Development and Validation of the Analytical method for the estimation of a combination of 5-fluorouracil and Imiquimod by RP-HPLC
Bhupender Tomar1, Ankita Sharma1*, Inder Kumar2, Sandeep Jain3, Pallavi Ahirrao4
1Department of Pharmaceutical Chemistry, Abhilashi College of Pharmacy, Ner - Chowk, Distt. Mandi (H.P)
2Department of Pharmaceutics, Abhilashi University Chail Chowk Mandi (H.P.)
3Research Scientist Oniosome Healthcare Pvt. Ltd.
4Department of Pharmaceutical Chemistry, Chandigarh College of Pharmacy, Landran, Mohali
*Corresponding Author E-mail: ankitasharma.anks@gmail.com
ABSTRACT:
A simple, precise, and accurate reverse phase high performance liquid chromatographic method (RP-HPLC) was developed and validated for the estimation of the combination of 5- Fluorouracil (5-FU) and Imiquimod in active pharmaceutical ingredients (APIs). The method was carried out on Phenomenex C18 (250 × 4.6mm I.D., 5𝜇m) using isocratic elution mode. The mobile phase was used as Acetonitrile: 10mM potassium dihydrogen orthophosphate: triethylamine (40:59.9:0.1, v/v, pH 4.5 with orthophosphoric acid) and Water: ACN (50:50 v/v) was used as a diluent. The concentration of solvents was 1-20µg/ml and the volume of injection was 20µl with the flow rate of 1.2ml/min. The retention times for 5-FU and Imiquimod were found to be 1.9±0.5 and 6.6±0.5 min respectively. The absorption maxima of 5FU and Imiquimod were found 267nm and 227nm respectively. The method was validated as per ICH guidelines. All the data were found within the specified limits. The limit of detection (LOD) and limit of quantification (LOQ) of 5- Fluorouracil were found to be 0.015μg/mL and 0.048 μg/mL, respectively, and Imiquimod was found to be 0.078μg/mL and 0.237μg/mL, respectively. The method developed in the present study was found to be sensitive, specific, and precise and can be applied for the simultaneous estimation of 5-FU and Imiquimod.
KEYWORDS: 5- Fluorouracil (5-FU), Imiquimod, Analytical Method, RP-HPLC, Validation.
INTRODUCTION:
5-Fluorouracil utilizes its cytotoxicity mostly following its conversion, to 5-. 5-FUMP is further transformed into 5-5-fluoro-2'-deoxyuridine-5'-O-monophosphate (5-FdUMP), an irreversible inhibitor of thymidylate synthase and results in deoxythymidine triphosphate (dTTP) starvation and subsequent apoptosis. 5-FU is primarily degraded to nontoxic ß-alanine by following the enzymatic pathways.5-5-FU is actively transported into mammalian cells. To avoid systemic toxicity of 5-FU, doses must be minimized. Therefore, In vivo has created vectors carrying genes encoding uracil phosphoribosyltransferase (UPRT), an enzyme found in prokaryotes and lower eukaryotes but absent from mammalian cells. UPRT catalyzes’ the direct conversion of 5-FU to 5-FUMP whereas in mammalian cells 5-FU is converted to 5-FUMP through the two highly regulated enzymes. Therefore, the expression of UPRT substantially increases 5-FU cytotoxicity in transfected cells. UPRT encoding genes available from InvivoGen are E. coli up gene and S. cerevisiae fur gene. Both genes were fused to cytosine deaminase genes to generate the following fusion genes: E. coli codA: up and S. cerevisiae.1,2
Imiquimod is white almost white crystalline powder and chemically it is 1-(2-methyl propyl)-1H-imidazo(4,5-c)quinolin-4-amine a non-nucleoside heterocyclic amine. Imiquimod acquired immunity by activating T-helper 1 cells and also stimulates the native immune response through the production of interferons. Generally, it is used for the treatment of lumps on the genital and anal areas. It doesn’t cure any new lumps during the treatments. Imiquimod only helps to relieve and control the production of warts as it does not fight to inhibit the virus that causes warts directly. It also helps to treat the various skin condition of faces like actiic Kerasotes and superficial basal carcinoma i.e. a skin cancer.1,3,4
(a) (b)
Figure 1: Chemical structure of (a) 5-Fluorouracil (b) Imiquimod
As per the literature, there are no combined methods for the development and validation of 5-FU and Imiquimod by RP-HPLC, therefore, our main objective is to develop and validate a simple, accurate, sensitive, and reproducible method for estimation of the combination of 5FU and Imiquimod by RP-HPLC.
MATERIAL AND METHODS:
5-Fluorouracil was purchased from the online from Sigma Aldrich and Imiquimod drug was obtained as a gift sample from Glenmark Pharma, Mumbai India. All other Chemicals like O-Phosphoric acid, Triethylamine, Potassium dihydrogen phosphate, and Sodium chloride were used of AR grade and solvents like Methanol, Acetonitrile and Water were used of HPLC grade.
Identification of Standard Drug:
Identification of bulk drugs (5-FU acid and Imiquimod) were carried out by melting point study infrared spectroscopic study, and solubility study.
Infra-red Spectroscopy:
Fourier transform infrared Spectroscopies of drugs were performed for the identification of that particular compound. FTIR Spectroscopy of 5-FU and Imiquimod was done using KBr pellets. Various peaks in FTIR Spectrum were interpreted for the identification of different groups in the structure of 5-FU and Imiquimod.
HPLC METHOD DEVELOPMENT:
Selection of Detection Wavelength:
In the present study, the 5-FU and Imiquimod solution of 10μg/ml was prepared in Methanol. This solution was then scanned in the UV region of 200-400nm and a spectrum was recorded.5,6
Selection of Mobile Phase:
Chromatographic separation was performed with a low-pressure gradient. Initially, the mobile phase was tried for 5-FU with Water: Acetonitrile (45:55), Methanol: Buffer (45:55), Acetonitrile: Buffer (40:60), Imiquimod tried for Water: Acetonitrile (45:55), Acetonitrile:0.1% glacial acetic acid (50:50), Acetonitrile: Buffer (30:70) and then finally with Acetonitrile: phosphate buffer (pH 2.45)(50:50). The following optimized parameters were used as a final method for the simultaneous estimation of 5-FU and Imiquimod.
Chromatographic condition:
In the optimized parameters, the stationary phase was Phenomenex C18 (250 × 4.6mm I.D., 5𝜇m) using isocratic elution mode. Acetonitrile: 10mM potassium dihydrogen orthophosphate: triethylamine (40:59.9:0.1, v/v, pH 4.5 with ortho-phosphoric acid) were used as mobile phase with Water: ACN (50:50 v/v) was used as a diluent. The concentration of solvents was 1-20µg/ml and the volume of injection was 20µl with the flow rate of 1.2ml/min. The retention times for 5-FU and Imiquimod were observed to be 1.9±0.5 and 6.6±0.5min respectively. The absorption maxima of 5FU and Imiquimod were set in 267nm and 227nm respectively.
Standard Stock Solution Preparation (100μg/ml):
An accurately weighed quantity of about 10mg of 5-FU and 10mg of Imiquimod in 1ml Eppendorf tube, about 1 ml of diluent was added and sonicate for 10 minutes to dissolve. From this stock solution, a suitable amount of sample further dilutes to form 100μg/ml.
Preparation of calibration curve of 5-FU and Imiquimod:
From the standard stock solution, appropriate aliquots were pipette out in 1ml Eppendorf tube, and dilutions were made with diluent to obtain working standard solution of concentration ranges from 1μg/ml to 10 μg/ml for5-FU and Imiquimod. HPLC Chromatogram was recorded of each concentration.
VALIDATION OF RP-HPLC METHOD:
Linearity:
Different concentrations of 5-FU and Imiquimod were selected for linearity. Selected linearity range for was 1-10μg/ml for both the drugs.7, 8
Accuracy:
In this method, the known concentration of standard drug was added to the assay sample at the level of 80%, 100%, and 120% respectively.9,10
Precision:
The intra-day and inter-day variation for determination of 5-FU and Imiquimod was carried out Six times in the same day and six consecutive days using concentration 5 μg/ml of 5-FU and Imiquimod. % RSD was calculated. The method was found to be precise due to the low values of the %RSD.11,12
Limit of Detection:
The limit of detection can be calculated using the following equation as per ICH guidelines.
LOD = 3.3 × N/S
Where N is the standard deviation and S is the slope of the regression line.13,14
Limit of Quantitation:
Limit of quantification can be calculated using the following equation:15,16
LOQ = 10 × N/S
Where N is the standard deviation and S is the slope of the regression line.
Robustness:
The robustness was studied by analyzing the same samples of 5-FU and Imiquimod concentration 5μg/ml by deliberate variation in the method parameters. The change in the responses of 5-FU and Imiquimod was noted in terms of %RSD.17
Ruggedness:
The ruggedness was studied by analyzing the same samples of 5-FU and Imiquimod concentration 5μg/ml by changing the analyst. The change in the responses of 5-FU and Imiquimod was noted in terms of %RSD.18,19
Specificity:
The separations of the analytes from the potential components were validated by the specificity method. A known volume of individual ingredients solution was injected and the chromatogram was recorded.20
RESULT AND DISCUSSION:
Identification of standard drugs:
5- Fluorouracil and Imiquimod were observed for organoleptic properties like physical appearance, odor, and melting point. The drugs were identified with the help of UV and FTIR and exhibited absorption maxima was 267nm for 5-FU and 227nm for Imiquimod when methanol was used as a solvent as mentioned in the literature
Method Development and Validation:
Linearity:
Different concentrations of 5-FU and Imiquimod were selected for linearity. Selected linearity range for was 1-10μg/ml for both the drugs. The peak area of both drugs were taken against concentration and calibration curve was prepared (Figure 2)
(a)
(b)
Figure 2: Average Linearity of 5-FU and Imiquimod
Figure 3: Chromatogram of 5-FU (9 μg/ml)
Figure 4: Chromatogram of (10μg/ml)
Accuracy:
The data indicate that the % recoveries were within the acceptance range of 80 – 120%, therefore, the method is accurate and it can be used for the simultaneous estimation of 5-FU and Imiquimod. (Table 1)
Method Precision:
The %RSD for the area of six replicate injections was found to be within the specified limits. Similarly, % RSD for Interday Precision and Intraday Precision was given below that was found to be within the specified limits. (Table 2-3)
Table 1: Accuracy results of 5-FU and Imiquimod
|
Accuracy results of 5-FU |
|||||||
|
Conc. level |
Area |
Mean |
Amount added |
Amount recovered |
% Recovery |
SD |
%RSD |
|
80% |
222691 |
226610.7 |
4 µg/ml |
3.98 |
99.72 |
3394.534 |
1.497959 |
|
228566 |
4 µg/ml |
4.09 |
102.27 |
||||
|
228575 |
4 µg/ml |
4.09 |
102.27 |
||||
|
100% |
273456 |
276092.7 |
5 µg/ml |
4.87 |
97.41 |
2686.832 |
0.973163 |
|
278827 |
5 µg/ml |
4.96 |
99.27 |
||||
|
275995 |
5 µg/ml |
4.91 |
98.29 |
||||
|
120% |
329103 |
327306.7 |
6 µg/ml |
5.83 |
97.28 |
1571.219 |
0.480045 |
|
326188 |
6 µg/ml |
5.78 |
96.43 |
||||
|
326629 |
6 µg/ml |
5.79 |
96.56 |
||||
|
Accuracy results of Imiquimod |
|||||||
|
80% |
617214 |
616326.7 |
4 µg/ml |
4.061 |
101.54 |
3977.93 |
0.645426 |
|
619786 |
4 µg/ml |
4.078 |
101.95 |
||||
|
611980 |
4 µg/ml |
4.027 |
100.69 |
||||
|
100% |
757214 |
754047.3 |
5 µg/ml |
4.96 |
99.37 |
2915.976 |
0.38671 |
|
751473 |
5 µg/ml |
4.93 |
98.63 |
||||
|
753455 |
5 µg/ml |
4.944 |
98.88 |
||||
|
120% |
909454 |
903975.3 |
6 µg/ml |
5.955 |
99.25 |
4746.132 |
0.525029 |
|
901354 |
6 µg/ml |
5.902 |
98.37 |
||||
|
901118 |
6 µg/ml |
5.901 |
98.35 |
||||
Table 2: Repeatability of 5-FU and Imiquimod
|
Sr. No. |
5-FU |
Imiquimod |
||
|
Conc. (µg/ml) |
Area |
Conc.(µg/ml) |
Area |
|
|
1 |
5 |
286917 |
5 |
833367 |
|
2 |
5 |
280206 |
5 |
838301 |
|
3 |
5 |
287373 |
5 |
837309 |
|
4 |
5 |
283621 |
5 |
835247 |
|
5 |
5 |
285022 |
5 |
834042 |
|
6 |
5 |
288078 |
5 |
833367 |
|
Mean |
285202.8 |
Mean |
835607.3 |
|
|
Standard Deviation |
2946.789 |
Standard Deviation |
1886.971 |
|
|
%RSD |
1.033226 |
%RSD |
0.22582 |
|
Table 3: Intraday Precision of 5-FU and Imiquimod
|
Sr. No. |
5-FU |
Imiquimod |
|||
|
Conc. (µg/ml) |
Area |
Conc.(µg/ml) |
Area |
||
|
1 |
5 |
283248 |
5 |
830024 |
|
|
2 |
5 |
286983 |
5 |
834581 |
|
|
3 |
5 |
286712 |
5 |
834334 |
|
|
4 |
5 |
287052 |
5 |
837830 |
|
|
5 |
5 |
288614 |
5 |
836869 |
|
|
6 |
5 |
281558 |
5 |
833677 |
|
|
Mean |
285694.5 |
Mean |
834552.5 |
||
|
Standard Deviation |
2689.157 |
Standard Deviation |
2736.075 |
||
|
%RSD |
0.94127 |
%RSD |
0.327849 |
||
|
Interday Precision of 5-FU and Imiquimod |
|||||
|
|
5-FU |
Imiquimod |
|||
|
|
Conc.(µg/ml) |
Area |
Conc.(µg/ml) |
Area |
|
|
1 |
5 |
277083 |
5 |
759593 |
|
|
2 |
5 |
278006 |
5 |
753095 |
|
|
3 |
5 |
279115 |
5 |
758196 |
|
|
4 |
5 |
274487 |
5 |
753071 |
|
|
5 |
5 |
277445 |
5 |
754280 |
|
|
6 |
5 |
278969 |
5 |
757692 |
|
|
Mean |
277517.5 |
1689.702 |
755987.8 |
||
|
Standard Deviation |
1689.702 |
277517.5 |
2848.526 |
||
|
%RSD |
0.608863 |
0.608863 |
0.376795 |
||
Limit of detection and Limit of quantitation:
The LOD was found to be 0.015µg/ml and 0.078µg/ml for 5-FU and Imiquimod respectively and LOQ were found to be 0.048µg/ml and 0.237µg/ml 5-FU and Imiquimod respectively which showed that sensitivity of the method was high.
Table 4: Robustness of 5-FU and Imiquimod at different flow rates and column temperature
|
Conc.(µg/ml) |
Change in flow Rate |
|||||
|
1.19 ml/min |
1.2 ml/min |
1.21 ml/min |
||||
|
Area |
Area |
Area |
||||
|
5-FU |
Imiquimod |
5-FU |
Imiquimod |
5-FU |
Imiquimod |
|
|
5 |
279511 |
801962 |
274206 |
751974 |
278002 |
807906 |
|
5 |
277813 |
806008 |
274922 |
752053 |
277026 |
807202 |
|
5 |
273022 |
800275 |
275313 |
751670 |
277209 |
809995 |
|
Mean |
276782 |
802748.3 |
274813.7 |
751899 |
277412.3 |
808367.7 |
|
SD |
3365.115 |
2946.279 |
561.395 |
202.2152 |
518.7989 |
1452.606 |
|
%RSD |
1.2158 |
0.367024 |
0.204282 |
0.026894 |
0.187014 |
0.179696 |
|
Change in column temperature |
||||||
|
Conc.(µg/ml) |
35 °C |
40 °C |
45 °C |
|||
|
Area |
Area |
Area |
||||
|
5-FU |
Imiquimod |
5-FU |
Imiquimod |
5-FU |
Imiquimod |
|
|
5 |
272443 |
800975 |
274206 |
751974 |
274197 |
804453 |
|
5 |
273686 |
805080 |
274922 |
752053 |
274103 |
801001 |
|
5 |
276469 |
802466 |
275313 |
751670 |
276771 |
806550 |
|
SD |
2061.505 |
2077.944 |
561.395 |
202.2152 |
1513.965 |
2801.937 |
|
Mean |
274199.3 |
802840.3 |
274813.7 |
751899 |
275023.7 |
804001.3 |
|
%RSD |
0.751827 |
0.258824 |
0.204282 |
0.026894 |
0.550485 |
0.348499 |
Change in mobile phase ratio
Table 5: Robustness of 5-FU and Imiquimod at different mobile phase ratio and change in wavelength
|
Conc.(µg/ml) |
ACN: Buffer 38:62 |
ACN: Buffer 40:60 |
ACN: Buffer 42:58 |
|||
|
Area |
Area |
Area |
||||
|
5-FU |
Imiquimod |
5-FU |
Imiquimod |
5-FU |
Imiquimod |
|
|
5, 0.5 |
275371 |
808878 |
274206 |
751974 |
282675 |
830935 |
|
5, 0.5 |
276282 |
804390 |
274922 |
752053 |
277354 |
837917 |
|
5, 0.5 |
278887 |
807872 |
275313 |
751670 |
275444 |
837014 |
|
SD |
1824.747 |
2355.083 |
561.395 |
202.2152 |
3747.188 |
3797.323 |
|
Mean |
276846.7 |
807046.7 |
274813.7 |
751899 |
278491 |
835288.7 |
|
%RSD |
0.659118 |
0.291815 |
0.204282 |
0.026894 |
1.345533 |
0.454612 |
|
Change in wavelength |
||||||
|
|
262, 222 nm |
267, 227 nm |
272,232 nm |
|||
|
|
Area |
Area |
Area |
|||
|
|
5-FU |
Imiquimod |
5-FU |
Imiquimod |
5-FU |
Imiquimod |
|
5 |
273880 |
744232 |
274206 |
751974 |
266444 |
640210 |
|
5 |
275603 |
740917 |
274922 |
752053 |
265553 |
641719 |
|
5 |
277837 |
743963 |
275313 |
751670 |
262226 |
645335 |
|
SD |
1983.992 |
1841.182 |
561.395 |
202.2152 |
2223.148 |
2633.697 |
|
Mean |
275773.3 |
743037.3 |
274813.7 |
751899 |
264741 |
642421.3 |
|
%RSD |
0.719428 |
0.247791 |
0.204282 |
0.026894 |
0.839745 |
0.409964 |
Robustness:
The results for the robustness study in Table 5-6 indicated that the small change in the conditions did not significantly affect the determination of 5-FU and Imiquimod.
Ruggedness:
The results were found within the specified limit, %RSD was less than 2. %RSD of analyst 1 and analyst 2 were found to be 0.372054 (5-FU), 0.2832 (Imiquimod) for analyst 1 and 0.300426 (5-FU), 0.297126 (Imiquimod) for analyst 2.
CONCLUSION:
The proposed developed method for 5-FU and Imiquimod was found to be sensitive, specific, and accurate for routine simultaneous analysis without prior separation and can be effectively applied for the simultaneous estimation of 5-FU and Imiquimod. Therefore, the present method could find practical application as an efficient and rapid quality-control tool with good separation for the simultaneous analysis of the two drugs from their combined dosage forms in both research and industrial quality-control laboratories.
CONFLICT OF INTEREST:
The authors declare no conflict of interest, financial or otherwise.
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Received on 13.05.2020 Modified on 15.07.2020
Accepted on 06.09.2020 © RJPT All right reserved
Research J. Pharm. and Tech. 2021; 14(6):3313-3318.
DOI: 10.52711/0974-360X.2021.00576