VALIDATION AND CALIBRATION OF HPLC
BY- Sakshi Garg
“Validation of an analytical procedure is the process by which it
is established, by laboratory studies, that the performance
characteristics of the procedure meet the requirements for the
intended analytical applications.”
A written plan stating how validation will be conducted and
defining acceptance criteria. For example, the protocol for a
manufacturing process identifies processing equipment,
critical process parameters/operating ranges, product
characteristics, sampling, test data to be collected, number of
validation runs, and acceptable test results.
Demonstrate that equipment used in validation studies is suitable
for use and is comparable to equipment used for routine analysis. 2
•Qualification: Action of proving and documenting that
equipment or ancillary systems are properly installed, work
correctly, and actually lead to the expected results.
•Qualification is part of validation, but the individual qualification
steps alone do not constitute process validation.
Parts Of The Qualification:
The entire process typically consists of four parts:
•Design qualification (DQ),
•Operational qualification (OQ), and
•Performance qualification (PQ).
A schematic diagram illustrating the timeline and documents in the
various stages in HPLC system qualification.
Basic Parameters for the Validation of Method:
Definition: The accuracy of an analytical procedure expresses the closeness of
agreement between the value that is accepted either as a conventional true
value or as an accepted reference value and the value found.
How To Determine??
• Known amounts of Related substances and the drug substance in placebo are
spiked to prepare an accuracy sample of known concentration of Related
• According to the ICH, accuracy should be determined using a minimum of
nine determinations over a minimum of three concentration levels covering
the range(from 50% of the ICH reporting level to 150% of the proposed shelf
life specification of the related substances) specified.
e.g. Level Accuracy
125% 99.6 +/- 0.2%
75% 100.3 +/- 0.8%
25% 99.2 +/- 0.7%
Determination of Voglibose by HPLC and validation of method)
Intrinsic accuracy indicates the bias caused by sample matrix and sample
o Sample preparation
o Calculation error
•Overall accuracy = -----------------------------------------
%relative substance (theory)
Definition : The Precision is a measure of the ability of the method to generate
reproducible results. The precision of a method is evaluated for repeatability,
intermediate precision, and reproducibility.
•Repeatability is a measure of the ability of the method to generate similar
results for multiple preparations of the same homogeneous sample by one
analyst using the same instrument in a short time duration (e.g., on the same
day). For instance, method repeatability for pharmaceutical assays may be
measured by making six sample determinations at 100% concentration, or by
preparing three samples at 80, 100, and 120% concentration levels each.
•Intermediate precision is a measure of the variability of method results where
samples are tested and compared using different analysts, different equipment,
and on different days, etc. This study is a measure of the intra-laboratory
variability and is a measure of the precision that can be expected within a
•Reproducibility is the precision obtained when samples are prepared and
compared between different testing sites. Method reproducibility is often
assessed during collaborative studies at the time of technology or method
transfer. %RSD= (s/µ)*100 9
Limit of Detection (LOD, DL)
– The LOD of an analytical procedure is the lowest amount
of analyte in sample which can be detected but not
necessarily quantitated as an exact value.
• Determination is usually based on
– Signal to noise ratio (~3:1) (baseline noise)
– Standard deviation of response (s) and Slope (S)
• 3.3 s/S
– SNR = H/h
Where H = height of the peak corresponding to the component
h = absolute value of the largest noise fluctuation from
the baseline of the chromatogram of a blank
Limit of Quantitation (LOQ, QL)
– The LOQ is the lowest amount of analyte in a sample
which can be quantitatively determined with suitable
precision and accuracy
– The quantitation limit is used particularly for the determination of
impurities and/or degradation products
• Determination is usually based on
– Signal to noise ratio (~10:1) (baseline noise)
– Standard deviation of response (s) and Slope (S)
• 10 s/S
- The Quantitation limit of a method is affected by both the detector
sensitivity and the accuracy of sample preparation.
Signal to Noise = 3:1
Signal to Noise = 10:1
LOD, LOQ and Signal to Noise Ratio (SNR)
ICH Definition: The range of an analytical procedure is the interval
between the upper and lower concentrations of analytes in the for
which it has been demonstrated that the analytical procedure has a
suitable level of precision, accuracy, and linearity.
Range For Different Tests:
80 to 120% of test concentration
• Content uniformity
70 to 130% of test concentration
Q-20% to 120%
Reporting level – 120% of specification limit (with respect to test
concentration of API)
• Assay & Impurities
Reporting level to 120% of assay specification
Definition : Linearity of an analytical procedure is its ability
(within a given range) to obtain test results which are
directly proportional to the concentration of analytes in the
•If there is a linear relationship test results should be
evaluated by appropriate statistical methods like,
•Slope of regression line
•Plot of the Data
•Method To Determine Linearity
•Direct Weigh Method
Linearity Ranges and Acceptance Criteria for Various
SYSTEM SUITABILITY TESTING (SST)
• System suitability testing (SST) is used to verify resolution,
column efficiency, and repeatability of the analysis system to
ensure its adequacy for performing the intended application
on a daily basis.
•Number of theoretical plates (efficiency)
•Separation (relative retention)
•Relative Standard Deviation (Precision)
•Plate number or number of theoretical plates (n)
n=L/H, where L is Length of Column
H is HETP or height of one theoretical plate
•Capacity factor (capacity ratio) k
k’= tr-tm/tm where tr is retention time
tm is dead time
•Separation Factor (relative retention)
α=k1/k2 where k1 is capacity factor of compound a
and k2 is capacity factor of compound b
•Tailing factor ,T
T=W/2f where W is width at 5% at peak height
f, distance between max and leading edge of the peak
Definition : Robustness is reliability of an analytical procedure with
respect to deliberate variations in method parameters.
•If measurements are susceptible to variations in analytical conditions
the analytical conditions should be suitably controlled or a
precautionary statement should be included in the procedure.
•PARAMETERS TO BE EVALUATED FOR ROBUSTNESS
•Three columns packed by bonded phases from three different
• pH (±0.1–0.2 units)
• Buffer concentration (±5–10mM)
• Percentage organic modifier (±1–2% MP)
•Injection volume or sample concentration
• Solvent strength for the final solution
•Column temperature (±5°C)
•Detector wavelength (±3nm) 19
Selectivity and Specificity :
•Selectivity is the ability to measure accurately and specifically the
analyte in the presence of components that may be expected to be
present in the sample matrix.
•Specificity for an assay ensures that the signal measured comes from
the substance of interest, and that there is no interference from
excipient s and/or degradation products and/or impurities.
Definition (ICH): The demonstration that a particular
instrument or device produces results within specified limits
by comparison with those produced by a reference or
traceable standard over an appropriate range of
Various Calibration parameters for HPLC are:
•Flow rate accuracy (Pump Module)
• Injector accuracy
• Injector linearity
• Gradient Performance Check
• Column oven temperature accuracy
Flow Rate Accuracy:
1. Prime all the solvent lines with water.
2. Set the flow rate to 0.500 ml/m.
3.Wait for about 15 m to stabilize the system and ensure that the pressure is stable.
4.Insert the outlet tubing into a 10 ml volumetric flask and start the stop watch
5. Stop the stopwatch when the lower meniscus reaches the 10 ml mark on the
6. Record the elapsed time.
7. Similarly check the flow for 1.0 ml/m and 2.0 ml/m.
The time taken to collect the water should be with in ± 2.0% of the actual value.
Pressure Test :
•The performance of the HPLC pump depends on the proper functioning
of the check valves and the proper connection of the tubing.
•For pump systems that output the pressure reading in the pump head
over time, a simple pressure test can be a useful qualitative test to check
the condition of the check valves and to determine whether or not there
are any leaks in the system.
•The first step of the pressure test is to plug the outlet of the pump using
a dead-nut and by setting the automatic pump shutdown pressure to
•The pump-head pressure signal output is connected to a recorder.
Pressurize the pump by pumping methanol at 1 mL/min. The pressure
inside the pump head increases quickly as the outlet of the pump is
blocked. As the pressure increases to about 3,000 psi, the flow rate is
reduced to 0.1 mL/min.
•The pressure will gradually rise to the shutdown pressure if the check
valves are able to hold the mobile phase in the pump chamber as would
be normally expected . If the check valve is not functioning properly, the
pressure will fluctuate at about 3,000 psi instead of reaching the
1. Connect the pump and detector inlet with union.
2. Prepare mobile phase consisting of a mixture of water and Methanol (70:30)
3. Set a flow rate of 0.5 ml/m and a run time of 1 m.
4. Set the column temperature at 25± 2°C.
5.Fill a standard HPLC vial to 2/3rd with water. Seal the vial properly with a
6. Weigh the vial and record the weight as W1 grams.
7. Place the vial in the chromatographic system and perform 6 injections of
50μl volume from this vial.
8. Weigh the vial again and note the weigh after the injections as W2 grams.
9. Calculate the mean volume injected per injection as follows:
•Mean injected volume (μl) = (W1 – W2) × 100/6
•Acceptance criteria: The mean injected volume should be 50.0±1.0 μl.
•Analytical Method Validation and Instrument Performance Verification
-Herman Lam and Y.C. Lec
•Small amounts of analyte may get carried over from the previous
injection and contaminate the next sample to be injected.
•The carryover will affect the accurate quantitation of the subsequent
•The problem is more serious when a dilute sample is injected after a
•In order to avoid cross contamination from the previous sample
injection, all the parts in the injector that come into contact with the
sample (the injection loop, the injection needle and the needle seat)
have to be cleaned effectively after the injection.
•The effectiveness of the cleaning can be evaluated by injecting a blank
after a sample that contains a high concentration of analyte.
•The response of the analyte found in the blank sample expressed as a
percentage of the response of the concentrated sample can be used to
determine the level of carryover. 26
(A)UV-Visible Detector Module:
1. Wavelength accuracy is defined as the deviation of the
wavelength reading at an absorption or emission band from the
known wavelength of the band.
2. The deuterium line at 656 nm and the absorption bands at 418,
453 and 536 nm in a Holmium oxide filter are often used.
(B) PDA Detector Accuracy:
•Select 3D mode and set the wavelength range as 200-400nm.
•Inject 20 μl Holonium oxide of standard preparation once into the
•Extract and record the chromatograms at wavelengths of 202 to 208nm
with an interval of 1nm and at 269 to 275 nm with an interval of 1nm.
•Note down the height or absorbance.
Acceptance criteria: The maximum absorbance should be at 205±2nm
You can Calibrate the PDA Detector for 5 Parameters:
1-Baseline Noise and PDA Drift.
2- Your Lamp Intensity.
3- You have to check for the Wavelength Accuracy.
• You can inject Pyrene with Methanol as eluent at a flow of 1 ml/min.
• The characteristically maximum of pyrene is determined at 333 nm and
compared to a theoretical value.
4- Lastly- You must check your PDA's Linearity
you can inject 5 caffeine solutions at different concentrations.
Concentrations and peak heights can be represented in a graph. The
regression coefficient of the resulting line and the deviations from it
indicate indicates the linearity- Usually you R square value should be
above 99.90% .
Automated Detector Calibration
Filter with a known
Filter with a known
Noise and Drift :
•Drift: upward &/or downward movement of base line.
•Noise : any unwanted signal recorded by the detector.
•Electronic, pump and photometric noise, poor lamp intensity,
dirty flow cell, and thermal instability contribute to the overall
noise and drift in the detector.
•Excessive noise can reduce the sensitivity of the detector and
hence affect the quantitation of low-level analytes.
•Detector drift may affect the baseline determination and peak
•Overcomes for avoiding Noise and Drift:
•The detector should be warmed up prior to the test, and any
temperature fluctuations should be avoided during the test.
•For a dynamic testing condition, methanol is passed through the
flow cell at 1 mL/min.
•A backpressure of about 500 psi is maintained to prevent bubble
Column Heating Module :
•The efficiency of a HPLC column varies with column temperature.
•capacity factor k’ decreases with temperature, and hence the
retention of the analysis decreases with temperature .
•Retention drops by 1 to 3% for each increase of 1◦C.
How To Check??
•The temperature accuracy of the column heater is evaluated by
placing a calibrated thermometer in the column compartment to
measure the actual compartment temperature.
•The thermometer readings are compared to the preset
temperature at 40 ◦C and 60◦C.