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www.chromatagraphyonlirie.com
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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.
250
300
350
400
450
500
600
550
650
700
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.
worth
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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Experimental
Acctonitrile was HPLC grade, purchased
from EMD (VWR, West Chester, Penn-
ATLANTH
Disk
For more information call 1-800-997-2997
or email us at info@honzoniechinc.com
Horizon
technology
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w w w . chroma t o g r a p h y o n f m e . c o m
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.
J,
_)
4.-1.
MACHEREY-NAGEL
Germany USA Switzerland France
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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.
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
Table I: Components of the AIQ process over the typical iaboratory instrument
life-cycle
Polymicro
TECHNOLOGIES
A Subsidiary oi
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.
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Would you
350
300
100% B*
250
10:90 A : B '
10:90 C:B*
200
10:90 D:B*
1 1
1
S 150
initiai 10-min
iinear gradient
100
!
3 /
50
0-
!1
90:10 C B '
90:10 D:E*
|^
blue
by Knauer
-50
10
20
30
40
50
60
Time (m n)
Figure 8: Typical gradient accuracy step height results for a quaternary gradient
system.
[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
A'
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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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,
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
%A
100
0
0
90
90
10
10
%B
0
100
100
10
10
90
90
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
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
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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
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
System performance
Pump
Extracolumn instrument
dispersion
No Pass/Fail specs
Record value
Compare to previous
values and to similar
HPLC systems
Flow stability
Drift NMT 1%
There should be no
outliers or indicators of
unstable flow
Gradient dwell
volume
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
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
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
Detector
Autosampler
www.chromatographyonllne.com
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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www. chromatographyonlme.com
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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Technology's
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(7) United States Pharmacopeia, Chapter <621 >.