J Ph & Bio Analysis

8/7/2019 J Ph & Bio Analysis

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BIOANALYTI CAL METHOD VALIDATION, SAMPLE ANALYSIS AND DATA REPORTING 91sensitive GC-MS instruments have greatlyenhanced the power of analytical chemists toproduce highly sensitive and specific methods.S tab i l i t y

To ensure that compound integrity is main-tained throughout the workup process, certainstability tests should be performed during thefinal stages of method development.

for injection at time 0, intermediate times andafter the expected maximum delay beforecompletion of a batch of samples, e.g. 24, 36,or 48 h. By pooling the extracted QCs at eachconcentration, the starting concentration forall aliquots is identical, and the comparison ofdetermined concentrations following storage tothe time 0 concentration is independent of theextraction reproducibility.

A n a ( v t e s t a b i l i t y . Published literature shouldbe investigated, or laboratory tests should beconducted to determine whether pure analyteand/or solutions of the analyte (e.g. drug,metabolite(s) and internal standard) are stableunder normal laboratory conditions of heat,humidity, light and air exposure.

I n p r o c e s s s t a b i li t y . It must be demonstratedthat drugs remain intact if left for several hoursat room temperature in the biological matrix.Certain analytes, e.g. captopril, acetyl salicylicacid, etc. undergo immediate changes/degrad-ation in the biological matrix. In such cases,appropriate additives, e.g. enzyme inhibitors,anti-oxidants and/or derivatizing agent may beadded. Reduction of the collecting devicetemperature might improve stability. It may benecessary to extract samples immediately aftercollection, and store the dried frozen residuefor later reconstitution and analysis. If thisprocedure is used, the calibration standardsand QC samples must be treated in the samemanner.It should also be noted that some matricesencountered at intermediate stages of sampleworkup such as buffered biological matrix andthe back-extraction medium may affect thestability. Again, if instability of analytes isobserved at any of, these stages, appropriateadditives should be considered, or processingtemperature reduced.

P r o c e s s e d s a m p l e s t a b i l i t y . Reconstitutedextracted samples must remain stable in thereconstitution solvent at the temperature of theauto-injector until they are injected, as well asat other lower temperature(s), e.g. 4C, toallow for storage of prepared samples thatcannot be immediately chromatographedowing to instrument problems. Processedsample stability should be examined in repli-cate at each QC concentration. After recon-stitution, replicate samples of each QC concen-tration should be pooled, mixed and aliquoted

R u g g e d n e s sIf it is discovered late in the validationprocess that a method is not easily transferredbetween systems, analysts, or analyticalcolumns of the same type, further developmentand revalidation becomes necessary. Tominimize the chances of this occurring, thefollowing should be investigated during thelatter stages of development.

C o l u m n e f f e c t . Analyses should be per-formed on at least two columns containing twodifferent lots of the identical packing material,preferably one column which was used formethod development and the other previouslyunused.

M o b i l e p h a s e e f f e c t. For HPLC methods, theeffect of small variations in the mobile phasesolvent ratio (i.e. less than 2% of the amountof each component), and buffer pH wereapplicable, should be examined and reported.For GC methods, other parameters such assmall variations in oven temperature and gasflow rates should be examined.Assessment of the above parameters shouldprovide an indication of the method's ability tomaintain critical separations when faced withexpected day to day mobile phase variations,and column to column variability.

R u g g e d n e s s a c c e p t a b i l i t y . Peak shapes andresolution from other peaks in the matrix mustremain visually acceptable. The limit of quan-titation must demonstrate a reproducibleresponse readily distinguishable from the noiselevel.

I n t e r n a l s t a n d a r d ( I S )Many bioanalytical methods are based onthe internal standard quantitation method.Internal standard is added at the earliest stageof sample preparation to minimize error bycompensation. It is commonly believed that agood IS should be structurally similar to the


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92 DARIOUSH DADGARet al.analyte under analysis. Although the abovestatement is generally true, there are occasionswhen it is not so. Such was the case in liquid-liquid extraction method followed by HPLCanalysis of the tricyclic antidepressant doxepin,and its metabolite, nordoxepin (Dadgar, un-published data). The structurally similar drug,imipramine did not minimize error by compen-sation for nordoxepin, whereas it successfullydid so for doxepin in spiked samples. This isbecause parent and metabolite differed fromeach other in their extractabilities under theextraction conditions used. Under this circum-stance, it was necessary to use two internalstandards, imipramine as IS for doxepin, anddesipramine as IS for nordoxepin.

Two other important criteria for choosing aninternal standard are:(i) it should elute close to the retention time

of the analyte of interest. Therefore, forsome multicomponent analyses two ormore internal standards are needed;(ii) it should normally elute after the parentdrug peak (where reversed-phasechromatography is used) so that thepossibility of interference with faster elut-ing more polar metabolites is obviated.

M e t h o d V a l i d a t i o nIn this section, commonly encounteredapplication issues in bioanalytical method

validation are discussed under the heading ofeach validation criterion.C a l i b r a t i o n c u r v e

The joint conference report [1] states thatbatch acceptance should be based on QCacceptance criteria. However, bioanalyticallaboratories react differently and somewhatarbitrarily regarding inclusion or exclusion of astandard point deviating greatly from the cali-bration curve. Some leave it in the curve andsome drop it. As it happens, there are timeswhen, for example, the LOQ standard maydeviate by, say 40%, but the next point in thecalibration curve demonstrates a minor devi-ation from nominal. However, dropping theLOQ standard causes the next point to deviategreatly from the nominal value.Another example of a commonly encoun-tered problem is where removal of a givenstandard causes a QC to fail to meet itsacceptance criteria when the QC would other-wise have been within the acceptable range. In

light of the above, inclusion/exclusion of pointsin the standard curve should be established ap r i o r i , and the following provides possibleguidelines for this.

Provided the calibration curve consists of atleast seven non-zero single standards, up totwo non-zero standrds may be removed fromthe calibration curve if at least one of thefollowing valid reasons exists and a minimumof five non-zero standards remain in the curve.(a) loss of sensivitity;(b) poor chromatography;(c) loss during sample processing;(d) if, when included in the calibration curve,

it clearly biases the QC results, and theback-calculated standard concentrationdeviates substantially from its nominalvalue.

In many bioanalytical laboratories, the cali-bration curves and QC samples are preparedsimultaneously and frozen for storage,normally at the same temperature as is in-tended for storage of biological samplesderived from clinical/toxicological samples.However, some laboratories prefer to preparecalibration curves fresh with each batch ofsamples analysed. The advantage of preparingand freezing calibration curves is that the effectof time and possible degradation of analyte(s)is the same for the calibration samples and thestudy samples.

In order to generate an accurate "analytical"calibration curve independent of possible timeeffect, the calibration curve may be preparedfresh with each batch of samples while QCsamples are prepared and stored frozen withstudy samples in order to account for the "timeeffect". A simple approach to using freshcalibration curves is to prepare a series ofworking calibration standards at concen-trations 10 or 20 times greater than thoseintended for biological standards, in a suitabledilution solution such as i:1 methanol-water.These working calibration standards may bestored provided stability is previously demon-strated over the maximum period over whichthey will be stored. Then, on a daily basis,blank biological matrix is spiked with theworking calibration standards in a ratio of, e.g.1 : 2 0 working standard:biological blank.Dilution of biological matrix with workingstandards will be compensated for by adding anequal volume of the working standard dilutionsolution (free of analyte(s)) to the studysamples.


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BIOANALYT ICAL METHOD VALIDATION, SAMPLE ANALYSIS AND DATA REPORTING 9 3Nu m ber o f ca libra tion curves

T h e q u e s t i o n o f h o w m a n y c a l ib r a t i o n c u r v e st o r u n w i t h e a c h b a t c h s h o u l d b e a n s w e r e d b yc o n s i d e r a t i o n o f w h e t h e r o r n o t s t u d y s a m p l e sa r e r u n , f o r e x a m p l e , s i n g l y , in d u p l i c a t e o r i nt r i p l i c a t e . I f s t u d y s a m p l e s a r e r u n s i n g l y , t h e na s i n g l e c a l i b r a t i o n c u r v e s h o u l d n o r m a l l y b eu s e d . I f r e p l i c a t e s o f s t u d y s a m p l e s a r ea n a l y s e d , t h e n i d e n t ic a l r e p l i c a ti o n o f s t a n d a r dc u r v e s i s d e s i r a b l e .

I n o r d e r n o t t o m a s k t h e a c c u r a c y o f t h em e t h o d , c a l i b r a t i o n c u r v e s s h o u l d b e p r o -c e s s e d i d e n t i c a ll y d u r i n g v a l i d a t io n a n d d u r i n gs t u d y s a m p l e a n a l y s i s .

Validat ion o f par t ia l or increased sa mp lev o l u meA c o m m o n d i f fi c u lt y a r is e s d u r i n g b i o l o g i ca l

s a m p l e a n a l y s i s w h e n l e s s t h a n t h e v a l i d a t e ds a m p l e v o l u m e i s a v a i l a b l e , a n d a p a r t i a lv o l u m e m u s t b e u s e d f o r a n a l y s i s . T h i s p r o -c e d u r e n e e d s t o b e v a l i d a t e d . I n a d d i t i o n ,s a m p l e s a n a l y s e d a n d f o u n d t o b e a b o v e t h ec a l i b r a t i o n c u r v e r a n g e r e q u i r e d i l u t i o n f o rr e a n a l y s i s .

A s i m p l e f o r m o f v a l id a t i o n f o r p a r ti a ls a m p l e v o l u m e i s t o u s e 1 /4 , 1 /3 a n d 1 /2 o fr e p l i c a te v o l u m e s o f Q C s a m p l e s o f a p p r o -p r i a t e c o n c e n t r a t i o n s . Q C c o n c e n t r a t i o n s a r ec h o s e n b o t h a b o v e t h e c a l i b r a t i o n c u r v e r a n g et o d e m o n s t r a t e a c c u r a t e d i l u t i o n t o w i t h i n t h er a n g e , a n d a t c o n c e n t r a t i o n s s u c h t h a t o n c ed i l u t e d , t h e y a r e n e a r , b u t n o t b e l o w t h e L O Q .T h e p a r t ia l s a m p l e v o l u m e is b r o u g h t u p t o t h ev a l i d a t e d v o l u m e b y a d d i t io n o f b l a n k m a t r i x ,a n d a n a l y s e d . T h e c a l c u l a t e d c o n c e n t r a t i o n s o fd i l u t e d Q C s , w h e n m u l t ip l i e d b y t h e i rr e s p e c t i v e d i l u t i o n f a c t o r s m u s t f a ll w i t h i n t h ed e f i n e d p r e c is i o n a n d a c c u r a c y c r i te r i a f o r th a tQ C .

O c c a s i o n a l l y , i t m a y b e d e s i r a b l e t o i n c r e a s et h e v o l u m e o f s a m p l e m a t ri x , c o m p a r e d w i tht h a t o f th e c a l i b r a t i o n s t a n d a r d s , s o t h a t t h ei n s t r u m e n t r e s p o n s e i s w i t h i n t h e s t a n d a r dc u r v e ra n g e . F o r e x a m p l e , w h e n a n a l y s i n gu r i n e , i t m a y b e n e c e s s a r y t o u s e a l a r g e rv o l u m e t o o b t a i n r e l i a b l e c o n c e n t r a t i o n s o w i n gt o a l a r g e v o i d v o l u m e . T h e i n t e r p o l a t e dc o n c e n t r a t i o n , c o r r e c t e d f o r v o l u m e , c a n b eb e l o w t h e L O Q c o n c e n t r a t i o n e s t a b l i s h e d f o rt h e a s s a y , p r o v i d e d t h a t t h e r e s p o n s e i s a b o v et h a t o f t h e L O Q s t a n d a r d . V a l i d a t i o n isn e c e s s a r y t o s h o w t h a t s e l e c t i v i t y is n o t c o m -p r o m i s e d a n d t h e p r e - d e f i n e d c r i t er i a f o r

a c c u r a c y a n d p r e c i s i o n a r e m e t w i t h th e l a r g e rs a m p l e v o l u m e .Q C s a m p l e s c a n b e p r e p a r e d a t , e . g . 1 /2 a n d

1 /4 t h e L O Q f o r re p l i c a t e e x t r a c t io n o f d o u b l ea n d q u a d r u p l e t h e u s u a l e x t r a c t i o n v o l u m e ,r e s p e c t iv e l y . T h e s e L O Q Q C s a r e a n a l y s e da g a i n s t t h e c a l i b r a t i o n c u r v e e x t r a c t e d u s i n gt h e u s u a l v o l u m e . T h e u s u a l p r e c i s i o n a n da c c u r a c y c r it e r ia s h o u l d b e m e t . I n a d d i t io n ,s e l ec t iv i t y t e st s s h o u l d b e c o n d u c t e d u s i n g t h ei n c r e a s e d b l a n k u r i n e v o l u m e s .Li mi t o f q u a n t im t i o n

T h e l im i t o f q u a n ti t a ti o n ( L O Q ) m u s t b ed i f f e r e n t i a t e d f r o m t h e l i m i t o f d e t e c t i o n( L O D ) . T h e v a l u e o f t h e L O D is t h e s m a l le s tc o n c e n t r a t i o n t h a t c a n b e d i s t i n g u i s h e d f r o mt h e b a c k g r o u n d n o i s e . T h e l i m it o f d e t e c ti o nc a n b e d e f i n e d i n d i f f e r e n t w a y s . H o w e v e r , n om a t t e r h o w t h e l im i t o f d e t e c t i o n is d e f i n e d ,t h e l im i t o f q u a n t i t a t i o n s h o u l d b e a t le a s tt w i ce th e r e s p o n s e o f t h e L O D , a n d w i t h i n th ep r e - d e f i n e d a c c u r a c y a n d p r e c i s io n b o u n -d a r i e s , n o r m a l l y w i t h in _ +2 0% o f n o m i n a l w i tha R S D - < 2 0 % .

I t i s i m p o r t a n t t h a t t h e a c c u r a c y a n d p r e -c is io n o f t h e L O Q b e o b t a i n e d u si ng L O Q Q Cs a m p l e s , i . e. i n d e p e n d e n t l y f r o m t h e c al i-b r a t i o n c u r v e , b e c a u s e t h e L O Q s t a n d a r dw h i c h is u s e d t o c o n s t r u c t t h e c a l i b ra t i o n c u r v ei n f l u e n c e s t h e r e g r e s s i o n e q u a t i o n . I n t h i sm a n n e r , t h e p r e c i s io n a n d a c c u r a c y o f t h eL O Q c a n b e d e f i n e d i n b o t h i n t e r - a n d i n t r a -b a t c h t e s t s. T h e p r a c t i c e o f p u s h i n g t h e L O Qt o t h e l im i t s o f th e L O D i n o r d e r t o c o m p e t ew i t h o t h e r m o r e s e n s i t i v e m e t h o d s s h o u l d b ea v o i d e d .Stabili ty

D a t a o b t a i n e d f r o m a n a l y s is o f s t u d ys a m p l e s a r e s u s p e c t i f n o t a c c o m p a n i e d b ys u p p o r t i n g d a t a a s s u r in g t h e s t ab i li ty o f t h ed r u g a n d m e t a b o l i t e s i n t h e m a t r i x . I t i si m p o r t a n t t o e m p h a s i z e t h a t s t a b i li ty t e st s m u s tb e d e s i g n e d i n s u c h a w a y a s t o d e t e c t a n yd e g r a d a t i o n , o v e r th e m a x i m u m p e r i o d o f t im et h a t s t u d y s a m p l e s w i l l b e s t o r e d p r i o r t oa n a l y s i s . O f t e n , s t a b i l i t y d a t a a r e b a s e d o nd u p l i c a t e o r t ri p l ic a t e d e t e r m i n a t i o n s o f t h ec o n c e n t r a t i o n s o f h ig h a n d l o w Q C s a m p l e s, a tm u l t ip l e t i m e p o i n t s a f t e r t h e s t a r t o f s t o r a g e t oa l l o w " t r e n d s " t o b e d e t e c t e d . H o w e v e r , t h ei s s u e i s n o t w h e t h e r t h e r e i s a t r e n d i nd e g r a d a t i o n , b u t w h e t h e r t h e s tu d y s a m p le sw i t h t h e l o n g e s t s t o r a g e t i m e a r e a d e q u a t e l y


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94 DARIOUSH DADGARet al.preserved at the time of analysis. For example,in order to demonstrate 12 month stability at-20C, the stored samples should be analysedwhen freshly collected or prepared, at inter-mediate times (e.g. 3 and 6 months followingpreparation) and after 12 months of storage.To show processed sample stability, forexample over 36 h (as discussed previously),samples should be analysed at time 0, atintermediate times such as 12 and 24 h, and at36 h.

If stability testing is conducted on QCsamples (as opposed to samples from dosedsubjects) it is recommended to directly com-pare responses of stored and fresh QCsanalysed at the same time, or to compareinterpolated values provided the values areinterpolated from the same calibration curve.

Two types of essential stability study havealready been discussed. Here, stability of theanalytes under prolonged storage conditionsand freeze- thaw will be discussed.

L o n g t e r m s t a b i l i t y . The stability andtendency for adsorption to the storage con-tainer should be assessed for all analytes in thebiological matrix using the exact type of con-tainer (e.g. glass, polypropylene) to be usedfor study sample storage. Stability must beproven over at least the maximum period ofstorage of study samples, under the tempera-ture conditions to be used for study samples.

Ideally, the control samples used for evalu-ation of long term stability, should be thoseobtained from dosed patients/volunteers,collected, pooled, aliquoted and stored at thesame time and in the same way as studysamples. This provides maximum assurance ofthe integrity of all analytes in study sample fora given study. However, for most drugs wheremetabolite reversion to the drug, or ongoingmetabolism in the frozen matrix are not prob-lems, blank matrix samples spiked at differentconcentrations are an acceptable alternativeand are usually used.Two procedures for the assessment of longterm frozen stability are discussed below, thefirst traditional, and the second developedrecently by some of us [8].(1) Replicates of stored and freshly preparedQC samples are analysed and theirresponses compared.(2) A batch of stablility samples is preparedand divided into two sub-batches, the firststored at the temperature intended for use

in storing study samples (typically -20C;the stability samples), and the second inliquid nitrogen (nominally at -


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B I O A N A L Y T I C A L M E T H O D V A L I D A T I O N , S A M P L E A N A L Y S I S A N D D A T A R E P O R T I N G 9 5f r e e z e - t h a w c y c l e s a t c o n c e n t r a t i o n s r e p r e s e n -t a t i v e o f t h e h i g h a n d l o w a n a l y t e c o n c e n -t r a t i o n s i n t h e m a t r i x s h o u l d b e e x a m i n e d i nr e p l ic a t e s . D a t a f o r f r e e z e - t h a w s t a b il i ty m a yb e o b t a i n e d i n t w o w a y s :( i) s t a b i l it y s a m p l e s a r e f r o z e n a n d t h a w e dt h r e e t i m e s a n d t h e n a n a l y s e d . T h i sm e t h o d i s l es s t i m e c o n s u m i n g , h o w e v e r , i fs a m p l e s a r e p r o v e n u n s t a b l e , t h e n e x p e r -

i m e n t d e s i g n , ( i i ) s h o u l d b e p e r f o r m e d .( ii ) Q u a l i t y c o n t r o l s a m p l e s a r e a n a l y s e d w i t h -o u t b e i n g f r o z e n a t f i r s t , a n d t h e n a f t e re a c h c y c l e o f f r e e z e - t h a w s o t h a t a t r e n d ,i f i t e x i s t s c a n b e s e e n . I f i t w a s p r o v e n , f o r

i n s t a n c e , t h a t Q C s a m p l e s a r e o n l y st a b lea f t e r tw o c y c l e s o f f r e e z e - t h a w , t h e n s t u d ys a m p l e s s h o u l d n o t b e f r o z e n a n d t h a w e dm o r e t h a n t w i c e .Recovery

H i g h r e c o v e r y o f a n a l y t e (s ) f r o m t h e m a t r ixi s d e s i r a b l e . H o w e v e r , s o m e t i m e s i t m a y b en e c e s s a r y t o i n t e n t i o n a l l y s a c r i f i c e h i g hr e c o v e r y i n o r d e r t o a c h i e v e b e t t e r s e l e c t i v it y ,a n d t h i s i s a c c e p t a b l e p r o v i d e d t h a t a d e q u a t es e n s i t i v i t y , p r e c i s i o n a n d a c c u r a c y a r ea c h i e v e d . S o l v e n t s s u c h a s e t h y l a c e t a t en o r m a l l y g i v e ri se t o h i g h r e c o v e r y o f a n a l y t e ,h o w e v e r t h i s s o l v e n t s i m u l t a n e o u s l y e x t r a c t sm a n y i n t e r f e r i n g c o m p o u n d s , t h e r e f o r e ,p r o v i d e d t h a t a n a d e q u a t e l y s e n s it iv e d e t e c t i o nl i m i t i s a t t a i n e d w i t h g o o d p r e c i s i o n a n da c c u r a c y , t h e e x t e n t o f re c o v e r y s h o u l d n o t b ec o n s i d e r e d a n i s s u e i n b i o a n a l y t i c a l m e t h o dd e v e l o p m e n t a n d v a l i d a t i o n .

R e c o v e r y i s b e s t t e s t e d b y d i r e c t l y c o m p a r -i n g r e s p o n s e s o f r e p l i c a t e s o f e x t r a c t e d Q Cs a m p l e s w i t h r e p l ic a t e s o f e x t r a c t e d b l a n km a t r i x t o w h i c h a n a l y t e h a s b e e n a d d e d a t t h es a m e n o m i n a l c o n c e n t r a t i o n . I n t h i s w a y , a n y

e f f e c t o f t h e m a t r i x o n , f o r e x a m p l e , p e a ks h a p e d o e s n o t c o m p l i c a t e i n t e r p r e t a t i o n o fr e c o v e r y d a ta . R e c o v e r y m a y b e d e t e rm i n e db y c o m p a r i s o n o f i n t e r p o l a t e d c o n c e n t r a t i o n so f e x t r a c t e d v s u n e x t r a c t e d Q C s p r o v i d e d t h e ya r e f r o m t h e s a m e c a l i b r a t i o n c u r v e .

R e v a l i d a t i o nW h e n i t i s n e c e s s a r y t o m a k e c h a n g e s t oc h r o m a t o g r a p h i c c o n d i t i o n s , o r t h e s a m p l ep r o c e s s in g p r o c e d u r e o f an a n a l y t i c a l m e t h o d ,r e v a t i d a t i o n m a y b e n e c e s s a ry . T h e d e c i si o n

r e g a r d i n g w h i c h p a r a m e t e r s r e q u i r e r e v a l i d -a t i o n s h o u l d b e b a s e d o n l o gi c al c o n s i d e r a t i o no f t h e s p e c i f i c v a l i d a t i o n p a r a m e t e r s l i k e ly t ob e a f f e c t e d b y t h e c h a n g e .

F o r e x a m p l e , c h a n g e s t o e x t ra c t i o n o r b a c k -e x t r a c t i o n m e d i a m a y b e e x p e c t e d t o a f f e c ts e l e c t i v i ty , r e c o v e r y , p r e c i s i o n a n d a c c u r a c y ,w i t h o u t a f f e c t i n g f r e e z e / t h a w s t a b i l i t y i n t h eb i o l o g i c a l m a t r i x . A c h a n g e t o t h e a n a l y t i c a lc o l u m n o r m o b i l e p h a s e m a y b e e x p e c t e d t oa f f e c t l i n e a r i t y a n d s e l e c t i v it y w i t h o u t a f f e c t i n gr e c o v e r y . A g u i d e l i n e t o r e v a l i d a t i o n i sp r e s e n t e d i n T a b l e 1 .

C r o s s - v a l i d a t i o nLinearity, intra-batch precision a nd accuracyC r o s s v a l i d a t i o n r e f e r s t o a p p l y i n g a v a l i d -a t e d m e t h o d i n a g i v e n b i o l o g i c a l m a t r i x t o t h es a m e t y p e o f m a t r i x f r o m a n o t h e r s p e c ie s , o r toa s i m i l a r m a t r i x ( e . g . p l a s m a a n d s e r u m ) f r o mt h e s a m e s p e c i e s , o r t o t h e s a m e m a t r i x w i t h ac h a n g e i n a n t i c o a g u l a n t .

C r o s s v a l i d a t i o n m a y b e c a r r i e d o u t a sf o l l o w s : a c a l i b r a t i o n c u r v e i s p r e p a r e d i n t h ev a l i d a t e d m a t r i x ; r e p l i c a t e s a t a l l Q C c o n c e n -T a b l e 1G u i d e l i n e s f o r r e v a l i d a t i o nM e t h o d p a r a m e t e r c h a n g e dE x t r a c t i o n s o l v e n t , b u f f e r , b a c k e x t r a c t i o n m a t r i xo r i n j e c t i o n s o l v e n t

C h r o m a t o g r a p h i c c o l u m n , m o b i l e p h a s e c o m p o -s i t i o n ( e . g . s i g n i f i c a n t c h a n g e i n r e t e n t i o n t i m e s ) ,d e t e c t o r t y p e o r w a v e l e n g t hE x t e n d i n g t h e u p p e r e n d , o r r e d u c i n g t h e l o w e r e n do f t h e c a l i b r a t i o n c u r v e r a n g e

P a r a m e t e r s t o r e v a l i d a t eL i n e a r i t y , r e c o v e r y , s e l e c t i v i t y , L O Q , i n t r a - b a t c h p r e c i s i o n a n da c c u r a c y , i n - p r o c e s s s t a b il i ty . A d d i t i o n a l l y , i f in j e c t i o n s o l v e n t i sc h a n g e d , p r o c e s s e d s a m p l e s t a b i li t y , b u t n o r e c o v e r y o r i n - p r o c e s ss t ab i l i t y .L i n e a r i t y , s e l e c t i v i t y , i n t r a - b a t c h p r e c i s i o n a n d a c c u r a c y f o r t h eL O Q a n d o t h e r Q C s

L i n e a r i t y , L O Q ( i f r e d u c e d ) , i n t r a - b a t c h p r e c i s i o n a n d a c c u r a c ya t r e v i s e d u p p e r a n d l o w e r l e v e l sI n t e r n a l s t a n d a r d S e l e c t iv i t y , i n t r a - b a t c h p r e c i s i o n a n d a c c u r a c y , a n d r e c o v e r y


8/9

96 D A R I O U S H D A D G A R e t a l .trations including the LOQ are prepared inboth the matrix from the validated method,and in the matrix to be validated; all QCsamples are back-calculated from the samecalibration curve in the validated matrix; themethod is considered cross validated if thedetermined concentration of QCs in the matrixto be validated satisfy the acceptance criteria.S t a b i l i t y

All types of stability studies previously dis-cussed (long term, freeze-thaw, in-process,processed sample) should be evaluated andreported under cross-validation.S e l e c t i v i t y

Selectivity should be evaluated in the revisedmatrix as interferences may differ betweenmatrices.

0 .80 .70 .60 .50.4e~p 0 .3

0. .20 .1

10 11 12 13 14 15 16 17 18 19 20A s s a y R S D ( % )

o O n e + T w o O T h r eeF i g u r e 1P r o b a b i l i t y o f r e j e c t in g a n a n a l y t i c a l b a t c h w h e n m e a s u r -i n g o n e , t w o a n d t h r e e a n a l y t e s .

A n a l y s i s o f S t u d y S a m p l e s a n d R e p o r t i n g o fS t u d y D a t aM u l t i a n a l y t e m e t h o d s

A recent conference report [1] recommendsbatch acceptance criteria defined as follows:duplicate QCs at three concentration levels(low, intermediate, high) with four of sixcalculated to be within +20% of theirrespective nominal values (no two at the sameconcentration level may be outside +20%).This approach is now generally accepted. Inpractice, using an analytical method character-ized by acceptable inter-batch precision(-


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B IOANAL YTIC AL ME THOD VAL IDATION, S AMP L E ANAL YS IS AND DATA R E P OR TING 9 7Reassay of samples

I n m o s t s t u d i e s , s o m e s a m p l e s w il l r e q u i r er e a s s a y . C r i t e r i a f o r i d e n t i f y in g t h e s e s a m p l e s ,t h e i r a n a l y s i s a n d r e p o r t i n g s h o u l d b e e s t a b -l i s h e d a priori. R e a s o n s f o r r e a n a l y s i n g b i o -a n a l y t i c a l s a m p l e s c a n b e s u m m a r i z e d a sf o l l o w s :

Results are analytically unacceptable. T h e s er e f e r t o s a m p l e s w i t h u n r e p o r t a b l e c o n c e n -t r a t i o n s d u e t o :

( i) e q u i p m e n t f a i lu r e ;( ii ) p o o r c h r o m a t o g r a p h y ;( ii i) l o s s d u r i n g s a m p l e p r o c e s s i n g ;( iv ) s a m p l e s w i t h c o n c e n t r a t i o n s b e l o w a n

e l e v a t e d L O Q ;( v) s a m p l e s w i t h c o n c e n t r a t i o n s a b o v e t h e

a c c e p t e d r a n g e o f t h e c a l i b r a t i o nc u r v e ;

( vi ) p r e - d o s e s a m p l e s w i t h o b s e r v e d c o n -c e n t r a t i o n s n e e d i n g c o n f i r m a t i o n ;

( v ii ) p r o c e s s i n g e r r o r s , s u c h a s i n c o r r e c ta d d i t i o n o f in t e r n a l s t a n d a r d o r o t h e rr e a g e n t s ;

( v ii i) r e j e c t e d b a t c h e s ;( ix ) l o w i n j e c t io n v o l u m e ( a n d th u s l o w

r e s p o n s e ) .S a m p l e s i n t h e s e c a t e g o r i e s s h o u l d b e r e -

a n a l y s e d s i n g l y , a n d p r o v i d e d t h a t t h e b a t c hm e e t s a c c e p t a n c e c r i t e r i a , t h e v a l u e f r o m t h er e a n a l y s i s i n e a c h c a s e i s r e p o r t e d .

Pharmacokinetic outliers. I t i s d e b a t a b l ew h e t h e r a s a m p l e w i t h a c o n c e n t r a t i o n i n-c o n g r u o u s w i t h t h e p h a r m a c o k i n e t i c p r o f i l es h o u l d b e r e a n a l y s e d . H o w e v e r , i f a d e c i s i o n ism a d e t o r e p e a t t h e a n a l y s i s , t h e r e a n a l y s i ss h o u l d b e c o n d u c t e d i n d u p l i c a t e .R e p o r t i n g a s i n g l e r e s u l t f r o m m u l t i p l ed e t e r m i n a t i o n s r e q u i r e s p r e - d e f i n e d c r i t e r i af o r d a t a s e l e c t i o n . S u c h c r i t e r i a a r e n o r m a l l yb a s e d o n t h e p e r c e n t a g e d i f f e r e n c e o f t h e m e a no f r e p e a t v a l u e s f r o m t h e o r i g i n a l v a l u e . B a s e do n t h e c r i t e r i a , t h e d e c i s i o n is m a d e t o r e p o r te i t h e r t h e o r i g i n a l s a m p l e v a l u e , o r a c h o i c e

b e t w e e n , f o r e x a m p l e , t h e m e a n o f du p l i ca t er e a n a l y s e s , a n d t h e m e d i a n v a l u e o f t h eo r i g i n a l a n d r e a n a l y s e s . I t i s h e l p f u l t o u s e af l o w c h a r t t o d e f i n e t h e d e c i s i o n m a k i n gp r o c e s s u s e d t o r e p o r t r e s u l t s f r o m m u l t i p l ed e t e r m i n a t i o n s , g i v e n d i f fe r e n t s c e n a r i o s o fo r i g i n a l a n d r e p e a t s a m p l e c o n c e n t r a t i o n sf o u n d .

C o n c l u s i o nI t a p p e a r s t h a t b i o a n a l y t i c a l v a l i d a t i o n p r o -

c e d u r e s a n d a c c e p t a n c e c r i t e r i a w il l c o n t i n u e t oe v o l v e . I t is l i k e l y , a n d d e s i r a b l e , t h a t f u t u r ec h a n g e s w i l l b e b a s e d m o r e o n s t a t i s t i c a lc o n s i d e r a t i o n s a n d t h e c a l c u l a t e d i m p a c t o fv a r i o u s c r i t e r ia o n t h e e n d u s e o f t h e d a t a ,r a t h e r t h a n o n " c o n s e n s u s " o f l e a d e r s i n t h ef i el d . T h e a u t h o r s o f th i s p a p e r a r e c o n d u c t i n gr e s e a r c h i n t h i s a r e a , t o b e t t e r l i n k t h e i m p a c to f v a l i da t i o n r e q u i r e m e n t s a n d a c c e p t a n c ec r i t e ri a o n t h e d e t e r m i n a t i o n o f b i o e q u i v a l -e n c e a n d o t h e r p h a r m a c o k i n e t i c e v a l u a t i o n s .

R e f e r e n c e s[1] V.P. S hah , K.K. M idha , S. Dighe, l.J. M cGilveray,J.P. S kelly, A. Yacobi, T. Layloff, C.T. Visw anathan,C.E. C ook, R.D. McD owall, K.A. Pittm an and S.Spector, J. Pharm. Sci. 81,309-312 (1992).[2] D. Dadgar and M.R . Sm ith , Trend Anal . ('hem. g,115-117 (1986).[3] V.P. Shah, Clin. Res. Pract. Drug. Reg. Affairs 5, 5 1 -60 (1987).[4] A.R. Bu ick , M .V. D oig , S .C . Jeal , G.S . Lan d andR.D. McDowall, J. Pharm. Biomed. Anal. 8,629-637(199(I).[ ,5 ] A . G . C a u s e y , H . M . H i l l a n d L . J . P h i l li p s , J. Pharm.

B i omed. Ana l . 8 , 6 2 5 - 6 2 8 ( 1 9 9 0 ) .[ 6 ] G . P . C a r r a n d J . C . W a h l i c h , J . Pharm. B i omed. Ana l .8, 613-618 (1990).[7] H.T. Karnes, G. Shiu and V.P. Sha h, Pharm. Res. 8,421-426 (1991).[8] J.W. H ooper, D . Lessard, K. Selinger and D. Dad gar,A new , more reliable method for the determination ofthe lon g term frozen s tability of dru gs in biologicalf lu ids . E i gh th In te rnat iona l A A P S Conference,Orlando, Florida (1993).

[Received for review 28 January 1994;revised manuscript received 20 May 1994]

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