Development and validation of RP-HPLC-PDA method for the quantification of eugenol in developed nanoemulsion gel and nanoparticles
© Pramod et al.; licensee Springer. 2013
Received: 18 May 2013
Accepted: 10 October 2013
Published: 28 October 2013
Eugenol is a potent phytochemical, and a plethora of delivery systems for this bioactive agent is being developed. Reversed-phase high-pressure liquid chromatography equipped with photodiode array detector (RP-HPLC-PDA) method is very useful in the quantification of the phytochemicals.
The RP-HPLC-PDA system with C18 reversed-phase column (250???4.6?mm, particle size 5??m) was used in this study. Acetonitrile and water in 1:1 (v/v) ratio was chosen as the mobile phase under a column temperature of 25?C. The detection wavelength was set at 280?nm with a flow rate of 1?mL/min. Method validation was performed according to the International Conference on Harmonization guidelines.
HPLC method for the quantification of eugenol was successfully developed and validated. The method was validated in terms of linearity and range, accuracy, precision, specificity, robustness, detection limit, and quantitation limit.
The developed RP-HPLC-PDA could be successfully employed for the quantification of eugenol in nanoemulsion gel and nanoparticles.
KeywordsAccuracy Precision Specificity Robustness ICH guidelines
A good number of novel delivery systems of eugenol (Chen et al. 2009; Gomes et al. 2011; Jadhav et al. 2004; Kriegel et al. 2010; Pokharkar et al. 2011) have been reported owing to its potent bioacitivity. Anti-inflammatory and anti-microbial actions are among other major pharmacological actions of eugenol (Pramod et al. 2010). Development of a suitable analytical method will be needed when eugenol is formulated in nanocarriers for targeted delivery.
Quantification of the pharmacologically active component in a dosage form is indispensable to the quality control of these systems. Quality control checks the suitability of a drug delivery system for the intended application. It serves as a marker for the consistency and predictability of the performance of dosage forms (Levi et al. 1964). High-pressure liquid chromatography (HPLC) methods are widely reported for the quantitative estimation of bioactive phytochemicals, but to this day, no reports are available on reversed-phase HPLC equipped with photodiode array detector (RP-HPLC-PDA) methods for the quantification of eugenol in nanostructured delivery systems such as nanoemulsion gel and nanoparticles. Besides this, none of the methods are available for eugenol quantification from formulations where a high specificity is required to overcome the probable interference of the excipients.
Nanoemulsion gel and nanoparticles have been developed as novel drug delivery systems of eugenol (Pramod et al. 2012 2013). The major aim of the present work was to develop a RP-HPLC-PDA method for the quantitative estimation of eugenol in these drug delivery systems for anti-inflammation and periodontal infection.
Chemicals and reagents
Eugenol (pure) was purchased from Central Drug House (Delhi, India). Poly-?-caprolactone (MW 14,000), chitosan, sodium alginate and Pluronic F-68 were purchased from Sigma-Aldrich Co. (MO, USA). Tween 80 and polyvinyl alcohol were purchased from Central Drug House (New Delhi, India). Carbopol 940 was a gift sample from Noveon Corporation (Cleveland, OH, USA). Tween 80 and triethanolamine were purchased from S D Fine-Chem Ltd. (Mumbai, India). Ethanol (99.9%) was purchased from Jiangsu Huax Co., Ltd. (Jiangsu, China). HPLC-grade water and acetonitrile were purchased from Merck (Mumbai, India).
Preparation of eugenol-loaded nanoemulsion gel and nanoparticles
Aqueous titration method was employed for the preparation of eugenol-loaded nanoemulsion. The formulated eugenol-loaded nanoemulsion was converted to nanoemulsion gel by dispersing 1% (w/w) Carbopol 940 in it. Tween 80 and ethanol were used as surfactant and co-surfactant, respectively. For S mix, a specific volume ratio of 4:1 (Tween 80/ethanol) was used (Pramod et al. 2012). For the preparation of the sample solution of nanoemulsion gel, an accurately weighed gel sample was taken in methanol, sonicated (Altrasonics, Mumbai, India) for 20?min, and filtered using a 0.2-?m syringe filter (Axiva Sichem Biotech, New Delhi, India).
Solvent displacement method was employed for the preparation of eugenol-loaded nanoparticles (Reis et al. 2006). Polycaprolactone (encapsulating polymer) and eugenol were dissolved in acetone (organic solvent phase) by mild heating. The solution of eugenol and polymer was injected dropwise into aqueous Pluronic F-68 (stabilizer) solution under magnetic stirring, and stirring was continued until complete evaporation of acetone occurred. Centrifugation of the suspension of nanoparticles thus obtained was carried out at 15,000?rpm for 1?h. The obtained nanoparticles were washed twice with distilled water and then freeze-dried (Pramod et al. 2013). For the preparation of the sample solution from nanoparticles, accurately weighed sample of dried nanoparticles was dissolved in 1?mL of acetone and then was added to 5?mL of methanol. Acetone was then evaporated. The sample was sonicated (Altrasonics, Mumbai, India) for 20?min, then was made up to 10?mL with methanol, and was filtered using a 0.2-?m syringe filter (Axiva Sichem Biotech, New Delhi, India) (Pramod et al. 2013).
HPLC instrumentation and chromatographic conditions
The HPLC method for the determination of eugenol was carried out on a Waters Alliance e2695 separating module (Waters Co., MA, USA) using a photodiode array detector (Waters 2998) with autosampler and column oven. The instrument was controlled by Empower 2 software (Europa Science, Ltd., Cambridge, UK) installed with equipment for data collection and acquisition. Compounds were separated on a C18 reversed-phase column (250???4.6?mm, particle size 5??m; Merck, Darmstadt, Germany) maintained at room temperature.
Acetonitrile and water in the ratio of 1:1 (v/v) was chosen as the mobile phase.
C18 reverse phase column (250???4.6?mm, particle size 5??m; Merck, Darmstadt, Germany)
PDA detector (Waters 2998)
Preparation of the mobile phase
HPLC-grade water was mixed with HPLC-grade acetonitrile in the volume ratio of 1:1. The prepared mobile phase was then filtered through a 0.45-?m nylon filter and sonicated in an ultrasonic bath for 15?min.
Linearity and range
A stock solution of eugenol (10?mg?mL?1) was prepared in methanol. Standard calibration solutions (5 to 1,000??g?mL?1) for the assessment of linearity were prepared from this stock solution using the mobile phase. The solutions were filtered through a 0.45-?m nylon filter. The filtered solution was then injected into the HPLC system. The data of peak area versus drug concentration were treated by linear least square regression.
Accuracy as recovery
Accuracy was determined by recovery studies using standard addition method. The pre-analyzed samples were spiked with extra 50%, 100%, and 150% of the standard eugenol, and the mixtures were analyzed by the proposed method. The experiment was conducted in triplicate.
The intraday (repeatability) and interday (intermediate precision) variations for the determination of eugenol was carried out at three concentration levels of 20, 100, and 600??g?mL?1. The determinations were carried out in triplicate.
The specificity of the method was ascertained by analyzing the standard drug and sample. The band for eugenol in nanoemulsion gel and nanoparticle samples was confirmed by comparing the R f values and spectra of the band with that of the standard. The peak purity of eugenol was assessed by comparing the spectra at three different levels, that is, peak start, peak apex, and peak end positions of the spectrum.
Robustness of the method was carried out by introducing very small changes in the analytical methodology at a single concentration level (100??g?mL?1). Robustness of the proposed method was determined in two different ways, i.e., by making deliberate changes in the mobile phase ratio, flow rate, and detection wavelength of analysis. The percentage of relative standard deviation (%RSD) of the experiment was calculated to assess the robustness of the method.
Detection and quantitation limits
where ? is the standard deviation of the intercept of the calibration plot and S is the slope of the calibration curve.
Results and discussion
Although limited HPLC methods for the determination of eugenol have been reported (Dighe et al. 2005; Li et al. 2004), none of them reported their specificity in quantifying eugenol in nanostructured delivery systems. Moreover, the application of PDA detector is an added advantage for the developed method. Detection of an entire spectrum simultaneously is possible with PDA detector. While UV?vis detectors visualize the obtained result in two dimensions (light intensity and time), only PDA adds the third dimension (wavelength). This is convenient to determine the most suitable wavelength without repeating analyses. No methods are available for eugenol quantification from formulations where a high specificity is required to overcome the probable interference of the excipients.
Linear regression data for the calibration curve ( n?=?3)
Linearity range (?g?mL?1)
5 to 1,000
Correlation coefficient (R2)
Accuracy as recovery
Recovery data for the accuracy of the HPLC method
Excess of eugenol added (%)
Concentration of sample (?g?mL ?1)
Theoretical concentration of spiked sample (?g?mL ?1)
Concentration of spiked sample???SD (?g?mL -1) (n?=?3)
Repeatability and intermediate precision of HPLC method
Concentration (?g?mL ?1)
Intermediate precision (n?=?3)
Mean peak area???SD
Mean peak area???SD
Robustness data of the HPLC method
%RSD of area
% RSD of R t
Mobile phase ratio (ACN/water)
Flow rate (mL?min?1)
Detection wavelength (nm)
Detection and quantitation limits
The DL and QL were determined as per the ICH Guidelines Q2(R1) (2005) and were found to be 0.44 and 1.34??g?mL?1, respectively.
The RP-HPLC-PDA system with C18 reversed-phase column (250???4.6?mm, particle size 5??m) was used in this study. Acetonitrile and water in the ratio of 1:1 (v/v) was chosen as the mobile phase, and a detection wavelength of 280?nm was used with a flow rate of 1?mL?min?1. The method validation was performed according to the guidelines of the International Conference on Harmonization (ICH). HPLC method for the quantification of eugenol was successfully developed and validated. The method was validated in terms of linearity and range, accuracy, precision, specificity, robustness, detection limit, and quantitation limit. The DL and QL were determined as per the ICH guidelines and were found to be 0.44 and 1.34??g?mL?1, respectively. The developed RP-HPLC-PDA could be successfully employed for the quantification of eugenol in its nanoemulsion gel and nanoparticles.
KP, SHA and JA proposed the idea and design the experiment. KP and JA carried out the preparation of nanoemulsion and nanoparticles. UKI assisted in framing the experiments. UKI prepared standards and samples for analysis. YTK and SA carried out the HPLC analysis of the samples and standard. All authors participated in the preparation of the manuscript. All authors read and approved the final manuscript.
Pramod K gratefully acknowledges Indian Council of Medical Research (ICMR), New Delhi, India, for providing Senior Research Fellowship (No. 35/3/10/NAN/BMS).
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