Eplerenone was subjected to the influence of ionizing radiation in the form of a high-energy electron beam (25–400 kGy), high temperature (90°C RH 0% and 60°C RH 76.4%), and light (6 mln lux h). An HPLC method was used to determine the content of eplerenone and to establish the impurity profile of all samples. As eplerenone was found to be a compound of great resistance to the above stress factors with the exception of high doses of ionizing radiation (≥200 kGy) when its degradation was above 1%, it is possible to sterilize eplerenone by radiation method with the standard dose of 25 kGy. Based on the analysis of impurities and degradation products, the mechanism of radiodegradation was demonstrated to differ from the mechanisms of photo- and thermodegradation. The observation that the DSC curves for the nondegraded and degraded samples of eplerenone were significantly different only under exposure to the electron beam confirmed the applicability of DSC for studies of radiolytic degradation of eplerenone.
Eplerenone (pregn-4-ene-7,21-dicarboxylic acid, 9,11-epoxy-17-hydroxy-3-oxo,
Structural formulas of eplerenone and its potential impurities [
What distinguishes it from spironolactone, the first aldosterone antagonist in use for over 50 years, is the epoxy bridge between positions 9
Previous studies of the stability of eplerenone [
The radiochemical stability of eplerenone has not been studied so far. Sterilization and decontamination of drugs by using gamma or e-beam ionizing irradiation has been recommended by the European Pharmacopoeia (Ph. Eur.) 7th Edition [
Although it is estimated that approximately 90% of medicinal substances in the solid state may be sterilized in that manner, it is necessary to determine whether the standard dose of 25 kGy does not damage the drug structure [
Research into drug irradiation stability is increasingly applying high doses of ionizing radiation (100–800 kGy) [
The resistance of steroid drugs to ionizing radiation has been investigated since the 1980s [
The purpose of this work was to investigate the radiochemical stability of eplerenone in order to determine whether it may be sterilized by means of irradiation. The use of doses exceeding 25 kGy (50–400 kGy) was designed to identify eplerenone degradation products and to compare them with those reported in the literature. The study involved the application of DSC to evaluate samples of eplerenone during radio-, thermo-, and photodegradation, with the aim of verifying the suitability of that method for a study of eplerenone stability.
Eplerenone and its impurities were obtained from Industriale Chimica s.r.l., Saronno, Italy. The structural formulas of impurities A–G are shown in Figure
All other chemicals and solvents were obtained from Merc KGaA (Germany) and were of analytical grade. High quality pure water was prepared using a Millipore purification system (model Exil SA 67120, Millipore, Molsheim, France).
Approximately 0.5 g of eplerenone was placed in 4 mL colourless glass jars closed with a plastic stopper and irradiated to 25, 50, 100, 200, and 400 kGy with the e-beam from a linear electron accelerator Elektronika 10/10. The energy of electrons was 9.96 MeV and the current intensity 6.2
Approximately 10 mg of eplerenone was placed in 4 mL colourless vials and illuminated with a SUNTEST CPS+ device (Heraeus, Germany). In the photodegradation studies that were consistent with the ICH Q1B guidelines the following conditions were applied: a 1500 W lamp, a 300–800 nm wavelength range, an ID65 solar filter, and an irradiation intensity of 250 Wm−2. Exposure times of 21.6 and 108 hours provided an overall illumination of not less than 1.2 million and 6 million lux hours, respectively. A 10 mg control sample of eplerenone in a glass vial was wrapped in aluminium foil.
10 mg samples of eplerenone were placed in 4 mL vials and put in heat chambers at 90°C (RH 0%) and 60°C (RH 76.4%). At specified time intervals (1, 3, 5, and 7 days), determined by the rate of degradation, the vials were removed and cooled to room temperature.
The analytical system (consisted of a quaternary pump L-7100, an autosampler L-7200, a column oven L-7360, and a diode array detector L-7455; all are Merck Hitachi products) was used for chromatographic separation of the impurities and degradation products of eplerenone samples. All the samples (2.5 mg/mL) were dissolved in the solvent mixture (methanol, acetonitrile, and water 25 : 25 : 50 V/V/V). An Inertsil ODS3 C18 (
The gradient system was as in Table
Mobile phase A | Mobile phase B | |
---|---|---|
0–25 min | 54 | 46 |
25–32 min | 54 |
46 |
32–45 min | 40 | 60 |
45–46 min | 40 |
60 |
46–56 min | 54 | 46 |
Degradation products of eplerenone [
Stress conditions and duration | Degradation of eplerenone |
Molecular formula | Molecular weight [Da] | Reference |
---|---|---|---|---|
0.5 mol/L NaOH, 1 h, 25°C | 20.3 | C24H34O8 | 450.52 | [ |
|
||||
1 mol/lL NaOH, 2 h, 100°C | 93.0 | C24H32O7 | 432.51 | [ |
|
||||
1 mol/lL HCl, 2 h, 100°C | 90.0 | C23H32O5 | 388.50 | [ |
C23H28O6 | 400.46 | [ | ||
C22H32O2 | 328.49 | [ |
UV detection was performed at 240 nm. The flow rate was 1.0 mL/min and the injection volume 20
Measurements were performed with DSC-50 Shimadzu, Japan. 2 mg samples were sealed in aluminium crucibles with pierced lids. The samples were thermally equilibrated at 20°C for 5 min and the measurements were performed at a heating rate of 5°C min−1 in a nitrogen atmosphere (30 mL min−1). For each sample, three independent measurements were performed and the results were averaged.
The eplerenone samples subjected to the influence of temperature (in dry air and at 76.4% RH), light, and ionizing radiation and the control sample (not exposed to the stress conditions) were analyzed chromatographically. The HPLC method, previously described [
Chromatograms of eplerenone and its 7 potential impurities.
Based on the chromatograms of the degraded and nondegraded samples (Figure
Eplerenone and impurities of known structure in degraded and nondegraded samples.
Conditions | Content [%] | ||||
---|---|---|---|---|---|
Eplerenone (10.23 min) | Imp. D (7.07 min) | Imp. A (7.47 min) | Imp. B (12.83 min) | Imp. F (15.15 min) | |
Nondegraded | 99.782 | — | — | — | 0.044 |
6 mln lux h | 99.657 | — | 0.008 | — | 0.031 |
90°C RH 0% | 99.598 | — | 0.007 | — | 0.022 |
60°C RH 76.4% | 99.737 | — | 0.007 | — | 0.051 |
25 kGy | 99.772 | 0.040 | — | — | — |
50 kGy | 99.559 | 0.070 | — | — | — |
100 kGy | 99.329 | 0.118 | — | 0.017 | — |
200 kGy | 98.758 | 0.204 | — | 0.032 | — |
400 kGy | 98.006 | 0.354 | — | 0.050 | — |
Eplerenone and impurities of unknown structure in degraded and nondegraded samples.
Conditions | Recovery* [%] | |||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
a |
b |
c |
d |
e |
f |
g |
h |
i |
j |
k |
l |
m |
n | |
Nondegraded | 0.015 | 0.012 | 0.030 | 0.024 | 0.036 | 0.031 | 0.01 | 0.018 | ||||||
6 mln lux h | 0.013 | 0.014 | 0.112 | 0.033 | 0.027 | 0.035 | 0.021 | 0.038 | 0.009 | 0.021 | ||||
90°C RH 0% | 0.010 | 0.014 | 0.009 | 0.034 | 0.028 | 0.035 | 0.139 | 0.017 | 0.011 | 0.023 | ||||
60°C RH 76.4% | 0.012 | 0.013 | 0.009 | 0.070 | 0.025 | 0.035 | 0.017 | 0.025 | ||||||
25 kGy | 0.022 | 0.011 | 0.038 | 0.011 | 0.012 | 0.019 | 0.035 | 0.021 | ||||||
50 kGy | 0.022 | 0.010 | 0.063 | 0.055 | 0.037 | 0.030 | 0.104 | 0.019 | ||||||
100 kGy | 0.026 | 0.018 | 0.021 | 0.093 | 0.106 | 0.024 | 0.021 | 0.183 | ||||||
200 kGy | 0.034 | 0.033 | 0.037 | 0.022 | 0.017 | 0.146 | 0.187 | 0.056 | 0.025 | 0.338 | ||||
400 kGy | 0.049 | 0.063 | 0.064 | 0.033 | 0.021 | 0.249 | 0.365 | 0.065 | 0.026 | 0.557 |
Chromatograms of eplerenone before (a) and after irradiation (b).
In the compounds of known chemical structure the presence of 4 impurities was detected: A, B, D, and F (identificated by comparison of retention times to standards). The nondegraded eplerenone sample was found to contain impurity F with a hydrolyzed lactone bond, whose small amount (0.022–0.051%) was also detected in the samples heated in dry air and those exposed to light. In the eplerenone samples exposed to light and heated trace amounts of impurity A (0.008%) were detected. Ionizing radiation proved to be the most destructive as only in the irradiated samples impurities D (≥25 kGy)—a compound with a hydrolyzed ester bond—and B (≥100 kGy)—an eplerenone isomer—were found.
14 compounds of unknown structure were found, of which 9 were present in the control sample that contained 99.78% eplerenone. After storage at 60°C (RH 76.4%) and 90°C (RH 0%) over a period of 1 week, the eplerenone content was slightly lower, 99.74% and 99.60%, respectively. Exposure to light for 110 h in the SUNTEST CPS+ chamber, simulating the energetic composition of sunlight, was equivalent to a dose of 6 mln lux h, which was 5 times greater than the minimum value recommended for photostability studies. According to the ICH guidelines [
Although the standard dose of sterilization irradiation also did not have a destructive effect on eplerenone, it was found that the highest dose (400 kGy) resulted a loss of 1.78%. It was also observed that different degradation products were formed than during termo- and photodegradation. For impurities B and D as well as the compounds of unknown structure, a relationship was identified between the content or recovery and the dose of ionizing radiation (Figure
Dependence of eplerenone radiodegradation product recovery on radiation dose.
The next stage of this work was to analyze the nondegraded samples of eplerenone and those affected by stress factors with the use of differential scanning calorimetry. The DSC curve for the control sample displayed a typical endothermic melting peak with a maximum at 248.8°C. After the thermo- and photodegradation of eplerenone, the DSC curves had similar melting peak parameters (Table
Results of DSC analysis. The values in parentheses are standard deviation.
Conditions | Temperature [°C] | Difference [°C] | ||||
---|---|---|---|---|---|---|
|
|
|
|
|
|
|
Nondegraded | 234.5 (3) | 248.5 (0) | 253.5 (3) | — | — | — |
6 mln lux h | 232.6 (0) | 248.3 (3) | 253.2 (0) |
|
|
|
90°C RH 0% | 232.3 (6) | 248.5 (0) | 253.7 (3) |
|
|
|
60°C RH 76.4% | 231.7 (3) | 248.2 (7) | 253.6 (7) |
|
|
|
25 kGy | 227.6 (7) | 246.1 (3) | 249.8 (3) |
|
|
|
50 kGy | 227.3 (3) | 245.7 (0) | 249.3 (7) |
|
|
|
100 kGy | 226.4 (3) | 244.1 (3) | 247.8 (3) |
|
|
|
200 kGy | 226.7 (3) | 242.0 (7) | 246.5 (0) |
|
|
|
400 kGy | 227.5 (0) | 239.6 (3) | 242.5 (7) |
|
|
|
The DSC curves for radiodegradation varied distinctly from that for the control sample (Figure
The DSC curves of degraded and nondegraded eplerenone.
Eplerenone is a compound exhibiting a high resistance to temperature, light, and ionizing radiation. That makes it suitable for irradiation sterilization and decontamination as a dose of 25 kGy caused merely a 0.13% loss of content, of which 0.04% was found to be impurity D—a derivative with a hydrolyzed ester bond. Its content was observed to increase in proportion to the growth of ionizing radiation and reached 0.35% at 400 kGy.
The method based on differential scanning calorimetry proved useful for the evaluation of eplerenone radiodegradation since a degradation level of 1.78% (400 kGy) was seen in the DSC curve as a shift of the endothermal melting peak maximum towards lower values by as much as 8.9°C.
The authors declare that there is no conflict of interests regarding the publication of this paper.
The help of Industriale Chimica s.r.l., Saronno, Italy, in providing eplerenone and its impurities used in this investigation is gratefully acknowledged.