Validation of an assay method for riboflavin hospital capsule preparation

B. Querin, A. Schweitzer Chaput, P. Ober, A. Fauqueur, A. Negbane, S. Cisternino, C. Cotteret
Hôpital Universitaire Necker - Enfants Malades, Paris, France

Vitamin B2 or riboflavin is a water-soluble vitamin that plays a fundamental role in the synthesis of erythrocytes and two cofactors required for the respiratory chain, as well as in growth and tissue repair. Riboflavin is used in the management of rare metabolic diseases such as mitochondrial cytopathies that can lead to unexplained comas in pediatric intensive care. The marketed form of riboflavin (Beflavine®) in tablet has been unavailable due to a marketing stop since October 2019. The availability of hospital preparations in the form of riboflavin capsules is essential to respond to vital pediatric emergencies. The European Pharmacopoeia requires control of the active ingredient content of hospital preparations as part of the release control. The aim of this study is to develop an analytical method for the assay of 50 mg riboflavin capsules by high performance liquid chromatography (HPLC).

Riboflavin capsules are composed of pharmaceutical grade riboflavin powder (Inresa®) combined with mannitol (Inresa®). A Thermo Scientific® Dionex Ultimate 3000 HPLC system with diode array detector (column: Agilent Polaris C18-A 250x4.6 mm, 5µm), and its Chromeleon® software were used. The mobile phase is composed of 97 mM citrate buffer (pH 3) and acetonitrile (85:15) with an isocratic flow rate at 1 mL/min. The quantification wavelength was set at 268 nm and the injection volume is 20 µL.
Analytical validation is performed according to the guidelines of the International Council for Harmonisation. A 5-point concentration range between 80 and 120 µg/mL (target concentration: 100 µg/mL), is performed at 3 different days and associated with quality controls (QC): QC1 = 85 µg/mL, QC2 = 100 µg/mL and QC3 = 115 µg/mL. Mean recovery rates for accuracy, coefficients of variation (CV) for repeatability, limits of detection (LOD) and quantification (LOQ) are determined.

The method is linear (r² 0.99), accurate (repeatability < 2%; intermediate fidelity CV < 2%) and precise (relative error < 2% on the different control levels). No matrix effect was found. The mean recoveries of QC1, QC2 and QC3 are 101.3% (± 0.1), 99.8% (± 0.1) and 99.8% (± 0.1), respectively. The CV of the repeatability for QC1, QC2 and QC3 are 1.7%, 1.1% and 1.2%, respectively. The LOD and LOQ are 10 µg/mL and 29 µg/mL, respectively.

The HPLC method developed allows the control of the uniformity of content required by the European Pharmacopoeia. It enables the preparation of riboflavin capsules in advance for immediate availability to health care services.

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