The effluent I worked with was from a high rate algal pond, ~1g/L TSS. Initially, I observed >90% recovery of Tetracycline (TET, 2 mg/L) in the effluent samples I used for batch degradation tests, but later only 25% recovery was observed, and experimentation showed that citric acid addition to the HPLC vials (after filtration of the effluent) improved recovery of detectable TET (4-epitetracycline and tetracycline were detectable by the HPLC method). The citric acid addition (0.2 mL of 0.1 M citric acid to 1mL filtered effluent with TET, final pH ~2.7) enhanced detection most for dilutions of effluent in MQ water, and also enhanced recovery of TET in control samples in 100% MQ water after 8 hours incubation in the dark.

I understand that Tetracycline (TET) is often difficult to analyse, due to the many epimers it can form, and I suspect this is part of the problem. Acids are often used in preparation for SPE analysis of TET, but I haven't found any information on using organic acids to improve detection of TET for direct HPLC analysis of filtered effluent samples. I think something in the WW effluent was catalyzing an epimer or hydrolysis equilibrium, since the effect of recovery of TET in the effluent improved after dropping the HRAP HRT from 7 days to 4 days, although the citric acid addition still improved detection for effluent samples diluted in MQ water.

Does anyone have experience working with tetracycline, and know what might be happening here? Also, I would greatly appreciate advice on whether others would consider this addition of citric acid buffer to filtered samples before HPLC analysis valid, or are there known problems that would cause the data to be suspect? Thanks for any help!