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A new, simple, and rapid high-performance thin-layer chromatographic method was developed and validated for quantitative determination of Acyclovir. Acyclovir was chromatographed on silica gel 60 F254 TLC plate using chloroform: methanol: formic acid (6.5 + 3.5 + 0.1 v/v/v) as mobile phase. Acyclovir was quantified by densitometric analysis at 259 nm. The method was found to give compact spots for the drug (Rf = 0.25±0.01). The linear regression analysis data for the calibration plots showed good linear relationship with r2 = 0.9996 in the concentration range 100–600 ng/spot. The method was validated for precision, recovery, repeatability, and robustness as per the International Conference on Harmonization guidelines. The minimum detectable amount was found to be 100 ng/spot, whereas the limit of quantitation was found to be 300 ng/spot. Statistical analysis of the data showed that the method is precise, accurate, reproducible, and selective for the routine quality control of Acyclovir.
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