Development of an efficient extraction method for oxytetracycline in animal manure for high performance liquid chromatography analysis

Abstract

Oxytetracycline (2-(amino-hydroxy-methylidene)-4-dimethylamino-5,6,10,11,12a-pentahydroxy-6-methyl-4,4a,5,5a-tetrahydrotetracene-1,3,12-trione) is a major member of the tetracycline antibiotics family of which are widely administered to animals in concentrated animal feeding operations for purposes of therapeutical treatment and health protection. With the disposal of animal manure as fertilizer into agricultural land, tetracyclines enter the environment. However, tetracyclines chelate with multivalent cations and proteins, resulting in low extraction efficiencies from animal manure for tetracycline residue analysis. In this study an efficient extraction method for oxytetracycline from steer manure using methanol/water solution amended with chelating organic acid was developed for the analysis of high performance liquid chromatography. The effect of species and amount of amendment acids, shaking time, methanol/water ratio, manure weight, and repeated times of extraction was investigated. It was optimized to amend 2.5 g citric acid and 1.1 g oxalic acid with 10.0 g manure sample in a 50-ml centrifuge tube and extract with 15 ml methanol/water (9:1 in volume) by vigorously shaking for 30 min in a reciprocating shaker. After centrifugation at 11,000 rpm, supernatant is collected. Sample was extracted for a total of 3 times. The developed extraction method was further applied to extract oxytetracycline from fresh and aged cow manure, swine and poultry manure, and soil. Satisfactory recoveries ranging from (84.1 ± 2.4) % to (102.0 ± 3.1) % were obtained, demonstrating that the optimized extraction method is robust for oxytetracycline from different manure sample matrixes.

Keywords:

• Oxytetracycline
• tetracyclines
• antibiotic
• extraction
• manure
• analysis
• high performance liquid chromatography

Acknowledgement

This project was partially supported by Delaware EPSCoR RII-2 Program (National Science Foundation grant # EPS-0814251). The authors sincerely appreciate the technical help from Ping Zhang for the part of work which was done in USDA/ARS Salinity Laboratory.

Notes

^a Measured at manure:water (in wet wt.) = 1:2

^b manure:water = 1:6 (in wet wt.)

^c manure(or soil):water = 1:3 (in wet wt.).