Method development and Validation for the Estimation of 5-Fluorouracil by using Reverse phase high-performance liquid chromatography

 

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

Narendra Kumar Pandey, Saurabh Singh

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

 

ABSTRACT:

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.

 

 

 

INTRODUCTION:

5-Fluorouracil (5-FU) is chemically 5-fluoro-1,2,3,4-tetrahydropyrimidine-2,4-dione^[1-2]. It is an analogue of uracil which is a component of ribonucleic acid. 5-FU is believed to function as an antimetabolite. During the cell division, it interferes in the process of DNA synthesis by blocking the conversion of deoxyuridylic acid to thymidylic acid with the cellular enzyme thymidylate synthetase^[3]. It is also believed that, 5-FU may interfere with synthesis of RNA. 5-FU may be used alone or in combination for the management and treatment of common malignancies specifically in the cancer of colon and breast^[3-4].

 

A simple and novel analytical method is required to quantify 5-FU, which can be utilized successfully during the quality control test of dosage forms. Till date there is no such simple, cheaper and sensitive method is available to estimate the amount of 5-FU. In this work a simple and sensitive method was developed and validated to estimate 5-FU.

 

MATERIALS AND METHODS:

Materials:

5-FU was procured from Molychem, Pvt. Ltd., India. All other chemicals and reagents used were of analytical grade and HPLC grade solvents were employed for the study. Triple distilled water was used throughout the study.

 

Method Development for the estimation of 5-FU using RP-HPLC:

The RP-HPLC system consisted of a mobile phase delivery pump (LC-20 AD; Shimadzu, Japan), a photodiode array detector (SPDM20A; Shimadzu, Japan), a 20µL loop fitted with manual Rheodyne injector and LC Solution software. A C-18 reverse-phase column (Nucleodur C18, 250mm × 4.6mm i.d., 5µ) was utilised as stationary phase for the estimation of 5-Fluorouracil, using 0.05M potassium dihydrogen orthophosphate buffer containing 0.1% Triethanolamine (TEA), as mobile phase. 5-Bromouracil (5-BU) was used as internal standard. The flow rate was 1.2 mL min⁻¹ and detection wavelength was 266nm. Standard dilutions (2, 4, 6, 8 and 10µg/mL) were prepared in mobile phase and analysed. The developed method was validated as per ICH Q2 (R1) guidelines^[5].

 

Method validation:

The standard analytical method validation parameters i.e., linearity and range, accuracy, precision, robustness, LOD and LOQ, tailing factor, peak purity index and system suitability parameters were performed.

 

Preparation of quality control standards:

The quality control standards were prepared at three different levels i.e., Lower Quality Control standard (LQC), Medium Quality Control standard (MQC) and Higher Quality Control standard (HQC) of calibration curve. Hence, 6µg/mL was kept as 100 % (MQC) level, 80 % of 6µg/mL (i.e., 4.8µg/mL) was used as LQC and 120 % of 6µg/mL (i.e., 7.2µg/mL) was kept as HQC levels. All the three concentrations were prepared in mobile phase.

 

Linearity and range:

The calibration curve was developed by plotting the graph between mean peak area of five replicates versus corresponding concentrations of 5-FU, and the regression equation was obtained.

 

Accuracy:

The accuracy of the method was determined through calculation of recovery of the drug from the quality control standard solutions prepared in mobile phase. The LQC, MQC and HQC standard solutions were injected five times to HPLC and its mean of response was recorded. Percentage recovery was calculated by dividing the actual recovery of drug with their respective theoretical concentrations and multiplying them by hundred (Eq.1). The mean of response was recorded and percentage relative standard deviation was calculated.

 

                                   Actual concentration recovered

Percent recovery = –––––––––––––––––––––––––––––––X 100

                                       Theoretical concentration

 

Precision:

Precision of the method was evaluated in terms of repeatability and intermediate precision. Repeatability was tested by injecting five times the samples of LQC, MQC and HQC on the same day and under same experimental conditions. The intermediate precision was evaluated by determining LQC, MQC and HQC samples five times on each of three different days (inter-day) as well as by the three different analysts (inter-analyst) under the same experimental conditions. The mean of response was recorded and percentage relative standard deviation was calculated.

 

Robustness:

In order to check the effect of small changes on robustness of the developed method, the study was carried out by varying the flow rate (0.8, 1.0 and 1.2 mL/min) and wavelength (264, 266 and, 268nm), respectively. Five replicates of medium concentration (6 µg/mL) were injected and their effect on area of the peak, recovery and retention time was observed and mean of response was recorded.

 

Estimation of LOD and LOQ:

LOD and LOQ were determined by standard deviation of response (sigma) and slope of calibration curve (S). Standard deviation of Y intercepts of regression line was used as standard deviation.

 

LOD = 3.3 σ/S

LOQ = 10 σ/S

 

System suitability:

In order to check the system suitability, 5 replicate injections of standard solution (6µg/mL) of 5-FU were injected to the HPLC and the system suitability parameters were calculated.

 

RESULTS AND DISCUSSION:

Selection of mobile phase for the estimation of 5-FU:

For the estimation of 5-FU, different trials by changing the mobile phase and its composition were performed such as acetonitrile-water, acetonitrile-potassium dihydrogen orthophosphate buffer, methanol-potassium dihydrogen orthophosphate buffer and potassium dihydrogen orthophosphate buffer containing 0.1 % TEA.

 

 

Fig. 1: Optimised chromatogram of 5-FU and 5-BU in potassium dihydogen orthophosphate buffer containing 0.1 % TEA

Out of the performed trials, the trial conducted with the mobile phase potassium dihydrogen orthophosphate buffer (0.05) containing 0.1% TEA showed better results in terms of resolution, sharpness of peak and, separation between the peak of 5-FU and the internal standard 5-BU. Since, there is a significant difference in the retention time of 5-FU (7.595 min) and 5-BU (20.238 min) peaks (Fig.1); the mobile phase composition was selected for validation.

 

 

Fig.2: Calibration curve of 5-FU

 

Linearity and Range:

The calibration curve was developed by plotting the graph between concentration and mean area. The curves were found to be linear in the range of 2-10 µg/mL with a correlation co‑efficient (r²) of 0.999 (Fig.2).

 

Accuracy:

The accuracy of the developed method was accessed by determining the mean percentage recovery of the LOQ, MQC and HQC solutions in mobile phase. The data revealed that for all the three levels, the mean percentage recovery in mobile phase was within the fixed limits of 95-105 % (Table 1). The accuracy of developed method was verified by percentage relative standard deviation which was less than 2 %. The results of accuracy study are summarised in Table 1.

 

Precision:

The precision of developed method was evaluated by calculating the percentage relative standard deviation for the five determinations of LQC, MQC and HQC solutions at interday, intraday and interanalyst level under the same experimental conditions. The observed percentage relative deviation was less than 2 % for all the samples (Table 2). These results clearly indicated that the developed method was satisfactorily précised. The results of precision study are summarised in Table 2.

 

Table 1: Results of accuracy studies

 

Table2: Results of precision studies

Table 3: Robustness results of various parameters tested for 5-FU

 

Robustness:

Robustness of developed method was studied by varying the flow rate (0.8, 1 and 1.2mL/min) of mobile phase and the detection wavelength (264, 266 and 268nm). The observed percentage relative deviation was found less than 2% for all the samples (Table 3), indicating that the developed method was satisfactorily robust and the responses were unaffected by these changes.

 

System suitability:

System suitability parameters i.e., Height Equivalent to Theoretical Plate (HETP), theoretical plate, theoretical plate/meter and, tailing factor of peak and peak purity index were calculated for the analytical method. The results of the study are summarised in Table 4.

 

Table 4: Results of system suitability parameters

 

CONCLUSION:

In the present study the estimation of 5-FU was carried out using RP-HPLC method. The reports of validation studies indicated that the method was accurate, precise, rugged and robust. This method can be successfully applied to estimate 5-FU and its concentration in various pharmaceutical formulations.

 

CONFLICT OF INTEREST:

The authors declare that there is no conflict of interest with a person or an organisation related this research work.

 

ACKNOWLEDGEMENT:

Authors are thankful to second International Conference of Pharmacy, held by School of Pharmaceutical Sciences, Lovely Professional University on September 13-14, 2019 to fund the publication of this manuscript.

 

REFERENCES:

1.      Straub JO. Combined Environmental Risk Assessment for 5-Fluorouracil and Capecitabine in Europe Integrated Environmental Assessment and Management. 2010; 6: 540-66.

2.      Hagmann W, Jesnowski R, Faissner R, Guo C, Lohr JM. ATP-binding cassette C transporters in human pancreatic carcinoma cell lines. Upregulation in 5‑fluorouracil‑resistant cells. Pancreatology. 2009; 9: 136-44.

3.      Summary of Product Characteristics (SmPC). 5-Fluorouracil 50mg/ml injection. Hospira UK. Ltd. Available from: https://www.medicines.org.uk/emc/product/3791/smpc

4.      5-Fluorouracil (5-FU). MACMILLAN Cancer Support (reviewed on 31 May 2018). Available from: https://www.macmillan.org.uk/information-and-support/treating/chemotherapy/drugs-and-combination-regimens/individual-drugs/fluorouracil.html

5.      ICH Validation of Analytical Procedure. Text and Methodology Q2 (R1). Geneva: International Conference on Harmonization, 2005.

 

 

 

Received on 12.12.2019           Modified on 10.02.2020

Accepted on 02.04.2020         © RJPT All right reserved