SOP for Verification of System Suitability Test

What is System Suitability?

• A system suitability test (SST) is a test to determine the suitability and effectiveness of a chromatographic system prior to use. The performance of any chromatographic system may continuously change during its regular use, which can affect the reliability of the analytical results. 
• The operation parameters of the whole chromatographic system can be checked with properly selected SST mixtures. These mixtures are used to establish characteristic chromatographic parameters, such as the number of effective theoretical plates, resolution, asymmetry, detection limit, and selectivity. 
• The system is then only declared suitable if the responses are within the given limits.
• 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.

System Suitability Test Criteria for Chromatographic Methods

Let’s now discuss this topic in detail with an example of a chromatographic system.

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• Obtaining true and precise chromatographic data is the sign of a well-behaved chromatographic system, e.g. 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. 
• 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. 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:

• 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 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 in 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.

SOP for Verification of System Suitability Test

PROCEDURE

Frequency: Once in 6 months.

• Follow system suitability procedure when system suitability test required in HPLC analytical method.
• Perform a system suitability test of all HPLC instruments prior to use and after completion of testing.
• The results of the different parameters that are included in system suitability should be within the limit as per the respective method of analysis.
• Operate the instrument as per the respective standard operating procedure of HPLC.
• Prepare the mobile phase consisting of 70% methanol: 30% water.
• Prepare a mixture of 0.1 % Benzene and 0.1% Toluene in methanol.
• Set flow rate at 1.0 ml/min.
• Allow the system to be saturated with the mobile phase for at least 15 minutes before injecting the test sample.
• Record the area and retention time of both Benzene and Toluene in methanol.
• Calculate the system suitability parameters with the help of software such as resolution, tailing factor, and theoretical plate.

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• Now calculate the tailing factor, resolution, and theoretical plate manually by the following formula:

Theoretical Plate:

• The efficiency of the column is expressed quantitatively by the number of plates. This can be calculated in various ways using different measures for peak broadening. In addition to the plate number determined, very often the plate number/meter values are also given. 
• In the SST report sometimes the height equivalent to a plate value is also indicated. A number of effective theoretical plates is defined in Eq.

Where 

t = Retention Time of the component

w = width of the component

As per software = _________________                       

Name of software: ___________

By manually (T) = 

= ________________ ( ± 10%)

Resolution (Rs):

• Rs is a measure for the ratio of the distance of two adjacent peak maxima and their widths. For complex sample mixtures, Rs should be determined for the critical pairs of components to characterize their separation. 
• The Rs of two components is calculated with Eq.

Where 

t R1 = Retention Time of 1st component

t R2 = Retention Time of 2nd component

W b1 = Width of 1st components

W b2 = Width of 2nd component

As per software = _________________                       

Name of software: ___________

2 (t R2 – t R1)

By manually: R =_________________      

W b1 + W b2

= _________________ ( +/- 10 %)

Tailing (T): 

• The performance of a column depends not only on the number of plates but also on the shape of the peaks separated. Skewed, asymmetrical peaks may overlap resulting in decreased resolution. The tailing of a peak can have a major impact on the performance of a quantitative method. 
• Depending on the properties of the column, the sample matrix and the analytes, peak asymmetry may vary over the lifetime of the column.
• The Asymmetry of a peak is given by comparing the fronting and tailing end of the peak. The calculation of T is given in Eq.

Where 

a is a front part

b is the back part of the line parallel to baseline at the 10 and 5% of the peak height, for asymmetry and tailing calculations, respectively.

• The performance of the software of the instrument is satisfactory if all the parameters of the system suitability test are within the acceptance criteria.

As per software = _________________                       

Name of software: ___________

By manually (T) = 

= ________________ ( ± 10%)

Remarks: Comply / Does not comply

Acceptance criteria: The difference should be within ± 10 %

ABBREVIATION

% = Percentage

ml = millilitre

REVISION HISTORY

Nil

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