2.1. MaterialsHard fat-Adeps solidus Ph. Eur. and Witepsol®H15 were purchased from Hänseler (Herisau, CH). Suppocire® AM and Suppocire® AS2X were purchased from Gattefossé
SAS (Saint Priest, France). Tween® 20, Tween® 80 and sodium laurylsulfate (SLS) were purchased from Hänseler (Herisau, CH). Cytotec® tablets 200 µg misoprostol were purchased from Pfizer (Zurich, CH). Benzophenone was purchase from Sigma Aldrich (Steinheim, Germany). Acetonitrile LC-MS Chromasolv, Fluka analytical was purchased from Sigma Aldrich (Steinheim, Germany). Pure misoprostol was purchased from Sigma Aldrich (Steinheim, Germany). HPCL-filtered water was obtained, in-house, from a Millipore model Synergy 185 (MA, USA).
2.2. MethodsSuppository preparation: Suppositories were manufactured according to the melting method by calculating the displacement value of the suppository bases. The 1000 µg misoprostol suppositories were prepared with different bases (Adeps solidus, Witepsol®H15, Suppocire® AM and Suppocire® AS2X) and different surfactants (Tween® 20, Tween® 80 and sodium laurylsulfate (SLS)) at concentrations of 0.5%, 1%, 2%, 3% or 5%. Suppositories were prepared by crushing five 200 µg misoprostol tablets in a mortar, melting the suppository base with the surfactant in a water bath at 37˚C and adding the crushed misoprostol. The mixture was transferred in suppository molds of 3 g.
In vitro release: A system was specially developed for the set-up of the in vitro release test. Three suppositories were put in a basket immersed in a 500 mL buffer bath at pH 7.2 containing SLS (from 0% to 5%) at 37˚C ± 1˚C with magnet agitation (100 rpm). A 900 µL volume was withdrawn from the medium at 2, 5, 10, 20 and 30 minutes and 100 µL of a 0.3 µg/mL solution of benzophenone (internal standard) was added. The solutions were analysed in triplicate by high-performance liquid chromatography (HPLC).
Stability study: Misoprostol stability in suppositories with the formula allowing the best release was assessed after conservation at 4˚C, 20˚C and 60˚C. Suppositories were tested immediately after preparation and at 7, 14, 30, 60, 90, 120, 150, 180 and 210 days. After the suppository was melted in a water bath at 37˚C ± 1˚C, 7.0 mL of acetonitrile was added and mixed for 2 min. with a Vortex-type mixer to extract the drug. The vortex mixer model SA7 was purchased from Stuard (UK). The mixture was centrifuged at 3000 rpm for 10 min. A 300 µL aliquot was mixed with 100 µL of a 30 µg/mL solution of benzophenone and 600 µL of acetonitrile. This solution was analysed in triplicate by HPLC.
HPLC method: Three HPLC methods were found in the literature for the analysis of misoprostol [21-23]. The method described by Hafirassou [23] was adapted and validated, following the ICH guidelines for the present study. The quantitative analyses were performed on an HPLC pump Serie 1100 with a binair pump model F 1312 A (Agilent, USA). The separation was achieved on a Zorbax Eclipse XDB C18 column (4.6 × 150 mm, 5 µm (Agilent, USA), maintained at 23˚C. The equipment also consisted of an automatic injector model G 1367 A (Agilent, USA) with a 20 µL loop and an equipped with an UV detector model DAD SL G 1315 C (Agilent, USA) set at 202 nm. The mobile phase was prepared by mixing acetonitrile and filtered water in the ratio of 60:40 (v:v). The flow rate was set at 1 mL/min.
No interfering peaks were identified with the lipophilic bases, the surfactants or the degradation products of misoprostol. The retention times for misoprostol and benzophenone were 3.77 and 5.59 minutes, respectively (Figure 1).
Standard solution and standard curve: The linearity, trueness and accuracy of the analytical method were evaluated for both studies (release and stability). A standard solution of misoprostol was prepared by dissolving pure misoprostol in acetonitrile (100 µg/mL = S). This solution was kept at −20˚C. This solution was further diluted in acetonitrile (10 µg/mL=S1). The standard curve was built by plotting the ratio of misoprostol peak area to that of benzophenone according to misoprostol concentration and used for measuring drug concentrations in samples. Three replicate standards at three different days and two replicate injections were used for the standard curve.
Misoprostol standard solutions for the release study were prepared by diluting S1 with acetonitrile to concentrations of 0.3, 3 and 7.2 µg/mL. Each solution contains 0.3 µg/mL of benzophenone (internal standard) diluted in acetonitrile. The standard curve was linear over the concentration range of 0.3 - 7.2 µg/mL. A linear relationship was plotted over the concentration range of 0.3 - 7.2 µg/mL. The intercept was found to be not significantly different from 0 (Student t test, α = 0.05). The intraday and interday relative standard deviations were found to be inferior to 6.2%.
Standard solutions for the stability study were prepared by diluting S solution to 3 different concentrations (28, 40 and 52 µg/mL) with benzophenone at 3 µg/mL in acetonitrile. This curve was also linear over the working range of 28 - 52 µg/mL. The plots intersected the origin at 0.08%. The intraday and interday coefficients of variation were ≤4.3% and 3.5%, respectively.
Data analysis: Misoprostol release was determined by calculating the percentage of misoprostol concentration liberated in the media according to time. Stability was determined by calculating the percentage of the initial concentration remaining after each time interval. Stability was defined as the retention of at least 90% from the initial concentration.
Facility of use: Because of this study the Swiss ethic committee was contacted, and it approved this project. The suppositories were proposed to the gynecology service of a regional hospital. They were used in 6 patients for the treatment of PPH as third-line treatment after oxytocin and methylergometrine instead of the 5 tablets of Cytotec® administrated rectally. The facility of use was evaluated through a questionnaire. These questionnaires were anonymously filled and sent to the pharmacy.
