Analytical Methodologie for Discovering and Profiling Degradation Impurities

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

Analytical methodologies fordiscovering and profilingdegradation-related impuritiesSteven W. Baertschi

The analytical technologies currently in use for profiling 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 profiling. 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.

ª 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

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: [email protected]

758

1. Introduction

There is currently a tremendous interest inprofiling of impurities in drugs, as evi-denced by numerous conferences, presen-tations, articles and books [1–3] devotedto this topic, including this issue of TrAC.In this article, the focus is on the topicdegradation-related impurity (DRI) profilingin drugs.

While the distinction between profilingof DRIs and process impurities may notseem significant from the viewpoint ofanalytical methodology, the strategiesemployed to develop DRI methods and tosolve DRI problems can be quite differentfrom analogous strategies for process-related impurities. There are a large numberof specialized analytical techniques avail-able for the characterization of the purity ofdrugs, so no one person can be qualified asan expert in all of these techniques.

Gorog [1a] has described the process ofsolving impurity problems via impurityprofiling as being analogous to the process

0165-9936/$ - see front matter ª 2006 Elsev

of a conductor leading an orchestra. It ishoped that the current article will helpthose laboratories dealing with DRI pro-filing to orchestrate solving impurity-related problems and perhaps to considernew ways of approaching the purity issuesthat regularly confront the pharmaceuti-cal researcher.

2. Overall strategy – in theory

In order to detect accurately and toquantify (i.e., ‘‘profile’’) DRIs, a stability-indicating analytical method is needed.Ideally, such a method should resolve allDRIs from the parent and from each other,and should detect and accurately quantifyall DRIs. As outlined by the InternationalConference on Harmonization (ICH) [4],the process for establishing a stability-indicating method involves stress testing:‘‘Stress testing of the drug substance canhelp identify the likely degradation prod-ucts, which can in turn help establish thedegradation pathways and the intrinsicstability of the molecule and validate thestability-indicating power of the analyticalprocedures used.’’

The overall strategy for developing astability-indicating method using stresstesting has been expressed simply (Fig. 1).This strategy involves:� identifying likely or ‘‘potential’’ DRIs

through stress-testing studies usinghighly-resolving or ‘‘discriminating’’methods;

� determining which of the potential DRIsare relevant (i.e., those DRI’s that formduring accelerated stability and long-term stability studies);

ier Ltd. All rights reserved. doi:10.1016/j.trac.2006.05.012

mailto:[email protected]

API Drug Product

API Stress Testing

Use discriminating methods Identify potential degradation products and pathways

Formulation Development

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

Drug Product Stress Testing

Use discriminating methods Identify potential degradation products and pathways not detected in drug substance stress testing

Accelerated Testing

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

minimize

Long-Term Testing

Determine degradation product levels Develop specifications Establish storage conditions and shelf

life

••

•••

•

••

••••

••

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

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

� developing ‘‘focused’’ stability-indicating impuritymethods (methods that resolve and detect the DRIsrelevant to real-world handling and storage condi-tions); and,

� quantifying DRI levels via long-term, formal stabilitystudies, and establishing specifications, storage condi-tions, 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 processseems fairly straightforward and deceptively simple. Inpractice, the details of carrying out the process providenumerous difficulties, complexities, and questions thatneed to be addressed. It is important to consider some ofthese difficulties.

3.1. Identifying likely [or ‘‘potential’’] DRIsThis step involves stressing samples of the drug sub-stance (alone and in the presence of the excipients in theformulation) under ‘‘relevant’’ conditions of heat,humidity, photostress (ultraviolet (UV) and visible (VIS)),oxidative conditions, and aqueous conditions across abroad pH range. It also involves choosing the analytical

technique(s), developing and using an appropriatemethod, and analyzing samples.

The first question that must be answered is: ‘‘What arethe relevant stress conditions and how much stress isenough?’’. Until recently, there was very little guidanceavailable to aid the analytical researcher in determiningthe answers to these questions, so stress-testing proce-dures varied tremendously [5]. The ICH stability guide-line provides some limited guidance [4]: ‘‘The testingshould include the effect of temperatures (in 10�Cincrements (e.g., 50�C, 60�C) above that for acceleratedtesting), humidity (e.g., 75% relative humidity or greater)where appropriate, oxidation, and photolysis on the drugsubstance. The testing should also evaluate the suscep-tibility of the drug substance to hydrolysis across a widerange 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 andscientific rationale for the choice of stress conditionsand endpoints, and the design of such studies. Since thistopic is covered in sufficient detail in these references, nofurther discussion will be provided here.

After the appropriate stressing of samples, questionsrelated to the analysis must be addressed. How do youanalyze the samples? What separation technique doyou choose? What detector? The inherent dilemma is

http://www.elsevier.com/locate/trac 759


how to measure the loss of parent and levels of theDRIs prior to development of a stability-indicatingimpurity method.

There are a couple of approaches generally taken toaddress this dilemma of measuring DRIs prior to havinga stability-indicating method. One approach is to use ageneric ‘‘screening’’ method, where the method may nothave been designed or optimized for a particular com-pound but is designed in general to be highly resolvingand broadly applicable [11]. Currently in the pharma-ceutical industry, the choice is overwhelmingly gradientreversed phase high-performance liquid chromatogra-phy (RP-HPLC) with UV detection (presumably at a lowwavelength for more universal detection, e.g., 210–220nm) [12,13]. Further confidence in the results of theanalyses can be gained by analyzing samples using asecond screening method that is orthogonal (i.e., non-overlapping) to the first screening method. Use of anorthogonal method increases the likelihood of resolvingand detecting all the DRIs.

A second approach is to optimize the screeningmethod to be specific for the compound of interest. Thus,after initial analysis of the stressed samples using ascreening method, an assessment is then made as towhich conditions caused degradation, and the samplesfrom these conditions are used for further methoddevelopment until the researcher concludes that themethod is stability-indicating (i.e., it resolves and detectsall the major DRIs observed during stress testing). Thisstability-indicating method can then be used to analyzeall of the stressed samples and to conduct other stability-related investigations during early drug development.

The problem facing these approaches is that they areboth ‘‘technique-oriented’’ – i.e., they rely on the ana-lytical technique to resolve and to detect all DRIsquantitatively. Since we do not currently have universaldetectors that respond equally to all compounds, we arefaced with the question of whether all the major deg-radation products have been detected in a [reasonably]quantitative manner. The technique-oriented approachwould focus on searching for unknown DRIs usingadditional separation and detection strategies. Forexample, the use of different separation techniquesand detectors should be considered. Determining whenthe investigation is complete (i.e., that all major DRIshave been accounted for) is the main issue with thisapproach.

It is worth considering in more detail what it means tobe confident that all DRIs have been accounted for. Thequestion is really one of ‘‘mass balance’’. That is, for par-tially-degraded stressed samples, does the increase in DRIlevels correspond (in ‘‘mass’’ or wt/wt%) to the decreasedlevel of the parent drug? This topic has been discussed indetail by Nussbaum et al. [14] and some of the mainconcepts are worth considering here. Nussbaum givesseven major causes of ‘‘poor’’ mass balance

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(i) DRI�s are not eluted from the HPLC column;(ii) DRI�s are not detected by the detector;

(iii) DRI�s are lost (i.e., not recovered/extracted, or lostdue to volatility) from the sample matrix;

(iv) parent compound lost (i.e., not recovered/extracted, or lost due to volatility) from the samplematrix;

(v) DRI�s co-elute with the parent compound;(vi) DRI�s are not integrated due to poor chromatography;

(vii) DRI�s have different response factors from theparent.

As outlined by Nussbaum, consideration of these sevencauses helps to guide the design of experiments andchoices of analytical techniques in investigating poormass-balance problems. The importance of stress testingalso becomes apparent upon consideration of the limita-tions of current analytical methodologies, (i.e., in thesearch for relevant DRIs of a particular compound, thestress-testing strategy targets a 5–20% level of degrada-tion [6,8]). High levels of degradation (e.g., 10% orgreater) are sometimes criticized as being ‘‘too harsh’’,possibly leading to secondary DRIs that may not beobserved at more realistic levels of perhaps 1–5% duringthe shelf-life of a compound. While it may be true thatsometimes non-relevant secondary and tertiary DRIs maybe formed at the 10–20% level of degradation, in practice,the DRI profile for drug substances is often very compa-rable to that observed at 1–5% degradation. It is alsoimportant to consider the ability to assess mass balance asan indicator of ‘‘missing’’ (i.e., non-detected) DRIs.

The ability to search for DRIs and to assess mass bal-ance is greatly enhanced with higher degradation levelsfor obvious reasons (higher degradation = higher levelsof DRIs = greater detectability). To assess mass balance,the loss of parent must be accurately measured andcompared to the increased levels of DRIs. The analyticalmeasurement of the parent (assay) depends on the pre-cision of the method. Gorog has pointed out that typicalassays have a relative standard deviation (analyticalerror) of 1% or greater [15], so, when the level of deg-radation of the parent drug is low (e.g., 1–3%), it isdifficult to assess ‘‘mass balance’’ because of assay var-iation. When degradation levels are low, quantificationof DRI increases are viewed as superior indicators ofdegradation levels [16], but this does not address theissue of mass balance, and it assumes an accuratestability-indicating method. Mass-balance issues aremost effectively investigated using more highly-degradedsamples obtained by stress testing.

As mentioned above, the use of different separationtechniques with varying ‘‘degrees of orthogonality’’ canincrease the likelihood of separating and detecting all themajor DRIs. Such a strategy fits well during investigativestress-testing studies or specific DRI investigations.Assuming the primary analytical technique is gradientRP-HPLC with UV detection, the search for unknown


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 fluidchromatography (SFC) [18], and using multiple detec-tors, such as photodiode array (PDA)-UV, MS, evapora-tive light-scattering (ELS), chemiluminescent nitrogen(CLN), refractive index (RI), flame ionization (FI), andthermal conductivity (TC), and multiple visualizationreagents for TLC [11].

Varying degrees of orthogonality in separation canalso be achieved within the same technique (e.g., RP-HPLC). Various articles have been written to describe‘‘orthogonal’’ separations for RP-HPLC [19–21]. Pellettet al. have recently proposed a general procedure forrapidly developing a separation that is orthogonal to apre-existing method for a given sample [22]. These re-cent articles are a great resource to guiding the processof searching for DRIs for particular drugs using thetechnique-oriented approach.

There is also the problem of quantifying DRIs usingcurrent analytical technologies. Since the most commondetector (UV) does not respond to mass but on the basisof absorbance at the monitoring wavelength, accuratequantification of individual impurity levels requiresknowledge of the response factor relative to that of theparent. Determining response factors can be a time-consuming and expensive undertaking, typicallyinvolving efforts to isolate and to purify individualimpurities. An alternative to this approach is to usedetectors that respond on the basis of the weight (ormore properly, mass) of the impurity.

Four HPLC detectors are viewed as giving a responsethat is proportional to mass – ELS, RI, CLN, and chargedaerosol (CA). Each has its strengths and weaknessesrelated to DRI profiling.

The ELS detector is widely used in the pharmaceuticaland chemical industry, especially for compounds that donot possess a good UV chromophore. The ELS detectorallows detection of most non-volatile substances, but theresponse depends on the quantity and the nature ofparticles produced upon evaporation of the mobilephase. Unfortunately, responses for diverse compoundscan vary significantly, and volatile compounds may notbe detected at all [23–25].

The RI detector is viewed as a universal detector thatresponds similarly to diverse compounds, but it suffersfrom variability in response depending on the mobile-phase composition, temperature, and dissolved gases,and it is relatively insensitive [26,27]. Because of theselimitations, the RI detector is not commonly employed asa routine detector for sensitive DRI profiling, especiallywith gradient RP-HPLC.

The CLN detector is applicable to nitrogen-containingcompounds and is quantitative with respect to moles ofnitrogen. The CLN has demonstrated utility in deter-

mining and calculating response factors directly,assuming the DRI has at least one nitrogen and themolecular formula is known [28,29]. The CLN is widelyused in the pharmaceutical industry, especially inpharmaceutical discovery research, to assess the purityof compounds in libraries used for activity screening[30,31], but it has also been employed in pharmaceuti-cal development [28,32]. The CLN is incompatible withnitrogen-containing HPLC mobile phases or additives(e.g., acetonitrile), and has a reputation for not beingrugged [32].

The CA detector (CAD) is relatively new and operatesby detecting charged particles that have a selected rangeof mobility. Studies have shown that the signal obtainedwith this technique depends primarily upon particle sizeand not significantly on the properties of the individualanalytes [33]. The response of the CAD should thereforebe similar for analytes regardless of the structure andGamache et al. have shown the response of the CAD to befairly uniform for a series of 16 different compounds [34].The CAD can therefore be used to estimate relative levelsof impurities without the use of a standard. The CAD hasbeen shown to be sensitive (limit of detection (LOD) 5–20ng), and reproducible, and to possess a wide dynamicrange of approximately four orders of magnitude. How-ever, the CAD is not without limitations. For example,mobile phases with non-volatile additives cannot be usedand its response is not linear, but rather must be fittedusing an exponential function. In addition, its responsealso depends greatly upon solvent composition, whichseverely limits its quantitative ability under gradient-elution conditions for compounds for which no standardis available. Nonetheless, this detector is a becoming avery useful tool in the analytical chemist�s arsenal [35].

In addition to the four detectors described above, MSand NMR can be used both as spectroscopic character-ization tools as well as detectors. MS detectors are oftenregarded as universal, but the response per unit weightdepends greatly on the ionization mode (e.g., positive ornegative ionization, electrospray, atmospheric pressurechemical ionization (APCI), or electron ionization) andon the ionization efficiency of the compound. Somecompounds can go undetected with a particular ioniza-tion type, so MS detectors cannot be relied upon foraccurate quantification of unknown DRIs. However, LC/MS has distinct advantages over the other detectorsdiscussed here by providing high sensitivity with theinherent selectivity afforded by recording individualmolecular and fragment ions. It has been pointed outthat LC/MS is an essential part of the comprehensivecharacterization of impurity profiles [36,37].

NMR is an extremely powerful ‘‘detector’’ and impuritycharacterization tool. DRIs can be isolated and intro-duced into the spectrometer via tubes or direct flowinjection [38]. Alternatively, DRIs can be introduced intothe spectrometer via HPLC or some other separation



technique such as SFC, capillary isotachophoresis, or CE[39–41]. Characterization of low-level DRIs in the pres-ence of the parent molecule using capillary or traditionalHPLC-NMR has been problematic due to poor signal-to-noise resulting from insufficient column-loading capacityand/or mismatch of the chromatographic peak volumewith that of the flow probe. Fortunately, most of thesehave been addressed by using capillary coil or cryogeni-cally-cooled NMR probes, and/or solid-phase extraction(SPE) techniques, resulting in substantial signal-to-noisegains [42,43]. Qualitative NMR applications often getcenter stage, so using NMR as a universal and quanti-tative detector is often overlooked [44]. Nonetheless,NMR has been used as a standard to which other quan-titative detectors are compared [45]. Accurate quantifi-cation using NMR usually requires isolated impurities[46]; however, quantitative detection of mixtures ofimpurities using HPLC-NMR is feasible in select cases[47]. Because of the high cost and significant technicalexpertise required, NMR has not yet evolved into a rou-tine quantitative detector for DRIs. In addition, resonanceoverlap can be a significant drawback to using NMR fordirect quantification of multiple DRIs, especially in thepresence of high levels of the parent drug.

3.2. Determining which potential DRIs are relevantThe DRIs observed during stress testing may or may notbe relevant to the actual storage and distribution con-ditions for the drug substance or product and cantherefore be thought of as ‘‘potential’’ DRIs. Determina-tion of the ‘‘relevant’’ DRIs can be accomplished byanalysis of samples stored under long-term andaccelerated stability-testing conditions using thescreening method(s) from step 1 (above). While this step

AB

C

D

E

F

B

C

D

E

Investigational/ScreeningMethod

B

CD

E

Parent

Final Control Method

H

Investigational/ScreeningMethod

Figure 2. Illustration of hypothetical chromatograms obtained from stress-screening method (middle) or an optimized ‘‘final control’’ method (lower


appears to be straightforward and without majorproblems, it depends upon the ability of the analyticaltechnique and methodology to detect the relevantDRIs accurately.

3.3. Developing ‘‘focused’’ stability-indicating impuritymethodsIf the relevant DRIs turn out to be a subset of the po-tential DRIs, the analytical methodology can be modifiedto focus on only the relevant DRIs. Such a strategyacknowledges the differences in goals of research anddevelopment (i.e., discovering what you do not know)and the goals for registration and marketing (i.e., con-trolling what you do know), as shown in Fig. 2.

The key drivers for analytical impurity methodologiesin early development are universal detection andresolving power. Impurity methods to be used as thecontrol methods for marketed products need to be wellvalidated, designed for speed, ruggedness, accuracy andprecision, and transferable (i.e., methods readily usablein multiple laboratories).

3.4. Quantifying DRI levels via long-term, formalstability studiesThe DRIs observed over the shelf-life of the drug sub-stance or product must be assessed quantitatively. Suchassessments are the basis of establishing storage condi-tions, packaging requirements, specifications, and shelf-life. In most circumstances, real-time data are notavailable from analysis of material stored under long-term storage conditions, so DRI levels must be statisti-cally predicted from trends observed in the long-termstability studies at intermediate points in time. In mostcases, the shelf-life of a drug will be limited by the

G

H

Parent

I

Parent

"Potential" Degradation Products (Stress Testing Results)

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

Final Method--designed for speed,robustness, and focused on "actual"degradation products

H

testing (upper), and accelerated or long-term stability studies using a).


accumulated levels of individual or total DRIs. Specifi-cations (which are tied to the analytical impuritymethod) for DRI levels are set based on safety consider-ations and the impurity thresholds delineated by the ICH[48,49].

An additional complication that can be encountered isa change in the rate of formation of DRIs during long-term stability studies. Such changes can result fromunexpected physical changes (e.g., form or phasechanges, solvation or desolvation, or deliquescence),autocatalytic reactions, or exhaustion of limitingreagents. In the case of limiting reagents, if the DRIs areforming from a reaction of the drug with low levels ofimpurities in the formulation (e.g., peroxides, and short-chain aldehydes) [50], the degradation reaction will slowor stop when the impurity(ies) is(are) consumed. Pre-dictions of future levels via growth over time may behighly inaccurate in such a scenario. Prediction in suchcases requires more knowledge about the chemistry andthe mechanism of the degradation reaction(s).

O

FOH

FHO

N

N

NH2

O

HCl

Gemcitabine hydrochloride

acidic aqueousdegradation

O

FOH

FHO

N

NH

O

O

of gemcitabine

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

4. Supplementing the technique-orientedapproach with the chemistry-guided approach

The problems and complexities outlined in the previoussections are the result of using a ‘‘technique-oriented’’approach with imperfect analytical techniques (i.e., weare relying on the analytical technique(s) to provideaccurate quantitative detection of DRIs, but we do nothave in our ‘‘analytical toolbox’’ truly universal detec-tors that respond equally to diverse compounds, nor dowe have separation technologies that can ensure sepa-ration and resolution of all DRIs prior to the detector).We cannot therefore be confident of the completeness ofstress-testing and DRI-profiling investigations using thetechnique-oriented approach with current tools. Theproblems associated with investigating and controllingimpurities with the technique-oriented approach havebeen discussed previously [11,51].

The ‘‘chemistry-guided’’ approach relies on scientificevaluation of the chemistry to guide the interpretation ofdata and the selection of appropriate analytical tech-niques. The first 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 degra-dation reactions? Will volatile compounds be formed bydegradation? Have degradation products or pathwaysbeen established for compounds with similar structure?What degradation reactions and products can bepredicted? What analytical techniques have been suc-cessfully used during synthesis and evaluation ofprocess-related impurities?

The next step is to evaluate the degradation pathways;however, one cannot evaluate degradation pathways by

merely observing discrete ‘‘peaks’’ detected in partially-degraded samples arising from particular stress condi-tions. Identification of a pathway involves both theconditions of degradation and the structures, which canlead to identification of the site of degradation and pos-sible mechanisms leading to the degradation product.The structures of DRIs and the associated pathways canpoint the researcher to appropriate analytical tech-niques. For example, if a degradation pathway impliesthe formation of a non-chromophoric product, the re-searcher is alerted to using a detector other than UV(e.g., MS, ELS, CA, RI). Similarly, if a pathway impliesthe formation of a volatile compound, the researchermay choose to monitor the headspace of an enclosedcontainer using GC/FID or GC/MS, for example. Relevantexamples to illustrate these concepts have been providedby Baertschi [51] and Nussbaum [14].

One example that illustrates the power of a chemistry-guided approach is that of gemcitabine [51,52]. In thisexample, stressed solutions of gemcitabine that exhibiteda significant loss of the parent showed only a very minorincrease in DRIs. The analytical method detected onlyone significant impurity, the b-uridine analog, a knowndegradation product of gemcitabine (Fig. 3). Applyingthe chemistry-guided approach, the researchers did asurvey of the literature on degradation of relatednucleosides. The mechanism proposed in the literaturefor 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 there-fore loss of the chromophore. This information led theresearchers to lower the wavelength of detection from275 nm to 205 nm, revealing two additional DRIs(Fig. 4). Further studies involved understanding thedegradation pathways and characterization of thestructures of the DRIs and led to the discovery that athird DRI was co-eluting with the parent peak. Thedegradation chemistry was then described in a proposedmechanism (Fig. 5) that, together with the analyticalresults, provided the confidence that the degradationpicture was complete, and that the mass-balance issuewas satisfactorily resolved.


AU

0.00

0.10

0.20

0.30

0.40

0.50

0.60

Minutes

2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00

275 nm

205 nm

B-uridine

Impurity eluting with parent

Gemcitabine

A

B C

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

O

FOH

F

HON

NH

NH2

OHO

O

FOH

F

HON

NH

OHO

O

O

FOH

F

HON

NH

O

O

FOH

F

N

NH

NH2

OO

NH2

O

FOH

F

N

NH

O

OO

O

FOH

F

HON

NH

O

O

O

FOH

F

HON

N

O

NH2

1 A and B

β-uridine analogue

H2O H2O

-NH3

H2O

-NH3

-H2O

H3O+

C

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


5. Future directions and approaches

5.1. Improving the technique-oriented approach5.1.1. Separations. Analytical separations are movingrapidly toward higher resolution, improved peak capaci-ties, and multi-dimensional approaches [53,54]. Utiliza-tion of HPLC columns with smaller and smaller particles,leading to higher and higher backpressures, is resultingin dramatically improved analysis time and improvedresolution. Increasing the column temperature cansignificantly decrease backpressure, speed up analytediffusivity, and can lead to significantly reduced analysistimes. These and further advances are discussed in somedetail in the article by Olsen et al. in the current issue [55].


Advances are needed in the area of coupled orthogonalseparation approaches to provide higher peak capacitiesto maximize the probability of resolving DRIs. Forexample, one could imagine the HPLC eluent being splitand further analyzed ‘‘on the fly’’ by a very rapidorthogonal technique, such as CE (e.g., using ‘‘lab on achip’’ technology, where the separations can be per-formed in seconds). Such multi-dimensional orthogonalapproaches can result in dramatic increases in peakcapacities, which increase the probability that DRIs willbe individually resolved in a given separation experiment.

5.1.2. Detection. While a combination of detectionmodes (e.g., PDA-UV, MS, CLN, CAD, and NMR) can


provide reasonable universality of detection (includingdetermination of response factors), volatile compoundscan still evade detection. Additionally, significant com-plexity and cost are also associated with using multipledetection strategies. Recent advances in the universal RIdetector have led to significant increases in sensitivityand stability for temperature and mobile-phase gradientsat the lHPLC scale [56]. If these advances prove appli-cable to routine HPLC and CE instrumentation, using RIas a universal detector for DRI profiling may increasegreatly. There is still a need for advances in detectors forrapid determination of response factors of unknownDRIs, and for selective detectors that have ‘‘dial in’’selectivity (e.g., for certain elements or functionalgroups). Such selective detectors would be especiallyrelevant for methods designed to detect specific DRIs atvery low levels (e.g., genotoxic impurities).

5.2. Improving the chemistry-guided approach5.2.1. Faster, more efficient means of obtaining chemicalstructure information. Improving the chemistry-guidedapproach will require faster structure-elucidation tools.For example, more sensitive NMR and LC/NMR tech-niques, and easier to use and more highly automated LC/NMR applications are needed. Continuing improvements(e.g., improved sensitivities, and automated SPE andisolation [57]) should help LC/NMR move from primarilyresearch applications to more routine use.

Automated preparative HPLC systems can dramati-cally speed up the isolation of DRIs to aid the spectro-scopic characterization process.

Computer-assisted structure elucidation (CASE) pro-grams, designed to aid in interpreting spectroscopic datato elucidate structures, continue to evolve and shouldeventually become widely used for assisting with struc-ture elucidation of unknown DRIs [58,59]. Accurate LC/MS data (obtained using time-of-flight or FT-ion cyclo-tron resonance MS systems) can provide the molecularformulas of unknown impurities even at very low levelsin complex mixtures [60], and that can greatly speed upthe process of elucidating unknown structures.

5.2.2. Improved forced degradation procedures. Althoughthere has been significant progress in developing pre-dictive procedures for stressing drug substances andproducts, there is a need for better, faster ways of pro-ducing DRIs that are relevant to real-world storage,distribution, and even patient in-use conditions for bothdrug substance and formulated products. Refinementsare needed in predicting both degradation profiles anddegradation rates from forced degradation studies.

5.2.3. Computational approaches. The field of drug deg-radation is ready for development of a software tool thatcan predict degradation pathways of drugs under vari-ous conditions of stress. The software ‘‘CAMEO’’

(computer assisted mechanistic evaluation of organicreactions) [61] is a tool that has been used for thispurpose with some success [62], but it was not designedfor predicting drug-degradation pathways, so significantexpertise in organic chemistry is needed to use it effec-tively. Mining published degradation pathways andstructures for specific drugs and organic functionalgroups to develop structure searchable databases wouldbe particularly useful. Coupling such a database withLC/MS data could provide a powerful tool.

Efforts have also been made in the area of computa-tional chemistry [63]. It is apparent that computationalchemistry can contribute significantly to the predictionof electrophilic and nucleophilic sites in a molecule, andto determination of pKs (i.e., protonation states).

Computational chemistry has been used to predictoxidative susceptibility of molecules [64]. Additionally,reactions can be mapped and assessed for their potentialto occur and for their mechanistic pathways. Unfortu-nately, such calculations still require significant exper-tise to set them up properly and it can be tricky tointerpret them properly. Continuing advances in com-puter speed and computational algorithms are likely tolead to new, more widely useful tools for predicting andexplaining drug-stability and degradation pathways.

6. Conclusions

Future directions in pharmaceutical DRI profiling willinvolve improvements in both the technique-orientedand the chemistry-guided approaches. As technologiescontinue to progress rapidly, significant improvements inanalytical techniques will lead to much faster DRI pro-filing, greatly reducing the risk of ‘‘missing’’ importantDRIs. Multi-dimensional approaches (e.g., coupledorthogonal separations with multiple detectors) arelikely to play a leading role in developing this area. Thechemistry-guided approaches will also advance as aresult of faster structure-elucidation technologies,improved computational tools, and better proceduresfor stress-testing and mechanistic investigations. Inaddition, publications about degradation chemistry inthe open literature will be of utmost importance todeveloping this burgeoning field.

Acknowledgement

I would like to thank Sandor Gorog for his invitation towrite this manuscript and for the inspiration he hasprovided by the numerous contributions he has made todrug-impurity profiling. Helpful discussions and ideasfrom Bernard A. Olsen, Timothy J. Wozniak, KallolBiswas, Andreas Kaerner, Todd A. Gillespie, William K.Smith, and Patrick J. Jansen are gratefully acknowledged.



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Analytical Methodologie for Discovering and Profiling Degradation Impurities

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