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1. Dr. Rajnikant B. Mardia
Senior Assistant Professor
Department of Pharmaceutical
Chemistry
Kamlesh Jivani
M.Pharm sem – II
ID No. 21MQPOS005
Dept. Pharmaceutical
Quality assurance
Faculty of Pharmacy, DDU, Nadiad
Guided by, Prepared by,
QUALIFICATION OF HPLC SYSTEM AND LC-MS SYSTEM

2. 2
QUALIFICATION OF HPLC SYSTEM

3. Introduction
Equipment qualification is a formal process that provides documented
evidence that an instrument is fit for its intended use.
The entire qualification process consists of four parts:
 Design qualification (DQ)
 Installation qualification (IQ)
 Operational qualification (OQ)
 Performance qualification (PQ)
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4. Design qualification (DQ)
• Design qualification (DQ) describes the user requirements and defines the
functional and operational specifications of the instrument.
Design elements Examples
Intended use Analysis of drug compounds and impurities
User requirement specification
for the HPLC analysis
 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
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5. Design elements Examples
Functional specifications
 Autosampler - 100 samples, 0.5 µl to 5 ml sample volume
 Column compartment - 15 to 60˚C, peltier controlled
 Detector - UV/Vis Diode-array, 190-900 nm
 Computer - System control, data acquisition for signals and
spectra, peak integration and quantitation, spectral evaluation
for peak purity and compound confirmation.
Operational specifications
Detector: Baseline noise < 5 X 10-5 AU
Sampler: Precision injection Volume
sample carry over: <0.5%
Maintenance
Vendor must deliver maintenance procedure and recommend
schedule
Training Vendor must provide familiarization and training
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6. Installation qualification (IQ)
Before installation
 Check the site for the fulfillment of the manufacturer’s recommendations
(utilities such as electricity, and environmental conditions such as humidity and
temperature).
 Allow sufficient shelf space for the equipment, SOPs, operating manuals and
software.
During installation
 Compare equipment as received, with purchase order (including software,
accessories, spare parts)
 Check documentation for completeness (operating manuals, maintenance
instructions, standard operating procedures for testing, safety and validation
certificates) 6

7.  Check equipment for any damage
 Install hardware (computer, equipment, fittings and tubings for fluid
connections, columns in HPLC, power cables, and instrument control
cables)
 Switch on the instruments and ensure that all modules power up and
perform an electronic self-test.
 Run test sample and compare chromatogram with reference chromatogram
 Prepare an installation report
Installation qualification (IQ)
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8. Performance qualification (PQ)
Parameter Procedure User Limit
Leak testing
Flow test by volume or
weight/time
± 5 %
Baseline drift ASTM Method <2 × 10-3 AU
Baseline noise ASTM Method <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
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ASTM Method → American Society for Testing and Material Method

9. Parameter Procedure User Limit
Detector linearity Inject 5 standards (caffeine solution)
>1.5 AU, 5%
RSD
Temperature accuracy
comparison with external measuring
device
± 1 °C
Temperature precision monitoring temperature over 20 min ± 0.25 °C
Auto sampler carry
over
Injection of blank solvent after large
concentration
< 0.5 %
Mobile phase
composition accuracy
Step gradients from 4 to 7 % B, step
heights relative to 100%, with acetone
tracer
± 1 %
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10.  BASELINE NOISE AND DRIFT
• Water is pumped at a flow rate of 1ml/min
• UV signal is recorded at 254nm
• To calculate noise the measuring signal is split into 20 intervals for 1 min
each.
• For each interval chromeleon calculates a regression based on measured
values, using the method of least square, then determine maximum
distance of two data points above and below the line.
• Limit should be between <2 x 10-3 AU
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11. 11
Figure: Long-term noise measurement
Figure: Short-term noise measurement

12. For drift
• Chromeleon calculates a regression line from all data points with in a
based on the method of least square.
• The slope of the straight line is expressed in absorbance units/hrs can
be provide an estimation of detector drift.
• Limit should be between <5 x 10—5 AU.
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Figure: Drift measurement

13.  DETECTOR LINEARITY
• A series of 5 traceable standards (caffeine solution of concentration about 0.00035 to
0.35mg/ml) are injected.
• The detector linearity is calculated by determining the peak area vs concentration.
• %RSD can also be calculated for checking the detector linearity.
• limit should be in between >1.5 AU, 5% RSD.
• 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.
 WAVELENGTH ACCURACY
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14.  TEMPERATURE ACCURACY
• 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 ± 1ºc
 TEMPERATURE PRECISION
• Monitor temperature for 20 minutes and limit should be in between ±0.25 º
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15.  AUTO SAMPLER 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.
• It Should be less than 0.5%
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16.  GRADIENT MOBILE PHASE COMPOSITION ACCURACY
• It is important for accurate quantitative analysis.
• Acetone tracer is used to determine gradient mobile phase accuracy,
stability and linearity.
• Make 6 compositions of methanol and acetone or caffeine 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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17. 17
Solnent channel A – Methanol
Solvent channel B - Caffeine

18. QUALIFICATION OF LC-MS SYSTEM
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19. The day-to-day performance of LC-MS or LC-MS/MS depends on its
calibration, tuning, system suitability test, and final overall validation.
Calibration Parameters
 Calibration parameters are instrument parameters whose values do not vary
with the type of experiment such as,
1. Peak width
2. Peak shape
3. Mass assignment
4. Resolution versus sensitivity
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20. 1. Peak width
• Peak width depends on the mass resolution.
• A resolution of 1 mass unit is sufficient to distinguish ions in most
qualitative/quantitative small molecule applications.
• Unit resolution → when the peak width at half-height is about 0.6 to 0.8 mass
unit.
• The profile scan of ions on a typical benchtop LC-MS has a bandwidth of about 1
mass unit (Figure 1).
Figure 1: Band width in a typical benchtop LC-MS 20

21. 2. Peak Shape and Profile Scan
• In a typical benchtop LC-MS, abundance measurements are collected at 0.10-
m/z increments.
• When these data are presented in a mass spectrum, a single line can be shown.
• The height and position are derived from the profile scan.
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22. 3. Mass Assignment
• It is performed using specific MS calibrants.
• Calibrants should be well-characterized reference materials.
• LC-MS qualitative analysis → Use reference materials
• LC-MS quantitative analysis → Use a combination of reference materials
and actual analyte standards as calibrants.
4. Resolution versus Sensitivity
• Mass resolution is a compromise between ion intensity and peak width.
• In general, as the resolution is increased, the ion intensity decreases.
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23. Calibration
• Comparison of a measurement standard or instrument of known accuracy
with another standard or instrument to eliminate deviations by adjustment.
• Manual, semiautomatic, and automatic LC-MS calibration require
introduction of the solution of the calibrant (calibration solution) into the
MS at a steady rate while the procedure is running.
• Calibration solution is introduced directly (infused) into the MS from a
syringe pump or through a loop injector connected to the LC pump.
• MS must be calibrated at least once every three months.
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24. Tuning Parameters
• Tuning parameters are instrument parameters whose values can vary with the type of
experiment.
• Manual, semiautomatic, and automatic tuning procedures require the introduction of a
tuning solution of the analyte of interest into the MS at a steady rate. This can be done
in following three different ways:
1. By introducing the solution directly from the syringe pump (direct infusion)
→ This method is good for tuning for experiments at a low flow rate.
2. By introducing the sample from the syringe pump into the effluent of the LC
by using a tee union
3. By injecting the sample into the effluent of the LC by using a loop injection
valve [flow injection analysis (FIA)]
→ second and third methods are useful for experiments at a higher flow rate.
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25. • Optimized parameter which affect the signal quality, change from
instrument model to instrument model & from brand to brand.
• Examples are Source, temperature, ionization voltages, gases (nebulization,
desolvation, and collision), ion path potentials (lens, multipoles, or stacked
rings), collision energy, solution, or mobile-phase flow rate.
• The potentials, RF values, and gas pressure affect the declustering,
focusing, fragmentation, and efficiency of ion transmission.
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26. System Suitability Testing
• Tests allows the determination of system performance by analysis of a
defined solution prior to running the analytical batch.
• System suitability should test the
- Entire analytical system
- Chromatographic performance
- Sensitivity of the mass spectrometer for the compounds of interest.
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27. Validation
• Final step to guarantee that a LC-MS system performs as expected.
• Validation includes instrument calibration, tuning, testing, and checking of the
documentation for completeness, correctness, and compliance with SOPs.
• Validation consists of four separate steps:
1. Validation of the instrument and the computer controlling it (computer system
validation or CSV)
2. Validation of the analytical method running on that equipment
3. System suitability testing, to test the equipment and the method together to
confirm expected performance
4. QA/QC review of sample analysis data collected on such a system
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28. MS CALIBRATION PARAMETER
• This is the window used by the calibration software to search for the most
intense peak.
• Increasing this window gives a greater chance of incorrect peak matching.
• It is important to ensure that the correct peak is located in the peak search
range; otherwise, a deviation in the calibration may arise.
• If an incorrect peak is located, the search peak range should be adjusted to
locate only the correct ion.
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1. Peak search range

29. 2. Peak threshold
• All peaks in the acquired spectrum below the intensity threshold
value (measured usually as a percentage of the most intense peak in
the spectrum) will not be used in the calibration procedure.
3. Peak Width
• This is a measure used to set the resolution, usually specified at 50%
of maximum intensity.
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30. Calibration solution
• Calibrants are required to calibrate the mass scale of any mass
spectrometer.
• Calibrants commonly used in electron ionization (EI) and chemical
ionization (CI), such as perfluorocarbons, are not applicable in the ESI
mode.
• The main calibrants used or still in use to calibrate ESI-MS can be divided
into the following categories:
polymers, perfluoroalkyl triazines, proteins, alkali metal salt clusters, poly
ethers, water clusters, and acetate salts.
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31. 1. Polymers
• polypropylene glycols (PPGs) and polyethylene glycols (PEGs) are
the preferred calibrant for many small molecule applications.
• PPG calibration solutions produce mostly singly charged ions over the
entire instrument mass range in both positive and negative ion mode.
• The PPG ions in general used for calibration in positive mode are: 59.0
(solvent and fragment ion), 175.1 (fragment ion), 616.5, 906.7, 1254.9,
1545.1, 2010.5, and 2242.6
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32. 32

33. 2. Perfluoroalkyl Triazines
• Perfluoroalkyl triazines such as Ultramark 1621 have mainly been used
as the calibrant for FAB and ESI-MS calibration.
• This standard is very sticky and is very difficult to remove from the ion
source. For this reason, Ultramark 1621 calibration solution should not be
used at flow rates above 10 µL/min, to avoid system contamination.
• Ultramark 1621 is in general used together with other calibrants to cover
the entire MS range.
• Calibration solutions containing caffeine, L-methionyl-arginyol-
phenylalanylalanine acetate H2O (MRFA) and Ultramark 1621 are
commonly employed for ESI-MS calibration.
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34. 34
caffeine: m/z 195; MRFA: m/z 524; and Ultramark 1621: m/z 1022, 1122, 1222, 1322,
1422, 1522, 1622, 1722, 1822.

35. 3. Proteins
• ESI-MS calibration was initially performed using solutions of gramicidin
S, cytochrome c, or myoglobin as calibrant.
• Proteins produce multiply charged species in ESI.
• When proteins are used as calibrants for low-resolution LC-MS and LC-
MS/MS instruments, it is not possible to resolve individual isotope peaks.
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36. 4. Alkali Metal Salt Clusters
• It cover a wide m/z range and are used to calibrate mass spectrometers in
ESI mode.
• Cesium iodide solutions produce singly and doubly charged species from
m/z 133 up to m/z 3510 or higher.
• Cesium iodide calibration solutions are not very commonly used due to
the following drawbacks:
1. sample suppression
2. persistence in the ESI source
3. possible cation attachment
4. The large spacing 260 amu between peaks.
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37. • A mixture of sodium iodide and rubidium iodide calibration solution is
able to cover the instrument’s full mass range from 23 to 3920.
• The peak at 23 is sodium, the 85 peak is rubidium, and the others are
clusters.
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38. 5. Polyethers
• Polyethers such as polyethylene oxide (PEO) and polypropylene oxide
(PPO) have been used for ESI-MS calibration.
• sodium attachment is frequently observed, due to traces of sodium in
solvents and glassware.
• Macrocyclic polyethers are also used as ESI-MS calibration.
• Derivatized polyethers such as polyether sulfate have been investigated for
both positive and negative-ion calibration.
• Lauryl sulfate ethoxy-lates were also used as calibrants for negative-ion
ESI.
• Polyether amines and quaternary ammonium salts were used as positive-
ion calibration solutions.
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39. 39
6. Acetate salts
• Sodium acetate and sodium trifluoracetate clusters were used
and produce useful reference peaks for both positive and negative
ESI.
• 0.5% acetic acid in ammonium acetate solutions can be used for
calibration in ESI-MS.

40. Reference
• Chung Chow Chan et al, Analytical method validation and instrument
performance verification, 2004, page no.173-184 and 197-216.
• Dr. Ludwig Huber qualification of high performance liquid
chromatography system, Bio pharm journal, vol 11 page number 1 to 9.
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41. T H A N K Y O U
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