Sulfonamide Antibiotics Research Articles

Yeast biomass-induced Co2P/biochar composite for sulfonamide antibiotics degradation through peroxymonosulfate activation.

Advanced oxidation processes (AOPs) based on peroxymonosulfate (PMS) activation have attracted increasing attention in recent years for organic pollutants removal. Herein, we put forward a facile method to form cobalt phosphide/carbon composite for PMS activation. Combining impregnation approach with pyrolysis treatment enabled the formation of Co2P/biochar composites using baker's yeast and Co2+ as precursors. The as-synthesized products exhibited excellent catalytic activity for sulfamethoxazole (SMX) degradation over the pH range 3.0-9.0by activating PMS. For example, 100% of SMX (20mgL-1) removal was achieved in 20min with catalyst dosage of 0.4gL-1 and PMS loading of 0.4gL-1. Near zero Co2+ leaching was observed during catalytic reaction, which remarkably lowered the toxic risk of transition metal ion in water. Meanwhile, the reusability of catalyst could be attained by thermal treatment. SMX degradation intermediates were identified by liquid chromatography-mass spectrometry (LC-MS), which facilitated the proposal of possible SMX degradation pathways. Ecological Structure Activity Relationships (ECOSAR) analysis indicated that SMX degradation intermediates may not pose ecological toxicity to the environment. Further investigation verified that Co2P/biochar composites could set off PMS activation not only for the degradation of SMX but also for other sulfonamides. In this study, we not only developed a facile method of utilizing environmental-benign biomass for transition metal phosphide/carbon composite formation, but also achieved highly efficient antibiotic elimination by PMS-based AOP.

Photochemistry of dissolved organic matter extracted from coastal seawater: Excited triplet-states and contents of phenolic moieties

Coastal seawater constitutes an important ecosystem receiving inputs of organic micropollutants (OMPs) such as sulfa antibiotics from land-based sources or mariculture activities. It is necessary to investigate photodegradation of OMPs in coastal seawaters for assessing their environmental fate and risks. However, effects of coastal seawater dissolved organic matter (S-DOM) on OMPs photodegradation are largely unknown, given that chemical compositions of S-DOM are different from those of freshwater DOM. Herein, photochemical characteristics of S-DOM extracted from Dalian coastal seawaters were investigated by simulating photochemical experiment adopting sulfachloropyridazine as a case. Results show that S-DOM accelerates the photodegradation mainly through excited triplet-state DOM (3DOM*) with an apparent rate constant (4.43 × 108 M−1 s−1) ten folds of that of freshwater DOM, which is mainly due to much lower phenol contents detected in the S-DOM (0.022 mg-Gallic acid mg-C−1). The S-DOM impacted by mariculture can photogenerate more high-energy 3DOM* than those less impacted by mariculture, further contributing to the high 3DOM* reactivity. The study shows that to accurately predict photolytic persistence of OMPs in field water bodies, it is of significance to determine the second-order reaction rate constants between 3DOM* and target OMPs using DOM extracted from relevant water bodies.

Multidrug-Resistant Hypervirulent Klebsiella pneumoniae Found Persisting Silently in Infant Gut Microbiota.

Since the spread of multidrug-resistant Klebsiella pneumoniae (MDRKP) strains is considered as a challenge for patients with weakened or suppressed immunity, the emergence of isolates carrying determinants of hypervirulent phenotypes in addition may become a serious problem even for healthy individuals. The aim of this study is an investigation of the nonoutbreak K. pneumoniae emergence occurred in early 2017 at a maternity hospital of Kazan, Russia. Ten bacterial isolates demonstrating multiple drug resistance phenotypes were collected from eight healthy full-term breastfed neonates, observed at the maternity hospital of Kazan, Russia. All the infants and their mothers were dismissed without symptoms or complaints, in a satisfactory condition. Whole-genome shotgun (WGS) sequencing was performed with the purpose to track down a possible spread source(s) and obtain detailed information about resistance determinants and pathogenic potential of the collected isolates. Microdilution tests have confirmed production of extended-spectrum β-lactamases (ESBL) and their resistance to aminoglycoside, β-lactam, fluoroquinolone, sulfonamide, nitrofurantoin, trimethoprim, and fosfomycin antibiotics and Klebsiella phage. The WGS analysis has revealed the genes that are resistant to aminoglycosides, fluoroquinolones, macrolides, sulfonamides, chloramphenicols, tetracyclines, and trimethoprim and ESBL determinants. The pangenome analysis had split the isolates into two phylogenetic clades. The first group, a more heterogeneous clade, was represented by 5 isolates with 4 different in silico multilocus sequence types (MLSTs). The second group contained 5 isolates from infants born vaginally with the single MLST ST23, positive for genes corresponding to hypervirulent phenotypes: yersiniabactin, aerobactin, salmochelin, colibactin, hypermucoid determinants, and specific alleles of K- and O-antigens. The source of the MDRKP spread was not defined. Infected infants have shown no developed disease symptoms.

Sulfonamide antibiotics inhibit RNAi by binding to human Argonaute protein 2 PAZ

We found that sulfisomidine, a sulfonamide antibiotic, potently binds to the Piwi/Argonaute/Zwille (PAZ) domain of human Argonaute protein 2 and inhibits RNA interference (RNAi). To elucidate the effect on RNAi of strong affinity of the 3′-ends in small interfering RNA (siRNA) to the PAZ domain, chemically modified siRNAs bearing sulfisomidine at the 3′-end were synthesized.

USB-Powered Coated Blade Spray Ion Source for On-Site Testing Using Transportable Mass Spectrometry.

On-site testing in food analysis using mass spectrometry (MS) requires miniaturization of vacuum systems, mass analyzers, sample cleanup, and ionization sources. In this study, a simple coated blade spray (CBS) ion source was developed that enables high voltage generation on the blade by ubiquitous certified (micro-)USB On-The-Go devices like smartphones, tablets, and power banks. CBS is capable of performing both analyte enrichment by solid-phase microextraction (SPME) material coated on the metal substrate and direct-spray ionization. The USB–CBS device was used on two different MS systems, a transportable single-quadrupole and a benchtop triple-quadrupole tandem MS. Various characteristics of the USB–CBS device, including high voltage generation and angular positioning, were studied. The potential of the newly developed device for food safety applications is demonstrated by banned and regulated veterinary drugs such as β-agonists and sulfonamide antibiotics, covering a wide range of molecular weights and polarities. The results highlight the potential of the developed, simplified, inexpensive (less than 10 USD), and universal vendor-independent USB-powered CBS ion source coupled with MS(/MS) systems for semiquantitative applications, in laboratories, and in future on-site food quality and safety testing. Apart from that, most likely on-site environmental, biomedical, and forensic testing will also benefit from this USB–CBS instrumental development that is compatible with any atmospheric inlet MS system.

Effects of sulfonamide antibiotics on aquatic microbial community composition and functions

Knowledge on interactions among microbial communities colonizing various streambed substrata (e.g. cobbles, sediment, leaf-litter etc.) is essential when investigating the functioning of stream ecosystems. However, these interactions are often forgotten when assessing the responses of aquatic microbial communities to chemical contamination. Using a stream microcosm approach, the respective impact of two sulfonamide antibiotics (sulfamethoxazole and sulfamethazine) on the ability of microbial heterotrophs to decompose alder leaves was investigated in the presence or absence of periphyton. Our hypothesis suggested that sulfonamides would negatively impair microbial litter decomposition and that periphyton could possibly alleviate this effect by stimulating microbial decomposer activity through a priming effect. Results showed that the presence of periphyton enriched water with oxygen and labile dissolved organic carbon forms. However, these labile organic carbon sources did not stimulate leaf-litter decomposition but mostly decoupled microbial decomposer activity from particulate organic matter to dissolved organic matter through negative priming. Also, the two sulfonamide molecules did not affect the leaf-litter decomposition process but significantly decreased bacterial biomass accrual on leaves. The reduction of bacteria was concomitant with an increase in biomass-specific β-glucosidase activity and this was attributed to a stress response from bacteria to sulfonamides. Further research looking at microbial interactions would provide for better assessment of chemical contamination effects in communities and processes in stream ecosystems.

The Forms, Distribution, and Risk Assessment of Sulfonamide Antibiotics in the Manure-Soil-Vegetable System of Feedlot Livestock.

Manure, soil, and vegetable samples were collected from different-sized livestock farms in Xinxiang, China. The residues of sulfadiazine, sulfamonomethoxine, and sulfamethoxazole were analyzed by high-performance liquid chromatography. The results indicated that the concentration ranges of the three total sulfonamides in manure, soil, and vegetables were 10.13-566.23µgkg-1, 7.60-176.26µgkg-1, and 0-32.70µgkg-1, respectively. The mean concentrations were 219.71µgkg-1, 70.73µgkg-1, and 7.08µgkg-1 for manure, soil, and vegetables, respectively. The mean concentrations in soil were lower than the ecotoxic effect trigger value (100µgkg-1), indicating a low risk for organisms in soil. The concentrations of the three sulfonamides varied significantly in different types of vegetables and all were lower than the acceptable daily intake value (50µg (kg day)-1). However, the potential ecotoxicity and danger to human and animal health via accumulation of the antibiotic in the food chain cannot be ignored.

Effect of sulfonamide class of antibiotics on a bacterial consortium isolated from an anaerobic digester of a sewage treatment plant

AbstractThe effect of the sulfonamide class of antibiotics, namely, trimethoprim and sulfamethoxazole (TMP–SMX) on growth and metabolism of a bacterial consortium isolated from an anaerobic digester of a sewage treatment plant, was investigated. The bacterial consortium was exposed to various concentrations of TMP–SMX. The results indicated that there was no effect of TMP and SMX on the bacterial consortium in terms of bacterial growth and carbon and nitrate removal even at the highest concentration tested at 250 mg/L. The carbon removal was more than 90%, and the nitrate removal was more than 87% in all culture conditions. A pure culture was isolated and identified as Leuconostoc mesenteroides from the consortium, which was able to use TMP and SMX as the sole source of carbon for growth. This pure culture has the sul1 gene to show resistance to these antibiotics. This research could be used in the anaerobic digestion process to degrade various classes of sulfonamide antibiotics before the treated sludge is discharged into the environment to reduce the concentration of TMP and SMX.

An assessment of Pleurotus ostreatus to remove sulfonamides, and its role as a biofilter based on its own spent mushroom substrate.

A double strategy based on the removal of sulfonamide antibiotics by Pleurotus ostreatus and adsorption on spent mushroom substrate was assessed to reclaim contaminated wastewater. P. ostreatus was firstly tested in a liquid medium fortified with five sulfonamides: sulfamethoxazole, sulfadiazine, sulfathiazole, sulfapyridine and sulfamethazine, to evaluate its capacity to remove them and to test for any adverse effects on fungal growth and for any reduction in residual antibiotic activity. P. ostreatus was effective in removing sulfonamides up to 83 to 91% of the applied doses over 14days. The antibiotic activity of the sulfonamide residues was reduced by 50%. Sulfamethoxazole transformation products by laccase were identified, and the degradation pathway was proposed. In addition, P. ostreatus growth on a semi-solid medium of spent mushroom substrate and malt extract agar was used to develop a biofilter for the removal of sulfonamides from real wastewater. The biofilter was able to remove more than 90% of the sulfonamide concentrations over 24h by combining adsorption and biodegradation mechanisms.

Exploring the decentralized treatment of sulfamethoxazole-contained poultry wastewater through vertical-flow multi-soil-layering systems in rural communities

Sulfamethoxazole (SMX) is the most widely distributed sulfonamide antibiotics detected in decentralized poultry wastewater in rural communities. As an economically-feasible and eco-friendly technology for decentralized wastewater treatment in rural areas, vertical-flow multi-soil-layering (MSL) system was promising to mitigate the ecological and human health risks from SMX in such areas. The treatment of SMX-contained poultry wastewater by using MSL systems was investigated for the first time, and the main and interactive effects of related multiple variables on system performance were explored through factorial analysis, including material of permeable layer, concentration of SMX, and pH of influent. Results indicated that SMX concentration and pH of influent showed significantly negative effects on SMX removal. Medical stone used in MSL systems with larger surface area could intensify the SMX removal compared to anthracite. MSL systems showed stable performances on SMX removal with the best SMX removal efficiency more than 91%. A novel stepwise-cluster inference (SCI) model was developed for the first time to map the multivariate numeric relationships between state variables and SMX removal under discrete and nonlinear complexities. It was demonstrated that the effect of SMX in wastewater with high concentration was significant on the differentiation of soil bacteria composition in MSL systems based on microbial diversity analysis. These results can help better understand the mechanism of SMX removal in MSL systems from perspectives of factorial analysis, numeric modeling, and microbiological change.

Improving sulfonamide antibiotics removal from swine wastewater by supplying a new pomelo peel derived biochar in an anaerobic membrane bioreactor

Sulfonamide antibiotics (SMs), as a class of antibiotics commonly used in swine industries, pose a serious threat to animal and human health. This study aims to evaluate the performance of an anaerobic membrane bioreactor (AnMBR) with and without supplying a new pomelo peel derived biochar to treat swine wastewater containing SMs. Results show that 0.5 g/L biochar addition could increase more than 30% of sulfadiazine (SDZ) and sulfamethazine (SMZ) removal in AnMBR. Approximately 95% of chemical oxygen demand (COD) was removed in the AnMBR at an influent organic loading rate (OLR) of 3.27 kg COD/(m3·d) while an average methane yield was 0.2 L/g CODremoved with slightly change at a small dose 0.5 g/L biochar addition. SMs inhibited the COD removal and methane production and increased membrane fouling. The addition of biochar could reduce the membrane fouling by reducing the concentration of SMP and EPS.

Key roles of electron cloud density and configuration in the adsorption of sulfonamide antibiotics on carbonaceous materials: Molecular dynamics and quantum chemical investigations

Electron transfer often drives the adsorption, but its role is difficult to determine by traditional experimental methods. In this work, two typical carbonaceous materials, graphene (GPE) and graphyne (GPY) were selected as adsorbents, and three sulfonamide antibiotics, sulfamethazine (SMT), sulfamethoxazole (SMX), and sulfamethizole (SMZ) were used as the model adsorbates. Molecular dynamics simulations and quantum chemical calculations were combined to explore the adsorption behavior and mechanisms. Molecular dynamics results showed that the antibiotic molecules were most likely to be adsorbed at a distance of 4–5 Å from the GPE and GPY surfaces. Subsequently, the energies and electronic information were analyzed based on quantum chemical calculations. The N-atom in the pyrimidine ring of SMT exhibited a stronger electron-donating ability than the O- and S-atoms in the heterocycles of SMX and SMZ, thereby enhancing its interaction with GPE and promoting its adsorption. GPE has a stronger π electron system and conjugation effect compared with GPY, and its triangular electron cloud configuration gives it a stronger adsorption ability. The electron cloud density and configuration played key roles in the adsorption. These results provide fundamental theoretical support for the structural design and optimization of carbonaceous materials and the efficient removal of sulfonamide antibiotics.

Comprehensive evaluation of adsorption performances of carbonaceous materials for sulfonamide antibiotics removal.

Sulfonamide antibiotics have highly toxic effects on humans and other organisms within the food chain. Adsorption by various carbonaceous materials is an effective method for removing them from the aqueous environment. Batch adsorption experiments were conducted between adsorbents and sulfamethoxazole (SMX) by studies of characterization, isotherm model, and kinetic model. The adsorption performances and mechanism of fifteen carbonaceous materials to remove SMX have been comprehensively evaluated. Results of the characterization showed that not only porosity, but also surface chemistry plays an important role in the adsorption process. Changes in the type and quantity of functional groups before and after adsorption are positive for the recyclability of carbonaceous materials. Moreover, kinetic studies showed that the adsorption process followed the pseudo-second-kinetic model and the intra-particle diffusion model. Four adsorbents (i.e., W-GAC, 3M-GAC, GP, and PAC) in this study have the best performance in each corresponding category in terms of the adsorption of SMX. Therefore, the results provide an indispensable reference for evaluating the adsorption performances of a variety of carbonaceous materials, and thus can support the selection of adsorbents for different applications. Graphical abstract.

Encompassment of phthalyl sulfacetamide in α- and β-cyclodextrin using ultrasonication: Physicochemical and computational modeling investigations

Phthalyl sulfacetamide (PSA) is an antibiotic sulfonamide. In this study, inclusion complexes (ICs) of α- (αCD) and β-cyclodextrin (βCD) were prepared in aqueous solution and in solid state with PSA using the ultrasonication (US) process. The structural and thermal characteristics of the US-assisted ICs were investigated by UV–Vis absorption, fluorescence, Fourier transform infrared (FT-IR) spectroscopy, proton nuclear magnetic resonance (1H NMR) spectroscopy, differential scanning calorimetry (DSC), powder X-ray diffraction (PXRD) and computational modeling techniques. The improved dissolution of PSA in the presence of CD was confirmed by UV–Vis absorption. The experimental and computational modeling studies proved that the stoichiometry of the ICs was 1:1 for PSA/CD-ICs. It was also observed that the IC formation between βCD and PSA was the strongest when compared to αCD host. The enhanced thermal stability of PSA was achieved for PSA/CD-ICs when compared to pure form of PSA. FT-IR, 1H NMR and computational modeling studies indicated that biologically active sulfonamide group (ring-A) of PSA was encapsulated in the hydrophobic CD cavity. The stability of ICs in terms of energetic, thermodynamic and electronic properties was verified and the PSA/βCD-IC revealed higher stability compared to that of PSA/αCD-IC, as determined by PM3 method.

Comparing the Effects of Types of Electrode on the Removal of Multiple Pharmaceuticals from Water by Electrochemical Methods

Considering the lack of information on simultaneously removing multiple pharmaceuticals from water or wastewater by electrochemical methods, this study aimed to investigate the removal of multiple pharmaceuticals by electro-coagulation and electro-oxidation based on two types of electrodes (aluminum and graphite). The synthetic wastewater contained a nonsteroidal anti-inflammatory drug (diclofenac), a sulfonamide antibiotic (sulfamethoxazole) and a β-blocker (atenolol). The pharmaceutical removal with electro-oxidation was much higher than those with the electro-coagulation process, which was obtained from a five-cell graphite electrode system, while the removal of pharmaceuticals with aluminum electrodes was about 20% (20 µM). In the electro-coagulation system, pharmaceutical removal was mainly influenced by the solubility or hydrophilicity of the compound. In the electro-oxidation system, the removal mechanism was influenced by the dissociation status of the compounds, which are attracted to the anode due to electrostatic forces and have a higher mass transformation rate with the electro-oxidation process. Therefore, atenolol, which was undissociated, cannot adequately be eliminated by electro-oxidation, unless the electrode’s surface is large enough to increase the mass diffusion rate.

Wide-scope screening for pharmaceutically active substances in a leafy vegetable cultivated under biogas slurry irrigation

The use of livestock waste for the production of biogas and the application of biogas slurry to agricultural soil can resolve livestock waste problems and reduce synthetic fertiliser use. However, the migration of veterinary drugs to land and crops resulting from biogas slurry irrigation is a potential food safety concern. This study employed an ultra-performance liquid chromatography coupled with quadrupole-time of flight high-resolution mass spectrometry system for wide-scope suspect screening of pharmaceutically active substances on crop cultivated under biogas slurry irrigation. Briefly, a total of 22 pak choi samples were obtained from a greenhouse farmed in tropical south Taiwan between March 2019 and March 2020. Molecular spectra and fragmented ions (between m/z 70 and 1100) were acquired. Ion features were searched and matched with a library consisting of 1068 compounds. The matrixes in the crop production environment including soil, livestock wastewater, biogas slurry, and groundwater were included in this study to elucidate potential sources of the pharmaceutically active substances. Results demonstrated 23 suspects were matched with high mass accuracy (mass error within ±5.0 ppm) in pak choi. The detection of both bufexamac and nandrolone were confirmed using standards, where a new system of identification points was applied. Nandrolone was detected throughout the pak choi samples as well as livestock wastewater. Tetracycline, macrolide, and sulfonamide antibiotics were presented in biogas slurry and soil but not pak choi. This is the first study to reveal the presence of multiclass pharmaceutically active substances in a crop supplied as food. Such findings suggest that anabolics and antibiotics should be closely monitored in the corps irrigated by biogas slurry in future.

Functionalized ionic liquids-supported metal organic frameworks for dispersive solid phase extraction of sulfonamide antibiotics in water samples

Sulfonamides antibiotic residues are commonly found in environmental samples, which is highly concerning for public safety and environmental protection. The detection of sulfonamides antibiotics (SAs) is quite important but challenging. In this work, functionalized Zr metal-organic frameworks (Zr-MOFs) and imidazole-based ionic liquids (ILs) were selected and designed according to the structures and properties of SAs. By supporting functionalized ILs into the water-stable Zr-MOFs, the novel ILs@Zr-MOFs nanocomposites were synthesized for the pretreatment of SAs. [H2Nmim][NTf2]@UiO-66-Br showed good selectivity for SAs with maximum adsorption capacity of 352.1 mg g−1 for sulfadiazine. The satisfied performance attributed to not only the large BET surface areas, but also the multiple interactions between the adsorbent and SAs, including electrostatic interaction, hydrogen bonding interaction and π-π interaction. The as-prepared nanocomposites were applied to the dispersive solid phase extraction of SAs in environmental water samples. Combined with high performance liquid chromatography-diode array detector (HPLC-DAD), the effective extraction and sensitive analysis of SAs was achieved by [H2Nmim][NTf2]@UiO-66-Br with enrichment factors higher than 270 and extraction recoveries between 90.5% and 101.9% in short extraction time (10 min). The detection limits were lower than 0.03 μg L−1.

Whole genome sequencing identifies genetic variants associated with co-trimoxazole hypersensitivity in Asians

Co-trimoxazole, a sulfonamide antibiotic, is used to treat a variety of infections worldwide, and it remains a common first-line medicine for prophylaxis against Pneumocystis jiroveci pneumonia. However, it can cause severe cutaneous adverse reaction (SCAR), including Stevens-Johnson syndrome, toxic epidermal necrolysis, and drug reaction with eosinophilia and systemic symptoms. The pathomechanism of co-trimoxazole-induced SCAR remains unclear. We aimed to investigate the genetic predisposition of co-trimoxazole-induced SCAR. We conducted a multicountry case-control association study that included 151 patients with of co-trimoxazole-induced SCAR and 4631 population controls from Taiwan, Thailand, and Malaysia, as well as 138 tolerant controls from Taiwan. Whole-genome sequencing was performed for the patients and population controls from Taiwan; it further validated the results from Thailand and Malaysia. The whole-genome sequencing study (43 case patients vs 507 controls) discovered that the single-nucleotide polymorphism rs41554616, which is located between the HLA-B and MICA loci, had the strongest association with co-trimoxazole-induced SCAR (P= 8.2×10-9; odds ratio [OR]= 7.7). There were weak associations of variants in co-trimoxazole-related metabolizing enzymes (CYP2D6, GSTP1, GCLC, N-acetyltransferase [NAT2], and CYP2C8). Areplication study using HLA genotyping revealed that HLA-B∗13:01 was strongly associated with co-trimoxazole-induced SCAR (the combined sample comprised 91 case patients vs 2545 controls [P= 7.2×10-21; OR= 8.7]). Astrong HLA association was also observed in the case patients from Thailand (P= 3.2×10-5; OR= 3.6) and Malaysia (P= .002; OR= 12.8), respectively. Ameta-analysis and phenotype stratification study further indicated a strong association between HLA-B∗13:01 and co-trimoxazole-induced drug reaction with eosinophilia and systemic symptoms (P= 4.2×10-23; OR= 40.1). This study identified HLA-B∗13:01 as an important genetic factor associated with co-trimoxazole-induced SCAR in Asians.

Influence of Sulfonamide Contamination Derived from Veterinary Antibiotics on Plant Growth and Development.

Veterinary antibiotics such as sulfonamides are widely used to increase feed efficiency and to protect against disease in livestock production. The sulfonamide antimicrobial mechanism involves the blocking of folate biosynthesis by inhibiting bacterial dihydropteroate synthase (DHPS) activity competitively. Interestingly, most treatment antibiotics can be released into the environment via manure and result in significant diffuse pollution in the environment. However, the physiological effects of sulfonamide during plant growth and development remain elusive because the plant response is dependent on folate biosynthesis and the concentration of antibiotics. Here, we present a chemical interaction docking model between Napa cabbage (Brassica campestris) DHPS and sulfamethoxazole and sulfamethazine, which are the most abundant sulfonamides detected in the environment. Furthermore, seedling growth inhibition was observed in lentil bean (Lens culinaris), rice (Oryza sativa), and Napa cabbage plants upon sulfonamide exposure. The results revealed that sulfonamide antibiotics target plant DHPS in a module similar to bacterial DHPS and affect early growth and the development of crop seedlings. Taking these results together, we suggest that sulfonamides act as pollutants in crop fields.

Lab-In-Syringe for automated double-stage sample preparation by coupling salting out liquid-liquid extraction with online solid-phase extraction and liquid chromatographic separation for sulfonamide antibiotics from urine

A double-stage Lab-In-Syringe automated extraction procedure coupled online to HPLC for the determination of four sulfonamides in urine has been developed. Our method is based on homogeneous liquid-liquid extraction at pH 3 using water-miscible acetonitrile with induction of phase separation by the addition of a saturated solution of kosmotropic salts MgSO4 and NaCl. The procedure allowed extraction of the moderately polar model analytes and the use of a solvent that is compatible with the used separation technique. The automated sample preparation system based on the stirring-assisted Lab-In-Syringe approach was coupled on-line with HPLC-UV for the subsequent separation of the sulfonamide antibiotics. To improve both preconcentration factor and extract cleanup, the analytes were trapped at pH 10 in an anion-exchange resin cartridge integrated into the HPLC injection loop thus achieving a double-stage sample clean-up. Analytes were eluted using an acidic HPLC mobile phase in gradient elution mode. Running the analytes separation and the two-step preparation of the following sample in parallel reduced the total time of analysis to mere 13.5 min. Limits of detection ranged from 5.0 to 7.5 μg/L with linear working ranges of 50–5000 μg/L (r2 > 0.9997) and RSD ≤ 5% (n = 6) at a concentration level of 50 μg/L. Average recovery values were 102.7 ± 7.4% after spiking of urine with sulfonamides at concentrations of 2.5 and 5 mg/L followed by 5 times dilution. To the best of our knowledge, the use of Lab-In-Syringe for the automation of coupled homogeneous liquid-liquid extraction and SPE for preparation of the complex matrices suitable for separation techniques is here presented for the first time.