Robustness for Assay by HPLC

1.0 OBJECTIVE:

To lay down the procedure for performing Robustness for Assay by HPLC

2.0 SCOPE:

This SOP is applicable for for performing Robustness for Assay by HPLC in company Name.

3.0 BACKGROUND:

NIL

4.0 RESPONSIBILITY:

4.1 QC-Chemist

4.2 QC- Executive

4.4 QC- Head

PROCEDURE:

5.1 ICH defines the robustness of an analytical procedure as a measure of its capacity to remain unaffected by small, but deliberate variations in method parameters. It provides an indication of the procedure’s reliability during normal usage.

5.2 Robustness tests examine the effect that operational parameters have on the analysis results. For the determination of a method’s robustness, a number of method parameters, such as pH, flow rate, column temperature, injection volume, detection wavelength or mobile phase composition, are varied within a realistic range, and the quantitative influence of the variables is determined. If the influence of the parameter is within a previously specified tolerance, the parameter is said to be within the method’s robustness range.

5.3 In the case of liquid chromatography, examples of typical variations are:

- influence of variations of pH in a mobile phase;

- influence of variations in mobile phase composition;

- column temperature;

- flow rate.

- detector wavelength

In the case of gas-chromatography, examples of typical variations are:

- temperature

- flow rate.

5.4 Experimental determination of Robustness

5.4.1 Mobile Phase/Buffer pH Variation:

Increase and decrease 0.10 units of pH of Mobile phase or Buffer

5.4.2 Mobile Phase Flow-Rate Variation:

Increase and decrease 10% flow rate of a mobile phase into the chromatographic system

5.4.3 Mobile Phase composition:

Decrease and increase in 10% of components. Example if the mobile phase contains two solvents then change the concentration to 90% of one solvent and 110% of the other and next time change first solvent to 110% and second to 90%.

5.4.4 Detector wavelength:

Change ± 2nm of the detector wavelength of the chromatographic system.

5.4.5 Temperature of a column:

± 2° of chromatographic column temperature

For evaluating above said changes in the method parameters, some follow case by case study and where as others follow pooling conditions study.

5.5 Pooling of Conditions:

5.5.1 Preparation I: -0.10 pH units of buffer, decrease in 10% flow rate, decrease in 2°C in column temperature, and first condition of mobile phase composition ( 90% of first component and 110% of second component)

5.5.2 Preparation II: +0.10 pH units of buffer, increase in 10% flow rate, increase in 2°C in column temperature, and second condition of mobile phase composition ( 110% of first component and 90% of second component)

5.5.3 Case by case study:

In this case analysts separately study each condition. For example for studying the effect of pH increase or decrease, prepare test solutions separately for each modification and for change in column temperature study this separately. This is laborious processes.

5.5.4 Chromatographic injections for Assay method:

This set of injections are applicable for pooling studies and case by case studies.

Name of the Preparation No of Injections

Diluent as Blank 1

Standard Preparation 5

Test Preparation-I 1

Test Preparation-II 1

Standard Preparation 1

5.5.5 Acceptance criteria for robustness testing for assay of single analyte:

The %RSD for the area of five replicate injections due to the major peak obtained from standard solution should not be more than 2.0.

Theoretical plates for the major peak from the first injection of standard solution should not be less than 3000.

Tailing Factor for the Major peak peak from the first injection of standard solution should not be more than 2.0.