SOP for Good Chromatography Practices in QC Laboratory

In short: Good chromatography practices (GChP) are the rules a QC laboratory follows so that every chromatographic result is accurate, traceable and reviewable, from mobile phase preparation to integration and data review. This is an illustrative example SOP for QC analysts and reviewers. It is a template: adapt it to your own quality system, validated methods and current pharmacopoeial text before use.

Illustrative template only. Numbers, limits and wording must be confirmed and approved by your own Quality unit before this is used as a controlled SOP.

See also: HPLC calibration Part 1 and HPLC troubleshooting steps for pharma.

What is good chromatography practice?

Chromatography separates the components of a mixture. The sample is dissolved and carried by a mobile phase through a stationary phase, and each component moves at a different speed. In a pharmaceutical laboratory, chromatography (mostly HPLC, also GC) is used for assay, related substances, content uniformity, dissolution and many other tests, so a result is only as reliable as the way the run was prepared, executed, processed and reviewed.

Good chromatography practices set out how analysts prepare solutions and columns, build a sequence, apply system suitability, integrate peaks, handle failures and document everything, so that the data stands up to review and inspection under data integrity expectations.

1.0 Objective

To describe the procedure for good chromatography practices in the Quality Control laboratory.

2.0 Scope

This SOP applies to all chromatographic analysis (HPLC and similar techniques) performed in the Quality Control laboratory.

3.0 Responsibility

• Executive / Officer, QC: carry out analysis and record findings as per this SOP; inform the Head QC or designee of any deviation.
• Section head / Manager, QC: ensure the SOP is followed, evaluate documentation and arrange training as needed.
• Head QA or designee: ensure the system is implemented and followed.

4.0 Procedure

4.1 General precautions

• Before starting, show on the status board the product or material, batch or A.R. number, stage, test, start time and analyst (sign and date).
• Make the entry in the instrument logbook.
• Handle the column with care and keep both ends closed after use.
• Change the flow rate stepwise and slowly.
• Use filtered and degassed HPLC-grade solvents and mobile phases. Use AR-grade solvents only if a chromatography grade is not available.
• Use high purity water suitable for chromatography (for example Elga or Milli-Q) to prepare mobile phase.
• Do not over-tighten fittings of the injector, column, pump or detector.
• Make sure the date and time of acquisition appear on every chromatogram and that chromatograms are compiled in sequence.
• Check system suitability before analysing samples.
• Solutions should be clear, homogeneous and free from particles. Filter solutions before use.
• Never mix the aqueous and organic portions in a measuring cylinder, as the volume change gives an incorrect composition.
• Wash the column before and after the sample set, and include the wash in the sequence.
• Flush the HPLC system with hot water (about 50 to 60 °C, or as suitable for the tubing) with a union in place of the column, at least weekly.
• When changing from reversed phase to normal phase or the reverse, follow the changeover steps in section 4.14.

4.2 Mobile phase and solvent preparation

• If the mobile phase contains a buffer, calculate the total volume needed for the whole analysis (including dilutions) and prepare the buffer accordingly.
• Adjust pH, if needed, with dilute acid or alkali, never with concentrated reagent.
• Filter the buffer through a 0.45 micron nylon filter or as stated in the test procedure. Filtering may also be done after the final mobile phase is mixed.
• Measure the buffer and the organic solvents separately, transfer to a clean, dry, stoppered bottle and mix well.
• Where a solvent forms 5 % or less of the mobile phase, use a suitably sized volumetric flask or pipette to measure it.
• Degas by sonication or vacuum for about 4 to 5 minutes only. Longer degassing of mixtures containing organic solvent can alter the composition. Loosen the bottle cap during sonication to avoid pressure build-up.
• For a reversed phase mobile phase, rinse the filtration assembly and receiver with water, then with the mobile phase, before filtering. For a normal phase mobile phase, rinse with the water-miscible component, then with the mobile phase.
• Degas by sonication, vacuum or helium sparging as the method allows, and filter aqueous buffers (for example 0.45 micron or as stated in the method) before use. Sonicate buffers only briefly, because prolonged sonication warms the solution and can change its composition. The original claim that sonication of acetate or phosphate buffers forms interfering complexes is not supported by any pharmacopoeial text and has been removed.

Record the preparation in the Mobile Phase Preparation Record (Annexure 1).

4.3 Column use, system set-up and sample analysis

• Inspect the column physically before connecting it.
• Wash the column with suitable solvents and equilibrate with the mobile phase for about 30 minutes or until the baseline is stable.
• The analyst prepares the injection sequence for each test. It should include, as a minimum: vial number, injection volume, a sample name that links each chromatogram to the sample (product, batch or A.R. number, stage), method set name, and chromatogram or injection number.
• Before the run, the reviewer or section head checks that the sequence and instrument method match the STP/GTP.
• Do not make trial injections to check suitability (other than the system suitability run defined in the STP/GTP). Run all injections as part of the main sequence.
• From the system suitability run or first standard injection, check peak shape, retention time, relative retention time, resolution, asymmetry, system pressure and theoretical plates.
• If a system modification is needed, it must be authorised first and must follow section 4.6.
• After a satisfactory system suitability run, inject blank (diluent or mobile phase) and standard, confirm system suitability, and then start the samples.
• If peak splitting or broadening occurs during the run, stop, wash the column properly and inject a bracketing standard or system suitability solution. If it passes, continue from the sample where the problem occurred.
• Enter system suitability data in the analytical worksheet.
• After the analysis, wash the column with suitable solvent for suitable time (longer, for example, with water if the buffer concentration is high or the column has been used for long), rinse the auto-injector and complete the column usage log and instrument usage logbook.
• All chromatograms form part of the final report. Enable the pressure trace when creating or editing the instrument method.

4.4 Change of column during analysis

If peak shape or system suitability still fails after allowed adjustments, the column may be changed, with this documentation:

• Enter the earlier column in the column usage log with the reason for discontinuation.
• Print all chromatograms from the earlier column, record the reason for change and the earlier column number on the chromatogram checklist, and attach them to the worksheet or report.

4.5 Change of mobile phase during analysis

If peak shape, resolution, plate count or tailing is not within limits even after allowed adjustments, the mobile phase may be changed:

• Record the reason in the instrument usage logbook.
• Repeat system suitability for every new mobile phase and column.
• Do not top up a running mobile phase with fresh mobile phase in the middle of an analysis.

Flow rate and retention time: any change in flow rate is an adjustment of the chromatographic system and is permitted only within the limits of the current USP <621> / Ph. Eur. 2.2.46 allowable adjustments (see section 4.6) and the validated robustness range of the method. The ± 10 % flow adjustment and the ± 1 minute (retention times up to 10 minutes) or ± 10 % (above 10 minutes) retention time tolerances used in the original SOP are in-house criteria, not pharmacopoeial limits. Example in-house criterion (not a pharmacopoeial requirement): confirm peak identity by comparing retention time with the standard within the tolerance defined in your approved SOP or method.

4.6 Allowable adjustments to the chromatographic system

Adjustments may be needed to meet system suitability but must not be used to hide column failure, analyst error or system malfunction. Adjustments are allowed only when suitable standards are available for all compounds in the suitability test and the adjusted system meets every system suitability requirement of the official procedure. Multiple adjustments can have a cumulative effect and must be considered carefully. Any change in the stationary phase chemistry (the “L” designation) is a method change and needs full validation. Gradient composition changes are not recommended because selectivity may change.

The permitted adjustments are those in the harmonised chapter, official since 1 December 2022 in USP–NF (<621> Chromatography) and since 1 January 2023 in the Ph. Eur. (2.2.46 Chromatographic separation techniques); the Japanese Pharmacopoeia carries the same text (2.00 Chromatography). They apply unless the monograph or validated procedure says otherwise. Key liquid chromatography allowances:

Where F = flow rate, dc = column internal diameter, dp = particle size, L = column length; subscript 1 = monograph, 2 = column used.

Once a procedure has been adjusted within these limits, no further adjustment is allowed without appropriate revalidation, and multiple adjustments call for a documented risk assessment and additional verification. Always check the current edition of the chapter your monograph calls before use.

Pharmacopoeial limits and site practice are different things. Anything in this SOP that is not taken from <621> or 2.2.46 is site practice and must be defined in your own approved SOP.

If the column temperature or sample temperature is not given in the method, or is “ambient”, the original SOP sets column temperature to 25 °C and sample temperature to 15 °C (in-house defaults, not pharmacopoeial requirements).

Note: older SOPs used a “±30 % relative / 10 % absolute” rule for mobile phase composition. That rule was replaced in the harmonised chapter by the system suitability and ±15 % retention time criterion shown above.

4.7 Duplicate standard and similarity factor

• Use a duplicate standard (a second standard preparation) for the assay test, whatever the product category.
• Prepare the second standard by the same or a different analyst. Inject the first standard as the system suitability standard, then inject the second standard in duplicate after system suitability and before the samples.
• Calculate the similarity factor between the two standards:

(mean area of standard 2 x weight of standard 1) / (mean area of standard 1 x weight of standard 2)

• Acceptance criterion: 0.98 to 1.02. Note: this similarity factor limit, and the practice of injecting a duplicate standard for every assay, are in-house rules, not pharmacopoeial requirements; your validated method or monograph may differ.
• If it fails, log a lab incident per the current Lab Incident SOP, investigate, and repeat with fresh standards, establishing the similarity factor before sample injection.
• Use the mean area of the first standard for calculation.

4.8 Bracketing standards

• Establish system suitability as per the test procedure.
• Inject a bracketing standard (single injection of standard preparation) after every 12 injections or every 3 hours since the last standard, whichever is earlier, and at the end of each test. Note: the “12 injections or 3 hours” interval is an in-house rule, not a pharmacopoeial requirement. Other intervals must be justified and documented.
• The area RSD of the last five standard injections, including bracketing standards, is used for system suitability, and the average standard area including bracketing standards is used in calculations.

Worked example: system suitability uses standard injections 1 to 5; sample A is injected as 6 and 7; the bracketing standard is injection 8; sample B is injections 9 and 10; and bracketing standard 11 follows.

• Result A: standard average area is the mean of injections 2, 3, 4, 5 and 8; sample A average area is the mean of 6 and 7.
• Result B: standard average area is the mean of injections 3, 4, 5, 8 and 11; sample B average area is the mean of 9 and 10.

The analysis is valid only if the bracketing standard RSD is within limits. If it fails, stop, log a lab incident per the current Lab Incident SOP, and investigate:

• If the failure is due to system instability, use results up to the last bracketing standard that meets criteria, and analyse the remaining samples after re-establishing system suitability as per the GTP.
• If one bracketing standard area changes significantly and the analysis continues, inject additional bracketing standards and justify the earlier variation.
• If a vial is missing, volume is low, areas vary between replicates, or an additional peak appears, re-injection from the same vial or solution is allowed with scientific justification.
• Extra injections of blank, diluent, placebo or impurity standard may be made to verify elution patterns. Attach the data and rationale to the analytical report.

4.9 Integration, review, calculation and documentation

Note 1. Process chromatograms within one working day of the end of the sequence. If this is exceeded, log a lab incident, investigate and justify before processing. Note 2. No chromatogram (blank, placebo, system check, standard, sample) may stay unprocessed.

• Before setting integration, the analyst checks peak shape and system suitability for all chromatograms in the sequence.
• Set integration parameters (width, threshold, area, height, scale) so that peaks are correctly marked. For tests other than related substances, degradation products and chromatographic purity, inhibit all other peaks except the principal peak.
• Use the same integration parameters for all chromatograms in a sequence or test. If different processing methods are used in one sequence, justify it.
• Manual integration is not allowed. Where the software cannot integrate properly, manual integration may be done with justification and only for the related substances test.
• In the system suitability chromatogram, identify peaks, RRT, system suitability parameters and peak shape.
• For related substances, identify each peak (blank/diluent, placebo, principal, unknown impurity, named known impurity). Inhibit peaks in the sample whose response is similar to that in the placebo or blank.
• If an unknown peak in the sample is larger than in the blank or placebo, integrate it in both and use the corrected area (sample minus blank or placebo).
• Attach an overlay of sample, blank and placebo for related substances.
• For in-house products where system suitability criteria are not met but the peak shape and elution pattern are good, send all data to the method development team to review and revise the criteria.
• Print integration parameters and all chromatograms. If degradation products or related substances are calculated from assay data, print the first injection of each sample with width and threshold set to detect all peaks, along with blank or placebo.
• Report system suitability data (plates, tailing, capacity factor and so on) in the worksheet.
• If reintegration is needed, identify, assess and justify all earlier integrations; attach the previous processing method printout, the audit trail for the change and the printouts to the sequence record.

Related substances and other low-level tests

• Zoom the baseline and check that all relevant peaks are integrated and marked correctly.
• Integrate incompletely separated peaks by valley-to-valley or tangential skim as appropriate.
• Set integration so that a peak of at least half of the disregard limit is integrated, and record this on the chromatogram checklist.
• Set the chromatogram scale so the shapes of all peaks of interest can be seen.
• Identify peaks on the chromatogram by retention time only; other identification goes in the peak table.

Assay, content uniformity, dissolution, preservative content

• Set integration for the principal peak.
• Set the scale so the principal peak is at least 70 % of full scale (or autoscale).

Disregard limits and impurity calculation

• In related substances and chromatographic purity, exclude diluent and placebo peaks.
• Ignore peaks below the disregard limit set in the GTP (based on LOQ from validation). Set the reporting and disregard thresholds per ICH Q3A(R2) (drug substance) or Q3B(R2) (drug product) and the product specification. The ICH reporting threshold depends on the maximum daily dose: for drug substance, 0.05 % at up to 2 g/day and 0.03 % above 2 g/day (Q3A(R2)); for drug product degradation products, 0.1 % at up to 1 g/day and 0.05 % above 1 g/day (Q3B(R2)). Where neither the GTP nor the specification gives a level, any site default (the original SOP used 0.03 % of the principal peak) is an in-house criterion, not a pharmacopoeial or ICH requirement.
• Area equivalent to the disregard limit T % by area normalisation = (T x area of principal peak in sample preparation 1) / 100.
• For the external standard method = (T x X x Z) / (Y x P), where T is the disregard limit (%), X is the theoretical drug substance concentration in the sample, Y the concentration of the external standard, Z its area and P its potency.
• For known and unknown impurities, disregard peaks below the LOQ-equivalent area = (L x A) / (C x P), where L is the LOQ of the impurity (ppm), C the impurity concentration in the standard (ppm), A the impurity area in the standard and P the potency of the impurity standard.
• Where an impurity standard is not injected and LOQ and response factor (RF) are given in the GTP: LOQ-equivalent area = (L x A x RF) / C, where C and A are the drug substance concentration (ppm) and area in sample preparation 1. Use the RF-corrected area in calculation where the GTP gives an RF.

4.10 Chromatogram checklist (Annexure 2)

• Fill in product or sample, test, reference (GTP, protocol or template number), batch number and A.R. number.
• Tick applicable points and write NA for others. Justify any non-compliant point under “Remark”, or give the deviation number.
• Bundle the checklist, mobile phase record, sequence printout, instrument and processing methods and all chromatograms with the worksheet or protocol, under the relevant batch or A.R. number.
• If the analysis is discontinued, state the reason in the instrument usage log and the checklist.
• Print all generated chromatograms, mark cancelled ones with justification, and attach them to the document.

4.11 General instructions

• The reviewer examines chromatographic systems at least weekly for unprocessed chromatograms, single injections, system check injections, system suitability injections, and partial or complete sequences not part of the original sequence. Record observations as per the Analytical Data Review SOP and log a lab incident for any such event.
• Identify, process and justify such runs, assess impact against the original sequence, and attach prints to it.
• Runs not used for evaluation (incomplete, discontinued, system suitability check and so on) are marked “Not Used” with justification, sign and date, and made part of the main sequence set.
• The “printed on” date and time must appear on the chromatogram report.
• The QC Head identifies instruments not compliant with 21 CFR Part 11 and derives an action plan with a timeline, referring to the Data Integrity SOP (see data integrity in the pharmaceutical industry).
• Samples for experimental analysis come from expired lots of finished product, lots prepared from working standard, or retention samples with Head QA approval.
• Record all service technician activities in the report and review and approve it as per the report approval procedure.
• Use a new cleaned vial for related substances and degradation product tests.

Run time and replicate injections

• Assay: run standard and sample chromatograms about 5 minutes beyond the principal peak elution, or as per the GTP/STP.
• Chromatographic purity, degradation products, related substances, stability samples: run for 2.5 times the retention time of the principal peak, or as specified.
• Specific impurity analysis: about 5 minutes after the principal peak.
• If the system is held running in the mobile phase and another sample is to be injected: if the gap is 2 hours or less, inject directly; if more than 2 hours, inject a bracketing standard and calculate the RSD of the last two initial system suitability injections plus the bracketing standard injections. Continue if satisfactory.
• At the end of the sequence, run a method that turns off the D2 lamp and reduces the flow to about 0.2 ml/min (or suitable) to keep the system stable in the mobile phase.
• Carry-over that does not affect the peak of interest may be accepted after justification and authorisation.
• Any unknown peak in assay, dissolution or content uniformity should not exceed the unknown impurity limit in related substances (which is derived from ICH daily dose criteria). Investigate any peak above it.
• During analysis, extra injections of blank, placebo or impurity solution (spiked or not) can be made with justification.
• If a failure of established system suitability, peak splitting or area variation occurs, correct it on line by altering the same sequence with justification.
• If an abnormality is seen in a sample, a freshly prepared sample may be injected with justification.
• If the system must be stopped for rectification, handle it per the Lab Incident SOP.

4.12 Specimen chromatograms

A file of specimen chromatograms is kept, product and test wise, to check reproducibility, to notice changes online during analysis, and to help during data review.

• If a reference chromatogram is available from the manufacturer or source laboratory, compare the first analysis or method transfer (or validation) chromatogram against it for peak shape, retention time, baseline (important in gradient work), scale, additional peaks and other system suitability parameters.
• Mark one chromatogram each of blank, resolution solution, standard, placebo and sample as “Specimen chromatogram”, signed and dated by the Head QC, and file them.
• During routine analysis the analyst compares chromatograms with the specimen. If a difference is found, inform the QC Head or designee, who may suggest an allowable modification (section 4.6).
• The reviewer repeats the comparison during raw data review.
• If the method changes, evaluate the impact on the specimen chromatogram.

4.13 Cancellation of chromatograms

Only the section head may cancel a chromatogram (for example after system failure, system suitability failure or poor chromatography). Stamp “Cancelled” on each, with justification, sign and date.

4.14 HPLC changeover between reversed phase and normal phase

A. Reversed phase to normal phase

1. After washing, remove the column and fit a union. Place all tubing in bottles of degassed HPLC water or solvent.
2. Dry prime: with 100 % HPLC water in the reservoirs, prime each channel for 5 minutes at 5 ml/min with the outlet valve open.
3. Wet prime: prime each channel for 3 minutes at 5 ml/min. The degasser should be off during priming (preferably for a quaternary pump).
4. Needle wash, seal wash and injector wash, for the default time.
5. Flush the system for 20 minutes, increasing the flow gradually from 1 to 5 ml/min with 25 % from each channel.
6. Repeat steps 3 and 4 with 100 % HPLC-grade methanol.
7. Repeat steps 3 and 4 with 100 % HPLC-grade isopropyl alcohol (IPA).
8. Use 100 % IPA or other non-polar solvent, as per GTP or template, for seal wash and needle wash during the analysis.

B. Normal phase to reversed phase

1. Remove the column after washing and fit a union. Place all tubing in bottles of degassed solvent.
2. Dry prime with 100 % IPA in the reservoirs, 5 minutes per channel at 5 ml/min, outlet valve open.
3. Wet prime for 3 minutes per channel at 5 ml/min (degasser off).
4. Needle, seal and injector wash for the default time.
5. Flush for 20 minutes, increasing flow gradually from 1 to 5 ml/min, 25 % from each channel.
6. Repeat steps 3 and 4 with 100 % HPLC-grade methanol.
7. Repeat steps 3 and 4 with 100 % HPLC-grade water.
8. Flush the system for 20 minutes, increasing flow from 1 to 5 ml/min with 25 % from each channel (quaternary pump).

5.0 Abbreviations

6.0 Annexures

Annexure 1: Mobile phase preparation record. Fields: product or material; batch or A.R. numbers; tests; mobile phase quantity; date of preparation; prepared by; date of use; quantity used; quantity discarded; discarded by (sign and date).

Annexure 2: Chromatogram checklist. Fields and checks: product or sample, A.R. number, test, batch number; integration parameters printed on the last standard chromatogram of system suitability (processing method printout attached); chromatograms in serial order from first injection (blank, standard, sample); cancellation of any chromatogram justified; each chromatogram properly identified; peak shape, baseline and peak identification checked; system suitability values entered in the worksheet and within limits; run time of 2.5 times the principal peak retention time for chromatographic purity, degradation, related substances and stability samples, or as specified; same method and integration parameters throughout; any reintegration authorised; samples bracketed by standards, or justification given; remarks.

Frequently asked questions

Is manual integration ever acceptable?

Regulators expect integration to be consistent and reproducible, and manual changes to be justified, reviewed and kept in the audit trail. This example SOP restricts it to related substances with justification; your own SOP must define what is permitted.

Can I make trial injections before the sequence?

Not as a routine. This SOP requires all injections, including system suitability, to be part of the main sequence. Unexplained trial injections are a common data integrity finding.

How much can I change the method to pass system suitability?

Only within the allowable adjustments of USP <621> (or the equivalent chapter your monograph calls), documented, and not to hide column failure, analyst error or system malfunction. See section 4.6 and check the current official text.

References

• US FDA Guidance for Industry: Data Integrity and Compliance With Drug CGMP, Questions and Answers (December 2018).
• US FDA, 21 CFR Part 11, Electronic Records; Electronic Signatures (1997), and FDA Guidance for Industry Part 11, Electronic Records; Electronic Signatures — Scope and Application (August 2003).
• USP–NF General Chapter <621> Chromatography (harmonised revision, official since 1 December 2022).
• European Pharmacopoeia 2.2.46 Chromatographic separation techniques (harmonised with USP <621>; use the edition in force).
• India, Drugs and Cosmetics Rules, Schedule L1 (Good Laboratory Practices) as cited in the original.
• ICH Q3A(R2) Impurities in New Drug Substances and ICH Q3B(R2) Impurities in New Drug Products (Attachment 1 thresholds).
• MHRA, GxP Data Integrity Guidance and Definitions (Revision 1, March 2018).