectrometry (LC–MS) has been developed for determination, in groundwater, of nitrobenzoic acids associated with 2,4,6-trinitrotoluene production. Pre-concentration on a co-polymer-based SPE cartridge enabled quantitative extraction of the analytes from water. Investigation of negative ion electrospray and atmospheric-pressure chemical ionization mass spectrometry indicated the sensitivity of APCI was more than twice that of ESI. An ¹⁵N-labeled internal standard was used to achieve more accurate quantitation and mass assignment. Recovery was better than 80% when 10 mL water was extracted with the SPE cartridge. Combination of SPE with LC–MS analysis resulted in method detection limits of less than 5 μg L⁻¹. The method has been used for analysis of groundwater samples collected from a site of a former ammunition plant. Contamination with nitrobenzoic acids was determined at μg L⁻¹ levels.     

1H-NMR study of polyisoprenes

¹H-NMR spectra of polyisoprene were assigned using polymers of isoprene-1,1,4,4-d₄, isoprene-1,1,5,5,5-d₅, and isoprene-4,4-d₂ polymerized with various catalysts. The methylene-proton signal at 2.1 ppm in cis-1,4 - and trans-1,4-polyisoprenes was divided into H₄- and H₁-proton signals; H₄ resonated at 2.21 ppm in both cis-1,4 and trans-1,4 units whereas H₁ resonated at 2.05, 2.21, and 2.15 ppm. Splitting due to the dyad sequences of 1,4 and 3,4 units was apparent. The methine-proton (H₃) in a 3,4 unit showed a broad peak centered around 1.5 ppm in C₆D₆. The overlapping of this signal with the methyl-proton signals at 1.73 and 1.63 ppm resulted in some uncertainty in the determination of the microstructure of polyisoprene which contained a considerable amount of 3,4 unit.     

Determination of olanzapine and desmethylolanzapine in the plasma of schizophrenic patients by means of an improved HPLC method with amperometric detection

Summary An improved HPLC method with electrochemical detection has been developed for the determination of olanzapine and its main metabolite, desmethylolanzapine, in human plasma. Chromatographic separation and analysis were performed on a C₈ reversed-phase column with a mixture of methanol, acetonitrile, and pH 3.7 phosphate buffer as mobile phase; 2-methylolanzapine was used as internal standard. Careful pretreatment of the plasma samples was implemented by means of solid phase extraction (SPE). Response was linearly dependent on concentration and precision was satisfactory over the concentration range 0.5–75.0 ng mL⁻¹ for both analytes. The limit of detection was 0.2 ng mL⁻¹ for both analytes. Application to plasma samples of patients treated with Zyprexa tablets gave good results. Because of its sensitivity and selectivity, and the need for small plasma samples, this method seems to be a useful tool for clinical monitoring.     

Determination of Abamectin Residues in Avocados by Microwave-Assisted Extraction and HPLC with Fluorescence Detection

In this article a new analytical method for the confirmation and quantification of abamectin residues in avocados is described. The method allows a fast analysis of abamectin homologues using microwave assisted extraction (MAE), solid-phase extraction (SPE) and high-performance liquid chromatography (HPLC) with fluorescence (FL) detection using trifluoroacetic anhydride (TFAA) and N-methylimidazole (NMIM) as derivatizing agents. The mobile phase consisted of water, methanol and acetonitrile (5:47.5:47.5 v/v/v) and was pumped at a rate of 1.1 mL min⁻¹ (isocratic elution). Homogenized avocado samples were extracted once with 20 mL acetonitrile:water 4:1 (v/v) in a microwave oven for 26 min at 700 W with a maximum temperature of 80 °C. MAE operational parameters were optimized by means of an experimental design. Extracts were cleaned using C₁₈ SPE cartridges. Average recoveries of the method at four spiked levels (0.005, 0.01, 0.10 and 1.0 mg kg⁻¹) were found to be in the range 90–100% with good precision (RSD < 12%). The limits of detection (LODs) and quantification (LOQs) of the whole method were 0.001 and 0.003 mg kg⁻¹, respectively, which are lower than the maximum residue limit (MRL) established by the Spanish and the European legislation in avocados (0.01 mg kg⁻¹). Several avocado samples previously treated with the pesticide were also analyzed.     

Comprehensive profiling of <Emphasis Type="Italic">N</Emphasis>-acylhomoserine lactones produced by <Emphasis Type="Italic">Yersinia pseudotuberculosis</Emphasis> using liquid chromatography coupled to hybrid quadrupole–linear ion trap mass spectrometry

A method for the comprehensive profiling of the N-acylhomoserine lactone (AHL) family of bacterial quorum-sensing molecules is presented using liquid chromatography (LC) coupled to hybrid quadrupole–linear ion trap (QqQLIT) mass spectrometry. Information-dependent acquisition (IDA), using triggered combinations of triple-quadrupole and linear ion trap modes in the same LC-MS/MS run, was used to simultaneously screen, quantify and identify multiple AHLs in a single sample. This MS method uses common AHL fragment ions attributed to the homoserine moiety and the 3-oxo-, 3-hydroxy- or unsubstituted acyl side chains, to identify unknown AHLs in cell-free culture supernatants in an unbiased manner. This LC-MS technique was applied to determine the relative molar ratios of AHLs produced by Yersinia pseudotuberculosis and the consequences of inactivating by mutation either or both of the AHL synthase genes (ypsI and ytbI) on AHL profile and concentration. The Y. pseudotuberculosis wild type but not the ypsI ytbI double mutant produced at least 24 different AHLs with acyl chains ranging from C4 to C15 with or without 3-oxo or 3-hydroxy substituents. YtbI, in contrast to YpsI, could direct the synthesis of all of the AHLs identified. The most abundant and hence most biologically relevant Y. pseudotuberculosis AHLs were found to be the 3-oxo-substituted C6, C7 and C8 AHLs and the unsubstituted C6 and C8 compounds. The LC-QqQLIT methodology is broadly applicable to quorum-sensing signal molecule analysis and can provide comprehensive AHL profiles and concentrations from a single sample and simultaneously collect confirmatory spectra for each AHL identified. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users. Catharine A. Ortori and Steve Atkinson made an equal contribution to the paper.     

CE Analysis of Cephalosporins in Environmental Waters

This study investigates an off-line solid phase extraction (SPE) for improving the sensitivity in the capillary electrophoretic (CE) analysis of four cephalosporins. Two sorbents—LiChrolut-C₁₈ and Oasis HLB—were used in a SPE process to detect cephalosporins in natural waters (tap, river and hospital sewage) and their performances were compared. By using Oasis HLB sorbent higher recoveries for river water were obtained (94–107% when 500 mL of sample were analyzed). The off-line SPE–CZE method was validated for river water with good detection limits (3 μg L⁻¹) and the linearity ranged between 5 and 200 μg L⁻¹.     

The quorum-sensing molecule <Emphasis Type="Italic">N</Emphasis>-3-oxododecanoyl homoserine lactone (3OC12-HSL) enhances the host defence by activating human polymorphonuclear neutrophils (PMN)

The P. aeruginosa quorum-sensing molecule N-3-oxododecanoyl homoserine lactone (3OC12-HSL) interacts not only with bacteria, but also with mammalian cells, among others with those of the immune defence system. We focussed on the possible interaction of 3OC12-HSL with human polymorphonuclear neutrophils (PMN), because these cells are the first to enter an infected site. We found that 3OC12-HSL attracts PMN, and up-regulates expression of receptors known to be involved in host defence, including the adhesion proteins CD11b/CD18 and the immunoglobulin receptors CD16 and CD64. Furthermore, the uptake of bacteria (phagocytosis), which is crucial for an efficient defence against infection, was enhanced. Thus, recognising and responding to 3OC12-HSL not only attracts the PMN to the site of a developing biofilm, but also reinforces their defence mechanisms, and hence could be a means to control the infection in an early stage and to prevent biofilm formation.     

Improving the sensitivity of the determination of ceftiofur by capillary electrophoresis in environmental water samples: in-line solid phase extraction and sample stacking techniques

This paper describes two different approaches for increasing the sensitivity for the analysis of ceftiofur by capillary electrophoresis (CE). Two different techniques based on the introduction of an enlarged volume of sample, namely large volume sample stacking (LVSS) and in-line solid phase extraction (SPE) were studied and compared. LVSS allowed the on-column electrophoretic preconcentration of ceftiofur without modification of the separation capillary. In-line SPE-CE was developed by using a home-made microcartridge that was filled with a reversed-phase sorbent (C₁₈). The microcartridge was coupled in-line near the inlet of the separation capillary. LVSS and in-line SPE-CE allowed automated operation and improved sensitivity for the analysis of ceftiofur with respect to conventional CE. When environmental water samples were analyzed, an additional pretreatment step based on off-line SPE was necessary in both cases to further decrease the detection limits. In terms of sensitivity for the determination of ceftiofur in river water samples, the combination of off-line SPE with in-line SPE-CE was found the most sensitive with a detection limit of 10 ng L⁻¹, whereas the method based on the use of off-line SPE with LVSS presented a detection limit of 100 ng L⁻¹.