Module 3 - Strategic case studies in practice

2.3.4. Internal Standards

Internal standards with high purity grades should be employed to account for any possible losses during workup or due to the thermal instability inherent to several N- nitrosamines. While analysis with conventional detectors (TEA, FID, UV-Vis, etc.) commonly utilise a synthetic N- nitrosamine (i.e. N- nitrosodiisopropylamine, NDIPA) that does not occur in nature, the typical internal standard for MS detection is an isotopic standard (e.g. NDMA-d 6 or NDEA-d 10 ) that show almost identical physicochemical behaviour during analysis as the analyte of interest. Non-isotopically labelled analogues should not be present in the internal standards to exclude false positive N- nitrosamine determinations by means of MS. Additionally, it should be noted that NDIPA has been found in valsartan of one CEP holder [RIS World-Online (2018)]. Therefore, any internal standard should be carefully chosen. Most of the utilized GC and LC laboratory systems in the OMCL network are equipped with mass spectrometric (MS) devices. The superior properties of GC- or LC-MS devices to provide molecular structure information by simultaneously maintaining highly sensitive detection limits meet the pinpointed analytical criteria and requirements for N- nitrosamine detection. For LC-MS applications, all devices were equipped with atmospheric pressure chemical ionization (APCI) sources to obtain tremendously higher ionization rates for NDMA and NDEA. It is worth mentioning that the ionisation principle used for MS plays a relevant role with regard to sensitivity and detectability. Electrospray ionisation (ESI), commonly used for LC-MS analysis of N- nitrosamines in the years after 2000, can be hampered by ion suppression due to matrix effects [Lee et al. (2013)]. Therefore, APCI (positive mode) is commonly used for analysis of N- nitrosamines nowadays where this effect is less relevant. Table 2.3.6-1 below gives an overview of the currently used methods in the OMCL network and in jurisdictions outside Europe. Details can be found on the website of the EDQM. Most of the laboratories use a direct extraction of the respective drug substance (DS) or drug product (DP) with a subsequent dilution and filtration step. Afterwards, the extracted supernatants are transferred to GC- or LC-MS (partly LC-UV) devices and measured via direct injection (DI). Another common method is GC headspace (HS)-MS dissolving the sample directly in either N- methylpyrrolidine (NMP) or dimethyl sulfoxide (DMSO). These short workup procedures were chosen to minimise any loss due to the volatile character of NDMA and NDEA. Table 2.3.6-1 Published methods of OMCLs to determine NDMA or NDEA. DCM = dichloromethane; DE = direct extraction; DI = direct injection; DMSO = dimethyl sulfoxide; DP = drug product; DS = drug substance; HS = headspace; LLE = liquid-liquid extraction; MeOH = methanol; NaOH = 1 M sodium hydroxide solution; NMP = N-methyl pyrrolidine 2.3.5. Advantages of mass spectrometric detection devices 2.3.6. Currently used methods in OMCLs

EMA/369136/2020

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