Validation of a stability-indicating HPLC-UV method: a prerequisite for conducting a stability study of an oral solution of isavuconazonium

Introduction
Isavuconazole (ISA) is a triazole antifungal agent indicated for invasive aspergillosis and mucormycosis for adults and children over 1 year old. The active substance (ISA) is present as a prodrug (isavuconazonium, ISN) in pharmaceuticals specialties. To date, ISA is available in two forms: 100mg capsules (the 40mg capsule is not marketed) and powder for solution for injection. The development of an extemporaneous oral liquid preparation at 10mg/mL is being considered to meet the needs of children (weight <37kg) and of patients with limited venous access and/or swallowing difficulties. The aim of the study is to develop and validate a stability-indicating HPLC-UV method to enable the stability study of this new extemporaneous ISN preparation.

Methods
The reverse-phase chromatographic conditions are: a C18 gravity NUCLEODUR column (150x4.6mm; 5 μm) maintained at 40°C and a mobile phase composed of a mixture of methanol and a 16mM pH 4.2 phosphate buffer (58-42% v/v) at a 1mL/min flow rate. Detection and quantification are performed at 280nm. Isocratic elution was selected for forced degradation analyses (analysis time of 40min). To optimize the duration of the other analyses, a gradient elusion was developed with an initial isocratic plateau under the same conditions until elution of the ISN, followed by an increase in eluent strength and a return to the initial conditions (analysis time of 16min).
We developed and validated a stability-indicating HPLC-UV method in accordance with ICH Q2(R2) guidelines. Validation consists of verifying linearity, accuracy, precision (reproducibility and repeatability) and specificity through forced degradation (under various stress conditions: acidic, basic, oxidative, thermal, UV).

Results
The stability-indicating method is linear (R²=0.998), precise (repeatability relative standard deviation (RSD) <0.47%, intermediate precision RSD <2.04%) and accurate (relative error between 0.27 and 4.52% across the three control levels).
The forced degradation study revealed degradation products that were well separated from the ISN molecule. ISN degrades rapidly in solution at room temperature, and particularly so under basic conditions. The injectable specialty must be stored in the refrigerator before reconstitution and in the freezer after reconstitution.

Discussion-Conclusion
ISN instability in solution at room temperature, and especially under basic conditions, points toward the formulation of a 10 mg/mL oral suspension expressed as ISA in an acidic suspending agent that remains to be validated. The validated HPLC method will make it possible to study the stability of this new oral formulation of ISN. To our knowledge, no HPLC analytical method for the assay of an oral formulation of ISN has been published.

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