Advantages of MS/MS/MS (MRM3) vs classic MRM quantification for complex environmental matrices: Analysis of beta-lactams in WWTP sludge.

When dealing with complex matrices such as wastewater treatment plant (WWTP) sludge or animal manure, usual MRM quantification may lack enough sensitivity or accuracy due to the presence of numerous interfering compounds co-extracted from the matrix. To circumvent the sensitivity and specificity loss, the method development can be focused on sample extraction, purification or/and optimisation of the detection. In this study, we propose an enhancement of a method for the analysis of five beta-lactams (Amoxicillin, Ampicillin, Cefapirin, Ceftiofur, and Cloxacillin) in WWTP, with the use of a hybrid triple quadrupole-Linear Ion Trap (LIT) spectrometer, enabling triple stage MS acquisition, namely MRM3, in place of the usual MS/MS detection. The adaptation of various parameters such as the secondary fragmentation energy, excitation, and accumulation times of the secondly generated ion are described. The method was then validated and enabled quantification limits between 0.8 and 14.7 ng g-1 associated with accurate quantification (between 98% and 113%). This method is the first to report the use of MRM3 acquisition in an environmental matrix as complex as sludge.

Gentamicin at sub-inhibitory concentrations selects for antibiotic resistance in the environment.

Antibiotics released into the environment at low (sub-inhibitory) concentrations could select for antibiotic resistance that might disseminate to the human microbiome. In this case, low-level anthropogenic sources of antibiotics would have a significant impact on human health risk. In order to provide data necessary for the evaluation of this risk, we implemented river water microcosms at both sub-inhibitory and inhibitory concentrations of gentamicin as determined previously based on bacterial growth in enriched media. Using metagenomic sequencing and qPCR/RT-qPCR, we assessed the effects of gentamicin on water bacterial communities and their resistome. A change in the composition of total and active communities, as well as a gentamicin resistance gene selection identified via mobile genetic elements, was observed during a two-day exposure. We demonstrated the effects of sub-inhibitory concentrations of gentamicin on bacterial communities and their associated resistome in microcosms (simulating in situ conditions). In addition, we established relationships between antibiotic dose and the magnitude of the community response in the environment. The scope of resistance selection under sub-inhibitory concentrations of antibiotics and the mechanisms underlying this process might provide the basis for understanding resistance dispersion and associated risks in relatively low impacted ecosystems.

Improvement of the QuEChERS extraction step by matrix-dispersion effect and application on beta-lactams analysis in wastewater sludge by LC-MS/MS.

In the last decade, beta-lactams use in veterinary and human medicine increased to represent today about 15% of the overall consumption. Beta-lactams tend to degrade and metabolize in the environment. Therefore, analytical methods must be sensitive enough to quantify low concentrations of the parent molecules and also allow detection of metabolites. This study presents the development of a modified QuEChERS method for the extraction of seven beta-lactams and one degradation product (Amoxicillin, Ampicillin, Cefapirin, Cefoperazone, Cefquinome, Ceftiofur, Cloxacillin, and Amoxicillin-Diketopiperazine) from sewage treatment plant sludge and their analysis by liquid chromatography coupled with tandem mass spectrometry. Before the QuEChERS extraction, a dispersion step of the sample with EDTA-treated sand was optimized and added, allowing to facilitate the exchanges between the matrix and the extraction solvent. Then, to decrease the interferences present in the extract, a fast and efficient pass-through SPE was implemented. The optimized method was validated and showed satisfactory performances, in adequacy with the analysis of beta-lactams in solid environmental matrices. Limits of quantification lower than 20 ng.g-1 for all analytes, high accuracy (96%-114% quantification on spiked samples nominal concentration) and interday precision (2%-12% RSD) were obtained. This method was then applied to eight sludge samples. Cefapirin and amoxicillin-diketopiperazine were detected in four samples each, at concentrations of 10.2-53.3 ng.g-1 and 3.0-9.5 ng.g-1 respectively. Thus, the developed method is very effective for the extraction of beta-lactams from environmental solid matrices.

Aminoglycosides analysis optimization using ion pairing liquid chromatography coupled to tandem mass spectrometry and application on wastewater samples.

Aminoglycosides are mostly used as veterinary antibiotics. In France, their consumption accounts for about 10% of all prescribed animal medicine. Due to their high polarity nature (log Kow < -3), they require chromatographic separation by hydrophilic interaction liquid chromatography or ion-pairing chromatography. This study presents the development of an ion pairing liquid chromatography with alkanesulfonates coupled to tandem mass spectrometry for the analysis of 10 aminoglycosides (spectinomycin, streptomycin, dihydrostreptomycin, kanamycin, apramycin, gentamicin, neomycin and sisomicin) in wastewater samples. The novelty of this method lies in the addition of the ion paring salt directly and only into the sample vial and not in the mobile phase, lowering the amount of salt added and consequently reducing signal inhibition. The optimized method was validated and showed satisfactory resolution, performances suitable with the analysis of aminoglycosides in wastewater samples, with limits of quantifications less than 10 ng/mL for most of the compounds, low matrix effects, high accuracy (85%-115% recoveries) and reproducibility (2%-12%RSD). It was then applied successfully to raw and treated wastewater samples.

Gentamicin Adsorption onto Soil Particles Prevents Overall Short-Term Effects on the Soil Microbiome and Resistome.

Antibiotics used in agriculture may reach the environment and stimulate the development and dissemination of antibiotic resistance in the soil microbiome. However, the scope of this phenomenon and the link to soil properties needs to be elucidated. This study compared the short-term effects of a range of gentamicin concentrations on the microbiome and resistome of bacterial enrichments and microcosms of an agricultural soil using a metagenomic approach. Gentamicin impact on bacterial biomass was roughly estimated by the number of 16SrRNA gene copies. In addition, the soil microbiome and resistome response to gentamicin pollution was evaluated by 16SrRNA gene and metagenomic sequencing, respectively. Finally, gentamicin bioavailability in soil was determined. While gentamicin pollution at the scale of µg/g strongly influenced the bacterial communities in soil enrichments, concentrations up to 1 mg/g were strongly adsorbed onto soil particles and did not cause significant changes in the microbiome and resistome of soil microcosms. This study demonstrates the differences between the response of bacterial communities to antibiotic pollution in enriched media and in their environmental matrix, and exposes the limitations of culture-based studies in antibiotic-resistance surveillance. Furthermore, establishing links between the effects of antibiotic pollution and soil properties is needed.

Simultaneous δ2 H and δ18 O analyses of water inclusions in halite with off-axis integrated cavity output spectroscopy.

Stable isotope compositions of ancient halite fluid inclusions have been recognized to be valuable tools for reconstructing past environments. Nevertheless, in order to better understand the genesis of halite deposits, it could be of great interest to combine both δ2 H and δ18 O measurements of the water trapped as inclusions in the defects of the mineral lattice. We developed a method combining off-axis integrated cavity output spectroscopy (OA-ICOS) connected on line with a modified elemental analyzer (EA-OA-ICOS) to perform those measurements. The first step was to test the method with synthetic halite crystals precipitated in the laboratory from isotopically calibrated waters. Water isotopic signatures have been measured with conventional techniques, equilibration for δ18 O and chromium reduction for δ2 H. Then, we modified and optimized a conventional EA to connect it online with an OA-ICOS instrument for H2 O measurements. The technique is first evaluated for calibrated free water samples. The technique is also evaluated for salt matrix effect, accuracy, and linearity for both isotopic signatures. Then, the technique is used to measure simultaneously δ2 H and δ18 O values of halite water inclusions precipitated from the evaporation experiments. Data generated with this new technique appeared to be comparable with those inferred from prior off-line technique studies. The advantages offered by the OA-ICOS technique are the simultaneous acquisition of both isotopic ratios and the substantial reduction of data acquisition time and sample aliquot size. Natural halite samples have been analyzed with this method. Natural halite samples as old as Precambrian have also been analyzed with this method.