Method Validation of Contact and ImmersionTLC-bioautography for Determination of Streptomycin Sulfate in Shrimp

INTRODUCTION: Contact and immersion Thin Layer Chromatography (TLC)-bioautography have been developed for identification and quantification of streptomycin sulfate in shrimp. The TLC of streptomycin sulfate standard solution has been carried out by using silica gel F254 and 7.5 % of KH2PO4 solution as stationary and a mobile phase respectively. The bioautograpy was performed by Escherichia coli ATCC 8739 as a test bacterial. Limit of Detection, linearity, accuracy, percentage recovery, and coefficient variation of the contact and immersion TLC-bioautography were employed as validation parameters.

METHODS: 1. Applying antimicrobial analytes on the TLC plate and eluted with KH2PO4 solution.
2. Contact TLC-bioautography method was performed by put the TLC chromatogram plate on the surface of the agar medium inoculated with the test bacteria, incubated for 24 hour.The antimicrobial activity was devoted by the inhibitory zone around the reservoir hole on the surface agar media or the spot position on the TLC-bioautogram plate.
3. The Immersion bioautography was performed by sprayed until the plate is covered by test media containing the test bacteria at temperature of 45oC.The plate then cooled to condense and allow for diffusion process.
RESULTS: Limit of Detection (LOD) of streptomycin sulfate obtained by the contact and immersion TLC-bioautography were 0.24 µg and 0.16 µg, respectively. Both methods gave r-value more than 0.999 and the Vxo value less than 2%. Percentage recoveryof contact and immersion were 86.93±1.60% and 96.42±0.65%, respectively, with coefficient of variation were 2.39±1.79% and 0.53±0.17 %, respectively.
DISCUSSION AND CONCLUSION: The method validated can be used to simultaneously determine the streptomycin sulfate and kanamycin sulfate in a common market frozen shrimp. Those simple methods are recommended for monitoring antibiotics abused in frozen foods, especially for streptomycin at the concentration of 0.16 µg.