The document discusses the qualification of HPLC systems, which consists of four parts: design qualification, installation qualification, operational qualification, and performance qualification. It provides details on the procedures and acceptance criteria for each qualification part. The design qualification establishes functional and operational requirements. The installation qualification verifies proper installation. The operational qualification demonstrates the system meets specifications under operating conditions. Test parameters like leak testing, precision, noise, drift and accuracy are evaluated.

1. Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721
QUALIFICATIN OF HPLC
A Seminar as a part of curricular requirement
for Master of Pharmacy, I Year - Ⅱ semester
Presented by
KHALID OSMAN
(20L81S0710)
Department of Pharmaceutical analysis

2. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721
Contents :
 Design Qualification
 Installation Qualification
 Operational Qualification
 Performance Qualification
 References
2

3. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721
Introduction :
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)
3

4. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721
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.
4

5. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721
Design qualification:
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
Intended use Analysis of drug components and
impurities.
5

6. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721
Cont.…
Design element Example
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 quantization 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
6

7. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721
Cont.…
Design element Example
Operational specification •Detector: base line noise:5 x 10-5 AU
•Sampler: precision inj. Volume :
<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
7

8. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721
Installation qualification :
 Installation qualification establishes that the instrument is received as
designed and specified.
 It establishes that instrument is properly installed in the selected
environment and this environment is suitable for the operation and use of
the instrument.
 IQ should include analysis of a test sample.
 A successful run of such a sample verifies correct installation of all
modules and electrical fluid connections.
8

9. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721
Before installation :
 Obtain manufacturers recommendations for installation site
requirements.
 Check the site for the fulfilment 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.
9

10. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721
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 tubing's, for fluid
connection, column in HPLC, power cables, data flow and instrument
control table).
10

11. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721
Cont.
➢ 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.
➢ Run test sample and compare chromatogram print out with reference
chromatogram.
➢ Prepare an installation report.
11

12. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721
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. E.g.: 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.
12

13. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721
Test parameters and acceptance criteria:
Parameter Procedure User limit
Leak testing Flow test by
volume or
weight/time
±5%
Baseline drift ASTM(American
Society for Testing
Material) Method
<2 x 10-3 AU
Baseline noise ASTM Method <5 x 10- 5AU
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 <1.5AU, 5% RSD
13

14. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721
Cont.…
Parameter Procedure User limit
WavelengthAccuracy Holmium oxide filter ±1 nm
TemperatureAccuracy 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%
14

15. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721
BASELINE NOISEAND 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.
 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.
 The flow cells free from gas bubbles.
 To measure the drift of a UV detector, also make sure that all measuring
conditions are constant.
15

16. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721
 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.
16

17. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721
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 recordedat 254nm.
 To calculate noise the measuring signal is split into 20
intervals for 1min each.
 For each interval chrome Leon calculates a regression based on measured
values , using the method of least square. The limit should be between <2 x
103AU.
 Tocalculate the drift, chrome Leon calculates a regression line from all data
points with in a range of 1-21mins based on the method of least square.
17

18. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721
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.
18

19. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721
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 >1.5AU, 5% RSD.
19

20. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721
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.
20

21. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 21

22. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721
Temperature accuracy :
 Temperature fluctuations of the solvent and column can result in
considerable retention time fluctuations.
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.
22

23. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721
Cont.…
 The achieved temperatures are compared to the set values.
 The difference indicates the temperature accuracy and the limit should be
in between ± 1oC.
23

24. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721
Autosampler carry over :
 It is the appearance of an analyte in a run when a blank containing no
analyte is injected.
Evaluating auto sampler 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.
 It should be less than 0.5%.
24

25. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721
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%.
25

26. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721
Cont.…
 All compositions accuracies are calculated as the absolute difference
between the mean composition at each set point and the theoretical
composition.
26

27. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721
Performance qualification :
 Performance Qualification (PQ) is the process of demonstrating that an
instrument consistently performs according to a specification
appropriate for its routine use.
 PQ should be performed on a daily basis or whenever the instrument is
used.
 The test frequency not only depends on the stability of the equipment but
on everything in the system that may contribute to analysis the result for a
liquid chromatography.
27

28. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721
Cont.…
 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.
 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.
28

29. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721
Cont.…
 If the limits of detection and quantifications 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.
29

30. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721
References :
 1. P.Bedson and M.Sargent, The development and application of guidance
on equipment qualification of analytical instruments, Accreditation and
Quality Assurance, 1 (6), 265/274 (1996) .
 L. Huber, Validation and qualification in analytical laboratories, Text book,
300 pages, 1998, Interpharm, Bufallo Grove, IL, USA, Nov. 1998
http://www.t-online.de/home/HUBERL/validat.htm (book) .
 L. Huber, Equipment Qualification in Practice LC/GC Magazine, Vol 16,
February 1998, 148-156 http://www.t-
online.de/home/HUBERL/validat.htm (book).
30

31. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721
Thank you
31