Stability Study of Finasteride: Stability-Indicating LC Method, In Silico and LC–ESI-MS Analysis of Major Degradation Product, and an In Vitro Biological Safety Study

Stability Study of Finasteride: Stability-Indicating LC Method, In Silico and LC–ESI-MS... Abstract Stability studies of the pharmaceutically important compound finasteride were conducted in order to evaluate decomposition of the drug under forced degradation conditions. A simple stability-indicating liquid chromatography method was developed and validated for the evaluation of finasteride and degradation products formed in pharmaceutical preparations and the raw material. Isocratic LC separation was achieved on a C18 column using a mobile phase of o-phosphoric acid (0.1% v/v), adjusted to pH 2.8 with triethylamine (10% v/v) and acetonitrile (52:48 v/v), with a flow rate of 1.0 mL min−1. The alkaline degradation kinetics of the drug were also evaluated and could be best described as second-order kinetics under the experimental conditions applied for the tablets and raw material. Based on in silico studies and molecular weight confirmation, a comprehensive degradation pathway for the drug and the identity of its major product could be suggested without complicated isolation or purification processes. Furthermore, a biological safety study was performed to evaluate the effect of the degraded sample in relation to the intact molecule. The results showed that the degraded sample affected the cell proliferation. Therefore, these studies show that special care must be taken during the manipulation, manufacture and storage of this pharmaceutical drug. Introduction Azasteroid finasteride, chemically known as N-(1,1-dimethylethyl)−3-oxo-(5α,17β)−4-azaandrost-1-ene-17-carboxamide (Figure 1), is a synthetic drug that is widely used for the treatment of androgenetic alopecia, benign prostatic hyperplasia, and prostate cancer. Finasteride is commercially available as both tablets and capsules (5 and 1 mg for both pharmaceutical formulations). Its mechanism of action is inhibition of 5-alpha-reductase, an intracellular enzyme that converts testosterone to dihydrotestosterone (DHT). For the therapeutic effect of this drug it is important to maintain its integrity and to evaluate the quality of bulk substances and formulations (1, 2). Figure 1. View largeDownload slide Chemical structure of finasteride. Figure 1. View largeDownload slide Chemical structure of finasteride. Official’s pharmacopeia methods have been described for the finasteride quality control in raw material and tablets (3–5). Different methods have been reported for analysis of this drug in pharmaceutical preparations and biological samples, including liquid chromatography (LC) (6–11), LC–MS (12–14), micellar electrokinetic chromatography (15), voltammetry (16), polarography (17), spectrophotometry (18–20), gas-chromatography (GC) (21) and thin-layer chromatography (TLC) (22). In some of these studies, the authors have developed stability-indicating methods to evaluate the forced degradation of the drug. These studies also evaluated the methods’ capability (specificity/selectivity) for drug determination in the presence of degradation products, which were generated under photolytic, acidic, oxidative and alkaline conditions. However, regarding finasteride, the forced degradation products were poorly studied (6, 11, 23). In all cases, the chromatographic systems of the developed methods were not able to separate and detect the major degradation product. Furthermore, there are few reports about the isolation and characterization, or toxicity assays of the finasteride degradation products (FDP) that were generated. Considering the mentioned above, the development of a new stability-indicating LC method for study of finasteride major degradation product is required, and also the biological safety study of the degraded sample. These studies are very important, and can help to understand the decomposition patterns of drug substances and drug products, which is valuable information about its stability and biological safety (24–28). Many factors can affect the stability of a pharmaceutical product, including the stability of the active ingredient, the manufacturing process, and the environmental conditions (such as heat, light and moisture during storage), as well as some chemical reactions like oxidation, reduction, hydrolysis and racemization (29, 30). Knowledge of the stability of the molecule helps in selecting the proper formulation, packaging, storage conditions and shelf life, which is essential for raw material and pharmaceutical product regulatory documentation. According to ICH guidelines, stress testing of drug substances can help to identify the likely degradation products, which helps to establish the degradation pathways, and to validate the stability-indicating procedures used (26, 31). In silico assessment is used to assist the experimental procedures performed to identify and determine the chemical or physical stability of organic compounds and drugs (32–34). There are several chemical functions that are subject to degradation, as well as many mechanisms of reactivity involved in these reactions. For each method of degradation, a different in silico method calculation is suggested, such as Fukui functions (FF) for alkaline or acid hydrolysis (34, 35), and bond dissociation energy or enthalpy (BDE) for autooxidation and photolytic reactions (32). Energies of frontier electron densities (FEDs) of the highest occupied molecular orbital (EHOMO) and the lowest unoccupied molecular orbital (ELUMO) are also used to predict the reactivity of drugs under photolytic environments (36). Quantum chemical studies using density functional theory (DFT) have been performed to understand and predict the structure degradation of drugs (34, 37). From DFT calculations it is possible to estimate the global and local reactivity indices of the compounds, reflecting their electrophilic/nucleophilic powers, from which is possible to calculate and detect the sites of the molecule that are prone to reacting with a nucleophilic agent (38). DFT of Becke3 (B3) exchange associated with the Lee, Yang and Parr (LYP) correlation process has been shown to be a satisfactory prediction method of chemical reactivity (39, 40), which allows study of the nucleophilic attack in hydrolysis reactions (41, 42). As previously mentioned, using the different stability-indicating drug determination methods, the drug instability under forced degradation was observed. Considering the susceptible degradation of finasteride, the aims of the present paper were: to study the drug stability in an alkaline medium by the development of an stability-indicating method using LC; to perform the prediction of chemical structure reactivity using DFT calculations and the FF; to assess the biological safety of the degraded sample. Furthermore, LC/MS was used to suggest the identity of the major degradation product generated in this stability study. Experimental Section Chemicals and reagents The finasteride reference substance and raw material were kindly provided by Multilab (Brazil). The pharmaceutical drug formulation was obtained commercially. The tablets were labeled as containing 5 mg of finasteride and the following inactive ingredients: lactose monohydrate, croscarmellose sodium, hypromellose, sodium lauryl sulfate, macrogol, titanium dioxide, magnesium stearate, yellow ferric oxide, polysorbate 80, povidone and silicon. LC-grade acetonitrile was purchased from J. T. Baker (USA) and analytical-grade ethanol was obtained from Fmaia (Brazil). All other reagents were acquired from Sigma Chemical Co. (USA). All aqueous solutions were prepared with purified water from a Milli-Q apparatus (Millipore®). LC method Instrumentation and conditions The LC system consisted of a Shimadzu® instrument equipped with a diode array detector (DAD). The separation was performed using a Supelco Ascentis® column (150 mm × 4.6 mm, 5 μm) at room temperature, and eluted at a flow rate of 1.0 mL min−1 with an injection volume of 20 μL, using an isocratic system. The mobile phase consisted of o-phosphoric acid (0.1% v/v adjusted to pH 2.8 using triethylamine) and acetonitrile (52:48 v/v). The detection of finasteride was achieved at 210 nm. The mobile phase was filtered through a 0.45 μm-thick nylon filter and degassed in an ultrasonic bath before use. The data obtained showed that the mobile phase was stable for at least 48 h when stored in a closed flask at room temperature. Calibration solutions A finasteride stock solution with a concentration of 500 μg mL−1 was prepared in a volumetric flask by dissolving the reference drug substance in ethanol. Aliquots of 0.4, 0.7, 1.0, 1.3 and 1.6 mL were transferred to volumetric flasks and diluted with ethanol to produce final concentrations of 20, 35, 50, 65 and 80 μg mL−1. Sample preparation solutions The average weight of 20 tablets was determined. The tablets were crushed to form a homogeneous powder and an accurately weighed amount, equivalent to 12.5 mg finasteride, was transferred to a 25 mL volumetric flask, extracted with ethanol (20 mL), sonicated for 5 min, and diluted to 25 mL with the same solvent. This solution was centrifuged for 30 min at 3,500 rpm, and an aliquot of the supernatant (1 mL) was transferred into a 10 mL volumetric flask, which was diluted with ethanol to give a final concentration of 50 μg mL−1. The solutions were filtered through a 0.45 μm-thick nylon filter before LC analysis. Method validation The developed stability-indicating LC analytical method was validated following ICH guidelines and USP requirements (5, 43). The linearity was evaluated by linear regression analysis, which was calculated using the least-squares regression method. The calibration curve was obtained with five concentrations: 20, 35, 50, 65 and 80 μg mL−1 (each prepared in triplicate). The specificity was estimated by forced degradation studies and the interference evaluation of the pharmaceutical formulations excipients. In order to determine whether the proposed LC method indicated stability, the finasteride active pharmaceutical ingredient (raw material) and pharmaceuticals formulations (tablets) were stressed under different conditions as part of the forced degradation studies (26, 44). The finasteride solutions for acid hydrolysis were prepared by dissolving the drug in a small volume of ethanol and diluting with aqueous hydrochloric acid to achieve a theoretical concentration of 500 μg mL−1. Acid hydrolysis was performed in 1 mol L−1 HCl at 70°C for 4 h under reflux, after which the samples were cooled to room temperature and neutralized with 1 mol L−1 NaOH. The study under alkaline condition was carried out in 1 mol L−1 NaOH at 70°C for 4 h under reflux, after which the samples were cooled to room temperature and neutralized with 1 mol L−1 HCl. An aliquot of each solution was diluted with ethanol to give a theoretical concentration of 50 μg mL−1. The stress degradation study with direct UV radiation (254 nm) was performed by exposing the finasteride solutions in acetonitrile (500 μg mL−1) to the UV beam for 3.5 h at room temperature in a photostability chamber (45, 46). The distance between the lamp and the sample was 10 cm. Afterward, the solutions were diluted to a theoretical concentration of 50 μg mL−1 with ethanol. Samples subjected to identical conditions, but protected from light, were used as a control. The oxidative reaction was performed in 30% H2O2 (5 mg mL−1) at 70°C for 4 h under reflux. An aliquot of this solution was diluted in ethanol to give a theoretical concentration of 50 μg mL−1. Peak purity tests were performed by the photodiode array detector, which showed that the analyte chromatographic peak did not contain more than one substance. The precision of the method was evaluated by repeatability (intra-day precision) and intermediate precision (inter-day precision) tests. The repeatability was tested by assaying six samples at the same concentration (50 μg mL−1) throughout one day under consistent experimental conditions. The intermediate precision of the method was assessed by carrying out the analysis on three different days and with a different analyst performing the analysis in the same laboratory (between-analyst precision). Data are expressed as a function of the relative standard deviation (RSD%) of a series of measurements. The accuracy was determined by a recovery test, which consisted of adding aliquots of the standard finasteride solution to placebo solutions, which gave final concentrations of the reference standards as 40, 50 and 60 μg mL−1 for the tests. Each solution was prepared in triplicate. The robustness of the method was determined by analyzing the same samples but with different method parameters, such as pH of the mobile phase (± 0.3 units), the flow rate (± 0.1 mL min−1), percentage of acetonitrile (± 2% organic phase), wavelength of detection (272 ± 3 nm), and the column (with the same specifications, but acquired from a different supplier). The five factors selected were examined in a Plackett–Burman design (N = 10). For each of the 10 experimental runs, two injections were performed for each solution. The effect (E) of each factor and the estimate experimental error (SE)e were calculated (47). The statistical interpretation provides a numerical limit value that makes it possible to define what is significant and what is not. This limit value to identify statistically significant effects is usually derived from the t-test statistical method, in accordance with the following equation: t=|Ex|(SE)e (1) An effect is considered significant at a given α level if t calculated > t critical. Alkaline degradation kinetics study The study was carried out with tablets and raw material solutions containing 500 μg mL−1 of finasteride. The solutions were prepared in 1 mol L−1 NaOH and inserted as rapidly as possible into a thermostatic water bath set at 70°C. At 60, 120, 180, 240 and 300 min, 1.0 mL from the reflux solutions was quantitatively transferred into a 10 mL volumetric flask and diluted with ethanol to give a final concentration of 50 μg mL−1 (N = 3). These solutions were protected from light and analyzed by LC, employing the developed and validated stability-indicating method. The regression coefficients (r) were determined and the best fit observed indicated the reaction order. The kinetic parameter constant (k) and t90% were also calculated. Majority degradation product evaluation In silico prediction study The computational analyses were performed using Spartan 08 version 116.2TM for Windows (Wavefunction, Inc., USA) and all of the initial structures were built using atoms and structural fragments from its molecular editor. Geometry optimization was carried out using the Merck Molecular Force Field (MMFF94) followed by the Austin Model (AM1), and re-optimized by the DFT method and the B3LYP/6.31 G* (d, f) basis set level of theory. At this step, the number of electrons in a natural atomic population analysis (NPA) was calculated using the single-point energy at the same level of theory of geometry optimization. From these data, the condensed FF derivatives values, positive (fj−) and negative (fj+) (fj+) were obtained using the following equations: fj−=qj(N)−qj(N−1),forelectrophilicattack; (2) fj+=qj(N+1)−qj(N),fornucleophilicattack; (3) fj0=½qj[(N+1)–qj(N−1)],forradicalattack. (4) In these equations, qj is the number of electrons (evaluated from NPA) at the jth atomic site in the neutral (N), anionic (N + 1), or cationic (N − 1) chemical species on the reference molecule, respectively. The dual descriptor Δf(r) of local reactivity, which allows us to obtain the preferred sites for nucleophilic attacks (Δf(r) > 0) and the preferably sites for electrophilic attacks (Δf(r) < 0) in the system at point r, was calculated for the chemical structure of finasteride using the following equation (48, 49): Δf(r)=f+(r)−f−(r) (5) LC–ESI-MS instrumentation and conditions The new LC–ESI-MS method was performed using a Shimadzu® instrument equipped with a MS detector. The separation was performed using a Supelco Ascentis® column (150 mm × 4.6 mm, 5 μm) at 40°C, and eluted at a flow rate of 0.4 mL min−1 with an injection sample volume of 4 μL. The mobile phase consisted of methanol and water (80:20 v/v), using an isocratic system. The solutions analyzed were the degraded samples of the kinetics study at 300 min (Alkaline degradation kinetics study). Mass spectra were acquired with an electrospray ionization interface in positive ionization mode, and the following conditions: capillary voltage 2.5 kV; source temperature 110°C; desolvation temperature 250°C; nitrogen desolvation flow 900 L h−1; nitrogen cone flow 90 L h−1. The full scan mass spectrum was acquired over a range of m/z 50–900. Toxicity study Human blood samples Peripheral blood was collected by venipuncture into sterile vials containing 68 I.U. of sodium heparin (BD Vacutainer®) per mL of blood. The vials were transferred to the laboratory, and whole-blood cultures were established. The blood samples were stored for up to 24 h at 4°C before culturing. This project was approved by the University’s Committee of Ethics in Research of Universidade Federal de Santa Maria (authorization n° 23081.012330/2006-94). Culture cell preparation The lymphocyte cultures were prepared with whole-blood samples and immediately transferred to 1 mL of culture medium containing RPMI 1640 supplemented with 10% fetal bovine serum and 1% streptomycin/penicillin, as previously described (50). The cells were then placed in a microaerophilic environment at 37°C for 72 h. The solutions under investigation were added to the blood at 10% concentration. The solutions analyzed included intact and degraded raw material (degraded by ∼50% under alkaline conditions; Alkaline degradation kinetics study) diluted in phosphate-buffered saline (PBS) at concentrations of 25, 50 and 100 ng mL−1. Each group consisted of three culture flasks. Genetic and oxidative parameters were analyzed after 72 h of growth. Analysis of genotoxic parameters To perform the genotoxicity tests, we first counted the total number of leukocytes in a Neubauer chamber (50). Viability was assessed by a loss of membrane integrity, which was indicated by trypan blue (51). Overall, we counted 300 cells. The genotoxicity test was conducted using the comet assay (52). Although the comet assay is not the only method for measuring oxidative DNA damage, it is one of the most sensitive and accurate and is relatively free of artifacts (53). We identified 100 cells in the slides that were submitted for analysis. The cells were visually scored according to tail length, with scores ranging from 0 (no migration) to 4 (maximal migration). Therefore, the damage index for cells ranged from 0 (all cells with no migration) to 400 (all cells with maximal migration). The tests were carried out in triplicate, and the data are presented as the mean ± standard error. The micronucleus (MN) frequency test was performed to evaluate the mutagenicity of the products. In this test, the cells were fixed with acetic acid and methanol (75:25, v/v), transferred onto clean microscope slides in duplicates, and then stained with 5% Giemsa. The criteria for scoring cells with MN were described in a previous report (54). One the cells are counted for each sample, and the results are expressed as the micronucleus frequency per 1000 cells. Statistical analysis Statistical software was used to perform all statistical analyses, which included an analysis of variance (ANOVA) followed by a post hoc Bonferroni test. P values <0.05 were considered statistically significant. Results Results of method validation The analytical method was validated for parameters such as linearity, specificity, precision, accuracy and robustness. Linearity was established by least-squares linear regression analysis of the calibration curve. The regression equation for finasteride was found by plotting the peak absorbance (y) versus the sample concentration (x). The representative linear equation was: y = 22704.2x – 7776.2 and the correlation coefficient (r = 0.9999) was highly significant. The validity of the assays was verified by means of ANOVA analysis (SAS 6.11 for Windows, SAS Institute Inc. CARY, NC, USA), which demonstrated significant linear regression and no significant linearity deviation (P < 0.05). The specificity test demonstrated that there was no interference in the LC determination of the drug. The forced degradation studies were conducted to evaluate the stability-indicating capability and selectivity of the proposed LC method using the finasteride raw material and the pharmaceutical formulation. Table I presents the extent of finasteride degradation under both stress conditions, and Figure 2 shows the chromatograms of the untreated solution and the forced degradation samples. It is important to note that although that several degraded products peaks can be observed, the peak of finasteride remains resolved. The chromatographic peak purity tool was applied to verify the finasteride peak, showing that it was 100% pure in all cases, indicating the specificity of the proposed method. It was observed that the finasteride peak presents appropriate resolution (Rs > 2) and selectivity (a > 1) in relation to the degradation products. The results indicate that the method indicates stability, and that the drug can be evaluated both qualitatively and quantitatively in the presence of degradation products and pharmaceutical excipients. Table I. Results of Finasteride Stability Under Force Degradation Conditions Condition Time (h) Degradation (%) Raw material Tablets Basic hydrolysis (1 mol L−1 NaOH) 4 51.58 32.52 Acid hydrolysis (1 mol L−1 HCl) 4 35.43 38.43 Photolysis 3.5 65.64 36.67 Oxidation (H2O2) 4 39.55 39.26 Condition Time (h) Degradation (%) Raw material Tablets Basic hydrolysis (1 mol L−1 NaOH) 4 51.58 32.52 Acid hydrolysis (1 mol L−1 HCl) 4 35.43 38.43 Photolysis 3.5 65.64 36.67 Oxidation (H2O2) 4 39.55 39.26 Table I. Results of Finasteride Stability Under Force Degradation Conditions Condition Time (h) Degradation (%) Raw material Tablets Basic hydrolysis (1 mol L−1 NaOH) 4 51.58 32.52 Acid hydrolysis (1 mol L−1 HCl) 4 35.43 38.43 Photolysis 3.5 65.64 36.67 Oxidation (H2O2) 4 39.55 39.26 Condition Time (h) Degradation (%) Raw material Tablets Basic hydrolysis (1 mol L−1 NaOH) 4 51.58 32.52 Acid hydrolysis (1 mol L−1 HCl) 4 35.43 38.43 Photolysis 3.5 65.64 36.67 Oxidation (H2O2) 4 39.55 39.26 Figure 2. View largeDownload slide (1) A and B are typical chromatograms for the raw material, and tablets in the selected experimental conditions, respectively, and C is the placebo chromatogram; (2) chromatogram of aciddegraded finasteride, and degradation products (2.7 and 2.9 min) for both samples; (3) chromatogram basedegraded finasteride, and degradation product (2.7 min) for both samples; (4) chromatogram of oxidatively degraded finasteride, and degradation product (4.1 min) for both samples; (5) chromatograms of photodegraded finasteride for raw material (A), and tablets (B). Figure 2. View largeDownload slide (1) A and B are typical chromatograms for the raw material, and tablets in the selected experimental conditions, respectively, and C is the placebo chromatogram; (2) chromatogram of aciddegraded finasteride, and degradation products (2.7 and 2.9 min) for both samples; (3) chromatogram basedegraded finasteride, and degradation product (2.7 min) for both samples; (4) chromatogram of oxidatively degraded finasteride, and degradation product (4.1 min) for both samples; (5) chromatograms of photodegraded finasteride for raw material (A), and tablets (B). Precision was determined by studying the repeatability and intermediate precision. Intra-day precision, performed by assaying the samples on 3 different days by different analysts, showed the following results: 97.67 ± 1.90%, 97.68 ± 0.61% and 97.48 ± 1.75% (mean ± RSD, N = 6). The RSD for inter-day precision was 1.44% (N = 3). The low variability of the results indicates the precision of the method. In the method accuracy assay, excellent mean percentage recovery values and low relative standard deviation values (RSD ≤ 1.5%) were found. At each level of the finasteride concentration, three determinations were performed. The mean recovery was 100.16% (RSD = 0.93%). These results reveal that any small change in the drug concentration in these solutions could be accurately determined by the proposed analytical method. In the robustness test, a number of responses can be determined from the performed experiments. For chromatographic methods, responses describing the quantity, such as peak areas or peak heights, and/or the content of the main substance and by-products are the most evident. In this study, the response determined in this test was the percentage of finasteride in the tablets. The SE of the percentage of finasteride in the tablet sample was 0.256%, which was used to perform the statistical test. The quantification of finasteride in the tablets can be considered robust because none of the factors studied had a significant effect (α = 0.05). Besides, the variation of robustness results (RSD = 0.53%) is in accordance with the results of the precision assay. Kinetic study The concentration, log and reciprocal concentration plots of the remaining drug versus time during the kinetic studies are shown in Figure 3. Degradation rate constant (k), half-life (t1/2) and t90 for finasteride in raw material and tablets solutions submitted to alkaline degradation, and determined by LC method are shown in Table II. Figure 3. View largeDownload slide Plots of concentration of remaining finasteride versus time—zero-order reaction (A); log of concentration of remaining finasteride versus time—first-order reaction (B); and reciprocal of concentration of remaining finasteride versus time—second-order reaction (C). Figure 3. View largeDownload slide Plots of concentration of remaining finasteride versus time—zero-order reaction (A); log of concentration of remaining finasteride versus time—first-order reaction (B); and reciprocal of concentration of remaining finasteride versus time—second-order reaction (C). Table II. Degradation Rate Constant (k), Half-Life (t1/2) and t90 for Finasteride in Raw Material and Tablets Solutions Submitted to Alkaline Degradation, and Determined by LC Method Samples k (min−1) t1/2 (min) t90 (min) Raw material 1.06 × 10-4 188.94 20.99 Tablets 0.94 × 10-4 211.86 23.54 Samples k (min−1) t1/2 (min) t90 (min) Raw material 1.06 × 10-4 188.94 20.99 Tablets 0.94 × 10-4 211.86 23.54 Table II. Degradation Rate Constant (k), Half-Life (t1/2) and t90 for Finasteride in Raw Material and Tablets Solutions Submitted to Alkaline Degradation, and Determined by LC Method Samples k (min−1) t1/2 (min) t90 (min) Raw material 1.06 × 10-4 188.94 20.99 Tablets 0.94 × 10-4 211.86 23.54 Samples k (min−1) t1/2 (min) t90 (min) Raw material 1.06 × 10-4 188.94 20.99 Tablets 0.94 × 10-4 211.86 23.54 Evaluation of the major degradation product The in silico results for finasteride are shown in Table III and the LC–ESI-MS chromatograms are shown in Figure 4. Table III. Values of the NPA (Neutral, Positive and Negative), Electrophilic f− and Nucleophilic f+ Condensed Fukui Functions and Δf(r) Over the Atoms of the Finasteride Molecule Calculated With the DFT/B3LYP and the 6.31 G* Basis Set Considering Equations (2)–(4) Atoms NPA NPA+ NPA− f+ f− Δf(r) C1 6.155034 6.364085 12.51912 0.209051 −6.36409 6.573136 C2 6.31507 6.421567 12.73664 0.106497 −6.42157 6.528064 C3 5.340941 5.427152 10.76809 0.086211 −5.42715 5.513363 N4 7.671368 7.703796 15.37516 0.032428 −7.7038 7.736224 C5 6.050351 6.042553 12.0929 −0.0078 -6.04255 6.034755 C6 6.08966 6.076132 12.16579 −0.01353 −6.07613 6.062604 C7 6.459159 6.451873 12.91103 −0.00729 −6.45187 6.444587 C8 6.449313 6.44338 12.89269 −0.00593 −6.44338 6.437447 C9 6.242572 6.243012 12.48558 0.00044 −6.24301 6.243452 C10 6.24425 6.241333 12.48558 −0.00292 −6.24133 6.238416 C11 6.447038 6.442907 12.88995 −0.00413 −6.44291 6.438776 C12 6.447758 6.445876 12.89363 −0.00188 −6.44588 6.443994 C13 6.042478 6.043273 12.08575 0.000795 −6.04327 6.044068 C14 6.24658 6.243506 12.49009 −0.00307 −6.24351 6.240432 C15 6.456839 6.45453 12.91137 −0.00231 −6.45453 6.452221 C16 6.454957 6.45363 12.90859 −0.00133 −6.45363 6.452303 C17 6.329044 6.326504 12.65555 −0.00254 −6.3265 6.323964 C18 6.684882 6.680537 13.36542 −0.00434 −6.68054 6.676192 C19 6.675568 6.669565 13.34513 −0.00600 −6.66957 6.663562 O20 8.605455 8.723617 17.32907 0.118162 −8.72362 8.841779 C21 5.295889 5.342884 10.63877 0.046995 −5.34288 5.389879 O22 8.641541 8.67851 17.32005 0.036969 −8.67851 8.715479 N23 7.661502 7.675312 15.33681 0.013810 −7.67531 7.689122 C24 5.862817 5.859824 11.72264 −0.00299 −5.85982 5.856831 C25 6.683708 6.680267 13.36398 −0.00344 −6.68027 6.676826 C26 6.683426 6.682354 13.36578 −0.00107 −6.68235 6.681282 C27 6.683198 6.681271 13.36447 −0.00193 −6.68127 6.679344 Atoms NPA NPA+ NPA− f+ f− Δf(r) C1 6.155034 6.364085 12.51912 0.209051 −6.36409 6.573136 C2 6.31507 6.421567 12.73664 0.106497 −6.42157 6.528064 C3 5.340941 5.427152 10.76809 0.086211 −5.42715 5.513363 N4 7.671368 7.703796 15.37516 0.032428 −7.7038 7.736224 C5 6.050351 6.042553 12.0929 −0.0078 -6.04255 6.034755 C6 6.08966 6.076132 12.16579 −0.01353 −6.07613 6.062604 C7 6.459159 6.451873 12.91103 −0.00729 −6.45187 6.444587 C8 6.449313 6.44338 12.89269 −0.00593 −6.44338 6.437447 C9 6.242572 6.243012 12.48558 0.00044 −6.24301 6.243452 C10 6.24425 6.241333 12.48558 −0.00292 −6.24133 6.238416 C11 6.447038 6.442907 12.88995 −0.00413 −6.44291 6.438776 C12 6.447758 6.445876 12.89363 −0.00188 −6.44588 6.443994 C13 6.042478 6.043273 12.08575 0.000795 −6.04327 6.044068 C14 6.24658 6.243506 12.49009 −0.00307 −6.24351 6.240432 C15 6.456839 6.45453 12.91137 −0.00231 −6.45453 6.452221 C16 6.454957 6.45363 12.90859 −0.00133 −6.45363 6.452303 C17 6.329044 6.326504 12.65555 −0.00254 −6.3265 6.323964 C18 6.684882 6.680537 13.36542 −0.00434 −6.68054 6.676192 C19 6.675568 6.669565 13.34513 −0.00600 −6.66957 6.663562 O20 8.605455 8.723617 17.32907 0.118162 −8.72362 8.841779 C21 5.295889 5.342884 10.63877 0.046995 −5.34288 5.389879 O22 8.641541 8.67851 17.32005 0.036969 −8.67851 8.715479 N23 7.661502 7.675312 15.33681 0.013810 −7.67531 7.689122 C24 5.862817 5.859824 11.72264 −0.00299 −5.85982 5.856831 C25 6.683708 6.680267 13.36398 −0.00344 −6.68027 6.676826 C26 6.683426 6.682354 13.36578 −0.00107 −6.68235 6.681282 C27 6.683198 6.681271 13.36447 −0.00193 −6.68127 6.679344 Table III. Values of the NPA (Neutral, Positive and Negative), Electrophilic f− and Nucleophilic f+ Condensed Fukui Functions and Δf(r) Over the Atoms of the Finasteride Molecule Calculated With the DFT/B3LYP and the 6.31 G* Basis Set Considering Equations (2)–(4) Atoms NPA NPA+ NPA− f+ f− Δf(r) C1 6.155034 6.364085 12.51912 0.209051 −6.36409 6.573136 C2 6.31507 6.421567 12.73664 0.106497 −6.42157 6.528064 C3 5.340941 5.427152 10.76809 0.086211 −5.42715 5.513363 N4 7.671368 7.703796 15.37516 0.032428 −7.7038 7.736224 C5 6.050351 6.042553 12.0929 −0.0078 -6.04255 6.034755 C6 6.08966 6.076132 12.16579 −0.01353 −6.07613 6.062604 C7 6.459159 6.451873 12.91103 −0.00729 −6.45187 6.444587 C8 6.449313 6.44338 12.89269 −0.00593 −6.44338 6.437447 C9 6.242572 6.243012 12.48558 0.00044 −6.24301 6.243452 C10 6.24425 6.241333 12.48558 −0.00292 −6.24133 6.238416 C11 6.447038 6.442907 12.88995 −0.00413 −6.44291 6.438776 C12 6.447758 6.445876 12.89363 −0.00188 −6.44588 6.443994 C13 6.042478 6.043273 12.08575 0.000795 −6.04327 6.044068 C14 6.24658 6.243506 12.49009 −0.00307 −6.24351 6.240432 C15 6.456839 6.45453 12.91137 −0.00231 −6.45453 6.452221 C16 6.454957 6.45363 12.90859 −0.00133 −6.45363 6.452303 C17 6.329044 6.326504 12.65555 −0.00254 −6.3265 6.323964 C18 6.684882 6.680537 13.36542 −0.00434 −6.68054 6.676192 C19 6.675568 6.669565 13.34513 −0.00600 −6.66957 6.663562 O20 8.605455 8.723617 17.32907 0.118162 −8.72362 8.841779 C21 5.295889 5.342884 10.63877 0.046995 −5.34288 5.389879 O22 8.641541 8.67851 17.32005 0.036969 −8.67851 8.715479 N23 7.661502 7.675312 15.33681 0.013810 −7.67531 7.689122 C24 5.862817 5.859824 11.72264 −0.00299 −5.85982 5.856831 C25 6.683708 6.680267 13.36398 −0.00344 −6.68027 6.676826 C26 6.683426 6.682354 13.36578 −0.00107 −6.68235 6.681282 C27 6.683198 6.681271 13.36447 −0.00193 −6.68127 6.679344 Atoms NPA NPA+ NPA− f+ f− Δf(r) C1 6.155034 6.364085 12.51912 0.209051 −6.36409 6.573136 C2 6.31507 6.421567 12.73664 0.106497 −6.42157 6.528064 C3 5.340941 5.427152 10.76809 0.086211 −5.42715 5.513363 N4 7.671368 7.703796 15.37516 0.032428 −7.7038 7.736224 C5 6.050351 6.042553 12.0929 −0.0078 -6.04255 6.034755 C6 6.08966 6.076132 12.16579 −0.01353 −6.07613 6.062604 C7 6.459159 6.451873 12.91103 −0.00729 −6.45187 6.444587 C8 6.449313 6.44338 12.89269 −0.00593 −6.44338 6.437447 C9 6.242572 6.243012 12.48558 0.00044 −6.24301 6.243452 C10 6.24425 6.241333 12.48558 −0.00292 −6.24133 6.238416 C11 6.447038 6.442907 12.88995 −0.00413 −6.44291 6.438776 C12 6.447758 6.445876 12.89363 −0.00188 −6.44588 6.443994 C13 6.042478 6.043273 12.08575 0.000795 −6.04327 6.044068 C14 6.24658 6.243506 12.49009 −0.00307 −6.24351 6.240432 C15 6.456839 6.45453 12.91137 −0.00231 −6.45453 6.452221 C16 6.454957 6.45363 12.90859 −0.00133 −6.45363 6.452303 C17 6.329044 6.326504 12.65555 −0.00254 −6.3265 6.323964 C18 6.684882 6.680537 13.36542 −0.00434 −6.68054 6.676192 C19 6.675568 6.669565 13.34513 −0.00600 −6.66957 6.663562 O20 8.605455 8.723617 17.32907 0.118162 −8.72362 8.841779 C21 5.295889 5.342884 10.63877 0.046995 −5.34288 5.389879 O22 8.641541 8.67851 17.32005 0.036969 −8.67851 8.715479 N23 7.661502 7.675312 15.33681 0.013810 −7.67531 7.689122 C24 5.862817 5.859824 11.72264 −0.00299 −5.85982 5.856831 C25 6.683708 6.680267 13.36398 −0.00344 −6.68027 6.676826 C26 6.683426 6.682354 13.36578 −0.00107 −6.68235 6.681282 C27 6.683198 6.681271 13.36447 −0.00193 −6.68127 6.679344 Figure 4. View largeDownload slide Representative LC–ESI-MS chromatogram of finasteride after alkaline degradation: FDP-1 at m/z 395, and FDP-2 at m/z 239. FDP, finasteride degradation product. Figure 4. View largeDownload slide Representative LC–ESI-MS chromatogram of finasteride after alkaline degradation: FDP-1 at m/z 395, and FDP-2 at m/z 239. FDP, finasteride degradation product. Toxicological study The results demonstrate that treating cultured human leukocytes with varying concentrations of degraded finasteride affected their biological parameters. Discussion LC method development and validation The development of an LC method requires the careful consideration of the polarity of the analyte, stationary phase and mobile phase in order to obtain good separation within a reasonable time. Thus, the LC procedure was optimized to develop a stability-indicating method so that the degradation products from the drug could be resolved. The chromatographic conditions were chosen after testing different mobile phases with different proportions of organic and aqueous solvents. During the development phase, acetonitrile and o-phosphoric acid (0.1% v/v) were used in the mobile phase at different ratios, which resulted in an asymmetric peak, and low retention times. To achieve a better separation, the pH aqueous phase was changed to optimize the finasteride retention time. Finally, a mobile phase containing o-phosphoric acid (0.1% v/v, adjusted to pH 2.8 by adding triethylamine) and acetonitrile (52:48 v/v), was adopted because of its low tailing factor, good capacity factor (k´) value, retention time, and its ability to separate the degradation products from finasteride with good peak parameters. Furthermore, the new chromatographic system was able to separate and detect the finasteride major degradation product. The chromatographic conditions developed are an innovation in terms of chromatography when compared to previous finasteride stability-indicating methods related in the literature. The system suitability test is an integral part of the analytical method, which confirms the suitability and effectiveness of the operating system. It was carried out to evaluate the reliability and reproducibility of the system for the analysis, using five replicate injections of a reference solution containing 50 μg mL−1 finasteride. The results were as follows: theoretical plates (N = 6209), tailing factor (T = 1.04), capacity factor (k´ = 4.29) and injection repeatability (RSD% < 2, N = 5). The values for these parameters were satisfactory and in accordance with previously published data (55). The method was validated, showing satisfactory data for all of the tested parameters. The validation testifies that the procedure is suitable for the intended purpose. Kinetic study The finasteride degradation profile was evaluated at different time intervals, under the same conditions described above. Approximately 50 and 45% drug degradation occurred during 300 min for the raw material and tablet solutions, respectively. One major degradation product was eluted at 2.7 min, which resulted from degradation of both analyzed samples. The shorter retention times of the degradation product compared to the finasteride retention time are due to more polar features. The degradation kinetics were calculated for both samples through the decrease in drug concentration over time. The finasteride concentration remaining was calculated at each time interval for the three replicates compared to the mean drug concentration of the standard solution. According to the evaluation of the correlation coefficients (Figure 3), it can be concluded that the finasteride degradation for both sample solutions shows second-order kinetics under the experimental conditions applied. By analyzing the straight-line slopes, it was possible to calculate the apparent second-order degradation rate constant k (1.06 × 10−4 min−1 for raw material; 0.94 × 10−4 min−1 for tablets), t1/2 (188.94 min for raw material; 211.86 min for tablets) and t90 (20.99 min for raw material; 23.54 min for tablets) for each sample solution tested (Table II). Evaluation of the major degradation product In an alkaline environment, as shown in Table I, there was some level of degradation of the finasteride structure. In silico studies of chemical reactivity were carried out by the prediction of the most likely position of structural hydrolysis. The indicated computational method to study the alkaline reaction conditions observed in stress degradation studies is FF (34), according by your two derivatives, f+ and f−, for nucleophilic and electrophilic attack, respectively. FF are considered the local reactivity descriptors that indicates the preferred regions where a chemical species will change its density when the number of electrons is modified. Thus, it indicates the susceptibility of the electronic density to deform at a given position upon accepting or donating electrons (56). In this context, Parr and Yang (57) showed that sites in chemical species with the largest values of Fukui function (f(r)) are those with higher reactivity . From the FF values reported in Table III, the reactivity atoms order for the electrophilic susceptibility was O20>O22>N4>N23, respectively. To nucleophilic, the reactivity order was C1>O20>C2>C3>O22>N4>N23. According to the dual descriptor Δf(r), the calculated order were O20>O22>N4>N24, with values indicating preference for the nucleophilic attacks (Δf(r) > 0) into the system at point r. The sites with higher values of FF and dual descriptor are located mainly at the dihydropyridinone ring and the lateral chain in the chemical structure of finasteride. In all FF calculations, the olefin binding, carbonyl and nitrogen atom moieties of cited ring were shown to be the most probable reaction sites in alkaline forced degradation studies. As previously mentioned, finasteride was found to be labile under alkaline conditions. After 300 min of exposure, an ~50% reduction in concentration was observed for both analyzed samples. Based on their retention times, the LC–ESI-MS studies indicated two major peaks with m/z 239 and m/z 395 (Figure 4), which were attributed to the FDP. The product FDP−1 with m/z 395 was formed by addition of 22 amu [M + 22]+ thus indicating a sodium adduct of finasteride. As previously seen, the in silico study was performed aiming to assist the comprehension of chemical reactivity by the prediction of the most likely position of hydrolysis. The obtained results showed that the reactive atoms are O20, O22, N4 and N23. Based on these results, we suggest that the finasteride hydrolysis to FDP-2 (m/z 239 [M + 1]+) involves opening of the dihydropyridinone ring and closing of the lateral chain. The proposed products generated from the original structure are shown in Figure 5. In a study of forced degradation of dutasteride, a compound with a similar structure to finasteride, breakage of the dihydropyridinone ring and oxidation of the carbonyl moiety was also reported, resulting in the generation of carboxylic acid at this structural position when submitted to alkaline stress-forced conditions (58). These data assist the understanding of finasteride degradation in analogous experimental conditions. Figure 5. View largeDownload slide Proposed alkaline degradation product (FDP-2) of finasteride based in sílico study and LC–ESI-MS results. FDP, finasteride degradation product, chemically characterized to (S)-4-((3aS,6 S)-octahydro-3amethyl-1H-inden-6-yl)pentanoic acid. Figure 5. View largeDownload slide Proposed alkaline degradation product (FDP-2) of finasteride based in sílico study and LC–ESI-MS results. FDP, finasteride degradation product, chemically characterized to (S)-4-((3aS,6 S)-octahydro-3amethyl-1H-inden-6-yl)pentanoic acid. Toxicological study In the kinetic study, the different samples tested (raw material and tablets) showed similar residual drug levels in the tested conditions (Figure 3). The chromatograms showed a decrease in the finasteride area and one additional major peak at 2.7 min, for both analyzed samples. In the identification study, the product was the same for both samples. Therefore, we decided to perform a degraded raw material toxicity assay, thus removing any possibility of pharmaceutical excipient interference (Figure 6). Regarding the proliferation of leukocytes, the only treatments that caused any significant interference were those with degraded samples. These treatments showed a decrease in proliferation at 16 ± 1.5% for the degraded sample at 25 ng mL−1, 31 ± 1.15% at 50 ng mL−1 and 27 ± 1.5% at 100 ng mL−1. The assay was performed to evaluate the effect of the degraded sample in relation to the intact molecule to foresee possible undesirable effects resulting from the degradation products. The results showed that the FDP affected the cell proliferation. Figure 6 indicates the cellular viabilities means ranged from 81.3 ± 1.5% (100 ng mL−1) to 92.7 ± 1.5% (25 ng mL−1) for the degraded sample. These data show a moderate decrease in the cellular viabilities, but no significant differences compared to the negative control. The micronuclei frequency and the DNA damage index data showed no genotoxic effects for the degraded sample. Figure 6. View largeDownload slide Markers of cell proliferation and genotoxic effects. The bar graphs indicate total leukocytes counted, percentage of cellular viabilities, frequency of micronuclei, and DNA damage by comet assay. NC, negative control (PBS, phosphate-buffered saline); PC, positive control (hydrogen peroxide: H2O2); IRW, intact raw material; DRW, degraded raw material. Data are presented as mean ± SD (n = 3). Letters indicate statistically significant differences. Figure 6. View largeDownload slide Markers of cell proliferation and genotoxic effects. The bar graphs indicate total leukocytes counted, percentage of cellular viabilities, frequency of micronuclei, and DNA damage by comet assay. NC, negative control (PBS, phosphate-buffered saline); PC, positive control (hydrogen peroxide: H2O2); IRW, intact raw material; DRW, degraded raw material. Data are presented as mean ± SD (n = 3). Letters indicate statistically significant differences. Conclusion This article presents a stability study of finasteride under forced degradation conditions. A simple stability-indicating LC method was validated for the determination of the drug and degradation products, showing satisfactory data for all of the tested parameters. The degradation of this drug during alkaline processing follows second-order reaction kinetics for tablets and the raw material. The kinetic parameters of the degradation rate constant, t1/2 and t90, can be predicted. Based on in silico and LC–ESI-MS studies, the identity of finasteride’s major alkaline degradation product could be suggested. Furthermore, the biological safety study showed that the degraded finasteride sample affected the cell proliferation. 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Oxford University Press , New York, NY , ( 1989 ); pp. 53 – 115 . 58 Chaudhari , R. , Mohanraj , K. , Shirsat , V. ; MS/MS and HPLC characterization of forced degradation products of dutasteride and tamsulosin hydrochloride ; International Journal of Pharmaceutical Sciences and Research , ( 2014 ); 5 : 2791 – 2806 . © The Author(s) 2018. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Chromatographic Science Oxford University Press

Stability Study of Finasteride: Stability-Indicating LC Method, In Silico and LC–ESI-MS Analysis of Major Degradation Product, and an In Vitro Biological Safety Study

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Oxford University Press
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© The Author(s) 2018. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com
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0021-9665
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10.1093/chromsci/bmy028
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Abstract

Abstract Stability studies of the pharmaceutically important compound finasteride were conducted in order to evaluate decomposition of the drug under forced degradation conditions. A simple stability-indicating liquid chromatography method was developed and validated for the evaluation of finasteride and degradation products formed in pharmaceutical preparations and the raw material. Isocratic LC separation was achieved on a C18 column using a mobile phase of o-phosphoric acid (0.1% v/v), adjusted to pH 2.8 with triethylamine (10% v/v) and acetonitrile (52:48 v/v), with a flow rate of 1.0 mL min−1. The alkaline degradation kinetics of the drug were also evaluated and could be best described as second-order kinetics under the experimental conditions applied for the tablets and raw material. Based on in silico studies and molecular weight confirmation, a comprehensive degradation pathway for the drug and the identity of its major product could be suggested without complicated isolation or purification processes. Furthermore, a biological safety study was performed to evaluate the effect of the degraded sample in relation to the intact molecule. The results showed that the degraded sample affected the cell proliferation. Therefore, these studies show that special care must be taken during the manipulation, manufacture and storage of this pharmaceutical drug. Introduction Azasteroid finasteride, chemically known as N-(1,1-dimethylethyl)−3-oxo-(5α,17β)−4-azaandrost-1-ene-17-carboxamide (Figure 1), is a synthetic drug that is widely used for the treatment of androgenetic alopecia, benign prostatic hyperplasia, and prostate cancer. Finasteride is commercially available as both tablets and capsules (5 and 1 mg for both pharmaceutical formulations). Its mechanism of action is inhibition of 5-alpha-reductase, an intracellular enzyme that converts testosterone to dihydrotestosterone (DHT). For the therapeutic effect of this drug it is important to maintain its integrity and to evaluate the quality of bulk substances and formulations (1, 2). Figure 1. View largeDownload slide Chemical structure of finasteride. Figure 1. View largeDownload slide Chemical structure of finasteride. Official’s pharmacopeia methods have been described for the finasteride quality control in raw material and tablets (3–5). Different methods have been reported for analysis of this drug in pharmaceutical preparations and biological samples, including liquid chromatography (LC) (6–11), LC–MS (12–14), micellar electrokinetic chromatography (15), voltammetry (16), polarography (17), spectrophotometry (18–20), gas-chromatography (GC) (21) and thin-layer chromatography (TLC) (22). In some of these studies, the authors have developed stability-indicating methods to evaluate the forced degradation of the drug. These studies also evaluated the methods’ capability (specificity/selectivity) for drug determination in the presence of degradation products, which were generated under photolytic, acidic, oxidative and alkaline conditions. However, regarding finasteride, the forced degradation products were poorly studied (6, 11, 23). In all cases, the chromatographic systems of the developed methods were not able to separate and detect the major degradation product. Furthermore, there are few reports about the isolation and characterization, or toxicity assays of the finasteride degradation products (FDP) that were generated. Considering the mentioned above, the development of a new stability-indicating LC method for study of finasteride major degradation product is required, and also the biological safety study of the degraded sample. These studies are very important, and can help to understand the decomposition patterns of drug substances and drug products, which is valuable information about its stability and biological safety (24–28). Many factors can affect the stability of a pharmaceutical product, including the stability of the active ingredient, the manufacturing process, and the environmental conditions (such as heat, light and moisture during storage), as well as some chemical reactions like oxidation, reduction, hydrolysis and racemization (29, 30). Knowledge of the stability of the molecule helps in selecting the proper formulation, packaging, storage conditions and shelf life, which is essential for raw material and pharmaceutical product regulatory documentation. According to ICH guidelines, stress testing of drug substances can help to identify the likely degradation products, which helps to establish the degradation pathways, and to validate the stability-indicating procedures used (26, 31). In silico assessment is used to assist the experimental procedures performed to identify and determine the chemical or physical stability of organic compounds and drugs (32–34). There are several chemical functions that are subject to degradation, as well as many mechanisms of reactivity involved in these reactions. For each method of degradation, a different in silico method calculation is suggested, such as Fukui functions (FF) for alkaline or acid hydrolysis (34, 35), and bond dissociation energy or enthalpy (BDE) for autooxidation and photolytic reactions (32). Energies of frontier electron densities (FEDs) of the highest occupied molecular orbital (EHOMO) and the lowest unoccupied molecular orbital (ELUMO) are also used to predict the reactivity of drugs under photolytic environments (36). Quantum chemical studies using density functional theory (DFT) have been performed to understand and predict the structure degradation of drugs (34, 37). From DFT calculations it is possible to estimate the global and local reactivity indices of the compounds, reflecting their electrophilic/nucleophilic powers, from which is possible to calculate and detect the sites of the molecule that are prone to reacting with a nucleophilic agent (38). DFT of Becke3 (B3) exchange associated with the Lee, Yang and Parr (LYP) correlation process has been shown to be a satisfactory prediction method of chemical reactivity (39, 40), which allows study of the nucleophilic attack in hydrolysis reactions (41, 42). As previously mentioned, using the different stability-indicating drug determination methods, the drug instability under forced degradation was observed. Considering the susceptible degradation of finasteride, the aims of the present paper were: to study the drug stability in an alkaline medium by the development of an stability-indicating method using LC; to perform the prediction of chemical structure reactivity using DFT calculations and the FF; to assess the biological safety of the degraded sample. Furthermore, LC/MS was used to suggest the identity of the major degradation product generated in this stability study. Experimental Section Chemicals and reagents The finasteride reference substance and raw material were kindly provided by Multilab (Brazil). The pharmaceutical drug formulation was obtained commercially. The tablets were labeled as containing 5 mg of finasteride and the following inactive ingredients: lactose monohydrate, croscarmellose sodium, hypromellose, sodium lauryl sulfate, macrogol, titanium dioxide, magnesium stearate, yellow ferric oxide, polysorbate 80, povidone and silicon. LC-grade acetonitrile was purchased from J. T. Baker (USA) and analytical-grade ethanol was obtained from Fmaia (Brazil). All other reagents were acquired from Sigma Chemical Co. (USA). All aqueous solutions were prepared with purified water from a Milli-Q apparatus (Millipore®). LC method Instrumentation and conditions The LC system consisted of a Shimadzu® instrument equipped with a diode array detector (DAD). The separation was performed using a Supelco Ascentis® column (150 mm × 4.6 mm, 5 μm) at room temperature, and eluted at a flow rate of 1.0 mL min−1 with an injection volume of 20 μL, using an isocratic system. The mobile phase consisted of o-phosphoric acid (0.1% v/v adjusted to pH 2.8 using triethylamine) and acetonitrile (52:48 v/v). The detection of finasteride was achieved at 210 nm. The mobile phase was filtered through a 0.45 μm-thick nylon filter and degassed in an ultrasonic bath before use. The data obtained showed that the mobile phase was stable for at least 48 h when stored in a closed flask at room temperature. Calibration solutions A finasteride stock solution with a concentration of 500 μg mL−1 was prepared in a volumetric flask by dissolving the reference drug substance in ethanol. Aliquots of 0.4, 0.7, 1.0, 1.3 and 1.6 mL were transferred to volumetric flasks and diluted with ethanol to produce final concentrations of 20, 35, 50, 65 and 80 μg mL−1. Sample preparation solutions The average weight of 20 tablets was determined. The tablets were crushed to form a homogeneous powder and an accurately weighed amount, equivalent to 12.5 mg finasteride, was transferred to a 25 mL volumetric flask, extracted with ethanol (20 mL), sonicated for 5 min, and diluted to 25 mL with the same solvent. This solution was centrifuged for 30 min at 3,500 rpm, and an aliquot of the supernatant (1 mL) was transferred into a 10 mL volumetric flask, which was diluted with ethanol to give a final concentration of 50 μg mL−1. The solutions were filtered through a 0.45 μm-thick nylon filter before LC analysis. Method validation The developed stability-indicating LC analytical method was validated following ICH guidelines and USP requirements (5, 43). The linearity was evaluated by linear regression analysis, which was calculated using the least-squares regression method. The calibration curve was obtained with five concentrations: 20, 35, 50, 65 and 80 μg mL−1 (each prepared in triplicate). The specificity was estimated by forced degradation studies and the interference evaluation of the pharmaceutical formulations excipients. In order to determine whether the proposed LC method indicated stability, the finasteride active pharmaceutical ingredient (raw material) and pharmaceuticals formulations (tablets) were stressed under different conditions as part of the forced degradation studies (26, 44). The finasteride solutions for acid hydrolysis were prepared by dissolving the drug in a small volume of ethanol and diluting with aqueous hydrochloric acid to achieve a theoretical concentration of 500 μg mL−1. Acid hydrolysis was performed in 1 mol L−1 HCl at 70°C for 4 h under reflux, after which the samples were cooled to room temperature and neutralized with 1 mol L−1 NaOH. The study under alkaline condition was carried out in 1 mol L−1 NaOH at 70°C for 4 h under reflux, after which the samples were cooled to room temperature and neutralized with 1 mol L−1 HCl. An aliquot of each solution was diluted with ethanol to give a theoretical concentration of 50 μg mL−1. The stress degradation study with direct UV radiation (254 nm) was performed by exposing the finasteride solutions in acetonitrile (500 μg mL−1) to the UV beam for 3.5 h at room temperature in a photostability chamber (45, 46). The distance between the lamp and the sample was 10 cm. Afterward, the solutions were diluted to a theoretical concentration of 50 μg mL−1 with ethanol. Samples subjected to identical conditions, but protected from light, were used as a control. The oxidative reaction was performed in 30% H2O2 (5 mg mL−1) at 70°C for 4 h under reflux. An aliquot of this solution was diluted in ethanol to give a theoretical concentration of 50 μg mL−1. Peak purity tests were performed by the photodiode array detector, which showed that the analyte chromatographic peak did not contain more than one substance. The precision of the method was evaluated by repeatability (intra-day precision) and intermediate precision (inter-day precision) tests. The repeatability was tested by assaying six samples at the same concentration (50 μg mL−1) throughout one day under consistent experimental conditions. The intermediate precision of the method was assessed by carrying out the analysis on three different days and with a different analyst performing the analysis in the same laboratory (between-analyst precision). Data are expressed as a function of the relative standard deviation (RSD%) of a series of measurements. The accuracy was determined by a recovery test, which consisted of adding aliquots of the standard finasteride solution to placebo solutions, which gave final concentrations of the reference standards as 40, 50 and 60 μg mL−1 for the tests. Each solution was prepared in triplicate. The robustness of the method was determined by analyzing the same samples but with different method parameters, such as pH of the mobile phase (± 0.3 units), the flow rate (± 0.1 mL min−1), percentage of acetonitrile (± 2% organic phase), wavelength of detection (272 ± 3 nm), and the column (with the same specifications, but acquired from a different supplier). The five factors selected were examined in a Plackett–Burman design (N = 10). For each of the 10 experimental runs, two injections were performed for each solution. The effect (E) of each factor and the estimate experimental error (SE)e were calculated (47). The statistical interpretation provides a numerical limit value that makes it possible to define what is significant and what is not. This limit value to identify statistically significant effects is usually derived from the t-test statistical method, in accordance with the following equation: t=|Ex|(SE)e (1) An effect is considered significant at a given α level if t calculated > t critical. Alkaline degradation kinetics study The study was carried out with tablets and raw material solutions containing 500 μg mL−1 of finasteride. The solutions were prepared in 1 mol L−1 NaOH and inserted as rapidly as possible into a thermostatic water bath set at 70°C. At 60, 120, 180, 240 and 300 min, 1.0 mL from the reflux solutions was quantitatively transferred into a 10 mL volumetric flask and diluted with ethanol to give a final concentration of 50 μg mL−1 (N = 3). These solutions were protected from light and analyzed by LC, employing the developed and validated stability-indicating method. The regression coefficients (r) were determined and the best fit observed indicated the reaction order. The kinetic parameter constant (k) and t90% were also calculated. Majority degradation product evaluation In silico prediction study The computational analyses were performed using Spartan 08 version 116.2TM for Windows (Wavefunction, Inc., USA) and all of the initial structures were built using atoms and structural fragments from its molecular editor. Geometry optimization was carried out using the Merck Molecular Force Field (MMFF94) followed by the Austin Model (AM1), and re-optimized by the DFT method and the B3LYP/6.31 G* (d, f) basis set level of theory. At this step, the number of electrons in a natural atomic population analysis (NPA) was calculated using the single-point energy at the same level of theory of geometry optimization. From these data, the condensed FF derivatives values, positive (fj−) and negative (fj+) (fj+) were obtained using the following equations: fj−=qj(N)−qj(N−1),forelectrophilicattack; (2) fj+=qj(N+1)−qj(N),fornucleophilicattack; (3) fj0=½qj[(N+1)–qj(N−1)],forradicalattack. (4) In these equations, qj is the number of electrons (evaluated from NPA) at the jth atomic site in the neutral (N), anionic (N + 1), or cationic (N − 1) chemical species on the reference molecule, respectively. The dual descriptor Δf(r) of local reactivity, which allows us to obtain the preferred sites for nucleophilic attacks (Δf(r) > 0) and the preferably sites for electrophilic attacks (Δf(r) < 0) in the system at point r, was calculated for the chemical structure of finasteride using the following equation (48, 49): Δf(r)=f+(r)−f−(r) (5) LC–ESI-MS instrumentation and conditions The new LC–ESI-MS method was performed using a Shimadzu® instrument equipped with a MS detector. The separation was performed using a Supelco Ascentis® column (150 mm × 4.6 mm, 5 μm) at 40°C, and eluted at a flow rate of 0.4 mL min−1 with an injection sample volume of 4 μL. The mobile phase consisted of methanol and water (80:20 v/v), using an isocratic system. The solutions analyzed were the degraded samples of the kinetics study at 300 min (Alkaline degradation kinetics study). Mass spectra were acquired with an electrospray ionization interface in positive ionization mode, and the following conditions: capillary voltage 2.5 kV; source temperature 110°C; desolvation temperature 250°C; nitrogen desolvation flow 900 L h−1; nitrogen cone flow 90 L h−1. The full scan mass spectrum was acquired over a range of m/z 50–900. Toxicity study Human blood samples Peripheral blood was collected by venipuncture into sterile vials containing 68 I.U. of sodium heparin (BD Vacutainer®) per mL of blood. The vials were transferred to the laboratory, and whole-blood cultures were established. The blood samples were stored for up to 24 h at 4°C before culturing. This project was approved by the University’s Committee of Ethics in Research of Universidade Federal de Santa Maria (authorization n° 23081.012330/2006-94). Culture cell preparation The lymphocyte cultures were prepared with whole-blood samples and immediately transferred to 1 mL of culture medium containing RPMI 1640 supplemented with 10% fetal bovine serum and 1% streptomycin/penicillin, as previously described (50). The cells were then placed in a microaerophilic environment at 37°C for 72 h. The solutions under investigation were added to the blood at 10% concentration. The solutions analyzed included intact and degraded raw material (degraded by ∼50% under alkaline conditions; Alkaline degradation kinetics study) diluted in phosphate-buffered saline (PBS) at concentrations of 25, 50 and 100 ng mL−1. Each group consisted of three culture flasks. Genetic and oxidative parameters were analyzed after 72 h of growth. Analysis of genotoxic parameters To perform the genotoxicity tests, we first counted the total number of leukocytes in a Neubauer chamber (50). Viability was assessed by a loss of membrane integrity, which was indicated by trypan blue (51). Overall, we counted 300 cells. The genotoxicity test was conducted using the comet assay (52). Although the comet assay is not the only method for measuring oxidative DNA damage, it is one of the most sensitive and accurate and is relatively free of artifacts (53). We identified 100 cells in the slides that were submitted for analysis. The cells were visually scored according to tail length, with scores ranging from 0 (no migration) to 4 (maximal migration). Therefore, the damage index for cells ranged from 0 (all cells with no migration) to 400 (all cells with maximal migration). The tests were carried out in triplicate, and the data are presented as the mean ± standard error. The micronucleus (MN) frequency test was performed to evaluate the mutagenicity of the products. In this test, the cells were fixed with acetic acid and methanol (75:25, v/v), transferred onto clean microscope slides in duplicates, and then stained with 5% Giemsa. The criteria for scoring cells with MN were described in a previous report (54). One the cells are counted for each sample, and the results are expressed as the micronucleus frequency per 1000 cells. Statistical analysis Statistical software was used to perform all statistical analyses, which included an analysis of variance (ANOVA) followed by a post hoc Bonferroni test. P values <0.05 were considered statistically significant. Results Results of method validation The analytical method was validated for parameters such as linearity, specificity, precision, accuracy and robustness. Linearity was established by least-squares linear regression analysis of the calibration curve. The regression equation for finasteride was found by plotting the peak absorbance (y) versus the sample concentration (x). The representative linear equation was: y = 22704.2x – 7776.2 and the correlation coefficient (r = 0.9999) was highly significant. The validity of the assays was verified by means of ANOVA analysis (SAS 6.11 for Windows, SAS Institute Inc. CARY, NC, USA), which demonstrated significant linear regression and no significant linearity deviation (P < 0.05). The specificity test demonstrated that there was no interference in the LC determination of the drug. The forced degradation studies were conducted to evaluate the stability-indicating capability and selectivity of the proposed LC method using the finasteride raw material and the pharmaceutical formulation. Table I presents the extent of finasteride degradation under both stress conditions, and Figure 2 shows the chromatograms of the untreated solution and the forced degradation samples. It is important to note that although that several degraded products peaks can be observed, the peak of finasteride remains resolved. The chromatographic peak purity tool was applied to verify the finasteride peak, showing that it was 100% pure in all cases, indicating the specificity of the proposed method. It was observed that the finasteride peak presents appropriate resolution (Rs > 2) and selectivity (a > 1) in relation to the degradation products. The results indicate that the method indicates stability, and that the drug can be evaluated both qualitatively and quantitatively in the presence of degradation products and pharmaceutical excipients. Table I. Results of Finasteride Stability Under Force Degradation Conditions Condition Time (h) Degradation (%) Raw material Tablets Basic hydrolysis (1 mol L−1 NaOH) 4 51.58 32.52 Acid hydrolysis (1 mol L−1 HCl) 4 35.43 38.43 Photolysis 3.5 65.64 36.67 Oxidation (H2O2) 4 39.55 39.26 Condition Time (h) Degradation (%) Raw material Tablets Basic hydrolysis (1 mol L−1 NaOH) 4 51.58 32.52 Acid hydrolysis (1 mol L−1 HCl) 4 35.43 38.43 Photolysis 3.5 65.64 36.67 Oxidation (H2O2) 4 39.55 39.26 Table I. Results of Finasteride Stability Under Force Degradation Conditions Condition Time (h) Degradation (%) Raw material Tablets Basic hydrolysis (1 mol L−1 NaOH) 4 51.58 32.52 Acid hydrolysis (1 mol L−1 HCl) 4 35.43 38.43 Photolysis 3.5 65.64 36.67 Oxidation (H2O2) 4 39.55 39.26 Condition Time (h) Degradation (%) Raw material Tablets Basic hydrolysis (1 mol L−1 NaOH) 4 51.58 32.52 Acid hydrolysis (1 mol L−1 HCl) 4 35.43 38.43 Photolysis 3.5 65.64 36.67 Oxidation (H2O2) 4 39.55 39.26 Figure 2. View largeDownload slide (1) A and B are typical chromatograms for the raw material, and tablets in the selected experimental conditions, respectively, and C is the placebo chromatogram; (2) chromatogram of aciddegraded finasteride, and degradation products (2.7 and 2.9 min) for both samples; (3) chromatogram basedegraded finasteride, and degradation product (2.7 min) for both samples; (4) chromatogram of oxidatively degraded finasteride, and degradation product (4.1 min) for both samples; (5) chromatograms of photodegraded finasteride for raw material (A), and tablets (B). Figure 2. View largeDownload slide (1) A and B are typical chromatograms for the raw material, and tablets in the selected experimental conditions, respectively, and C is the placebo chromatogram; (2) chromatogram of aciddegraded finasteride, and degradation products (2.7 and 2.9 min) for both samples; (3) chromatogram basedegraded finasteride, and degradation product (2.7 min) for both samples; (4) chromatogram of oxidatively degraded finasteride, and degradation product (4.1 min) for both samples; (5) chromatograms of photodegraded finasteride for raw material (A), and tablets (B). Precision was determined by studying the repeatability and intermediate precision. Intra-day precision, performed by assaying the samples on 3 different days by different analysts, showed the following results: 97.67 ± 1.90%, 97.68 ± 0.61% and 97.48 ± 1.75% (mean ± RSD, N = 6). The RSD for inter-day precision was 1.44% (N = 3). The low variability of the results indicates the precision of the method. In the method accuracy assay, excellent mean percentage recovery values and low relative standard deviation values (RSD ≤ 1.5%) were found. At each level of the finasteride concentration, three determinations were performed. The mean recovery was 100.16% (RSD = 0.93%). These results reveal that any small change in the drug concentration in these solutions could be accurately determined by the proposed analytical method. In the robustness test, a number of responses can be determined from the performed experiments. For chromatographic methods, responses describing the quantity, such as peak areas or peak heights, and/or the content of the main substance and by-products are the most evident. In this study, the response determined in this test was the percentage of finasteride in the tablets. The SE of the percentage of finasteride in the tablet sample was 0.256%, which was used to perform the statistical test. The quantification of finasteride in the tablets can be considered robust because none of the factors studied had a significant effect (α = 0.05). Besides, the variation of robustness results (RSD = 0.53%) is in accordance with the results of the precision assay. Kinetic study The concentration, log and reciprocal concentration plots of the remaining drug versus time during the kinetic studies are shown in Figure 3. Degradation rate constant (k), half-life (t1/2) and t90 for finasteride in raw material and tablets solutions submitted to alkaline degradation, and determined by LC method are shown in Table II. Figure 3. View largeDownload slide Plots of concentration of remaining finasteride versus time—zero-order reaction (A); log of concentration of remaining finasteride versus time—first-order reaction (B); and reciprocal of concentration of remaining finasteride versus time—second-order reaction (C). Figure 3. View largeDownload slide Plots of concentration of remaining finasteride versus time—zero-order reaction (A); log of concentration of remaining finasteride versus time—first-order reaction (B); and reciprocal of concentration of remaining finasteride versus time—second-order reaction (C). Table II. Degradation Rate Constant (k), Half-Life (t1/2) and t90 for Finasteride in Raw Material and Tablets Solutions Submitted to Alkaline Degradation, and Determined by LC Method Samples k (min−1) t1/2 (min) t90 (min) Raw material 1.06 × 10-4 188.94 20.99 Tablets 0.94 × 10-4 211.86 23.54 Samples k (min−1) t1/2 (min) t90 (min) Raw material 1.06 × 10-4 188.94 20.99 Tablets 0.94 × 10-4 211.86 23.54 Table II. Degradation Rate Constant (k), Half-Life (t1/2) and t90 for Finasteride in Raw Material and Tablets Solutions Submitted to Alkaline Degradation, and Determined by LC Method Samples k (min−1) t1/2 (min) t90 (min) Raw material 1.06 × 10-4 188.94 20.99 Tablets 0.94 × 10-4 211.86 23.54 Samples k (min−1) t1/2 (min) t90 (min) Raw material 1.06 × 10-4 188.94 20.99 Tablets 0.94 × 10-4 211.86 23.54 Evaluation of the major degradation product The in silico results for finasteride are shown in Table III and the LC–ESI-MS chromatograms are shown in Figure 4. Table III. Values of the NPA (Neutral, Positive and Negative), Electrophilic f− and Nucleophilic f+ Condensed Fukui Functions and Δf(r) Over the Atoms of the Finasteride Molecule Calculated With the DFT/B3LYP and the 6.31 G* Basis Set Considering Equations (2)–(4) Atoms NPA NPA+ NPA− f+ f− Δf(r) C1 6.155034 6.364085 12.51912 0.209051 −6.36409 6.573136 C2 6.31507 6.421567 12.73664 0.106497 −6.42157 6.528064 C3 5.340941 5.427152 10.76809 0.086211 −5.42715 5.513363 N4 7.671368 7.703796 15.37516 0.032428 −7.7038 7.736224 C5 6.050351 6.042553 12.0929 −0.0078 -6.04255 6.034755 C6 6.08966 6.076132 12.16579 −0.01353 −6.07613 6.062604 C7 6.459159 6.451873 12.91103 −0.00729 −6.45187 6.444587 C8 6.449313 6.44338 12.89269 −0.00593 −6.44338 6.437447 C9 6.242572 6.243012 12.48558 0.00044 −6.24301 6.243452 C10 6.24425 6.241333 12.48558 −0.00292 −6.24133 6.238416 C11 6.447038 6.442907 12.88995 −0.00413 −6.44291 6.438776 C12 6.447758 6.445876 12.89363 −0.00188 −6.44588 6.443994 C13 6.042478 6.043273 12.08575 0.000795 −6.04327 6.044068 C14 6.24658 6.243506 12.49009 −0.00307 −6.24351 6.240432 C15 6.456839 6.45453 12.91137 −0.00231 −6.45453 6.452221 C16 6.454957 6.45363 12.90859 −0.00133 −6.45363 6.452303 C17 6.329044 6.326504 12.65555 −0.00254 −6.3265 6.323964 C18 6.684882 6.680537 13.36542 −0.00434 −6.68054 6.676192 C19 6.675568 6.669565 13.34513 −0.00600 −6.66957 6.663562 O20 8.605455 8.723617 17.32907 0.118162 −8.72362 8.841779 C21 5.295889 5.342884 10.63877 0.046995 −5.34288 5.389879 O22 8.641541 8.67851 17.32005 0.036969 −8.67851 8.715479 N23 7.661502 7.675312 15.33681 0.013810 −7.67531 7.689122 C24 5.862817 5.859824 11.72264 −0.00299 −5.85982 5.856831 C25 6.683708 6.680267 13.36398 −0.00344 −6.68027 6.676826 C26 6.683426 6.682354 13.36578 −0.00107 −6.68235 6.681282 C27 6.683198 6.681271 13.36447 −0.00193 −6.68127 6.679344 Atoms NPA NPA+ NPA− f+ f− Δf(r) C1 6.155034 6.364085 12.51912 0.209051 −6.36409 6.573136 C2 6.31507 6.421567 12.73664 0.106497 −6.42157 6.528064 C3 5.340941 5.427152 10.76809 0.086211 −5.42715 5.513363 N4 7.671368 7.703796 15.37516 0.032428 −7.7038 7.736224 C5 6.050351 6.042553 12.0929 −0.0078 -6.04255 6.034755 C6 6.08966 6.076132 12.16579 −0.01353 −6.07613 6.062604 C7 6.459159 6.451873 12.91103 −0.00729 −6.45187 6.444587 C8 6.449313 6.44338 12.89269 −0.00593 −6.44338 6.437447 C9 6.242572 6.243012 12.48558 0.00044 −6.24301 6.243452 C10 6.24425 6.241333 12.48558 −0.00292 −6.24133 6.238416 C11 6.447038 6.442907 12.88995 −0.00413 −6.44291 6.438776 C12 6.447758 6.445876 12.89363 −0.00188 −6.44588 6.443994 C13 6.042478 6.043273 12.08575 0.000795 −6.04327 6.044068 C14 6.24658 6.243506 12.49009 −0.00307 −6.24351 6.240432 C15 6.456839 6.45453 12.91137 −0.00231 −6.45453 6.452221 C16 6.454957 6.45363 12.90859 −0.00133 −6.45363 6.452303 C17 6.329044 6.326504 12.65555 −0.00254 −6.3265 6.323964 C18 6.684882 6.680537 13.36542 −0.00434 −6.68054 6.676192 C19 6.675568 6.669565 13.34513 −0.00600 −6.66957 6.663562 O20 8.605455 8.723617 17.32907 0.118162 −8.72362 8.841779 C21 5.295889 5.342884 10.63877 0.046995 −5.34288 5.389879 O22 8.641541 8.67851 17.32005 0.036969 −8.67851 8.715479 N23 7.661502 7.675312 15.33681 0.013810 −7.67531 7.689122 C24 5.862817 5.859824 11.72264 −0.00299 −5.85982 5.856831 C25 6.683708 6.680267 13.36398 −0.00344 −6.68027 6.676826 C26 6.683426 6.682354 13.36578 −0.00107 −6.68235 6.681282 C27 6.683198 6.681271 13.36447 −0.00193 −6.68127 6.679344 Table III. Values of the NPA (Neutral, Positive and Negative), Electrophilic f− and Nucleophilic f+ Condensed Fukui Functions and Δf(r) Over the Atoms of the Finasteride Molecule Calculated With the DFT/B3LYP and the 6.31 G* Basis Set Considering Equations (2)–(4) Atoms NPA NPA+ NPA− f+ f− Δf(r) C1 6.155034 6.364085 12.51912 0.209051 −6.36409 6.573136 C2 6.31507 6.421567 12.73664 0.106497 −6.42157 6.528064 C3 5.340941 5.427152 10.76809 0.086211 −5.42715 5.513363 N4 7.671368 7.703796 15.37516 0.032428 −7.7038 7.736224 C5 6.050351 6.042553 12.0929 −0.0078 -6.04255 6.034755 C6 6.08966 6.076132 12.16579 −0.01353 −6.07613 6.062604 C7 6.459159 6.451873 12.91103 −0.00729 −6.45187 6.444587 C8 6.449313 6.44338 12.89269 −0.00593 −6.44338 6.437447 C9 6.242572 6.243012 12.48558 0.00044 −6.24301 6.243452 C10 6.24425 6.241333 12.48558 −0.00292 −6.24133 6.238416 C11 6.447038 6.442907 12.88995 −0.00413 −6.44291 6.438776 C12 6.447758 6.445876 12.89363 −0.00188 −6.44588 6.443994 C13 6.042478 6.043273 12.08575 0.000795 −6.04327 6.044068 C14 6.24658 6.243506 12.49009 −0.00307 −6.24351 6.240432 C15 6.456839 6.45453 12.91137 −0.00231 −6.45453 6.452221 C16 6.454957 6.45363 12.90859 −0.00133 −6.45363 6.452303 C17 6.329044 6.326504 12.65555 −0.00254 −6.3265 6.323964 C18 6.684882 6.680537 13.36542 −0.00434 −6.68054 6.676192 C19 6.675568 6.669565 13.34513 −0.00600 −6.66957 6.663562 O20 8.605455 8.723617 17.32907 0.118162 −8.72362 8.841779 C21 5.295889 5.342884 10.63877 0.046995 −5.34288 5.389879 O22 8.641541 8.67851 17.32005 0.036969 −8.67851 8.715479 N23 7.661502 7.675312 15.33681 0.013810 −7.67531 7.689122 C24 5.862817 5.859824 11.72264 −0.00299 −5.85982 5.856831 C25 6.683708 6.680267 13.36398 −0.00344 −6.68027 6.676826 C26 6.683426 6.682354 13.36578 −0.00107 −6.68235 6.681282 C27 6.683198 6.681271 13.36447 −0.00193 −6.68127 6.679344 Atoms NPA NPA+ NPA− f+ f− Δf(r) C1 6.155034 6.364085 12.51912 0.209051 −6.36409 6.573136 C2 6.31507 6.421567 12.73664 0.106497 −6.42157 6.528064 C3 5.340941 5.427152 10.76809 0.086211 −5.42715 5.513363 N4 7.671368 7.703796 15.37516 0.032428 −7.7038 7.736224 C5 6.050351 6.042553 12.0929 −0.0078 -6.04255 6.034755 C6 6.08966 6.076132 12.16579 −0.01353 −6.07613 6.062604 C7 6.459159 6.451873 12.91103 −0.00729 −6.45187 6.444587 C8 6.449313 6.44338 12.89269 −0.00593 −6.44338 6.437447 C9 6.242572 6.243012 12.48558 0.00044 −6.24301 6.243452 C10 6.24425 6.241333 12.48558 −0.00292 −6.24133 6.238416 C11 6.447038 6.442907 12.88995 −0.00413 −6.44291 6.438776 C12 6.447758 6.445876 12.89363 −0.00188 −6.44588 6.443994 C13 6.042478 6.043273 12.08575 0.000795 −6.04327 6.044068 C14 6.24658 6.243506 12.49009 −0.00307 −6.24351 6.240432 C15 6.456839 6.45453 12.91137 −0.00231 −6.45453 6.452221 C16 6.454957 6.45363 12.90859 −0.00133 −6.45363 6.452303 C17 6.329044 6.326504 12.65555 −0.00254 −6.3265 6.323964 C18 6.684882 6.680537 13.36542 −0.00434 −6.68054 6.676192 C19 6.675568 6.669565 13.34513 −0.00600 −6.66957 6.663562 O20 8.605455 8.723617 17.32907 0.118162 −8.72362 8.841779 C21 5.295889 5.342884 10.63877 0.046995 −5.34288 5.389879 O22 8.641541 8.67851 17.32005 0.036969 −8.67851 8.715479 N23 7.661502 7.675312 15.33681 0.013810 −7.67531 7.689122 C24 5.862817 5.859824 11.72264 −0.00299 −5.85982 5.856831 C25 6.683708 6.680267 13.36398 −0.00344 −6.68027 6.676826 C26 6.683426 6.682354 13.36578 −0.00107 −6.68235 6.681282 C27 6.683198 6.681271 13.36447 −0.00193 −6.68127 6.679344 Figure 4. View largeDownload slide Representative LC–ESI-MS chromatogram of finasteride after alkaline degradation: FDP-1 at m/z 395, and FDP-2 at m/z 239. FDP, finasteride degradation product. Figure 4. View largeDownload slide Representative LC–ESI-MS chromatogram of finasteride after alkaline degradation: FDP-1 at m/z 395, and FDP-2 at m/z 239. FDP, finasteride degradation product. Toxicological study The results demonstrate that treating cultured human leukocytes with varying concentrations of degraded finasteride affected their biological parameters. Discussion LC method development and validation The development of an LC method requires the careful consideration of the polarity of the analyte, stationary phase and mobile phase in order to obtain good separation within a reasonable time. Thus, the LC procedure was optimized to develop a stability-indicating method so that the degradation products from the drug could be resolved. The chromatographic conditions were chosen after testing different mobile phases with different proportions of organic and aqueous solvents. During the development phase, acetonitrile and o-phosphoric acid (0.1% v/v) were used in the mobile phase at different ratios, which resulted in an asymmetric peak, and low retention times. To achieve a better separation, the pH aqueous phase was changed to optimize the finasteride retention time. Finally, a mobile phase containing o-phosphoric acid (0.1% v/v, adjusted to pH 2.8 by adding triethylamine) and acetonitrile (52:48 v/v), was adopted because of its low tailing factor, good capacity factor (k´) value, retention time, and its ability to separate the degradation products from finasteride with good peak parameters. Furthermore, the new chromatographic system was able to separate and detect the finasteride major degradation product. The chromatographic conditions developed are an innovation in terms of chromatography when compared to previous finasteride stability-indicating methods related in the literature. The system suitability test is an integral part of the analytical method, which confirms the suitability and effectiveness of the operating system. It was carried out to evaluate the reliability and reproducibility of the system for the analysis, using five replicate injections of a reference solution containing 50 μg mL−1 finasteride. The results were as follows: theoretical plates (N = 6209), tailing factor (T = 1.04), capacity factor (k´ = 4.29) and injection repeatability (RSD% < 2, N = 5). The values for these parameters were satisfactory and in accordance with previously published data (55). The method was validated, showing satisfactory data for all of the tested parameters. The validation testifies that the procedure is suitable for the intended purpose. Kinetic study The finasteride degradation profile was evaluated at different time intervals, under the same conditions described above. Approximately 50 and 45% drug degradation occurred during 300 min for the raw material and tablet solutions, respectively. One major degradation product was eluted at 2.7 min, which resulted from degradation of both analyzed samples. The shorter retention times of the degradation product compared to the finasteride retention time are due to more polar features. The degradation kinetics were calculated for both samples through the decrease in drug concentration over time. The finasteride concentration remaining was calculated at each time interval for the three replicates compared to the mean drug concentration of the standard solution. According to the evaluation of the correlation coefficients (Figure 3), it can be concluded that the finasteride degradation for both sample solutions shows second-order kinetics under the experimental conditions applied. By analyzing the straight-line slopes, it was possible to calculate the apparent second-order degradation rate constant k (1.06 × 10−4 min−1 for raw material; 0.94 × 10−4 min−1 for tablets), t1/2 (188.94 min for raw material; 211.86 min for tablets) and t90 (20.99 min for raw material; 23.54 min for tablets) for each sample solution tested (Table II). Evaluation of the major degradation product In an alkaline environment, as shown in Table I, there was some level of degradation of the finasteride structure. In silico studies of chemical reactivity were carried out by the prediction of the most likely position of structural hydrolysis. The indicated computational method to study the alkaline reaction conditions observed in stress degradation studies is FF (34), according by your two derivatives, f+ and f−, for nucleophilic and electrophilic attack, respectively. FF are considered the local reactivity descriptors that indicates the preferred regions where a chemical species will change its density when the number of electrons is modified. Thus, it indicates the susceptibility of the electronic density to deform at a given position upon accepting or donating electrons (56). In this context, Parr and Yang (57) showed that sites in chemical species with the largest values of Fukui function (f(r)) are those with higher reactivity . From the FF values reported in Table III, the reactivity atoms order for the electrophilic susceptibility was O20>O22>N4>N23, respectively. To nucleophilic, the reactivity order was C1>O20>C2>C3>O22>N4>N23. According to the dual descriptor Δf(r), the calculated order were O20>O22>N4>N24, with values indicating preference for the nucleophilic attacks (Δf(r) > 0) into the system at point r. The sites with higher values of FF and dual descriptor are located mainly at the dihydropyridinone ring and the lateral chain in the chemical structure of finasteride. In all FF calculations, the olefin binding, carbonyl and nitrogen atom moieties of cited ring were shown to be the most probable reaction sites in alkaline forced degradation studies. As previously mentioned, finasteride was found to be labile under alkaline conditions. After 300 min of exposure, an ~50% reduction in concentration was observed for both analyzed samples. Based on their retention times, the LC–ESI-MS studies indicated two major peaks with m/z 239 and m/z 395 (Figure 4), which were attributed to the FDP. The product FDP−1 with m/z 395 was formed by addition of 22 amu [M + 22]+ thus indicating a sodium adduct of finasteride. As previously seen, the in silico study was performed aiming to assist the comprehension of chemical reactivity by the prediction of the most likely position of hydrolysis. The obtained results showed that the reactive atoms are O20, O22, N4 and N23. Based on these results, we suggest that the finasteride hydrolysis to FDP-2 (m/z 239 [M + 1]+) involves opening of the dihydropyridinone ring and closing of the lateral chain. The proposed products generated from the original structure are shown in Figure 5. In a study of forced degradation of dutasteride, a compound with a similar structure to finasteride, breakage of the dihydropyridinone ring and oxidation of the carbonyl moiety was also reported, resulting in the generation of carboxylic acid at this structural position when submitted to alkaline stress-forced conditions (58). These data assist the understanding of finasteride degradation in analogous experimental conditions. Figure 5. View largeDownload slide Proposed alkaline degradation product (FDP-2) of finasteride based in sílico study and LC–ESI-MS results. FDP, finasteride degradation product, chemically characterized to (S)-4-((3aS,6 S)-octahydro-3amethyl-1H-inden-6-yl)pentanoic acid. Figure 5. View largeDownload slide Proposed alkaline degradation product (FDP-2) of finasteride based in sílico study and LC–ESI-MS results. FDP, finasteride degradation product, chemically characterized to (S)-4-((3aS,6 S)-octahydro-3amethyl-1H-inden-6-yl)pentanoic acid. Toxicological study In the kinetic study, the different samples tested (raw material and tablets) showed similar residual drug levels in the tested conditions (Figure 3). The chromatograms showed a decrease in the finasteride area and one additional major peak at 2.7 min, for both analyzed samples. In the identification study, the product was the same for both samples. Therefore, we decided to perform a degraded raw material toxicity assay, thus removing any possibility of pharmaceutical excipient interference (Figure 6). Regarding the proliferation of leukocytes, the only treatments that caused any significant interference were those with degraded samples. These treatments showed a decrease in proliferation at 16 ± 1.5% for the degraded sample at 25 ng mL−1, 31 ± 1.15% at 50 ng mL−1 and 27 ± 1.5% at 100 ng mL−1. The assay was performed to evaluate the effect of the degraded sample in relation to the intact molecule to foresee possible undesirable effects resulting from the degradation products. The results showed that the FDP affected the cell proliferation. Figure 6 indicates the cellular viabilities means ranged from 81.3 ± 1.5% (100 ng mL−1) to 92.7 ± 1.5% (25 ng mL−1) for the degraded sample. These data show a moderate decrease in the cellular viabilities, but no significant differences compared to the negative control. The micronuclei frequency and the DNA damage index data showed no genotoxic effects for the degraded sample. Figure 6. View largeDownload slide Markers of cell proliferation and genotoxic effects. The bar graphs indicate total leukocytes counted, percentage of cellular viabilities, frequency of micronuclei, and DNA damage by comet assay. NC, negative control (PBS, phosphate-buffered saline); PC, positive control (hydrogen peroxide: H2O2); IRW, intact raw material; DRW, degraded raw material. Data are presented as mean ± SD (n = 3). Letters indicate statistically significant differences. Figure 6. View largeDownload slide Markers of cell proliferation and genotoxic effects. The bar graphs indicate total leukocytes counted, percentage of cellular viabilities, frequency of micronuclei, and DNA damage by comet assay. NC, negative control (PBS, phosphate-buffered saline); PC, positive control (hydrogen peroxide: H2O2); IRW, intact raw material; DRW, degraded raw material. Data are presented as mean ± SD (n = 3). Letters indicate statistically significant differences. Conclusion This article presents a stability study of finasteride under forced degradation conditions. A simple stability-indicating LC method was validated for the determination of the drug and degradation products, showing satisfactory data for all of the tested parameters. The degradation of this drug during alkaline processing follows second-order reaction kinetics for tablets and the raw material. The kinetic parameters of the degradation rate constant, t1/2 and t90, can be predicted. Based on in silico and LC–ESI-MS studies, the identity of finasteride’s major alkaline degradation product could be suggested. Furthermore, the biological safety study showed that the degraded finasteride sample affected the cell proliferation. 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Journal of Chromatographic ScienceOxford University Press

Published: Apr 9, 2018

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