Are You Sure You Understand USP <621>?

The updated version of United States Pharmacopoeia (USP) general chapter <621> Chromatography has changes that impact chromatography parameters. In this column, we look at the current version and the update of USP <621> on high-performance liquid chromatography (HPLC) that becomes effective 1st May 2025. Do you fully understand the changes and how they will impact your laboratory?

Chromatographic analysis in regulated Good Manufacturing Practice (GMP) laboratories is under the control of pharmacopoeial general chapters. These should define the qualification parameters for instrumentation and System Suitability Test (SST) parameters to demonstrate the analytical system is under control. The changes are a result of the harmonization process between USP, Japanese Pharmacopoeia (JP) and European Pharmacopoeia (Eur. Ph.). The harmonized USP <621> became official in December 2022; however, a few subsections of the SST section were delayed and will become official in May 2025 (1). Are you prepared?

We also look and revisit the use of SSTs for Analytical Instrument Qualification (AIQ) that were discussed in three earlier “Questions of Quality” columns.

Before providing an overview of the changes in USP <621>, we compare the structure of the chromatography general chapter against the USP spectroscopic general chapters shown in Figure 1.

USP Chromatography and Spectroscopy General Chapters

USP chapter numbers below 1000 are mandatory and applicable, and those between 1000 and 1999 are informational and strong guidance for the industry. These are shown in different colors in Figure 1. USP <621> currently covers many existing chromatography modalities in a single chapter with few exceptions, such as supercritical fluid chromatography (SFC) and Capillary Electrophoresis. Historically, the same approach was used with spectroscopic techniques originally featured in USP <851>. However, USP has now split each spectroscopic technique into separate technique and applicable chapters (USP <852> to <858>) along with informational chapters covering fundamentals and applications (USP <1852>–<1854> and <1856>–<1858>) as described by Burgess and Hammond (2). This is shown for ultraviolet–visible (UV–vis) and near-infrared (NIR) spectroscopy on the right of Figure 1.

In contrast, the European Pharmacopoeia has an overarching general chapter 2.2.46 (3) that is linked to six general chapters for specific chromatography techniques shown in Figure 2.

Given the importance of chromatography in pharmaceutical analysis, is the update of USP <621> overdue? Division into separate technique general chapters similar to the EP and the USP spectroscopic general chapters could be more didactic and effective. In fact, this was proposed for the USP 2005–2010 cycle (4), but no progress has been made yet. Why?

Timeline of USP <621>

USP has included a general chapter related to chromatography since at least 1955, and the first publication of the chapter as USP <621> was in 1980. USP <621> is one of the most important and accessed USP general chapters for the pharmaceutical industry. The scale of USP <621> is evident when searching for “<621>” in the current United States Pharmacopeia–National Formulary (USP–NF) electronic edition, which yields 3,980 results. Therefore, the scale of the changes required if <621> was to be split (which is required to align with other instrumental related general chapters) could potentially explain why USP has been reluctant to implement the change.

Because of the broad scope of USP <621>, it has undergone many revisions and updates over the years. Between 2002 and 2017, 20 entries related to changes were published in Pharmacopeial Forum (PF). This included successive PF updates; for example, four updates were made in 2002 and five were made in 2004. Additionally, the changes included in the many PF versions of <621> can be difficult to trace into released updates of the general chapter, possibly compounding uncertainty over changes. To be clear, effective August 1st 2014, <621> mapped the “allowable adjustments” that can be made to USP monograph methods without having to re-validate the method. Prior to this, as introduced in 2009, changes to isocratic high performance liquid chromatography (HPLC) methods were only possible to achieve system suitability. Major changes over this time are summarized in Table I.

It is possible that a chapter or monograph goes to PF with changes but they are either not approved or it takes too long for the approval to take place. This means that not every change in PF will necessary become official in the USP-NF. This is why it is difficult to track draft proposals in PF to issued changes to <621>. Most of the changes between 2002–2017 resulted from the Pharmacopoeial Discussion Group (PDG) taking actions to finalize harmonization details.

In 2017, PF43(5), the USP published the Stage 4 Harmonization version of <621>, with it becoming official in the USP-NF on December 1st 2022. With this update, as was previously allowed for LC methods based on isocratic elution, modification to LC methods based on gradient elution could also be made without revalidation, provided that System Suitability requirements are met. Additional verification tests may still be required. Based on stakeholder feedback, the USP proposed postponing a few sections to allow the industry additional time to conduct risk assessment and evaluate the impact of the changes regarding two proposed new SST requirements. Below are examples are two Intent to Revise notices:

• System Suitability Section Aug 26, 2022:
  • The purpose of this revision is to delay the implementation of the sections titled “System Sensitivity’ and “Peak Symmetry” under System Suitability: https://www.uspnf.com/notices-gc-621-nitr-20220826
• System Suitability Section Aug 25, 2023:
  • Based on the comments received from stakeholders, the committee considered it appropriate to introduce a change in the text under the subsections “System sensitivity” and “Peak Symmetry.”
  • This new PF proposal would require an additional step to align the official date for the two sections proposed to be revised with the target official date of the PF proposal, namely changing it from December 1, 2023 to May 1, 2025. Chromatography USP-NF (uspnf.com).

The version of <621> effective May 1st, 2025 includes these postponed changes (1).

Detailed Structure of USP <621>

As USP <621> has evolved, so have the sections contained within it. The new version includes the sections shown in Table II.

Based on the extensive chromatographic theory/explanation included in USP <621>, the different structure becomes more evident when compared with the modernised spectroscopy general chapters. There are now 19 pages for USP <621> compared to just nine pages for USP <857> on UV–vis spectrometry (5).

Interpreting USP <621>

When reading any pharmacopoeial General Chapter, it is important to understand that it does not exist in a vacuum. There is interaction between it, the General Notices (6) (the bit you skip over to get to an analytical general chapter), and the monograph for the substance or product being analyzed, as shown in Figure 3.

The hierarchy means that the general chapter must be followed unless there is a statement in the monograph that overrules the general chapter. You will often read the statement, “unless specified in the monograph,” in a general chapter. Additional information for an analysis is given in the General Notices. For example, if the monograph says, “Weigh about 100 mg,” Section 8.20 of the General Notices states, “‘About’ indicates a quantity within 10% (6).”

New System Suitability Changes in USP <621>

The two changes to SST sections are the inclusion of new requirements for system sensitivity (signal-to-noise ratio) and peak symmetry; the current and new definitions are shown in Table III.

Comparing the old and new definitions can give the impression of slight changes, but like regulations—and USP <621> is a mandatory general chapter—the devil is in the details. Here, the pharmacopoeia says what you must comply with, but there are individual interpretations of how a laboratory or company complies with it.

Let us look in detail at system sensitivity:

• Does a Monograph Specify a Limit? A corollary of Sod’s Law is when all else fails, read the manual, or in this case, the monograph. Does it specify a reporting threshold? If not, don’t measure the signal-to-noise (S/N) ratio. However, do not forget, this is strongly recommended to be established as control strategy for impurities procedures.
• When to Measure (S/N): The first part of interpreting the new definition is to define when this SST requirement must be used. It should be self-evident that if you are measuring S/N, then you will be determining impurities at or near to limits of quantification. Apparently not! Therefore, the first part of the new definition is to make an explicit statement that system sensitivity is when you measure impurities. Consider, if you are measuring an active pharmaceutical ingredient, why on earth would you measure S/N? The main peak will be orders of magnitude above any noise. This is legislation for idiots.
• S/N is an SST parameter: The corollary is that sensitivity is not a test for instrument qualification because S/N is dependent on the analytical procedure used. We will discuss why SSTs are not a substitute for instrument qualification later in this column.
• I nstructions for Measuring S/N: Here comes the interpretation. Your method validation experiments will determine the limit of quantification (LOQ), but not on a day of routine analysis. That is why a point-of-use measurement is essential to ensure the chromatography is fit for its intended purpose on the day and not subject to variances because of the instrument, column, mobile phase preparation, or other factors. Always use the pharmacopoeial reference standard for the measurement, never a sample. However, a laboratory still has to interpret other factors. How many injections? Are we looking at range of concentrations or just one? Should we determine S/N throughout the run, at the start and end, or just at the start?
• Acceptance Criterion: The LOQ is based on a S/N of 10 and is related to the monograph.

Although <621> applies to drug monograph testing, it is not unusual for laboratories to ask if chromatographic calculations performed during analytical instrument qualification, such as S/N, “comply” with USP <621>. The short answer is, of course, yes. This is because the <621> only applies to drug monograph testing.

However, this is an important subject, and at the heart of it is the need to understand the role of the tests performed during instrument qualification life cycle stages and SSTs during routine use. Typically, this question is most often asked in relation to the injection precision tests (see System Repeatability section in <621>). However, it should be noted that injection precision is analytical method-specific, so unless a drug monograph test is performed during an operational qualification (OQ), the OQ test is not designed to meet the stringent performance criteria expressed in <621> and does not need to satisfy this criterion. However, this is where the role of PQ rears its head again!

Historically, for impurity analysis, column-to-column variation meant that one of the best ways to ensure suitable chromatography, sensitivity of the system, and resolution of critical trace impurities eluting in the tail of a larger peak, was to prepare a designated “typical sample” and examine the integration and peak shapes as part of approving the SST of the methodology on the day. Use of inappropriate SSTs using sample injections and without acceptance criteria can find its way into a Warning Letter (7), as well as the requirement that SSTs are supported by validation report (8).

Unfortunately, the evolution of fraudulent data integrity practices also means that it is no longer acceptable to do this because it would be classed as trial injections and testing into compliance (9) (unless the material used was well characterised and could be readily differentiated from a “typical batch”). The FDA has a Q&A on trial injections on their website that will allow evaluation injections under strict criteria only using standard solutions–see Q17 (10).

Additionally, there is a regulatory expectation that failure to meet SST requirements (for example, at the end of a run) will be monitored and investigated because of data integrity and OOS risks. Peak integration and manipulation of SST injections to invalidate a failing run was discussed in an earlier “Questions of Quality” column (11).

Adjustment of Chromatographic Conditions

Changes to <621> over time include evolution of the role of System Suitability for chromatography methods. Used to verify the reproducibility and resolution of the chromatography system, adjustments to analytical method operating parameters could be made to meet system suitability requirements. Historically, for non-monograph testing, there was always a risk that successive changes could result in the analytical method “drifting” outside of its registration and validation limits.

Over time, USP have addressed this subject in <621> through iterations of changes in PF. Originally, for HPLC, only changes could be made to isocratic methods, but the need to address the requirements to be able to transfer methods from traditional columns to more modern columns and ultrahigh-pressure liquid chromatography (UHPLC) systems led to the current situation as defined by clear guidance in the harmonized USP <621> published in 2022 (1).

A summary is shown in Table IV. This shows the maximum variance allowed (unless otherwise directed in the monograph). A change is allowed, provided SST requirements are met, and the selectivity and elution order of the specified impurities to be controlled are demonstrated to be equivalent, though additional verification may be needed. Changes other than those indicated would require revalidation of the procedure. It should be noted that for some parameters, limits must be calculated (based on the equations provided in the chapter). It is worth mentioning that the decision to implement these changes is not made by an individual. A risk assessment needs to be conducted, and various departments and individuals should be involved and held accountable for the decision. Don’t forget that when making changes of this nature, they need to be traceable, controlled, approved, and documented. Changes may impact the overall method performance, which may be identified when results are trended, and you’ll need to identify the possible cause.

USP <621> Frequently Asked Questions

The USP provides a Frequently Asked Questions (FAQ) page for USP <621>, with examples of questions and responses being shown in Table V.

It should be noted that in addition to the provision that changes to monograph methods can be made within the ranges specified, there are some additional provisions which need to be understood (14):

• Multiple adjustments can have a cumulative effect on the performance of the system and must be properly evaluated by users.
• If adjustments are made to a pharmacopeial procedure, additional verification tests may be required.
• Compliance with the system suitability test criteria is required to verify that conditions for satisfactory performance of the test or assay are achieved.
• Adjustment of conditions with gradient elution HPLC is more critical than with isocratic HPLC, since it may shift some peaks to a different step of the gradient, potentially causing partial or complete coelution of adjacent peaks or peak inversion. Thus, this could lead to the incorrect assignment of peaks and to the masking of peaks or a shift, such that elution occurs beyond the prescribed elution time.
• These changes are acceptable, provided that system suitability criteria are fulfilled, and selectivity and elution order of the specified impurities to be controlled are demonstrated to be equivalent.

Therefore, caution is required.

USP <1058> Data Quality Triangle

USP <1058> on Analytical Instrument Qualification contains the Data Quality Triangle (13) consisting of four layers, stating at the bottom:

• Analytical Instrument Qualification (AIQ): This is the foundation of all analytical work and is a major contributor to data quality: do you believe your instrument? AIQ is independent of an analytical procedure and calibrated and traceable test equipment and reference standards. AIQ should enable efficient technology transfer as a chromatograph can be shown to be equivalent of another one.
• Analytical Procedure Validation: Using a qualified instrument, a procedure from sampling to a reportable result is developed and validated. The instrument control parameters for acquisition, processing (especially the peak integration [11, 15, 16]), calculation, and reporting are defined and validated. In addition, the specific SSTs to confirm that the system is ready to analyse are selected while the acceptance criteria are defined and verified.
• System Suitability Tests: To determine if the chromatographic system can analyse samples, an SST or point-of-use check is performed. The FDA’s Out of Specification (OOS) guidance (17) specifically says that if SST injections are outside of their acceptance criteria, then the run can be disregarded. Be careful, as some “enterprising’ chromatographers have used this to invalidate OOS results (11), which generally means that regulators will take a strong interest in runs which fail SST. Additionally, persistent SST failures may result in the performance of the method being challenged, and you may have a bigger problem–defending all the decisions you have made using this method.
• Quality Control Checks: These will not be discussed as the topic is not pertinent.

USP <621> Has No Instrument Qualification Criteria

The problem with USP <621> is that, unlike the technique general chapters, there are no instrument parameters with acceptance criteria to qualify a chromatograph. For a liquid chromatograph, there are no criteria for pump flow accuracy, gradient mixing (either high- or low-pressure), autosampler precision and accuracy, detector accuracy, and more. This is a failing that needs to be rectified in an updated USP <621>.

In contrast, if we look at USP <857> for UV–vis spectrometry, there is a section on Qualification that presents Installation Qualification (IQ) requirements and Operational Qualification (OQ) tests. The latter includes wavelength accuracy, absorbance, and stray light, with acceptance criteria that take up about half of the general chapter (5).

SSTs Are Not Instrument Qualification Tests

The SST and instrument qualification have different purposes. This was covered in some depth in three Questions of Quality columns in 2010–2012 (18-20), and key points are summarised below:

• SSTs are intended to demonstrate adequate performance of the whole analytical system (chromatograph, column, mobile phase, and data system) on the day of analysis.
• SSTs are not instrument qualification tests and are method-specific.
• Acceptance criteria for SSTs are verified and documented in the validation report for each analytical procedure.

You will notice from the data quality triangle that the AIQ and SST layers are separated from each other by the procedure validation because they have different functions. Qualifying an HPLC pump will use a calibrated digital flow meter and a recognised wavelength standard, such as holmium perchlorate solution, to check the wavelength accuracy of the detector. However, there will not necessarily be a check for S/N. This is because S/N is a holistic system check for any impurity analysis LOQ on the day.

Where SSTs can be used, in part, to indirectly confirm the correct operation of a chromatograph is in the Performance Qualification (PQ) phase of the instrument’s life cycle. An example is the use of some SST parameters, such as retention time, to infer that the pump is performing as intended (21).

Summary

This article started out as a review of changes to USP <621> that become official on December 1st 2024 and the ones to become official on May 2025 (such as SST acceptance criteria for sensitivity and peak tailing check). However, as we investigated the subject, it became apparent that a more detailed discussion was required because of the criticality of USP <621> and potential confusion that surrounds appropriate interpretation of this general chapter. Because chromatography tends to be a focus area during regulatory inspections and audits, even this represents a regulatory risk which could potentially involve compliance issues.

Do you fully understand the changes and how they impact your laboratory?

Recent changes to the Definitions section are still relatively new and require careful understanding to ensure compliance. Use of new examples of chromatograms in USP <621> helps make these changes stand out.

A significant proportion of USP <621> covers explanation and theory–which now contrasts strongly with the structure of spectroscopy general chapters. Additional contrast and limitations are evident when Analytical Instrument Qualification requirements are considered for chromatography systems. The idea of splitting USP <621> into specific general chapters for each chromatographic technique is strongly recommended by the authors. Common theory and explanation could still be grouped into an appropriate overarching informational general chapter, so it remains a collective. This would enable focused clarification of instrument requirements and limits for each specific technique and allow inclusion of instrument qualification requirements to align with USP <1058>.

As pharmacopoeial chapters change, it is important that your CDS application is upgraded to incorporate and validate the new changes unless you want to have an entry into the Museum of Analytical Antiquities (22).

Acknowledgments

The authors thank Amanda Guiraldelli Mahr for her comprehensive and thorough reviews of this column.

References

(1) USP General Chapter <621> Chromatography. United States Pharmacopoeia Convention Inc., Rockville, MD.

(2) Burgess, C.; Hammond, J. P. Quality Matters: Modernisation of the spectroscopy General Chapters in the United States Pharmacopoeia (USP). Spectroscopy Europe 2015, 27 (1), 26–29. DOI: 10.1255/sew.2015.a1

(3) Eur. Ph. 2.2.46 Chromatographic Separation Techniques. European Pharmacopoeia: Strasbourg, France.

(4) Williams, R. L. US Pharmacopeia Council of Experts 2005-2010: Work Plans, New Revision Approaches, and Other Enhancements. AAPS J. 2006, 8 (4), E661–E664. DOI: 10.1208/aapsj080475

(5) USP General Chapter <857> Ultra Violet Spectroscopy. United States Pharmacopoeia Convention Inc., Rockville, MD.

(6) USP General Notices and Requirements. United States Pharmacopoeia Convention Inc., Rockville, MD.

(7) US Food and Drug Administration, FDA Warning Letter Grupo Quimico SRL. (FDA, Silver Spring, MD, 2024).

(8) US Food and Drug Administration, FDA Warning Letter Centaur Pharmaceuticals Private Ltd. (FDA, Silver Spring, MD, 2023).

(9) US Food and Drug Administration, FDA Warning Letter Aspire Pharmaceuticals, Inc. (FDA, Silver Spring, MD, 2022).

(10) Questions and Answers on Current Good Manufacturing Practices—Laboratory Controls. FDA 2019. https://www.fda.gov/drugs/guidances-drugs/questions-and-answers-current-good-manufacturing-practice-requirements-laboratory-controls (accessed 2024-08-21)

(11) McDowall, R. D. Ingenious Ways to Manipulate Peak Integration? LCGC International 2024, 1 (2), 20–26. DOI: 10.56530/lcgc.int.lb8988h2

(12) Heering, A.; et al. Improved pH measurement of Mobile Phases in Reversed-Phase Liquid Chromatography. Analyst 2024, 149, 1481–1488. DOI: 10.1039/D3AN02029K

(13) USP General Chapter <1058> Analytical Instrument Qualification. United States Pharmacopoeia Convention Inc.: Rockville, MD.

(14) Taylor, T. Allowable Changes to Chromatography Methods for HPLC – More Freedom for Chromatographers Granted by the Pharmacopeias? MJH Life Sciences 2023. https://www.chromatographyonline.com/view/allowable-changes-to-chromatography-methods-for-hplc-more-freedom-for-chromatographers-granted-by-the-pharmacopoeias- (accessed 2024-08-21)

(15) McDowall, R. D. Where Can I Draw The Line? LCGC Europe 2015, 28 (6), 336–342.

(16) Longden, H.; McDowall, R. D. Can We Continue to Draw the Line? LCGC Europe 2019, 21 (12), 641–651.

(17) US Food and Drug Administration, FDA Guidance for Industry, Investigating Out-of-Specification (OOS) Test Results for Pharmaceutical Production. (FDA, Silver Spring, MD, 2022).

(18) McDowall, R. D. Why System Suitability Tests Are Not A Substitute for Analytical Instrument Qualification or Calibration (Part 1). LCGC Europe 2010, 23 (7), 369–374.

(19) McDowall, R. D. Why System Suitability Tests Are Not A Substitute for Analytical Instrument Qualification or Calibration (Part 2). LCGC Europe 2010, 23 (11), 585–589.

(20) Wätzig, H.; et al. Why System Suitability Tests Are Not a Substitute for Analytical Instrument Qualification (Part 3): Performance Qualification (PQ). LCGC Europe 2011, 24 (8), 418-422.

(21) Smith, P.; McDowall, R. D. Data Integrity and USP <1058>: Part 3: Monitoring and Requalification. LCGC Europe 2019, 32 (1), 28–32.

(22) Smith, P.; McDowall, R. D. Museum of Analytical Antiquities. Technology Networks 2024. In press.

About the Authors

Paul Smith is Director of Compliance2425 Ltd., an independent laboratory compliance consultancy, helping regulated laboratories understand instrument qualification, data integrity and the latest regulatory trends. Before this, he was Global Strategic Compliance Specialist at Agilent Technologies. After 17 years in the pharmaceutical industry, Paul moved into laboratory consultancy work. He is a former spectroscopist, laboratory manager, and university lecturer.

R.D. McDowall is Director at R.D. McDowall Ltd, Bromley, Kent, UK. A company involved in process redesign, the specification, implementation and validation of computerized systems, laboratory digitalization, data integrity assessment and remediation, training in these areas and auditing regulated organisations in the pharmaceutical and allied industries. He is also a member of the LCGC International editorial advisory board (EAB).