What are system suitability tests (SST) of analytical methods?
In today’s blog article we will learn about the System Suitability Testing of analytical methods in the pharmaceutical field, its importance in quality control (QC) of drugs and how it differs from the Analytical Instrument Qualification.
The System Suitability Testing (SST) is used to verify that an analytical method was suitable for its intended purpose the day the analysis was done. It is an essential parameter to ensure the quality of the method for correct measurements. An SST is run each time an analysis is performed and each SST is specific to an individual method with pre-defined acceptance criteria for certain parameters e.g. absorbance values being between 0.2 and 1.0 for a photometric content determination method or some resolution factors for chromatographic methods.
System Suitability Testing versus Analytical Instrument Qualification
A notable point to mention here is that SSTs must not be confused with analytical instrument qualification (AIQ). A laboratory should absolutely not skip the SST because of having already an AIQ procedure in place. This is a big mistake as both the United States Pharmacopoeia (USP) as well as the European Pharmacopoeia (Ph. Eur.) have strong recommendations about SST performance (e.g. for chromatographic methods check USP <621> or Ph. Eur. chapter 2.2.46) and FDA warning letters are issued in case of incorrect behavior as can be seen in this example. In case an SST fails, the method can’t be used for the analysis of the corresponding samples, check also USP chapter <1034>: "If an assay (or a run) fails system suitability, the entire assay (or run) is discarded and no results are reported other than that the assay (or run) failed.". Although different, both ensure the quality of obtained results in a QC lab.
AIQ can be regarded as the basis for all analytical procedures. It proves that the instrument is operating as intended by the manufacturer across the operating ranges defined by the lab. It is done initially and later on in regular intervals. Thus it is related to the instrument while an SST is method related. An SST is run each time immediately before or in parallel to the analysis of the samples to be investigated. The basis for the SST working reliably is that the instrument has previously been appropriately qualified and the method has been validated. USP chapter <1058> defines SST as “Verify that the system will perform in accordance with the criteria set forth in the procedure. These tests are performed along with the sample analyses to ensure that the system's performance is acceptable at the time of the test.“. This statement actually questions if the method used on the system is working as expected the day the samples are analyzed.
System suitability test criteria for chromatographic methods
Let’s now discuss this topic in detail with an example of a chromatographic system.
Obtaining true and precise chromatographic data is the sign of a well behaved chromatographic system, like e.g. a HPLC system. There are multiple factors in a chromatogram that can be assessed as a part of the SST if appropriate. Some examples are as follows:
- Precision or injection repeatability: This demonstrates the performance of the system within the defined environment, plumbing conditions and column usage. Unless otherwise stated in a specific monograph, 5 replicates of a standard are used if a relative standard deviation (RSD) of max 2.0% is required and 6 replicates for an RSD >2.0%. A calculation for the maximum permitted RSD is also provided in USP <621>. It should be considered that measured sample values should not differ from the ones of the reference standard for more than the obtained RSD of the reference standard replicate testing described above. In contrast, the Ph. Eur. imposes stricter requirements for repeatability, which is particularly useful for narrow specification limits. The requirements are based on a formula that takes into account both the specification upper limit and the number of replicates to be injected (3 to 6). A maximum repeatability of 1.27% is allowed when B = 3.0 (i.e. a specification upper limit of 103.0%) and 6 replicates are injected.
- Signal-to-noise ratio (S/N): This parameter can be useful as SST.
- Relative retention (r): This is an important tool to have when dealing with impurity determinations. Relative retention measures the relative location of two individual peaks (with one being the compound of interest and the other one the one the corresponding reference).
- Resolution (RS): This parameter is essential for quantitation and measures how well the two peaks are separated by considering their retention times and widths at the bases. This parameter comes handy when dealing with potential interference peaks. A clean separation between the peaks is always desired for quantitation.
- Capacity factor (also known as retention factor k): It is the relation of the amount (or time) of the substance in the stationary phase against the one in the mobile phase. This essentially means the location of the main peak with respect to the void volume. The peaks must be absolutely free from void.
- Tailing factor (also called symmetry factor AS): Peak tailing is a notorious phenomenon and can affect the accuracy estimation of a chromatographic system as peak integration based on where the peak ends could be very challenging. It is essential to determine the location of the upslope and downslope, failing which the accuracy may drop.
Establishment of a SST
If not performed earlier (e.g. during method qualification), the SST criteria are established during method validation. For the establishment of the STT, some useful points should be considered:
- If possible, the sample and the reference standard should be dissolved in mobile phase or in similar amount of organic solvent.
- The concentration of the sample and the reference standard should be comparable.
- When filtering samples, it must be taken into account that apart from removing particulates adhesion of the analyte to the filter might also occur, especially at lower analyte concentrations.
Examples of SSTs of other methods
For an API manufacturer wishing to recombinantly produce its drug substance in E.coli, it is necessary to test the E.coli strains for the presence of the coding plasmid by checking their antibiotic resistance. In this test, the bacteria are plated on media containing antibiotics. SSTs to be carried along may comprise the simultaneous plating of a positive control as well as of a plasmid-free strain as negative control. In order to prove the viability of the plasmid-free strain, it must be incubated in parallel without selection pressure.
The use of a positive control also makes sense as SST in an E.coli identification test using selective chromogenic media. In case of non-existent growth, it detects a lack of quality of the nutrient medium and on the other hand it helps to distinguish between colored and colorless colonies.
For SDS-PAGE, a clear separation of the bands of a molecular size marker carried along in the gel is a common possibility for a SST. Furthermore, it is possible to define where the bands of a reference standard must be located, since their sizes are known. If a quantification is to be carried out for which different concentrations of the reference standard are applied, the coefficient of determination of the linearity determined therefrom can also be a SST criterion.
For photometric protein determination, e.g. several measurements of a reference standard of known concentration are made and the standard deviation of the measured values is not allowed to fluctuate more than a defined value. In addition, for a successful SST, the mean of the measurements of the reference standard could be in a certain range around the known concentration, e.g. ± 5% of the nominal value.
In case of a ready to use ELISA kit e.g. to determine the host cell protein (HCP) concentration, it may be sufficient as SST to check whether the means of the smallest and highest standard are within the manufacturer's specification.
These examples demonstrate how diverse and method specific SSTs can be.
With strong instrument qualification, correct method validation, and strict system suitability criteria, the reliability of the generated data can be ensured. Only with trustworthy validated methods, data that are generated during release and stability testing, are reliable.
Recently, the FDA has also published an answer to the question of the material to be used for SSTs of chromatographic methods. It is expected that high pure primary or secondary reference standards will be used, which were previously qualified against the former reference standard. In addition, they are not allowed to be originated from the same batch as the samples to be tested. Furthermore, written instructions are expected to be complied with and, in terms of data integrity, completeness of the records and their review.
Tags: method validationtest methodssystem suitability testSST
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HPLC chromatography technique is used in pharmaceutical industries for research and testing purposes. Most of the pharmaceutical companies follow a three-step approach to check the functioning of their High performance liquid chromatography systems- Initial System Qualification, Method Validation and Suitability Testing (SST).
System Suitability Test (SST)
Selecting a proper System Suitability Testing mixture is essential to check the specifications of a liquid chromatographic system. System Suitability Testing limits are acceptance criteria that must be met prior to sample analysis. Some of the common chromatographic systems used in pharmaceutical sample analyses are:
- Active ingredient assays
- Impurity determinations
- Dissolution testing
- Retention time
- Column efficiency
- Plate Number
- Tailing factor
- Signal-to-noise ratio
Let us look at some of these parameters.
Resolution:One of the most important parameters. Resolution is used to check whether the critical separation is feasible under the given conditions.
Retention time:Retention time is one of the easy-to-identify parameters. The resolution should be fairly constant. This is because values outside the retention-time window might go unreported by the system under test.
Pressure:The suitability testing must be carried out within set pressure limits. This is to ensure that wearing of system components is reduced.
The column is efficiency-Also known as band broadening.
Repeatability:Successive measurements taken under the same measurement conditions should give the same results.
Plate number:The Plate number is a theoretical plate index(N)used to determine column efficiency. The plate number is arrived at by using the plate theory. Plate number changes depending on the type of analysis carried out.
Tailing factor:This is also called as Symmetry factor. Tailing factor becomes important if the peak tailing has chances of affecting the method’s performance. Similar to Plate number, the Tailing factor also depends upon the type of analysis.
Signal-to-Noise Ratio:S/N ratio is a measure of the system’s performance at the lower end.
It is not enough to perform System Suitability Testing at the beginning of the assay alone. For the purpose of periodic monitoring, it is necessary to do the System Suitability Testing at regular intervals. This is because the performance of the system under test may change due to continuous use.
The System Suitability Tests mentioned in this article are not exhaustive. The System Suitability Tests to be performed are decided by the pharmaceutical scientists or analysts, considering various factors of the system under test. Pharmaceutical scientists use statistical analyses to set realistic System Suitability Testing limits. An optimum system should provide the maximum information out of a minimum number of tests. The System Suitability Tests should be designed such that the requirements are met easily when the system is functioning correctly and fails if there is a problem.
An interesting article from the USP experts group "Small Molecules" has been published in the Pharmacopoeial Forum 39(5). It deals with USP's future requirements regarding system suitability tests (SST).
SSTs are performed each time an analytical method is used. Together with instruments qualification and methods validation, the SST ensures the quality of analytical test results. The SST shows that a procedure and an instrumental system are performing as they did when the procedure was validated and that the method is thus "fit for purpose" for the intended use.
General requirements can be found in the USP Chapter <621> Chromatography which also contains provisions and acceptance criteria for individual parameters of the SST. Nevertheless, parameters and acceptance criteria laid down in specific monographs always take priority over the general provisions provided in General Chapter <621>.
In the article, the USP Expert Committee has noted that because of many different sponsors who have submitted a number of various monographs to the USP, inconsistencies with regard to SSTs in the USP have arisen.
That's why the USP Expert Committee has described in this article which data are required on SSTs for new monographs or monographs to be updated. The following descriptions are provided:
- Content Uniformity
You can find all information on the USP website of the Pharmacopeial Forums (PF).
An interesting article from the USP experts group has been published in the Pharmacopoeial Forum 39(5). It deals with small molecules and describes the requirements regarding system suitability tests (SST) for chromatographic procedures submitted in new or revised monographs.
System suitability tests are performed each time a chromatographic method is used. They thus belong to the scientific working practices in the laboratory beside analytical instruments qualification, and methods validation.
For many years, general acceptance criteria for the SST have been described in USP General Chapter <621>. Nevertheless, parameters and acceptance criteria laid down in specific monographs always take priority over the general provisions provided in General Chapter <621>.
Because at the USP a wide range of proposals are submitted by different sponsors, this leads to apparent inconsistencies in chromatographic system suitability requirements for monographs. Moreover, many SST requirements are now obsolete and no longer the state of the art.
The article thus describes the future expectations regarding the parameters and acceptance criteria for both HPLC and GC methods. The following tests are listed:
- Dissolution and Content Uniformity.
You can find all information on the USP website of the Pharmacopeial Forums (PF).
Source: USP - http://www.usp.org
System suitability usp
Change to read:
The resolution, R, [NOTEAll terms and symbols are defined in the Glossary of Symbols] is a function of column efficiency, N, and is specified to ensure that closely eluting compounds are resolved from each other, to establish the general resolving power of the system, and to ensure that internal standards are resolved from the drug. Column efficiency may be specified also as a system suitability requirement, especially if there is only one peak of interest in the chromatogram; however, it is a less reliable means to ensure resolution than direct measurement. Column efficiency is a measure of peak sharpness, which is important for the detection of trace components.
Replicate injections of a standard preparation used in the assay or other standard solution are compared to ascertain whether requirements for precision are met. Unless otherwise specified in the individual monograph, data from five replicate injections of the analyte are used to calculate the relative standard deviation, SR, if the requirement is 2.0% or less; data from six replicate injections are used if the relative standard deviation requirement is more than 2.0%.
The tailing factor, T, a measure of peak symmetry, is unity for perfectly symmetrical peaks and its value increases as tailing becomes more pronounced (see Figure 2). In some cases, values less than unity may be observed. As peak asymmetry increases, integration, and hence precision, becomes less reliable.
These tests are performed by collecting data from replicate injections of standard or other solutions as specified in the individual monograph. The specification of definitive parameters in a monograph does not preclude the use of other suitable operating conditions (see Procedures under Tests and Assays in the General Notices). Adjustments of operating conditions to meet system suitability requirements may be necessary.
Unless otherwise directed in the monograph, system suitability parameters are determined from the analyte peak.
To ascertain the effectiveness of the final operating system, it should be subjected to suitability testing. Replicate injections of the standard preparation required to demonstrate adequate system precision may be made before the injection of samples or may be interspersed among sample injections. System suitability must be demonstrated throughout the run by injection of an appropriate control preparation at appropriate intervals. The control preparation can be a standard preparation or a solution containing a known amount of analyte and any additional materials useful in the control of the analytical system, such as excipients or impurities. Whenever there is a significant change in equipment or in a critical reagent, suitability testing should be performed before the injection of samples. No sample analysis is acceptable unless the requirements of system suitability have been met. Sample analyses obtained while the system fails requirements are unacceptable.
La USP says to run 5 replicate injections. These injections must to be consecutive or not? I think the SS must to be run at the beginning in order to be sure that the system is OK or the system precision is OK (RSD < 2.0%). But some people says that these injections could not to be consecutive, it means that they can use std injections of the bracket (std's after samples injections) in order to arrive to RSD<2.0%. For example, if in the SS for the 5 first std injections the RSD>2.0%, they can use the other std injections after the samples injections for to arrive RSD<2.0%.
So the question is to do the SS in order to validate the HPLC system before inject the samples or only for to find the precision of the method?
First, USP has harmonized their requirement with Ph Eur with regard to RSD for assays.
Two, FDA has been in favour of bracketing standards/SSTs for at least ten years, USP has been slow here.
Three, have had several auditors including FDA (last one summer 2012), still stuck in the old thinking ie 5-6 injections prior to samples, realizings after some thinking or contacting home office that recommendations has changed.
Four. I would recommend doing at least 3 injections prior to samples to keep an Eye on RSD, assay sequences tends to be long (especially content of uniformity) so to get an indication if you run overnight (not unusual). Bracketing the other ones, one last in sequence.
Five. While harmonized USP is more fuzzy, Ph Eur chapter 2.2.46 is more clear and scientific based on bracketing in general.
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