Module 3 - Strategic case studies in practice

2.3. Consideration for analytical method development to identify and quantify N-nitrosamines in APIs and finished products This section is based on all available data, including a literature survey on analytical methods for N- nitrosamines and addresses current analytical methods for N- nitrosamines recommended by the OMCL network and EDQM.

2.3.1. Analytical Methods

Since the awareness of the carcinogenic potential of N- nitrosamines, many detection methods have been developed in the field of food, cosmetic, rubber, pharmaceutical/toxicological, and environmental analysis. While the early analytical methods employed had been polarography, spectrophotometry, and thin- layer chromatography (TLC), gas chromatography (GC) with a special chemiluminescence detector (also called thermal energy analyser (TEA)) was commonly used for about three decades. This detector catalytically pyrolysis the N- nitrosamines previously separated by GC. The N-NO bond is cleaved releasing the nitrosyl radical NO• that is separated from organic fragments and other gaseous products typically by cold traps. The nitrosyl radical is then oxidised with ozone leading to electronically excited nitrogen dioxide (NO 2 * ) that decays back to the ground state emitting a characteristic wavelength in the near IR (NIR). The TEA is highly selective and sensitive down to the picomole range. However, organic nitrites, N- nitramines, C-nitroso, nitrates, and inorganic nitrite may also respond, too [Perera (2006)]. Thus, subsequent confirmation is needed to exclude false-positive results. The use of mass-selective (mass-spectrometry, MS) detection in conjunction with GC or (ultra) high- performance liquid chromatography ((U)HPLC) allows analyte-specific detection based on both retention time and structurally specific fragmentation information in conjunction with high sensitivity. Therefore, GC-MS, GC-MS/MS, and LC-MS/MS are nowadays commonly used for analysis of N- nitrosamine in all types of materials. Nevertheless, a recent publication [Kodamantani et al. (2018)] described a special version of the TEA detector in an LC system with a post-column anion exchange module followed by photochemical reactor and chemiluminescence detection (HPLC-AEM-PR-CL) for wastewater analysis looking for N- nitrosamines. Without any pre-concentration a LOQ of about 1 ng/L (depending on the concrete analyte) using 200 µL sample volume (i.e. about 0.2 pg absolute) was reached. The anion exchange module was used to generate hydroxide ions for the photochemical reactor from anions present in the eluate. The method of choice has to guarantee the unambiguous determination of N- nitrosamine in accordance with scientifically recognized guidelines [ICH Q2(R1) (1995); EMEA/CHMP/EWP/192217/2009 Rev. 1 Corr. 2 (2012).]. Due to their physicochemical properties and, in some cases, their low molecular weight, thoroughly performed method evaluation is necessary to discriminate N- nitrosamine from other compounds. Considering specificity and selectivity of the method, identification criteria should be gained during the determination procedure as much as possible to corroborate the presence of N- nitrosamine. Published review articles about recently implemented N- nitrosamine analytical procedures particularly highlight the benefits of combining chromatographic separations with highly sensitive detection methods determining N- nitrosamine in traces [Perera (2006); Wiltschko et al. (1998); Parr et al. (2019)]. Their character as volatile or non-volatile molecules make N- nitrosamines more or less suitable for different chromatographic techniques (LC, GC) . The main candidates of interest, namely

EMA/369136/2020

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