s14 Pharmaceutical Technology ANALYTICAL METHODS 2006 www.pharmtech.com

rug manufacturers invest atremendous amount of timeand money to identify, quan-

tify, and minimize impurities relatedto their drug products so that theUS Food and Drug Administrationcan make informed decisions re-garding drug product purity andsafety. An area of increasing concernand scrutiny for FDA’s Center forDrug Evaluation and Research(CDER) is the potential adulterationof drug products by extractable andleachable compounds that enter adrug from a container, closure sys-tem, disposable, or device.

Addressing this concern, 21 CFR211.94 a) states that:

Drug product containers and clo-sures shall not be reactive, additive,or absorptive so as to alter thesafety, identity, strength, quality, orpurity of the drug beyond the offi-cial or established requirements (1).

FDA provides guidance for protec-tion against extractables and leach-ables in various documents (2, 3).

Therefore, the qualification andquality control of all components

Identification andRisk-Assessment ofExtractables andLeachablesJon S. Kauffman

D

Extractable and leachableissues should be resolvedearly in the drug manu-facturing process. Opencommunication is impera-tive for a successful study.

LA

NC

AS

TE

R L

AB

OR

AT

OR

IES

Jon S. Kauffman, PhD,is the director of methoddevelopment and validation and biopharmaceutical servicesfor Lancaster Laboratories, 2425 New Holland Pike, PO Box 12425, Lancaster, PA17605-2425,tel. 717.656.2300, fax 717.656.2681, jskauffman@lancasterlabs.com.

©2006 Advanstar Communications, Inc.Used by permission.

NONDESTRUCTIVE ANALYTICAL METHODS

s16 Pharmaceutical Technology ANALYTICAL METHODS 2006 www.pharmtech.com

coming into con-tact with the drugformulation is anintegral part of anyFDA applicationprocess.

Extractables andleachables issuesoften are not ad-dressed up frontand ultimately cancause regulatorydelays for the drugmanufacturer. Thedevelopment ofunique packaging,novel formulations and delivery sys-tems, and drug-coated medical de-vices has exacerbated this issue be-cause of the increasingopportunities for foreign materialsto come into contact with drugproducts. In addition, the increasingpopularity of single-use disposablessuch as filters, tubing, and bags forbiopharmaceuticals can introduceunwanted extractables into the finalproduct. (For the rest of the article,the word components will refer tocontainer closures, labels, drug de-livery systems, packaging materials,devices, disposables, and so forth).

The development, validation, andtesting of these components must becarried out under International Con-ference on Harmonization andUnited States Pharmacopeia guide-lines in a laboratory that complieswith current good manufacturingpractices. These activities may be timeconsuming and require expertise anda wide array of analytical techniques.Drug manufacturers may not have

the resources available or may want tokeep these resources focused on de-velopment of new products. There-fore, it is common for drug manufac-turers to outsource these activities tocontract laboratories.

What are extractablesand leachables?Extractables are compounds that canbe extracted from a componentunder extreme conditions such asthe presence of harsh solvents or ele-vated temperatures. These com-pounds can contaminate the drugproduct. Leachables are compoundsthat leach into the drug product for-mulation from the component as aresult of direct contact with the for-mulation under normal conditions.Leachables are typically a subset ofextractables. Sources of these com-pounds include plastic components,elastomers, coatings, accelerants, an-tioxidants, inks, and vulcanizingagents. Phthalates are one specificexample. These carcinogens are

NONDESTRUCTIVE ANALYTICAL METHODS

ToxicologistRisk assessment

safe exposure levels

etc.

requirements

VendorInformation about materials used in

containers,disposables, etc.

LaboratoryAnalytical testing

and data.

Collaborative approach

Figure 1. The collaborative approach to studying extractables andleachables.

Pharmaceutical Technology ANALYTICAL METHODS 2006 s17

added to plastics to make them moreflexible and can be found throughoutthe manufacturing process and inpackaging materials. Other examplesare nitrosamines and polynuclear aro-matic hydrocarbons (PAHs), whichare classes of carcinogenic com-pounds found in rubber. Many drugproducts are distributed or adminis-tered in packages made of plastic andrubber components, and, therefore,phthalates, PAHs, or nitrosaminescould potentially come into contactwith the drug product and be passedon to the patient. Metered-dose in-halers (MDIs), dry-powder inhalers(DPIs), and nebulizers can be com-plex because they may be constructedfrom a myriad of plastic, rubber, andstainless steel components. Neverthe-less, these devices have many advan-tages (e.g., rapid absorption and onsetof activity and reduced dosing) for ef-fective drug delivery.

In summary, extractables must becontrolled to the extent that compo-nents used are appropriate. Leach-ables must be controlled so that thedrug products are not adulterated.

Team approachFor an extractables and leachablesevaluation to be meaningful andsuccessful, it must be a collaborativeapproach on the part of the drugmanufacturer, component vendor,analytical laboratory, toxicologist,and regulatory agency. Each partyhas critical input and is a significantcontributor to the overall process(see Figure 1). The laboratory canonly perform appropriate testing ifall relevant information is provided.

It is critical that vendors providedata on their potential extractablesso that the manufacturer can choosethe appropriate components early inthe process. Vendors often do notmake extraction data available todrug manufacturers, however. Ide-ally, after the extractables study, atoxicologist reviews the data to assessrisk and set maximum levels basedon total daily intake, as defined bythe product dosing. This part of theprocess is typically beyond the scopeof the analytical laboratory. Specificextractables are chosen to be moni-tored in the components and asleachables in the drug product. Atthis point, the laboratory can pro-ceed to the other phases of the study.

Design of studySingle-standard, regulatory-acceptedprocedures are not available. Eachproject is unique. Designing a studyfor a vial is much different than onefor an MDI composed of many parts.In addition, the formulation must beconsidered. Injection products pose agreater safety risk than topical prod-ucts. Studies are carried out in phases(see sidebar,“Various phases ofstudy”) that may be separated by longperiods of time, during which analy-sis, data reduction, and risk assess-ment are performed. Each phase typi-cally is described in a protocol.

Extractable characterization iscarried out first and is probably themost critical because all other deci-sions and testing are based upon it.Lancaster Laboratories (Lancaster,PA) has attempted to formalize aconsistent approach, which is de-

www.pharmtech.com

NONDESTRUCTIVE ANALYTICAL METHODS

s18 Pharmaceutical Technology ANALYTICAL METHODS 2006

scribed in more detail later in thisarticle. The process starts with gath-ering information and continueswith the profiling and characterizingthe extractables. In general, prepara-tion requires both nonpolar and polarsolvents. The instrumentation usedincludes high-performance liquidchromatography–photo-diode array detection–mass spectrometry(HPLC–PDA–MS) for organicsanalysis; gas chromatography–massspectrometry (GC–MS) for organicsanalysis; inductively coupledplasma–optical emission spec-troscopy or mass spectrometry(ICP–OES or ICP–MS) for metalsanalysis; and sometimes ion chro-matography (IC) for inorganics andion analysis. These techniques arecomplementary and provide awealth of information needed toprofile the extractables and leach-ables that may come from the com-ponent. Mass spectrometry is usedbecause it is a powerful tool that elu-cidates structure. Sometimes moreadvanced MS detectors such astime-of-flight (TOF) are used to ob-tain accurate mass information. Itshould be noted, however, that un-ambiguous identification is not al-ways possible. This issue will be dis-cussed later in the article. Once theextraction profile is established, it iscrucial that the toxicologists reviewthe data, perform risk assessment,and propose maximum levels basedon the total daily intake defined bythe product dosing.

After extractables have been char-acterized and qualified, the analyticalmethods are optimized and validated

for the compounds of concern. Then,these methods are used for analysis ofthe components. Typically, severalbatches of components are tested,and suitable specifications are pro-posed for controlling consistency inthe quality of these materials.

Once appropriate components arechosen, analytical methods forleachables in the drug product aredeveloped and validated. Samples ofthe drug products, which have beenin contact with the components foran extended length of time, aretested. In case such samples are notavailable, the drug product andcomponent are stressed under ap-propriate conditions to generate theleachables. Note that the leachablesmay be the same compounds asthose identified during extractablestudies or their chemical identitymay be different from the extracta-bles because of drug product inter-action. All major compounds andtarget compounds that do not haveorigins in the drug substance or ex-cipients are identified and quanti-fied. These goals can be accom-plished by making a formulation inan inert glass container to excludethe leachables originating from thecomponents.

• extractable characterization (or controlledextraction study);

• extractable method optimization;• extractable method validation;• routine extractable testing;• leachable method development;• leachable method validation;• leachable testing (stability samples).

Various phases of study

www.pharmtech.com

NONDESTRUCTIVE ANALYTICAL METHODS

s20 Pharmaceutical Technology ANALYTICAL METHODS 2006

The remainder of the article willdiscuss this critical first phase.

Protocol for extractablecharacterizationTo develop an accurate risk assess-ment of compounds that can be ex-tracted or leached from a givencomponent, the component mustfirst be exposed to extreme solventsand conditions to generate every po-tential extractable. The goal is toquantify and qualify these com-pounds if it is analytically possible.Lancaster Laboratories has been fol-lowing a consistent protocol whenapplicable. The general protocol isdescribed below and is modifiedbased on the component and drugproduct to be investigated.

For organics analyses, each com-ponent is prepared so that the maxi-mum exposure of the component tothe extraction solvent can be attain-ed. United States Pharmacopeia Gen-eral Chapter ^88& Biological ReactivityTests recommends that the total sur-face area (both sides combined) tovolume of solvent ratio be 60 cm2:20mL (4). The components often arecut or crushed to maximize surfacearea. They undergo a variety of sol-vent extractions, typically employingeither a Soxhlet extractor or sonica-tion. For example, components maybe exposed to hexane (i.e., nonpolar,organic), alcohol (i.e., polar, organic)and water (i.e., aqueous) and heatedfor a specified amount of time. Afterextraction, internal standards areadded to monitor system perform-ance and to define lower detectionlimits for the analyses.

For metals analysis, samples areprepared for ICP by placing aknown amount of the component ina beaker and then adding deionizedwater and concentrated nitric acid.The sample is covered and heated atspecified temperatures for a speci-fied amount of time. Then, the sam-ples are diluted into deionized waterand analyzed by ICP–OES orICP–MS.

For each set of preparations,blank solutions containing the sol-vents without the component areprepared and analyzed in the samemanner as the test samples. Thisprocess ensures that no contamina-tion from the labware or reagents ispresent that could be interpreted asan extractable. Postextraction stepsmay include filtration, concentra-tion, and exchange of solvent to im-prove detection.

Hexane and alcohol extractionsare analyzed by GC–MS. Blanksspiked with internal standard arefirst injected five times, and the per-cent relative standard deviation (%RSD) is determined to ensure thesystem is operating properly. In addi-tion, the response is used to establishthe detection limit. The mass spectraof detected extractable compoundsare compared with the NIST98Kdatabase for identification. Althoughthe database is used to assist in iden-tifying the extractable components,the identification of each componentcannot be guaranteed. All extractablepeaks are quantitated on the basis ofthe average area response for the in-ternal standard, assuming equivalentresponse factors.

Pharmaceutical Technology ANALYTICAL METHODS 2006 s21

Water and alcohol extractions areanalyzed by HPLC. The HPLC sys-tem is configured with PDA and MSdetection and the mobile phase iswater–acetonitrile or methanol. ThePDA is used to screen the samples.Again, blanks spiked with internalstandard are first injected five timesand the % RSD is determined to en-sure the system is operating prop-erly. The response is used to estab-lish the detection limit. The MS isset first in the electrospray positivemode, then the negative mode, andfinally chemical ionization in thepositive mode.

The identification of the ex-tractable compounds is attemptedbased on the molecular weight of thecompound and compared to the mo-lecular weight of known chemicalcompounds. The industry currentlyhas no comprehensive databases forassistance in identifying liquid chro-matography (LC)–MS-generatedmass spectra. If a compound cannotbe identified, an attempt is made toidentify a class of compound for theextractable. Sometimes an accurate

mass determination can be madeusing an LC–MS TOF. All extractablepeaks are quantitated based on theaverage area response for the internalstandard, assuming equivalent re-sponse factors.

The acid-digested sample is ana-lyzed by ICP–MS. Once digested, thesample can be analyzed for morethan 25 elements simultaneouslywith detection limits in the part-per-trillion to part-per-billion range.

Mass spectroscopic identificationAs mentioned previously, mass spec-trometry is a powerful tool forstructural elucidation. Still, it is notalways possible to make an indis-putable identification of every peak(or compound) detected. Each ofthe different mass spectrometrytechniques has its advantages anddisadvantages. GC–MS data consistof fingerprint patterns that can becompared with large databases oforganic compounds. Identificationoften can be performed with a highdegree of confidence, and certifiedstandards can be purchased for con-

• aluminum;• antimony;• bismuth;• butylated hydroxy

toluene;• calcium;• cobalt;• decalin;

• dibutylphenols;• ethylbenzene;• iron;• magnesium;• miscellaneous

alkanes;• palladium;• palmitic acid and

esters;• phthalates;• platinum;• selenium;• silicon;• siloxane;• stearic acid and

esters;

• strontium;• styrene dimers;• styrene trimers;• titanium;• vitamin E;• xylenes;• zinc;• zirconium.

Common extractables and leachablesThe list below contains common extractables that have been identified in components made of plastic,rubber, films, and so forth.

www.pharmtech.com

NONDESTRUCTIVE ANALYTICAL METHODS

s22 Pharmaceutical Technology ANALYTICAL METHODS 2006

firmation. The compoundsamenable to GC, however, onlymake up a minority of the possibleorganic compounds that may bepresent in a component. Further-more, some classes of compoundssuch as alkanes yield very similarfingerprint patterns or fragments,and thus a specific molecular entitycannot be identified.

LC–MS data typically only providea few ions, not a fingerprint pattern asthose observed with GC–MS. Otheradvanced techniques such as tandemmass spectrometry (MS–MS) andTOF can provide more informationsuch as molecular fragments and ac-curate mass. Database matching,however, is not achievable as it is withGC–MS. Orthogonal approachessuch as nuclear magnetic resonance(NMR) and Fourier transform in-frared (FTIR) spectroscopy also canbe used to try to solve this sometimescomplex puzzle.

At some point, the toxicologistshould perform a risk assessment.These other analytical techniquescan be time-consuming and expen-sive and do not always yield answers.Sometimes the data must be lookedat from a different perspective. Forexample, an extractable is detectedat 50 ppb from a component sampleexposed to a strong solvent at hightemperature for an extended periodof time. The drug product is formu-lated in an aqueous buffer, exposedto this component for a relativelyshort amount of time, and must bekept refrigerated. Therefore, the ex-tractable would probably not leachinto the final product. The effort put

into method development, analyticaltesting and identification must beweighed against the risk.

ConclusionQualification and quality control ofcomponents that come into contactwith the drug formulation is an in-tegral part of any US Food and DrugAdministration application. There-fore, extractable and leachable issuesshould be investigated and resolvedearly in the process. For a successfulstudy, it is imperative that the com-ponent vendors, laboratories, toxi-cologists, and the regulatory agencyhave open, effective, and timelycommunication. As new delivery de-vices, disposables, and medical de-vices are developed, it is certain thatFDA will continue to demand infor-mation about these components sothat the agency can make informeddecisions on risk and safety.

References1. Code of Federal Regulations, Title 21,

(Office of Federal Register, Washing-ton, DC, April 2005), Part 211.94,p. 128.

2. US Food and Drug Administration,Guidance for Industry: Container Clo-sures Systems for Packaging HumanDrugs and Biologics (FDA, Rockville,MD, May 1999), pp. 1–56.

3. FDA, Guidance for Industry: MeteredDose Inhaler (MDI) and Dry Powder(DPI) Drug Products (draft, FDA,Rockville, MD, October 1998), pp.1–65.

4. United States Pharmacopeia 28—National Formulary 23, (US Pharma-copeial Convention, Rockville, MD,2005) p. 2269. PT