A BRIEF KNOWLEDGE ON QUALIFICATION OF HPLC

1. PRESENTATION ON
QUALIFICATION OF HIGH PERFORMANCE LIQUID
CHROMATOGRAPHY
PRESENTED BY
HUZAIFA NAAZ
H.T.NO.636217885002
M. pharmacy 1stYear
Under The Guidance Of
Dr. SANDIP SEN
Department Of Pharmaceutical Analysis
SRIKRUPA INSTITUTE OF PHARMACEUTICAL SCIENCES
{Approved by AICTE, PCI }
{Affiliated to OSMANIA University} 1

2. QUALIFICATION OF HIGH PERFORMANCE LIQUID
CHROMATOGRAPHY.
QUALIFICATION: Action of proving and documenting that
equipment or ancillary systems are properly installed, work
correctly, and actually lead to the expected results. The entire
qualification consists of four parts:
1. Design qualification(DQ).
2. Installation qualification(IQ).
3. Operational qualification(OQ).
4. Performance qualification(PQ).
1.DESIGN QUALIFICATION: It describe the user requirements and
defines the functional and operational specifications of the
instrument
• DQ should ensure that instrument to be purchased have the
necessary functions and performance that will enable for suitable
intended application.
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4. Design element Example:
Intended use Analysis of drug components and
impurities.
User requirements specification for the
HPLC analysis
•Up to 100 samples/day
•Automated over night analysis.
•Limit of quantitation:0.1%
•Automated confirmation of peak
identity and purity with diode-array
detection
•Automated compound quantitation and
printing of report.
FUNCTIONAL SPECIFICATION:
•Pump
•Detector
•Auto sampler
•Column compartment
•Computer
•Binary or higher gradient
•UV/VIS Diode array,190-900nm
•100 samples, 0.5µl to 5ml sample
volume
•15 to 60ºc controlled.
•System control, data acquisition for
signals and spectra, peak integration
and quantitation
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5. Design element Example
Spectral evaluation for peak purity and
compound confirmation .
Electronically save all chromatograms
generated by the system.
Operational specification •Detector: base line noise:<5 x 10-5
AU
•Sampler: precision inj. Volume :
<0.5% RSD. Sample carry over:<0.5%
•Pump: precision of retain time: <0.5%
RSD.
User instruction •Operation manual on paper
•Computer based tutorial
Qualification The vendor must provide procedures
and services for IQ and OQ
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6. Design element Example
Maintenance •Vendor must deliver maintenance
procedure and recommended schedule
•Instrument must include early
maintenance feedback for timely
exchange of most important
maintenance parts.
•Maintenance procedure must be
supplied on multimedia CD ROM
Training •Vendor must provide familiarization and
training
2.INSTALLATION QUALIFICATION: Installation qualification establishes that the
instrument is received as design and specified.
•It establishes that instrument is properly installed in the selected environment and
that environment should be suitable for operation of the instrument.
•Run of test samples verifies correct installation of all modules, electrical and fluid
connections
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7. BEFORE INSTALLATION ?
Obtain manufacturers recommendations for installation site requirements
Check the site for the fulfillment of the manufacturers recommendation (utilities
such as electricity and environment condition such as humidity and temperature)
Allow sufficient shelf space for the equipment, SOPs, operating manual and
software.
DURING INSTALLATION ?
Compare equipment as received , with purchase order (including software,
accessories, spare parts).
Check documentation for completeness (operating manuals, maintenance
instruction, standard operating procedure for testing , safety and validation
certificate)
Check equipment for any damage.
Install hardware( computer, equipment, fittings and tubings, for fluid
connections , column in HPLC , power cables , data flow and instrument control
table)
Switch on the instrument and ensure that all modules power up and perform an
electronic self test.
Identify and a make a list with a description of all hardware, include drawings
where necessary 7

8. Run test sample and compare chromatogram print out with reference
chromatogram.
Prepare an installation report.
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3.OPERATIONAL QUALIFICATION:
 It is the process of demonstrating that an instrument will function according to
its operational specification in the selected environment . It verifies that the HPLC
system compiles with key function and operational requirements as specified in
the design qualification .
In operational specification the supplier must define exactly the conditions that
must be observed with varying conditions. Eg: different ambient temperature.
Before performing all other test first perform leak test if, it is failed then most of
the remaining test will get failed.

9. Parameter Procedure User limit
Leak testing Flow test by volume or
weight/time
±5%
Baseline drift ASTM (American society
for testing material)
method E19.09, 20 min.
<2 x 10-3 AU
Baseline noise ASTM method E19.09, 20
min x 1
<5 x 10—5 AU
Precision of injection
volume
6 x injection of caffeine
standard, RSD of peak
areas
0.3% RSD
Precision of flow rate 6 x injection of caffeine
standard, RSD of
retention times
0.5% RSD
Detector linearity Inject 5 standards >1.5 AU, 5% RSD
Wavelength accuracy Holmium oxide filter ±1 nm
Temperature accuracy Comparison with external
measuring device
± 1º c
TEST PARAMETERS AND ACCEPTANCE CRITERIA :
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10. Parameter Procedure User limit
Temperature precision Monitoring temperature
over 20 mins
±0.25 ºc
Auto sampler carry over Injection of large sample
after large concentration
< 0.5%
Mobile phase
composition accuracy
Step gradient from 4 to
7% B, Step heights
relative to 100% with
acetone tracer
± 1%
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BASELINE NOISE AND DRIFT:
•Drift and baseline noise are important factors for UV detectors. Increased
baseline noise considerably reduces the sensitivity, as it is not possible to
distinguish between low-level signals and noise. With increased drift, it is more
difficult to integrate the signals correctly because the less stable the baseline is,
the more inaccurate is integration.
•The baseline noise of the detector mainly depends on the lamp. There is a
considerable increase in noise if an old lamp with poor light intensity is used. This
is also true when the flow cells is dirty. In addition make sure that the flow cells
free from gas bubbles.
•To measure the drift of a UV detector, also make sure that all measuring
conditions are constant. In addition, it is very important that the lamp has been
burning for several hours in the detector environment, avoid direct sunlight.
•The lamp intensity decreases while the lamp is burning. Besides, the lamp
ages when it is turned on and off very often.

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EVALUATING BASELINE NOISE AND DRIFT:
•TO check noise, drift water is pumped through the cell at a flow rate of
1ml/min. The UV signal is recorded at 254nm.
•To calculate noise the measuring signal is split into 20 intervals for 1min each.
For each interval chromeleon calculates a regression based on measured
values, using the method of least square. The limit should be between <2 x 10-
3 AU.
•To calculate the drift, chromeleon calculates a regression line from all data
points with in a range of 1-21mins based on the method of least square. The
slope of the regression line is the calculated drift. The limit should be between
<5 x 10—5 AU.
PRECISION OF INJECTION VOLUME:
•Precision of injection volume is an important parameter for accuracy of
quantitation.
EVALUATING PRECISION OF INJECTION VOLUME:
•Inject 6 standard caffeine solution and calculate height, area, average height,
average area, %RSD of height and %RSD of area which gives precision of
volume and the limit should be in between 0.3% RSD.

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DETECTOR LINEARITY:
•Linearity of a detector is a critical parameter to establish for reliable and
accurate quantitative results.
EVALUATING DETECTOR LINEARITY:
•A series of 5 traceable standards (caffeine solution of concentration about
0.00035 to 0.35mg/ml) are injected and evaluated. The detector linearity is
calculated by determining the peak area vs concentration. %RSD can also be
calculated for checking the detector linearity. The limit should be in between
>1.5 AU, 5% RSD.
WAVELENGTH ACCURACY:
•It is an important parameter for accuracy of quantitative and qualitative
analysis.
EVALUATING WAVELENGTH ACCURACY:
•Traceable caffeine standard is used to determine the wavelength accuracy.
Caffeine is trapped in the flow cell and a programmable timetable is used to
determine the wavelength maxima (205nm) and minima (273nm). The
wavelength accuracy is determined as the absolute difference between the
measured and certified wavelength values.

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TEMPERATURE ACCURACY:
•Temperature fluctuations of the solvent and column can result in considerable
retention time fluctuations. Therefore, accuracy of the temperature is important.
EVALUATING TEMPERATURE ACCURACY:
•4 measuring points are used to check the temperature accuracy of the column
compartment. The check is performed with column oven sequence. The
achieved temperature is measured with external calibrated thermometer.
•The achieved temperatures are compared to the set values. The difference
indicates the temperature accuracy and the limit should be in between ± 1º c
TEMPERATURE PRECISION:
•Monitor temperature for 20minutes and limit should be in between ±0.25 ºc
AUTOSAMPLER CARRY OVER:
•After a highly concentrated sample, a sample containing only solvent is
injected. Ideally only the signal for the solvent is displayed in the chromatogram.
However, if a signal for the sample is displayed, this indicates the carry over by
the autosampler.
EVALUATING AUTOSAMPLER CARRY OVER:
Run the sample containing only solvent. The signal for solvent will be displayed
. If other signals are displayed it is due to auto sample carry over. Should be
less than 0.5%

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GRADIENT MOBILE PHASE COMPOSITION ACCURACY:
It is important for accurate quantitative analysis.
EVALUATING GRADIENT MOBILE PHASE COMPOSITION ACCURACY:
An Acetone tracer is used to determine gradient mobile phase accuracy, stability
and linearity.
Make 6 compositions of water+acetone in concentration of
0%,20%,40%,60%,80% and 100% (20% increment).
Linear ramp down from 100% to 0% is performed where the composition
linearity is determined between ranges of 95,75 and 25%.
All compositions accuracies are calculated as the absolute difference between
the mean composition at each set point and the theoretical composition.

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4.PERFORMANCE QUALIFICATION:
 It is the process of demonstrating that an instrument consistently performs
according to a specification appropriate for its routine use.
Important here is the word consistently. The test frequency is much higher
than for OQ. Another difference is that PQ should always be performed under
conditions that are similar to routine sample analysis. For a chromatogram this
means using the same column, the same analysis conditions and the same or
similar test compounds.
PQ should be performed on a daily basis or whenever the instrument is
used.
The test frequency of only depends on the stability of the equipment but on
everything in the system that may contribute to analysis the result
For a liquid chromatograph, this may be the chromatographic column or a
detectors lamp
The test criteria and the frequency should be determined during the
development and the validation of the analytical method.
In practice PQ mean system stability testing , where critical key system
performance characteristic are measured and compared with documented ,
preset limit.

18. For example a well characterized standard can be injected 5 or 6 times and the
standard deviation of amounts are then compared with predefined value
PQ test are application specific
If the limits of detection and quantitisation are critical, the lamps intensity profile
or the base line should be tested
They should use the same column and chemicals for the real sample.
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Test should include:
•Precision of the amounts
•Precision of retention times
•Resolution between two peaks
•Peak width at half height
•Peak tailing
•Baseline noise
•Wavelength accuracy of the uv/vis wavelength detector, preferably using built-in
holmium – oxide filters

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RETENTION TIME
Problem cause correction

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RESOLUTION BETWEEN PEAKS:
PROBLEM CAUSE CORRECTION

21. Documentation : on completion of equipment qualification , documentation
should be available that consists of :
 Design qualification document
IQ document (includes description of hardware and software)
Procedures for OQ testing
OQ test reports ( includes test parameters, acceptance criteria and actual
results.
PQ test procedures and representative results.
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REFERENCES:
•Ph.Eur.2.2.35 chromatography; High
performance liquid chromatography
•Guidance on equipment qualification of
analytical instruments
•Journal of Perkin Elmer life & analytical
science

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