RP-HPLC Method Development and Validation of Synthesized Codrug in Combination with Indomethacin, Paracetamol, and Famotidine

Background Indomethacin is considered a potent nonsteroidal anti-inflammatory drug that could be combined with Paracetamol to have superior and synergist activity to manage pain and inflammation. To reduce the gastric side effect, they could be combined with Famotidine. Methodology. A codrug of Indomethacin and Paracetamol was synthesized and combined in solution with Famotidine. The quantification of the pharmaceutically active ingredients is pivotal in the development of pharmaceutical formulations. Therefore, a novel reverse-phase high-performance liquid chromatography (RP-HPLC) method was developed and validated according to the International Council for Harmonization (ICH) Q2R1 guidelines. A reverse phase C18 column with a mobile phase acetonitrile: sodium acetate buffer 60 : 40 at a flow rate of 1.4 mL/min and pH 5 was utilized. Results The developed method showed good separation of the four tested drugs with a linear range of 0.01–0.1 mg/mL (R2 > 0.99). The LODs for FAM, PAR, IND, and codrug were 3.076 × 10−9, 3.868 × 10−10, 1.066 × 10−9, and 4.402 × 10−9 mg/mL respectively. While the LOQs were 9.322 × 10−9, 1.172 × 10−10, 3.232 × 10−9, and 1.334 × 10−8 mg/mL, respectively. Furthermore, the method was precise, accurate, selective, and robust with values of relative standard deviation (RSD) less than 2%. Moreover, the developed method was applied to study the in vitro hydrolysis and conversion of codrug into Indomethacin and Paracetamol. Conclusion The codrug of Indomethacin and Paracetamol was successfully synthesized for the first time. Moreover, the developed analytical method, to our knowledge, is the first of its kind to simultaneously quantify four solutions containing the following active ingredients of codrug, Indomethacin, Paracetamol, and Famotidine mixture with added pharmaceutical inactive ingredients in one HPLC run.


Introduction
Nonsteroidal anti-inflammatory drugs (NSAIDs) are among the most consumed and prescribed drugs for both pain and inflammation worldwide [1]. eir blockage of prostaglandin synthesis by inhibiting cyclooxygenase (COX) is responsible for both the desired anti-inflammatory effects and the undesired gastrointestinal effects [2][3][4]. Based on COX selectivity, NSAIDs are divided into two families: nonselective NSAIDs that block both cyclooxygenase I & II and selective cyclooxygenase II inhibitors [5][6][7].
Indomethacin (IND) is an example of a potent nonselective COX inhibitor that showed efficient analgesia with antipyretic and anti-inflammatory activities [8]. It is classified as an indole-acetic acid derivative according to the NSAIDs chemical classification with the chemical name of 1-(p-chlorobenzoyl)-5-methoxy-2-methylindole-3-acetic acid [9]. It is a poorly soluble class II compound with a half-life of 4-5 h [10,11]. It is utilized to treat rheumatoid diseases by elevating the inflammation. Moreover, it can decrease the risk of colon cancer by providing chemoprotective effects against tumors [12,13]. Like other NSAIDs, it appeared to have gastrointestinal, renal, and other side effects [14]. e gastric side effect could be reversed by the administration of Famotidine (FAM), which is a competitive histamine H2-receptor antagonist that inhibits the secretion of gastric acid and also increases collagen secretions [15,16].
Recently, there has been a trend for combining NSAIDs with Paracetamol (PAR) as this often provides a synergic analgesic effect and reduces the adverse effects resulting from NSAIDs [17]. Paracetamol, N-acetyl-p-aminophenol (also known as acetaminophen), is utilized globally as an analgesic and antipyretic drug. Regarding the mechanism of action which is considered to be a weak inhibitor of the synthesis of prostaglandins (PGs), their effects in vivo are similar to these COX-2 inhibitors [18]. Although their analgesic effects are often weaker than NSAIDs, it has better tolerance, and accordingly, it is often preferred [19].
Indomethacin is considered strong and potent antiinflammatory activity against rheumatoid arthritis and other inflammatory diseases and Paracetamol is considered as the first-choice medication for both acute and chronic pain [20]. erefore, the combination of Indomethacin with Paracetamol provides excellent anti-inflammatory and analgesic activities with a reduction of the Indomethacin side effects. Seidman and Melander reported equianalgesic activity with milder side effects upon the administration of Paracetamol with a low dose of Indomethacin in comparison to the high dose of Indomethacin alone for the treatment of rheumatoid arthritis [21]. Famotidine is considered the most potent H2 antagonist for the treatment of peptic ulcers and was found to be effective for prevention of Indomethacin-induced gastric injury even in the lowest dose [22,23]. erefore, we aim to synthesize a novel codrug of Indomethacin and Paracetamol (IND-PAR) through a hydrolyzable ester bond combined in solution with Famotidine.
Reverse phase-high performance chromatography (RP-HPLC) is considered one of the most common analytical techniques used for the development and characterization of pharmaceutical products [24,25]. Moreover, HPLC provides a rapid, sensitive, and precise technique to separate and identify the analyzed drugs in combination or the used pharmaceutical dosage forms. erefore, It is necessary to validate the developed HPLC method according to the International Council for Harmonization (ICH) and the United States Pharmacopeia (USP) requirements [26,27].
Moreover, a simple and universal RP-HPLC method of analysis was developed and validated for the successful separation of a mixture containing four components: codrug, Indomethacin, Paracetamol, and Famotidine in the formulation. e developed method was used to study the hydrolysis profile of the codrug in the presence of the esterase enzyme.

Instrumentations.
High-Performance liquid chromatography (Waters 1525, Singapore) binary HPLC pump and waters 2298 photodiode Array Detector were used. Nuclear Magnetic Resonance (NMR) spectrum was recorded on Bruker 500 MHz-Avance III, Switzerland. e high-resolution mass spectrum (HRMS) was recorded on a Shimadzu LCMS-IT-TOF utilizing ESI (+) method.
en the crude product was purified using flash chromatography on silica gel eluted with a mobile phase of Hex: EtOAc 1 : 2 to provide a yellow solid product with a yield 70% (220 mg) and R f � 0.63 (Hex: EtOAc 1 : 2). 1

Prepared Solutions
(1) Buffer Solution pH 6. 13.6 g of sodium acetate trihydrate was dissolved in 750 mL HPLC water, then 1 mL of Et 3 N was added, diluted with HPLC water to 1 L, and adjusted to pH 6.0 with glacial acetic acid. e mobile phase was firstly prepared using a mixture of sodium acetate Buffer:ACN, 93 : 7 [28].
(2) Diluent 1. 6.8 g of potassium dihydrogen phosphate was dissolved in 1 L HPLC water and adjusted to a pH 6.0 using glacial acetic acid.

pH, Mobile Ratio, and Diluents Used in Method
Development Trials. Different mobile phase composition, pH, and diluents were tried throughout the analytical method development. e used mobile phases and diluents at different pH are summarized in Table 1.

Analytical Method Validation.
e analytical method was developed according to USP and ICHQ2R1 guidelines and validated using the following parameters: linearity, range, accuracy, precision, robustness, and ruggedness [29]. All prepared parameters were in triplicates.

Linearity and
Range. Linearity was measured by preparing a serial five concentrations in the range of 0.01-0.1 mg/mL form a preprepared stock solution of 1 mg/ mL. e calibration curves were built by plotting the mean area under the curve (AUC) obtained from the HPLC against concentrations. e regression equation and the squared correlation coefficient (R 2 ) were calculated for each ingredient curve.

Accuracy.
Accuracy and selectivity validation parameters were calculated by preparing a standard solution of a mixture of four drugs, having a concentration of 0.24 mg/ mL for each drug. ree concentration levels of 80%, 100%, and 120% of the standard concentration were made. e three solutions were prepared containing different excipients: microcrystalline cellulose, magnesium stearate, aerosol, and Ac-Di-Sol. e accuracy was evaluated by calculating the percentage of recovery.

Selectivity.
e selectivity of the developed method was examined as the eluted peaks are well separated and not affected by any of the added excipients.

Precision.
Precision was performed at different levels. At first, instrument precision was done by injecting the standard mixture 9 times; the % RSD of the generated peaks of the chromatogram was calculated. An intermediate precision including interday and between analyst precision was examined on 0.08 mg/mL and 0.1 mg/mL concentration, respectively. e percentage relative standard deviation was calculated for both mixtures.

Robustness.
e robustness of the developed method was performed by doing minor modifications on the method parameters, including detection wavelength, different mobile phase pH, and flow rate [30]. e studied robustness parameters were the pH effect of the mobile phase (4.9, 5.0, and 5.1), the detection wavelength (273, 275, and 277 nm), and the effect of the mobile phase flow rate (1.2, and 1.4 mL/min).

Detection and Quantification Limit (LOD & LOQ).
Limit of detection (LOD) and limit of quantification (LOQ) is an indication of the analytical method sensitivity. Signal to noise ratio in the HPLC chromatogram was used to calculate these two parameters for each compound. e LOD and LOQ value of the compound was determined when the signal to noise ratio is 3 : 1 and 1 : 10, respectively.

Hydrolysis of Codrug.
e synthesized codrug was incubated with an esterase enzyme to study its hydrolysis to its parent drugs (IND & PAR). is was achieved by incubating 1 mg of codrug into 10 mL phosphate buffer saline solution (pH 7.4) containing 1 mg of esterase enzyme (10 U) at 37°C for 1 h [31][32][33]. At different time intervals, aliquots of 1 mL were obtained, and then the concentrations were analyzed by the developed HPLC method.

Statistical Analysis.
All prepared parameters, including the in vitro hydrolysis of the codrug, were performed in triplicates. e data were expressed as means ± relative standard deviation. Statistical analysis was performed on robustness parameters using the ANOVA test. Statistically, a significant difference was considered when the p value was <0.05.

Synthesis of IND-PAR Codrug.
Herein, we aim to synthesize a codrug of Indomethacin and Paracetamol to obtain a synergistic analgesic, antipyretic, and anti-inflammatory activities. e synthesis of the codrug was achieved through the formation of the ester bond between IND and PAR using EDC as a coupling agent and 4-(Dimethylamino)pyridine as a base, as shown in Scheme 1. e codrug was successfully synthesized with a high yield of 70%. e structure of the codrug was confirmed by NMR and HRMS.

Method Development.
e RP-HPLC analytical method for codrug, IND, PAR, and FAM was developed and validated according to the ICH guidelines [34]. e analytical method development was mainly based on the USP International Journal of Analytical Chemistry analytical method of FAM and IND and was then optimized for the best separation for the component mixture [35]. At first, FAM and IND mixture was injected using the USP analytical method for the Famotidine tablet. e produced HPLC chromatogram showed only one peak of FAM, while the IND was not eluted even after 40 minutes. e late elution of IND is probably due to its high lipophilicity and the high hydrophobicity of the used mobile phase (ACN: sodium acetate buffer 7 : 93). Further modification of the mobile phase pH was done in order to reduce the retention time of IND by making the mobile phase more acidic. Different pH mobile phases were tried, including 5, 5.5, and 6. e results showed that the most acidic pH mobile (pH 5) achieved early elution of IND to less than 10 min. In order to get the best separation, the mobile phase was further optimized at different solvent compositions. Lastly, the best separation was achieved at the mobile phase composition (ACN: sodium acetate buffer 60 : 40). e codrug was added to the above mixture of IND and FAM using pure ACN as the diluent. e final HPLC chromatographic conditions of the developed method were by using XTERRA ® MS C18, 5 μm, 4.6 × 250 mm analytical column with a flow rate of 1.4 mL/min and a detection wavelength of 275 nm. e operation temperature of the column was set at 25°C. e injection volume was 10 μL and the run time was 20 minutes. e developed method showed well-separated peaks for the component mixture. e resulted peaks were symmetrical with narrow broadening eluted at different retention times: 3.220, 3.624, 7.751, and 14.034 min for FAM, PAR, IND, and codrug, respectively, as shown in Figure 1.

Linearity and Range.
e quality of an analytical method is profoundly dependent on the linearity of the calibration curve. e main characteristics of a calibration curve are the slope line, the regression, and the correlation. e linearity of the method was measured by plotting the area under the curve obtained from the HPLC of each drug against the corresponding concentrations. e linearity was demonstrated over the concentration range (0.01-0.1 mg/ mL) for FAM, PAR, IND, and codrug, respectively. e obtained goodness-of-fit (R 2 ) was more than 0.99 that confirms the linearity between the concentration and the area under the peak. e slopes of the regression line for FAM, PAR, IND, and codrug are shown in Figure 2.

Selectivity.
e four drugs components (FAM, PAR, IND, and codrug) were formulated with the following inactive ingredients: microcrystalline cellulose, magnesium stearate, aerosol, and Ac-Di-Sol to study selectivity of the developed analytical method [36].
is parameter was investigated to show that there is no possible interference of the added tablet formula excipients on the separation and measurements of peak areas for the ingredient mixture ( Figure 3).

Accuracy.
e method showed great accuracy within the tested concentration range (0.08-0.12). e percentage of RSD and percentage of recovery for all tested solutions are within the acceptable limits (100% ± 2%); the data are shown in Table 2.

Precision.
e precision of a method is the degree of agreement among individual test results when the procedure is applied repeatedly to multiple samplings. e method precision was examined at different levels; system precision was examined by injecting 0.1 mg/mL nine times on HPLC and the % RSD was found to be less than 2.0 for all tested compounds. e intermediate precision validation parameter at different days (intraday precision) was studied by performing three replicates measurements at two different concentrations (0.08 and 0.1 mg/mL). e results showed that the percentage relative standard deviation of the triplicate of each concentration was less than 2.0. Moreover, the repeatability was tested for different analysts by doing three replicates measurements of the mixture at 0.12 mg/mL and the result % RSD was also less than 2.0. e precision results at different precision levels are illustrated in Table 3.

Detection and Quantification Limit (LOD & LOQ).
e detection limit or LOD is the lowest amount of analyte in a sample that can be detected but not necessarily quantified. However, the limit of quantification or LOQ is the lowest amount of analyte in a sample that can be determined quantitatively with convenient precision and accuracy. e result showed that the calculated LODs for FAM, PAR, IND, and codrug were found to be 3.076 × 10 −9 , 3.868 × 10 −10 , 1.066 × 10 −9 , and 4.402 × 10 −9 mg/mL, respectively, while the calculated LOQs were 9.322 × 10 −9 , 1.172 × 10 −10 , 3.232 × 10 −9 , and 1.334 × 10 −8 mg/mL, respectively.

Robustness.
Robustness is the capacity of a method to remain unaffected by minor variations in method conditions; in other words, it is a measure of the reliability of a method. e robustness of an analytical procedure was tested by measuring its capacity of the developed method to remain unaffected by small but deliberate variations in the method parameters and providing an indication of its reliability during the normal use. For this study, the flow rate, wavelength, and pH parameters were changed for a mixture of 0.1 mg/mL. e results are summarized in Table 4. As can be observed, the % RSD values in all tested and varied parameters were less than 2.0 which indicates the good robustness of the developed analytical method. Moreover, the ANOVA test shows no significant difference for the tested compounds at different robustness validation parameters (p value >0.05).

System Suitability.
System suitability tests are utilized to justify that a system is performing sufficiently to guarantee confidence in the analytical method and the obtained results.
e developed method showed that all of the standard system suitability parameters, including the resolution (R), the symmetry of the peaks theoretical plates (N), and retention factor (K), are within acceptable limits as exhibited in Figure 4. e system suitability tests are summarized in Table 5.
An acidic mobile phase was used in the analytical method (pH � 5) and the results of the system suitability showed good results of the tested parameters indicating the method still performs very well under the acidic pH conditions. Moreover, the method was tested at lower pH (4.9) as a part of ruggedness and robustness validation, and results were not affected by this intended lowering of the mobile phase pH.

In Vitro Hydrolysis of Codrug.
e developed analytical method was applied to investigate the in vitro conversion of the codrug to its parent drugs (IND and PAR) in the presence of esterase enzyme (1 U/mL) in PBS (pH 7) at 37°C.

Conclusion
In this study, IND and PAR codrug was successfully synthesized. RP-HPLC method was developed and validated according to the ICH Q2R1 and USP guidelines for the successful separation of a mixture containing four components formula: FAM, PAR, IND, and codrug. e tested parameters, including linearity, accuracy, selectivity, precision, limits of detection and quantification, and robustness, were found to be within the recommended guideline ranges. Moreover, the developed method was successfully applied to quantify the in vitro hydrolysis and conversion of codrug into its parent drugs. To the best of our knowledge, this is the first study that provides an easy and direct quantification method of a combination formula of different chemical compounds (FAM, PAR, IND, and codrug).

Data Availability
e data used to support the findings of this study are included within the article.

Conflicts of Interest
e authors declare that there are no conflicts of interest regarding the publication of this paper.