Study of the microbiological stability and preservative efficiency of a 2 mg/mL hydrochlorothiazide oral suspension in Inorpha^®

Introduction
Hydrochlorothiazide (HCTZ) is a diuretic used in pediatrics for which no suitable commercial form exists. The CHSF pharmacy plans to make drinkable preparations, facilitating dosage adjustment. We selected a 2 mg/mL oral suspension in Inorpha^®, which has shown 3-month physico-chemical stability at 4±2°C¹ and 2 months at 20±2°C².

Objective
The study aimed at assessing the microbiological stability and antimicrobial preservative efficiency of the 2 mg/mL HCTZ oral suspension in Inorpha®, to establish its appropriate shelf life and ensure safety in clinical use.

Methods
Method applicability was evaluated to determine the smallest dilution neutralizing potassium sorbate, the preservative in Inorpha^®. Multiple dilutions were tested for microbial enumeration (Ph.Eur 2.6.12) and specified microorganism (MO) detection: E.coli (Ph.Eur 2.6.13). The microbiological stability study was then conducted on 6 batches (30 bottles): half bottles stored at room temperature (25±2°C) and half refrigerated (4±2°C). Microbial enumeration and E.coli detection were performed at Day 0, Day 21, Month 2, and Month 3 on 3 batches of unopened bottles (stability before opening) and 3 batches opened at Day 0 (stability after opening). Daily sampling by oral syringe simulated real use of the opened bottles. Lastly, antimicrobial preservative efficiency (Ph.Eur 5.1.3) was evaluated on 5 bottles of oral suspension by inoculating each with 5 different MO strains, with results evaluated at D0, D14, and D28.

Results
The smallest dilution, 1/10th, was validated during the applicability test, showing less than a twofold difference in colony-forming units (CFU) between the diluted samples and positive controls for the recommended MOs. Microbiological quality (Ph.Eur 5.1.4) was maintained for 3 months, both before and after opening, under both tested conditions. At Day 0, 17 CFU/mL were observed in opened bottles stored at 25±2°C, and 33 CFU/mL in unopened bottles stored at 4±2°C, both corresponding to B.subtilis. CFU counts remained below Ph.Eur limits (200 CFU/mL for bacteria and 20 CFU/mL for fungi), with asbsence of E.coli throughout the study. The antimicrobial preservative efficiency test demonstrated significant logarithmic reductions, exceeding expectations (>3 for bacteria and >1 for fungi at Day 14, with no increases observed at Day 28). Consequently, the test has been validated.

Discussion-Conclusion
The concentration of potassium sorbate in Inorpha^® provides effective antimicrobial preservation. The HCTZ oral suspension demonstrates robust microbiological stability, remaining viable for 3 months at both 25±2°C and 4±2°C, before and after opening. This evidence supports routine validation of a shelf life of 3 months before and 2 months after opening for bottles stored at 4±2°C.

¹ Mise au point d’une SB d’hydrochlorothiazide, Dr ROBIN, Poitiers 2014
² Stabilité d’une suspension d’HCTZ à 2mg/ml, Laure NAPOLY, APHIF 2014

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