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Trends in Analytical Chemistry, Vol. 25, No. 8, 2006

Analytical methodologies for discovering and proling degradation-related impurities

Steven W. Baertschi
The analytical technologies currently in use for proling degradation-related impurities (DRIs) are powerful, but not without their limitations. This article critically assesses the technologies, their strengths and limitations, and recommends strategies for successful DRI proling. Improvements in analytical separations and detection technologies are leading to strategies involving multiple orthogonal separations and detectors. Advances in knowledge of degradation chemistry and in computer-prediction tools are needed for the future. 2006 Elsevier Ltd. All rights reserved.
Keywords: Analytical technology; Chemistry-oriented; Degradation pathway; Degradation product; Degradation-related impurity; Detector; DRI; Forced degradation; Impurity; Method development; Profiling; Stability; Stability indicating; Stress testing; Structure elucidation; Technique-oriented

of a conductor leading an orchestra. It is hoped that the current article will help those laboratories dealing with DRI proling to orchestrate solving impurityrelated problems and perhaps to consider new ways of approaching the purity issues that regularly confront the pharmaceutical researcher.

2. Overall strategy in theory In order to detect accurately and to quantify (i.e., prole) DRIs, a stabilityindicating analytical method is needed. Ideally, such a method should resolve all DRIs from the parent and from each other, and should detect and accurately quantify all DRIs. As outlined by the International Conference on Harmonization (ICH) [4], the process for establishing a stabilityindicating method involves stress testing: Stress testing of the drug substance can help identify the likely degradation products, which can in turn help establish the degradation pathways and the intrinsic stability of the molecule and validate the stability-indicating power of the analytical procedures used. The overall strategy for developing a stability-indicating method using stress testing has been expressed simply (Fig. 1). This strategy involves:  identifying likely or potential DRIs through stress-testing studies using highly-resolving or discriminating methods;  determining which of the potential DRIs are relevant (i.e., those DRIs that form during accelerated stability and longterm stability studies);

1. Introduction
Steven W. Baertschi* Analytical Sciences Research & Development, Eli Lilly and Company, Indianapolis, IN 46285-3811, USA

Tel.: +1 317 276 1388; Fax: +1 317 277 2154; E-mail: Baertschi@Lilly.com

There is currently a tremendous interest in proling of impurities in drugs, as evidenced by numerous conferences, presentations, articles and books [13] devoted to this topic, including this issue of TrAC. In this article, the focus is on the topic degradation-related impurity (DRI) proling in drugs. While the distinction between proling of DRIs and process impurities may not seem signicant from the viewpoint of analytical methodology, the strategies employed to develop DRI methods and to solve DRI problems can be quite different from analogous strategies for processrelated impurities. There are a large number of specialized analytical techniques available for the characterization of the purity of drugs, so no one person can be qualied as an expert in all of these techniques. Gorog [1a] has described the process of solving impurity problems via impurity proling as being analogous to the process

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0165-9936/$ - see front matter 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.trac.2006.05.012

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API
API Stress Testing

Drug Product
Formulation Development

Use discriminating methods Identify potential degradation products

and pathways

Use information from stress testing Use discriminating methods Perform drug-excip.compatibility studies Test trial formulations
to aid development

Accelerated Testing

Drug Product Stress Testing

Determine significant degradation products Develop focused methods Identify containers/conditions to

minimize

Use discriminating methods Identify potential degradation products

and pathways not detected in drug substance stress testing

Long-Term Testing

Determine degradation product Develop specifications Establish storage conditions and shelf
life levels

Figure 1. Overall strategy for the prediction, identication and control of stability-related issues.

 developing focused stability-indicating impurity methods (methods that resolve and detect the DRIs relevant to real-world handling and storage conditions); and,  quantifying DRI levels via long-term, formal stability studies, and establishing specications, storage conditions, and shelf-life. These studies are typically performed on both the drug substance and the formulated product.

3. Overall strategy in practice As shown in the four steps described above, the process seems fairly straightforward and deceptively simple. In practice, the details of carrying out the process provide numerous difculties, complexities, and questions that need to be addressed. It is important to consider some of these difculties. 3.1. Identifying likely [or potential] DRIs This step involves stressing samples of the drug substance (alone and in the presence of the excipients in the formulation) under relevant conditions of heat, humidity, photostress (ultraviolet (UV) and visible (VIS)), oxidative conditions, and aqueous conditions across a broad pH range. It also involves choosing the analytical

technique(s), developing and using an appropriate method, and analyzing samples. The rst question that must be answered is: What are the relevant stress conditions and how much stress is enough?. Until recently, there was very little guidance available to aid the analytical researcher in determining the answers to these questions, so stress-testing procedures varied tremendously [5]. The ICH stability guideline provides some limited guidance [4]: The testing should include the effect of temperatures (in 10C increments (e.g., 50C, 60C) above that for accelerated testing), humidity (e.g., 75% relative humidity or greater) where appropriate, oxidation, and photolysis on the drug substance. The testing should also evaluate the susceptibility of the drug substance to hydrolysis across a wide range of pH values when in solution or suspension. Recent contributions from Baertschi et al. [5,6], Alsante et al. [7], Reynolds et al. [8] and Thatcher et al. [9,10] have provided benchmarking information and scientic rationale for the choice of stress conditions and endpoints, and the design of such studies. Since this topic is covered in sufcient detail in these references, no further discussion will be provided here. After the appropriate stressing of samples, questions related to the analysis must be addressed. How do you analyze the samples? What separation technique do you choose? What detector? The inherent dilemma is 759

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how to measure the loss of parent and levels of the DRIs prior to development of a stability-indicating impurity method. There are a couple of approaches generally taken to address this dilemma of measuring DRIs prior to having a stability-indicating method. One approach is to use a generic screening method, where the method may not have been designed or optimized for a particular compound but is designed in general to be highly resolving and broadly applicable [11]. Currently in the pharmaceutical industry, the choice is overwhelmingly gradient reversed phase high-performance liquid chromatography (RP-HPLC) with UV detection (presumably at a low wavelength for more universal detection, e.g., 210220 nm) [12,13]. Further condence in the results of the analyses can be gained by analyzing samples using a second screening method that is orthogonal (i.e., nonoverlapping) to the rst screening method. Use of an orthogonal method increases the likelihood of resolving and detecting all the DRIs. A second approach is to optimize the screening method to be specic for the compound of interest. Thus, after initial analysis of the stressed samples using a screening method, an assessment is then made as to which conditions caused degradation, and the samples from these conditions are used for further method development until the researcher concludes that the method is stability-indicating (i.e., it resolves and detects all the major DRIs observed during stress testing). This stability-indicating method can then be used to analyze all of the stressed samples and to conduct other stabilityrelated investigations during early drug development. The problem facing these approaches is that they are both technique-oriented i.e., they rely on the analytical technique to resolve and to detect all DRIs quantitatively. Since we do not currently have universal detectors that respond equally to all compounds, we are faced with the question of whether all the major degradation products have been detected in a [reasonably] quantitative manner. The technique-oriented approach would focus on searching for unknown DRIs using additional separation and detection strategies. For example, the use of different separation techniques and detectors should be considered. Determining when the investigation is complete (i.e., that all major DRIs have been accounted for) is the main issue with this approach. It is worth considering in more detail what it means to be condent that all DRIs have been accounted for. The question is really one of mass balance. That is, for partially-degraded stressed samples, does the increase in DRI levels correspond (in mass or wt/wt%) to the decreased level of the parent drug? This topic has been discussed in detail by Nussbaum et al. [14] and some of the main concepts are worth considering here. Nussbaum gives seven major causes of poor mass balance 760
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(i) DRIs are not eluted from the HPLC column; (ii) DRIs are not detected by the detector; (iii) DRIs are lost (i.e., not recovered/extracted, or lost due to volatility) from the sample matrix; (iv) parent compound lost (i.e., not recovered/ extracted, or lost due to volatility) from the sample matrix; (v) DRIs co-elute with the parent compound; (vi) DRIs are not integrated due to poor chromatography; (vii) DRIs have different response factors from the parent. As outlined by Nussbaum, consideration of these seven causes helps to guide the design of experiments and choices of analytical techniques in investigating poor mass-balance problems. The importance of stress testing also becomes apparent upon consideration of the limitations of current analytical methodologies, (i.e., in the search for relevant DRIs of a particular compound, the stress-testing strategy targets a 520% level of degradation [6,8]). High levels of degradation (e.g., 10% or greater) are sometimes criticized as being too harsh, possibly leading to secondary DRIs that may not be observed at more realistic levels of perhaps 15% during the shelf-life of a compound. While it may be true that sometimes non-relevant secondary and tertiary DRIs may be formed at the 1020% level of degradation, in practice, the DRI prole for drug substances is often very comparable to that observed at 15% degradation. It is also important to consider the ability to assess mass balance as an indicator of missing (i.e., non-detected) DRIs. The ability to search for DRIs and to assess mass balance is greatly enhanced with higher degradation levels for obvious reasons (higher degradation = higher levels of DRIs = greater detectability). To assess mass balance, the loss of parent must be accurately measured and compared to the increased levels of DRIs. The analytical measurement of the parent (assay) depends on the precision of the method. Go g has pointed out that typical ro assays have a relative standard deviation (analytical error) of 1% or greater [15], so, when the level of degradation of the parent drug is low (e.g., 13%), it is difcult to assess mass balance because of assay variation. When degradation levels are low, quantication of DRI increases are viewed as superior indicators of degradation levels [16], but this does not address the issue of mass balance, and it assumes an accurate stability-indicating method. Mass-balance issues are most effectively investigated using more highly-degraded samples obtained by stress testing. As mentioned above, the use of different separation techniques with varying degrees of orthogonality can increase the likelihood of separating and detecting all the major DRIs. Such a strategy ts well during investigative stress-testing studies or specic DRI investigations. Assuming the primary analytical technique is gradient RP-HPLC with UV detection, the search for unknown

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DRIs might involve orthogonal separation techniques, such as hydrophilic interaction chromatography [17], normal-phase HPLC, capillary electrophoresis (CE), GC, thin layer chromatography (TLC), and supercritical uid chromatography (SFC) [18], and using multiple detectors, such as photodiode array (PDA)-UV, MS, evaporative light-scattering (ELS), chemiluminescent nitrogen (CLN), refractive index (RI), ame ionization (FI), and thermal conductivity (TC), and multiple visualization reagents for TLC [11]. Varying degrees of orthogonality in separation can also be achieved within the same technique (e.g., RPHPLC). Various articles have been written to describe orthogonal separations for RP-HPLC [1921]. Pellett et al. have recently proposed a general procedure for rapidly developing a separation that is orthogonal to a pre-existing method for a given sample [22]. These recent articles are a great resource to guiding the process of searching for DRIs for particular drugs using the technique-oriented approach. There is also the problem of quantifying DRIs using current analytical technologies. Since the most common detector (UV) does not respond to mass but on the basis of absorbance at the monitoring wavelength, accurate quantication of individual impurity levels requires knowledge of the response factor relative to that of the parent. Determining response factors can be a timeconsuming and expensive undertaking, typically involving efforts to isolate and to purify individual impurities. An alternative to this approach is to use detectors that respond on the basis of the weight (or more properly, mass) of the impurity. Four HPLC detectors are viewed as giving a response that is proportional to mass ELS, RI, CLN, and charged aerosol (CA). Each has its strengths and weaknesses related to DRI proling. The ELS detector is widely used in the pharmaceutical and chemical industry, especially for compounds that do not possess a good UV chromophore. The ELS detector allows detection of most non-volatile substances, but the response depends on the quantity and the nature of particles produced upon evaporation of the mobile phase. Unfortunately, responses for diverse compounds can vary signicantly, and volatile compounds may not be detected at all [2325]. The RI detector is viewed as a universal detector that responds similarly to diverse compounds, but it suffers from variability in response depending on the mobilephase composition, temperature, and dissolved gases, and it is relatively insensitive [26,27]. Because of these limitations, the RI detector is not commonly employed as a routine detector for sensitive DRI proling, especially with gradient RP-HPLC. The CLN detector is applicable to nitrogen-containing compounds and is quantitative with respect to moles of nitrogen. The CLN has demonstrated utility in deter-

mining and calculating response factors directly, assuming the DRI has at least one nitrogen and the molecular formula is known [28,29]. The CLN is widely used in the pharmaceutical industry, especially in pharmaceutical discovery research, to assess the purity of compounds in libraries used for activity screening [30,31], but it has also been employed in pharmaceutical development [28,32]. The CLN is incompatible with nitrogen-containing HPLC mobile phases or additives (e.g., acetonitrile), and has a reputation for not being rugged [32]. The CA detector (CAD) is relatively new and operates by detecting charged particles that have a selected range of mobility. Studies have shown that the signal obtained with this technique depends primarily upon particle size and not signicantly on the properties of the individual analytes [33]. The response of the CAD should therefore be similar for analytes regardless of the structure and Gamache et al. have shown the response of the CAD to be fairly uniform for a series of 16 different compounds [34]. The CAD can therefore be used to estimate relative levels of impurities without the use of a standard. The CAD has been shown to be sensitive (limit of detection (LOD) 520 ng), and reproducible, and to possess a wide dynamic range of approximately four orders of magnitude. However, the CAD is not without limitations. For example, mobile phases with non-volatile additives cannot be used and its response is not linear, but rather must be tted using an exponential function. In addition, its response also depends greatly upon solvent composition, which severely limits its quantitative ability under gradientelution conditions for compounds for which no standard is available. Nonetheless, this detector is a becoming a very useful tool in the analytical chemists arsenal [35]. In addition to the four detectors described above, MS and NMR can be used both as spectroscopic characterization tools as well as detectors. MS detectors are often regarded as universal, but the response per unit weight depends greatly on the ionization mode (e.g., positive or negative ionization, electrospray, atmospheric pressure chemical ionization (APCI), or electron ionization) and on the ionization efciency of the compound. Some compounds can go undetected with a particular ionization type, so MS detectors cannot be relied upon for accurate quantication of unknown DRIs. However, LC/ MS has distinct advantages over the other detectors discussed here by providing high sensitivity with the inherent selectivity afforded by recording individual molecular and fragment ions. It has been pointed out that LC/MS is an essential part of the comprehensive characterization of impurity proles [36,37]. NMR is an extremely powerful detector and impurity characterization tool. DRIs can be isolated and introduced into the spectrometer via tubes or direct ow injection [38]. Alternatively, DRIs can be introduced into the spectrometer via HPLC or some other separation
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technique such as SFC, capillary isotachophoresis, or CE [3941]. Characterization of low-level DRIs in the presence of the parent molecule using capillary or traditional HPLC-NMR has been problematic due to poor signal-tonoise resulting from insufcient column-loading capacity and/or mismatch of the chromatographic peak volume with that of the ow probe. Fortunately, most of these have been addressed by using capillary coil or cryogenically-cooled NMR probes, and/or solid-phase extraction (SPE) techniques, resulting in substantial signal-to-noise gains [42,43]. Qualitative NMR applications often get center stage, so using NMR as a universal and quantitative detector is often overlooked [44]. Nonetheless, NMR has been used as a standard to which other quantitative detectors are compared [45]. Accurate quantication using NMR usually requires isolated impurities [46]; however, quantitative detection of mixtures of impurities using HPLC-NMR is feasible in select cases [47]. Because of the high cost and signicant technical expertise required, NMR has not yet evolved into a routine quantitative detector for DRIs. In addition, resonance overlap can be a signicant drawback to using NMR for direct quantication of multiple DRIs, especially in the presence of high levels of the parent drug. 3.2. Determining which potential DRIs are relevant The DRIs observed during stress testing may or may not be relevant to the actual storage and distribution conditions for the drug substance or product and can therefore be thought of as potential DRIs. Determination of the relevant DRIs can be accomplished by analysis of samples stored under long-term and accelerated stability-testing conditions using the screening method(s) from step 1 (above). While this step

appears to be straightforward and without major problems, it depends upon the ability of the analytical technique and methodology to detect the relevant DRIs accurately. 3.3. Developing focused stability-indicating impurity methods If the relevant DRIs turn out to be a subset of the potential DRIs, the analytical methodology can be modied to focus on only the relevant DRIs. Such a strategy acknowledges the differences in goals of research and development (i.e., discovering what you do not know) and the goals for registration and marketing (i.e., controlling what you do know), as shown in Fig. 2. The key drivers for analytical impurity methodologies in early development are universal detection and resolving power. Impurity methods to be used as the control methods for marketed products need to be well validated, designed for speed, ruggedness, accuracy and precision, and transferable (i.e., methods readily usable in multiple laboratories). 3.4. Quantifying DRI levels via long-term, formal stability studies The DRIs observed over the shelf-life of the drug substance or product must be assessed quantitatively. Such assessments are the basis of establishing storage conditions, packaging requirements, specications, and shelflife. In most circumstances, real-time data are not available from analysis of material stored under longterm storage conditions, so DRI levels must be statistically predicted from trends observed in the long-term stability studies at intermediate points in time. In most cases, the shelf-life of a drug will be limited by the

Parent

Investigational/Screening Method C A B D

"Potential" Degradation Products G (Stress Testing Results) H F I

Parent

Investigational/Screening Method C B D

"Actual" Degradation Products (Accel. / Long-Term RT Stability) H

Parent

Final Control Method C B D E H Final Method--designed for speed, robustness, and focused on "actual" degradation products

Figure 2. Illustration of hypothetical chromatograms obtained from stress-testing (upper), and accelerated or long-term stability studies using a screening method (middle) or an optimized nal control method (lower).

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accumulated levels of individual or total DRIs. Specications (which are tied to the analytical impurity method) for DRI levels are set based on safety considerations and the impurity thresholds delineated by the ICH [48,49]. An additional complication that can be encountered is a change in the rate of formation of DRIs during longterm stability studies. Such changes can result from unexpected physical changes (e.g., form or phase changes, solvation or desolvation, or deliquescence), autocatalytic reactions, or exhaustion of limiting reagents. In the case of limiting reagents, if the DRIs are forming from a reaction of the drug with low levels of impurities in the formulation (e.g., peroxides, and shortchain aldehydes) [50], the degradation reaction will slow or stop when the impurity(ies) is(are) consumed. Predictions of future levels via growth over time may be highly inaccurate in such a scenario. Prediction in such cases requires more knowledge about the chemistry and the mechanism of the degradation reaction(s).

4. Supplementing the technique-oriented approach with the chemistry-guided approach The problems and complexities outlined in the previous sections are the result of using a technique-oriented approach with imperfect analytical techniques (i.e., we are relying on the analytical technique(s) to provide accurate quantitative detection of DRIs, but we do not have in our analytical toolbox truly universal detectors that respond equally to diverse compounds, nor do we have separation technologies that can ensure separation and resolution of all DRIs prior to the detector). We cannot therefore be condent of the completeness of stress-testing and DRI-proling investigations using the technique-oriented approach with current tools. The problems associated with investigating and controlling impurities with the technique-oriented approach have been discussed previously [11,51]. The chemistry-guided approach relies on scientic evaluation of the chemistry to guide the interpretation of data and the selection of appropriate analytical techniques. The rst step is to evaluate the parent compound, asking: Does it have a chromophore? How volatile is it? Does it have ionizable functional groups (i.e., any pKs)? Could the chromophore be destroyed by simple degradation reactions? Will volatile compounds be formed by degradation? Have degradation products or pathways been established for compounds with similar structure? What degradation reactions and products can be predicted? What analytical techniques have been successfully used during synthesis and evaluation of process-related impurities? The next step is to evaluate the degradation pathways; however, one cannot evaluate degradation pathways by

merely observing discrete peaks detected in partiallydegraded samples arising from particular stress conditions. Identication of a pathway involves both the conditions of degradation and the structures, which can lead to identication of the site of degradation and possible mechanisms leading to the degradation product. The structures of DRIs and the associated pathways can point the researcher to appropriate analytical techniques. For example, if a degradation pathway implies the formation of a non-chromophoric product, the researcher is alerted to using a detector other than UV (e.g., MS, ELS, CA, RI). Similarly, if a pathway implies the formation of a volatile compound, the researcher may choose to monitor the headspace of an enclosed container using GC/FID or GC/MS, for example. Relevant examples to illustrate these concepts have been provided by Baertschi [51] and Nussbaum [14]. One example that illustrates the power of a chemistryguided approach is that of gemcitabine [51,52]. In this example, stressed solutions of gemcitabine that exhibited a signicant loss of the parent showed only a very minor increase in DRIs. The analytical method detected only one signicant impurity, the b-uridine analog, a known degradation product of gemcitabine (Fig. 3). Applying the chemistry-guided approach, the researchers did a survey of the literature on degradation of related nucleosides. The mechanism proposed in the literature for deamination of cytidine (leading to the b-uridine) involved intermediates in which a nucleophile (e.g., water) is added to position six of the cytidine moiety, resulting in the loss of the 5,6-double bond, and therefore loss of the chromophore. This information led the researchers to lower the wavelength of detection from 275 nm to 205 nm, revealing two additional DRIs (Fig. 4). Further studies involved understanding the degradation pathways and characterization of the structures of the DRIs and led to the discovery that a third DRI was co-eluting with the parent peak. The degradation chemistry was then described in a proposed mechanism (Fig. 5) that, together with the analytical results, provided the condence that the degradation picture was complete, and that the mass-balance issue was satisfactorily resolved.
NH2 N N HO O F OH F HCl O acidic aqueous degradation N HO O F OH F O NH O

Gemcitabine hydrochloride

of gemcitabine

Figure 3. Structures of gemcitabine hydrochloride and the b-uridine degradation product.

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Gemcitabine
0.60

0.50

A Impurity eluting with parent

0.40

AU

0.30

B
0.20

B-uridine

275 nm
0.10

0.00

205 nm

2.00

4.00

6.00

8.00

10.00

12.00

14.00

16.00

18.00

20.00

Minutes

Figure 4. RP-HPLC chromatograms obtained on a gemcitabine hydrochloride in pH 3.2 acetate buffer stressed at 70C for 2 days. Upper trace shows analytical results from UV detection at 275 nm, while the lower trace shows the same sample analyzed at 205 nm.

NH2 N N HO O F OH F OH O H3O+ HO O F F

NH2 NH N O H2O HO O F OH F HO

NH2 NH N O H2O -NH3 HO O F OH F HO

O NH N O

A and B
-H2O NH2 NH O O F OH F OH N O H2O -NH3 O O F F OH N O NH O HO O F F N O NH O

-uridine analogue

Figure 5. Proposed deamination mechanism of gemcitabine in acidic aqueous solution.

5. Future directions and approaches 5.1. Improving the technique-oriented approach 5.1.1. Separations. Analytical separations are moving rapidly toward higher resolution, improved peak capacities, and multi-dimensional approaches [53,54]. Utilization of HPLC columns with smaller and smaller particles, leading to higher and higher backpressures, is resulting in dramatically improved analysis time and improved resolution. Increasing the column temperature can signicantly decrease backpressure, speed up analyte diffusivity, and can lead to signicantly reduced analysis times. These and further advances are discussed in some detail in the article by Olsen et al. in the current issue [55]. 764

Advances are needed in the area of coupled orthogonal separation approaches to provide higher peak capacities to maximize the probability of resolving DRIs. For example, one could imagine the HPLC eluent being split and further analyzed on the y by a very rapid orthogonal technique, such as CE (e.g., using lab on a chip technology, where the separations can be performed in seconds). Such multi-dimensional orthogonal approaches can result in dramatic increases in peak capacities, which increase the probability that DRIs will be individually resolved in a given separation experiment. 5.1.2. Detection. While a combination of detection modes (e.g., PDA-UV, MS, CLN, CAD, and NMR) can

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provide reasonable universality of detection (including determination of response factors), volatile compounds can still evade detection. Additionally, signicant complexity and cost are also associated with using multiple detection strategies. Recent advances in the universal RI detector have led to signicant increases in sensitivity and stability for temperature and mobile-phase gradients at the lHPLC scale [56]. If these advances prove applicable to routine HPLC and CE instrumentation, using RI as a universal detector for DRI proling may increase greatly. There is still a need for advances in detectors for rapid determination of response factors of unknown DRIs, and for selective detectors that have dial in selectivity (e.g., for certain elements or functional groups). Such selective detectors would be especially relevant for methods designed to detect specic DRIs at very low levels (e.g., genotoxic impurities). 5.2. Improving the chemistry-guided approach 5.2.1. Faster, more efcient means of obtaining chemical structure information. Improving the chemistry-guided approach will require faster structure-elucidation tools. For example, more sensitive NMR and LC/NMR techniques, and easier to use and more highly automated LC/ NMR applications are needed. Continuing improvements (e.g., improved sensitivities, and automated SPE and isolation [57]) should help LC/NMR move from primarily research applications to more routine use. Automated preparative HPLC systems can dramatically speed up the isolation of DRIs to aid the spectroscopic characterization process. Computer-assisted structure elucidation (CASE) programs, designed to aid in interpreting spectroscopic data to elucidate structures, continue to evolve and should eventually become widely used for assisting with structure elucidation of unknown DRIs [58,59]. Accurate LC/ MS data (obtained using time-of-ight or FT-ion cyclotron resonance MS systems) can provide the molecular formulas of unknown impurities even at very low levels in complex mixtures [60], and that can greatly speed up the process of elucidating unknown structures. 5.2.2. Improved forced degradation procedures. Although there has been signicant progress in developing predictive procedures for stressing drug substances and products, there is a need for better, faster ways of producing DRIs that are relevant to real-world storage, distribution, and even patient in-use conditions for both drug substance and formulated products. Renements are needed in predicting both degradation proles and degradation rates from forced degradation studies. 5.2.3. Computational approaches. The eld of drug degradation is ready for development of a software tool that can predict degradation pathways of drugs under various conditions of stress. The software CAMEO

(computer assisted mechanistic evaluation of organic reactions) [61] is a tool that has been used for this purpose with some success [62], but it was not designed for predicting drug-degradation pathways, so signicant expertise in organic chemistry is needed to use it effectively. Mining published degradation pathways and structures for specic drugs and organic functional groups to develop structure searchable databases would be particularly useful. Coupling such a database with LC/MS data could provide a powerful tool. Efforts have also been made in the area of computational chemistry [63]. It is apparent that computational chemistry can contribute signicantly to the prediction of electrophilic and nucleophilic sites in a molecule, and to determination of pKs (i.e., protonation states). Computational chemistry has been used to predict oxidative susceptibility of molecules [64]. Additionally, reactions can be mapped and assessed for their potential to occur and for their mechanistic pathways. Unfortunately, such calculations still require signicant expertise to set them up properly and it can be tricky to interpret them properly. Continuing advances in computer speed and computational algorithms are likely to lead to new, more widely useful tools for predicting and explaining drug-stability and degradation pathways.

6. Conclusions Future directions in pharmaceutical DRI proling will involve improvements in both the technique-oriented and the chemistry-guided approaches. As technologies continue to progress rapidly, signicant improvements in analytical techniques will lead to much faster DRI proling, greatly reducing the risk of missing important DRIs. Multi-dimensional approaches (e.g., coupled orthogonal separations with multiple detectors) are likely to play a leading role in developing this area. The chemistry-guided approaches will also advance as a result of faster structure-elucidation technologies, improved computational tools, and better procedures for stress-testing and mechanistic investigations. In addition, publications about degradation chemistry in the open literature will be of utmost importance to developing this burgeoning eld.

Acknowledgement I would like to thank Sandor Gorog for his invitation to write this manuscript and for the inspiration he has provided by the numerous contributions he has made to drug-impurity profiling. Helpful discussions and ideas from Bernard A. Olsen, Timothy J. Wozniak, Kallol Biswas, Andreas Kaerner, Todd A. Gillespie, William K. Smith, and Patrick J. Jansen are gratefully acknowledged. 765

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