Development and validation of an assay method for Tramadol and Nefopam in solution to study the stability of experimental drugs

The Drug Preparation Unit was asked to manufacture treatment units (TU) for an institutional clinical study evaluating multimodal analgesic management with Tramadol (T), Nefopam (N), Ketamine (K) and Remifentanil (R) in mechanically ventilated intensive care patients. Inclusion must be carried out within 24 hours of intubation, and the treatment duration was 72 hours, either a production of 43 TU per patient. In view of the time required for inclusion and the production volume, the manufacture of kits for 72h of treatment was envisaged. Stability data in the literature at the desired concentrations for K and R were sufficient (over 14 days), unlike those for N and T (N 48h ¹ / T 14 days ²). Thus, the aim of this work was to develop and validate a common stability-indicating assay method for solutions of T (8.33 mg.ml-^-1), of N (2.5 mg.ml-^-1), and of the mixture of T and N (0.943- 0.377 mg.ml^-1) in 0.9% sodium chloride solution.

The analytical method developed was performed by HPLC: Gravity C18 column (152x4.6 mm, 5µm). The mobile phase consisted of 80% phosphate buffer (pH 3) and 20% acetonitrile, with a pump flow rate of 2 ml.min-1. The injection volume was 10 µl. A calibration range of T and N in mixture was prepared : T (0.6 à 1.35 mg.ml^-1) and N (0.125 à 0.5625 mg.ml^-1). Six replicates of the assay for each product (T, N and the T-N mixture) were performed to assess repeatability. Subsequently, 6 runs with alternating manipulators between each run (1 run per day, 3 separate manipulators) were used to validate intermediate fidelity. Forced degradation (FD) tests were carried out to verify the detection of any degradation products : acid (1N HCl, 60°C, 4h) and basic (1N NaOH, 60°C, 4h) hydrolysis oxidation (3% H2O2, 60°C, 4h), UV light (365 nm, 4h).

Retention times (RT) for T and N were 6.39 min and 18.3 min respectively. The analytical method proved linear over the established calibration range, with correlation coefficients R > 0.999 for N and T. The repeatability parameters (CV 0.04% for T and 0.07% for N) and intermediate precision (CV 0.60% for T and 0.37% for N) were validated. DF tests revealed the sensitivity of the 2 molecules to acid hydrolysis: 2 degradation products (DP) were observed for T with relative retention times (RRT) of 0.79 and 4.99, and 1 for N (RRT 0.65). Under oxidizing conditions, PD were found with a TRR of 1.18 (T) and 1.16 (N). The other DF tests didn’t show PD.

The stability indicating dosing method has been validated in accordance to recommendations of GERPAC ³ guide. The development of a common method for the simultaneous determination of T and N, for a stability study, is an asset, enabling us in particular to optimize handling time.

¹ D’Huart. et al. Etude de stabilité physico-chimique d’une solution de néfopam et d’un mélange néfopam-dropéridol dilués en seringues polypropylène pour les services de soins intensifs. Hopipharm, 2019.
² Gu J. et al. Long term stability of Tramadol and Ketamine solutions for patient-controlled analgesia delivery. Med Sci Monit, 2015.
³ SFPC et GERPAC. Guide méthodologique des études de stabilité des préparations. 1ère édition, avril 2013.

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