ORIGINAL RESEARCH PAPER A simple, rapid and sensitive high-performance thin-layer chromatographic method for the simultaneous estimation of berberine and 5-fluorouracil in rabbit plasma Preeti K. Tamane 1 & Rajasree G. Pai 1 & Varsha B. Pokharkar 2 Received: 11 October 2019 /Accepted: 31 December 2019 # Akadémiai Kiadó, Budapest, Hungary 2020 Abstract The purpose of the present study was to develop a simple, rapid and sensitive high-performance thin-layer chromatography (HPTLC) method for the simultaneous estimation of 5-fluorouracil (5FU) and berberine (BER) in rabbit plasma. BER and 5-FU are currently under extensive studies for their synergistic effects in cancer chemotherapy. The HPTLC method was developed using TLC aluminum plates precoated with silica gel 60 F 254 as the stationary phase. A mixture of toluene:methanol:ethyl acetate:formic acid (6:2:6:1, v/v) was used as the solvent system for development and detection was carried out at 266 nm. The method was designed to obtain the most optimum retardation factors (Rf) with best resolution. The Rf values of BER and 5- FU were 0.28 ± 0.02 and 0.57 ± 0.02, respectively. The plasma extraction was optimized using ethyl acetate. The method was validated in compliance with the USFDA guideline for bioanalytical method validation. The method was found to be linear in the range of 20 ng/spot to 300 ng/spot with R 2 of 0.9964 for 5-FU and 0.9943 for BER. The lower limit for quantification (LLOQ) of BER and 5-FU in rabbit plasma were obtained as 29 ng/spot and 26 ng/spot, respectively. The stability of BER and 5-FU in plasma were confirmed during freeze-thaw cycles (-20 °C), on bench for 5 h and long term for 21 days. The proposed method was validated statistically and was precise, accurate and sensitive for the estimation of BER and 5-FU in rabbit plasma. This method can be extended for therapeutic drug monitoring and pharmacokinetic studies in rabbits. Keywords 5-fluorouracil . Berberine . High-performance thin-layer chromatography . Rabbit plasma . Liquid-liquid extraction 1 Introduction Bioanalytical methods are developed for the quantitative de- termination of drugs and their metabolites in biological matri- ces. These methods play a substantial role in the estimation and interpretation of bioequivalence, toxicokinetic and phar- macokinetic profile of drugs in the body [1]. The bioanalytical procedure comprises sampling, sample preparation, analysis, calibration and validation. Cancer is a group of diseases involving abnormal growth of cells having a potential to spread to other body parts. It is one of the leading causes of death worldwide accounting for about 9.6 million deaths in 2018. Chemotherapy is one of the pri- mary treatment options for any type of cancer. It comprises the use of one or more antineoplastic or cytotoxic drugs along with immunomodulatory agents [2]. 5-FU, a pyrimidine analogue (Fig. 1a) has been used exten- sively as an antineoplastic, for the treatment of multiple solid tumours like rectal, breast, colon, ovarian, gastric, etc. 5-FU is a part of a chemotherapy drug group known as antimetabolites that irreversibly inhibits the activity of thymidylate synthase to result in decreased intracellular concentrations of deoxy thymi- dine monophosphate and thus the DNA damage [3]. 5-FU hin- ders cell development and DNA repair; thereby preventing growth and multiplication [4]. The drug is administered as a single agent or in combination with other anticancer drugs for synergistic effect. For determination of 5-FU in plasma, ultravi- olet (UV), high-performance liquid chromatography (HPLC), * Varsha B. Pokharkar varsha.pokharkar@bharatividyapeeth.edu 1 Department of Drug Regulatory Affairs, Bharati Vidyapeeth (Deemed to be) University, Poona College of Pharmacy, Erandwane, Pune, Maharashtra 411038, India 2 Department of Pharmaceutics, Bharati Vidyapeeth (Deemed to be) University, Poona College of Pharmacy, Erandwane, Pune, Maharashtra 411038, India JPC – Journal of Planar Chromatography – Modern TLC https://doi.org/10.1007/s00764-020-00013-4