Good Chromatographic Practices_GMP_Dr.A.Amsavel

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Good Chromatographic Practices

• Dr. A. Amsavel

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Content

• Introduction
• Solvents Reagent Quality
• Mobile phase preparation usage
• Buffer, Filtration, Degassing
• Storage usage
• Preparation for analysis
• Sample preparation System suitability
• Adjustment allowed as per USP
• Column management
• Washing and change of mobile phase
• Analysis and integration
• Documentation
• Problems and reporting
• Qualification and Calibration

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Introduction

• Chromatography analysis (HPLC, GC TLC etc ) of
  material in pharmaceuticals QC Lab is inevitable
• Chromatographic technique is used for
  Identification, determination of assay, purity,
  related substances and residual solvents etc.
• Purpose of Chromatography analysis is for
  final product , intermediate, reaction
  monitoring, process validation, cleaning
  validation, cleaning verification, stability
  testing etc
• Chromatographic analysis has to be performed and
  documented as per GMP and to meet the regulatory
  requirements
• There are challenges, while performing analysis
  and compliance

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Qualification And Validation

• HPLC GC instruments shall be qualified (URS,
  DQ, IQ, OQ and PQ)
• Software used shall meet the 21CFR Part-11
  compliance
• Hardware Software shall be qualified as per
  Computer system validation (CSV)requirement (
  GAMP-5)
• Instruments shall be calibrated appropriately at
  defined interval
• Test method shall be validated, compendia method
  shall be verified, and or method transfer as
  applicable
• Analyst shall be qualified and trained
• Ref Qualification Of Equipment Annex 1
  Qualification Of Liquid Chromatography Equipment

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• What are the challenges faced while
  Chromatographic analysis?
• What are the possible errors
• occur during analysis?

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• Problems or challenges !!!
• How can you prevent the problems?
• How can it be controlled?
• How errors can be eliminated or minimized?

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• Follow Good Chromatographic practices
• SOP for Good Chromatographic practices
• Use Qualified the Instruments
• Validated / verified and appropriate test method
  
• Perform periodic Calibration Preventive
  maintenance
• Right Quality solvents, proper sample
  preparation, system suitability
• Train and Qualify the analyst
• Let us discuss on HPLC analysis

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HPLC Analysis Flow
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Water for HPLC analysis

• Always use only Ultra pure / Milli-Q water for
  HPLC analysis
• Ultra pure HPLC water of 18M? resistivity
•  Do not use RO water/de-ionised water for HPLC
  analysis. It will have organic and in-organics
  impurities
• If water contains impurities, it will have higher
  absorption and lead to poor baseline , drift ,
  ghost peak and less accurate
• Note
• Use fresh water if possible or store properly
• Store HPLC grade water in glass containers.
• Plastic containers may leach the additives in
  the plastic into the water

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Solvents Reagents

• All reagents and solvents should be highest
  quality.
• HPLC grade reagents solvents are high purity
  will have low UV absorbance
• Low grade solvent contain impurities to produce
  spurious peaks, poor peak , high baseline etc
• Reagent / acid for buffer preparation should be
  highest purity.
• Use specific quality of reagent as stated in
  Pharmacopiea / test method as required
• Eg. Different grade of Ammonium acetate based on
  the purity
• TEA TFA are expected to be contaminated from
  the lab environment and should be refrigerated
  during storage.

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Impact of Quality of Solvent
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Buffers

• Preferably all buffers should be prepared freshly
  on the day of use .
• Buffer pH will affected by prolonged storage and
  prone to microbial growth both will affect badly
  in chromatography.
• If buffer solutions are used for long or
  stored establish the shelf life and use
• Where possible use reagents that without
  stabilizer. Eg Sodium metabisulphite is used as
  stabilizing agent
• These stabilizing agents often may affect the
  optical and chromatographic behavior.
• Avoid the repeated / multiple opening use of
  solid reagent since may easily contaminate
• Based on requirement, purchase in low pack size
  to reduce the exposure.

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Use of buffer

• Do not leave the buffer solutions for long in
  system, to avoidcrystallization. This will
  affect the system.
• Effect on pump - damage plunger and seal
• Effect on column - creation of column voids
• Effect on flow line - corrosion of stainless
  steel lines
• Long storage may develop a bacterial growth eg
  phosphate buffers it is good medium for
  bacterial and fungus growth. Hence, prepared
  freshly and avoid storage.
• All the solutions should be clear, homogenous
  free from particulate matter.
• Buffer solutions must be filtered through 0.45µm
  filter
• Note small amount of sodium azide to the aqueous
  solvent to inhibit microbial growth.

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Filtration

• All HPLC solvents or mobile phase should be
  filtered through a 0.45 µm filter before use.
• Removes particulate matter that may cause
  blockages of column and HPLC system.
• Do not keep mobile phase under the suction for
  more than 5 minutes. composition of mobile phase
  change if use highly volatile solvents
• Filtration prevents the wear tear of parts.
• Pump plungers, seals and check valves will
  perform better and lifetimes will be maximized.
• Also column performance and life will be
  maximised
• Filtered solvents should be stored in a covered
  reservoir to prevent evaporation dust
  contamination etc.

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Changing Over of Mobile Phase- RP

• Changing Over of Mobile Phase one analysis to
  another analysis is important for smooth
  chromatographic analysis.
• Before changing to totally organic phase in the
  RP system, the whole system should be flushed
  with water organic (11) mixture to remove the
  buffers used.
• Prevent the crystallisation when the organics
  come into contact with leftover buffers in
  system.
• Eg 100 Acetonitrile use will lead to precipitate
  the salt in the buffer solution

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Changing Over of Mobile Phase .

• To convert a normal phase system/column to a
  reversed phase, flush with a solvent that is
  miscible with both the current normal phase
  solvents
• If the final reversed phase solvents include a
  buffer, then it is advisable to move from the
  100 methanol flush to a 50 aqueous methanol
  flush. For example
• Normal phase Hexane/Ethyl acetate
• Flush IPA (isopropenyl acetate) then
  Methanol
  Finally (5050) Methanol/Water
• Reversed phase Buffered aqueous methanol

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Changing Over of Mobile Phase.

• To convert a reversed phase system/column to a
  normal phase follow a similar way but in
  reverse,
• For example,Reversed Phase Buffered Aqueous
  MethanolFlush 5050 Methanol/Water
  finally Methanol then IPANormal Phase
  Hexane/Ethyl Acetate

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Mobile phase preparation- Isocratic

• Different solvents has to be premixed for
  isocratic systems
• Preparation tips Measure the solvents
  separately for accuracy and mix. E.g. To prepare
  1000ml of 5050 mixture of water/methanol, Measur
  e 500ml of milliQ water, transfer into a bottle
  transfer 500ml of Methanol and mix sonicate
  for about 10-15 minutes.
• Protection of mobile phase storage
• Use glass bottle
• Use Plastic bottle for high sensitivity inorganic
  analysis.
• Always cover the container to prevent evaporation
  of solvents dust freeNote Do not cover the
  mobile phase bottle with parafilm. Theleachable
  from the parafilm may contaminate the analysis.

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Mobile phase preservation usage

• Do not mix old mobile phase with new mobile phase
• Do not expose the bottle to direct sunlight or
  wind.
• Use always suction filter
• Filter and sonicate the mobile phase.
• Keep reservoir above solvent delivery system
• Keep suction filter 2 cm above the bottom to
  prevent particulates from reaching pumps
• Periodically clean the suction filter by
  sonicating in isopropanol or 1N nitric acid and
  water
• Use mobile phase after reach the room temperature

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Degassing

• Mobile phase should be thoroughly degassed to
  remove all dissolved gasses.
• Dissolved gas can be removed from solution by
  Bubbling with helium Sonication / Vacuum
  filtrationIf the mobile phase is not degassed,
  air bubbles can form in the high-pressure system
  resulting in problems with system instability,
  spurious baseline peaks etc.
• Do not use Online degasser while THF as mobile
  phase due to degradation of Teflon in vacuum
  chamber.

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Advantage of Degassing
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Suction filter

• All solvent line shall be fitted with an inlet
  filter to prevent the particles contamination
• The filters should be kept clean to prevent cross
  contamination.
• When system is not being used, store the
  filters in a solution of 50 Acetonitrile /50
  water and in closed condition.
• This is to prevent the microbial growth, dust and
  dirt to clog the filter pores.
• The solvent lines should be clean, clog free, no
  air bubble and should not have sharp bends or
  creases in them.
• The solvent lines and filters should be of
  sufficient length to reach the bottom of the
  solvent reservoir
• Solvent reservoirs should be placed higher than
  the pump inlet manifold.

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Equilibration of Column

• Column volume
• This column volume is more correctly called as
  void volume (Vm).
• The amount of mobile phase required to fill the
  column.
• Otherway the volume of mobile phase which should
  be flushed through a column before it is ready to
  use
• When a column has just installed on a reverse
  phase HPLC system then it will typically require
  10 to 20 column volumes.(exceptions use of
  pairing agents chiral methods)
• This is typically approximately 70of the total
  column tube volume.

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Equilibration of Column
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HPLC Column Management

• Do not store HPLC columns in buffers. A buffer
  may precipitate inside the column, it will clog
  and affects the packing material.
• Mobile phases with 100 or close to 100 buffer
  may lead to bacterial growth, which can block the
  column frit and packing material.
• Bacteria may also affect the analyte, and organic
  products from the dead bacteria may cause "ghost
  peaks" in chromatograms.
• Do not store HPLC columns in solvents that
  degrade easily tetrahydrofuran (THF),
  triethylamine (TEA), trifluoroacetic acid (TFA).
• Unstabilized THF can form peroxides which may
  degrade the column
• All buffers should be washed out of the column
  before flushing with Acetonitrile.
• Gradually start the column washing Eg Starts
  the flow with 0.2 ml/min and increases gradually
  to 2.0 ml/min and continues for 20min to 30 min
  or as per procedure
• Have Dedicated Columns for each Method / each
  product

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Washing of HPLC System Columns

• Flush the system thoroughly with strong
  solventBecause switching to pure organic
  solvent, especially acetonitrile, can cause
  buffers or salts to precipitate.
• Recommend switching to non-buffered mobile phase
  first. Just replace the buffer bottle with water
  and run the gradient again.
• Run the gradient to 100 strong solvent (usually
  methanol or acetonitrile) and hold it at 100 for
  1015 min to thoroughlyflush the column and
  equipment
• Do not shut off a system that contains buffers or
  salts

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Washing Storage of HPLC Columns

• Contamination may affects the chromatography and
  column performance.
• Short term storage, i.e. over night, columns can
  be stored in the eluent used in last analysis.
• Middle term storage, i.e. 2 days or over the
  weekend, columns should be flushed with pure
  water to prevent microbial growth.
• Long term storage, silica based columns should be
  stored in an aprotic solvent.
• The best storing solvent is Acetonitrile water
  content should not be higher than 50.
• Buffer salts will precipitate in Acetonitrile and
  can block the capillaries and the column.

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Column cleaning Regeneration

• Column cleaning has to be used 40 60 column
  volumes appropriate solvent or as per procedure
  .
• Check the column efficiency, capacity factor
  etc. Before and after clean-up procedure to
  confirm the improvement of column performance
• Solvent can be used as follows for regenration
• Normal phase media
• flush with tetrahydrofuran
• flush with methanol
• flush with tetrahydrofuran
• flush with methylene chloride
• flush with benzene-free n-hexane
• Reversed phase media
• flush with HPLC water inject 4 aliquots of 200
  µL DMSO during this flush
• flush with methanol
• flush with chloroform
• flush with methanol

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System preparedness

• Needle and plunger seals shall be washed before
  starting the analysis.
• Use wash solutions, which are miscible with the
  mobile phase to wash and plunger seal
• Use separate solutions and containers for plunger
  seal wash and needle wash.
• Use of one solution for both functions may
  compromise the effectiveness because the
  functions of these solutions differ.

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Seal wash - Composition

• Over a period of time small amounts of mobile
  phase solvents seep through the seal to the back
  of the pump head.
• If these solvents contain buffers then the salts
  may precipitate out forming deposits which can
  shorten the life of the seal.
• Seal wash can start with aqueous solvent to
  dissolve buffers.
• A small amount of organic solvent is added to
  prevent bacteria growth and also to reduce the
  surface tension of the water (this helps the wash
  solvent cling to surfaces).
• Typical seal wash composition is 80 water and
  20 organic solvent.
• The organic solvent may be methanol, acetonitrile
  or isopropyl alcohol (IPA)

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Needle wash

• The needle-wash required by flushing to prevent
  carryover of sample between injections.
• To extends the life of the injector seals by
  removing buffer sample
• Use Extended needle wash if the carry over is
  expected, especially analysis of Related
  substances.
• The composition of the needle wash needs to be
  matched to the sample.
• Proportions of aqueous and organic solvents in
  the mobile phase will be appropriate.

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Injector Maintenance

• Purge the flow line at least once a day.
• Purge all the flow lines with mobile phase to
  ensure the solvent tubing is completely filled
  with mobile phase
• Change purge liquid frequently.
• Never use salt solutions as purge liquid.
• Use good quality septum's for sample vial and
  do not reuse .
• Do not fill vials to the brim.
• Filter sample with 0.2µm or 0.45µm filters before
  injection.
• If the sample contains a lot of solid, centrifuge
  it, and the filter the solution.

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Fitting plumbing

• Excess or short tube fitting will affect peak
  shape.
• Estimate the length required and fix properly .
• Allow extra length if the tubing is to go around
  corners as sharp bends in the tubing will distort
  the inside bore
• Note Care should s be taken when tightening
  fittings.
• Over-tightening can cause damage to the fitting
  threads, ferrules etc,
• Lead to leak or break off in the housing

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Guard Columns and or cartridges

• Protect the analytical column from sample and
  system debris and contaminants for better column
  performance and efficiency.
• Guard columns or cartridges are one of the most
  cost effective and efficient ways of trapping
  these unwanted system components.

Note Guards are designed to be disposable.
Should be replaced once contaminated Recommend
that the guard is replaced every 50 100
injections or based on user
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Precaution

• Avoid pressure shocks on the column.
• Pressure shocks lead to channeling in the column,
  whichresults in peak splitting in corresponding
  chromatogram.
• Always keep both ends of the column closed, after
  usage.
• Keep the columns in the designated column
  cabinets after use.
• Use the pH range of 2 to 8 or follow the
  manufacturersinstructions

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Sample Preparation

• Sample preparation is not only dissolution of a
  solid in a liquid.
• If Samples require use filtration, extraction or
  derivative etc
• Accurate weighing and/or dissolution or make up
  volume for assay.
• Filtrate the sample -By syringe filter or using
  on-line pre-column filter
• Sample solvents should match the mobile phase to
  avoid baseline errors and spurious peaks
• The sample should be clean for long life of
  column and reliable
• If sample contaminates the column, we may spend
  more time money
• Important If using a membrane filter make sure
  that it is compatible with all the solvent used
  

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Definition

• Dwell volume (D) Also known as gradient delay
  volume, is the volume between the point at which
  the eluents meetand the top of the column.
• Hold-up volume (VM) The volume of mobile phase
  required for elution of an unretained component.
• Resolution (RS) The resolution is the separation
  of two components in a mixture,
• Retention volume (VR) The volume of mobile phase
  required for elution of a component (Vm
  retention time (TR) X flow rate (F)
• Separation factor (a) The relative retention
  calculated for two adjacent peaks (k2/k1)
• Theoretical plates (N) A measure of column
  efficiency based on RT PW

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System Suitability

• Perform system suitability (SS) test to verify
  the chromatographic system is adequate for the
  intended analysis.
• SS is based the performance equipment,
  electronics, analytical operations, and sample
  etc .
• The following factors may affect chromatographic
  behavior
• Composition, ionic strength, temperature, and pH
  of the mobile phase
• Flow rate, column dimensions, column temperature,
  and pressure
• Stationary phase characteristics- type of column
  (particle-based or monolithic), particle or pore
  size, porosity, and specific surface area
• Reverse-phase and other surface modification of
  the stationary phases, the extent of chemical
  modification (as expressed by end-capping, carbon
  loading, and others)

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System Suitability Parameters

• Resolution between specified peaks ( gt2)
• Theoretical plate (Ngt2000)
• Precision / injection repeatability RSD of
  replicate injection unless otherwise specified
• RSD for five replicate injections of the
  analyte- 2.0
• RSD for five replicate injections of the
  analyte- gt 2.0
• (Can vary based on the purpose assay , RC, etc
  from 0.7 to 10)
• Tailing factor (T lt2)
• Capacity factor ( k' gt 2 )

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Resolution

• Resolution is defined as the distance between two
  adjacent peak apexes, divided by the average base
  width of both peaks.
• Resolution is dependant on three other variables,
  the column efficiency (N), capacity factor (k)
  selectivity (a).
• Increasing N increases resolution because peak
  width decreases. Decreasing N decreases the
  resolution due to broad peak width
• Decreasing k sharpens the peaks but decreases
  resolution. Increasing k broadens the peaks but
  improves resolution.
• Increasing a increases resolution. One peak moves
  relative to the other. Likewise, decreasing a
  decreases resolution.

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Capacity factor

• Capacity factor (N) is affected by changes in
  mobile phase, operating temperature, analyte
  retention characteristics and changes to the
  surface chemistry of the column.
• Changes in N that occur both with standard and
  sample mixes are likely to be due to changes in
  the column, temperature or mobile phase
  composition.
• Changes in N that occur only in the sample mix
  and not the standard mix are most likely to be
  due to the composition of the sample.
• Note Capacity factor will change by up to 10
  for a 5C rise in column temperature.
• Selectivity is a measure of the relative
  retention of two adjacent peaks in a chromatogram
  

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Band spreading

• Broad peaks with change in retention time,
  indicate band spreading.
• HPLC column or incorrect system plumbing will
  affects band spreading
• Measure the band spreading due to the HPLC
  system.Remove the HPLC column from the system
  and replace with a zero dead volume union. Inject
  column efficiency solution and calculate
• Band Spread (µL) Peak Width x (1/20) x 1 x 1000
  Confirm column effects or plumbing .

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Solvent property

• Understand the property of solvent, which may
  affect the HPLC analysis
• Properties like viscosity, Polarity, RI,
  UV-cutoff, miscibility number etc
• Know the miscibility numbers, to predict the
  miscibility of solvents.
• If the smaller miscibility number is subtracted
  from the larger and the difference is 15 units or
  less, then the two liquids are soluble in all
  proportions at 15C.
• Difference in Smaller miscibility number is lt 15
  mixer of solvents lead to poor chromatographic
  analysis and less accurate.

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Solvent properties
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Impact of PH

• Reversed phase HPLC The retention of analytes is
  related to their hydrophobicity. The more
  hydrophobic the analyte are retained longer .
  When an analyte is ionized, it becomes less
  hydrophobic and retention decreases.
• When separating mixtures containing acids and/or
  bases it is necessary to control the pH of the
  mobile phase of buffer in order to achieve
  reproducible results.
• Eg Resolution changes to 1.4 from 3.0 due to less
  of 0.1pH

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Adjustment allowed for HPLC condition
Property USP General Chapter 621 Ph.Eur.  Gen. Chapter 2.2.46
Column length 70 70
Particle size Reduction by 50 Reduction by 50
Particle size No increase No increase
Internal diameter Can be adapted as long as the linear flow velocity remains the same 25
Flow rate 50 or more, provided the linear flow velocity remains the same 50
Column temp. 10 C 10 C, maximum 60 C
pH -mobile phase 0.2 units 0.2 units (1 for neutral subs)
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Adjustment allowed for HPLC condition
Property USP General Chapter 621 Ph.Eur.  Gen. Chapter 2.2.46
Injection volume Reduction allowed as far as precision and detection limit acceptable. No increase. Reduction allowed as far as precision and detection limit acceptable. No increase.
Salt conc. of the buffer 10, as far as the allowed change in pH value 10
Composition of mobile phase Minor components 30, if not more than 10 absolute Minor components 30,if not more than 2 absolute (greater value accepted)
Wavelength Not permitted , can be Max 3nm based on the validation
For gradient separation, a change of the mobile phase is not recommended For gradient separation, a change of the mobile phase is not recommended For gradient separation, a change of the mobile phase is not recommended
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Starting HPLC analysis

• Test method shall be created and verified
• Sequence can be as below or as per test method
• Blank, system suitability, reference solution ,
  test, and bracketing standard . Bracketing
  standard at defined number of sample injections
• Data file shall be continuous (do not repeat the
  file no/name)
• Load the sample and run the system suitability
  and once SS passes run the test
• After completion, process the chromatograms
• Print the method, sequence and data
• Perform audit trail before batch release
• Store the data and keep back up

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Integration

• Do not integrate any peak by manually.
• Integrate all the sample sets by batch wise.
• Always use same processing method for processing
  ofblank, standard sample chromatograms in case
  of Assay related substances, etc.
• Verify the processing parameters like
• Threshold,
• Width,
• System suitability,
• Peak names etc.
• Save the processing method

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Integration

• Integrate all the injections including entire
  set of failures.
• For System suitability injection for which
  Tailing factor,Resolution or Theoretical plates
  are to be checked,consider only the first
  injection of the sequence.
• Note Do not consider this criteria for average
  of all the standard injections unless otherwise
  specified.
• Re-integration
• Do not re-integrate the chromatograms
  withoutdocumenting.
• Document reason for reintegration.

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Common problem in HPLC analysis

• System failures may occur during analysis due to
• System over pressure
• Communication error
• Failure of system suitability
• Peak splitting/ negative peak
• Bracketing standard failure

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Common problem in HPLC analysis

• How to handle the failure ?
• SOP shall be available and shall address the
  handling of Lab deviation/ incidents.
• SOP shall define clearly the deviation/ incident
  , reporting investigation, CAPA and
  documentation
• Record all the deviation/ incident happened in
  chromatographic analysis
• Process all the injections including the invalid
  injection and report and store the data along
  with Raw data.

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Common problem in HPLC analysis

• How to handle the failure ?
• Do not omit any injection
• Investigate the deviation/ incident
• Find the root cause for the sample set failure.
• Rectify the problem, take appropriate CAPA and
  document
• Repeat complete sample set of injections in case
  of sample injection failure

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How to handle the problem if any

• In case of interruption, due to power failure,
  computer interruption, time gap due to sample
  preparation or due to injection for 4 hours and
  if system is in continuous state of equilibrium
  
• Inject bracketing standard and proceed otherwise
  restart
• Deviation in RT for sample or std, is gt 15 of
  specified RT
• Inhibit the peak upto void volume
• Use the same integration parameter for entire set
• Reprocess shall be at the same time for entire
  sequence

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Documentation

• Ensure are followed contemporaneously
• Instrument Use Log
• Routine Maintenance Log
• Problem Log
• Column History Log

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PQ of HPLC
Ensure the below as parameter during PQ as min,
but not limited to
Component Parameter Acceptance Criteria
Pump Pump Flow Accuracy 0.5ml (0.475 to 0.525) 5.0ml (4.75 to 5.25)
Pump Pump flow precision RT RSDNMT0.50
Pump Gradient composition in 20, 40, 60, 80 2.0
Column oven Column Temperature Accuracy Column Oven lt 2.0
Column oven Column Temperature Stability Column Oven lt 1.0
Sample oven Sample temperature Accuracy Set temperature 4C gt -2.00 and lt 5.00
UV Detector Wavelength Accuracy (201nm to 209nm) 205 nm lt 2
UV Detector Wavelength Accuracy (241nm to 249nm) 245 nm lt 2
UV Detector Wavelength Accuracy (269nm to 277nm) 273 nm lt 2
UV Detector Noise and Drift Noise lt 0.040 mAU Drift lt 0.500 mAU/Hr
UV Detector Signal to Noise gt 3000
UV Detector Response Linearity (Resp.Factors) Correlation coefficient gt 0.99900
Lamp Intensity 1000
Sampler / Injectors Injector Precision Volume Delivery - Linearity RSD for Area lt 1.0 RSD for Height lt 2.00
Sampler / Injectors Injector Precision Volume Delivery - Linearity Correlation coefficient NLT0.99
Sampler / Injectors Injection Carryover Carryover for Area lt 0.20
Sampler / Injectors Injection Carryover Carryover for Area lt 0.40
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Calibration of HPLC
Component Calibration test Acceptance Criteria
Pump Leak Test  No leak
Pump Flow rate Accuracy 0.5ml (0.49 to 0.51) 1.0ml (0.98 to 1.02) 2.0ml (1.96 to 2.04)
Pump Flow stability RT RSDNMT1.0
Pump Gradient Delivery Accuracy in 20, 40, 60, 80 2.0
Column oven Sampler Temperature Accuracy by Calibration of Thermocouple and Air temperature  Column Oven 25C/40C/60C 2.0 
UV Detector Wavelength Accuracy(266nm to 276nm) 271 to 273nm
UV Detector Dynamic Short-term Noise (Single to Noise Ratio) Noise0.04 mAU or less Drift5.0mAU/hr or less
UV Detector Response Linearity Correlation coefficient NLT0.99
UV Detector Lamp energy Low intensity (gt 200) Average intensity (gt 5000) Highest intensity (gt 10000)
Sampler / Injectors Volume Precision RSD NMT 1.0
Sampler / Injectors Volume Delivery - Linearity Correlation coefficientNLT0.99
Sampler / Injectors Injector Carryover NMT 0.1
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Reference

• WHO 791 Guideline for GOOD CHROMOTOGRAPHY
  PRACTICES (February 2019)
• Troubleshooting LC Systems, John W Dolan Lloyd
  R Snyder, Humana Press
• Qualification Of Equipment Annex 1 Qualification
  Of Liquid Chromatography Equipment

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• Thank you