Development and Validation of an RP-UHPLC Method for Quantitative Determination of Eugenol as per ICH Guidelines

When there are no definitive techniques are present, new methodologies are being progressed for evaluation of the novel product. Analytical method could be spectral, chromatographic, electrochemical, hyphenated or miscellaneous. Analytical method development is the process of selecting an accurate assay procedure to determine the composition of a formulation. It is the process of proving that an analytical method is acceptable for use in laboratory to measure the concentration of subsequent samples. An analytical procedure is developed to test a defined characteristic of the substance against established acceptance criteria for that characteristic. In the development of a new analytical procedure, the choice of analytical instrumentation and methodology should be based on the intended purpose and scope of the analytical method. The important parameters that may be evaluated during method development are specificity, linearity, limits of detection (LOD) and quantitation limits (LOQ), range, accuracy.

Eugenol is a naturally occurring component of numerous plant essential oils, including those of cloves (Szygium aromaticum, Eugenia aromatica,or Eugenia caryophyllus), cinnamon (Cinnamomum spp.), basil(Ocimum spp.), allspice(Pimenta dioica), bay laurel(Laurus nobilis), turmeric(Curcuma longa), and other plants. Eugenol is a naturally occurring phenolic molecule found in several plants such as cinnamon, clove, and bay leaves. It has been used as a topical antiseptic as a counter-irritant and in dental preparations with zinc oxide for root canal sealing and pain control.

MATERIAL AND METHODOLOGY:

Eugenol (pure) was purchased from Scientific Systems & Chemicals(p). Ltd.,Bhopal. HPLC                grade Acetonitrile, HPLC grade Methanol and HPLC-grade water were obtained from SIRT- Pharmacy, Bhopal Lab.

 Preparation of solutions:

• Standard Eugenol stock solutions:

Accurately measured 10 ml of Eugenol and was transferred to a 50 ml volumetric flask, sufficient volume of methanol HPLC Grade was added to it so as to dissolve the drug, and sonicated for 5 minutes. Then the volume was make upto the mark with methanol to produce 10 ml of 1000 μg/ml stock solution.

• Standard Eugenol sub-stock solutions:

From the above mentioned stock solution, 1 ml of the solution was withdrawn and was transferred to a 50 ml of volumetric flask. The volume was made upto 10 ml with methanol so as to produce 10 ml of 100μg/ml of the sub-stock solution. This sub-stock was used to prepare further dilutions.

•Working Standard Solution of Euegnol :

0.2, 0.4, 0.6, 0.8, 1.0 from substock solution were taken separately in 10 ml volumetric flask and volume made up tp 10 ml with methanol.this gave the solution of  2μg/ml, 4μg/ml, 6μg/ml, 8μg/ml, 10μg/ml respectively.        

Chromatographic system :

The chromatographic system is composed of the following (Table 1): Chromatographic conditions.

Table 1: UHPLC Chromatographic optimized condition

Figure 1: Chromatogram of Standard Eugenol at  285 nm

Method Validation:

Linearity :

To establish the linearity of a proposed method series of dilution ranging from 0.2µg/ml to 10ug/ml were made separately from stock solution of drug (1000µg/ml). Sample were filtered through 0.22 m PTFE filter and injected; chromatograms were recorded (Figure 1). Table 2 shows the results.

Figure 2:  Calibration curve of standard eugenol

Accuracy

For the evaluation of accuracy of the developed method the recovery study was perform. Three replicates of each 10,20 and 30 pg/ml of drug solutions were injected into the UHPLC system and all three recovery levels were recorded and recovery was calculated and shown in table 3.

Precision

Repeatability and reproducibility of the method were assessed by performing replicated the analysis of standard solutions in the mobile phase. Repeatability were characterized for different concentrations and given by mean recovery and RSD.

Repeatability

Intra-day precision - Three replicates of each 10,20 and 30 pg/ml of drug solutions were injected into the UHPLC system at a different time on the same day. The % RSD recovery was calculated and shown in table 4.

Reproducibility

Inter-day precision: Three replicates of each 10. 20 and 30 ppm of drug solutions were injected into the UHPLC system at different days. The % RSD of recovery was calculated and given in table 5.

Range

Range of the method was determined from the data of linearity, recovery and precision experiments.

Specificity

The specificity of the developed method was determined by injecting the blank (mobile phase) and standard drug solution (10µg/ml) into the UHPLC system.

 Robustness

The robustness of the developed method were evaluated by doing minute variations in the optimized method parameters. Performed by altering following chromatographic conditions: 2% variation in mobile phase, variations in the flow rate.Triplicate injections of a standard drug solution µg/ml) were analyzed as per the procedure in each altered condition and chromatograms were recorded. The robustness was determined from the data obtained from the recovery study. Results shown in table 6 and 7.

6.5.7 System Suitability Parameters

The USP suggests that system suitability tests should be performed prior to analysis. The parameters include tailing factor, retention time (RT), theoretical plate number (N), asymmetry factor, selectivity and %RSD of peak height or area for repetitive injections.Typically, at least two of these criteria are required to demonstrate system suitability for the proposed method. Separation variables were set and mobile phase ACN: Water 60:40 was allowed to saturate the column at flow rate 0.8 ml/min and plotted to get sharp base line.Six replicates of reference standard of curcumin 10 µg/ml were injected separately. Peak report and column performance report were recorded for all chromatogram.

RESULT AND DISCUSSION:

Calibration curve

A representative chromatogram of eugenol in the developed UHPLC method is shown in Figure 1. A retention time of 4.930 min can be observed from the UHPLC chromatogram in Figure 1. The calibration curve for eugenol by the developed UHPLC method is shown in Figure 2. The linear regression data for the calibration curve demonstrated a good linear relationship over the concentration range of 2 to 10 μg mL−1. A good linearity was established by a correlation coefficient (R2) value of 0.999 (Table 2). Correlation coefficient is a statistical tool used to measure the degree or strength of this type of relationship, and here, a high correlation coefficient value (a value very close to 1.0) indicates a high level of linear relationship between the concentration of eugenol and peak area.

Table : 2 Linearity data for validation of eugnol

Accuracy

Accuracy was investigated by analyzing three concentrations of drug solution. The recovery studies were carried out to check the sensitivity of the method to estimate eugenol. The average percentage recovery was calculated as 99.47 which is considered to be within acceptance limit (80-120%). This confirms that the method is accurate. The values of percentage recovery and %RSD are displayed in Table 3. The mean percentage recovery values, close to 100%, and their low %RSD values indicated high accuracy of the analytical method.

Table 3 :Accuracy data for validation of eugenol

Precision

Repeatability: The % RSD of the % recovery was calculated from the recovery data and was found to be 0.269 which is within the given range. This confirms that the method is precise in terms of intra-day precision.         

Table 4: Intra-day precision data for validation of eugenol

Reproducibility: The %RSD of the %recovery was found as 0.411 which is less than 2 ,It indicates that the method is precise in term of inter-day precision.

Table 5: Inter-day precision data for validation of eugenol

Specificity

The developed method was found to be specific for the determination of the drug eugenol since no interference of the mobile phase was found with the detection of the drug.

Robustness

Robustness data for mobile phase: The method developed is robust for the mobile phase since the %RSD is 0.690 which is in the acceptance criteria (less than 2).

Table 6: Data for robustness of eugenol by alteration in mobile phase

Robustness for flow rate : The developed method is robust for change in flow rate since the % RSD value is less than 2.

Table 7: Data for robustness of eugenol by alteration in flow rate

System suitability parameters:

The system suitability parameters were determined for parameters such as retention time, area under the curve, no. of theoretical plates and tailing factor, and the system was found suitable since the % RSD value was found to be less than 2.