Enhancing Extraction and Detection of Veterinary Antibiotics in Solid and Liquid Fractions of Manure

Abstract

Analysis of veterinary antibiotics in separated liquid and solid fractions of animal manures is vital because of wide variations in the composition of agriculturally applied manure. Differentiation of antibiotic concentrations is important between liquid and solid manures, as their sorption onto the solid fraction depends on physicochemical properties of each antibiotic and manure composition (e.g., organic content, pH) and because each fraction may be treated and reused differently. Here, an efficient and sensitive method for the analysis of 22 veterinary antibiotics in the liquid and solid fractions of manure is reported. Tetracycline (TC), macrolide, and sulfonamide antibiotics were extracted from liquid manure by liquid-liquid extraction (LLE) with methanol following acidification with acetic acid. Extraction from solids was performed by sonication with acetonitrile, methanol, and 0.1 M EDTA-McIlvaine buffer. Cleanup of extracts was achieved by solid-phase extraction with hydrophilic-lipophilic balance (HLB) cartridges or tandem amino (NH2) and HLB cartridges. Quantification of antibiotics was performed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) under wrong-way-round (WWR) ionization for sulfonamides and TCs and right-way-round ionization for macrolides. Recoveries of 58 to 94.7% and 62 to 94.3% were obtained in liquid and solid manure, respectively. Method detection limits range from 1.2 to 12 ng L and 0.5 to 7.9 μg kg dry wt. in liquids and solids, respectively. This method allows for extraction and analysis of both mobile antibiotics in liquid phase and hydrophobic antibiotics adsorbed on the solids. Without separate analysis, antibiotic concentrations may be improperly estimated by analyzing whole manure, as reported in many studies to date.

Metagenomic profiling of historic Colorado Front Range flood impact on distribution of riverine antibiotic resistance genes

“…A separate LC-MS/MS method was adapted from Wallace and Aga42 for macrolide antibiotics to enhance sensitivity. All analytes were normalized to the internal standard d 10 -carbamazepine (sulfonamides and macrolides) or minocycline (tetracyclines).…”

On‐farm screw press and rotary drum treatment of dairy manure‐associated antibiotic residues and resistance

“…Samples were analyzed for nine sulfonamide, seven tetracycline, and nine macrolide antibiotic residues using liquid chromatography with tandem mass spectrometry (LC-MS/MS), following the methods of Wallace and Aga (2016). While β-lactams were used in the herd, these antibiotics were not analyzed based on our preliminary studies that showed non-detection in manure.…”

“…Studies have shown some persistent antibiotic residues (e.g. tetracyclines, macrolides) adsorb to organics and are detected at higher concentrations in dairy manure solids (Wallace & Aga, 2016;Hurst et al, 2018). There is also evidence that SLS technologies can reduce the occurrence of bovine pathogens (Liu et al, 2017), and ARGs (Tien et al, 2017;Wallace et al, 2018).…”

<i>Antibiotics and antibiotic resistant bacteria and genes in northeastern dairy manure management systems � Project overview and preliminary findings from an 11 farm case study</i>

“…Antibiotic residues were extracted and analyzed for select Sulfonamides, Macrolides, and Tetracycles similarly to the methods of Wallace & Aga (2016). Frozen manure samples were freeze dried, homogenized and 100 mg of dried manure solids were added to a polypropylene centrifuge tube.…”