Team:IISER-Tirupati India/Protocols

Protocols

HPLC/MS

Instrument parameters:
The ‘SHIMADZU’ HPLC system was used for carrying out the experiments. The recorded output data were analyzed using the ‘Lab solution’ software. The C-18 column which is a reverse-phase column was used. The column temperature and flow rate were maintained at 25℃ and 1ml/min respectively. The injection volume was 20µL.
Excreta sample collection and storage: The excreta samples were collected from a nearby poultry farm having around 5000 chickens using falcon tubes. Methanol was added at the site of the collection itself to ensure biosafety. These samples were then stored at -20℃ until further experimentation. Standardization of HPLC method for sulfonamides: Sample preparation - To obtain a standard curve for sulfonamides, sulfa drugs were bought from a pharmacy. These sulfa drugs contained sulfadiazine compounds as an active ingredient. The drugs were suspended in methanol, filtered via a PVDF filter to remove the unwanted binders, and then run through the C-18 column. Procedure - Multiple methods were screened from literature and a final protocol was laid out after suitable modifications were incorporated according to the sample. The method set for running the mobile phase involved using a high-pressure gradient system. The solvents used were 0.1% acetic acid (A), pure methanol (B), and pure acetonitrile (C) with varying (v:v) ratios at different time points as mentioned in Table 1. Run time - The total run time was 35 min. The retention times obtained were taken as the standard for future experiments. The eluted fractions were collected and confirmed by MS.