Estimation of 5-fluorouracil by high-performance liquid Chromatography Reversed-phase Validated method

 

Deep Shikha Sharma, Sheetu Wadhwa, Sachin Kumar Singh*, Arya Ramanunny, Rajan Kumar

School of Pharmaceutical Sciences, Lovely Professional University, Phagwara, Punjab (India)–144411.

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

 

ABSTRACT:

The main intention of the current work is the development of a suitable, simple, precise, reproducible, and accurate high-performance liquid chromatography reversed-phase validated method which can be used for the estimation of 5-Fluorouracil.The estimation was done by high-performance liquid chromatography (HPLC) method. In this suitable stationary phase and optimum composition of the mobile phase was selected which provides good resolution and short run time for the estimation of 5-Fluorouracil (5-FU). Stationary phase used was Nucleodur C18 column (Reverse phase, 250mm Χ 4.6mm i.d., particle having 5 micron size) and mobile phase consists of combination of Ortho-Phosphoric Acid (OPA) (0.5%) and methanol having ratio 95:5, v/v were used in an isocratic mode of elution. The mobile phase used with a flow rate of 0.8 mL/min and volume of the injection for 5-FU was 20μL. The eluent observed at 266nm for measurement of 5-FU. The validation of this method was carried out with the help of various parameters such as sensitivity, selectivity, system suitability, precision (inter-day and intra-day), accuracy, and linearity according to International Conference on Harmonization guidelines i.e. ICH Q2 (R1). The 5-FU showing retention time at 7.2 min. The responses showing linearity in the concentration range between 2-10μg/mL with correlation coefficient 0.99. The % mean recovery of 5-FU was calculated at three different levels i.e Lower Quality control Concentration (LQC), Middle Quality control Concentration (MQC), High Quality control Concentration (LQC) whose results falls within the range i.e. 95% to 105%, which indicates the accuracy of this method. The % relative standard deviation (RSD) precision (intraday and intermediate) at 3 different levels was < 2% which indicated the precision of this method. LOD i.e. Limit of Detection and LOQ i.e. Limit of Quantification was found to be 0.870277 and 2.637202 respectively for 5-FU.

 

KEYWORDS: 5-Fluorouracil, RP-HPLC, ICH Guidelines, System suitability, Accuracy, Precision.

 

 


INTRODUCTION:

5-Fluorouracil (5-FU) is a pyrimidine analogue (shown in Figure 1 below) and used as anticancer and antimetabolite. 5-FU blocks the thymidylate synthetase enzyme activity which is required for the conversion of deoxyuridylic acid to thymidylic required for synthesis of DNA [1,4]. For estimation of 5-FU a suitable, simple, precise, reproducible, and accurate high-performance liquid chromatography (HPLC) reversed-phase (RP) validated method is required which can be further used for the bioanalytical estimation of 5-FU.

 

Figure 1: 5-FU Chemical Structure

 

The importance of analytical method validation in drug development process:

HPLC developed and validated method helps in identification as well as quantification of compounds. As per GMP (Good Manufacturing Practice) requirements different pharmaceutical companies have validation policy to determine the purity of the drug, presence of relative substances, and stability profile of drug [2]. The current validated method also helps in estimation of drug in biological fluids during pharmacokinetic, toxicokinetic, and drug distribution study [3]. It also plays an important role in determination of drug loading, and drug dissolution studies [4].

 

In present research work, different parameters related to the validation of this method such as selectivity, sensitivity, system suitability, precision (inter-day and intra-day), accuracy, and linearity was well explained according to ICH Q2 (R1) guidelines [5]. Validation of method plays a very important role to interpret the data.

 

The main intention of this research work is development of a suitable, simple, precise, reproducible, and accurate high-performance liquid chromatography reversed-phase validated method which can be used for the estimation of 5-FU [6]. A bioanalytical method could be developed using the same chromatographic conditions for pharmacokinetic study of 5-FU in preclinical and clinical subjects [7].

 

MATERIAL AND METHODS:

Chemicals and reagents:

5-Fu was procured from LOBA Chemie Pvt. Ltd. Mumbai. Methanol and O-Phosphoric acid (OPA) were of analytical grade and were purchased from Dee-Jay Corporation, Jalandhar. A 0.45΅m nylon filter (Pall life Sciences, Mumbai, India) was used. All other chemicals and reagents used were analytical grade unless otherwise indicated.

 

Instrumentation:

Method Development on RP-HPLC:

HPLC is an instrument which can be used to identify the drug, determine the quantity of drug, and isolation of different components present in mixtures. The HPLC system consists of delivery pump for mobile phase (LC-20 AD; Shimadzu, Japan), a detector (PDA-photodiode array) (SPDM20A; Shimadzu, Japan), a 20μL loop (Rheodyne), and software LC Solution [12]. A Nucleodur C18 column (Reverse phase, 250mm Χ 4.6 mm i.d., particle having 5 micron size) used as stationary phase and combination of Ortho-Phosphoric Acid (OPA) (0.5%) and methanol having ratio 95:5, v/v used as mobile phase in an isocratic mode of elution [8]. The solvent sample mixture (drug solution) and mobile phase passes through an HPLC column (stationary phase) and then into a detector, where an electronic output is given as a chromatograph signal [9].

 

Estimation of 5-FU on RP-HPLC:

Different composition of mobile phase solvents system being used, out of which one reliable ratio was selected which provides good resolution and short run time for the estimation of 5-FU. The waste was collected in a vessel outside the machine.

 

The mobile phase used with flow rate of 0.8mL/min and volume of the injection of 5-FU drug solution was 20 μL. The eluent was observed at 266nm. The retention time of 5-FU was found to be 7.2 min in 20 min run time of HPLC. The peaks of 5-FU in a chromatogram are shown in Figure 2 below. The blank (without drug) was also injected under the same conditions and HPLC was run for 20 min. in which one peak was observed at 4.405 min as shown in Figure 3 below which indicates that drug having a very sharp peak at 7.2 min. only.

 

The chromatograph signals area i.e. mean peak area was calculated for repeated samples of the same drug solution. For validation of this developed method different parameters were estimated such as selectivity, sensitivity, system suitability, precision (inter-day and intra-day), accuracy, and linearity according to International Conference on Harmonization guidelines i.e. ICH Q2 (R1) [10]..

 

Figure 2: HPLC chromatogram of 5-FU

 

Figure 3: HPLC chromatogram of Blank (Distilled water)

 

Stock solution preparation:

Stock solution of 5-FU was prepared by accurately weighing drug (10mg) on digital weighing balance and dissolving it in 2mL of distilled water in 10 mL volumetric flask by using vortex. Then make up the volume up to 10mL with distilled water to prepare 1 mg/mL stock solution. Sonicate the stock solution for 10 min. to completely dissolve the drug.

 

Then, 1 mL of this solution was taken and transferred into another 100mL volumetric flask and diluted up to 100mL with distilled water. Sonicate this solution for 10 minutes to form 10μg/mL concentration. Similarly, other concentrations were prepared such as 2, 4, 6, and 8 μg/mL from the stock solution in different volumetric flasks [11].

 

Development of calibration curve:

From the above prepared standard drug stock solution 2, 4, 6 and 8 and 10mL was transferred into different flasks (10mL) with the help of pipette and make up the volume up to 10mL with purified water. Sonicate all the samples for 10 minutes. Peak area of all the samples was determined by using HPLC method. Six injections were given for each concentration. The peak of the drug was observed at 266nm and the average peak area was calculated from all six peaks of different concentrations. The calibration curve was plotted between concentrations of 5-FU between 2-10μg/mL drug (x-axis) versus the area of the peak (y-axis). The regression coefficient was found to be 0.99 which shows linearity of the curve [12]. This calibration curve was used further to determine different parameters for validation of the developed method of HPLC.

 

The proposed work was carried out on a Shimadzu UV-visible spectrophotometer (model UV-1800 series), which possesses a double beam double detector configuration with a1 cm quartz matched cell. All weighing was done on electronic balance (Sansui-vibra DJ-150S-S). A Fast clean ultrasonicate cleaner (India) was used for degassing the mobile phase.

 

VALIDATION OF METHOD:

Validation was done for the developed method by determining system suitability, linearity, accuracy, precision, LOD, LOQ, precision (inter-day and intra-day), and specificity study as per ICH Q2 (R1) guidelines.

 

System suitability:

To check system suitability, 6 times injections was given of standard solutions (6μg/mL) of 5-FU was injected to HPLC. The theoretical plates number, peak asymmetry, HETP i.e. height equivalent to theoretical plate and retention time were also measured [13].

 

Linearity and Range:

The calibration curve was plotted between concentrations of 5-FU between 2-10μg/mL drug (x-axis) versus area of the peak (y-axis) to determine the linearity and range. The regression coefficient was calculated which represents linearity of the curve (4). The range of the analytical procedure was given by the interval between the upper and lower limit of drug concentration. This calibration curve was used further to determine other parameters for HPLC developed method validation [14].

 

Accuracy:

The accuracy of an analytical method describes the closeness of result between the value which is accepted either as a conventional true value or an accepted reference value and the value found. Accuracy of the experiment was established by using recovery studies from the selected concentration range of calibration curve from 2-10μg/mL, mid concentration of the drug (6 μg/mL) was taken as MQC [100%]. Similarly, LQC [80%] and HQC [120%] of 6μg/mL were also prepared from the stock solution. Suitable aliquots of 4.8, 6.0, and 7.2mL were withdrawn from stock solution and transferred individually into different flasks (10mL) and make up the volume up to 10mL to prepare LQC, MQC and HQC respectively.

 

Six injections were given repeatedly and the mean area of the observed peaks were calculated for all six injections. For the determination of accuracy of this method mean percentage recovery of the drug was calculated from all these three concentrations. % absolute recovery was calculated by dividing the actual recovery of drug to their theoretical concentration and multiplying them by hundred (Eq.1) [15].

 

--(1)

 

The data revealed that for all the three levels, the mean % recovery was within the fixed limits of 95-105%. Moreover, the % RSD was also calculated and it was coming in range i.e. < 2%. This indicates accuracy of this method.

 

Precision:

The precision of an analytical method defined the closeness of results between measurements obtained from multiple sampling of the same homogeneous sample under the prescribed conditions. For determining precision of this method % RSD was calculated for the six observations of LQC, MQC, and HQC solutions at interday (all three dilutions made in three different days), intraday (all three dilutions made within same day) and interanalyst (all three dilutions made by three different analysts) with same experimental conditions. The % RSD for all the samples was within the limit i.e. RSD < 2%. This proved this method was sufficiently precise [16].

 

Estimation of LOD and LOQ:

The LOD i.e. Limit of Detection of an analytical method is defined as the lowest concentration of sample or drug which can be detected. The LOQ i.e. Limit of Quantification of an analytical method is defined as the lowest concentration of sample or drug which can be determined quantitatively. Both were calculated to determine sensitivity of this method by using standard deviation of response (σ) and slope of standard curve (S). Standard deviation of Y intercepts of regression line was used as standard deviation [17]. Eq. 2 and Eq. 3 for LOD and LOQ, respectively as follow:

 

———–––––––––––––––––––––––––––(2)

——–––––––––––––––––––––––––––––(3)

 

APPLICATION OF VALIDATED METHOD:

The developed and validated method will be used for estimation of percentage of drug loaded in Nano Lipid Carriers i.e. NLCs, assay of tablet formulation, percentage of drug released during dissolution with respect to time, and drug diffused through goat cornea. The estimation of drug in biological fluids during pharmacokinetic, toxicokinetic, drug distribution study can be done by using this validated method [18].

 

RESULTS AND DISCUSSION:

HPLC method development for 5-FU estimation:

Different compositions of mobile phase in varying ratios and flow rate were used. Among them, the compositions having mobile phase combination of OPA (0.5%) and methanol with ratio 95:5, v/v and 0.8 mL flow rate which provide acceptable chromatograms. Hence, it was decided to develop chromatograms with these conditions. The retention time for 5-FU was observed at 7.2 min in the obtained chromatogram with the help of HPLC [19].

 

System suitability:

Number of theoretical plates, tailing factor, peak asymmetry, HETP, and resolution were measured. All the parameters were found within the limits as shown in Table 1 below.

 

Table 1: System suitability parameters

Parameters

5-FU

HETP

18.883

Theoretical plate

7943.607

Tailing factor

1.294

Resolution

7.201

 

Linearity and Range:

The graph was plotted between the concentration of drug and mean peak area to obtain the calibration curve [20]. 5-FU calibration curve showing linearity in the concentration range from 2-10μg/mL having 0.99 regression coefficient as shown in Figure 4 below.

 

Figure 4: Standard curve of 5-FU

 

Accuracy

Accuracy was observed by calculating mean % recovery of the drug from HQC, MQC, and LQC solutions containing 6 μg/mL of sample solution. The data revealed that for all the three levels, the mean % recovery was within the fixed limits of 95-105 % (Table 2). Moreover, the % relative standard deviation (RSD) was < 2%. This indicates the accuracy of the developed method.

 

Table 2: Precision data of the proposed HPLC method from the standard solution of 5-FU

Level

Conc (μg/mL)

Mean peak area (N=6)

% RSD

% recovery

Mean % recovery

LQC

4.8

418793.3

1.16

95.05

96.92

MQC

6.0

520793.8

1.84

95.17

HQC

7.2

656759.0

1.62

100.56

 

Precision:

Precision of this method was observed by calculating the % RSD for the 6 injections of the HQC, MQC, and LQC solutions at inter day, intraday and inter analyst by using same experimental conditions. The calculated % RSD for all the samples showing < 2% RSD (Table 3). This proves that the developed method was sufficiently precise.


 

Table 3: Precision data of the proposed HPLC method from the standard solution of 5-FU

Parameters

Levels

Conc. (μg/mL)

1

2

3

4

5

6

Mean area (N=6)

Standard Deviation

% RSD

% Recovery

Repeatability (Intraday precision)

 

LQC

4.8

426409

421031

413124

415398

420817

415981

418793.3

4871.952

1.16

95.05

 

MQC

6.0

520272

507850

511819

531648

522721

530453

520793.8

9624.855

1.84

95.17

 

HQC

7.2

647909

645153

665459

648195

667899

665939

656759.0

10681.070

1.62

100.56

Intermediate Precision (Interday)

Day 1

LQC

4.8

427504

427571

421298

420149

420813

420945

423046.7

3498.514

0.82

96.04

 

MQC

6.0

578362

577859

567830

575905

576280

578247

575747.2

4013.395

0.69

105.00

 

HQC

7.2

641328

623472

632932

635493

632796

621622

631273.8

7456.281

1.18

96.50

Day 2

LQC

4.8

427484

423376

420025

421107

420280

420553

422137.5

2633.264

0.62

95.83

 

MQC

6.0

529125

521914

526259

526612

526735

526735

526230.0

2352.263

0.44

96.10

 

HQC

7.2

658315

652603

653486

640909

647474

663877

652777.3

8042.184

1.23

99.94

Day 3

LQC

4.8

426409

421031

413124

415398

420817

415981

418793.3

4871.952

1.16

95.05

 

MQC

6.0

520272

507850

511819

531648

522721

530453

520793.8

9624.855

1.84

95.17

 

HQC

7.2

647909

645153

665459

648195

667899

665939

656759.0

10681.07

1.62

100.56

Intermediate Precision (Inter-analyst)

Analyst 1

LQC

4.8

422626

425254

418589

422869

411314

416989

419606.8

5065.722

1.20

95.20

 

MQC

6.0

576697

578655

567866

577069

576541

553699

571754.5

9636.609

1.68

104.70

 

HQC

7.2

668853

657830

657326

639130

651552

644430

653186.8

10593.110

1.62

100.00

Analyst 2

LQC

4.8

421879

422209

424847

422832

420616

421308

422281.8

1467.903

0.34

95.86

 

MQC

6.0

526072

529951

528316

514351

523912

518729

523555.2

5967.287

1.13

95.69

 

HQC

7.2

625902

622961

627159

622389

624441

624331

624530.5

1782.537

0.28

95.50

Analyst 3

LQC

4.8

417612

424204

423769

420214

429046

415422

421711.2

4956.752

1.17

95.73

 

MQC

6.0

535344

524297

522601

531344

527763

515741

526181.7

6906.219

1.31

96.18

 

HQC

7.2

658743

663460

664909

652526

662644

661976

660709.7

4502.980

0.68

101.18

 


Estimation of LOD and LOQ:

LOD and LOQ were determined by the slope of standard curve (S) and standard deviation of response (σ) were as follow [20]:

 

CONCLUSION:

In this research, a successful RP-HPLC method was developed for the estimation of 5-FU. The developed method was validated for linearity, range, precision, accuracy, system suitability, LOD, and LOQ as per ICH guidelines. The selected method conditions and mobile phase composition provides good resolution and short run time (around 7 min.) for estimation of 5-FU. The results of this work reflected that current method is free from interference of the impurities during the estimation of 5-FU. The low RSD values for all parameters confirmed the validity and reliability of method. All the results manifested that developed method is selective, precise, accurate and linear over the concentration range of 2-10 μg/mL. The method was found to be appropriate for estimation of 5-FU in bulk and pharmaceuticals. In future, a bioanalytical method could be developed using the same chromatographic conditions for pharmacokinetic study of 5-FU in preclinical and clinical subjects.

 

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.

 

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

 

REFERENCES:

1.      Parrozzani R, Frizziero L, Trainiti S, and Testi L, et al. Topical 1% 5-fluoruracil as a sole treatment of corneo conjunctival ocular surface squamous neoplasia: long-term study. British Journal of Ophthalmology. 2017; 101:1094–1099.

2.      Youssof AM, Salem-Bekhit MM, Shakeel F, Alanazi FK, Haq N. Analysis of anti-neoplastic drug in bacterial ghost matrix, w/o/w double nanoemulsion and w/o nanoemulsion by a validated ‘green’ liquid chromatographic method. Talanta. 2016; 154:292-2981. DOI: 10.1016/j.talanta.2016.03.086

3.      Mishra V, Thakur S, Patil A, Shukla A. Quality by design (QbD) approaches in current pharmaceutical set-up. Expert Opinion on Drug Delivery. 2018; 15 (8):737-758. doi: 10.1080/17425247.2018.1504768.

4.      Cohn NS. Similar cytological effects of hydroxylamine and 5-FUDR, agents with different modes of action. Experientia. 1964; 20 (3): 158-61,March 1964.

5.      Sameer A, Abdulrahman M. Sensitive and selective spectrophotometric assay of gabapentin in capsules using sodium 1, 2-naphthoquinone-4-sulfonate. Drug Testing and Analysis. 2011;10.

6.      Saka, Chromatographic Methods for Determination of Drugs Used in Prostate Cancer in Biological and Pharmacological Samples. Critical Reviews in Analytical Chemistry. 2019; 49 (1) 78-99.https://doi.org/10.1080/10408347.2018.1487776

7.      Ravisankar P, Navyal N, Pravallika V, NavyaSri D, A Review on Step-by-Step Analytical Method Validation. IOSR Journal Of Pharmacy. 2015; 5 (10):7-19.

8.      Sharma P, Singh SK, Pandey N, Kumar NR, et al. Impact of solid carriers and spray drying on pre/post-compression properties, dissolution rate and bioavailability of solid self nano emulsifying drug delivery system loaded with simvastatin. Powder Technology. 2018: 836-846.

9.      Mujeeb U, Rahman U., Rathore A, Siddqui AA, Parveen G, Yar MS, Determination of the Stabilities of New Quinazoline Derivatives by HPLC. Journal of Liquid Chromatography and Related Technologies. 2014; 29 (5): 733-43. doi: 10.3109/14756366

10.   Jyoti J, Anandhakrishnan NK, Singh SK, Kumar B, et al. A three-pronged formulation approach to improve oral bioavailability and therapeutic efficacy of two lipophilic drugs with gastric lability. Drug Delivery and Translational. 2019:1-18. doi: 10.1007/s13346-019-00635-0

11.   Hassain Y, Gupta VK. Analysis of Melatonin in Dosage Formulation by Capillary Electrophoresis, Journal of Liquid Chromatography and Related Technologies. 2007;30 (4):545-556.

12.   Garg VK, Singh S, Pandey NK, Bhatia A, et al. Impact of spray drying over conventional surface adsorption technique for improvement in micromeritic and biopharmaceutical characteristics of self-nanoemulsifying powder loaded with two lipophilic as well as gastrointestinal labile drugs. Powder Technology. 2018; 326:425-442.

13.   Jin Y, Rong LF, Lu XW, Huang Y, et al. Pharmacokinetics and tissue distribution of 5-fluorouracil encapsulated by galactosy lceramide liposomes in mice. Acta Pharmacologica Sinica, 2005; 26 (2) 250-6.

14.   Mohanta S, Singh SK, Kumar B, Gulati M, et al. Solidification of liquid Modified Apple Polysaccharide by its adsorption on solid porous carriers through spray drying and evaluation of its potential as binding agent for tablets. International Journal of Biological Macromolecules, 2018; 120:1975-1998..

15.   Mittal S, Alexander KS, Dollimore D. A High-Performance Liquid Chromatography Assay for Yohimbine HCl Analysis. Drug Development and Industrial Pharmacy. 2000;26 (10);1059-65.

16.   Sharma AK, Ponery AS, Lawrence PA, Ahmed I, et al. Effect of αtocopherol supplementation on the ultrastructural abnormalities of peripheral nerves in experimental diabetes. Journal of the Peripheral Nervous System. 2001;6 (1):33-9.

17.   Awad M, Hammad MA, Omar MA. Utility of Von Pechman synthesis of coumarin reaction for development of spectrofluorimetric method for quantitation of salmeterol xinafoate in pharmaceutical preparations and human plasma. Luminescence. 2018;33 (5): 913-918. doi: 10.1002/bio.3490.

18.   Kim W, Yousaf AM, Li DX, et al. Development of RP-HPLC method for simultaneous determination of docetaxel and curcumin in rat plasma: Validation and stability. Asian Journal of Pharmaceutical Sciences. 2017;12(1):105-113.

19.   Omar MA, Hammad MA, Awad M. Utility of Europium ion characteristic peak for quantitation of Fenoterol hydrobromide and Salmeterol xinafoate in different matrices; application to stability studies. Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy. 2019;217:182-189.

20.   Kamalakkannan V, Puratchikody A, Ramanathan L, Jayapraba S. Development and validation of a dissolution test with reversed-phase high performance liquid chromatographic analysis for Candesartan cilexetil in tablet dosage forms. Arabian Journal of Chemistry. 2016;9 (1): S867-S873.

 

 

Received on 19.11.2019            Modified on 08.01.2020

Accepted on 10.02.2020           © RJPT All right reserved

Research J. Pharm. and Tech 2020; 13(9):4249-4254.

DOI: 10.5958/0974-360X.2020.00750.7