A stability-indicating method was validated for the determination in pharmaceutical forms of idebenone a coenzyme Q10-like compound. The assay was achieved by liquid chromatography analysis using a reversed-phase C18 column and a detector set at 480 nm. The optimized mobile phase consisted of isocratic flow rate at 1.0 mL/min for 3 min with methanol. The linearity of the assay was demonstrated in the range of 3.0 to 8.0 mg/mL with a correlation coefficient
Idebenone (2-(10-hydroxydecyl)-5,6-dimethoxy-3-methyl-cyclohexa-2,5-diene-1,4-dione) (Figure
Chemical structure of idebenone.
Idebenone pharmaceutical-quality grade powder was supplied by Inresa (Bartenheim, France, lot PR140151B). The pharmaceutical dosage tablets were purchased from Intsel Chimos (Mnesis, St. Cloud, France). HPLC-grade methanol was provided by Merck KGaA (Lichrosolv, Darmstadt, Germany).
A Dionex Ultimate 3000 system (Thermo Scientific, Villebon sur Yvette, France) was used for the assay. This HPLC system contained an integrated solvent and degasser SRD-3200, an analytical pump HPG-3200SD, a thermostated autosampler WPS-3000TSL, a thermostated column compartment TCC-3000SD, and a diode array detector MWD-3000. Data acquisition was carried out using in-line Chromeleon software (v6.80 SP2) (Thermo Scientific).
The eluent was monitored at 480 nm. Chromatographic separation was achieved at 25°C using a Nova-Pak C18 column (Waters, Guyancourt, France) with 4
Idebenone stock solution (100 mg/mL) was prepared by accurately weighing 10 g and transferring to 100 mL volumetric flask. Volume was made up to the mark with methanol. The stock solution was stored at 2–8°C during 5 days.
Two idebenone 45 mg tablets were crushed to a fine powder in a glass mortar and 5 mL of methanol was added. The suspension was transferred to a 10 mL volumetric flask and made up to 10 mL with methanol to achieve a final idebenone 9 mg/mL concentration. The recovery of idebenone from tablets was
Calibration standards at 3.0, 4.0, 5.0, 6.0, 7.0, and 8.0 mg/mL were freshly prepared using the stock solution. Quality control solutions at 3.5, 4.5, and 6.5 mg/mL were prepared extemporaneously.
Validation studies were performed according to the ICH guidelines [
The specificity was determined by analyzing the chromatograms of tablets in comparison with those obtained for idebenone standard solution aiming at confirming that none of the ingredients interfere with the quantitation of the drug.
The linearity was determined by a least-square linear regression routine using the compound peak area and concentration of the working standard solutions prepared at six concentration levels (3.0, 4.0, 5.0, 6.0, 7.0, and 8.0 mg/mL). Six replicates of each concentration were independently prepared and injected in triplicate into the chromatograph. The method was evaluated by determination of the correlation coefficient and intercept values according to the ICH guidelines.
LOD and LOQ were determined by calibration curve method. Solutions of idebenone were prepared in linearity range and injected in triplicate. Average peak area of three analyses was plotted against concentration. LOD and LOQ were expressed as 3.3 × Syx/
The precision was assessed at intraday and interday precision. The intraday precision was determined by measuring quality control samples of 3.5, 4.5, and 6.5 mg/mL of idebenone, injected six times on the same day. The intermediate (interday) precision was estimated by injecting quality control samples prepared at the same concentrations on three different days by different operators. Results were reported in terms of relative standard deviation (RSD).
The accuracy was investigated by using the standard addition method at different levels: 20, 40, 80, 100, and 120%. The mean recovery of idebenone of the target concentration (4 mg/mL) was calculated and accepted with
To determine the robustness, experimental HPLC conditions were purposely modified to check the reproducibility of the method. The evaluation of robustness was based on RSD values obtained by changing analytical setting such as isocratic flow rate (1.1 to 1.3 mL/min), wavelength detection (481 to 483 nm), temperature of analytical column (23 to 30°C), and composition of mobile phase (1 and 2% deionised water).
Forced degradation studies were carried out to provide some information about the drug stability and to validate the specificity of the idebenone quantification of the assay. The standard solution of idebenone was exposed to accelerated degradation by alkaline, acid, and oxidative and direct exposure to sunlight conditions.
To perform the alkaline hydrolysis, 2 mL of idebenone stock solution (100 mg/mL) was mixed in a 4 mL tube with 800
To investigate oxidative degradation, 2 mL of idebenone stock solution was mixed in a 4 mL tube with 1 mL of 3% hydrogen peroxide for 48 h at 50°C in the dark. This solution was completed with methanol to reach a targeted concentration of 5 mg/mL before injection into the HPLC system.
For the photolytic degradation, 2 mL of idebenone stock solution was transferred in a 4 mL tube and directly exposed to sunlight for 5 days. The solution was completed with methanol to reach a targeted concentration of 5 mg/mL.
To achieve a quick and effective separation, idebenone was injected into different mobile phase solutions mixing deionised water and methanol at different proportions. Due to the insolubility of idebenone in water, the various assays demonstrated that the proportion of 100% methanol was more appropriate for the assay and an idebenone elution was observed at 1.70 min. Column temperature was found to be not a critical factor of the analysis. The optimum drug absorbance was obtained at 480 nm, as there was no interference from excipients present in commercial tablets. A typical chromatogram obtained with the present method is shown in Figure
Representative HPLC chromatogram of idebenone standard (4 mg/mL) in methanol mobile phase; flow rate: 1.0 mL/min; detection wavelength: 480 nm; column temperature:
The proposed method was validated by determining its performance characteristics regarding specificity, linearity, LOD, LOQ, accuracy, precision, and robustness.
Specificity was estimated by comparing the chromatograms of blank methanol solution, idebenone standard solution, and excipients solution from tablets as per assay method (Figure
HPLC chromatograms obtained from excipients compounded from tablets.
The linearity range of idebenone was demonstrated in the interval of 3.0 to 8.0 mg/mL. The mean linear regression equation obtained was
Linearity data of the developed method.
Initial conc. | Mean peak area ± SD | Actual conc. | % assay |
---|---|---|---|
(mg/mL) | ( |
(mg/mL) | |
3.0 | 40.16 ± 0.08 | 3.02 ± 0.04 | 100.7 |
4.0 | 52.14 ± 0.57 | 3.94 ± 0.00 | 98.4 |
5.0 | 65.59 ± 0.28 | 4.96 ± 0.02 | 99.3 |
6.0 | 80.35 ± 0.54 | 6.09 ± 0.00 | 101.5 |
7.0 | 92.91 ± 0.25 | 7.05 ± 0.02 | 100.7 |
8.0 | 104.39 ± 0.56 | 7.93 ± 0.00 | 99.1 |
|
LOD and LOQ were 0.031 and 0.047 mg/mL, respectively.
The results obtained for the intraday and interday precision are shown in Table
Precision study of the method.
Nominal conc. |
Intraday precision | Interday precision | ||
---|---|---|---|---|
Calculated conc. |
RSD (%) | Calculated conc. |
RSD (%) | |
3.5 | 3.46 ± 0.03 | 0.95 | 3.45 ± 0.02 | 0.58 |
4.5 | 4.53 ± 0.03 | 0.76 | 4.58 ± 0.03 | 0.69 |
6.5 | 6.51 ± 0.05 | 0.81 | 6.47 ± 0.04 | 0.65 |
The mean recoveries were found to be 98.6 to 101.5% (Table
Accuracy of the method.
Standard |
Added | Found (mg/mL) | % recovery | RSD | |
---|---|---|---|---|---|
% | mg/mL | Mean ± SD, |
Mean | ||
4.0 | 20 | 4.8 | 4.86 ± 0.02 | 101.45 | 0.43 |
4.0 | 40 | 5.6 | 5.52 ± 0.03 | 98.67 | 0.55 |
4.0 | 80 | 7.2 | 7.16 ± 0.04 | 99.52 | 0.52 |
4.0 | 100 | 8.0 | 8.01 ± 0.03 | 100.11 | 0.41 |
4.0 | 120 | 8.6 | 8.62 ± 0.03 | 100.34 | 0.34 |
The percent recoveries of idebenone were good under most conditions except for the flow rate at 1.3 mL/min (Table
Robustness.
Parameters | Modification | RT (min) | % recovery |
---|---|---|---|
Flow rate (mL/min) | 1.1 | 1.558 | 99.9 |
1.2 | 1.425 | 91.2 | |
1.3 | 1.317 | 85.2 | |
|
|||
Wavelength of detection (nm) | 481 | 1.71 | 101.7 |
482 | 1.70 | 101.3 | |
483 | 1.70 | 98.2 | |
|
|||
Column temperature (°C) | 23 | 1.72 | 100.7 |
27 | 1.70 | 100.3 | |
30 | 1.69 | 100.6 | |
|
|||
Deionised water in mobile phase | 1% | 1.71 | 100.9 |
2% | 1.75 | 101.9 |
As shown in Table
Forced degradation study.
Stress conditions | % remaining | % degradation | Retention time of degraded products |
---|---|---|---|
Acidic stress (1N HCl, 50°C, 1 h) | 51.8 | 48.2 | — |
Alkaline stress (1N NaOH, 50°C, 1 h) | 16.6 | 83.4 | — |
Oxidative stress (3%, 50°C, 48 h) | 84.6 | 15.4 | — |
Thermal stress (50°C, 8 days) | 99.3 | 0.7 | — |
Direct sunlight (8 days) | 95.8 | 4.2 | — |
Ambient temperature (15 days) | 92.0 | 8.0 | — |
Chromatograms of 1 N HCl (a), 1 N NaOH (b), and oxidative (c) stress conditions.
A simple and rapid stability-indicating high-performance liquid chromatographic method was developed and validated for the determination of idebenone in pharmaceutical dosage forms. The analytical method is specific, linear, precise, accurate, and robust for a rapid determination of this drug and can be used for studying the stability and degradation kinetics of idebenone.
The authors confirm that this paper’s content has no conflict of interests.