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Year : 2021, Volume : 14, Issue : 1
First page : ( 185) Last page : ( 188)
Print ISSN : 0974-3618. Online ISSN : 0974-360X.
Article DOI : 10.5958/0974-360X.2021.00032.9

Method development and validation for the estimation of 5-Fluorouracil by using reverse phase high-performance liquid chromatography

Kumar Ankit, Singh Sachin Kumar*, Gulati Monica, Kumar Bimlesh, Prasher Rahul, Gupta Jayprakash, Mankotia Diksha, Pandey Narendra Kumar, Singh Saurabh

School of Pharmaceutical Sciences, Lovely Professional University, Punjab-144411, India.

*Corresponding Author E-mail: singhsachin23@gmail.com

Online published on 22 April, 2021.


An analytical method was developed to quantify 5-Fluorouracil (5-FU) using reverse phase high performance liquid chromatograph (RP-HPLC). A C-18 reverse-phase column was used as stationary phase for the separation of 5-FU. Potassium dihydrogen orthophosphate buffer (0.05 M) containing 0.1% of Triethanolamine (TEA) was use as a mobile phase to facilitate the elution. The flow rate was 1.2mL min−1 and the chromatogram of 5-FU was detected at wavelength of 266nm. 5-Bromouracil (5-BU) was used as internal standard. Method was validated as per ICH Q2 (R1) guidelines. The retention time of 5-FU was found to be 7.568 min and 5-BU was 20.067 min. The developed method was found to be linear at a concentration range of 2–10μg/mL with r2 of 0.9996. The mean percentage recovery of 5-FU was found within 95–105% at all the levels which indicated that the method was accurate. The percentage relative standard deviation was found less than 2% which indicated that method was satisfactorily précised. The LOD and LOQ for 5-FU were found to be 3.3 and 0.83 respectively. The method was found to be robust as there was no significant change in response with variation in the flow rate and wavelength. It was concluded that the developed method has passed all the validation tests and can be successfully applied to estimate the presence of 5-FU in bulk as well as in pharmaceutical formulations.



5-Fluorouracil, RP-HPLC, Linearity, Validation, Precision.


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