4 6 4 LCGC NORTH AMERICA VOLUME 26 NUMBERS MAY Z008 www.chromatagraphyonlirie.com

Performance Qualification of HPLCInstrumentation in RegulatedLaboratories

Jonathan Crovrther*, JeremyDowling+, Richard Hartwick*, andBill Ciccone+*

*Ortho Clinical Diagnostics, Raritan, NewJerseytPharmAssist Analytical Laboratory, SouthNew Berlin, New YorkttMicroSolv Technology Corporation,Eatontown, New Jersey

Piease direct correspondence to RichardHartwick at rhartwick@pharma55lstlab.com

With the forthcoming USP monograph <1058>, many laboratories are inthe process of reexamining their high performance liquidchromatography (HPLC) instrumentation qualification practices. Thisarticle demystifies the qualification procedures and proposes a welldesigned, easy and simple set of experiments upon which to establishinternal standard operating procedures (SOPs) for the completequalification of HPLC instruments. A key concept is the development ofa consistent test system, comprised of premade test solutions, aprequalified HPLC column, standardized protocols, and validatedsoftware that can be prepared in-house or purchased commercially as akit. This system can be applied to any HPLC system worldwide, toproduce comparable test results under uniform conditions. The testsystem is designed to be rapid, with a comprehensive performancequalification being completed in about 2 h for isocratic, and 3 h forquaternary gradient systems.

T he generation of high-quality, reli-able analytical data is grounded onthree fundamental components:

instrument qualification, method valida-tion, and user training (1,2). For thepharmaceutical industry, these activitiesfall under cGMP/GLP regulations.Although the specific regulations can varyfor the environmental or other industries,the principles remain the same.

The first of these, instrument qualifi-cation, is the focus of this article. A labo-ratory plan for analytical instrumentqualification (AIQ) is a requirement forall cGMP/GLP laboratories. The pend-ing USP guidance document <1O58> (3)reflects the evolving accepted practicesfor the introduction and qualification ofanalytical instrumentation into the regu-lated laboratory environment.

In.struments must be maintained sys-tematically and proven to be precise andaccurate for their intended use on anongoing basis (4,5). However, the specificqualification procedures are, appropri-ately, not predetermined by regulation.Instead, the laboratory management is

responsible for developing a scientificallysound, risk-based plan for the periodicmaintenance and qualification of theiranalytical instruments. Nonetheless, theapproach is subject to FDA review.

A sound instrument qualification pro-gram should be both scientifically rigorousand straightforward to use. It must be suf-ficiendy comprehensive to capture aber-rant instrument performance, yet be rapidenough to promptly return instruments toservice after the maintenance or repairshave been completed. Development ofthestandard operating procedures (SOPs) andtesting materials tor the qualification pro-gram can be a daunting, and sometimesconfusing task. This article presents anapproach that we have developed and tri-aled over many years, which provides acomprehensive, rapid performance qualifi-cation for high performance liquid chro-matography (HPLC) instruments.

Who Should Perform theQualification?The proposed USP AIQ monograph<1O58> states that, "Users are ultimately

4 6 6 LCGC NORTH AMERICA VOLUME 26 NUMBtB S MAT 2008 www.zhromatographyonUne.con]

230 270 310 350 390 430 470 510 550 590 630 670

Wavelength (nm)

Figure 1: UV-vis spectrum of 4% holmium oxide in 10% perchloric acid, NIST SRM2034. Preferred absorbance bands are shown above peaks 1, 3, 4, 7, 9, 10, 13, and 14.

250 275 300 325Wavelength (nm)

Figure 2: Spectrum of caffeine in method diluent (mobile phase), showing the spectralmaxima at 205 nm and 273 nm.

responsible for instrument operarions anddata quality. The user's group encompassesanaJysts, their supervisors, and organizationmanagement." It further states that:

"Users should also be responsihle forqualifying their instruments, because theirtraining and expertise in the use of instru-ments make them the best-qualified groupsto design the instrument lest(s) and specifi-cation(s) necessary for successful AIQ."

This view of user responsibility isshared by the FDA (6).

The AIQ ProcessThe AIQ process is often summarized as"The Four Qs" — that is, the design,installation, operational, and performancequalifications, referred to as DQ, IQ, OQ,and PQ. For a new installation, the instru-ment vendor often will be responsible forthe IQ and O Q procedures, albeit underlaboratory SOPs governing this operation.Depending upon the vendor, some abbre-viated form of a system check also might beperformed. Some vendors refer to this as aperformance verification {VV). The exactprocedures Lised will vary with each manu-facturer. This IQ-OQ-PV process essen-tially is performed once per installation.Upon completion, responsibility for rou-tine maintenance and periodic qualifica-tion is transferred to the user, even if out-side contractors are employed for futurepreventative maintenance and .service. lx>g-icaliy, because each instrLiment vendor hastheir own particular qualification routines,a master-level set of iabomrory PQ testingprotocols must be created to generate uni-form performance data across the differentbrands ot HPLC systems within the labo-ratory, while specifying measures that arerequired due to instrument maintenance,repair, or change. Such protocols must beconsistent with the company's overallchange control policy.

Table I summarizes the AJQ process andtypical situations in which each stage mightbe applied. Specific choices will vary fromlaboratory to laboratory, and this is notpurported to be a comprehensive plan forall contingencies. However, some rational,prewritten plan must exist to govern what-ever procedures are decided upon.

Note that unlike the OQ, PQ testing isperformed frequently, for many differentevents. System suitability of a particularmethod is not a substitute for a PQ,although its procedures might be incorpo-

4 6 8 LCGC NORTHAMERia VOLUME 26 NUMBER 5 MAY 2008 www.chroma tographyo nllne.com

250 300 350 400 450 500 550

Expected wavelength (nm)

600 650 700

Figure 3: Linear regression of the found vs. expected wavelengths for the combinedresults of the holmium oxide (squares) and caffeine (triangles) wavelengthqualification.

so

D

c

3M

HD

w J v . ; V J V0.J 1.0 i ! j~ lio

M 1,8 illTlmt (mIn)

Figure 4: Injection of the resolution test mixture for system suitability (upper figure)and L3 caffeine solution with uracil as a void volume marker, for injector precisiondetermination.

rated into a PQ testing protocol. A systemsuitability is method specific, and serves avery difierent purpose than a fiili PQ.

O Q and PQ also have different pur-poses, although similarities often exist intests used for these qualification steps. Adistinguishing feature of the O Q is itsfocus on testing the individual instru-ment tnodule, and often is driven by themanufacturers design specifications. PQtesting on the other hand, is holistic, anddocuments the performance ofthe work-ing system, which, thus includes bothhardware and software issues.

This difference is refleaed in the devel-opment of reasonable acceptance criteria.While OQ requirements are influenced bythe design Hmics of each particular mod-ule, PQ acceptance criteria reflect the min-imum acceptable performance levelsrequired for all instruments of similartypes in the laboratory. The assignment ofreasonable, acceptance criteria can be oneof the more difficult aspects of the entirePQ process. When available, acceptancecriteria are taken from compendial orother official sources, for example, USP<621>. Otherwise, scientifically reason-able, defensible criteria are assigned.

A word of caution is in order here. Userssometimes will look to the specificationssection oF an instrument manual foracceptance values. In our experience, thosevalues for wavelengtli accuracy, noise, sta-bility, and so forth are instrument-specific.Moreover, they sometimes are obtainedunder ideal conditions that cannot he repli-cated easily in the laboratory. Assignmentof such vendor-specific values to all labora-tory instruments invites unwarranted test-ing failures. PQ tesring is coming from theopposite direction, and seeks to define anacceptable performance level for all instru-ments in the laboratory. It is thus expectedthat most instruments will perform signifi-cantly better than the limits set by the PQ.Under that umbrella, the perfomiance his-tory of each individual instrumentbecomes its signature, and is of greatervalue than its absolute performance. Therationale for die assigned values for some ofthe acceptance criteria are discussed morefully in the "Results" secrion.

Logical and clearly written ongoingmaintenance procedures are equallyimportant co a successftil PQ program.Selected tests from the suite of test proto-cols can he performed, depending upon

www.chramatog raphyon line.com MAV 2008 L C G : NORTH AMERICA VOLUME 26 NUMBERS 4 6 9

the situation. For example, ifa pump sealfails midway before the next scheduledpreventative maintenance due to normalwear, it is not necessary to requalify theentire LC system. After investigation anddocumentation of the cause (and itsimpact on any data already generated),only the pressure leak check and flow raterequalification normally would he per-formed. A similar logic would be appliedto lamp changes. As part of the rootcause assignment for the failure however,ihe laboratory should review its preven-tative maintenance frequency, perhapsincreasing it it such failures are common.On the other hand, relocation of theinstrument to a new bench or adjacentlaboratory would initiate a fiill PQ, plusupdates to the logbook and database doc-umenting the new location. Shipmenc ofche instrument ro a new location wouldbe treated like a new installation, trigger-ing the entire IQ-OQ-PQ process, andperhaps a DQ if warranted.

The dcHnition of instrument portabilitymust be explicidy addressed in the SOR Anexample ofthis might be an HPLC systemon a portable care that is used periodicallyfor LC-mass spectrometry (MS) work.Without a ciear defmitJon of portability inthe relevant SOP, an overaealous auditormight conclude that rolling the instriunentover to the mass spectrometer constitutes amove, requiring a requalification, as hashappened to one of the authors on occa-sion. Clearly written SOPs are essential toanticipate and forestall such issues.

Development of a PQ TestMethodWe have developed a stiite of test methodsto fiilly evaluate the instrument under real-istic conditions, yet be as rapid and as auto-mated as possible. These test solutions haveproven to be chemically stable at roomtemperature for at least two years. Wemaintain all test components as a single,convenient kit, which includes the solu-tions, a prequalified base-deactivated PQtest column, test protocols, and validatedExcel template for data analysis. This cre-ates a closed, reproducible system, in whichthe only variable is the mobile phase. A sys-lem suitability solution is included to con-firm proper mobile phase preparation andcolumn performance. With this approach,the PQ test system hecomes an independ-ent, universal measuring tool that can be

reproduced easily in any laboratory world-wide on any brand of instrument.

Table 11 summarizes the PQ test systemcomponents. These solutions can be pre-pared and qualified in the laboratory underNIST-traceable conditions, or can be pur-chased commercially in kit form. Themobile phase stability is 60 days, so that itcan be prepared in large batches and storedfor multiple instrument qualifications.

The retention time window for caffeineis set at 1.0-1.5 min. A comprehensive iso-cratic qualification for al! test protocolsrequires about 50 injections and is com-

pleted in as little as 1.5 h. Additional gra-dient qualification requires ahout 35 minfor a binary system, or 65 min for a qua-ternary. Selected partial qualification testprotocols such as autosampler precisionand pump stability are completed in lessthan 30 min. Detector wavelength accu-racy, column oven, refrigerated autosam-pler temperature accuracy, and flow-rateverification are manual operations. Theremaining qualification tests are com-pleted within a single injection sequence.Only three methods are required; one of 3-min length for the resolution test mixture

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Figure 5: Extracolumn volume dispersion as measured from a typical resolution testmixture chromatogram.

and system suitability testing, a secondmethod of about 1.8 min for the bulk ofthe test injeaions atid a gradient method(if applicable) for dwell volume and accu-racy determination.

ExperimentalAcctonitrile was HPLC grade, purchasedfrom EMD (VWR, West Chester, Penn-

sylvania). Water was purified in-house,meeting USP reagent-grade specifica-tions. Caffeine, uracil, theophylline, and8-chlorotheophylline were ACS reagent-grade or better, and were purchased fromSigma-Aldrich (St. Louis, Missouri). Thecertified PQ test column, 75 mm X 4.6mm. Cogent C8, 5-|jLm particle size, wasobtained from MicroSolv Technology

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Corporation (Eatontown, New Jersey).The mobile phase was filtered anddegassed using a 0.45-|xm nylon filter.

A validated Excel template was usedfor calculations and graphing. The tem-plate required only entry ofthe raw data,with all subsequent calculations andgraphing being performed automaticallyby the spreadsheet. In addition todetailed results and graphs, the spread-sheet provides a single-page summarysheet for review and signoff.

For flow accuracy and leak-testing, adevice was constructed from a back-pres-sure regulator with a qualified pressuregauge. A Tescom (Elk River, Minnesota)mode! 26-1721-24-084 back-pressuit reg-ulator was used, with a rating of 10,000 psi.Appropriate tubing and male-unions werefitted, so that the colutnn itilet tubingcould be connected to the union. Dyna-seal variable stop depth fittings were sup-plied by Sonntek {Upper Saddle River,New Jersey) to ensure secure leak-tight fit-tings without regard to stop-depths. Theprinciple author can be contacted electron-ically if further details regarding construc-tion of this device are required.

In practice, the back-pressure regulatorassembly is fitted in lieu of the normalcolumn. Any desited back pressure isapplied by the regulator knob of theback-pressure regulator, at any flow rate.Flow accuracy measurements at variousflow rates are made at a constant 1000psi backpressure. The HPLC pressurereadout is checked against the gauge atincrements of 1000 psi, up to the maxi-mum instrument pressure, typically justunder 6000 psi. This hack-pressure regu-lator system has proven to be anextremely useful and versatile tool tohave available in the laboratory. It is usedto diagnose pump check valve problemsby observing pressure fluctuations. It hasproven robust enough that it also can beused to perform leak checks, such thatthe system can be pressurized at anydesired level, and the rate of pressure lossdirectly observed on the gauge.

Chromatograms in the figures wereproduced using an Agilent 1100 diodearray system (Santa Clara, California)with a quaternary gradient. The flow cellwas the standard analytical cell.

The test kit used in this article, completewith column, solutions, SOPs, and vali-dated software, is available commercially

www. chroma tog raphyon fme .com MAV 2008 LCGC NORTH AMERICA VOLUME 26 NUMBERS 471

ftom MicroSolv Technology Corporation as[he High Speed Qualification (HSQ) Idt.

Results and DiscussionOnce the laboratory SOPs have beenwritten and approved, and the instru-ment methods and sequences assembled,routine PQ testing is quite straightfor-ward. The following shows typical resultsfor some ofthe test protocols, along witha discussion of acceptance criteria.

Manual operations — pump accuracy,column oven temperature: Pump flowaccuracy and stability are the foundationupon which the subsequent testing isbuilt, because gradient dwell volume andother tests will use this value in their cal-culation. Most laboratories use dry volu-metric flasks of different volumes, with aNIST-traceable calibrated stopwatch tomeasure flow rates. We use our back-pres-sure regulator system (see Experimental)to impose a constant pressure of 1000 psi,and measure the fiow rates at nominalsettings of 0.5, 1.0, and 5.0 mL/min. Thequalification flow range should encom-pass all methods in the laboratory andcan he modified as desired. Given thepotential errors in drop collection and

1000-

0.00 0.05 0.10 0.15 0.20 0.25

Concentration (mg/mL)

0.30 0.35 0.40

Figure 6; Typical detector linearity produced by six caffeine solutions over theconcentration range of 0.00035-0.35 mg/mL, under the test method conditions. Theplot was generated from an Excel template.

timing, an accuracy specification of ± 5 %is assigned for this test.

Column oven temperature is meas-ured by using a NIST-traceable digitalthermometer, inserting a flexible ther-mocouple into the compartment, takingcare not to allow it to test on any metalsupports. The air temperature is meas-ured over a temperature range encom-passing all laboratory methods, withacceptance criteria of ± 5 "C.

Column oven designs vary widely intheir preheating designs and efficiency,and the actual mobile phase tempera-ture might deviate substantially fromthe oven air temperature. There is sim-ply no easy way to measure accuratelythe true internal column fluid tempera-ture, which will vary with hoth radialand axial position within the columndue to frictional heating (especially forultrahigh-pressure LC), and other

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70

'app

SO

40

20

-100.00 0.25 0,50 0.75 1.00

Time (min)1.25 1.50 1.75

Figure 7: Determination of gradient dwell voiume from the injection of the uracil-spiked mobiie phase, tg is the time for the nonretained uracil peak, and t^pp is theapparent time of the first visible onset of the gradient.

issues. In a similar vein, refrigeratedautosamplers also are qualified usingair, rather than solution temperatures,to the same specification.

Wavelength QualificationThe qualification of wavelength accuracy isa critically important test. Holmium oxidesolution in 10% perchloric acid (NISTSRM 2034) is an internationally acceptedwavelength standard, covering the range of241-641 nm. Caffeine is used as a second-ary standard, with two bands at 205 and273 nm, in this diluent. Thus, both stan-dards can be used in combination to qualifythe detector over the range of 205-641 nm(or to 56] nm for the UV range only). Thewavelength accuracy specification is set at± 3 nm, in accord with USP <621> (7).

Both variable-wavelength and diode-array detectors can be qualified by first

autozeroing the detector with diluent inthe flow cell, then pulling the solutionsthrough the cell with a spring-loadedsyringe and tubing, with a finger-tight fit-ting securing it to the detector outlet(never attempt to push the solutions, forsafety reasons). Release the vacuum beforetesting to eliminate bubbles and produce astable signal. For diode-array detectors, aspectrum of the flow-cell contents is taken,and the maxima are determined with theresident instrument software. While thereare 14 available bands for holmium oxide,it is only necessary to measure three or fourof these over the spectral region of interest(UV, visible, or both). For variable wave-length instruments, the wavelength is setsequentiaUy in 1 -nm increments, for a fewnanometers before and after tbeabsorbance band of interest. Theabsorbance reading is recorded, and the

wavelength maximum found by interpola-tion. Alternatively, a series of no-injection,no-flow methods can be written, each witha different wavelength, stepping across thespectral region to fmd the maximum. Fig-ure 1 shows the hoimium oxide spectrum.For the UV range, the 241-. 287-, and361-nm bands are convenient, while the451-, 537-, and 64l-nm bands can beadded for visible-wavelength detectors.

The found absorbance maxima are com-pared against the official published NISTvalues (8). The consensus spectral valuesare published for spectral bandwidths of0.1, 1.0, and 3.0 nm. For most HPLCdetectors, comparison against the 3.0 nmspectral bandwidth would be appropriate.

Figure 2 shows the spectrum of caffeinein tbe mediod diluent, u.sed to extend thequalification to 205 nm. Note that the 273nm maximLim overlaps with the holmiumoxide spectral range, thtis, linking it to theNIST standard values. These spearal max-ima have been confirmed independently tobe accurate with this mobile phase anddiluent (9). For diode-array systems, thecaffeine spcxtrum can be acquired conve-niently during the main injection sequencefrom of any of the mid-concentration solu-tions producing a good signal-to-noiseratio (S/N). Figure 3 shows the combinedresults for a typical wavelength accuracydetermination using the combined resultsof both the holmium oxide and caffeinesolutions, as automatically generated by thevalidated software. The slope of the regres-sion should equal one, with a statisticallynonsignificant intercept. While not usedfor qualification purposes, the regressionline can be extrapolated to 200 and 700nm, to show any inaccuracy trends at wave-lengths outside the qualification range.

The combined solutions, with their nar-row absorbance maxima across a widewavelength range, produce what is cur-rently a state-of-the-art wavelength accu-racy qualification. However, some labora-tories might have methods that usewavelengths falling outside of this range.The question frequently arises as towhether it is valid to use a method beyondthe qualified wavelength range of the detec-tor. Some feel that if a method of say, 200nm is tised, that the detector qualificationmust include that wavelength. The otherview is that if the wavelength accuracy isconfirmed at several points across a broadregion of the spectrum, the detector can be

www.chromatograp hyon Une.com MAY2008 LCGC NORIHAMERICA VOLUME 26 NUMBERS 4 7 3

Table I: Components of the AIQ process over the typical iaboratory instrumentlife-cycle

presumed to be in reasonably good operat-ing condition and is, thus, qualified for useacross its entire wavelength design range.We support this second view.

In support of this argument, note thatfor UV-vis spectropboto meter wavelengthqualifications, most compendia! agencies(including the USP, BP, and EP) specifyhoimium oxide as a suitable wavelengthreference standard, with its limited rangeof 241-641 nm. None of these agenciesstate that a spectrophotometer cannot beused beyond this qualified range, whichwould be the inference from the firstapproach. By confirming operation at sev-eral discrete wavelengths across a broadswath of its design range, one assumes thatthe instrument will function as designedacross its entire range. This i.s an assump-tion, but it is not unreasonable. Indeed,even within the qualification wavelengthrange, there is no proof that the mono-chrometer is not malfunctioning at someparticular wavelength between the check-points. Tbe system suitability of eachmethod run on the instrument is designedto provide such additional run-time assur-ance of the system performance.

Isocratic HPLC QualificationSystem suitability, noise, and extracol-umn dispersion: Once the wavelengthqualification, flow accuracy, and columnoven temperature checks have been com-pleted, all remaining tests are accomplishedby a single fully automated injection.sequence on the HPLC system. Figure 4shows typical injections of the resolutiontest mixture solution, along with the (L3)caffeine solution (also containing uracil)used for injector precision and pump flowstability. A system suitability test is per-formed to demonstrate correct retentiontimes and column efficiency and that theinstrument is operating properly and readyto perform the required test protocols.

Dynamic noise: Short-term dynamicnoise is determined for a blank injection atthe method wavelength of 273 nm, thus,documenting total instrument noise levelsunder realistic operating conditions.Because all method conditions are con-stant, this noise value can be compared tohistorical levels for the same instrument, aswell as to other instruments in the labora-tory. A minimum S/N ratio of ^10 isrequired for die LI peak (generated in themethod injection sequence), which is 0.1 %of the highest concentration solution.Unusually high noise levels can indicate afailing lamp, a dirty flow cell, pump noise,or other problems. The noise values givenby the manufeaurers in their instrumentspecifications are usually for the detectoralone under ideal conditions that cannot bereplicated easily in the laboratory. Such aspecific noise measurement might be per-formed as part of the OQwhen an instru-ment is first put into service. The dynamicprocedure described here has the advantageof providing a realistic noise measurementunder standardized operating conditions,so that various laboratory HPLC systemscan be compared directly. Note that thedeteaor-data system time constant affectsthe noise and, thus, should be documentedas part of the run method.

Extracolumn dispersion: Extracolumnvolume dispersion results in the loss ofsharpness of the eluted peak due to theinjeaor, conneaing tubing, and flow cell.It is the limiting parameter that determineswhether small-particle, low-volumecolumns can be successfully used in a giveninstrument. It is not recommended that anacceptance criteria for extracolumn disper-sion be part of the PQ. However, it is a veryusefiil instrument characteristic to know,and should be documented during thecourse of the PQ.

The most accurate way to measureextracolumn dispersion is to measure the

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350

300

250

200

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100

50

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30 40 50 60

Time (m n)

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Figure 8: Typical gradient accuracy step height results for a quaternary gradientsystem.

dispersion with a series of jumper rubesin lieu of the column, and extrapolate tozero length. However, a simpler, yet suf-ficiently accurate way is to plot the peakvariances against the square of the reten-tion volumes (10) as follows.

Because variances are additive, theobserved total system variance is

system2

= CTc

[1]

The variance in voliime units is calcu-lated from the retention volume and themeasured peak efficiency as follows:

[2]

N

The retention volume is simply the flowrate times the observed retention times forthe resolution test mixture peaks, thus

where f̂ Is the retention time (in min-utes) of the peaks in the resolution test mix-ture (assuming the extracolumn volumedelay is negligible relative to the peak reten-tion times), F'n the flow rate (as microlitersper minute), and N^.^^^,,, is the measuredefficiency for each peak. Regression yieldsan intercept of the extra column dispersion(a^^) in units of |xL2 (mm'') with a slopeof the reciprocal of the true column effi-ciency, exclusive of extracolumn effects.The test mixture was designed specificallyto match the dispersion levels found inmodem analytical HPLC systems. Figure 5shows a typical plot for determination ofextracolumn dispersion. The exrracolumndispersion data is obtained fi'om the injec-tion of the Resolution Test Mbcture, shared

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Table II; Summary of qualification test system components*

Resolution test mixture (RTM)

Caffeine: (linearity solutions;

Gradient visualization soiution (GVS)

Composition: ca. 0.05 mg/mL in each. Adjustto produce about equai peak heights at 273nm for ali components.UraciiTheophyliineCaffeine8-ChiorotheophyiiineLabeled: "RTM"

Six solutions covering three orders, produc-ing signals within the range of the detector:Labeled: "L I " through "L6"(0.00035-0.35 mg/mL)Note: This concentration range will producepeak heights from about 1.2 to 1500 mAUwith an 8-10 (JL injection volume and a 10-mm flow cell. Adjust injection volume to pro-duce the desired absorbance range, if desired,

Uracil (Stock solution, 1 mg/mL). Spikemobile phase with GVS at 6 mL/L to produceabout 0.006 mg/mL.Labeled: "GVS"

Column: HPLC tes-t column, 5-|jm C8, 75 mm x 4,6 mm — prequaiified for PQ testing

Mobile Phase-diluent: 13:87:1 acetoni-trile-water-acetic acid

Test Conditions— Isocratic:Flow rate: 2 mL/minWavelength: 273 nmInjection volume: 8 pL (adjust asrequired)Column temperature: 20-30 "CGradient test profile:

*Mobile phase B is spiked with GVS at 6mL/L of mobile phase

Injert spiked mobile phase B with nogradient delay time.

Initial 10-min linear gradient, hold at100% B for 5 min, then steps of 10% and90% of each solvent vs. the B spikedmobile phase.

Time (min)0101517232530

%A1000090901010

%B010010010109090

Repeat the 10%/90% steps for GB andD/B fluid circuits if present

Table III: Ranges of extra-column dispersion observed forinstruments in the laboratories of the authors

9

36

81

144

1444

1600

3

6

9

12

38

40

various analytical HPLC

Manufacturer A - LC 1

Manufacturer A - LC 2

Manufacturer A - LC 3

Manufacturer B - LC 4

Manufacturer B - LC 5

Manufacturer C - LC 6

iis pai[ of System Suitability. Because the the USF haif-height or tangent efficiencytest conditions arc defined and limited, and techniques. These dispersion values usuaiiy

peak tailing due to the column is tighdy differ by only a few microliters as comparedcontroUed, this method yields sufficiendy with the more rigorous statistical momentaccurate dispersion values even when using efiFiciency method. The volume standard

deviation (simply the square root of" thevolume variance), often is used as a moreconvenient term for the extracolumn dis-persion produced by an instrument, and issomedmes misleadingly referred to as extra-column volume. It is the volume dispersionthat is being measured, not the physicalvolume ofthe connecdng tubes.

The greatest value of this test is thecomparison of relative dispersion levelsof various instruments within the labora-tory, as well as deviations from the histor-ical norms established for a single instru-ment. The range of measuredextracolumn dispersion for six differentanalytical HPLC systems from three ven-dors is summarized in Table III.

Note that the dispersion can vary sig-nificantly even for two instruments ofthe same design, due to differences in theconnecting tubing and instrument setup.While extracolumn dispersion measure-ments should be considered accurateonly to 1-2 significant figures, they arcstill very informative. For example, onlythe first instrument would be even mar-ginally compatible with the newer 1.8-|j,m particle columns (assuming 10% lossfor a peak at k' - 1). Instruments 1-3would be suitabie generally for 3—p.mcolumns of at least 3 mm diameter andmoderate length, while instruments 4-6should be restricted to conventional 5-(xm or greater column technology.

The extracolumn dispersion measure-ment is not part of the written PQrequirements in our laboratories and,thus, no formal acceptance criteria areassigned. However, the value is recordedin the instrument logbook and pastedonto the instrument face, along withother critical performance data, to facili-tate rapid comparisons between instru-ments within the laboratory. It is a veryuseful parameter to have available whentransferring methods to other laborato-ries or to troubleshoot problems for agiven instrument, for example if largediameter tubing were substituted insome part of the critical flow path, or asemipreparative flow cell was mistakenlyleft in a detector, without proper docu-mentation in the logbook.

Detector linearity: Linearity ofresponse across at least three orders can beexpected for most modern HPLC detec-tors (USP <621 >) and is necessary for sin-gle dilution purity methods at the 0.1%

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concentration level. The six caffeine solu-tions with concentrations from about0.00035 to 0.35 mg/mL are designed toproduce peak heights falling within thelinear range of most modern HPLCdetectors, using the typical 10-mm path-length for an analytical flow cell. Theentire absorbance range can be shifted upor down to produce the desiredabsorbance for different flow cell volumesor instruments, by adjusting the nominalinjection volume to produce the desiredresponse range. Each solution is injectedin triplicate. Linear regression analysis isperformed on the entire data set of 18injections. A typical detector linearityplot is presented in Figure 6. Not shownis the plot of the residuals, which isprinted along with the linear regression,ro help illuminate nonlinearity.

Injector carryover: Immediately fol-lowing the last injection of the highestconcentration (L6) standard, we injectblanks of clean mobile phase. Anydetectable peak at the retention time ofcaffeine is the result of injector carrj'over.The percent carryover is calculated readilyas the area of the carryover peak dividedby the average of tbe L6 solution area. Itshould be noted whether a wash vial isused or not. We typically inject tbreeblanks, using tbe first to calculate carry-over. Tbe remaining two serve to docu-ment how quickly the injector is able tocleanse itself if carryover is observed.

The instrument manufacturer's specifi-cations sbould be consulted wben settingtbe acceptance criteria for tbis test. A max-imum carryover of ^ 0 . 1 % is common.However, tbis represents fairly substantialcarryover, and in most cases, values ofunder 0.03% sbould be expected from awell designed autosampler in good repair.Excessive carryover can be caused by wornrotot or needle seals, altbougb instrumentdesign also plays a large role.

Autosampler volume linearity(optional): If tbe autosamplet Is capableof variable injection volumes, the linear-ity and precision of volume deliveryacross a range can be used as a generalindicator of the condition of tbeautosampler delivery syringe and seals.Because tbe linear range of tbe detectoralready has been establisbed, tbe volumedelivery of tbe autosampler can bemeasured by selecting a test solute con-centration tbat will produce peak areas

falling within the establisbed detectorlinear range. Using tbe L2 caffeine solu-tion (0.0035 mg/niL), injections from5-100 (xL will produce peak beigbtsand areas witbin tbis demonstratedrange. These volumes can be modifiedto cover tbe injector design range. TheL2 solution is injected in triplicate ateacb of five volumes covering tbe injec-tor volume range. Linear regression isperformed as previously sbown for cbedetector linearity. In addition to goodlinearity, a nonsignificant interceptwitbin tbe 95% CL is expected.

Gradient qualification — dwell vol-ume and delivery accuracy: Gradientqualification consists of measurmg tbegradient dwell volume, the shape of ashort linear gradient, and tbe deliveryaccuracy at 10% and 90% of eacb fluidcombination. Tbis is accompHsbed byspiking one of tbe mobile pbases witbtbe nonretained uracil (fluid circuit B isused in tbis illustration), to produce amid-range absotbance signal at 273 nm(see Table II). An aliquot of tbis spikedmobile pbase is injected, starting the gra-dient witb no delay time.

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Table IV: Suggested HPLC performance qualification tests criteria

Manual Qualification Tests

Component

Pump

Column ovenRefrigeratedautosampler

PQ Test

Leak check

Flow-rate accuracy

Temperatureaccuracy

Procedures Suggested AcceptanceCnterta

Back-pressure regulatorwith gauge, or plugged

outlet

Flowmeter, or timedvolume collection for

three flow ratesover pump range

Calibratedthermocouple, air temp

No visible leaks.Loss of pressure <100

psi/mrn over 5 min

±5%

UV detector Wavelengthaccuracy

Fill flow cell withholmium oxide solution(241-641 nm); caffeine

(205 nm, 273 nm)

±3 nm

Automated Tests (results produced within single injection sequence)

System performance

Pump

Extracolumn instrumentdispersion

Flow stability

Gradient dwellvolume

Gradient deliveryaccuracy

Detector

Dynamic short termnoise

Linearity

Inject Rs test mixture,calculate dispersion

Retention time driftover 10 consecutive

injections of test solute(combined with

autosampler precision).

Inject spiked mobile phaseblank at start of gradient

Measure accuracy ofstep gradient of A vs. B*;Cvs, B*, D vs, B* at 10%and 90%, compared to100% B*. where B* is

spiked with uracil

Measure baseline noiseunder flow over 1-2 min

interval

Triplicate injections ofsolute over concentration

range of at least threeorders. Adjust upperabsorbance range to

1.2-1.5 AU.

No Pass/Fail specsRecord value

Drift NMT 1%

No Pass/Fail specsRecord value

±1% absolute

Record valueS/N for LI ^ 10

R2 ^ 0.999Residuals random

Autosampler

Precision

Volume deliverylinearity

(optional)

Injector carryover

10 consecutive injectionsof test solute

Triplicate injections oftest solute at 5 volumes

over selected range,within detector linear

range

Injea three blanksfollowing highest

concentration sample inDetector Linearity.

Use back-pressureregulator with gauge.

Test at moderate pressure,ca. 1000 psi with

back-pressure regulator

Test over anticipatedrange of use

Regress expectedvs. found to look

for trends

Compare to previousvalues and to similar

HPLC systems

There should be nooutliers or indicators of

unstable flow

Compare to previousvalues and to similar

HPLC systems

Observe sharpness oftransitions and any

anomalies in deliveryprofiles

Compare to previousvalues and similar

HPLC systems

Range may adjusted toproduce desired

absorbance values

%RSD ^ 1 %

fi2 ^ 0.999Residuals random

<«0,l% for first blankinjection, or as per instru-

ment specifications.

There should beno outliers

Optional. Can beperformed initially uponinstrument installation as

part of OQ along withvolume accuracy.

May be performed withor without wash vial,

or both.

www.chromatographyonllne.com MAV 2008 LCGC NORTHAMERia VOLUME 26 NUMBERS 4 7 9

A single gradient method is written,consisting of an initial 10-min lineargradient from 0 to 100% B. After a 5-min hold at 100% B to establish themaximum signal height, delivery accu-racy is measured at 10% and 90% ofeach combination of the other fluid cir-cuits with the spiked B circuit, using a 3-min hold ac each level. The hold timescan be varied if required to produce sta-ble signals at each level.

To start rhe qualification, an aliquot otthe uracil-spiked mobile phase is injectedand the gradient profile is started. Theuracil is eluced in the column void vol-ume, while the onset ot the gradientbegins later, delayed by the gradientdwell time. The dwell volume is calcu-lated using the previously calibratedpump flow rate, multiplied by the delaytime. An example of a dwell volumedetermitiation is shown in Figure 7.

Figure 8 shows the full chromatogramof a typical gradient qualification profilefor a quaternary gradient system.

This general technique based uponuracil offers several significant advantagesover using a jumper tube in lieu of a col-umn. First, this procedure measures the

operation ofthe entire gradient HPLCsystem under actual operating conditionsot pressure and flow with a column inplace. Second, the entire procedure isfully automated, and can be pro-grammed as part ofthe overall PQ injec-tion sequence, so that no manual substi-tution of jumper tubes is required. It issimple, and the steps of 10% and 90%delivery for each solvent combinationadequately test the system near theextremes of its solvent delivery and mix-ing system. More-complex profiles can ofcourse be generated if desired.

No acceptance criteria are assigned tothe gradient dwell volume, because this isa design feature of each particular instru-ment. However, it is a critically importantparameter to know for a gradient instru-ment (11,12) and its value is recorded inthe logbook, and noted on the face of theinstrument. For critical gradient methods,certain instruments might need to beflagged as being not suitable if they pro-duce excessive dwell volumes or indistinctgradient profiles. During method develop-ment and validation, instruments with dif-ferent gradient dwell volumes can beselected deliberately for robustness testing.

During method transfer and troubleshoot-ing, the gradient dwell volume of thereceiving laboratory should be noted.

The general shape of the initial linearsegment is documented, but no quantita-tive acceptance criteria are applied. For thedelivery accuracy, an acceptance criterionof ± 1 % absolute is used, (equivalent to± 10% relative ofthe lower delivery range).

Table IV summarizes the test protocolsthat have been discussed previously,along with typical acceptance criteria.

Final documentation: If any ofthe PQtests foil, an investigation is initiated, andthe cause ofthe failure is determined andcorrected before retesting, as per our inter-nal SOPs. After qualification, the sum-mary results are entered into the labora-tory database. We also affix to theinstrument a brief tabular summary ot theactual values obtained from the qualiflca-tion, such 2S the dynamic noise, extracol-umn dispersion and gradient dwell vol-ume, in addition to the normalqualification sticker. This way, the per-formance ofthe various HPLC systems inthe laboratory can be compared readily, ascan changes in the performance of a giveninstrument compared to its historical

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record. This information can be extremelyvaluable when selecting instruments forintermediate precision, and of coursewhen comparing instruments duringmethod transfer and similar activities.

ConclusionsImplementing a comprehensive HPLCinstrumentation maintenance and qualifi-cation program is a cGMP requirement.The use of a standardized PQ test system,complete with a prequalified column,greatly facilitates the routine testing of lab-oratory HPLC systems. The test systemhas been designed to be robust, while pro-viding fast separations to reduce overalltime to perform a qualification. A compre-hensive suite of test protocols can be com-pleted within a few hours. Having such asimple, yet universal test system availablein the laboratory has many advantages:

In-house instrument qualification:The mosc obvious advantages ot using anin-house performance qualification sys-tem are tbose of quality, time and money.In our experience, HPLC qualificationsare often of higher quality when per-formed by trained laboratory personnel.Not only does the laboratory develop an

intimate feel for the operating character-istics of each of its HPLC systems, butmore time can be taken to investigatesmall aberrations in performance. Withclear, well written SOPs, routine PQs canbe completed within hours, at a largecost savings, with the added flexibility ofbeing able to complete instrument test-ing on the laboratory's schedule ratherthan the instrument vendor's schedule.

Trending the performance of asingle HPLC instrument: A major advan-tage of using a standardized test methodfor HPLC qualification is that one canobtain trends in the historical performancerecord of a single HPLC system. The accu-mulated performance history of eachHPLC system is readily available, and anychanges in the instrument performancesuch as after repairs or moving to a newlocation (precision, noise, efficiency, sensi-tivity) are immediately obviotis.

Comparison of HPLC performance lab-oratory-wide and worldwide: A stan-dardized, complete PQ test system permitsdirect comparison of che critical perform-ance characteristics of all HPLC systemswith and between laboratories under iden-tical test conditions, so that valid compar-

www. chromatographyonlme.com

isons of performance can be made betweeninstruments. This information can be use-ful when selecting equivalent (or non-equivalent) instruments for robustnessevaluation during method validation.

Instrument and method trou-bleshooting: If a problem is suspectedduring routine instrument operation, aquick PQ test wilt readily distinguishwhether the problem lies with the testmethod, the user's understanding ofthemethod or the instrument performanceitself If the test solutions are maintainedin [he laboratory, instrument problemsoften can be isolated quickly, savingweeks of troubleshooting and reducingunnecessary travel to remote sites.

Method transfer: Before method trans-fer, the recipient laboratory anywhere inthe world can test their HPLC conditionsidentical to the originating laboratory. Inthe event of method transfer problems(which can be common), instrument per-formance characteristics (gradient dwellvolume and delivery accuracy, injector pre-cision and carryover, noise, sensitivity, andso forth) Gin be compared fbr equivalencyand suitability before transfer. For methodtroubleshooting, one can go back quickly

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to [he PQ conditions to rule out Instrii-mennil problems.

Training: This test system also makes anexcellent training and evaluation tool fotanalysts new to HPLC. The analyst is giventhe materials and procedures in the kit,using an instrument that has been previ-ously qualified, and is instructed to makethe mobile phase and run the variou.s testprotocols. Because the separation and thequality of the expected data ate alreadyknown, the new analyst becomes theunknown. This exetcise can help the stu-dent learn all aspects of the operation of theinstrument under both isocracic and gradi-ent conditions, including how to use thedata system and selea suitable integrationparameters, as weU as proper preparation ofthe mobile phase and column installation.

Ensuring and documenting the qualityof analytical data is a fundamental respon-sibility of all analytical laboratories. Instru-ment qualification is one of the basic com-ponents required to achieve thi.s goal. Evenif preventative maintenance is contractedCO an outside vendor, final qualification byin-house personnel is highly recom-mended. The accumulated historical dataacquired under universal test conditions

becomes a vety strong pillar, complement-ing method validation, and training.

Ack now ledg mentsThe authors would like to thank Mr,Frank Harris of Vintage Pharmaceuti-cals, Inc., and Mr. Nilesh Patel of ActavisPharmaceutical Company for their inputand assistance.

References1I) H. Lam, in Analytical Method Validation and

Instrument Perfomiance Verification, C C .

Chan, Y.C. Lee. H. Lam, and X.M, Zhang,,

Eds. (Wiley-Interscicnce, Hoboken, New

Jersey, 2004), pp. 173-185.

(2) J. Crowther, M.L Jimidar. N. Niemeijer,

and P. Salomons, in Analytical Chemistry in a

GMP Environment, J.M. Miller and J.B.

Crowcher, Eds. (Wiley-lnterscience, New

York, 2000), pp. 423-458.

(3) United States Pharmacopeial Forum, Vol.

32(2). pp. 595-605, <I058>, Analytical

Instrumeni Qualification, Mar.-Apr. 2006,

(4) V. Grisanti and E.J. Jachowski, LCGC

20(4), 356-362 (2002).

(5) C. Hall and J.W. Dolan, LCGC 20(9),

842-848 (2002).

(6) W,B, Furman, T.P. Ijyloff, and R.F. Ter̂ lalT.

http://www. fda.gov/ora/science_ref/priv_la

b/co mp_l i q_ch ro/jaoac.htm.

(7) United States Pharmacopeia, Chapter <621 >.

(8) J,C, Travis, J.C. Acosta, G. Andor, J, Bastie,

V. Blattner, CJ. Chunnilall, S.C Crosson,

D,L. Ducwcr, E.A. Early, F. Hengstberger,

CS. Kim, L. Liedquist, F. Manoocheri. F.

Mercader, L,A.G. Monard, S, Nevas, A.

Mito, M. Niisson, M. Noel, A.C

Rodriguez. A. Ruiz, A. Schirmacher, M.V.

Smith, G. Valencia, N. van Tonder, and J.

Zwirikds,/ Phys. Chem. Ref. Data 34(1),

41-56(2005).

(9) S. TomeLlini, Univ, Of New Hampshire, pri-

vate communications.

(10) K.W. Freebairn and J.H. Knox, Chro-

matographia 19. 37-47 (1985).

(11) L.R. Snyder, J.J. Kirkland, and J.L. Glajch,

in Practical HPLC Method Development

(Wiley, New York. 1997), pp. 386-394,

(12) J.J. Gilroy and J.W. Dolan, LCGC 24(7),

662-668 (2006). •

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