What Is System Suitability Test (SST) In HPLC And GC Analysis: 11 Minutes Easy Learning

System Suitability Test (SST) is a critical step in ensuring that chromatographic systems – whether HPLC, GC, LCMS, GCMS, or TOC – are operating optimally for sample analysis. Before performing routine sample or standard injections, a system suitability solution is first injected into the system, and the results are evaluated against pre-defined criteria. Only if these criteria are met can further analysis proceed.

In chromatographic techniques like HPLC and GC, SST is more than just a regulatory requirement; it’s a safeguard for data accuracy and system performance. During method development, deciding on appropriate SST criteria can be a challenging task for chromatographers. A small error in this process can result in major failures, especially in industries like pharmaceuticals, where even minor mistakes can lead to costly regulatory issues. It’s not uncommon to see observations, such as the dreaded FDA 483 forms, or deficiency letters, due to improper SST practices.

In this article, I aim to demystify the complexities of SST, drawing from my experience to guide you through the process. I’ll walk you through key concepts such as how to establish SST criteria, the role of system suitability solutions and markers, the acceptance criteria for SST, retention time markers, and common pitfalls to avoid. I’ll also provide real-world case studies and answer some frequently asked questions, giving you the tools you need to confidently determine SST during method development and make your chromatography analysis more reliable.

By the end, you’ll be equipped with the knowledge to not only meet industry standards but also to improve your method development process, ensuring your analyses are accurate, reliable, and regulatory-compliant.

System Suitability Test

The System Suitability Test (SST) is a key procedure that evaluates whether a chromatographic system is capable of producing accurate and reliable results. During method development, several critical parameters are assessed, including the elution order of components, peak shape, resolution, and reproducibility. The SST ensures that the system, comprising the column, detector, and other components, meets predefined criteria to deliver consistent and precise measurements. Key chromatographic parameters that influence system performance, such as retention time, peak symmetry, and signal sensitivity, are carefully considered when establishing the SST acceptance criteria.

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System Suitability Test Parameters

The following chromatographic parameters are used to define the system suitability test. It is denoted by t.

• Retention time (RT): It tells whether the analyte elutes in the chromatogram.
• Relative retention time (RRT): It tells about the relative location of the peak with void volume peak. It is denoted by RRT.
• Resolution (R): It tells about the separation between two adjacent peaks. For example, in HPLC, you want to ensure that peaks are well-separated and don’t overlap.
• Theoretical plate or column efficiency (N). It tells about the narrowness/sharpness of the peak.
• Tailing factor (T): It tells about the symmetry of the peak. This is particularly important for impurity testing and for ensuring accurate quantification. It is calculated by the following formula give in the following chromatogram:

• Signal-to-noise ratio (S/N). It tells about blank interference in the chromatogran and method sensitivity.
• Precision: This could include replicate injections of a standard solution and evaluating the %RSD (Relative Standard Deviation) of the retention time and peak area.
• Detection Limit (DL): It tells about the method’s sensitivity and is very helpful in integrating the peak. It is denoted by DL. It is calculated by the following formula:

• Quantitation Limit (QL): It tells about the method’s sensitivity and is very helpful in integrating the peak and reporting the result. It is denoted by QL and calculated by the following formula:

• Capacity factor (K): The capacity factor is a measure of where the peak of interest is located relative to the void volume peak or unretained peak. It is also called the Retention factor.
  

Selection of Analytes

The analyte used for the system suitability test must have the following characteristics:

• Easily available
• Stable
• Cost effective
• Good UV absorbance or good response
• Good solubility in the common solvents
• Related to the process
• Polarity

Number of components in the System suitability test

The system suitability test must contain at least two chromatographic parameters. Generally, 2 to 5 chromatographic parameters are kept in the system suitability test. The system suitability test solution is prepared by using an individual analyte standard. The SST marker standard is also used to prepare the system suitability solution.

System Suitability Solution

The SST solution is prepared as a method and is injected into the chromatographic system to assess its performance. For instance, in pharmaceutical analysis, the SST solution might contain known amounts of active pharmaceutical ingredients (APIs) and related impurities.

System suitability test marker

System suitability marker contains all the components on which sst is decided.

What is the retention time (RT) marker?

If the process contains impurities of different polarities (polar, intermediate polar and nonpolar ) in that case retention time (RT) marker is used as a system suitability test standard. I personally recommend to use RT marker as an SST standard since this approach is cost effective and it avoids all confusion related to variation in the retention time.

Establishment of system suitability test criteria

• System suitability test may be method specific (e.g. assay method, impurity profile method and content test method)
• System suitability is decided based on the process impurities and their elution pattern.
• If the process contains a close eluting impurity peak, in that case, these close eluting analytes must be included in SST acceptance criteria, and Resolution will be one of the system suitability test parameters
• If the process does not containa close eluting impurity peak. In that case ,system suitability will be decided based on column efficiency (N) and tailing factor (T)

Other chromatographic parameters, like precision and signal-to-noise ratio, can be included in the system suitability test.

System Suitability Test Acceptance Criteria

• There must be base-to-base separation between the peaks. If the analyte chromatogram contains a close eluting peak, in that case resolution (R) will be one of the system suitability acceptance criteria.
• The minimum resolution should be not less than 1.5 between adjacent peaks. If 1.5 resolution is not achieved in that case lower resolution can be considered with suitable justification.
• The general limit of the USP tailing factor or symmetry factor is between 0.8 to 1.8. If USP tailing factor or symmetry factor is not achieved in this range in that case lower or higher value can be considered with suitable justification.
• The acceptance criteria of S/N ratio or signal to noise ratio for sensitivity or QL solutop should be more than or equal to 10
• The limit of theoretical plate, tailing factor, and capacity factor should be decided based on tend data of several batches

The typical example of a reporting pattern

A typical example to report retention time (RT) and relative retention time (RRT)

*Note: Refer to this post to handle variation in RTs and RRTs

7 Best practices of system suitability test

Use the following best practices for the system suitability test :

1. Keep at least two system suitability test criteria in the SST
2. Keep resolution in the system suitability test if two peaks elute close to each other in the chromatogram
3. Does not keep resolution in the system suitability test if two peaks elute far away to each other in the chromatogram
4. Keep either column efficiency (N) or tailing factor in all cases while deciding the system suitability test criteria
5. Keep the Quantitation limit or sensitivity in the related substances, impurity profile and content test
6. Keep precision test in the assay, related substances by the external standard method and content test
7. Don’t keep the quantitation test (QL) or the sensitivity test in the assay test

Regulatory requirements on the System suitability test

Regulatory agencies like the FDA, KFDA, and TGA are more cautious about the system suitability testing. System suitability test must be decided scientifically and based on the trend data from multiple lots. Method must contain at least two system suitability parameters

SST Case Studies

System suitability test in the impurity profile test: A drug substance A contains 3 impurities. Main impurity elutes at 5 minutes, impurity B elutes at 6.5 minutes, impurity C elutes at 10 minutes, and impurity D elutes at 25 minutes. What should be the system suitability criteria?

Since the main analyte A and impurity B elute adjacent to each other. Hence, one of the system suitability parameters will be resolution (R). Column efficiency or tailing factor may be kept second system suitability test parameter. Since this is the impurity profile method and hence Quantitation limit will also be part of the system suitability test and acceptance criteria will be decided based on the S/N ratio. The acceptance criteria of the system suitability will be decided based on the trend data of several lots.

System suitability test in Assay test: In a drug substance X, having assay limit 98 to 100%w/w. Drug substance X elutes at 6 minutes and its impurity A elutes at 30 minutes. What should be system suitability criteria?

Since impurity A peak elutes far away from the drug substance peak X and therefore Resolution can not be kept in the system suitability test. The system suitability can be decided using column efficiency and tailing factor. Precision will also be part of the system suitability

Inadequate system suitability test and its impact

If the system suitability test is inadequate, it may

• impact Quality of pharmaceuticals or drug products
• lead to warning letter from regulatory agencies and
• lead to deficiency letters from regulatory agencies

Conclusion

System Suitability Testing (SST) is crucial for assessing the performance and reliability of chromatographic systems, ensuring that your method delivers accurate and consistent results. Deciding on the appropriate SST criteria requires both a strong foundation of knowledge and practical experience. I hope this article has clarified any uncertainties and empowered you to confidently define SST acceptance criteria for any chromatographic method.

If you have any feedback, questions, or suggestions related to this article, feel free to share them in the comments section below. For further assistance or personalised guidance, don’t hesitate to reach out via the contact form.

You may also want to check out other articles on my blog, such as:

• What is the Analytical Method Validation?
• The 7 Key Differences Between Validation and Verification

FAQs

What is the system Suitability?

The system suitability test tells whether the chromatographic system is suitable or not to give the correct result. SST contains two or more than two analytes. SST acceptance criteria.should be decided using at least two chromatographic parameters

What is the system suitability solution?

The system suitability test solution is prepared by using two or more than two analytes. In some of the method SST marker standard is used to prepare the system suitability solution.

What is the USP limit for the tailing factor?

The USP limit for the tailing factor is between 0.8 to 1.8

What is system suitability test marker?

System suitability marker contains all the analytes which decide system suitability acceptance criteria. It may be prepared in the lab or commercial SST marker can be used (if available)

How many chromatographic parameters are included in the system suitability test?

System suitability test acceptance criteria must contains at least two chromatographic parameters

What happens if the SST fails?

You can explain the troubleshooting steps (e.g., checking the system for leaks, optimising flow rate, or column replacement) while using HPLC

How often should SST be performed?

Is it just during method development, or should it be checked periodically throughout routine analyses?

Can SST be automated?

Many modern instruments come with built-in SST checks.

References

• JP pharmacopeia
• USP pharmacopoeia, chapter <621>
• https://www.fda.gov/media/163682/download
• https://www.pharmaguideline.net/fda-warning-letter-shilpa-medicare-limited/

Abbreviations

• RTs: Retention time
• RRTs: Relative retention time
• GC: Gas chromatography
• HPLC: High pressure liquid chromatograph
• R: Resolution
• N: Theoretical plate
• T: Tailing factor
• RSD: Relative standard deviation
• SST: system suitability test