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4 6 4 LCGC NORTH AMERICA VOLUME 26 NUMBERS MAY Z008

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Performance Qualification of HPLC


Instrumentation in Regulated
Laboratories
With the forthcoming USP monograph <1058>, many laboratories are in
the process of reexamining their high performance liquid
chromatography (HPLC) instrumentation qualification practices. This
article demystifies the qualification procedures and proposes a well
designed, easy and simple set of experiments upon which to establish
internal standard operating procedures (SOPs) for the complete
qualification of HPLC instruments. A key concept is the development of
a consistent test system, comprised of premade test solutions, a
prequalified HPLC column, standardized protocols, and validated
software that can be prepared in-house or purchased commercially as a
kit. This system can be applied to any HPLC system worldwide, to
produce comparable test results under uniform conditions. The test
system is designed to be rapid, with a comprehensive performance
qualification being completed in about 2 h for isocratic, and 3 h for
quaternary gradient systems.

Jonathan Crovrther*, Jeremy


Dowling+, Richard Hartwick*, and
Bill Ciccone+*
*Ortho Clinical Diagnostics, Raritan, New
Jersey
tPharmAssist Analytical Laboratory, South
New Berlin, New York
ttMicroSolv Technology Corporation,
Eatontown, New Jersey
Piease direct correspondence to Richard
Hartwick at rhartwick@pharma55lstlab.com

he generation of high-quality, reliable analytical data is grounded on


three fundamental components:
instrument qualification, method validation, and user training (1,2). For the
pharmaceutical industry, these activities
fall under cGMP/GLP regulations.
Although the specific regulations can vary
for the environmental or other industries,
the principles remain the same.
The first of these, instrument qualification, is the focus of this article. A laboratory plan for analytical instrument
qualification (AIQ) is a requirement for
all cGMP/GLP laboratories. The pending USP guidance document <1O58> (3)
reflects the evolving accepted practices
for the introduction and qualification of
analytical instrumentation into the regulated laboratory environment.
In.struments must be maintained systematically and proven to be precise and
accurate for their intended use on an
ongoing basis (4,5). However, the specific
qualification procedures are, appropriately, not predetermined by regulation.
Instead, the laboratory management is

responsible for developing a scientifically


sound, risk-based plan for the periodic
maintenance and qualification of their
analytical instruments. Nonetheless, the
approach is subject to FDA review.
A sound instrument qualification program should be both scientifically rigorous
and straightforward to use. It must be sufficiendy comprehensive to capture aberrant instrument performance, yet be rapid
enough to promptly return instruments to
service after the maintenance or repairs
have been completed. Development ofthe
standard operating procedures (SOPs) and
testing materials tor the qualification program can be a daunting, and sometimes
confusing task. This article presents an
approach that we have developed and trialed over many years, which provides a
comprehensive, rapid performance qualification for high performance liquid chromatography (HPLC) instruments.
Who Should Perform the
Qualification?
The proposed USP AIQ monograph
<1O58> states that, "Users are ultimately

4 6 6 LCGC NORTH AMERICA VOLUME 26 NUMBtB S MAT 2008

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responsible for instrument operarions and


data quality. The user's group encompasses
anaJysts, their supervisors, and organization
management." It further states that:
"Users should also be responsihle for
qualifying their instruments, because their
training and expertise in the use of instruments make them the best-qualified groups
to design the instrument lest(s) and specification(s) necessary for successful AIQ."
This view of user responsibility is
shared by the FDA (6).

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 SRM
2034. Preferred absorbance bands are shown above peaks 1, 3, 4, 7, 9, 10, 13, and 14.

250

275
300
Wavelength (nm)

325

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

The AIQ Process


The AIQ process is often summarized as
"The Four Qs" that is, the design,
installation, operational, and performance
qualifications, referred to as DQ, IQ, OQ,
and PQ. For a new installation, the instrument vendor often will be responsible for
the IQ and O Q procedures, albeit under
laboratory SOPs governing this operation.
Depending upon the vendor, some abbreviated form of a system check also might be
performed. Some vendors refer to this as a
performance verification {VV). The exact
procedures Lised will vary with each manufacturer. This IQ-OQ-PV process essentially is performed once per installation.
Upon completion, responsibility for routine maintenance and periodic qualification is transferred to the user, even if outside contractors are employed for future
preventative maintenance and .service. lx>gicaliy, because each instrLiment vendor has
their own particular qualification routines,
a master-level set of iabomrory PQ testing
protocols must be created to generate uniform performance data across the different
brands ot HPLC systems within the laboratory, while specifying measures that are
required due to instrument maintenance,
repair, or change. Such protocols must be
consistent with the company's overall
change control policy.
Table I summarizes the AJQ process and
typical situations in which each stage might
be applied. Specific choices will vary from
laboratory to laboratory, and this is not
purported to be a comprehensive plan for
all contingencies. However, some rational,
prewritten plan must exist to govern whatever procedures are decided upon.
Note that unlike the OQ, PQ testing is
performed frequently, for many different
events. System suitability of a particular
method is not a substitute for a PQ,
although its procedures might be incorpo-

4 6 8 LCGC NORTHAMERia VOLUME 26 NUMBER 5 MAY 2008

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rated into a PQ testing protocol. A system


suitability is method specific, and serves a
very difierent purpose than a fiili PQ.
O Q and P Q also have different purposes, although similarities often exist in
tests used for these qualification steps. A
distinguishing feature of the O Q is its
focus on testing the individual instrument tnodule, and often is driven by the
manufacturers design specifications. PQ
testing on the other hand, is holistic, and
documents the performance ofthe working system, which, thus includes both
hardware and software issues.

250

300

350

400

450

500

600

550

650

700

Expected wavelength (nm)

Figure 3: Linear regression of the found vs. expected wavelengths for the combined
results of the holmium oxide (squares) and caffeine (triangles) wavelength
qualification.

so

Jv.

0.J

1.0

1,8

;V

J V
i!j~

lio

3M

HD

ill
Tlmt (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 precision
determination.

This difference is refleaed in the development of reasonable acceptance criteria.


While O Q requirements are influenced by
the design Hmics of each particular module, PQ acceptance criteria reflect the minimum acceptable performance levels
required for all instruments of similar
types in the laboratory. The assignment of
reasonable, acceptance criteria can be one
of the more difficult aspects of the entire
PQ process. When available, acceptance
criteria are taken from compendial or
other official sources, for example, USP
<621>. Otherwise, scientifically reasonable, defensible criteria are assigned.
A word of caution is in order here. Users
sometimes will look to the specifications
section oF an instrument manual for
acceptance values. In our experience, those
values for wavelengtli accuracy, noise, stability, and so forth are instrument-specific.
Moreover, they sometimes are obtained
under ideal conditions that cannot he replicated easily in the laboratory. Assignment
of such vendor-specific values to all laboratory instruments invites unwarranted testing failures. P Q tesring is coming from the
opposite direction, and seeks to define an
acceptable performance level for all instruments in the laboratory. It is thus expected
that most instruments will perform significantly better than the limits set by the PQ.
Under that umbrella, the perfomiance history of each individual instrument
becomes its signature, and is of greater
value than its absolute performance. The
rationale for die assigned values for some of
the acceptance criteria are discussed more
fully in the "Results" secrion.
Logical and clearly written ongoing
maintenance procedures are equally
important co a successftil P Q program.
Selected tests from the suite of test protocols can he performed, depending upon

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the situation. For example, ifa pump seal


fails midway before the next scheduled
preventative maintenance due to normal
wear, it is not necessary to requalify the
entire LC system. After investigation and
documentation of the cause (and its
impact on any data already generated),
only the pressure leak check and flow rate
requalification normally would he performed. A similar logic would be applied
to lamp changes. As part of the root
cause assignment for the failure however,
ihe laboratory should review its preventative maintenance frequency, perhaps
increasing it it such failures are common.
On the other hand, relocation of the
instrument to a new bench or adjacent
laboratory would initiate a fiill PQ, plus
updates to the logbook and database documenting the new location. Shipmenc of
che instrument ro a new location would
be treated like a new installation, triggering the entire I Q - O Q - P Q process, and
perhaps a D Q if warranted.
The dcHnition of instrument portability
must be explicidy addressed in the SOR An
example ofthis might be an HPLC system
on a portable care that is used periodically
for LC-mass spectrometry (MS) work.
Without a ciear defmitJon of portability in
the relevant SOP, an overaealous auditor
might conclude that rolling the instriunent
over to the mass spectrometer constitutes a
move, requiring a requalification, as has
happened to one of the authors on occasion. Clearly written SOPs are essential to
anticipate and forestall such issues.

MAV 2008 LCG: NORTH AMERICA VOLUME 26 NUMBERS 4 6 9

reproduced easily in any laboratory worldwide on any brand of instrument.


Table 11 summarizes the PQ test system
components. These solutions can be prepared and qualified in the laboratory under
NIST-traceable conditions, or can be purchased commercially in kit form. The
mobile phase stability is 60 days, so that it
can be prepared in large batches and stored
for multiple instrument qualifications.
The retention time window for caffeine
is set at 1.0-1.5 min. A comprehensive isocratic qualification for al! test protocols
requires about 50 injections and is com-

pleted in as little as 1.5 h. Additional gradient qualification requires ahout 35 min


for a binary system, or 65 min for a quaternary. Selected partial qualification test
protocols such as autosampler precision
and pump stability are completed in less
than 30 min. Detector wavelength accuracy, column oven, refrigerated autosampler temperature accuracy, and flow-rate
verification are manual operations. The
remaining qualification tests are completed within a single injection sequence.
Only three methods are required; one of 3min length for the resolution test mixture

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Development of a PQ Test
Method
We have developed a stiite of test methods
tofiillyevaluate the instrument under realistic conditions, yet be as rapid and as automated as possible. These test solutions have
proven to be chemically stable at room
temperature for at least two years. We
maintain all test components as a single,
convenient kit, which includes the solutions, a prequalified base-deactivated PQ
test column, test protocols, and validated
Excel template for data analysis. This creates a closed, reproducible system, in which
the only variable is the mobile phase. A syslem suitability solution is included to confirm proper mobile phase preparation and
column performance. With this approach,
the PQ test system hecomes an independent, universal measuring tool that can be

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Corporation (Eatontown, New Jersey).


The mobile phase was filtered and
degassed using a 0.45-|xm nylon filter.
A validated Excel template was used
for calculations and graphing. The template required only entry ofthe raw data,
with all subsequent calculations and
graphing being performed automatically
by the spreadsheet. In addition to
detailed results and graphs, the spreadsheet provides a single-page summary
sheet for review and signoff.

Figure 5: Extracolumn volume dispersion as measured from a typical resolution test


mixture chromatogram.
and system suitability testing, a second
method of about 1.8 min for the bulk of
the test injeaions atid a gradient method
(if applicable) for dwell volume and accuracy determination.

Experimental
Acctonitrile was HPLC grade, purchased
from EMD (VWR, West Chester, Penn-

sylvania). Water was purified in-house,


meeting USP reagent-grade specifications. Caffeine, uracil, theophylline, and
8-chlorotheophylline were ACS reagentgrade or better, and were purchased from
Sigma-Aldrich (St. Louis, Missouri). The
certified PQ test column, 75 mm X 4.6
mm. Cogent C8, 5-|jLm particle size, was
obtained from MicroSolv Technology

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For flow accuracy and leak-testing, a


device was constructed from a back-pressure regulator with a qualified pressure
gauge. A Tescom (Elk River, Minnesota)
mode! 26-1721-24-084 back-pressuit regulator was used, with a rating of 10,000 psi.
Appropriate tubing and male-unions were
fitted, so that the colutnn itilet tubing
could be connected to the union. Dynaseal variable stop depth fittings were supplied by Sonntek {Upper Saddle River,
New Jersey) to ensure secure leak-tight fittings without regard to stop-depths. The
principle author can be contacted electronically if further details regarding construction of this device are required.
In practice, the back-pressure regulator
assembly is fitted in lieu of the normal
column. Any desited back pressure is
applied by the regulator knob of the
back-pressure regulator, at any flow rate.
Flow accuracy measurements at various
flow rates are made at a constant 1000
psi backpressure. The HPLC pressure
readout is checked against the gauge at
increments of 1000 psi, up to the maximum instrument pressure, typically just
under 6000 psi. This hack-pressure regulator system has proven to be an
extremely useful and versatile tool to
have available in the laboratory. It is used
to diagnose pump check valve problems
by observing pressure fluctuations. It has
proven robust enough that it also can be
used to perform leak checks, such that
the system can be pressurized at any
desired level, and the rate of pressure loss
directly observed on the gauge.
Chromatograms in the figures were
produced using an Agilent 1100 diode
array system (Santa Clara, California)
with a quaternary gradient. The flow cell
was the standard analytical cell.
The test kit used in this article, complete
with column, solutions, SOPs, and validated software, is available commercially

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MAV 2008 LCGC NORTH AMERICA VOLUME 26 NUMBERS 471

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[he High Speed Qualification (HSQ) Idt.
Results and Discussion
Once the laboratory SOPs have been
written and approved, and the instrument methods and sequences assembled,
routine PQ testing is quite straightforward. The following shows typical results
for some ofthe test protocols, along with
a discussion of acceptance criteria.
Manual operations pump accuracy,
column oven temperature: Pump flow
accuracy and stability are the foundation
upon which the subsequent testing is
built, because gradient dwell volume and
other tests will use this value in their calculation. Most laboratories use dry volumetric flasks of different volumes, with a
NIST-traceable calibrated stopwatch to
measure flow rates. We use our back-pressure regulator system (see Experimental)
to impose a constant pressure of 1000 psi,
and measure the fiow rates at nominal
settings of 0.5, 1.0, and 5.0 mL/min. The
qualification flow range should encompass all methods in the laboratory and
can he modified as desired. Given the
potential errors in drop collection and

1000-

0.00

0.05 0.10

0.15

0.20 0.25

0.30

0.35 0.40

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

timing, an accuracy specification of 5 %


is assigned for this test.
Column oven temperature is measured by using a NIST-traceable digital
thermometer, inserting a flexible thermocouple into the compartment, taking
care not to allow it to test on any metal
supports. The air temperature is measured over a temperature range encompassing all laboratory methods, with
acceptance criteria of 5 "C.

Column oven designs vary widely in


their preheating designs and efficiency,
and the actual mobile phase temperature might deviate substantially from
the oven air temperature. There is simply no easy way to measure accurately
the true internal column fluid temperature, which will vary with hoth radial
and axial position within the column
due to frictional heating (especially for
ultrahigh-pressure LC), and other

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wavelength maximum found by interpolation. Alternatively, a series of no-injection,


no-flow methods can be written, each with
a different wavelength, stepping across the
spectral region to fmd the maximum. Figure 1 shows the hoimium oxide spectrum.
For the UV range, the 241-. 287-, and
361-nm bands are convenient, while the
451-, 537-, and 64l-nm bands can be
added for visible-wavelength detectors.

70

'app
SO

40

20

-10
0.00

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 uracilspiked mobiie phase, tg is the time for the nonretained uracil peak, and t^pp is the
apparent time of the first visible onset of the gradient.

issues. In a similar vein, refrigerated


autosamplers also are qualified using
air, rather than solution temperatures,
to the same specification.
Wavelength Qualification
The qualification of wavelength accuracy is
a critically important test. Holmium oxide
solution in 10% perchloric acid (NIST
SRM 2034) is an internationally accepted
wavelength standard, covering the range of
241-641 nm. Caffeine is used as a secondary standard, with two bands at 205 and
273 nm, in this diluent. Thus, both standards can be used in combination to qualify
the detector over the range of 205-641 nm
(or to 56] nm for the UV range only). The
wavelength accuracy specification is set at
3 nm, in accord with USP <621> (7).
Both variable-wavelength and diodearray detectors can be qualified by first

autozeroing the detector with diluent in


the flow cell, then pulling the solutions
through the cell with a spring-loaded
syringe and tubing, with a finger-tight fitting securing it to the detector outlet
(never attempt to push the solutions, for
safety reasons). Release the vacuum before
testing to eliminate bubbles and produce a
stable signal. For diode-array detectors, a
spectrum of the flow-cell contents is taken,
and the maxima are determined with the
resident instrument software. While there
are 14 available bands for holmium oxide,
it is only necessary to measure three or four
of these over the spectral region of interest
(UV, visible, or both). For variable wavelength instruments, the wavelength is set
sequentiaUy in 1 -nm increments, for a few
nanometers before and after tbe
absorbance band of interest. The
absorbance reading is recorded, and the

The found absorbance maxima are compared against the official published NIST
values (8). The consensus spectral values
are published for spectral bandwidths of
0.1, 1.0, and 3.0 nm. For most HPLC
detectors, comparison against the 3.0 nm
spectral bandwidth would be appropriate.
Figure 2 shows the spectrum of caffeine
in tbe mediod diluent, u.sed to extend the
qualification to 205 nm. Note that the 273
nm maximLim overlaps with the holmium
oxide spectral range, thtis, linking it to the
NIST standard values. These spearal maxima have been confirmed independently to
be accurate with this mobile phase and
diluent (9). For diode-array systems, the
caffeine spcxtrum can be acquired conveniently during the main injection sequence
from of any of the mid-concentration solutions producing a good signal-to-noise
ratio (S/N). Figure 3 shows the combined
results for a typical wavelength accuracy
determination using the combined results
of both the holmium oxide and caffeine
solutions, as automatically generated by the
validated software. The slope of the regression should equal one, with a statistically
nonsignificant intercept. While not used
for qualification purposes, the regression
line can be extrapolated to 200 and 700
nm, to show any inaccuracy trends at wavelengths outside the qualification range.
The combined solutions, with their narrow absorbance maxima across a wide
wavelength range, produce what is currently a state-of-the-art wavelength accuracy qualification. However, some laboratories might have methods that use
wavelengths falling outside of this range.
The question frequently arises as to
whether it is valid to use a method beyond
the qualified wavelength range of the detector. Some feel that if a method of say, 200
nm is tised, that the detector qualification
must include that wavelength. The other
view is that if the wavelength accuracy is
confirmed at several points across a broad
region of the spectrum, the detector can be

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

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presumed to be in reasonably good operating condition and is, thus, qualified for use
across its entire wavelength design range.
We support this second view.
In support of this argument, note that
for UV-vis spectropboto meter wavelength
qualifications, most compendia! agencies
(including the USP, BP, and EP) specify
hoimium oxide as a suitable wavelength
reference standard, with its limited range
of 241-641 nm. None of these agencies
state that a spectrophotometer cannot be
used beyond this qualified range, which
would be the inference from the first
approach. By confirming operation at several discrete wavelengths across a broad
swath of its design range, one assumes that
the instrument will function as designed
across its entire range. This i.s an assumption, but it is not unreasonable. Indeed,
even within the qualification wavelength
range, there is no proof that the monochrometer is not malfunctioning at some
particular wavelength between the checkpoints. Tbe system suitability of each
method run on the instrument is designed
to provide such additional run-time assurance of the system performance.

Isocratic HPLC Qualification


System suitability, noise, and extracolumn dispersion: Once the wavelength
qualification, flow accuracy, and column
oven temperature checks have been completed, all remaining tests are accomplished
by a single fully automated injection
.sequence on the HPLC system. Figure 4
shows typical injections of the resolution
test mixture solution, along with the (L3)
caffeine solution (also containing uracil)
used for injector precision and pump flow
stability. A system suitability test is performed to demonstrate correct retention
times and column efficiency and that the
instrument is operating properly and ready
to perform the required test protocols.

Dynamic noise: Short-term dynamic


noise is determined for a blank injection at
the method wavelength of 273 nm, thus,
documenting total instrument noise levels
under realistic operating conditions.
Because all method conditions are constant, this noise value can be compared to
historical levels for the same instrument, as
well as to other instruments in the laboratory. A minimum S/N ratio of ^ 1 0 is
required for die LI peak (generated in the
method injection sequence), which is 0.1 %
of the highest concentration solution.
Unusually high noise levels can indicate a
failing lamp, a dirty flow cell, pump noise,
or other problems. The noise values given
by the manufeaurers in their instrument
specifications are usually for the detector
alone under ideal conditions that cannot be
replicated easily in the laboratory. Such a
specific noise measurement might be performed as part of the OQwhen an instrument is first put into service. The dynamic
procedure described here has the advantage
of providing a realistic noise measurement
under standardized operating conditions,
so that various laboratory HPLC systems
can be compared directly. Note that the
deteaor-data system time constant affects
the noise and, thus, should be documented
as part of the run method.
Extracolumn dispersion: Extracolumn
volume dispersion results in the loss of
sharpness of the eluted peak due to the
injeaor, conneaing tubing, and flow cell.
It is the limiting parameter that determines
whether small-particle,
low-volume
columns can be successfully used in a given
instrument. It is not recommended that an
acceptance criteria for extracolumn dispersion be part of the PQ. However, it is a very
usefiil instrument characteristic to know,
and should be documented during the
course of the PQ.
The most accurate way to measure
extracolumn dispersion is to measure the

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350

300

100% B*

250

10:90 A : B '

10:90 C:B*

200

10:90 D:B*

1 1
1

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iinear gradient

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90:10 C B '

90:10 D:E*

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-50

10

20

30

40

50

60

Time (m n)

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

dispersion with a series of jumper rubes


in lieu of the column, and extrapolate to
zero length. However, a simpler, yet sufficiently accurate way is to plot the peak
variances against the square of the retention volumes (10) as follows.
Because variances are additive, the
observed total system variance is
2

[1]

system = CT

The variance in voliime units is calculated from the retention volume and the
measured peak efficiency as follows:
[2]
N
The retention volume is simply the flow
rate times the observed retention times for
the resolution test mixture peaks, thus

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where f^ Is the retention time (in minutes) of the peaks in the resolution test mixture (assuming the extracolumn volume
delay is negligible relative to the peak retention times), F'n theflowrate (as microliters
per minute), and N^.^^^,,, is the measured
efficiency for each peak. Regression yields
an intercept of the extra column dispersion
(a^^) in units of |xL2 (mm'') with a slope
of the reciprocal of the true column efficiency, exclusive of extracolumn effects.
The test mixture was designed specifically
to match the dispersion levels found in
modem analytical HPLC systems. Figure 5
shows a typical plot for determination of
extracolumn dispersion. The exrracolumn
dispersion data is obtained fi'om the injection of the Resolution Test Mbcture, shared

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4 7 6 LCGC NORTH AMERICA VOLUME 26 NUMBER 5 MAY 2008

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Table II; Summary of qualification test system components*

Composition: ca. 0.05 mg/mL in each. Adjust


to produce about equai peak heights at 273
nm for ali components.
Uracii
Theophyliine
Caffeine
8-Chiorotheophyiiine
Labeled: "RTM"

Resolution test mixture (RTM)

Caffeine: (linearity solutions;

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

Gradient visualization soiution (GVS)

Uracil (Stock solution, 1 mg/mL). Spike


mobile phase with GVS at 6 mL/L to produce
about 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 acetonitrile-water-acetic acid
Test Conditions Isocratic:
Flow rate:
2 mL/min
Wavelength:
273 nm
Injection volume: 8 pL (adjust as
required)
Column temperature: 20-30 "C
Gradient test profile:

Time (min)
0
10
15
17
23
25
30

*Mobile phase B is spiked with GVS at 6


mL/L of mobile phase
Injert spiked mobile phase B with no
gradient delay time.
Initial 10-min linear gradient, hold at
100% B for 5 min, then steps of 10% and
90% of each solvent vs. the B spiked
mobile phase.

%A
100
0

0
90
90
10
10

%B
0
100
100

10
10
90
90

Repeat the 10%/90% steps for GB and


D/B fluid circuits if present

Table III: Ranges of extra-column dispersion observed for various analytical HPLC
instruments in the laboratories of the authors

Manufacturer A - LC 1

36

Manufacturer A - LC 2

81

Manufacturer A - LC 3

144

12

Manufacturer B - LC 4

1444

38

Manufacturer B - LC 5

1600

40

Manufacturer C - LC 6

iis pai[ of System Suitability. Because the


test conditions arc defined and limited, and
peak tailing due to the column is tighdy
controUed, this method yields sufficiendy
accurate dispersion values even when using

the USF haif-height or tangent efficiency


techniques. These dispersion values usuaiiy
differ by only a few microliters as compared
with the more rigorous statistical moment
efiFiciency method. The volume standard

deviation (simply the square root of" the


volume variance), often is used as a more
convenient term for the extracolumn dispersion produced by an instrument, and is
somedmes misleadingly referred to as extracolumn volume. It is the volume dispersion
that is being measured, not the physical
volume ofthe connecdng tubes.
The greatest value of this test is the
comparison of relative dispersion levels
of various instruments within the laboratory, as well as deviations from the historical norms established for a single instrument. The range of measured
extracolumn dispersion for six different
analytical HPLC systems from three vendors is summarized in Table III.
Note that the dispersion can vary significantly even for two instruments of
the same design, due to differences in the
connecting tubing and instrument setup.
While extracolumn dispersion measurements should be considered accurate
only to 1-2 significant figures, they arc
still very informative. For example, only
the first instrument would be even marginally compatible with the newer 1.8|j,m particle columns (assuming 10% loss
for a peak at k' - 1). Instruments 1-3
would be suitabie generally for 3p.m
columns of at least 3 mm diameter and
moderate length, while instruments 4-6
should be restricted to conventional 5(xm or greater column technology.
The extracolumn dispersion measurement is not part of the written PQ
requirements in our laboratories and,
thus, no formal acceptance criteria are
assigned. However, the value is recorded
in the instrument logbook and pasted
onto the instrument face, along with
other critical performance data, to facilitate rapid comparisons between instruments within the laboratory. It is a very
useful parameter to have available when
transferring methods to other laboratories or to troubleshoot problems for a
given instrument, for example if large
diameter tubing were substituted in
some part of the critical flow path, or a
semipreparative flow cell was mistakenly
left in a detector, without proper documentation in the logbook.
Detector linearity: Linearity of
response across at least three orders can be
expected for most modern HPLC detectors (USP <621 >) and is necessary for single dilution purity methods at the 0.1%

MAY 2008 LCGC NORTH AMERICA LUM 26 NUMHSR S 4 7 7

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concentration level. The six caffeine solutions with concentrations from about
0.00035 to 0.35 mg/mL are designed to
produce peak heights falling within the
linear range of most modern HPLC
detectors, using the typical 10-mm pathlength for an analytical flow cell. The
entire absorbance range can be shifted up
or down to produce the desired
absorbance for different flow cell volumes
or instruments, by adjusting the nominal
injection volume to produce the desired
response range. Each solution is injected
in triplicate. Linear regression analysis is
performed on the entire data set of 18
injections. A typical detector linearity
plot is presented in Figure 6. Not shown
is the plot of the residuals, which is
printed along with the linear regression,
ro help illuminate nonlinearity.
Injector carryover: Immediately following the last injection of the highest
concentration (L6) standard, we inject
blanks of clean mobile phase. Any
detectable peak at the retention time of
caffeine is the result of injector carrj'over.
The percent carryover is calculated readily
as the area of the carryover peak divided
by the average of tbe L6 solution area. It
should be noted whether a wash vial is
used or not. We typically inject tbree
blanks, using tbe first to calculate carryover. Tbe remaining two serve to document how quickly the injector is able to
cleanse itself if carryover is observed.
The instrument manufacturer's specifications sbould be consulted wben setting
tbe acceptance criteria for tbis test. A maximum carryover of ^ 0 . 1 % is common.
However, tbis represents fairly substantial
carryover, and in most cases, values of
under 0.03% sbould be expected from a
well designed autosampler in good repair.
Excessive carryover can be caused by worn
rotot or needle seals, altbougb instrument
design also plays a large role.
Autosampler
volume
linearity
(optional): If tbe autosamplet Is capable
of variable injection volumes, the linearity and precision of volume delivery
across a range can be used as a general
indicator of the condition of tbe
autosampler delivery syringe and seals.
Because tbe linear range of tbe detector
already has been establisbed, tbe volume
delivery of tbe autosampler can be
measured by selecting a test solute concentration tbat will produce peak areas

falling within the establisbed detector


linear range. Using tbe L2 caffeine solution (0.0035 mg/niL), injections from
5-100 (xL will produce peak beigbts
and areas witbin tbis demonstrated
range. These volumes can be modified
to cover tbe injector design range. The
L2 solution is injected in triplicate at
eacb of five volumes covering tbe injector volume range. Linear regression is
performed as previously sbown for cbe
detector linearity. In addition to good
linearity, a nonsignificant intercept
witbin tbe 95% CL is expected.

Gradient qualification dwell volume and delivery accuracy: Gradient


qualification consists of measurmg tbe
gradient dwell volume, the shape of a
short linear gradient, and tbe delivery
accuracy at 10% and 90% of eacb fluid
combination. Tbis is accompHsbed by
spiking one of tbe mobile pbases witb
tbe nonretained uracil (fluid circuit B is
used in tbis illustration), to produce a
mid-range absotbance signal at 273 nm
(see Table II). An aliquot of tbis spiked
mobile pbase is injected, starting the gradient witb no delay time.

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Table IV: Suggested HPLC performance qualification tests criteria


Manual Qualification Tests
PQ Test

Procedures

Suggested Acceptance
Cnterta

Leak check

Back-pressure regulator
with gauge, or plugged
outlet

No visible leaks.
Loss of pressure <100
psi/mrn over 5 min

Flow-rate accuracy

Flowmeter, or timed
volume collection for
three flow rates
over pump range

Column oven
Refrigerated
autosampler

Temperature
accuracy

Calibrated
thermocouple, air temp

UV detector

Wavelength
accuracy

Fill flow cell with


holmium oxide solution
(241-641 nm); caffeine
(205 nm, 273 nm)

Component

Pump

5%

Use back-pressure
regulator with gauge.
Test at moderate pressure,
ca. 1000 psi with
back-pressure regulator
Test over anticipated
range of use

3 nm

Regress expected
vs. found to look
for trends

Automated Tests (results produced within single injection sequence)

System performance

Pump

Extracolumn instrument
dispersion

Inject Rs test mixture,


calculate dispersion

No Pass/Fail specs
Record value

Compare to previous
values and to similar
HPLC systems

Flow stability

Retention time drift


over 10 consecutive
injections of test solute
(combined with
autosampler precision).

Drift NMT 1%

There should be no
outliers or indicators of
unstable flow

Gradient dwell
volume

Inject spiked mobile phase


blank at start of gradient

No Pass/Fail specs
Record value

Compare to previous
values and to similar
HPLC systems

Gradient delivery
accuracy

Measure accuracy of
step gradient of A vs. B*;
Cvs, B*, D vs, B* at 10%
and 90%, compared to
100% B*. where B* is
spiked with uracil

1% absolute

Observe sharpness of
transitions and any
anomalies in delivery
profiles

Dynamic short term


noise

Measure baseline noise


under flow over 1-2 min
interval

Record value
S/N for LI ^ 10

Compare to previous
values and similar
HPLC systems

Linearity

Triplicate injections of
solute over concentration
range of at least three
orders. Adjust upper
absorbance range to
1.2-1.5 AU.

R2 ^ 0.999
Residuals random

Range may adjusted to


produce desired
absorbance values

Precision

10 consecutive injections
of test solute

%RSD ^ 1 %

There should be
no outliers

Volume delivery
linearity
(optional)

Triplicate injections of
test solute at 5 volumes
over selected range,
within detector linear
range

fi2 ^ 0.999
Residuals random

Optional. Can be
performed initially upon
instrument installation as
part of OQ along with
volume accuracy.

Injector carryover

Injea three blanks


following highest
concentration sample in
Detector Linearity.

<0,l% for first blank


injection, or as per instrument specifications.

May be performed with


or without wash vial,
or both.

Detector

Autosampler

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A single gradient method is written,


consisting of an initial 10-min linear
gradient from 0 to 100% B. After a 5min hold at 100% B to establish the
maximum signal height, delivery accuracy is measured at 10% and 90% of
each combination of the other fluid circuits with the spiked B circuit, using a 3min hold ac each level. The hold times
can be varied if required to produce stable signals at each level.
To start rhe qualification, an aliquot ot
the uracil-spiked mobile phase is injected
and the gradient profile is started. The
uracil is eluced in the column void volume, while the onset ot the gradient
begins later, delayed by the gradient
dwell time. The dwell volume is calculated using the previously calibrated
pump flow rate, multiplied by the delay
time. An example of a dwell volume
determitiation is shown in Figure 7.
Figure 8 shows the full chromatogram
of a typical gradient qualification profile
for a quaternary gradient system.
This general technique based upon
uracil offers several significant advantages
over using a jumper tube in lieu of a column. First, this procedure measures the

MAV 2008 LCGC NORTHAMERia VOLUME 26 NUMBERS 4 7 9

operation ofthe entire gradient HPLC


system under actual operating conditions
ot pressure and flow with a column in
place. Second, the entire procedure is
fully automated, and can be programmed as part ofthe overall PQ injection sequence, so that no manual substitution of jumper tubes is required. It is
simple, and the steps of 10% and 90%
delivery for each solvent combination
adequately test the system near the
extremes of its solvent delivery and mixing system. More-complex profiles can of
course be generated if desired.
No acceptance criteria are assigned to
the gradient dwell volume, because this is
a design feature of each particular instrument. However, it is a critically important
parameter to know for a gradient instrument (11,12) and its value is recorded in
the logbook, and noted on the face of the
instrument. For critical gradient methods,
certain instruments might need to be
flagged as being not suitable if they produce excessive dwell volumes or indistinct
gradient profiles. During method development and validation, instruments with different gradient dwell volumes can be
selected deliberately for robustness testing.

During method transfer and troubleshooting, the gradient dwell volume of the
receiving laboratory should be noted.
The general shape of the initial linear
segment is documented, but no quantitative acceptance criteria are applied. For the
delivery accuracy, an acceptance criterion
of 1 % absolute is used, (equivalent to
10% relative ofthe lower delivery range).
Table IV summarizes the test protocols
that have been discussed previously,
along with typical acceptance criteria.
Final documentation: If any ofthe PQ
tests foil, an investigation is initiated, and
the cause ofthe failure is determined and
corrected before retesting, as per our internal SOPs. After qualification, the summary results are entered into the laboratory database. We also affix to the
instrument a brief tabular summary ot the
actual values obtained from the qualiflcation, such 2S the dynamic noise, extracolumn dispersion and gradient dwell volume, in addition to the normal
qualification sticker. This way, the performance ofthe various HPLC systems in
the laboratory can be compared readily, as
can changes in the performance of a given
instrument compared to its historical

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record. This information can be extremely


valuable when selecting instruments for
intermediate precision, and of course
when comparing instruments during
method transfer and similar activities.
Conclusions
Implementing a comprehensive HPLC
instrumentation maintenance and qualification program is a cGMP requirement.
The use of a standardized PQ test system,
complete with a prequalified column,
greatly facilitates the routine testing of laboratory HPLC systems. The test system
has been designed to be robust, while providing fast separations to reduce overall
time to perform a qualification. A comprehensive suite of test protocols can be completed within a few hours. Having such a
simple, yet universal test system available
in the laboratory has many advantages:
In-house instrument qualification:
The mosc obvious advantages ot using an
in-house performance qualification system are tbose of quality, time and money.
In our experience, HPLC qualifications
are often of higher quality when performed by trained laboratory personnel.
Not only does the laboratory develop an

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intimate feel for the operating characteristics of each of its HPLC systems, but
more time can be taken to investigate
small aberrations in performance. With
clear, well written SOPs, routine PQs can
be completed within hours, at a large
cost savings, with the added flexibility of
being able to complete instrument testing on the laboratory's schedule rather
than the instrument vendor's schedule.
Trending the performance of a
single HPLC instrument: A major advantage of using a standardized test method
for HPLC qualification is that one can
obtain trends in the historical performance
record of a single HPLC system. The accumulated performance history of each
HPLC system is readily available, and any
changes in the instrument performance
such as after repairs or moving to a new
location (precision, noise, efficiency, sensitivity) are immediately obviotis.
Comparison of HPLC performance laboratory-wide and worldwide: A standardized, complete PQ test system permits
direct comparison of che critical performance characteristics of all HPLC systems
with and between laboratories under identical test conditions, so that valid compar-

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instruments. This information can be useful when selecting equivalent (or nonequivalent) instruments for robustness
evaluation during method validation.
Instrument and method troubleshooting: If a problem is suspected
during routine instrument operation, a
quick PQ test wilt readily distinguish
whether the problem lies with the test
method, the user's understanding ofthe
method or the instrument performance
itself If the test solutions are maintained
in [he laboratory, instrument problems
often can be isolated quickly, saving
weeks of troubleshooting and reducing
unnecessary travel to remote sites.
Method transfer: Before method transfer, the recipient laboratory anywhere in
the world can test their HPLC conditions
identical to the originating laboratory. In
the event of method transfer problems
(which can be common), instrument performance characteristics (gradient dwell
volume and delivery accuracy, injector precision and carryover, noise, sensitivity, and
so forth) Gin be compared fbr equivalency
and suitability before transfer. For method
troubleshooting, one can go back quickly

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to [he PQ conditions to rule out Instriimennil problems.


Training: This test system also makes an
excellent training and evaluation tool fot
analysts new to HPLC. The analyst is given
the materials and procedures in the kit,
using an instrument that has been previously qualified, and is instructed to make
the mobile phase and run the variou.s test
protocols. Because the separation and the
quality of the expected data ate already
known, the new analyst becomes the
unknown. This exetcise can help the student learn all aspects of the operation of the
instrument under both isocracic and gradient conditions, including how to use the
data system and selea suitable integration
parameters, as weU as proper preparation of
the mobile phase and column installation.
Ensuring and documenting the quality
of analytical data is a fundamental responsibility of all analytical laboratories. Instrument qualification is one of the basic components required to achieve thi.s goal. Even
if preventative maintenance is contracted
CO an outside vendor, final qualification by
in-house personnel is highly recommended. The accumulated historical data
acquired under universal test conditions

becomes a vety strong pillar, complementing method validation, and training.

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 >.

Ack now ledg ments


The authors would like to thank Mr,
Frank Harris of Vintage Pharmaceuticals, Inc., and Mr. Nilesh Patel of Actavis
Pharmaceutical Company for their input
and assistance.
References
1I) 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).

(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, private communications.
(10) K.W. Freebairn and J.H. Knox, Chromatographia 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).

For more information on this topic,


please visit
www.chromatographyonline.com/
Ic/hpic

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

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