Development and validation of a stability-indicating assay method for a hospital-compounded oral suspension of ferrous fumarate for preterm neonates

Introduction
Iron deficiency anaemia affects 25 to 85% of preterm neonates [1]. The only oral liquid formulation commercially available in France is Ferrostrane^® Nourrissons. However, it is unsuitable for premature newborns due to the presence of 4 excipients with known effects and an osmolality > 4000 mOsm/L. In this context, a 5 mg/mL oral suspension of ferrous fumarate was developed as a hospital stock preparation to meet the specific needs of these infants. The aim of this study is to develop and validate a stability-indicating assay method and to establish the degradation profiles of ferrous fumarate, alone and within the formulation matrix.

Materials and methods
A reverse-phase high-performance liquid chromatography (HPLC) method (LC 2030+, Shimadzu) with UV detection (λ = 282 nm) and a complexation of ferrous iron (Fe2+) with bathophenanthroline [2]. was developed. A TSKgel Super ODS column (50×4.6 mm, 2 µm; Tosoh Bioscience) was used with a water:ACN mobile phase (40:60, 0.06 M MSA; 35 °C; 1 mL·min⁻¹; 10 µL) under isocratic conditions. Method validation, assessment of the matrix effect and forced degradation were performed in accordance with the international ICH Q2(R2) and SFSTP guidelines.

Results and discussion
The method was successfully validated for both ferrous fumarate alone and the final formulation. The method was specific (Tr [Fe-Bphen] = 2.3 min, no matrix interference), linear (R² > 0.99; ANOVA and Cochran’s test) over a range of 1.696 to 3.957 µg/mL. No matrix effect was observed, based on comparison of slopes and intercepts (p > 0.05). The method showed good accuracy (mean recovery: 100.01%; 95% CI: [98.82–101.20]), repeatability (CV = 1.13%), and intermediate precision (CV = 0.85%). The limits of quantification and detection were established at 0.29 µg/mL and 0.10 µg/mL, respectively. Forced degradation studies demonstrated immediate susceptibility to peroxidation, consistent with the Fenton reaction [3], rapid degradation under alkaline conditions, and significant photosensitivity, probably related to a photo-Fenton effect [4]. The effect of CuCl₂ was observed exclusively in the formulation matrix, suggesting a metal–excipient interaction. No degradation products were identified. Degradation was reflected by a decrease in the AUC of the [Fe–Bphen] complex.

Conclusion
A stability-indicating HPLC assay method was developed and validated for a hospital stock preparation of ferrous fumarate suspension. The main degradation pathways were identified and will guide the selection of the most appropriate container type and storage conditions. This work is ongoing with a stability study of the in-house developed formulation.

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