validación bioanálisis

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Editorial

When we report nal bioanalytical quantitativedata we occasionally eel some degree o anxiety.Are our data correct? Have the guidelines criteria

rom authorities been ullled and did we ollowall our internal standard operation procedures?Are all study data and peripheral data traceableand properly archived? Would the data pass aninspection by authorities i it were to be scruti-nized? To a certain degree, anxiety can be ruit-ul as a tool or quality improvement. However,it must not supersede sound science. Below, someissues are discussed in this respect.

In drug development, rom the regulatory pre-clinical saety toxicology through to the clini-cal investigational phase, validated bioanalyticalmethods are mandatory when quantitative mea-

surements o drug or/and active metabolite(s) areperormed. Validation is not just an investiga-tion and description o a methods perormance,there are also a number o predened criteria(specications) that must be met, or example,the methods precision must be better than 15%and its accuracy must be within 15%. Herewe nd a similarity between GLP and GMP. InGMP there are well-dened specications ordrug substances, or example, detection limitsor impurities (identied and unidentied), deg-radation products or residue solvent. However,

we can also see a dierence: in GMP, a method(monograph) is validated exclusively or thecharacterization and release o a starting mate-rial, a drug substance or a drug product, whereasin bioanalysis a method is oten developed andvalidated or a broader use, or example, or pre-clinical saety and toxicology pharmacokinet-ics (PK), clinical Phase I PK proling, clinicalPhase II PK versus pharmacodynamics, clinicalbioequivalence, clinical Phase III populationPK and so on. O course, this multipurpose useo a method leads to diculties in ormulating

validation criteria suitable or all applications.

When specied validation criteria are at hand,we ulll those without considerations, but whenno specied criteria are available we tend to be

anxious and try to cover every pitall that we canimagine, sometimes even rather ar-etched ones.However, it is important to investigate what isrelevant and scientically sound to validate or,so that we do not end up validating whether ourmethods are infuenced by the absence or thepresence o the ull moon.

Precision better than 15% & accuracywithin 15%These method validation criteria are not onlywidespread and widely applied, they are alsomandatory in all GLP laboratories. However,

we should bear in mind that it might not alwaysbe enough to blindly ulll criteria without ur-ther considerations (oddly enough, it is not rec-ommended to use a method with a precision o20% and improve nal data by analyzing dupli-cates or triplicates). Good precision in combina-tion with poor accuracy constitutes a bias in amethod. Assume the ollowing measurements:accuracy: 98, 115, 115, 116, 116, 117, 117, 118and 120%. The average is 114.7% and the rela-tive standard deviation is 5.6%, such that thecriteria are ullled. In this data set there are

clear indications o a systematic error (bias), theonly counter indication is the relatively smallnumber o data points. Further investigationsshould be carried out and maybe method cor-rections should thereater be implemented.Furthermore, i a method really has an accu-racy o 114.7%, we will in most analytical runslose hal o our quality control (QC) samplesi we use the widely accepted rule 2/3 o theQC samples within 15% o nominal con-15% o nominal con-15% o nominal con-centration. This discrepancy also points to aninconsistency in our handling o data: when we

validate a method we treat accuracy as a normal

Sound science versus anxiety: validation of

bioanalytical assays for small molecules

Leif D Svensson

Active Biotech AB, Box 724,

SE-220 07 Lund, SwedenTel.: +46 (0)46 192000Fax: +46 (0)46 191100E-mail: leif.svensson@

activebiotech.com

Keywords: anxiety n data n regulatory n sound science n validation

Where shall we place the burden of proof when designing validations? We need some guidance in deciding what to

investigate, because we have neither the time nor the money to investigate everything.

SpEcialFocuS: rEid B ioanalyticalForum

2685ISSN 1757-618010.4155/BIO.11.241 2011 Future Science Ltd Bioanalysis(2011) 3(24), 26852689


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distribution characterized by an average and astandard deviation and when we look at QCsamples we treat accuracy as individual values.In addition, when we use the rule 2/3 o the QCsamples within 15% o nominal concentration,

precision worse than 15% is overlooked, as in theollowing example: 98, 102, 106, 107, 202 and311 are acceptable QC % values. Hence, there issometimes room or anxiety even when the cri-teria are ullled. Another example o inconsis-tency in these criteria is that or ligand-bindingassays the acceptance criteria limits are increasedto 20 and 20%. This increase is probably notdue to a belie that PK data or biologicals shouldbe less important than or small molecules, butrather due to pragmatic considerations o whatis achievable. In analogy, it is also said that or

determination at the lower limit o quantica-tion, a precision o


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Stability tests at three concentrationlevelsIn many laboratories, the stability o the analytein plasma is tested at three dierent levels inanalogy with QC samples at three levels o con-

centration (low, medium and high). Hopeully,nobody ever expected an analyte to be stableonly at the medium concentration range or, evenworse, obtained this type o result. Even i sta-bility were tested at two levels o concentrationit would be surprising i stability results woulddiverge because o concentration. I we had datao this kind it would be o common interest toshare them with the bioanalytical communityso we could ormulate a mechanism and try tounderstand this alleged concentration-depen-dent instability phenomenon and determine

whether it was general or an exception. It shouldbe remembered that bioanalysis in most casesdeals with very low analyte concentrations, nomatter whether we dene them as low, mediumor high. I we assume that plasma is composedo water to at least 50%, then the molecules sur-rounding the analyte are predominantly water(with the addition o proteins, ats and salts),because Avogadros constant is a huge numberand it is unlikely that our analyte would meethis brother and be aected by him i it did. Iwe accept this as a reasonable argument it mustalso be applicable to co-administrated drugs

(see below).

Stability tested when a drugis co-administered Years ago when detectors and chromatogra-phy in bioanalysis were not as selective as, orexample, todays UPLCMS/MS technique, itwas sometimes suggested that method selectivityor analytes towards common drugs should betested. Today, at conerence dinners and in cor-ridors, we can hear a similar anxious question:Is the analyte stable in plasma when another

drug is co-administered? Let us assume thata co-administered drug present in plasma to aconcentration in the nanomolar range wouldhave an impact on our analytes stability, whatare the obvious consequences? Our biology doesnot know what substances we happen to deneas drugs and what substances are o dierentorigin. We obtain plasma samples rom smokers,vegetarians and whiskey connoisseurs, amongothers, that wil l contain components not oundin well-controlled healthy volunteers plasmas;a co-administered drug is just the tip o a huge

iceberg and we know very little o the whole

scenery. Can we propose a mechanism in whicha co-administered drug would have an impact onthe stability o our analyte? Do we have any datathat suggest that the presence o a co-admin-istered drug aects the stability o an analyte?

Would this type o investigation really improvethe quality o PK studies, or should we deeatthis anxiety and concentrate on more relevantinvestigations?

Stability in plasma at -20Cversus -70CIt has been discussed [1] whether separate sta-bility investigations are required at -70C istability already has been shown at -20C. Therationale or a requirement o this kind could ocourse not be based on the Arrhenius principle

o chemical reactivity, which states lower rateso chemical reactivity occurring at lower tem-peratures. This is independent o whether theanalyte is regarded as a substrate or a reagent, oris exposed to other reagents, which o course alsoollows the same principle. No, the rationale orsuch a requirement would be based upon obser-vations that plasma samples rozen at -70C ver-sus -20C have dierent consistencies. However,to date there are no such observations and nodata have been presented, nor has any methodor the measurement o plasma consistency beensuggested. However, a proposed mechanism is

that a lower temperature could cause denatur-ation or precipitation o matrix proteins and thatthis could aect protein binding [non-covalent,authors comment] or the ability to extract thedrug rom the matrix [1]. Reversible protein(albumin) binding is a well-known phenome-non and the binding is complicated to measure,owing to weak interaction (membrane dialysisor ultracentriugations are used or the separa-tion o bound and ree raction), so it is notlikely that this would cause a methodologicalproblem. Extraction yield could be aected i

the proteins precipitate in such a way that drugmolecules are entrapped in the plasma precip-itate. We must also keep in mind that manymethods today are based on protein crash (pre-cipitation with organic solvent), which severelydenatures the proteins by aecting the proteinstertiary and quaternary structures. However,despite the risk o entrapment, crashes providecondent extraction yields. However, and mostimportant, we are not now discussing a stabil-ity problem but a yield extraction problem; sowe must also assume that the denaturing eect

that occurs when storage conditions are changed

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rom -20C to -70C is correlated to storagetime, otherwise this could be investigated as asimple reezethaw experiment.

Are expiration dates superior to

QC samples?In the GLP laboratory we label chemicals,reagents and solvents with expiration dates.When a supplier provides a chemical labeledwith an expiration date, this is oten respectedin GLP laboratories due to practical reasons.However, expiration dates are not at all relatedto the intended applications in a laboratory, butto the suppliers specications, and are based onthe act that the specications are met when theproduct is reanalyzed a ter storage. For example,the specications or a methanol product could

be dened with the ollowing:nAssay (GC), minimum 99.9%;

nWater (Karl Fischer), maximum 0.01%;

nAcidity, maximum 0.0005 meq/g;

nEvaporation residue, maximum 0.0005%.

The recommended expiration date could be3 years ater production date or 3 years ateropening. This means that the supplier hasreanalyzed the product ater 3 years o storageor opening and has met the specications. Thedurability ater more than 3 years is unknown

it is not known which parameters will not meettheir specications (and to what extent) ater theexpiration date. Furthermore, a statement to theeect that a product will keep or a speciednumber o years ater opening is convenient orthe user but innite stability beore opening hasprobably not been tested.

In the laboratory, the methanol product inthe above example could be used or analyzingthe water content o a compound (Karl Fischer)or preparing a working standard solution oras a component in a methanol/water HPLC

mobile phase. An open container with meth-anol will allow absorption o water rom theatmosphere because methanol is hygroscopic.Hence, to claim that this methanol will stillbe suitable or water determination accordingto Karl Fischer 3 years ater opening would bequestionable. On the other hand, i the watercontent exceeds the specication with a ac-tor o ten, the methanol would still be suit-able as a mobile phase component. I think thisexample illustrates that in the laboratory, expi-ration dates or solvents should be applied in

relation to intended use and not to suppliers

specications. However, it is inconvenient tolabel the same container with dierent expira-tion dates depending on use, so why not rely oninternal method controls? When determiningthe water content with Karl Fischer, reerence

samples (with known and well-dened watercontent) and blanks should be included in theanalytical run and i these ulll predened cri-teria; this should be superior to rely on, ratherthan the expiration dates o the solvents used.In the same way, calibration, blanks and QCsamples are included in a bioanalytical run andare supposed to refect the reliability o the ana-lysis. Would the use (by mistake) o an expiredsolvent in a mobile phase supersede the resultsrom a successul suitability test, an excellentcalibration graph, brilliant blanks and accurate

QC samples? I so, do we need analytical con-trols or would it not be better to ignore genericstability data or solvents and chemicals and relyon calibration and QC samples? Alternatively, iwe (by mistake) have validated a method withexpired chemicals and all criteria or a robustmethod are ullled during the validation,does this mean that the method could not beused with nonexpired chemicals? Would it beo general interest to randomly select a numbero published LCMS/MS methods (preerablywith stable-isotope internal standards) and thenrun them with expired chemicals to evaluate

the quality impact o expiration dates? Nobodyquestions the relevance o expiration dates orchemicals in GMP production, but is this reallyapplicable in bioanalysis? We should also bearin mind that unnecessary production and dis-posal o chemicals has a negative eect on ourenvironment. Maybe this should cause us someanxiety?

It is paramount that our investigations

should be driven by sound science rather

than by anxiety.

Closing remarksWhere shall we place the burden o proo whendesigning validations? We need some guidancein deciding what to investigate, because we haveneither the time nor the money to investigateeverything. What should be considered worthyo investigation? Is a vague hunch or a meretheoretical possibility without supporting dataenough to initiate investigations? Or, ought weinstead to go or the other extreme and inves-

tigate only proven issues? It is paramount that

Editorial| Svensson

Bioanalysis(2011) 3(24)2688 future science group


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our investigations should be driven by soundscience rather than by anxiety. It would not beoptimal i certain types o investigations wereto be carried out at some laboratories and apraxis were to be created and propagated with-

out thorough discussion and evaluation withinthe bioanalytical community. It would be help-ul i the bioanalytical community could agreeupon where sound science ends and anxietybegins.

Financial & competing interests disclosure

The author has no relevant aliations or nancial involve-

ment with any organization or entity with a nancial inter-

est in or nancial confict with the subject matter or materi-

als discussed in the manuscript. This includes employment,

consultancies, honoraria, stock ownership or options, expert

testimony, grants or patents received or pending, or

royalties.

No writing assistance was utilized in the production o

this manuscript.

References1 Viswanathan CT, Bansal S, Booth B et al.

Workshop/conerence report quantitative

bioanalytical methods validation and

implementation: best practices or chromatographic

and ligand-binding assays.AAPS J. 9(1), E30E42

(2007).

2 Sennbro CJ, Knutsson M, van Amsterdam P,

Timmerman P. Anticoagulant counter ion impact on

bioanalytical LCMS/MS a ssays: results rom

discussions and experiments within the European

Bioanalysis Forum. Bioanalysis3(21), 23932399

(2011).

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