Analysis of fluoroquinolones in dusts from intensive livestock farming and the co-occurrence of fluoroquinolone-resistant Escherichia coli.

Fluoroquinolones are important therapeutics in human and veterinary medicine. This study aimed to retrospectively analyse sedimentation dusts from intensive-livestock-farming barns for fluoroquinolones and investigate the association between resistant Escherichia coli and the detected drugs. Sedimentation-dust samples (n = 125) collected (1980-2009) at 14 barns of unknown-treatment status were analysed by HPLC and tandem-mass spectroscopy to detect enrofloxacin, ciprofloxacin, marbofloxacin, and difloxacin. Recent microbiological data were included to investigate the relationship between fluoroquinolone presence and fluoroquinolone-resistant E. coli. Fifty-nine dust samples (47%) from seven barns contained fluoroquinolone residues. Up to three different fluoroquinolones were detected in pig and broiler barns. Fluoroquinolone concentrations ranged from 10-pg/mg to 46-ng/mg dust. Fluoroquinolone-resistant E. coli were isolated from four barns. Of all the dust samples, 22% contained non-susceptible isolates. Non-susceptible isolate presence in the dust was significantly associated (p = 0.0283) with detecting the drugs, while drug detection increased the odds (4-fold) of finding non-susceptible E. coli (odds ratio = 3.9877, 95% CI: 1.2854-12.3712). This retrospective study shows that fluoroquinolone usage leads to dust contamination. We conclude that farmers and animals inhale/swallow fluoroquinolones and fluoroquinolone-resistant bacteria due to drug application. Furthermore, uncontrolled drug emissions via air exhausted from the barns can be assumed.

Determination of fluoroquinolones in chicken feces - a new liquid-liquid extraction method combined with LC-MS/MS.

The application of antibiotics including fluoroquinolones to farming animals is widespread and may lead to the development of antibiotic resistance and other environmental effects. To calculate environmental loads and for a proper risk assessment it is necessary to determine the antibiotic concentration in feces. Therefore, a new liquid-liquid extraction method combined with HPLC-MS/MS for the detection of marbofloxacin, ciprofloxacin, enrofloxacin and difloxacin in chicken feces was developed. Recoveries ranged from 51.0% to 83.5%. LOQs were between 0.10 and 1.09µg/kg. Feces of chickens treated with an enrofloxacin dosage of 10mg/kg bodyweight revealed maximum enrofloxacin and ciprofloxacin concentrations of 61.3 and 18.8mg/kg. Both antibiotics could be detected in feces up to two days after the last application in notable amounts (∼1mg/kg). Thus, feces of recently medicated chickens should not be used as a fertilizer without any further processing.

Comparison of different solid-phase extraction materials for the determination of fluoroquinolones in chicken plasma by LC-MS/MS.

Fluoroquinolones are synthetic antibiotics which are frequently used in veterinary medicine e.g. for the treatment of poultry. Their specific importance is based on the fact that they are regarded as antibiotics of last resort because of their broad spectrum of action against Gram-negative and -positive bacteria. Here, a new and sensitive method for the simultaneous determination of four fluoroquinolones (marbofloxacin, ciprofloxacin, enrofloxacin and difloxacin) in chicken plasma by LC-MS/MS was developed. Solid-phase extraction was chosen for sample preparation because a selective sample clean-up is combined with an effective extraction. Various solid-phase extraction materials including polymer-based reversed-phase, silica-based reversed-phase and mixed-mode sorbents were compared. Selection criteria were analyte recovery, sample extract purity and economical aspects (analysis time and elution solvent volume). Best recoveries and minimized elution solvent volumes were achieved using polymeric reversed-phase cartridges. However, post-column infusion experiments revealed that the analysis is influenced by co-eluting matrix components. Hence, a combination of a mixed-mode anion-exchange cartridge and a mixed-mode cation-exchange cartridge was used as final extraction method. This method yield slightly lower analyte recoveries compared to polymeric-reversed-phase cartridges but exhibit no matrix effects. Recoveries of spiked chicken plasma ranged from 61.9% to 84.8% with an inter-day precision of generally less than 12%. LODs are between 0.03 and 0.05µg/L; LOQs are between 0.08 and 0.16µg/L. Maximum plasma concentrations of chickens medicated with an enrofloxacin dosage of 3mg/kg bodyweight were 38.9µg/L for enrofloxacin and 3.3µg/L for its main metabolite ciprofloxacin.

Regional, annual, and individual variations in the dihydroxyacetone content of the nectar of maÌ nuka (Leptospermum scoparium) in New Zealand.

A method was designed and validated for the analysis of dihydroxyacetone in the floral nectar of ma̅nuka (Leptospermum scoparium). The method was applied to samples collected from different regions of the North Island and the Nelson region of the upper South Island of New Zealand during the period 2009-2012 as well as to nectar samples from some Australian Leptospermum species. The ratio of dihydroxyacetone to total sugar (DHA/Tsugar) was classified as low (<0.001 mg/mg), moderate (0.001-0.002 mg/mg), or high (>0.002 mg/mg). Inter- and intraregional variation were observed as well as interannual variation with variation from low to high classification occurring within one region and from low to moderate between years. Australian species also demonstrated elevated levels of dihydroxyacetone in the nectar. Some garden cultivars were shown to produce very high nectar DHA/Tsugar, and a survey of cultivars was undertaken; cultivars with single-flowered red or pink flowers were the most common producers of very high nectar DHA/Tsugar.

Evaluation and subsequent minimization of matrix effects caused by phospholipids in LC-MS analysis of biological samples.

BACKGROUND: The influence of matrix effects in LC-MS/MS analysis of biological samples can be enormous and has to be evaluated during method development. Phospholipids, which are present in considerable quantities in biological fluids, are supposed to cause matrix effects when co-eluting with analytes. Therefore, the reduction of phospholipids should lead to the minimization of matrix effects. METHODOLOGY & RESULTS: Here, a polymeric reversed-phase (PRP) SPE cartridge was compared with a combination of mixed-mode-anion-exchange (MAX) and mixed-mode-cation-exchange (MCX) SPE cartridges regarding elimination of matrix effects during sample clean-up. For evaluation of matrix effects post-column infusion experiments were performed. Phospholipid amount in the sample extract and matrix effects are enhanced using PRP in contrast to the combination of MAX/MCX. CONCLUSION: For an efficient elimination of phospholipids during sample preparation and to improve method accuracy and precision it is advisable to use a combination of MAX/MCX SPE cartridges.