Development of a stability-indicating assay and study of the physico-chemical stability of Etoposide phosphate in a 0.9% sodium chloride solution

Introduction
Macrophage activation syndrome (MAS) is a rare but life-threatening complication of some haemopathies, characterised by tissue infiltration by activated macrophages. The gold standard treatment is intravenous administration of Etoposide phosphate (EP), a topoisomerase II inhibitor, at 150 mg/m². Managing this therapeutic emergency requires the immediate availability of EP, which must be previously reconstituted and diluted, without any risk of contamination for staff or the environment. One way of dealing with this is to set up standard doses.
The aim of this work is to design a stability-indicating dosing method, and to test the physico-chemical stability through time of a solution of EP at 1 mg/mL in bags of 0.9% sodium chloride, in order to have standard doses available at any time.

Methods
A stability-indicating method using reverse-phase high-performance liquid chromatography coupled with a diode-array UV detector was developed and validated in accordance with ICH Q2 R1 guidelines. Forced degradation tests were carried out under acidic, alkaline, oxidative and thermal conditions. The EP, after reconstitution according to the Product Characteristics Summary, was diluted in a 0.9% polyolefin sodium chloride bag at a concentration of 1 mg/mL. A stability study was carried out in two storage conditions: 25°C ± 3°C (n=3) and 4°C ± 3°C (n=3). The EP content, pH and organoleptic properties were monitored at D3, D7, D10, D14, D21 and D28.

Results, discussion
The method is linear (R² = 0.999), precise (repeatability <1%; intermediate reliability CV <2%), and accurate (relative error <5% on the different control levels). The mobile phase consisted of pH 3.2 phosphate buffer (80%) and acetonitrile (20%) with a flow rate of 1 mL/min, and an injection volume of 20µL. The wavelength of detection and quantification was set at 210 nm. Etoposide, the active metabolite of EP, was detected at a distinct retention time (T2 = 10.5 min versus T1 = 5.2 min for EP).The forced degradation assay showed significant degradation in a basic environment and moderate degradation in an acidic environment, without interference from degradation products. The stability study showed a variation in EP content below 5% at room temperature and 4°C for 3 days. No modification of pH or organoleptic properties (visual examination, color) was observed. This study is still ongoing.

Conclusion
The EP injectable solution diluted in a 0.9% sodium chloride polyolefin bag to 1 mg/mL is stable for 3 days at room temperature and +4°C. Continuation of the study over an extended period will allow us to determine if the stability of EP is high enough for standard doses to be used in the rapid management of MAS.

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