INTRODUCTION:

Estimation of drug is a key practice in the research and development of any drug delivery system, and the formulation scientist need an appropriate, precise, and highly sensitive analytical method for this purpose. Since analysis is such an essential and fundamental part of the formulation process, it has a substantial impact on the product development process. The simple, quick, accurate, exact, and stable approach will accelerate the product development process and increase success. Before beginning design and drug delivery system development, it is critical to create an analytical technique for every drug molecule.

In the pharmaceutical sector, reversed-phase high performance liquid chromatography (RP-HPLC) is the analytical technique that is most frequently utilized. A mobile phase is allowed to move through the porous silica column which act as stationary phase and a solution of the component which is to be separated, quantified, and qualified is injected into the column. Compounds with lesser affinity towards the stationary phase move more quickly and elute out first and the sample compounds with a higher affinity to the stationary layer moves slowly and elute later^1,2,3,4.

Figure 1: Chemical structure of methotrexate

Figure 2: Chemical structure of naringenin

Methotrexate, chemically (2S)-2-[(4-{[(2,4-Diaminopteridin-6-yl) methyl (methyl)amino} benzoyl) amino] pentane-dioic acid (Fig 1) is used to treat cancer and autoimmune diseases. It is a folic acid inhibitor that works by inhibiting the enzyme dihydrofolate reductase⁵. It predominantly suppresses DNA synthesis by acting at the S-phase of the cell cycle. In psoriasis, it blocks the epidermal mitosis thereby resolving the psoriatic scales and plaques⁶. It has dose-related adverse effects such as stomach discomfort, severe liver damage, diarrhea, decreased red, white blood cells, and platelet count, which restrict its usage. Topical application of the drug has been recommended to avoid these adverse effects, and a few trials have shown promising outcomes⁷. Naringenin, chemically (2S)-5,7-dihydroxy-2-(4-hydroxyphenyl)-3,4-dihydro-2H-1-benzopyran-4-one (Fig 2) is an active phytochemical compound found in citrus fruits as a glycoside conjugate and has a number of therapeutic attributes, including inhibitory and anti-inflammatory effect on chemokine production, which is linked to the pathophysiology of psoriasis. Literature suggests that it operates by inhibiting the production of NF-B, TNF-, IL-1, IL-6, and IL-8 while increasing the expression of VEGF and TGF-β, which facilitates tissue healing in animal models⁸.

Many methods for determining methotrexate and naringenin alone and in combination with other medicines have been developed and are available in the literature. However, no HPLC approach for simultaneous quantification of these drugs in combination dose forms has been developed and reported. The goal of this study was to create an RP-HPLC technique with UV detection for determining methotrexate and naringenin in pharmaceutical dose form at the same time. The current RP-HPLC technique was verified in accordance with the ICH criteria.

MATERIAL AND METHODS:

Methotrexate was received as a gift sample from Indian Pharmacopoeia Commission, India and naringenin was purchased from TCS chemicals (India) Pvt. Ltd. Acetonitrile (HPLC grade), di-potassium hydrogen phosphate, and orthophophosphoric (analytical grade) were obtauned from Finar Chemicals. Milli‑Qsystem (Millipore, Milford, MA, USA) water purification unit provided Ultra‑purified HPLC grade water.

Instrumentation and chromatographic conditions:

Simultaneous estimation of methotrexate and naringenin was carried using Dionex/ThermoUltiMate 3000 UHPLC System with UV-VIS detector. Cromleon^TM6.8 Software version was used for the data acquisition. Inertsil ODS-3 (15 cm × 4.6mm, 5μm) was the stationary phase. Injection volume of sample solution was 10μl and the run time was 32min. The temperature in the column was kept at room temperature and the eluents were detected through a UV detector at 302nm. Flow rate was 1ml/min with gradient programming given below.

Table 1: Chromatographic conditions for method development

Time (Minute)	Flow Rate (ml/min)	Mobile phase A (%)	Mobile phase B (%)
0	1	95	5
3	1	95	5
6	1	67	33
22	1	67	33
25	1	95	5
32	1	95	5

Mobile Phase Preparation:

A buffer solution of 3.4g di-potassium hydrogen phosphate in 1000ml water was the mobile phase A. The pH was modified to 6 which was achieved through dilute ortho-phosphoric system, filtered through 0.45µm filter followed by sonication for 20mins. Acetonitrile constituted mobile phase B. Mobile phase A and mobile Phase B in ratio of 1:2 was used as diluent in sample preparation which was filtered through 0.45µm filter followed by sonication for 20mins.

Standard Stock Solution:

To prepare the standard stock solution, 25mg of methotrexate and 50mg of naringenin were combined in a volumetric flask (25mL) followed by dilution with diluent. After sonicating the solution for 15 minutes, it was cooled down and the volume was adjusted with diluent. The solution was diluted further with the diluent to achieve concentrations of 100ppm for methotrexate and 200ppm for naringenin. Before analyzing, the solution was subjected to filtration with the help of a 0.45m filter.

Selection of wavelength:

Different wavelengths were screened for best system suitability parameters like asymmetry, theoretical plates, and resolution of methotrexate and naringenin at 302nm was found suitable for the simultaneous estimation both the drugs.

Preparation of Calibration Curve:

Calibration curves were established by dividing standard stock into suitable aliquots solution of methotrexate and naringenin in different volumetric flasks (10 mL) followed by dilution using the mobile phase to obtain final concentrations of 50, 80, 100, 120, 150ppm of methotrexate and 100, 160, 200, 240, 300ppm of naringenin. The chromatograms were acquired using a 1.0ml/min gradient flow rate after injecting the standard solution. At 302nm, the effluent was monitored, and calibration curves were established by graphing the average peak area versus concentration.

Method Validation:

The method was validated according to ICH guidelines for Range, Linearity, Specificity, Precision, Robustness, Accuracy, Limit of quantification (LOQ) and Limit of detection (LOD)⁹.

Specificity:

For investigating the specificity of the method, topical gel containing methotrexate and naringenin was formulated and investigated by injecting the blank (Diluent) and placebo to demonstrate the lack of elution interference of methotrexate and naringenin in gel formulation at concentration of 100 ppm and 200 ppm for methotrexate and naringenin respectively. The test and standard solution chromatograms were compared and the recovery percentage for the analytes was studied^9,10,11.

Figure 3: Chromatogram for methotrexate and naringenin test sample

Figure 4: Chromatogram for methotrexate and naringenin reference sample

Linearity and Range:

To investigate the linearity and range of the analytical process, successive dilutions of the stock solution were done by removing appropriate aliquots, yielding calibration curves spanning the concentration ranges of 50-150ppm for methotrexate and 100-300ppm for naringenin, respectively. The calibration plot was prepared using the triplicates of each concentration^12-16.

Precision:

The proposed analytical method's precision was established by computing the intra-day and inter-day relative standard deviation (RSD) values with acceptability criteria of less than 2%.

Percentage RSD after five replicate injections of each concertation 50ppm, 100ppm and 150ppm is determined for methotrexate and for naringenin. %RSD was determined after 5 replicate injections of each concentration of 100ppm, 200ppm and 300ppm^17-19.

Accuracy:

It was ascertained by calculating the recovery at level 80%, 100% and 120% for both the drugs. Three different test samples were assayed for each concentration 80ppm, 100ppm and 120ppm for methotrexate and 160ppm, 100ppm and 240ppm for naringenin.

Robustness:

To establish robustness, influence of small purposeful changes in the chromatographic parameters was examined^14-15. Influence of flow rate, buffer pH and temperature on percentage recovery and retention duration were investigated at concentrations of 100ppm and 200ppm for methotrexate and naringenin, respectively.

Sensitivity:

The sensitivity of the analytical procedure was calculated using the limit of quantification and limit detection (LOQ) and (LOD). The LOD and LOQ of the developed method was established by injecting small amounts of the standard solutions utilizing the described procedures. The LOD is the lowest analyte concentration detectable with a signal-to-noise ratio of (1:3), while the LOQ is the lowest concentration that can be quantified with acceptable precision and accuracy with a signal-to-noise ratio of (1:10)^20-21.

Assay in gel formulation:

In a volumetric flask, gel containing methotrexate (10 mg) and naringenin (20 mg) was placed along with the diluent, and the solution was sonicated for 20 mins in order to extract the active pharmaceutical ingredients from the formulated gel. A chilled extract solution was filtered using a nylon filter and the final concentrations of methotrexate and naringenin were 100g/mL and 200g/mL respectively. Using the peak areas mean values of the calibration curve the drug content in the gel was determined

RESULTS AND DISCUSSIONS:

Specificity:

The chromatograms in Figures 3 and 4 indicate the specificity of the HPLC technique established for the measurement of methotrexate and naringenin. Selectivity and specificity were investigated to detect the interaction of endogenous substances using a working solution comprising methotrexate and naringenin produced with diluent. It was affirmed by the results that there was no difference in retention period for standard and test formulations. None of the contaminants interfered with the test, as seen in the figure 3 and 4, methotrexate and naringenin were free of interference from other excipients used in the formulation. This shows that the approach has been chosen and is particular for determining methotrexate and naringenin at the same time.

Linearity and Range:

A calibration curve was generated by plotting an area versus concentration graph. Linear regression analysis was performed to assess the linear nature of the curve. The linearity validation procedure further revealed that the assay showed linear behavior over the studied concentration range with regression coefficients of R²=0.999 for methotrexate and R²=0.997 for naringenin. The regression equation for methotrexate and naringenin was Y= 0.599x + 5.113 and Y= 1.114x + 8.989 respectively. For both HPLC systems used in the validation, R2 values of more than 0.990 were obtained when the proposed analytical technique's linearity was tested. Therefore, linearity of the method makes it appropriate for quantifying methotrexate and naringenin in pharmaceutical dose forms.

Figure 5: Calibration curve of naringenin

Figure 6: Calibration curve for methotrexate

Precision:

RSD values were used to indicate the method's intra-day and inter-day accuracy. The RSD values for intra- and inter-day assay of QC samples did not surpass 2%, showing that the method have excellent accuracy¹². Table 2 summarizes the findings.

Table 2: Precision data of methotrexate and naringenin in gel using the described HPLC method

Compound	Concentration (ppm)	n	Interday Precision		Intraday Precision
Compound	Concentration (ppm)	n	Mean	% RSD	Mean	% RSD
Methotrexate	50	5	49.68	0.73	49.51	0.74
	100		99.87	0.47	99.72	0.40
	150		150.20	0.18	149.78	0.42
Naringenin	100	5	99.88	0.30	99.68	0.36
	200		199.87	0.23	199.72	0.20
	300		299.73	0.16	299.97	0.10

Accuracy:

Accuracy may be represented in terms of percent bias, that is the difference of percentage error between a reference and measured value. The high recoveries of the conventional addition procedure demonstrated that the proposed approaches were accurate. The procedure can be contemplated accurate as the % RSD of replicate assay of each test was <2% for all determinations.

Table 3: Results for Accuracy

Drug	Level (%)	Concentration (ppm)	(n) Amount Recovered (ppm)	% Recovery	% RSD
Methotrexate	80	80	80.22	100.27	0.38
	100	100	99.99	99.99	0.11
	120	120	120.26	100.22	0.21
Naringenin	80	160	160.13	100.08	0.58
	100	200	199.82	99.91	0.15
	120	240	240.26	100.11	0.11

Robustness:

The results in Table 4 demonstrate that small modifications to the chromatographic elements have no impact on the different chromatographic parameters, suggesting the robustness of the method.

Table 4: Results for Robustness

Compound	Level	Retention Time (mins)		% Recovery
Compound	Level	Methotrexate	Naringenin	Methotrexate	Naringenin
Flow Rate (ml/min)	1.2	6.415	20.917	100.15	100.05
Flow Rate (ml/min)	0.8	7.514	21.819	99.73	99.88
pH	6.2	7.056	21.515	100.03	100.12
pH	5.8	6.755	21.118	99.24	100.17
Temperature (ºC)	23	6.998	21.325	100.26	99.95
Temperature (ºC)	27	6.969	21.309	100.13	100.07
λ_max (nm)	304	6.981	21.319	100.55	100.19
λ_max (nm)	300	6.983	21.316	99.56	100.11

Sensitivity:

LOQ and LOD were determined using the signal to noise ratio approach. LODs of methotrexate and naringenin were affirmed to be 0.1g/mL and 0.2g/mL, distinctively. Methotrexate and naringenin LOQs were determined to be 1g/mL and 2g/mL, respectively.

Assay of Formulation:

The relevance of the method was evaluated through a laboratory-developed nano-emulgel formulation for measuring methotrexate and naringenin. Table 5 shows the data, which indicate low percent RSD values and high percentage recoveries.

Table 5: Results of Assay of nano-emulgel

Name of API	Amount Added (mg)	N	Amount Observed (mg)	% Mean Recovery	% RSD
Methotrexate	100	5	99.99	99.99	0.11
Naringenin	200	5	199.82	99.91	0.15

CONCLUSIONS:

The developed method for the simultaneous estimation of methotrexate and naringenin is simple, sensitive, and consistent, it may be utilized for determining methotrexate and naringenin in pharmaceutical formulations. Statistical examination of the findings indicated that the devised approach has excellent precision and accuracy. The RSD values for all parameters and asymmetry peaks were less than 2 and the theoretical plates for both methotrexate and naringenin was greater than 5000, indicating that the method is valid and that the test findings obtained using this approach are consistent. Therefore, the developed method can be used to estimate methotrexate and naringenin concentrations in medicinal formulations.

CONFLICT OF INTEREST:

The authors affirm that they do not have any competing interests.

ACKNOWLEDGMENT:

The facilities needed to carry out this investigation were generously provided by the Delhi Pharmaceutical Sciences and Research University (DPSRU), Delhi Institute of Pharmaceutical Sciences and Research (DIPSAR), and Indian Pharmaceutical Commission (IPC), for which the authors are grateful.

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Received on 06.09.2023 Modified on 08.12.2023

Research J. Pharm. and Tech 2024; 17(7):3050-3054.

DOI: 10.52711/0974-360X.2024.00477