Quantification of Genotoxic Impurities in Active Pharmaceutical Ingredients

The presence of genotoxic impurities (GIs) within pharmaceutical products has been a subject of considerable recent interest following the issuance of guidelines from European [1,2] and US [3] regulatory agencies These guidelines typically mandate routine control of both documented GIs and potential genotoxic impurities (PGIs) at the parts-per-million level until suitable scientific data are acquired to dismiss the 101 A Brief Introduction to Genotoxic Impurities 294 102 Quantitative Analysis of Genotoxic Impurities 296 1021 Method Development 296 1022 Sample Preparation and Derivatization 298 1023 Gas Chromatography 299 1024 High-Performance Liquid Chromatography 302 10241 Multiple Reaction Monitoring 303 10242 High-Resolution Mass Spectrometry 309 1025 Ion Chromatography 309 1026 Element-Specific Analysis by High-Resolution Inductively Coupled Plasma-Mass Spectrometry 310 1027 Sulfur, Phosphorus, Chlorine, Bromine, and Iodine Detection 310 1028 Instrumental Limit of Detection 311 10281 Comparison of GC-FID, GC-MS, and HR-ICP-MS 311 1029 Acceptable Elemental Limits Calculation for Genotoxic Impurity Alerts 311 10210 Validation 314 103 Future Outlook and Conclusions 315 References 315 concern, significantly adding to the number of compounds impacted by these guidelines [4-6] This chapter describes the challenges surrounding the trace analysis of GIs; however, the majority of the principles presented are similarly applicable to the analysis of PGIs Responding to this new GI trace analysis mandate has required considerable innovation in the creation of new methods for pharmaceutical synthesis, purification, and analysis In particular, the accurate and reproducible trace analysis of GIs has been especially challenging, as many of the traditional methods used for routine pharmaceutical analysis are simply not suitable for GI analysis at the parts-per-million level This problem is further exacerbated by the fact that many GIs owe their potential genotoxicity to an ability to chemically react with DNA This propensity toward reactivity renders these GIs extremely difficult to analyze, as samples can easily degrade under standard sample preparation or analysis protocols Nevertheless, analytical chemists within the pharmaceutical industry and academia have risen to the challenge, creating new methods and approaches for accurately measuring GIs-a vital first step to inform the development of a suitable control strategy Excellent reviews of current GI methods based on alerting functional groups in the chemical structure of analytes have been published [7-12] In this chapter, we survey the state of the art of this rapidly evolving analysis field, pointing out a number of current challenges, existing solutions, and still unmet needs All active pharmaceutical intermediates (APIs) will always contain impurities as a result of the imperfect nature of chemical reactivity Typically, these API impurities originate from incomplete consumption of starting materials and intermediates in chemical reactions, formation of by-products, or decomposition of reagents and intermediates Among this host of potential impurities, a few known as GIs are of special concern owing to their ability to react with DNA and cause genetic mutations Additionally, genotoxins may also produce their effects by interfering with DNA replication, potentially leading to tumor development or other major health problems When this damage can be transferred from cell to cell, or from generation to generation, the compounds are said to be mutagenic [5] At very low levels of exposure, protective mechanisms can sometimes “correct” DNA damage, resulting in threshold effects [13] However, demonstration of such a threshold of activity for genotoxic compounds is very difficult; hence, for most compounds a linear dosereactivity relationship is assumed To ensure the safety of patients and healthy volunteers in clinical trials for developmental new drugs, the levels of impurities present in APIs must be controlled within appropriate limits International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) guidelines Q3A and Q3B were collaboratively created by industry and regulators to address control of impurities in APIs and drug products, respectively [14,15] These guidelines define the reporting, identification, and qualification thresholds of impurities in APIs and final drug products based on the projected clinical dose, route of administration, and additional mitigating factors These guidelines recognize that the ICH-defined impurity thresholds are not appropriate guidance for impurities that are “unusually toxic” such as GIs To address this shortcoming, the Committee for Proprietary Medicinal Products of the European Medicines Evaluation Agency released a guidance document in 2007, which defined a new framework to address GIs in APIs [1] The EMEA guidelines recommended that the structures of impurities detected in APIs, as well as those highly likely to form in APIs via chemistry arguments, are assessed in silico for PGI structural alerts with respect to an established public access database The genotoxicity of these identified PGIs can be confirmed using a suitable bacterial reverse mutation test like the Ames test, but long-term in vivo studies in animals are required to determine whether a compound is a carcinogen [5,16,17] For compounds with sufficient evidence for a threshold-related mechanism, limits can be calculated following the “permitted daily exposure” (PDE) approach as per ICH guideline Q3C for residual solvents [18] This approach calculates a PDE derived from the “no observed effect level” or the “lowest observed effect level” For compounds without sufficient evidence for a threshold-related mechanism, the EMEA guideline recommends that all due diligence is performed to avoid the presence of genotoxic compounds in an API, either by eliminating the particular reagent of concern from the synthesis or by using alternative synthetic processes to avoid the formation of the GI This approach can often be difficult to implement, as the highly reactive nature of many synthetic intermediates and reagents (eg, benzyl bromides and epoxides) is essential to their function in the chemical synthesis, but is often also the underlying cause of their genotoxicity During early development of a new API, where understanding of chemical processes and impurity control are still relatively limited, such efforts are further complicated and become impractical In cases where the presence of the GI cannot be avoided, levels should be reduced using reaction workup steps and/or purification technologies [5,19] To establish acceptable levels of GIs in APIs, the EMEA guidance based its approach on a previous approach used by the Food and Drug Administration (FDA) to establish acceptable levels of contaminants leaching from food packaging [20], renaming it the “threshold of toxicological concern” (TTC) The TTC approach was established so as to set acceptable limits for GIs in APIs, which will not expose patients to a significantly increased risk of developing cancer during a lifetime of taking a given medication Based on the carcinogenic potency in rodents of over 700 carcinogens, exposures of less than 015 µg/day were estimated to be a “virtually safe dose,” unlikely to increase a lifetime cancer risk by more than 1 in 106, for all but the most potent carcinogens Using this rationale, the EMEA recommended a limit of 15 µg/day for chronic exposure to a GI/PGI, representing an excess lifetime cancer risk of 1 in 105 This small level of added patient risk was justified by the significant positive health benefits received by the patient taking the medicine The acceptable concentration of the GI in the API is calculated based on the expected dose as follows: ( ) ( )( ) = µ Concentration limit ppm TTC g day Dose g day (101) The EMEA guidance acknowledges that lower TTC values should be used for compounds of high potency and that higher TTC values can be justified for shortterm exposure The EMEA guidance does not provide specific guidance for the limits of PGIs in investigational APIs during shorter duration clinical trials, leaving its applicability to development somewhat open to interpretation To address this issue, the Pharmaceutical Research and Manufacturers of America (PhRMA) proposed a staged TTC approach, which extrapolated the allowable daily intake (ADI) for GIs from lifetime levels [21] This approach targeted a 1 in 106 level of added patient risk for early clinical trials less than 1 year in duration, since healthy volunteers do not receive any benefits from exposure to the API For longer exposures, the risk is kept as 1 in 105 as only patients receiving a benefit from exposure to the API are likely to be used in such long-term clinical studies The ADIs were therefore set as shown in Table 101 The staged TTC approach was adopted by the EMEA in 2010 [2] The daily allowable levels were calculated in a way similar to what was proposed by the PhRMA group, but a factor of 2 was introduced to account for deviations from the linear model A similar TTC approach was also considered acceptable by the FDA in a draft guidance document in 2008 [3] Trace level (parts-per-million) quantitation of GIs presents the pharmaceutical analytical chemist with many technical challenges First, an analytical technique appropriate for the properties of a GI (volatility, thermal stability, presence of a chromophore, hydrophobicity, etc) must be selected as the basis for the analysis method to be developed Second, the reactive nature and stability of the GI must be adequately addressed during method development to ensure that requisite reproducibility and accuracy are achieved Third, the clinical dose and duration of the study must be understood well to guide the development of an adequately sensitive analysis method meeting project needs The targeted allowable GI limit will directly impact the selection of critical parameters of the analysis method to be developed, including choice of detection technique (ultraviolet [UV], light scattering, electrochemical detection, mass spectrometry, etc) Additionally, certain components of the sample matrix may also present substantial method development challenges due to analytical interference from the API itself, process impurities, or degradation products The elimination of these interfering matrix components is typically achieved by (1) isolation of the analyte of interest by sample preparation, (2) chromatographic resolution, or (3) using a more selective detector A wide range of analytical techniques can be used to analyze GIs, depending on the properties of the analyte Due to the high structural diversity of GIs, and the complexity of the sample matrix, no single approach is applicable to address all problems However, analytical laboratories within the pharmaceutical industry and academia have developed systematic strategies to guide GI method development, which have proved to be quite useful [5,6,11,22-25] The majority of these strategies TA B LE 1 0. 1 D ai ly A llo w ab le L im it s fo r G Is a s Sp ec ifi ed b y Ph R M A ,E M EA ,a nd F D A consist of the following two steps: (1) evaluation of the volatility of the analyte, which typically informs the choice of chromatographic technique to be used, and (2) evaluation of the detection technique based on the properties of the analyte (eg, presence of a chromophore and presence of a halogen atom within the molecule) Traditionally, API impurity analysis in pharmaceutical laboratories has been carried out using high-performance liquid chromatography-ultraviolet (HPLC-UV) for nonvolatile compounds and gas chromatography-flame ionization detection (GC-FID) for volatile compounds; therefore, method development for GIs also typically begins from a chromatographic analysis dictated in large part by analyte volatility For low parts-per-million detection of GIs, the use of nonspecific detectors such as flame ionization detection (FID) or UV may not be feasible owing to insufficient detector sensitivity and artifacts produced by minor interferences from the sample matrix Hyphenated mass spectrometry techniques such as gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS) have gained popularity in GI analysis due to their superior sensitivity and selectivity [25] For GC-MS, both electron ionization (EI) and chemical ionization ionization modes have been employed, with EI being the more popular approach [24,26] Ionization modes for LC-MS include electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) For some nonpolar and low-polarity compounds that are not efficiently ionized by either ESI or APCI, atmospheric pressure photoionization (APPI) may be used Selectivity is usually improved by conducting selected ion monitoring (SIM) on a single quadrupole mass spectrometry (MS) or multiple reaction monitoring (MRM) on an MS-MS instrument LC-MS-MS methods are typically used in the analysis of very complex samples where masses are likely to overlap despite the high resolution of the MS instrument MRM mode provides a better signal to noise (S/N) ratio and reduced baseline offset as compared to SIM mode, resulting in better sensitivity and selectivity [24] For situations where the chromatographic resolution of the analyte from the sample matrix is not sufficient for the development of a suitably sensitive and selective method, sample preparation is critical Several extraction/preconcentration techniques to isolate and/or concentrate the analyte of interest from the sample matrix have been reported In particular, techniques relying on the partitioning of the analyte of interest between two phases to enrich one of the phases in the analyte have been investigated The simplest of these techniques, liquid-liquid extraction (LLE), where the analyte is portioned between two immiscible liquids, often requires an additional concentration step before analysis Liquid-phase microextraction (LPME) uses a capillary hollow membrane filled with microliters of an extracting solvent, which is introduced in a liquid sample prepared in an immiscible solvent The analyte is concentrated in the extracting solvent, which typically can be directly analyzed via chromatography Additionally, extraction can be to the analyte of interest and other sample components between a and a liquid phases are to including a large of and ion phases alternative to microextraction is a extraction technique in which a is to a gas or liquid sample The analyte is between the sample matrix and the and at a high in a gas chromatography or in a solvent to analysis by or These techniques can be to the analysis of GIs but typically method and to address matrix effects before routine to samples of often these techniques difficult to between samples Derivatization may be used to analyte properties by volatility or stability, or the extraction However, development of requires of the reaction parameters solvent, etc) to stability of the and selectivity of the reaction A that the for a given of GIs can be used, as in the method reported by and where the is from this the shown in the for the of or of In this a understanding of the impurities that may be present in the sample is required to ensure that the from the analysis can be used to the level of GIs present in the sample Several in have been to during the sample required for the sample preparation include the in of or of reagents of the parameters can be very complex as must be suitable for both the and the with is used in the analysis of volatile GIs For were analyzed in a drug using a method with a limit of quantitation of ppm in the drug the GI has sufficient or is usually to the of the or by such as APIs [25] an a published the analysis of the in of drug using Development of a method requires of the parameters solvent, and duration of but the resulting method can often be to a range of similar such as In cases where the GI is not volatile for techniques may be used In such of the is as the of API impurities and thermal degradation products can with GI quantitation In the thermal stability of GIs must be assessed and for to quantitation For GIs a halogen such as and benzyl electron detectors have been investigated due to the halogen selectivity of these detectors This technique is useful due to its no other of the sample matrix halogen but the sensitivity of the approach typically that of for compounds The sensitivity of detection can significantly depending on the nature and number of halogen present on the hence, most published methods for MS detection The use of a specific also known as a specific has also been reported for the analysis of trace in to the with have been used in the quantitation of volatile GIs other detectors do not the required sensitivity and are used in the synthesis of active pharmaceutical as or as to form to or control pharmaceutical properties in solvent can potentially to the formation of which are GIs [21] the presence of other (eg, in solvent can to the formation of which are also considered GIs [21] These volatile and small have been analyzed using very sensitive methods such as the one shown in 102 This method an and used in the SIM mode can detection limits as low as 1 ppm Excellent and and values have all been reported has also been used for the quantitation of at levels as low as ppm In one approach developed by and was first to an by in with which was analyzed by parts-per-million levels of and have all been analyzed by as well is used as a technique for the analysis of nonvolatile GIs The most used mode is For extremely liquid chromatography can be used to sufficient in with low a that is to form a at the of the is achieved by partitioning the analytes between the and the the The use of has been reported for the analysis of which were either compounds of interest or products and For the detection of analytes a chromophore, the UV detector is the first choice for and In particular, liquid chromatography-ultraviolet techniques can be very useful to control GIs during the early of developing an API where clinical studies are shorter in duration and GI may be and not quantitation at low parts-per-million limits However, UV detection is often sensitive selective to low parts-per-million levels of detection a detectors such as light detectors or detectors may be used, as to the analysis of carcinogenic in products However, the of using or for the analysis of GIs in APIs are quite owing to the of and/or selectivity for GI analysis major of is that the can be highly on analyte volatility and is also on analyte volatility is more popular due to its more wide and The use of a detector has also been reported for the analysis of two GIs, and as shown in 103 LC-MS and LC-MS-MS have been used in of nonvolatile GIs UV or do not the required sensitivity or can often be easily detected by atmospheric pressure ionization LC-MS in the positive ion mode due to their high analytes can often be detected in the ion mode via with groups are better ionized by mode than ESI [24] For ion can be used to and in the positive ion mode [24] can also be efficiently used to and analytes For very reactive the compound of interest is often to a easily ionized to analysis analysis of GIs such as and by in SIM mode has been reported [25] and can be with and the can be analyzed by LC-MS using ESI in the ion mode [25] the analysis of 1 ppm of and [25] and are highly sensitive techniques for the analysis of but many of the have very high and sensitivity LC-MS has been as a highly sensitive analytical with limits of detection less than ppm being easily 105 the and of at 1 ppm concentration with respect to the API The MS SIM in the positive ion electrospray mode, monitoring and of The between SIM at appropriate in the chromatographic to the all be a specific ion by the of the instrument and the sensitivity and are observed Multiple Reaction Monitoring the of and quadrupole mass are uses in the of GIs MRM the of compounds based on their a known as a This highly selective detection technique can interference from chromatography and significantly signal to and therefore detection limits quadrupole mass can be to both and and in both cases can the analysis of GIs in complex In the and observed can be used as of similar In we the selectivity and sensitivity of a quadrupole mass an MRM A number of were detected to per levels with a very chromatographic In the was that with the of the an of such a is shown in 106 A further of the MRM the of a The method was only of a GI detection limit of with a API concentration of 2 Monitoring the using an MRM from to using a mass the detection limit to be reduced to 1 ppm with very method development The in selectivity significant in chromatographic was no ion with In this a than was achieved in a methods for have been using as a The reaction is shown in The nature of the is such that any potential reactive will be This of the sample preparation that all potential in the sample matrix will react and therefore be not those targeted from a of the synthetic route The resulting sensitivity in and can be easily from the sample matrix using The of a number of GIs are shown in Table 102 with the and can be using a ion or a of which is of the the of and or a of can be assumed to have with the and are a potential The to this approach is that with the but cannot be since the resulting are However, of the synthetic process a structure to be proposed TA B LE 1 0.