Stability of 5 mg/mL Nitrendipine Oral Suspension in Syrspend^® SF PH4

Reagents

Nitrendipine (Nidrel^®) 20 mg tablets were bought to UCB Pharma (Colombes, France) and Syrspend^® SF PH4 to Fagron (Thiais, France). The composition of the suspension base is as follows: sucralose (sweetener), citric acid and sodium citrate (acidifier/buffer), modified food starch (suspending agent), sodium benzoate (<0.1 %, preservative), malic agent (buffer), simethicone (anti-foam agent), and purified water (approximately 95 %). Methanol and acetonitrile were of HPLC grade and were procured respectively from VWR Chemicals (Fontenay-sous-Bois, France) and from Fisher Chemical (Illkirch-Graffenstaden, France). Water for HPLC was distilled and passed through a reverse osmosis system. Sodium hydroxide (NaOH) and hydrochloric acid (HCl) solutions used for the degradation study were purchased from Merck (Darmstadt, Germany) and oxygen peroxide (H₂O₂) 10 volumes to Gifrer (Decines-Charpieu, France).

Formulations preparation

Nitrendipine oral suspension (5 mg/mL) was prepared, according to the Good Manufacturing Practices (7), using nitrendipine 20 mg tablets (Nidrel^®) and the suspending vehicle Syrspend^® SF PH4. Tablets were ground to fine powder and combined with the vehicle. These preparations were packaged in amber glass bottles because, like most 1,4-dihydropyridines derivatives, nitrendipine shows high photosensitivity (8, 9, 10). The products were stored at room temperature (24 °C ± 1 °C) because nitrendipine is not a thermolabile active ingredient [11].

Instrumentation and chromatographic conditions

Nitrendipine concentrations of each sample were measured by HPLC with UV detection. The chromatographic system included a 515 HPLC pump, an autosampler 717plus and a 2487 UV detector (Waters, Milford, USA). The separation of the analytes was performed on a C-18 reversed phase column Atlantis T3 column (4.6×150 mm, 5 µm) that was kept as 25 °C. The mobile phase was prepared by a mixture of methanol/water/acetonitrile (40:20:40 v/v). The injection volume of sample was 10 μL, the mobile phase flow rate was set at 1 mL/min, and detection was done at 280 nm. Data collection and analysis were performed using Empower^® Software (Waters, Milford, USA). All the HPLC equipment is available in the control laboratory of the hospital pharmacy: this facilitates the preparations’ control extemporaneously.

Analytical validation

The analytical validation was performed according to the recommendations of ICH Q2R1 [3] including the assessment of system linearity, accuracy, precision (repeatability, intermediate precision) and specificity [12].

The linearity was checked for nitrendipine assay in the concentration range from 75 to 175 µg/mL (75 µg/mL, 100 µg/mL, 125 µg/mL, 150 µg/mL, 175 µg/mL). In order to check if there were interactions of excipients, two types of range were realized: one containing only the API and one reconstituted with the suspending vehicle. In summary, six different curves were performed on three different days. The method was considered as linear if the correlation coefficient was over 0.99 for the mean standard curve.

To assess the accuracy, the results obtained during the evaluation of the linearity were used. Accuracy values were obtained by calculating relative error (report between theoretical and calculated concentration) for three quality control samples (75 µg/mL, 125 µg/mL, 175 µg/mL). This report allows to access to a recovery rate. To be accepted, this rate has to be less than 5 %.

To study the repeatability of the method, six determinations of the 125 µg/mL solutions were achieved. To evaluate intermediate precision, the protocol applied to the study of repeatability was reproduced two other days by different operators. Repeatability and intermediate precision were determined using relative standard deviation and the threshold value for acceptability was 5 %.

A stability-indicating method is a method able to distinguish the API from its degradation products. The method has to be sufficiently sensitive to detect these degradation products in low quantity and sufficiently resolute to distinguish products with potentially close structures [13, 14]. Different degradative conditions were tested: alkaline, acidic, thermal and oxidative [15]. Alkaline hydrolysis was studied by adding different concentration of NaOH (0.1 M, 0.5 M, 1 M, and 12 M) in the nitrendipine solution (1:4 v/v). After one hour, neutralization was performed with respectively 0.1 M, 0.5 M, 1 M and 12 M HCl solutions. The acidic hydrolysis was performed by doing the opposite procedure. Thermal degradation was assessed by placing the solution at 80 °C during 1 hour. The oxidative degradation was studied by adding a H₂O₂ 10 volumes solution to the nitrendipine solution.

Stability of oral suspension

Physical stability

A visual examination was performed along the stability study in order to detect a precipitation, color change or other macroscopic manifestations (e. g. smell change). The preparation was considered physically stable if those characteristics were not changed.

Microbiological stability

According to the European Pharmacopoeia [11], the microbiological quality of oral preparations must satisfy the following tests: enumeration of total viable aerobic germs with a maximum of 10³ bacteria and 10² molds and yeasts per milliliter and absence of Escherichia coli. Microbiological stability was assessed at each study day by manual counting of colony-forming units on media plates. Because the suspensions were purposed for use as a multiple dose regimen, it was necessary to determine the microbiological stability of the preparations whether they retained the quality once the containers were opened under ambient conditions. Previously, a fertility test was performed in order to ensure that one of the products present in the vehicle Syrspend SF PH4 did not inhibit microbial sprouting.

Statistical analysis/data analysis

The results are represented as the mean value of nine independent experiments ± 95 % confidence interval. All statistical tests were performed by the Excel^® software (Microsoft Office, USA, 2007) with a risk α of 5 %. Statistical significance was defined as p<0.05.

Analytical validation

Specificity

The retention time of nitrendipine was approximatively 3.6 min (Figure 1A). In this method, alkaline degradation seems to be the major route of degradation. We observed a major degradation product at 3.2 min (Figure 1B). The degradation product was well-resolved from the nitrendipine peak. Resolution between nitrendipine peak and the major degradation product peak identified was 1.6. This resolution value, greater than 1.5, ensures that nitrendipine and his degradation product are well separated.

Figure 1:

Representative chromatograms of nitrendipine standard solution, in test samples (A) and under stressed conditions (B).

Stability of oral suspension

pH and osmolality

During the whole study, all pH measurements were comprised between 4.20 and 4.27 (Figure 2). No significant variation was observed. In the same way, osmolality were not modified, varying between 44 and 48 mOsm/kg (Figure 3).

Figure 2:

pH evolution of nitrendipine oral suspension as a function of time (n=3).

Figure 3:

Osmolality evolution of nitrendipine oral suspension as a function of time (n=3).

Physical stability

Visual inspections revealed no change in color during the studied period (all suspensions maintained their initial yellow color). No precipitation or macroscopic changes were observed.

Microbiological stability

No antimicrobial properties were observed with the suspending vehicle. Nitrendipine suspensions prepared using good manufacture practice was found to be free of germs (no colony-forming units were found during the study). Those results show that the microbiological quality of the suspensions is not impaired during the repeated opening of the bottle over a period of 90 days, allowing the manufacture of a multidose vial.