Formulation, stability testing, and analytical characterization of melatonin-based preparation for clinical trial

Abstract

A new institutional clinical trial assessed the improvement of sleep disorders in 40 children with autism treated by immediate-release melatonin formulation in different regimens (0.5 mg, 2 mg, and 6 mg daily) for one month. The objectives of present study were to (i) prepare low-dose melatonin hard capsules for pediatric use controlled by two complementary methods and (ii) carry out a stability study in order to determine a use-by-date. Validation of preparation process was claimed as ascertained by mass uniformity of hard capsules. Multicomponent analysis by attenuated total reflectance Fourier transformed infrared (ATR-FTIR) of melatonin/microcrystalline cellulose mixture allowed to identify and quantify relative content of active pharmaceutical ingredients and excipients. Absolute melatonin content analysis by high performance liquid chromatography in 0.5 mg and 6 mg melatonin capsules was 93.6%±4.1% and 98.7%±6.9% of theoretical value, respectively. Forced degradation study showed a good separation of melatonin and its degradation products. The capability of the method was 15, confirming a risk of false negative <0.01%. Stability test and dissolution test were compliant over 18 months of storage with European Pharmacopoeia. Preparation of melatonin hard capsules was completed manually and melatonin in hard capsules was stable for 18 months, in spite of low doses of active ingredient. ATR-FTIR offers a real alternative to HPLC for quality control of high-dose melatonin hard capsules before the release of clinical batches.

Keywords: Autism; Clinical trial; High-performance liquid chromatography; Melatonin; Multicomponent infrared analysis; Stability-indicating method.

Graphical abstract

Fig. 1

Chemical structure of melatonin.

Fig. 2

Chromatograms of (A) melatonin solution, (B) melatonin/ microcrystalline cellulose suspension and (C) Circadin^® LP, 2 mg obtained by HPLC.

Fig. 3

Chromatograms of melatonin after forced degradation by (A) 5 M HCl solution at 70 °C for 30 min, (B) 2 M NaOH solution for 30 min at 70 °C, (C) 3% H₂O₂ solution at 80 °C for 10 min and (D) UV (λ=365 nm) for 5 h.

Fig. 4

Multi-wavelength detection of melatonin by HPLC.

Fig. 5

(A) Simultaneous determination of melatonin and microcrystalline cellulose relative content in a 6 mg melatonin hard capsule by ATR-FTIR. Upon treatment of melatonin/microcrystalline cellulose spectrum, melatonin and microcrystalline cellulose were identified separately and matched closely original components (melatonin: 94%; microcrystalline cellulose: 73%) and the relative content of melatonin and microcrystalline cellulose in mixture was resolved (melatonin:microcrystalline cellulose: 9:91, m/m). (B) A linear relationship was found between the relative content of melatonin/microcrystalline cellulose determined by ATR-FTIR and the mass ratio of melatonin (0%–100%; R = 0.9989) and microcrystalline cellulose (0%–100%; R = 0.9984). Each data point is the mean±standard deviation of three determinations.