Development and validation of a stability-indicating analytical method for evaluating the physico- chemical stability of amoxicillin/clavulanic acid capsules for oral provocation tests

Introduction
Amoxicillin (A) and clavulanic acid (CA) are among the antibiotics associated with a high prevalence of suspected hypersensitivity reactions. For diagnostic purposes, oral provocation tests (OPTs) are performed in the hospital. Currently, capsules are prepared by the hospital pharmacy with a beyond-use date of one month (M) (extemporaneous preparations from crushed 500 mg/62.5 mg A/CA tablets and lactose monohydrate, in size 2 non-opaque blister packs). Driven by the increasing demand, the clinical unit requested the development of a hospital-compounded preparation for floor stock.

The objective of this work was to develop a stability indicating method for both active pharmaceutical ingredients (APIs) and to evaluate the physico-chemical stability of the capsules when stored at room temperature and protected from light.

Materials and Methods
The stability-indicating method for both APIs was developed using a forced degradation study by high-performance liquid chromatography coupled with a diode array UV detector (HPLC-DAD), using a C18 column (100x2.1 mm, 2.6 µm) and a mobile phase consisting of 25 mM phosphate buffer at pH 2/MeOH (99/1, % v/v). Validation was performed according to the SFSTP and ICH Q2 guidelines. Accuracy profiles were established for each API. Physico-chemical stability was evaluated for two formulations (A/CA: 100/12.5 mg and 10/1.25 mg). The API content (mean ± standard deviation) and the presence of degradation products were assessed at Day (D) 0, D7, D14, M1 and M2.

Results
The validated 20-min method allowed the separation of CA (tR = 2.8 min), A (tR = 9.9 min) and their potential degradation products. No changes in the first derivative spectra at the tR of CA and A were observed during the forced degradation study, confirming the selectivity of the method. To prevent the hydrolysis of CA in an acidic medium while ensuring the solubilization of amoxicillin, the pH of the solubilization medium (25 mM phosphate buffer/MeOH 99/1 (% v/v)) was adjusted to 5.5. Due to the A/CA ratio (10 :1), specific dilution steps were required after capsule dissolution to ensure that the concentration fell within the respective calibration ranges. This allowed proper detection while preventing signal saturation. At M2, the 100 mg (A)/12.5 mg (CA) capsules remained stable (100.77 ± 4.20 %/101.72±4.05%), whereas instability of CA was demonstrated for the 10 mg (A)/1.25 mg (CA) capsules (97.28±4.10%/82.35±1.60%).

Discussion/conclusion
The developed stability-indicating analytical method demonstrated instability at M2 for the 10 mg (A)/1.25 mg (CA) capsules, the cause of which remains to be determined. Stability monitoring of the 100 mg (A)/12.5 mg (CA) capsules will be continued at M3. This study highlights the importance of assessing the stability of compounded preparations containing labile active ingredients.

Keywords: Drug stability, Amoxicillin-Potassium clavulanate Combination, High Performance Liquid Chromatography

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