Eco-friendly RP-HPLC method for concurrent estimation of a promising combination of methocarbamol and etoricoxib in rat plasma

2.1 Chemicals and reagents

This study utilized chemicals such as methanol, sodium dihydrogen phosphate, sodium hydroxide, and orthophosphoric acid procured from reputable suppliers like Merck, Scharlau, and Fischer. MTC of purity 100.07% was purchased by Gennex Laboratories Limited, Telangana, India, while Aurore Life Sciences, a Surat-based company, acquired ETC of purity 99.33%. CFA was acquired from Sigma Chemical Co. in Missouri, USA, while methotrexate (MTX) was sourced from Shanghai Xinyi Pharmaceutical Co., Ltd. in Shanghai, China.

2.2 Chromatographic conditions

The Agilent 1200 Series HPLC system, manufactured by Agilent Technologies (Santa Clara, CA, USA), was employed to develop a novel method for elution in an isocratic mode. A 4.6 mm × 250 mm Hypersil BDS C8 column with a 5 µm particle size was used in the experimental setup. The analysis was maintained at a wavelength of 274 nm and a flow rate of 1.5 mL·min⁻¹. Methanol and buffer pH 2.5 were combined in the mobile phase at a volumetric ratio of 34:66 v/v. To prepare the buffer solution, 1 mL of phosphoric acid was dissolved in 1,000 mL of water (Solution 1), and 1.6 g of sodium dihydrogen phosphate monohydrate was dissolved in 1,000 mL of water (Solution 2). Equal volumes of Solution 1 and Solution 2 were mixed thoroughly. The resulting buffer solution contained 0.05 M sodium dihydrogen phosphate, and the pH was adjusted to 2.5 using phosphoric acid or sodium hydroxide as required.

2.3 Diluent

The diluent employed in the study comprised distilled water and methanol combined in a 50:50 volumetric proportion.

2.4 Stock solutions

The MTC, ETC, and IS solutions were meticulously prepared with the utmost care and precision at 1 mg·mL⁻¹ concentration. Accurately measuring 100 mg of each component and dissolving them in 50 mL of a suitable diluent, we filled the flasks to the mark with the same diluent to produce three working solutions. One working solution has 10 μg·mL⁻¹ of MTC, one with 10 μg·mL⁻¹ of ETC, and one with 100 μg·mL⁻¹ of the IS solution. The solutions are stored at −20°C in the following step, ensuring their stability and reliability for future use.

2.5 Establishing of calibration curves

ETC and MTC were diluted to respective working solutions (10 µg·mL⁻¹) using a pair of 10 mL volumetric flasks to generate varying concentrations within the 1–50 µg·mL⁻¹ range. Methanol was added to the remaining volume of each flask after 1 mL of blank plasma was added. After vertexing for 1 min and centrifugation for 10 min at 4,000 rpm, the filtrate was transferred into separate pairs of volumetric flasks. The contents were then evaporated until all the liquid had evaporated, resulting in dry residue. In each flask, 0.1 mL of the working solution was added after the residue was dissolved in 2 mL of methanol. Following that, a mobile phase combination was added to increase flask capacity. We accurately applied 20 μL of each solution in triplicate using the autosampler. Under specified chromatographic conditions, the operation was conducted. A 10 µg·mL⁻¹ concentration was added to the calibration standards for both ETC and MTC. Plotting the area under the peaks versus the concentration in µg·mL⁻¹ for the calibration curves. After such calibration curves were obtained, regression equations were calculated.

The quality control samples (QCSs) also had 4, 8, and 12 µg·mL⁻¹ concentrations. After that, HPLC was executed on each sample based on a volume of 20 μL. Calibration standards and samples were maintained at −20°C immediately after preparation.

2.6 Experimental animals

Twenty male Wistar rats weighing 160–200 g in adulthood were carefully selected for the study. The animals were observed for 2 weeks to ensure no simultaneous infections before initiating the experiment. The rats were housed in polypropylene enclosures, calibrated to maintain a humidity level of approximately 55.5%, a room temperature of 22°C, and a light–dark cycle of 12 h. They had unlimited access to rodent food and water. The postgraduate studies for advanced sciences faculty at Beni-Suef University in Egypt conducted the study following all applicable laws and regulations governing animal welfare and ethics for regional experimental animals. All the animal handling, feeding, watering, dosing, and weighting were approved according to the ethical committee for Animal Care & Use Committee of Beni-Suef University (Institutional Animal Care and USE Committee; IACUC) with approval number BSU.022.461 and also according to the ARRIVE guidelines for animal research studies.

1. Ethical approval: The research related to animal use has been complied with all the relevant national regulations and institutional policies for the care and use of animals, and has been approved by the ethical committee for Animal Care & Use Committee of Beni-Suef University (Institutional Animal Care and USE Committee; IACUC) with approval number BSU.022.461.

2.7 Induction of rheumatoid arthritis

In the experiment, arthritis was induced by administering 0.1 mL of CFA solution into the right hind paw footpad on two separate days.

2.8 Animal grouping

After inducing RA in adult male rats using Freund’s adjuvant (CFA), they were divided into four groups of ten each. After inducing RA with CFA, twenty adult male rats were divided into four groups of ten rodents each. Group 1 (normal control rats) received the same quantity of vehicle (saline) intra-pedal and orally. Group 2 (arthritis control group) was injected with equal quantities of saline and given orally as a positive control group. The MTX group as standard treatment were received intragastric administration of MTX (2.5 mg/tablet once per day; 20150403; Shanghai Xinyi Pharmaceutical Co., Ltd.), and treatment rats via oral administrations of MTC/ETC combinations, at doses of 135 mg·kg⁻¹ b.wt and 1.8 mg·kg⁻¹ b.wt., respectively. Before CFA injection, at zero time after injection, 1, 2, 3, and 4 h after rheumatoid arthritis induction, measurements were obtained. Using an electronic micrometer, the volume of the rear limb was measured. The measurements were taken before administering the CFA injection, immediately after injection (zero time), and at 1, 2, 3, and 4 h following arthritis induction. The posterior limb volume was measured using an electronic micrometer.

2.9 Assessment of paw edema

As part of the paw edema and swelling rate evaluation, paw volume is measured in each group to monitor arthritis progression. All animals used in this study were ethically treated throughout the experimental time and before scarification via ketamine (90 mg·kg⁻¹) and xylazine (5 mg·kg⁻¹) combination by ratio of 1:1 and 0.1 mL/100 g rat, intraperitoneal. Four measurements were taken before and after injection of CFA, as well as 1, 2, 3, and 4 h after injection. A micrometer was used to measure the rear limb’s volume.

2.10 Biochemical investigations

This study used specific ELISA kits to measure c-reactive protein (CRP) levels in serum, liver, and kidney function in compliance with the manufacturer’s guidelines.

2.11 In vivo sample preparation

Gastric gavage was used to deliver ETC and MTC combinations simultaneously. A blood sample was taken from the median orbital plexus of the rat’s eye to determine the blood and plasma levels. After oral administration, 30 min, 1, 2, 4, 6, 8, 12, and 24 h were chosen as the time intervals. Six rats from the ten total numbers per group were used for sample analysis after excluding the very thick and dead rats. We obtained the rodents from the Beni-Suef University Faculty of Veterinary Medicine. Each animal was weighed, given anesthesia, imbibed, and petted under standard conditions. The approval number was obtained from Beni-Suef University's animal care and ethical committee, carrying the number 022-461. Laboratory animals were handled and used according to their guidelines, as well as the ARRIVE guidelines for animal research studies. These guidelines are incorporated into the IACUC guidelines. The frozen plasma samples underwent thawing at room temperature before the study. The next step was to transfer 100 μL of each sample into a centrifuge tube and mix it with 1 mL of methanol to precipitate the plasma proteins. After vortex mixing the samples for 1 min, they were centrifuged at 4,000 revolutions per minute for 20 min to separate the precipitated plasma proteins. The clear liquid part of each sample was then transferred entirely to a separate sterile tube and left to dry until completely evaporated. Then, the samples were reconstituted using 0.5 mL of methanol. A 10 μL sample of DIC was mixed with the diluent to make a final volume of 1 mL. This was used to make the working solution. This was the last step. Twenty microliters of each sample were added three times to the HPLC machine, following the steps for the HPLC procedure.

2.12 Statistical analysis

The data were analyzed using Statistics for the Social Sciences, version 22 (SPSS, Chicago, IL, USA). A one-way analysis of variance with Tukey’s post hoc multiple comparison test was used to compare the means of the different groups. Additionally, the mean standard error of the mean was calculated to reflect the statistical measure of variability in a sample mean. The outcome obtained from the study showed statistical significance with a p-value of less than 0.05.

3.1 AMVI metric

The AMVI technique is extensively acknowledged as the most reliable and robust tool for assessing chromatographic procedures with a high degree of professionalism. It guarantees accurate quantification of the solvents and waste generated during the analysis cycle. It takes a specific HPLC analysis to get precise results when making an active microbial volatile organic compound index (AMVI). To determine the total amount of solvents used, it is significant to record all solvents and compounds used during the collection of samples and chromatographic procedures. The given equation offers a simple method for calculating how much solvent the analytical approach will use and guarantees accuracy with no room for error. Solvent consumption during HPLC and sample preparation is added to determine a method’s total solvent consumption. The overall amount of solvent consumed by a technique is equal to the sum of the solvent consumption during HPLC and the solvent consumption during sample preparation. A complete HPLC analysis requires “number of injections” divided by the number of analytes and a flow rate to estimate the solvent consumption (mL) [23]:

Total solvent consumption = (amount of solvent for standard preparation) + (volume of sample preparation × number of sample preparations) + (volume of system suit preparations).

Multiply the total solvent usage by the number of peak regions of interest to get the AMVI value. A lower AMVI number means that sustainability is more likely to be attained. Analytical methods can be evaluated professionally using this equation. To calculate the volume intensity, divide the total method solvent consumption by the number of analytes:

Technique consumption of solvents = % HPLC consumption of solvent and total method use of solvents.

The solvent consumption sample preparation process is the entire amount of solvent consumed divided by the number of solvent consumption sample preparations.

3.2 Analytical eco-scale assessments

Using the ESA metric is essential when appraising sustainability. As a result, a penalty score is assigned based on parameters such as reagent usage, the potential for risks, the amount of energy consumed, and the amount of waste generated [24]. These scores are summed to determine a comprehensive sustainability level. Penalty points will reduce the score if a perfect 100 is assumed [25]. A system can be classified as “superior green” if its score exceeds 75 points, as “Ideal green” if it scores 50–75 points, or as “subpar green” if it scores lower than 50 points.

3.3 AGREE tool

The complete assessment method employed in AGREE incorporates both general and ecological sustainability factors. Green analytical chemistries (GAC's) 12 core principles determine the numerical score of each principle, ranging from 0 to 1. Each principle contributes to ecological sustainability and, therefore, receives a score of 1. The accomplishment grades for each standard are shown as red, yellow, and green hues on the graph, with each region’s size comparable to the evaluated parameter [26]. AGREE is most used to assess an organization’s environmental sustainability. This methodology is based on the 12 GAC principles.

4.1 Methods development and optimization

After performing various trials, we found that manipulating the mobile phase composition was crucial. We first tried methanol and water in a 50:50 v/v ratio. We then tried a combination of buffer phosphate (pH 4.0) and methanol in the same ratio. Finally, we found the best conditions by using a mobile phase consisting of a buffer solution with a pH of 2.5 and methanol in a volumetric ratio of 34:66. We also used different columns with varying packing materials and lengths, including C18, phenyl, and cyano columns ranging from 100 to 250 mm in length. After trying all of them, we found that the 5 µm Hypersil C8 column with 250 mm × 4.6 mm dimensions was the best choice. This column showed improved resolution and rapid separation, as shown in Figure 1. We also tested various wavelengths in the 200–400 nm range and found that a wavelength of 274 nm gave a chromatogram with distinct peaks and high resolution. Therefore, the optimal chromatographic conditions for obtaining well-defined, distinct peaks with high resolution were a Hypersil BDS C8 column with 4.6 mm × 250 mm dimensions and a particle size of 5 µm, a flow rate of 1.5 mL·min⁻¹, a detection wavelength of 274 nm, and a mobile phase consisting of a mixture of buffer pH 2.5 and methanol in a volumetric ratio of 34:66. The column temperature was maintained at 25°C.

Figure 1

HPLC chromatograms for three different samples: (a) blank plasma, (b) blank plasma spiked with MTC, ETC, and DIC (IS) medicines, and (c) sample obtained after oral administration to rats for 1 h.

4.2 In vivo study

Considering the reported pharmacokinetic properties, the recommended administration schedule is 6-h intervals. In hemodialysis patients, the duration of elimination does not exhibit any significant variation compared to the control group. Nevertheless, it was observed that the renal clearance in hemodialysis patients was diminished [32]. In individuals diagnosed with cirrhosis, there is a decrease in clearance, but there is currently no specific recommendation for a dose change. According to a study conducted by researchers, individuals diagnosed with cirrhosis could tolerate a dosage of 500 mg delivered twice daily [33]. This study assesses CRP as a measure of systemic inflammation in RA [34]. Rodents with arthritis had noticeably increased serum levels of CRP, which is matched with the results of Arjumand et al. [35]. On the other hand, MTC and ETC supplementation dramatically reduced CRP serum levels in arthritic animals, suggesting that MTC and ETC are anti-inflammatory.

4.3 Effect of MTC and ETC on gross lesions of the paw and ankle joint (induced RA)

On the surface, rats with arthritis induced by CFA exhibited swelling, edema, and erythema on their right rear feet and ankles. As a result of the MTC and ETC treatment, these visible lesions significantly decreased in the arthritic group (Figure 2).

Figure 2

Different groups of rats show visceral edema and inflammation in their right rear feet and ankles; normal control rats, arthritic rats, and standard-treated rats treated with MTC and ETC suggest their anti-inflammatory and antiarthritic macroscopic properties.

4.4 Effect of MTC and ETC on right hind paws volume

CFA-induced arthritis caused rats to have edematous paws as compared with controls (p < 0.05). Rats with arthritis had smaller feet that were treated with MTC and ETC (Figure 3). In addition to measuring the circumference of the right hind foot and ankles, digital calipers were used to visualize edema and inflammation in the right rear feet of rodents from various groups. An experimental group of normal rats and arthritic rats received MTC and ETC, as well as an experimental group of normal rats and arthritic rats.

Figure 3

CFA-induced arthritis rats’ right-hand paw size in response to MTC–ETC combination. The symbols at p < 0.05 indicate significance.

4.5 Effect of MTC and ETC on serum CRP levels

As shown in Figure 4, MTC and ETC affect serum levels of CRP. Rodents given CFA showed significant increases in CRP serum levels (p < 0.05). As compared to the arthritis group, MTC and ETC significantly decreased CRP serum levels in arthritic rodents (p < 0.05).

Figure 4

Efficacy of MTC and ETC in reducing serum CRP levels. Statistically significant differences (p > 0.05) between parameters are indicated using different superscript alphabets (a) and (b).

4.6 Safety study about MTC and ETC combinations on complete blood picture (CBC), liver (glutamate pyruvate transaminase [GPT] and glutamate oxaloacetate transaminase [GOT]), and kidney functions (creatinine and urea)

In the current study, no statistically significant alterations (p > 0.05) were seen in the levels of hemoglobin and total erythrocyte count among the different groups of rats. Similarly, there were no significant changes in packed cell volume and TEC, as depicted in Figure 5. Concerning the differential cell count, as depicted in Figure 5, there were no notable disparities found across the treatment groups. The total leucocytic count did not significantly change (p > 0.05) in control-positive arthritic rats; however, the platelet count did decrease modestly, as shown in Figure 5.

Figure 5

Blood pictures were taken in (a) normal, (b) arthritic, (c) standard, and (d) treated rats.

MTC and ETC reduce the elevated levels of hepatic enzymes in untreated, positive rodents with elevated ALT, AST, and GPT/GOT or GPT/GOT activity, which indicates a hepatotoxic effect in RA patients (Figure 6). Accordingly, MTC and ETC reduce the elevated renal enzymes depicted in Figure 6 as well as the blood urea levels, suggesting a nephrotoxic effect of CFA or renal injury in RA patients.

Figure 6

Different groups had differing liver enzyme (GPT and GOT) and kidney enzyme (creatinine and urea) activity. Superscript alphabets (a)–(c) indicate statistically significant parameter differences.

As a result, our findings showed how effective MTC and ETC are at lowering inflammatory responses within tissues and suppressing inflammatory responses and arthritis-related paw edema. Because of their inhibitory effects in reducing tissue inflammation and restricting the edematous features of arthritis, our results thus indicated the anti-inflammatory activity of MTC and ETC therapies as therapeutic targets for RA.

The results of our study jointly provide evidence and elucidate the effectiveness of MTC and ETC as medicines for the treatment of RA, exhibiting anti-arthritic and anti-inflammatory properties without causing toxicity. Our results are supported by the observed normal liver and kidney functions in the subjects of our investigation. The study found a significant decrease in peptic ulcers among individuals who received treatment with ETC compared to those treated with ibuprofen [36]. The comparative analysis of ETC with other NSAIDs has demonstrated its efficacy as a therapeutic alternative for those suffering from osteoarthritis and RA. However, it is essential to note that ETC is associated with a relatively elevated likelihood of gastrointestinal problems. According to a comparative analysis between ETC and other NSAIDs, ETC demonstrates greater efficacy as a treatment option for patients suffering from osteoarthritis, RA, ankylosing spondylitis, or gout who exhibit limited cardiovascular risk factors and a relatively elevated susceptibility to gastrointestinal complications. MTC toxicity in isolation, without numerous drug exposures, is rare and improbable to result in fatality [37].

4.7 Method validation

The proposed methodology was rigorously evaluated as an integral part of the validation procedure for the pharmaceutical compounds under investigation, adhering strictly to the procedures issued by the United States Food and Drug Administration (FDA) for validating bioanalytical methods in an industrial setting for parameters specific to biological matrices (e.g., matrix effect, recovery, stability in plasma) [38] and ICH Q2(R1) guidelines for analytical validation criteria, including specificity, linearity, accuracy, precision, limit of detection (LOD), and limit of quantification (LOQ) [38,39]. This dual approach ensures compliance with international standards for both bioanalytical and pharmaceutical quality control (QC) applications [39].

4.8 Linearity and range

Calibration curves were constructed utilizing a linear regression approach to graphically represent the peak-area ratio of each drug relative to the IS as a function of their respective concentrations. The HPLC approach involved the examination of concentrations within the range of 1–50 µg·mL⁻¹. Using the HPLC method, it was possible to find influential correlation coefficients, with values of 0.9999 for MTC and 0.9998 for ETC. Regression equation parameters are given in Table 1.

4.9 Limits of quantification

4.10 QCSs

Three QCSs were used to validate the procedure (spiked plasma samples). The QCSs are categorized into three groups: low QC (LQC) samples, middle QC (MQC) samples, and high-quality (HQC) samples. The LQC samples have a concentration three times higher than the LLOQ. The MQC samples have concentrations that fall within the middle range of the calibration graph. The HQC samples have concentrations that are below the upper limits of quantitation. Both drugs were examined using HPLC methods with 4, 8, and 12 µg·mL⁻¹ QC concentrations.

4.11 Accuracy and precision

The method’s accuracy was assessed by employing QCSs and following the linearity technique. After performing the regression analysis, the concentrations were computed. The acceptable accuracy limit for QC should not surpass 15%, whereas the LLOQ should not exceed 20% accuracy. The accuracy and precision of a previous QCS were evaluated by performing three separate analyses on the same day. Additionally, the precision and accuracy between runs were evaluated by conducting a consecutive 3-day examination of the same samples. It was considered less accurate if the RSD (or CV) value was higher than 15%. In comparison, a bias value within 15% for the measured concentrations was considered good enough. The intraday HPLC method was found to have a remarkably low relative standard deviation (% RSD), as shown in Table 2. The values obtained for recovery fell within the acceptable range of 100 ± 15%, indicating that the established procedures are reliable and accurate, as demonstrated by the finding in Table 2. These findings confirm the precision and reproducibility of the simultaneous determination of the researched substances using the established procedures.

4.12 Selectivity

Selectivity in the plasma matrix pertains to the efficacy with which the used procedures may effectively differentiate and isolate the investigated medicines from one another. The QCSs underwent spiking with the study medications and the IS to assess the presence of endogenous interferences. Subsequently, a comparison was made between the chromatograms obtained from the plasma samples and those derived from blank plasma. Based on the chromatograms of blank plasma samples and plasma samples containing the investigated medications, as depicted in Figure 1a–c, it can be concluded that there was no significant interference from endogenous plasma constituents.

4.13 Matrix effect and extraction recovery

This experiment aimed to assess the potential impact of the plasma matrix on the recovery of the compounds under investigation. This was achieved by comparing the peak areas acquired from plasma samples spiked with pure pharmaceuticals to those obtained from pure standard solutions of the same concentration. Testing was conducted on the QCSs. Because the extraction recovery calculation was used, the MTC and ETC values were between 103.76% and 105.31% for MTC and between 103.56% and 105.09% for ETC. Following the finding displayed in Table S1, the presence of the plasma matrix had almost no effect on the drug being extracted from the plasma matrix.

4.14 Stability

Various conditions were employed to assess the stability of the investigated drugs in the endogenous plasma matrix. These conditions encompassed storing the samples at room temperature (25°C) for 6 h to evaluate bench-top stability, maintaining the samples at −20°C for 6 weeks to examine long-term stability, subjecting them to three freeze–thaw cycles at temperatures ranging from 20°C to room temperature to assess freeze and thaw stability, and finally leaving them at 25°C for 2 days at an autosampler to evaluate post-preparative stability. Additionally, the samples were subjected to three freeze-thaw cycles. The samples were evaluated in triplicate per the instructions for each respective procedure. The stability of the analyte was appraised by analyzing the plasma concentrations and calculating the average recovery rate, which was found to be 100 ± 15%. The results shown in Table 3 show that the stability of QCs was carefully studied in several different conditions. The test conditions did not have a noticeable effect on the plasma samples. Therefore, it can be confidently concluded that the QCs remained stable across different settings.

4.15 System suitability

The system’s appropriateness was thoroughly evaluated based on several parameters, including retention time, peak area, asymmetry factor, theoretical plates, and tailing factor for both ETC and MTC. To ensure that the system is appropriate, it is strongly recommended to maintain the RSD below 1% and ensure that the asymmetry factor exceeds a value of 2 and the number of theoretical plates is above 2,000. The optimal values are displayed in Table S2.

4.16 Assay of medicinal form

An HPLC method was used to analyze the assay sample. Six samples were evaluated in depth. The results show no problem with the medication’s assay form. A summary of the outcomes is provided in Table S3.

5.1 Greenness assessment of the suggested approach

5.1.3 AGREE tool

Green profiles are evaluated using AGREE, a technique based on 12 principles of GAC, as shown in Figure S2a. Various green color combinations are shown in Figure 7a, indicating various levels of eco-friendliness, along with AGREE pictograms and a score of 0.57 in the middle.

Figure 7

The pictograms utilized for assessing the environmental sustainability of the suggested HPLC method encompass the following: (a) AGREE, (b) AGREEprep, (c) GAPI, (d) ComplexGAPI, (e) AMGS, and (f) HPLC EAT.