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T HE FDA REGULATES THE PRESENCE of impurities in APIs,formulated drug products, food ingredients and cos-metics, and sets thresholds at which these impurities

must be identified or adequately tested in safety and clinicalstudies. Investigation of these impurities must be initiatedduring the early stages of drug or product development,such as the pre-investigational new drug (pre-IND) stage.

Modern mass spectrometry (MS) and nuclear magnetic reso-nance (NMR) techniques can have a tremendous impact on thespeed and sample requirements for structure elucidation oftrace impurities or metabolites. A rapid protocol used for theisolation and characterization of trace impurities in drug sub-stances and formulated drug products has been developed. Theapproach has been successfully employed for the characteriza-tion of several hundred impurities frommore than 130 differentsubstances covering a wide variety of structural classes.

The sources of impurities vary greatly. These couldinclude starting materials, intermediates, by-products of thesynthesis of an API, or degradation products of the API or itsimpurities arising during manufacture or storage. Impuritiesmay also be present that are not related to the API, arisingfrom the synthetic or extractive process or as a result of con-tamination from unrelated chemicals. The stability of APIs is

determined using short- and long-term stability studies withresulting degradants often requiring identification. In addi-tion, new impurities can suddenly appear during drug orproduct development due to changes in the synthetic proto-col, starting materials, source of starting materials, or evenvariability as processes are scaled up.

The International Conference on Harmonization (ICH)sets standards for the purity of drug substances1 and drugproducts.2 These guidelines set levels at which impuritiesmust be reported, identified, or qualified and vary depen-dant on the dosage. For example, for drug substances to begiven at levels of less than two grams per day, the guidancestates that impurities between 0.10% and 0.15% should beidentified and those that reach 0.05% must be reported.Impurities present at levels greater than or equal to 0.15%must be evaluated according to the ICH standards.

Dennis J. Milanowski is senior scientist II, Structural Analysis atAlbany Molecular Research, Inc., Bothell Research Center. He canbe reached at dennis.milanowski@amriglobal.com. Ulla Mocek issection head, Structural Analysis at the same AMRI center. She canbe reached at ulla.mocek@amriglobal.com.

Trace ImpurityIdentification

Trace ImpurityIdentification

A combination of spectrometric and spectroscopic techniques

By Dennis J. Milanowski and Ulla MocekAlbany Molecular Research, Inc.

Photo

courtesy

ofAMRI

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Similar regulations provide specifications for food andcosmetic ingredients, which set thresholds at which impuri-ties need to be reported, identified, and qualified. The earlyisolation and identification of impurities often allowsimprovements or modifications in the synthetic pathway orpurification process, which can prevent the formation of animpurity or reduce it to sub-threshold levels.

The Impurity ID Isolation andCharacterization ProcessOur rapid protocol used for the isolation and characteriza-tion of trace impurities in drug substances and formulateddrug products uses a combination of spectrometric and spec-troscopic techniques to analyze impurities during and afterisolation, which minimizes the total analysis time. The appli-cation of capillary NMR facilitates this process by reducingthe amount of an impurity that must be isolated to allowacquisition of NMR data, which is traditionally the techniquerequiring the most sample.

Capillary NMR is a powerful tool for structural elucidationdue to the ease of use and the fact that only approximately20 to 50 micrograms of an impurity need to be isolateddepending on the set of NMR experiments required. Thisneed to isolate only minute amounts for MS and NMR analy-sis accelerates the isolation and structure elucidationprocess. The reduced sample requirements of capillary NMRare also advantageous in cases where the amount of animpurity that can be isolated is limited due to limitations ofthe starting material. The following impurity identificationprotocol has been applied in the isolation and characteriza-tion of a large number of impurities in drug substances andproducts as well as impurities in food ingredients and ofdrug metabolites. This capability also logically extends tothe analysis of Schedule I to Schedule V controlled sub-stances and potent compounds.

An outline of the impurity characterization workflow isprovided in Figure 1. API or drug product is provided con-taining the impurity or impurities of interest. Typically any-where from one to five impurities are present in the sampleat the 0.01% to 0.5% (w/w) level. An analytical liquid chro-matography (LC) method is also commonly available and, ifamenable, this analytical method is used to acquire MS databy LC/MS and to follow the scaled-up isolation of the impu-rities. The MS data can often provide the molecular weightof the impurity, which is typically the first piece of structur-al information obtained. The use of non-volatile mobilephase additives, such as ion-pairing agents or phosphatebuffers, which was common before the routine availability ofLC/MS, precludes the in-line acquisition of MS data.

If the analytical LC method is not compatible with MS,either an alternate LC/MS compatible analytical methodmay be developed or the acquisition of MS data will beundertaken after isolation of the impurity. Tracking the isola-tion by LC/MS allows confirmation that the correct com-pound has been isolated and that the impurity is stable.Many impurities are found to be unstable during isolationand require special handling during isolation and concentra-tion, such as limiting exposure to light or moisture, orremoval of acidic mobile phase modifiers. In extreme cases itmay be necessary to isolate and characterize the degradant ofan impurity, which can then allow the structure of the origi-nal impurity to be inferred.

To facilitate isolation of the target impurity, the analyticalmethod may be scaled up. Method development or modifi-cation is often necessary at this stage if the analytical methodis not easily applicable at the larger scale. This typicallyinvolves the substitution of non-volatile mobile phase addi-tives, an optimization of the separation between the impuri-ty of interest and other components in the sample, and/or areduction of the total separation time.

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Figure 1: General outline of the impurity isolation andstructure elucidation work flow.

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Formulated drug products also often require additionalworkup at initial stages to separate the API and relatedsubstances from excipients present in the formulation. Thistypically involves dissolving or suspending solid formula-tions in a suitable solvent, followed by filtration or centrifu-gation to remove insoluble excipients, which may be fol-lowed by one or more solvent or solid phase extraction steps.

Once a suitable LC method has been determined, the nextstep is to enrich the impurities by depleting the sample of themajor component followed by additional purification of theindividual impurities as needed. One process used is reverseor normal phase high-pressure liquid chromatography(HPLC) at the semi-preparative or preparative scale for iso-lation. Once an isolation method has been optimized, thepurification of the impurity can be automated using a WatersAutopurification system, which allows peak collection trig-gered by mass, ultraviolet, or evaporative light scattering(ELS) detection.

The typical structural characterization methods includeLC/MS, LC with tandem MS detection (LC/MS/MS), highresolution mass spectrometry (HRMS), and 1D and 2D NMRexperiments using either a capillary or standard 5 millimetergradient NMR probe. MS data can often be acquired duringthe isolation procedure on the starting material or partiallypurified fractions, which speeds up the overall process.Preliminary NMR data is used to monitor purity with afull NMR dataset being acquired once a sample of sufficientpurity and quantity has been obtained. In cases of unstableor volatile compounds LC/NMR can be the method of choice.

It is an important tool to generate NMR data in-line afterseparation of mixtures, but its drawbacks include limitedLC methods available due to the need to use deuteratedsolvents, as well as the potential overlap or masking ofcritical signals in the NMR spectrum with the mobile phasecomponents.

Initial characterization by LC/MS facilitates the isolationof the selected impurities, as well as provides preliminarystructural information. Ionization can be achieved for mostsubstances using electrospray (ESI) or atmospheric pressurechemical ionization (APCI). An example is provided by therecently completed characterization of five impurities (SeeFigure 2, Compounds 2-6) of AMRI’s ALB109654(a),Compound 1, a novel tubulin inhibitor under develop-ment.3,4 LC and MS analysis provided the molecular weightfor each component. Based on the MS data, Compound 2 wastentatively assigned as vinblastine, which is a starting mate-rial in the synthesis, and Compound 3 was determined to bean isomer of the parent Compound 1.

Acquisition of HRMS data can allow a determination ofthe molecular formula in most cases or at a minimum narrowthe list of possibilities. In Figure 2, for example, the molecu-lar weight and isotope pattern observed for Compound 4 byLC/MS suggested substitution with bromine, which wasconfirmed by accurate mass determination. Similarly,Compound 5 was found to result from the substitution withiodine, and Compound 6 was found to result from loss of amethyl group. Fragmentation data obtained fromLC/MS/MS experiments often yields information on theregion of the molecule that has been changed and is crucialfor structure identification. A comparison of the fragmenta-tion pattern of the parent compound with that ofCompounds 4-6 indicated that each of these impurities wasmodified in the lower indoline moiety.

In less complex structures, MS and MS/MS data may allowa structure to be proposed without the need to isolate materialfor NMR analysis. In more complex structures, such as theexample described here, NMR data is also required to completeor confirm the structural assignment. The structure ofCompound 2 was confirmed by a comparison of the 1H NMRspectrum with that of an authentic sample of vinblastine.The use of a capillary NMR probe required isolation of lessthan 50 µg of pure compound that was obtained by semi-preparative HPLC in a single run from 8 mg of substrate.Similarly, capillary NMR analysis of Compound 3 allowedits structure to be determined as the positional isomer of thetubulin inhibitor, Compound 1, which has the SCH3 sub-stituent at C-13’. NMR data was also used to determinethat both Compounds 4 and 5 had the halogen substituent atC-17. Depending on the impurity, NMR datasets can

Figure 2: Impurity characterization work carried out during thedevelopment of one novel tubulin inhibitor (1) identified fourimpurities (2-5). A fifth impurity (6) was identified as adegradant, which was observed to form during subsequentstability studies.4

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include 1D 1H NMR, 13C NMR, and DEPT spectra as well as2D 1H-1H COSY, TOCSY, HSQC, HMBC, NOESY or ROESY,and HSQC-TOCSY spectra. MS and NMR data of the drugsubstance, starting materials, and intermediates are alsoacquired during the process to provide a set of comparisondata for analysis of the structure of the impurity.

Degradants arising from the manufacture or storage of adrug are characterized in the same manner. Compound 6 wasfirst observed during an accelerated stability study of theparent compound. Subsequent isolation and characterizationindicated that this degradant arises by hydrolysis of theester at C-3. Once the structures of the impurities havebeen determined it is often possible to suggest plausible routesof formation relating the impurities to each other andthe substance under study. This information can then beused to alter the synthetic (or storage) conditions to reducetheir accumulation.

Most of the impurities we have identified were present indrug substances or drug products prepared by syntheticprocesses or in some cases by fermentation. However, our pro-tocol is also applicable within the food and beverage industry.For example, it is well known that the major constituents of theleaves of S. rebaudiana are the remarkably high-potency, low-calorie sweeteners stevioside, rebaudiosides A and C, and dul-coside A; glycosides of the diterpene steviol, ent-13-hydrox-ykaur-16-en-19-oic acid.6 Extracts of the leaves of S. rebaudianahave been used for centuries to sweeten food and beverages inJapan, South America, and China.7 Purification of the crudeextract obtained from the leaves of S. rebaudiana resulted in theisolation of two new minor diterpene glycosides, along withthe known steviol glycosides stevioside, rebaudiosides A-F,and dulcoside A. During another study,8 the viability of thesweetener monatin9 was tested in a model lemon-lime bever-age system. A total of seven compounds were identified asmajor degradation products of monatin, which included twonovel structures.

In addition to the isolation and characterization of impuri-ties, our protocol is applicable to characterization of drugmetabolites. A number of metabolites have been isolated andidentified frommixtures generated by either human or rat livermicrosome preparations, or isolated from plasma samples orurine.10 Unlike other sources, the amount of a metabolitepreparation or extractable biological fluid is often limited,requiring isolation and identification of metabolites, which canonly be isolated in µg quantities.

In conclusion, modern MS, NMR, and separation tech-niques can have a tremendous impact on the identification andstructure elucidation of trace impurities or metabolites. Wehave developed and employed an impurity ID protocol to

characterize several hundred impurities from more than 130different substances covering a wide variety of structuralclasses. Our impurity ID protocol has proven useful in the iso-lation and characterization of a large number of impurities indrug substances and drug products as well as impurities infood ingredients and drug metabolites.

With continuing advances in separation and spectroscopictechniques, modern technology has allowed a formerly ardu-ous and difficult process to become easier to achieve in a rela-tively straightforward and rapid series of steps. �

References1 International Conference on Harmonization (ICH), Guidelinesfor Industry, Q3A, Impurities in New Drug Substances(Revision 2), June 2008.

2 International Conference on Harmonization (ICH), Guidelinesfor Industry, Q3B, Impurities in New Drug Product (Revision2), July 2006.

3 Voss ME, Ralph JM, Xie D, Manning DD, Chen X, Frank AJ,Leyhane .J, Liu L, Stevens JM, Budde C, Surman MD, FriedrichT, Peace D, Scott .L,Wolf M, Johnson R, Bioorg. Med. Chem.Lett. 2009; 19: 1245-1249.

4 Milanowski DJ, Keilman, J, Guo, C, Mocek, U, J. Pharm. Biomed.Anal., 2011; 55: 366-372.

5 Chaturvedula SP, Rhea J, Milanowski D, Mocek U, Prakash I,Nat. Prod. Comm., 2011; 6: 175-178.

6 Brandle JE, Starrratt AN, Gijen M, Can. J. Plant Sciences, 1998;78: 527-536.

7 Genus JM. Phytochemistry, 2003; 64: 913-921.

8 Upreti M, Somayajula KV, Milanowski DJ, Kowalenko P, MocekU, San Miguel R, Prakash I, Food Chemistry, 2012; 131: 413-421.

9 Vleggaar R,Ackerman, LG J, Steyn PS, J. Chem. Soc., PerkinTransactions 1: Organic and Bio-Organic Chemistry (1972-1999), 1992; 22: 3095-3098.

10 Unpublished results.

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