Pharmaceutical validation

QUALIFICATION OF ANALYTICAL
EQUIPMENT
Presented By:
Miss. Mohini Tawade,
First Year M. Pharmacy,
Department of Quality Assurance,
Dr. D.Y. Patil College of Pharmacy, Akurdi, Pune.

QUALIFICATION
 Action of proving and documenting that equipment or ancillary systems are
properly installed, work correctly, and lead to the expected results.
 Qualification is part of validation, but the individual qualification steps alone
do not constitute process validation.
 Types Of Qualification:
Design Qualification
Installation Qualification
Operational Qualification
Performance Qualification

Design
Qualification
Performance
Qualification
Operational
Qualification
Installation
Qualification
Before
purchasing a
new instrument
At documented
installation of a
new or existing
instrument
After
installation
After major
change
e.g., repair,
update
After regular
interval
Whenever the
instrument is
used
e.g., daily
Or
Periodically at
specified
intervals for
each instrument

 Design Qualification (D.Q):
Documented evidence shows that the plant design agrees with the design specification of the
customer.
 Installation Qualification (I.Q):
The purpose of I.Q. is to check the installation site/ environment, confirms equipment
specifications, and verify the condition of installed equipment.
 Operational Qualification (O.Q):
O.Q. includes procedures and documentation of the O.Q. of the analytical instrument.
When all procedures are executed and all items pass the inspection, it is verified that the
system operates to satisfy the intended purpose.
 Performance Qualification (P.Q):
The objective is to ensure that the instrument is performing within specified limits.
Hence documented verification that the equipment and ancillary systems, as connected, can
perform effectively and reproducibly based on the approved process method and
specifications.

 Apart from URS, DQ, IQ, OQ, and PQ; some other parameters are to be
performed such as Factory acceptance test (FAT) and site acceptance test (SAT)
where applicable.
 When the instrument undergoes major repairs or modifications, this should be
evaluated using change control.
 Relevant IQ, OQ, and/or PQ tests should be repeated to verify that the
instrument continues to operate satisfactorily.
 If an instrument is moved to another location, an assessment should be made of
what, if any, qualification stage should be repeated

Qualification of FTIR
 Fourier transfer infrared spectroscopy (FTIR) is a technique that is used to obtain
an infrared spectrum of absorption, emission, photoconductivity, or Raman
scattering of a solid, liquid, or gas.
 An FTIR spectrometer simultaneously collects spectral data in a wide spectral
range. This confers a significant advantage over a dispersive spectrometer which
measures intensity over a narrow range of wavelengths at a time.

WAVE-NUMBER ACCURACY
The wave number scale is usually calibrated by the use of several characteristic wave
numbers of a polystyrene film.
3060.0(+/-1.5) cm-1
2849.5(+/-1.5) cm-1
1942.9(+/-1.5) cm-1
1601.2(+/-1.0) cm-1
1583.0(+/-1.0) cm-1
1154.5(+/-1.0) cm-1
1028.3(+/-1.0) cm-1
The software then judges whether the values are within the allowable range.
The program labels the results “PASS” if all the peak numbers are within the range.
In the case of the dispersive spectrophotometer, the permissible level of frequency at
1601.2 cm-1 and at 1028.3 cm-1 should be within +/- 2.0 cm-1

DETECTOR ENERGY RATIO
Method:
Record the minimum energy ratio value for at least one of the following
measurement points and compare it to the vendor’s specifications: -
Energy at 3990 cm-1 / energy at 2000 cm-1
Limits:
Energy ratio test specifications vary for each spectrometer configuration.
The optical bench shall include a DTGS detector with a frequency range of 7400
to 350 cm-1

SIGNAL/NOICE RATIO
Method: Record the maximum noise level for each of the following regions:
Peak-to-peak noise between:
4050 cm-1 and 3950 cm-1
2050 cm-1 and 1950 cm-1
1050 cm-1 and 950 cm-1
550 cm-1 and 450 cm-1
(systems with DTGS detector only)
RMS (root mean square) noise between:
4050 cm-1 and 3950 cm-1
2050 cm-1 and 1950 cm-1
1050 cm-1 and 950 cm-1
550 cm-1 and 450 cm-1
(systems with DTGS detector only)
Limits (% T):
Noise level test specifications vary for each spectrometer configuration.

RESOLUTION
Materials:
Certified polystyrene film of approximately 35 µm in thickness.
Method:
For instruments having a monochromator, record the spectrum of the polystyrene film.
For Fourier-transform instruments, use suitable instrument resolution with the appropriate application
prescribed by the manufacturer. The resolution is checked by suitable means, for example by recording the
spectrum of a polystyrene film approximately 35 µm in thickness.
Limits:
 Difference between the absorbance at the absorption minimum at 2870 cm-1 and the absorption maximum at
2849.5 cm-1 > 0.33.
 Difference between the absorbance at the absorption minimum at 1589 cm-1 and the absorption maximum at
1583 cm-1 > 0.08.

ZERO TEST
Method:
 When using a polystyrene film of approximately 35 µm in thickness as standard
at the wavelength of 2925 cm-1 and 700 cm-1, almost complete absorption of
the irradiated energy can be observed.
 With this test, the remaining transmission is measured. As the maximum
absorption can be observed at 700 cm-1 negative values may be observed.
 The objective of the test is to evaluate if, despite the fact that there is almost
complete absorption, energy is still detectable.
 Non-valid results are an indication of the non-linear behavior of the detector
and the electronic system.

CALIBRATION
WAVE NUMBER PRECISION:
 This is performed for substances with well-known peak wave number(s) positions such as carbon
dioxide, water vapor, polystyrene, and ammonia.
 The test is performed to determine whether the exact peak wave numbers are shown during
validation.
Thus the result is obtained from the difference between the peak wave numbers position for a
substance with well-known peak wave numbers and the values indicated by the system.
0% TRANSMITTANCE:
 A sample that does not allow light transmission is measured to investigate the 0% transmittance.
 This test thus can be used to find out errors caused by stray light and secondary emission spectra.

CALIBRATION
100% TRANSMITTANCE
This is investigated by performing analysis without a sample. By performing
analysis without the sample 100% transmittance can be investigated.
LINEARITY OF CURVE
A calibration curve for the % transmittance and concentration and the linearity of
the inspected are created.
REPRODUCIBILITY
A stable sample is measured twice within a short period and confirmed whether
the variation in the measurement values such as wave numbers and transmittance
is obtained.

CALIBRATION
VALIDATION OF FTIR
 To perform FTIR validation and to confirm that it is operating properly, diverse IR
inspection was performed by measuring the spectra of polystyrene film.
POWER SPECTRUM
 The power spectrum gives the plot of a portion of the signal’s power(energy per unit
time) falling within the given frequency bins.
 This test estimates the intensity of the power spectrum at a specified wave number.
 The test is passed when the measured intensity is equal to or larger than the criterion
value.

Qualification of GC
Instrument module Parameter to be
checked
Typical tolerance
limit
Inlet System
Injector leak test Pressure drop ≤ 15 kPa Within
5 minutes.
Pressure/flow accuracy and
stability
Flow rate ±10% of set flow.
Repeatability of injection
 In split mode
 In split less mode
RSD ≤ 3.0%
RSD ≤ 3.0%
Injector temperature accuracy
and stability
Relative retention times from
two consecutive injections
should be ≤ 1.0% ≤ 0.2%

OVEN Repeatability of oven
temperature characteristic
Within ±2°C
FID Detector Linearity r² ≥ 0.999
Constant Detector Response RSD of the peak areas should
be ≤ 3.0%
Noise NMT:-100 μV
Drift NMT:-2500μV/Hr

GC Inlet System
Injector Leak Test
Method: If not otherwise specified by the instrument manufacturer, the leak test is carried out according to the
procedure laid down in the instrument manual or by the built in automatic leak check procedure of the
instrument.
Otherwise use the test described below:
 Disconnect the column from the injector and close the injector outlet with a sealed cap.
 Close the septum purge and the bypass.
 Adjust the flow and pressure controller to the maximal possible value of the pressure gauge.
 Adjust the flow controller to zero.
 Read the pressure after 1 minute and record the value. Record the pressure after 5 minutes.
Limits: Pressure drop ≤ 15 kPa within 5 minutes.

Inlet Pressure / Flow Accuracy And Stability
 Connect the digital flow meter to detector outlet port.
 Set the carrier gas(Helium)flow and wait till it reaches to the set flow.
 Note the observed the flow in replicate.
 Repeat the procedure for other carrier gas such as hydrogen and air.
 Record the result in GC calibration protocol.
Limits: Flow rate ±10% of set flow.
Sr. No Carrier Gas Acceptance Criteria in ml/m
1 Helium 125
2 Hydrogen 40
3 air 400

Repeatability of Injection
Method:
 Carry out 6 consecutive injections of the test solution and calculate the RSD of the different
peak areas and retention times.
Limits:
 Retention time repeatability: the RSD of the retention times should be ≤ 2.0%
 Peak area precision (split and split less mode): the RSD of the peak areas should be ≤ 3.0

OVEN
Repeatability of oven temperature
Method:
 Connect the column to the detector port.
 Place the thermometer probe in the column oven and set the column oven temperature at
40°C.Wait till the temperature stabilizes.
 Note the observed temperature as read by the probe in triplicate over a period of 10 min.
 Repeat the procedure for 100°C, 150°C and 190°C.
Limit:
The resulting oven temperature from the thermometer display should be within ±2°C of the set
temperature.

FID DETECTOR
Fid Detector Linearity
 Increasing amounts of analyte are injected and a linear response should be obtained.
Limits: r² ≥ 0.999
Constant Fid Detector Response
 The proper and reproducible functioning of the FID can be demonstrated by checking the peak
areas obtained from a predefined standard solution.
Limit: the RSD of the peak areas should be ≤ 3.0%

FID Detector Noise and Drift
If the instrument has a built-in automatic system for the verification of the noise and drift, follow the
manufacturer’s instructions and apply the defined acceptance criteria.
Otherwise, use the test described below:
 Column installed
 Suitable flow, depending on column length/diameter
 No injection
 Oven temperature: 40°C
 Detector on and heated at working temperature (270- 300°C)

Method: After stabilization of the system, record the signal for 15 minutes.
Noise: evaluate 10 periods of 1 minute and calculate the mean value.
Drift: Evaluate the slope of the baseline over the 15 minutes.
Limits: The acceptance criteria for these parameters have to be chosen in accordance
with the instrument vendor’s instructions and the intended use of the instrument.
If no instructions are given, the user has to pre-define these acceptance criteria by
taking into account the previous experience and the intended use of the instrument.

Qualification of HPLC
Design elements Examples
Intended use Analysis of drug component and
impurities
User requirement 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 Binary or high gradient
Detector UV/VIS diode array, 190-900nm
Auto sampler 100 samples, 0.5µl to 5 ml sample volume
Column components 15 to 60°c controlled
Computer System control, data acquisition for signals and
spectra, peak integration and quantification
Operational specifications Detector:
base line noise: <5x 10-5 AU
Sampler:
Precision injection volume: <0.5% RSD
Sample carry over: <0.5% RSD
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
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

Test parameters and acceptance criteria
Parameter Procedure User Limit
Leak test 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
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%

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 are
dirty. In addition, make sure that the flow cells are 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, and 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.

Evaluating baseline noise and drift
 To check the 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
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 calculates a regression line from all data points within 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
The precision of injection volume is an important parameter for the accuracy of
quantitation.
Evaluating Precision Of Injection Volume:
Inject 6 standard caffeine solutions and calculate the height, area, average height,
average area, %RSD of height, and %RSD of the area which gives the precision
of volume, and the limit should be in between 0.3% RSD.

Detector linearity
The linearity of a detector is a critical parameter to establish 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) is 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 between >1.5
AU and 5% RSD.

Wavelength accuracy
It is an important parameter for the accuracy of quantitative and qualitative
analysis.
Evaluating Wavelength Accuracy:
A 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.

Temperature accuracy
Temperature fluctuation of the solvent and column can result in considerable
retention time fluctuations. Therefore, the accuracy of the temperature is
important.
Evaluating Temperature Accuracy:
Measuring points are used to check the temperature accuracy of the column
compartment. The check is performed with the column oven sequence. The
achieved temperature is measured with an external calibrated thermometer.
The achieved temperatures are compared to the set values. The difference
indicates the temperature accuracy and the limit should be between ± 1º c
Temperature Precision:
Monitor the temperature for 20 minutes and the limit should be between ±0.25 ºc

Autosampler carryover
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 carryover 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 carryover. Should be less
than 0.5%

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 concentrations 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 composition accuracies are calculated as the absolute difference between the mean
composition at each set point and the theoretical composition.

Qualification HPTLC
Linearity of spotting:
Apply 2 µl, 4µl, 6µl, 8µl and 10 µl of solution on the HPTLC plate with a spotter.
Allow the plate to run in the mobile phase.
Dry the plate with a drier.
Scan the plate with the scanner.
Check the linearity and correlation coefficient between the spots.
How to check linearity and correlation coefficient b/w the spots?? Correlation Coefficient:
A number that gives you a good idea about how closely one variable(spot) is related to another variable.
Acceptance limit:
Correlation coefficient = Not less than 0.9900

Reproducibility of spotting:
Apply 10µl solution on the HPTLC plate six times in sequence.
Allow the plate to run in the mobile phase.
Dry the plate with a drier.
Scan the plate with the scanner.
Calculate the Relative Standard Deviation(RSD) for six tracks.
Acceptance limit:
RSD Limit: NMT 3.0%

Detection capacity:
Requirements
i. Alumina glass plate
ii. Sodium salicylate
iii. 96% v/v alcohol
Preparation of stock solution:
Stock solution-1: Weigh 500mg of Sodium salicylate and transfer it into a 250ml
volumetric flask dissolve and dilute with 96% v/v alcohol.
Stock solution-2: Weigh 100mg of Sodium salicylate and transfer it into a 250ml
volumetric flask dissolve and dilute with 96% v/v alcohol.

Procedure:
Spot 5 microliter of each solution observes at 254nm and 366nm.
Acceptance:
1. The spot shall be comparable intensity-wise.
2. Spot due to stock solution-2 shall be visible at 254nm.
3. Spot due to stock solution-1 shall be visible at 366nm

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