95-_

Automated Tablet Processing:Sample Preparation and HPLC with

Photodiode Array Detection

Patricia Young and Raymond McGuirk

Waters Corporation

• Today we are goingto discussuseof an automatedinstrumentforcontentuniformityand compositeanalysistestingof soliddosageforms.

• We are goingto discussthe advantagesof photodiodearraydetection.

DiscussionOutline

• Review Content Uniformity and CompositeAssay Testing

• Discuss Advantages of Automated TabletProcessing

• Discuss Utility of Photodiode Array Detection

• We will briefly overview what contentuniformity and compositeanalysis testing is.

• We will focus on why it is an advantage to automate these analyses.• Then we will move on to discussthe uses of photodiode array

detection and how PDA can give you more information about yoursample. This type of detection can be very useful in stability analysis.

Solid Dosage Form Testing

" • Dissolution-Time/Release Study

• Assay- CompositeSample

• Content Uniformity-IndividualTests

• Shownhere are three essentialteststhateach soliddosageformsoldinthe US and otherhighlyregulatedcountriesmustgo through.

• Waters was amongthe firstto automatethe entireprocessofdissolutiontesting withHPLC,analysis.We continueinthisarea andwillbe expanding ourofferingsinautomateddissolutiontesting.Dissolutiontestingmonitorsthe rateof releaseof the activecomponentunderhighlycontrolledconditions.These conditionsaredesignedto simulatethedissolutionof the "tablet"inthe body.

• We willfocuson contentuniformityandcompositeassayanalysis.• With Content Uniformitytestingwe wantto quantifythe amountof

activecomponentin each tabletbyindividuallyassayingeach oneforcontent.

• Compositeanalysispoolsas manyas 20 individualdosageformsprovidinga meansto compensatefor anyvariabilitybetweensamples. This typeof testingisespeciallyusedfor stabilitytesting.

Analytical TestingTime & Effort

Assay 15%Impurities 10%

Identification 8%

Physical 2% Dissolution 25%

Content Uniformity 40%

• (note:thisgraph was obtainedfrom a customerand has been agreedwithby others)

• ContentUniformity& Compositeassaytake upto 55% of the time inthe Formulationtestinglab.

Problems in Today's PharmaceuticalFormulation Testing Laboratory

• No automated solutionsfor the entire process ofcontentuniformitytesting

• Increase in regulatorycompliance issues

• Training laboratory personnel

• Tracking and trending product quality

• (Bullets1&2) ContentUniformity(CU) testinginvolvesmanysteps.Due tothe regulatednatureof theseanalyses,a completerecordmustbe maintained. Increasinglythe industryis movingto HPLCanalysis(ratherthan UV detectionin a bench-topspec.)VVTPSistheonly fullyautomatedsystemfortheseanalyses.

• (Bullet3) Thosethatdo theseanalysesmanuallywillbe the firstto tellyouthattheyare boring. The personnelmustbe highlytrainedtoperformthese routineanalyseswithgoodprecisionand accuracy.The operatorhasto be ableto fullyrecordalldata at each stepof theprocess.Alsothere isthe ever-presentneedforthe supervisortofullydocumentthe trainingofthese individualsperformingthe tests.

• (Bullet4) It is alwaysimportantto be able to trackand trendproductqualityovera periodof time. Thismaybe data for the annualreportor data to showthe FDA. Bystoringalldata ina commondatabase,we can morecompletelytrack& trendproductquality.

WTPS

Combines All St_ps

• The Waters Tablet ProcessingSystemcombineall the steps. Fromtabletto reportinone system.

• Firstyouweighthe sample,themyougrindit, diluteit, filter& injectsample,analyzeresultsandgeneratereports.

Waters Tablet Processing SystemWTPS

_- Joint Development and Marketing Program

=.Source For Automation

,-Waters Corporation

• WTPS representsa jointeffortbetween Waters and Source ForAutomatio. SourceForAutomationhas placedunitsinpharmaceuticalhousesinthe US, PuertoRico,Eumpeand Australia.SourceFor Automationhas combinedtheirexpertisein roboticswithWatersworldwide experienceand supportfor HPLC and ResultsManagement.

Waters Tablet Processing System• Integrates

• Waters HPLC Modules

• Millennium2010 ChromatographyManager• Solid Dosage Assay Systemfrom

Source ForAutomation

,, Proven Performance• Ruggedness• Reproducibility• Reliability• Flexibility

• A full Waters HPLC with Millenniumcontrol. Samp[le prep done bythe SDAS from Source For Automation

• Ruggedness - the ability to run consistently under different users anddifferent environment conditions

• Reproducibility - The ability to obtain results reproducible over time• Reliability - consistent uptime of unit• Flexibility- the ability to do more than just tablets in a single overnightrun.

PHOTOS

• Complete Unit• Solvent and Rack storage• LC cabinet interior

• Three slidesfollowwith instrumentphotos. It is it'sown lab space. Itcan readilybe set upanywhere;suchas themanufacturingfloor.

• 1) The overallWTPS shot: PointoutSDAS and HPLC modules.• 2) The VentilatedSolventStorageArea: Show:1) the flexibilityfor

solventbottlesizes,2) storageareas fordifferentsampleracks,suchas composites.3) there is an in-boardsolventwaste containerwithdedicatedwastepumpthatpumpswasteoutto an external reservoir.(There shouldnotbe toomuchwastebecauseofthe solventrecyclerand we don'thaveto repeatedlyrinsea commonextractionvessel)

• 3) The LC cabinetinteriorwith : 600-pumpand controller,996photodiodearraydetector,in-lineeluentdegasser, and the computerWHERE ALL DATA IS STORED ON A RELATIONAL DATABASE.

WTPS Main Screen

• This is the WTPS main screen. It'salmost as easy as ready, set, go• Point out the securityof log-inprivilegesthat can permit the methods

development chemist full access to all methods developmentsections, system controls,and SOP writing,while limitingthe routineuser to only run samples, butnot have access to methodsdevelopment.

Photo

Shows sample prep with LC

• Here we see the sample prepgoingon while the sample is runon theLC. No waiting: the samplethatwasjust preparedfor LC isrunningonthe LC whilethe nextsampleisbeingpreparedfor injection.

• This is a goodtimeto pointoutallthe pumps,cannulaand extractor• - The 3 pumpsin a rowon thefar rightare solventdeliverypumpsso

it ispossibleto use 3 differentsolventsduringa singlerun. You canuse 1 solventfor initialextractionanotherforthe finaldilutionOR setupto do one dosageformwithone set ofsolventsand anotherdosageformwitha differentactivewithanothersolvent.

• - The sampleis onthe cannulastation.There isa dedicatedwashpumpforwashingthe cannula+ a syringeflushthatflushesthecannulastationinternally.

• - The extractorstationiscoveredbythe washingcupand ithas 2pumpsthat wasfromthetopand bottom, respectively.

• -Alsoyoucan see thewaste reservoirpump.

WTPS

Reporting Results

• All Sample Preparation and AnalysisResults Linked Together

• Customized Reports• System Suitability• Tracking and Trending• Control Charts

• We tie all the sample prep resultswith the LC resultsto providetheuserwithfamiliarreportsthatare recognizedby regulatoryagencies.

• These reportscan be customizedfor specificneedsofthe individualuser.

• The relational database permits ready access for tracking andtrending product quality.

Eight Steps of MethodValidation_r Precision

i=,..._r

( Limitof Detection |

Method =Validation =_ Selectivity l

_{ Ruggedness ],,,..._f"

• The main reason to show this slide isto introducethe Ruggednessexperiment

• It is important to show this in the contextof Method validationbecause it is we must be able to transfer methods between units.We want to place VV'I'PSin methodsdevelopmenttabs with996-detection and let them knowthat method transfer to QC or otherManufacturing sites can be done.

• Millennium has many differentcapabilitiesthat facilitate methodsvalidation.

Experimental Protocol for Ruggedness TestingWaters Tablet Processing System

• 3 VVTPSUnits- 10 ContentUniformity Analyses• VVTPS-A Waters 486 TUV Detector (275 nm)• VVTPS-B Waters 996 Photodiode Array Detector

230-450 nm, 1.2 nm resolution• WTPS-C Waters 486 TUV Detector (275 nm)

• Sample: Generic TriActive Pain Reliever• Eluent: Water:Methanol:Acetic Acid (81:16:3, v/v),- Flow: 0.8 ml/min

• Temp: 30 C• Column: Nova-Pak C18 (3.9 mm x 150 mm)

• Self explanatory methods slide• PDA detection vs. Single wavelength detection on 486-detector.

Overlay:Threeo.soi! High (1=,/o=aim)....... Levels ofo.50 i Medium (100% claim) Standard

Low (50% claim) Iniecti ond_

°-=1 !g -U

u

_ 0.3:)

_ < _o 0.20 o ,_

<: _u _um fJ

0.10 "5_

, _ _-,-....... ................,i__,- ....,o.00

o._ 4._ 8.oo " i21_ ' _S._ 2o.00

TimeImin.)

• Typically, we injectstandards equivalent to 100% LABEL CLAIM,and establish lower and upper calibrationvalues at 50 % and 150%label claim.

• Here we have overlaid standard injectionsof acetaminophen,caffeine, acetylsalicylicacid (aspirin) and salicylicacid (degradationproduct)for quantitation and spectral characterization.

WTPS-B WTPS Reproducibility® Content Uniformity (n=10)¢

_o= _ TriActive Pain Reliever

0.00

'0 10 2O

Time(_n.)

• Overlay of 10 CU runs on the PDA-based WTPS (unit B)• We have good reproducibilitywith retentiontime and amount

quantified. %RSD< 2.0• There is no salicylicacid because there was no degradation.

TriActive4th sample from each separauon:0.20 unit wTPSA...... 3 WTPS Units

_PsB 10 CU Samples per UnitWTPSC

!

0.00 _

0 10 20 30

Time (rain.)

• Here we haverun 10each ofthetri-activetabletson w'rPS A, B. andC.We have overlaiddatafromthe4th sampleofeach ofthe 10 CUruns.

• When all 30 CU data pointsare averagedwe get reproducibility%RSD< 2.0 % betweenunitsfor amountand RT.

• The absorbency,at 275 nmforthe PDA-deriveddataVVTPSB isequivalenttothe data fromsingle-wavelengthdetectionon the 486detector. You can indeedhave spectraldatawithoutsacrificingsensitivity.

• Also,the methoddevelopmentchemistcan developmethodsusingthe PDA andwithgoodsuccessexpectto transferthe methodto QCwhere singlewavelengthdetectionmaybe used.

Precision: Data from 3 WTPS Units

0.60 --

i Caffeine (n=30)

0.30

0.00 !L

.o_, ._

• Typical;PrecisiondataforCaffeinefromthe 30 CU analysisfrom the3 separateW'i'PSunits.

• ShowingAmountandRTandanyotherfavoriteSystemSuitabilityParameter.

ContentUniformityControlChart: Acetaminophenin TriAcUveComparison of Results from 3 Wi'PS units

WTPS-A • r/Ul )erConfidenceUmitWTPS- B El,

E 110- VVTPS-C ._mtO Tar .=t(100% Label Claim)- /o,,Qm 10o- "_" 'IB---J n 13 .c • • .

" . l=• a 13 • •o

13 . E!0. 90 I El

_Lo 'erConfidence Urrdt

80-

1 2 3 4 5 6 7 8 9 10

Tablet Number

• Another way of looking at the data form the 3 x 10 CU runs from 3different WTPS units.

• For acetaminophen we have plottedthe individualdata points fromeach of the 10 individualtablets. WTPS-A is the white circle,VVTPS-B isthe square and W'I'PS -C isthe yellow star.

• This illustratesthe reproducibilitybetweenthe 3 units (as well asshowing a controlchart)

Photodiode

Array Detectionabsorbanc

wavelength

absorbance z _ Chromatogram

e

wavelengtzh_ v _Spectrum

• Now let's spendsometime talkingaboutPhotodiodeArray Detection(PDA).

• Thinkof it as a 3-D cube ofdatawithtimeon 1-side,absorbencyonanotherandwavelengthon anotherface.

• (Upperpanel) Ifwe take a sectionalongthewavelengthface we geta chromatogramjust likewhatwe get fromsinglewavelengthdetection.

• (LowerPanel) Ifwe take a sectionfromthe cubeat a discretetimepointwe get a spectrum.Spectraldatacan be usedto give us a lotofinformationregardingour samplesand ourseparations.

Spectrum Index Plot for Triactive Drugwith Aspirin Degradation Product: Standards

Absoorban_(275nm) WavelengthRange(nm)¢n o

- • ................. ' ' " ....... -- Apex

____rm Spectrum

2.00 " "........... "nophen ... Ubrary• Match

-.4.oo-

-..._ Caffeine=D

r,_,(m) e.oo _' "......._'-'_--_*__

". IsaMcytic

8.00

10.0( _ Salicylic

.... • .... • - . . , .... , .....

Chromatogram at 275 nm Spectral data from 230-400nm

• A good way of orientingyourself to spectral data is the correlate itwith an extracted c'gram in Spectrum Index Plot.

• On the left panel we have a c'gram extracted from the 3-D cube at275 nm.

• On the right-sidewe have spectra from each of the individualpeaks.• We have asked it to plotthe apex spectrafrom each peak along with

the spectra from the best librarymatch.• As we see, not all componentsof the tri-active separation have

maximum absorbency at 275 nm. Caffeine does have anabsorbance max near 275 nm.

i

4

Photodiode Array Detection

C°mp°und C°nfirmation !

Peak. I Copoun!H°m°gene'tyI

@ @

• You can furtherformulationvalidationbyaddingphotodiodearraydetection. PDA can measurepeak homogeneityof a peakto ensureitsone component.

• Librarysearchcan be usedto identifyknownstabilityor reactionbyproducts.

Increased Signal-to-Noise Ratiowith Maxplot

Maxplot (230.400 nm)( 0.57 AUFS)

....... Extracted Chromatogram0Ao- _ 275 nm (0.1z AUFS)

O

m 0.20- _. __

o.oo_"3 _:'......."''/_'.............":'"'t}:t""','-""_0.00 10.00

Time (min)

• This can be clearly illustrated in the MaxPIotwhere wehave our extracted chromatogram at 275 nm (in blueor dashed ) and our MaxPIot (in white or solid).

• MaxPIot plots each data point in the 3-D data file at itsmaximum wavelength.

• The caffeine peak represented at 275 nm vs. its theMaxPIot are not different in response. However, othercomponents of the mixture, notably salicylic acid andacetylsalicylic acid are shown at much higherabsorbency. While MaxPIotwould not typically beused for quantitation, it is an excellent way of learningmore about the presence of any possible contaminantsor breakdown products.

Spectral Library Matching

,-MatchingStandard Unknowncompares theunknown apex

® spectrumofco the peak with

a reference= spectrum in aJ_

./ /.Time Time

Waters

21,719

• SpectrallibrarymatchingusesSpectralContrastto comparethe apexspectrumof an unknownchromatographicpeakwitha referencespectrumstoredin a usercreatedlibraryin Millennium.

Peak Purity (Homogeneity) Analysis

• Peak Purityanalyzes allspectra within a

0(_ peak

J_

3 • Apex spectrum

. Ill•= is the reference

I spectrumTime Waters

21,720

• Peak purityor peak homogeneityanalysiscomparesspectrawithinthe samepeak.

• Allthe spectrafrompeak liftoffto touchdownare analyzedforspectraldifferences.

• The apex spectrumisthe referencespectrum. Eachof the otherspectraare comparedtothe apex spectrum.

• If thespectraare all similar,the peak isspectrallypureorhomogeneous.(Thisis notproofofchemicalpurity.)

Symmetrical Chromatographic Peaks mayhave Co-Eluti_urities

-- _Y A _=_4 Noise

6O

0.164O

0.08

(R@

o.ooO

0 10

_o= B v

0.20 60 =

40

0.10

0.00 0.01.25 1.35 1.45 1.55 1.75

Tmle (ram)

• Panel A shows the acetaminophenpeakelutingwell beforethecaffeinepeak as we have beendepictingin all the previousseparations. The caffeinepeak cameoutabout3 minlater in panelA. The apex spectrum(showninyellow)was usedto ascertainspectralhomogeneitythroughoutthe peak. The purityangle (0.30)was lessthan the puritythresholdindicatingthatthe peak washomogenous.When requested,a maximumimpurityspectrum(thespectrumhavingthe mostspectraldifferencefromthe apex spectrum) was determined.Sincetherewas nosignificantimpuritypresenta"default"sectioninthe baselinewas selected.

• In Panel B, the mobilephasecompositionwas alteredsuchthatcaffeineand Acetaminophennowco-elutewitha verynicesymmetricalpeak shape. The PurityAnglewas 9.2 andwhen posted, the maximumimpuritywas identifiedonthe descendingslopeofthepeak. We can takethismaximumimpurityspectrumintoSpectrumReview for identification.

Library Spectrum Match

Softwareuses

SpectralComparisonto IdentifyImpurities

hi I=OAlilal_ I_mmlls ii

Match Match 19pectrumlLibrarYlMatChll'* all _" I'==hl 1Anmll rl"hflllhold I Natal I Nlml I Fllla I • I RMS IErmrt I

111 o.30ol 1.049 ICliffetrle ITrl ilctl IYeslo.o0001 I Ii

IJ. I I I I i I I I l _Il

• Here we have usingLibrarymatchingto identifypossibleimpurities.

d

Conclusion: Ruggedness TestingWaters Tablet Processing System

• Reproducibility within a Single Unit

• Reproducibility between Units for Method Transfer

• Advanced Detection with Photodiode Array (PDA)• Sensitivity Equal to Single Wavelength Detection• Spectral Data for Purity and Spectral matching• MaxPIot to Aid in Methods Development

• We havedemonstratedthatautomationcan be a significantadvantagetoCU andcompositeanalysistesting.

• We haveexcellentreproducibilitywithina singleunit.• We havereproducibilitybetweenWTPS unitstofacilitatemethods

transfer.• PDAhas-1) sensitivityequivalenttosinglewavelengthdetection

2) SpectraldataforLibraryMatchingandPurityassessmentand3) MaxPIottobetterreportunknowncomponentsinyoursample.