VALIDATION OF HPLC METHOD AND ULTRA VIOLET DETECTION FOR THE QUANTIFICATION OF ATROPINE SULFATE IN AQUEOUS SAMPLES

Atrium

Objective. To validate a USP HPLC method for the quantification of Atropine Sulfate (AS) in aqueous samples. Background. AS IM injection is the recommended antidote for management of organophosphate toxicity. We hypothesized that AS sublingual administration can result in similar pharmacokinetic profile as AS IM injection. Methods. An HPLC system and ultra violet detector (Perkin Elmer, MA, USA) were used for the separation and quantification of AS according to USP method for AS injection. AS stock solution (2mg/mL in water) was used to prepare several standard calibration curves ranging from 5¼g/mL to 200¼g/mL at various days to calculate the intra- and inter-assay variability. An increasing injection volumes ranging from 10¼L to 100¼L were injected to test for injection linearity. To test for method and instrument accuracy, the lowest (5¼g/mL) and highest (200¼g/mL) standards were injected 5 times. Mean, standard deviation, and relative standard deviation (RSD%) have been calculated for each validation test. Results. The Calibration curves were linear with a mean (SD) R2 of 0.9966±0.00085 (n=6). The RSD% (n=2) intra-and inter-assay at 5 ¼g/mL were 7.24% and 7.69%, respectively, and at 200 ¼g/mL were 0.076% and 0.83%, respectively. RSD% (n=5) of instrument and method accuracy for the lowest and highest standards (5 ¼g/mL and 200 ¼g/mL) were 3.915% and 0.359%, respectively. The instrument injection was linear with R2 of 0.99998 and the minimum detection limit was 50ng/mL with RSD% (n=5) of 0.86%. Conclusion. A reproducible and sensitive USP HPLC method was validated for the quantification of AS from Aqueous samples. Grants. This study was funded by Saudi Arabian Cultural Mission Grant