Simultaneous Quantification of 5-Fluorouracil and Leucovorin in Pharmaceutical Dosage Form and Human Spiked Plasma by Using RP- HPLC Method

Back ground: 5-Fluorouracil (5-FU) is one of the widely used chemotherapeutic drugs targeting various cancers including colon cancer. Leucovorin is not a chemotherapy drug, but it is often given as part of chemotherapy. This drug is used to make the chemotherapy more effective and also reduce the risk of side effects. The addition of Leucovorin (LV) to 5-Fuorouracil (5-FU) in advanced colorectal cancer treatment, has shown improved tumor response rates in many trials. Limited data is available for the optimal dosage regimen of both drugs. Purpose: The purpose of this study was to develop and validate a new method i.e. RP-HPLC for the simultaneous identification and quantification of 5-Fluorouracil (5-FU) and Leucovorin in pharmaceutical dosage form as well as in human spiked plasma. Method and Results: An HPLC method for the determination of 5-Fluorouracil (5-FU) and Leucovorin in pharmaceutical dosage form in the human spiked plasma, each for 5-Fluorouracil (5-FU) and Leucovorin was developed by using the mobile phase of 20 mM KH 2 PO 4 buffer and methanol at a ratio of (80:20) with pH 5.4 at a flow rate of 1.0 ml min -1 . 5-Fluorouracil (5-FU) and Leucovorin were eluted and detected at 242 nm with the retention time of 2.67 and 6.01 min, respectively. The limit of quantification (LOQ) and limit of detection (LOD) values of 5-Fluorouracil (5-FU) were 2.5 and 1.25 ng ml -1 , respectively. Similarly, The LOQ and LOD values of Leucovorin were 25 and 12.5 ng ml -1 , respectively. Both drugs were eluted through C18 BDS Hypersil column of 150×4.6 mm id with 5 µ particle size. Diclofenac sodium was also co-eluted at 8.61 minutes as an internal standard. The method was found linear for 5-Fluorouracil (5-FU) and Leucovorin in the range of 12.5 to 500 and 25 to 1000 ng ml -1 respectively. Similarly, values of % CV were well within the prescribed limits of ICH guidelines. Conclusion: The results indicated that the method is sensitive and reliable for the quantification of two drugs simultaneously in pharmaceutical dosage form and in human spiked plasma.