Development and Validation of RP-HPLC Method for Estimation of Winter green Oil in Pharmaceutical Formulation

Introduction

Since the beginning of time, medicinal plants have been employed for their healing abilities. They contain vital This work is licensed under a Creative Commons Attribution-NonCommercial 4.0. components for eradicating infections, relieving pain, curing ailments, and maintaining overall well-being. The therapeutic properties of plants offer several options for the development of a wide range of drugs.¹ One such medicinal plant, Gaultheria procumbens, is a member of the Ericaceae family and contains the essential oil, wintergreen oil (WO). According to contemporary studies, the aforementioned herb exhibits antiinflammatory, antioxidant, antibacterial, and analgesic properties.² According to reports, the biological activity of dried alcoholic extract of Gaultheria procumbens L. indicated methyl salicylate up to 96.9% as the predominant active element. This aspirin-like chemical has been shown to have anti-inflammatory, antirheumatic, and other analgesic properties.³ As a result, Gaultheria species is used to treat inflammatory diseases, rheumatoid arthritis, chronic tracheitis, common cold, both acute and chronic prostatitis, and for alleviating pain associated with swelling. These salicylates act by preventing cyclooxygenase (COX), a crucial enzyme in the biosynthesis of pro-inflammatory cytokines.⁴

A comprehensive literature survey revealed few methods for the estimation of methyl salicylate in WO, which include simple liquid chromatography,⁵ gas chromatography-mass spectrometry (GC-MS),⁶ headspace (HS) chromatography along with gas chromatography (GC)⁷ and ultraviolet-visible (UVVIS) spectrophotometry.⁸ Due to their complex composition, developing a simple analytical approach for the quantification of oils in pharmaceutical dosage forms is a difficult task. Furthermore, development of analytical method for the analysis of the same is required due to demand for simple analytical methods for routine estimation of WO in pharmaceutical dosage forms. This led to an endeavour to develop and validate the Reversedphase high-performance liquid chromatography (RPHPLC) method for quantifying WO using methyl salicylate as the reference standard.

Materials and Methods

Materials

Wintergreen oil (WO) was purchased from Falcon, Exporters of 100% Pure & Essential oils, Bangalore, India. Methyl salicylate (pure ≥ 99%), a reference standard, was purchased from Sigma-Aldrich. Acetonitrile (HPLC grade), the solvent of choice, was purchased from Thomas Baker (Chemicals) Pvt. Ltd. Mumbai. From Himedia Laboratories Pvt. Ltd., Thane, methanol (HPLC grade) and glacial acetic acid (analytical grade) were purchased. The analytical grade of orthophosphoric acid was purchased from Karnataka Fine Chemicals, Bangalore. The Millipore purification system was used to provide water that is suitable for HPLC. During the experiment, Class A glassware was used.

Method Development

Instrumentation

Shimadzu LC-20AD, equipped with a column made by Phenomenex Gemini NX C18 (250 mm X 4.6 mm) 5 µm, and operating in isocratic pump mode, was used to develop an RP-HPLC technique.

Chromatographic conditions

An injection volume of 20 µL, flow rate of 1.5 mL/ min, and UV detection wavelength of 268 nm were used (Figure 1). Acetonitrile and water were used as the mobile phase in the ratio of 50:50 v/v, with a pH of 2.9. The procedure was developed at room temperature with a 10 minute runtime.

Preparation of standard stock solution

Accurately weighed 150 mg of methyl salicylate was transferred to a 100 mL volumetric flask. It was dissolved in a diluent (a mixture of acetonitrile: glacial acetic acid 98:2% v/v), and the volume was made up to the mark with the same to obtain the concentration of 1500 µg/ mL. After 10 minutes of sonication, this solution was filtered using 0.45 µ Millipore membrane filter

Preparation of working standard solution

Working standard solutions were made from standard stock solutions at concentrations ranging from 300 to 1500 µg/mL by transferring aliquots of 5 mL, 10 mL, 12.5 mL, 15 mL, 18.75 mL, and 25 mL to a 25 mL volumetric flask, and then manually diluted each volume to the appropriate level. All aliquot dilutions were sonicated for 10 minutes and then filtered using a 0.45 µ Millipore filter.

Preparation of mobile phase A mixture of 50% water and 50% HPLC grade acetonitrile were used to prepare the mobile phase. Orthophosphoric acid was used to bring the pH to a level of 2.9. After being sonicated for 10 minutes, the prepared mobile phase was filtered through a 0.45 µ Millipore membrane filter.

Preparation of sample solution

One gram of WO was transferred into volumetric flasks measuring 25 mL, and the remaining volume was filled with diluent to yield a concentration of 40 mg/mL. 1 mL of the above solution was pipetted into a 10 mL volumetric flask, and 10 mL of diluent (stock solution II) was added to bring the volume to 10 mL, yielding a concentration of 4 mg/mL. 1 mL of the stock II solution was pipetted into a 10 mL volumetric flask, diluted to a volume to yield the concentration of 400 µg/mL, and sonicated for 10 minutes and filtered through a 0.45 µ Millipore filter.

The suitability of the established method was evaluated based on system suitability factors like, resolution (Rs), USP tailing factor (T), asymmetry, and the number of theoretical plates (N). These factors also included injection precision for retention time (min), injection precision for peak area (n=6), and injection precision for peak height.

Method validation

The developed HPLC method for the analysis of WO has been validated in accordance with International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) guidelines, taking into consideration of linearity, limit of detection, limit of quantification, precision, accuracy, ruggedness, and robustness.⁹

Linearity

Regression analysis was applied to statistically examine the calibration curve method, which included six different concentrations of methyl salicylate ranging from 300 µg/mL to 1500 µg/mL to determine the linearity of the developed method. A series of standard methyl salicylate dilutions at concentrations of 300 µg/mL, 600 µg/mL, 750 µg/mL, 900 µg/mL, 1125 µg/mL, and 1500 µg/mL were made from the aliquots of the working standard solution. Each of the standard methyl salicylate dilutions were injected into the chromatograph in an amount of 20 µL. Plotting the concentration of methyl salicylate on x-axis and the peak area on y-axis allowed for the construction of standard curve, the peak area being recorded.

Precision

Repeatability, inter-day, and intraday measures were used to determine precision. By analyzing the dilution containing 750 µg/mL of methyl salicylate (n=6), the repeatability, inter-day, and intraday precision of the analytical method were assessed. Six repetitions' peak areas were recorded and % relative standard deviation (RSD) was determined.

Accuracy

By estimating the known amounts of analyte added, the accuracy of the proposed approach was assessed by calculating the percentage recovery of analyte. By spiking WO with the usual amounts of methyl salicylate at concentrations of 600 µ/mL, 750 µ/mL, and 900 µ/ mL at three different levels of 80%, 100%, and 120%, respectively, it was carried out at three different levels of concentrations: Lower quality control, middle-quality control, and higher quality control. Each measurement was carried out in triplicates, and the recovery was quantified in terms of the analyte discovered in spiked samples.

Ruggedness and robustness

The ability of the technique to stay unaffected by intentional changes to the method conditions is what is meant by the terms "ruggedness" and "robustness." This was tested by altering the pH (6.0±1.0), detection wavelength, flow rate (1±0.2 mL), and mobile phase ratio. The peak area was measured, and the RSD percentage was calculated.

Specificity

The capacity of the method to differentiate methyl salicylate peak from the other peaks of the oil is known as specificity. This was determined by contrasting the chromatograms of the blank (mobile phase) methyl salicylate, and WO.

Sensitivity

The limit of detection (LOD) and the limit of quantification (LOQ) were used to calculate the method's sensitivity. The formulae provided below were used to calculate LOD and LOQ.

LOD =3.3σ/S

LOQ=10σ/S

Where, σ - Standard deviation

            S - Slope of the curve

Estimation of methyl salicylate in WO

By mixing 0.1 mg/mL of oil with 0.1 mg/mL of diluted methyl salicylate solution and injecting the resulting mixture in triplicates into the chromatogram, the amount of methyl salicylate present in the WO was estimated. The following equation was used to determine the amount of methyl salicylate in WO, where the standard's purity is 99%.

Assay = (Sample area)/(std area) x (std wt)/(Sample wt) x (Sample dilution)/(std dilution) x Purity/100

Determination of WO content in pharmaceutical formulation

The WO content in WO-loaded pharmaceutical formulations was determined using the validated method. The chosen pharmaceutical formulation included nanosponges, which were weighed and dissolved in 10 mL of dichloromethane using a sonicator for six hours. The nanosponges were equivalent to 10 mg of WO. The amount of WO was estimated from this solution using the validated HPLC method, after it was thoroughly filtered through a 0.45 µm membrane filter.

Results

Figure 1 shows the UV spectrum of Wintergreen Oil. The established concentration range observed was 300-1500 μg/mL, and data from the regression line (Y=2154x+27071), with an R2 value of 0.9983, ensured significant linearity between the response and analyte concentration (Figure 2).

The precision measurements were given as a percentage of RSD. The calculated % RSD of repeatability and intermediate precision (Table 1) was determined to be <2%, indicating that the procedure is precise. The accuracy of this approach can be seen in the recovery percentage, which ranged from 100.4 to 100.8% for 80 to 100%. Standard deviation, RSD, and retention time did not significantly alter despite deliberate changes to the method's set parameters, which highlighted the method's robustness (Table 2).

Methyl salicylate's retention time was found to be 5.6 min (Figure 3), and the peak with RT 5.6±5 (min) was almost identical with WO, and WO-loaded nanosponges (Figure 3), compared to the chromatogram of a blank (Figure 3). The LOD and LOQ values were 0.082 μg/mL and 0.25μg/mL, respectively. In WO, methyl salicylate was found to be 95%. Nanosponge formulations were found to have entrapment efficiencies and loading capacities that ranged from 73.7% to 97.4% and 6.85% to 0.07%, respectively.

Discussion

The in-house development of the HPLC method took into consideration the solubility of methyl salicylate in various solvent systems, the method's applicability, cost, and its applicability for different purposes. Acetonitrile and water combined in a ratio of 50:50 v/v was chosen as an appropriate mobile phase system based on preliminary experiments carried utilizing several solvent mixture systems with changing proportions. By analysing the system suitability parameters, such as theoretical plates, height equivalent to a theoretical plate (HETP), and tailing factor, the method's suitability was analysed. All these parameters were found to be within the acceptable range.

According to ICH requirements, the newly developed method was validated for specificity, linearity, precision, accuracy, and robustness. The capacity of a method to quantify specifically an analyte of interest in the presence of other components is known as specificity. Methyl salicylate's retention time was found to be almost identical with WO, and WO-loaded nanosponges, compared to the chromatogram of a blank. This demonstrates both the complexity of WO composition and the specificity of the approach. The linearity across the concentration range supported the relationship between the analyte and the response. The established concentration range and data from the regression line, with an R2 value of 0.9983, ensured significant linearity between the response and analyte concentration. The LOD and LOQ values confirm that the approach is sensitive enough to identify and quantify the methyl salicylate concentration in the nanosponge formulation. The precision measurements were given as a percentage of RSD. The calculated %RSD of repeatability and intermediate precision were determined to be <2%, indicating that the procedure was precise. The accuracy of this approach can be seen in the recovery percentages and method was found to be robust. In WO, methyl salicylate was found to be 95%. Hence, using methyl salicylate as a reference standard, the approach was successful in assessing the entrapment efficiency and loading capacity of nanosponges. Nanosponge formulations were found to have entrapment efficiencies and loading capacities that ranged from 73.7% to 97.4% and 6.85% to 0.07%, respectively.

Conclusion

The developed RP-HPLC technique for the determination of methyl salicylate in WO was demonstrated to be successful by validating the method in terms of the validation parameters as per ICH guidelines. It was proved that the procedure is simple, specific, sensitive, precise, accurate, and robust. Hence, the routine analysis of WO in nanosponge formulations was carried out using the proposed method.

Conflict of interest

The authors declare they have no conflicts of interest.

Acknowledgment

The authors are thankful to the Government College of Pharmacy, Bengaluru, for providing the required facilities to carry out research.