System suitability testing

System suitability testing

• 1.
  System suitability testfor HPLC Dr. Vrushali Tambe PES Modern College of Pharmacy (for ladies), Moshi
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  • System suitabilitytests are based on the concept that the equipment, electronics, analytical operations, and samples to be analyzed constitute an integral system that can be evaluated as such. • System suitability test parameters to be established for a particular procedure depend on the type of procedure being evaluated. • Ensures that both methodology and instrumentation are performing within expectations prior to the analysis of the test samples • Used to verify column efficiency and repeatability of chromatographic system and electronics • The performance of any chromatographic system may continuously change during their regular use, which can affect the reliability of the analytical results. Its criteria should be established during the method validation The produced data by the system should be reliable.
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  • Recommended byICH and USP • Used during assay, impurity determination, dissolution testing and bioassay • Performed before and throughout the regulated assays (eg. 5 SSS injections before and one SSS injection every 10 assays) whenever instrument malfunctions. • Assessed with any significant change in equipment or in a critical reagent • It represents minimum acceptance performance 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
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  Constitution of Systemsuitability solution At least the major analyte of interest and, ideally, a closely eluted component or components that could be found in actual samples at known levels Analyte should be sensitive to operating conditions and should indicate slight deterioration of the system Standard solution can be used Single component calibration standard is not adequate for SST as system separation capability is not tested SST solution should consist of main component to be tested and related impurities critical to separate from API
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  Typically, the SSTinvolves performance parameters, such as • Number of Theoretical plates/ Column efficiency • Resolution • Asymmetry/ Tailing factor • Retention time/ Relative retention time/ Retention Volume • Detection limit • Selectivity • Capacity factor Most of the performance parameters are calculated automatically by data evaluation software of sophisticated chromatography equipment The system is then only declared suitable if the responses are within given limits.
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  Resolution (Rs) Measure forthe 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 component to characterize their separation. 𝑅𝑠 = 2 (𝑡𝑅2 − 𝑡𝑅1) 𝑊𝑏1 + 𝑊𝑏2 • where, tR2 and tR1 are the retention times of closely eluted compounds, and • Wb1 and Wb2 are their peak widths at the base.
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  Resolution- • Function ofcolumn efficiency • Measure of resolving power of system • NLT 2 • Most closely eluting species
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  Column efficiency Theoretical plates •The plate theory concept assumes that the chromatographic column comprises a large number of imaginary separation layers called theoretical plates. Equilibrium of the sample takes place between the stationary and the mobile phase in these imaginary plates. The analyte moves down the column by transfer of equilibriated mobile phase from one plate to the next. • Column efficiency is expressed in terms of theoretical plates(N).High resolution means greater number of plates in a given length of column • N=16 (tR/w)2 Where W is the peak at base • Theoretical plates should not fall below 2000
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  Precision • Replicate injectionsof a standard preparation are used to ascertain if requirements of precision are met • Data from five replicate injections are used Relative standard deviation should be less than 1%.
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  Asymmetry or Tailingfactor (As) An ideal chromatographic peak should be of symmetrical Gaussian shape but due to various factors the shape often deviates. Peak tailing is the commonly observed peak deformation. It is mainly due to occurrence of more than one mechanism of analyte retention. Tailing can be reduced by changing mobile phase pH or end-capping of stationary phase. where A and B are peak widths at 10% of the height for leading and tailing ends of the peak Ideal peak has As =1 but values in the range 0.9 – 1.1 are acceptable Tailing becomes apparent when asymmetry factor As equals to or exceeds 1.2 T should be less than or equal to 2 to satisfy the system suitability requirement.
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  Asymmetry is mostsensitive indicator of system contamination
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  Retention factor (k’)/Capacity factor • Retention factor (k’) or partition ratio or capacity factor is the relation of time spent by a compound in stationary phase to the time it spends in the mobile phase. • k’ is a unitless quantity • k′=tr–tm/tm • tr: retention time of analyte • tm: retention time of unretained species • Higher the value of k’ greater is the retention of a compound on a column • Ideally k’ should be greater than 2.0 • It tells about how much interaction analyte have along with stationary phase
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  Retention time: Theresolution should be fairly constant. Retention volume: It is volume of mobile phase required for elution of compound Vr= tR * F Relative retention time (RRT) is the ratio of the retention time of analyte peak relative to that of another used as a reference obtained under identical conditions. Pressure: The suitability testing must be carried out within set pressure limits. This is to ensure that wearing of system components is reduced. Signal-to-Noise Ratio: S/N ratio is a measure of the system's performance at the lower end.
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  For dissolution orrelease profile test methods using an external standard method, k', T and % RSD are minimum recommended system suitability tests. For acceptance, release, stability, or impurities degradation methods using external or internal standards k’, T, Rs, and %RSD are recommended as minimum system suitability testing parameters
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  When SST fails,if possible the assay sequence should be stopped immediately Analyst diagnose the system problem, make necessary adjustments and repairs Perform SST again If SST passes further analysis can be done
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  Reasons for SSTfailure • Poor precision of autosampler • Aged column • Pump problem • Mobile phase preparation error • Air bubbles • SST is early warning to reduce sample retesting
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  Thank You…