Forced degradation of fentanyl: Identification and analysis of impurities and degradants

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Abstract

Fentanyl, N-(1-phenethylpiperidin-4-yl)-N-phenylpropionamide is a rapid-acting, powerful opioid analgesic used extensively for anesthesia and chronic pain management. A forced degradation study of fentanyl active pharmaceutical ingredient (API) was performed using light, acid, base, heat and oxidation. Under acidic conditions, fentanyl was shown to degrade to N-phenyl-1-(2-phenylethyl)-piperidin-4-amine (PPA1). Fentanyl was stable to light exposure and base treatment with no degradation observed. Oxidation with hydrogen peroxide produced fentanyl N-oxide by rapidly oxidizing the nitrogen on the piperidine ring. Five degradants were formed during thermal degradation of fentanyl. The two known degradants included propionanilide (PRP2) and norfentanyl (NRF3). The three unknown degradants were first identified by mass using LC/MS, and postulated compounds were synthesized and confirmed by LC/MS and 1H NMR. These degradants were identified as 1-phenethylpyridinium salt (1-PEP4), 1-phenethyl-1H-pyridin-2-one (1-PPO5), and 1-styryl-1H-pyridin-2-one (1-SPO6). In addition to the seven degradants, three known process impurities, acetyl fentanyl, pyruvyl fentanyl and butyryl fentanyl were also detected by reverse-phase high performance liquid chromatography (HPLC) with UV detection. All degradants and impurities were identified and confirmed using authentic materials. Method validation was performed for the assay of fentanyl and its related compounds in accordance to ICH guideline Q2(R1), and the method was demonstrated to be specific, linear (r > 0.999 for fentanyl assay and r > 0.996 for related compounds), accurate (recovery > 99.6% for fentanyl assay and recovery > 91.0 for related compounds), precise (%RSD < 0.8% for fentanyl assay and <4.8% for related compounds), sensitive (limit of detection = 0.08 μg/mL or 0.016% of nominal concentration), robust and suitable for its intended use. The chemical structures for the degradants and impurities were submitted to three in silico toxicity programs to identify any structural alerts.

Introduction

Fentanyl is a controlled substance and has been categorized as a Schedule II drug under the “Controlled Substance Act”. Forced degradation samples produced seven degradants, phenethylpyridinium salt (1-PEP), norfentanyl (NRF), propionanilide (PRP), N-phenyl-1-(2-phenylethyl)-piperidin-4-amine (PPA), 1-phenethyl-1H-pyridin-2-one (1-PPO), fentanyl N-oxide, and 1-styryl-1H-pyridin-2-one (1-SPO). In addition, three known process impurities, acetyl, pyruvyl and butyryl fentanyl were also monitored. Structures for fentanyl degradants and process impurities are depicted in Table 1.

Previously, Rabinowitz et al. [1] compared the purity of fentanyl starting material (>99%) to fentanyl powder heated on a hot plate at 300 °C, which showed approximately 30% degradation. Two major and three minor peaks were observed in the chromatogram for the fentanyl material heated on a hot plate, but no identification was provided.

A forced degradation study on fentanyl was reported by Lambropoulos et al. [2] using light, acid, base, heat and oxidation. PPA formation was reported under acidic conditions, but unknown peaks generated by light, base, heat and oxidation were not identified. Chen et al. [3] developed an analytical method for the quantitation of PPA, which was suggested as a potential genotoxic compound due to the aniline moiety in the structure. The European Pharmacopoeia (EP) has designated fentanyl impurities by letters [4], where A: N-oxide, B: NRF, C: acetyl, D: PPA, E: benzaldehyde, F: aniline, and G: PRP. EP also describes a fentanyl HPLC method where acetonitrile and ammonium carbonate (pH 9.0) is used with tetrahydrofuran as an additive. The EP method has low sensitivity since fentanyl is practically insoluble at pH 9.0. No other information on forced degradation of fentanyl was found in the literature.

There is an increased interest in the identification and control of potentially genotoxic impurities. In 2007, the European Medicines Agency (EMEA) issued guidelines on the limits of genotoxic impurities [5], and the Food and Drug Administration (FDA) issued draft guidance on the same subject in December of 2008 [6]. Because of the structure of fentanyl, and the possibility of generating aromatic amines or amine derivatives via degradation, we identified the impurities resulting from common degradation pathways, and validated a stability-indicating method to separate fentanyl from its process impurities and degradants.

Section snippets

Materials

Fentanyl base was purchased from Mallinckrodt (Hazelwood, MO, USA). USP standard fentanyl citrate was used for HPLC quantitation. All impurities and degradants including 1-PPO, 1-SPO, 1-PEP, PRP, NRF, acetyl, pyruvyl and butyryl fentanyl were synthesized and qualified as standards at Alexza Pharmaceuticals Inc. PRP and NRF were also available from Sigma Aldrich (St. Louis, MO, USA). HPLC grade acetonitrile from J.T. Baker (Phillipsburg, NJ, USA) was used in the mobile phase. HPLC grade methanol

Forced degradation studies

The forced degradation results for fentanyl API are summarized in Table 1. The structures of fentanyl impurities and degradants are shown in Table 2. For each set of samples, a methanol blank, resolution solution (see Fig. 1) and fentanyl API controls were injected. The peak purity angles of the fentanyl in the fentanyl API controls were much less than the thresholds and the mass spectra across the peaks were homogenous. This demonstrated that the fentanyl peak was pure and free of co-eluting

Conclusions

A forced degradation study for fentanyl was performed using light, acid, base, and oxidation degradation on fentanyl API. Fentanyl was very stable to light and base treatment, no degradation was seen. Oxidation selectively produced diastereomers of fentanyl N-oxide. Acid degradation exclusively generated PPA. Thermal degradation produced the degradants 1-PEP, NRF, PRP, 1-PPO, and 1-SPO. Unknown degradants 1-PEP, 1-PPO, and 1-SPO were synthesized and identified using LC/MS and 1H NMR

Acknowledgements

We wish to acknowledge the Analytical Research and Development and PK/ADME groups at Alexza Pharmaceuticals Inc. We also thank Glenn Myatt from Leadscope for providing the output from the Leadscope program; and Pranas Japertas and Kiril Lanevskij from ACD/Labs for providing the output from the ACD/Tox Suite and discussions on the results.

Supporting Information Available

Synthesis information for fentanyl related substances is available from the corresponding author.

References (12)

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Parts of this work were accepted to be presented in poster form at AAPS Annual Meeting 2009.

1

N-Phenyl-1-(2-phenylethyl)-piperidin-4-amine.

2

Propionanilide.

3

Norfentanyl.

4

1-Phenethylpyridinium salt.

5

1-Phenethyl-1H-pyridin-2-one.

6

1-Styryl-1H-pyridin-2-one.

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