Related Substances (RS) Calculation in HPLC

Related substances of a pharmaceutical product refer to any impurities, degradation products, or contaminants present in a drug substance or drug product. These substances can arise from the synthesis or manufacturing process, storage conditions, or the natural degradation of the active pharmaceutical ingredient (API).

When it comes to calculate the % related substances or impurity by area normalization one can do it by taking the individual area of the impurity divided by the total area and multiply that by 100 to get the % of impurity.

Area Normalization = (Impurity area/Total Area) × 100%

There are mostly two formula for RS calculation in HPLC:

1) Calculating known impurity Level,

2) Calculating unknown impurity level.

% of known impurity = (Ri /Rs) x 100 x RF

% of unknown impurity = (Ri /Rs) x 100


  • Ri = Area of each impurity Peak in the chromatogram of the sample solution,
  • Rs = Sum of areas of drug product and all impurity Peaks in the chromatogram of the sample solution,
  • RF = Response Factor
  • Total Impurities = Sum of known impurities + Sum of unknown impurities.

Calculation of percentage of related substances as follows:

Percent individual related substance by HPLC = (Ai × 100) / At

Percent total related substances by HPLC = (A × 100) / At


  • Ai = Area of the individual related substance peak;
  • A = the sum of areas of all peaks minus the area due to the API peak and solvent front peak; and
  • At = the sum of areas of all peaks in the chromatogram excluding the solvent peak.

But it should be developed an external standard methods for RS by well establishing the relative response factors (RRF’s) for each known impurities may it be single or multiple drug combination. This approach with proper justifications and required validations is well accepted by regulatory authorities.

Relative response factor is the ratio of the response of the impurity and active pharmaceutical ingredient (API) under the identical chromatographic condition (chromatographic column, temperature, mobile phase, flow rate etc.).

Whereas major RRF difference is there for impurities then also Area normalization can apply with RRF.

Area normalization with RRF = (Impurity Area/Total Area)*(1/RRF)*100


  • Total Area=Main peak area + (Impurity Area/Impurity RRF) + sum of all unspecified impurities

The presence of related substances in a pharmaceutical product can affect the quality, safety, and efficacy of the product, and it is important to monitor and control their levels.

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