A systematic study of determination and validation of finasteride impurities using liquid chromatography

2.1 Reagents

All the chemicals and solvents used in this study were of best analytical reagent grade available. Finasteride and its impurities (compounds) were obtained from Cipla, Inc. (Mumbai, India). Dilaprost® film-coated tablets were obtained from Biofarma Pharmaceutical Industry Co. Inc. (Istanbul, Turkey). Acetonitrile and tetrahydrofuran were of LC-grade and procured from J. T. Baker, Inc. (Amhem, The Netherlands). LC-grade water was obtained using a GFL-2004 unit and the Milli-Q® Reference Water Purification System (Istanbul, Turkey).

2.2 Chromatographic conditions and instrumentation

The LC system consisted of a G1311A quaternary pomp, an 1100 Variable Wavelength Detector, a 1100 G13222A degasser, a G1313A ALS autosampler, a 1100 G1316A column thermostat, and an original loop (all from Agilent Scientific Instruments, Santa Clara, CA, USA). The loop volume was 100 μL. A Nova Pak C₁₈ (60 Å, 4 μm, 250 × 4.6 mm) LC Cartridge Column was used with a water + acetonitrile + tetrahydrofuran (80:10:10, v/v/v) mobile phase at a flow rate of 2.0 mL min⁻¹, and the column was maintained at a temparature of 60 ± 1 °C. The UV detection was performed at 210 nm using a Shimadzu 2100 spectrophotometer and Helma quartz cells with a 1 cm pathway.

All reagents were used ‘as received’ without any further purification. All glasswares and pipettes were calibrated with distilled water at ambient temperature prior to the use. Flasks were protected from light, and the diluted solutions were freshly prepared at the time of use. Whatman® white filter paper (125 mm, Schleicher & Schuell) was used to filter tablet powder. The solutions were filtered through a Millipore Millex-HV filter with a pore size of 0.45 μm.

2.3 Calibration curves

The stock solutions of the impurities A, B, and C were prepared by dissolving 5.0 mg of each compound in 100 mL of acetonitrile + water (1:1, v/v). The stock solutions of finasteride for the HPLC analysis were prepared by dissolving 50 mg of the compound in 100 mL of acetonitrile + water (1:1, v/v). All samples (15 μL each) were injected and chromatographed under the above noted experimental conditions. A calibration curve was prepared by plotting the peak areas against the concentrations of the finasteride and its three impurities. Triplicate injections were made. The calibration curve was plotted over the concentration range of 0.0015–0.0045 mg mL⁻¹.

3.1 Assessments of impurities in the preparations

A total of 10 Dilaprost® tablets were crushed in a porcelain mortar, and 10 mg was transferred into a 10 mL volumetric flask. The mixture was shaken vigorously in acetonitrile + water solution (1:1, v/v). The solution was filtered and passed through a 0.45 µm NyLon filter and a 15 µL of this was injected into the LC system. The amount of active substance was calculated from the peak areas obtained in several analyses.

3.2 Validation parameters

The LC assay was validated by several experimental parameters such as selectivity, linearity, accuracy, precision (reproducibility, repeatability, and intermediate precision), robustness, limit of detection (LOD), limit of quantification (LOQ) and stability (as briefly summarized below).

3.3 Linearity

The linearity of the method was determined at five concentration levels, i.e., 50%, 75%, 100%, 125% and 150% for each impurities of finasteride. Linear regression analyses were used to calculate the correlation between the analytical results obtained and the theoretical concentrations used. The mean area values against the concentration used were calculated utilizing the following equations: $$\text{y}=0.1961\text{x}+2.708\left({\text{r}}^2=0.9807\right)\text{for}\text{impurity}\text{A,}$$ $$\text{y}=0.5884\text{x}+0.9806\left({\text{r}}^2=0.9983\right)\text{for}\text{impurity}\text{B}\text{and}$$ $$\text{y}=0.4866\text{x}+0.6740\left({\text{r}}^2=0.9966\right)\text{for}\text{impurity}\text{C}$$

3.4 Selectivity

Chromatograms of a placebo solution, the impurity solutions, and of a sample solution were recorded. Fig. 3 depicts the chromatogram of a placebo solution, which indicates that there are no speaks close to the retention times of the finasteride impurities under our investigational and chromatographic conditions.

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Fig. 3

The HPLC chromatogram of a placebo solution. Chromatographic conditions: A Nova Pak C₁₈ (60 Å, 4 μm, 250 × 4.6 mm) column; mobile phase: water + acetonitrile + tetrahydrofuran (80:10:10, v/v/v); flow rate: 2.0 mL min⁻¹; injection volume: 15 μL; UV detection at 210 nm; column temperature (60 ± 1 °C). Concentration of finasteride was 0.5 mg mL⁻¹.

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3.5 Accuracy

The recovery experiments were performed to investigate the deviations of the obtained results from the actually reported values. The degree of deviation from the actual value was used to confirm the accuracy of the proposed method. Three different concentration levels (80%, 100%, and 120%) were added to the pre-analyzed placebo samples in triplicate. The accuracy of the method was demonstrated by the satisfactory recoveries thus obtained. The percentage recoveries of the impurities at each level (in triplicates) were determined. The mean percentage recovery and the percentage relative standard deviation (RSD) were calculated. The impurities A, B and C [stock solutions; 3 mg diluted to 100 mL acetonitrile + water (1:1, v/v)]. Placebo (1,490 mg) and stock solutions (1 mL) were added to 10 mL acetonitrile + water (1:1, v/v) to obtain a concentration of 100%. Three sample solutions were prepared for each concentration. The individual recoveries were excellent in the range of 99.24–102.89% and the RSDs were up to 1.029%, which indicate that the method is accurate. Table 1 summarizes the recovery data of the assays for the three impurities of finasteride.

3.6 Precision (intermediate precision, reproducibility, repeatability)

3.7 The limits of detection and quantification

As part of the sensitivity assessments, the LOD and the LOQ were determined for finasteride and its three impurities at a signal-to-noise ratio of 3:1 and 10:1, respectively, by injecting a series of stock solutions diluted to varying levels. The values thus obtained for the LOD and the LOQ of finasteride and its impurities are summarized in Table 3.

3.8 Robustness

In order to evaluate the robustness of the assay method, the influence of small and intended chromatographic variations of the analytical parameters on the retention times of finasteride and its three impurities was examined. In this robustness experiments, the LC columns from the different lots were used. The analyses of the impurity solutions were carried out at the column temperatures of 55 °C, 60 °C, and 65 °C, which were adjusted by changing the system temperatures, and the respective RSD results thus obtained are presented in Table 4. While no significant effect was observed on the system’s suitability parameters, the retention times for each impurity changed with varying temperatures; however, the impurity results are generally within the acceptable analytical range (Table 4). Notably, when the ratio of water + acetonitrile + tetrahydrofuran in the mobile phase was changed from 80:10:10 to 10:80:10, there was no peak observed related to the place.

3.9 Stability

In order to determine the stability of finasteride and its three impurities in the mobile phase, the drug was stored in the mobile phase for 48 h and chromatographed at the ambient temperature. The solutions were stable during the 48 h of experimentation and no significant alterations were observed in the percent recovery of impurities. Replicate injections (n = 5) of finasteride solutions were performed and the RSD of the peak area was determined to be within 0–0.01% range.

In order to assure the selectivity and to provide properties (values) of the proposed method pertaining to stability, forced degradation experiments were also conducted under various stress conditions. Appropriate amounts of powdered tablets equivalent to the average tablet weight (298 mg) were stressed with 10 N NaOH, 0.1 N NaOH, and 0.1 N MeOH hydrochloride. After 72 h mixing, the solutions were tested again and the values for the finasteride impurities resulting from the accelerated degradation studies were monitored and the results thus obtained are summarized in Table 5. As reported in the recent peer-reviewed literature (Srinivas et al., 2011; Reddy et al., 2012; Mohanty et al., 2014), our experience also suggests that finasteride must be protected from extensive heat, light, and oxidizing agents. The forgoing experimental observations are valuable to the quality assessments of finasteride and its three main impurities as the technique offers efficient chromatographic separation with improved sensitivity, specificity, and turn-around-time.