High-performance liquid chromatography method for the simultaneous determination of sulfamethoxazole and trimethoprim in bovine milk using an on-line clean-up column

• Published in: Journal of chromatography. B, Analytical technologies in the biomedical and life sciences Vol. 826; no. 1; pp. 139 - 146
• Main Authors: Pereira, A.V., Cass, Q.B.
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 05-11-2005; Elsevier Science
• Subjects: Analysis; Analytical, structural and metabolic biochemistry; Animals; Biological and medical sciences; Bovine milk; Cattle; Chromatography, High Pressure Liquid - methods; Column-switching; Direct injection; Drug Stability; Female; Freezing; Fundamental and applied biological sciences. Psychology; General pharmacology; Medical sciences; Milk - chemistry; Online Systems; Pharmacology. Drug treatments; Reproducibility of Results; Restricted access media (RAM); Sensitivity and Specificity; Sulfamethoxazole; Sulfamethoxazole - analysis; Sulfonamide; Trimethoprim; Trimethoprim - analysis; Trimethoprim, Sulfamethoxazole Drug Combination - therapeutic use; Restricted access media (RAM); Sulfamethoxazole; Sulfonamide; Column-switching; Bovine milk; Trimethoprim; Direct injection; Sulfanilamide derivatives; Accessibility; HPLC chromatography; Ox; Switching; Antifolate; Sulfonamide: Sulfamethoxazole; Vertebrata; Mammalia; Animal; Simultaneous measurement; Pyrimidine derivatives; Sheep; Antiinfectious; Artiodactyla; Antibacterial agent; Column chromatography; Ungulata; Restricted access media (RAM): Column-switching
• ISSN: 1570-0232; 1873-376X
• DOI: 10.1016/j.jchromb.2005.08.006

A bidimensional HPLC method for the simultaneous determination of sulfamethoxazole (SMX) and trimethoprim (TMP) in bovine milk has been developed and validated. After centrifugation, aliquots (150
μl) of milk samples were directly injected to a column-switching HPLC system. At the first step a RAM octyl-BSA column was employed to automatically remove proteins that otherwise would interfere with milk analysis. The mobile phase 0.01
M phosphate buffer pH 6.0:acetonitrile (95:5, v/v) was used in the first 5
min for the elution of milk proteins and then 0.01
M phosphate buffer pH 6.0:acetonitrile (83:17, v/v) for transfer SMX and TMP to the analytical column. The separation of SMX and TMP from one another and from other remaining milk components was performed on an octyl column using the mobile phase 0.01
M phosphate buffer pH 5.0:acetonitrile (82:18, v/v), which were detected by UV at 265
nm. The calibration graphs were linear in the concentration ranges of 25–800
ng/ml and 50–400
ng/ml for SMX and TMP, respectively. The intra- and inter-assay coefficients of variation were less than 15% for both drugs. The validated method was applied to the analysis of milk samples of twelve (two groups of six) cows after administration (intramuscular or subcutaneous) of a single recommended therapeutic dose of the SMX–TMP combination.