Estimation of RRF for Known Impurities:

The impurity standards are very costly and not available with the required quantities. Hence
pharmaceutical companies tend to use RRF value instead of using impurity standard during related
substances analysis. Relative Response factor (RRF) is used to correct the difference in detector
response of impurities with analyte peak. RRF is established by slope method with linear range of
solutions.

Estimation of RRF: Prepare the standard solutions not less than five preparations having concentration
preferably in the range of 0.1% to 1.0% (e.g. 0.1%, 0.3%, 0.5%, 0.7%, and 1.0%) of test preparation
concentration using drug substance working standard or reference standard. Prepare the individual
impurity standard solutions not less than five preparations having concentration preferably in the range
of 0.1% to 1.0% (e.g. 0.1%, 0.3%, 0.5%, 0.7%, and 1.0%) of test preparation concentration. Inject the
blank solution, resolution solution, or standard solution as per test method and establish the system
suitability parameters. Inject individual impurity solutions in chromatographic system and measure the
area of impurity peak(s). Inject individual standard solutions in chromatographic system and measure
the area of analyte peak(s).
Note: Mixture of drug substance and impurity shall also be injected if the drug substance and impurities
are well separated from each other and one material shall not interfere at other retention time.

Calculate the concentration of solutions for each level for all the known impurities (Ci) in µg/mL as
follows.

Wi × DFi × Pi × 1000

Ci = ----------------------------------------
100

Where,

Weight of impurity standard in mg.

Dilution factor for each level of impurity solution.

Potency of impurity standard.

Calculate the concentration of solution for each level for drug substance (Cs) in µg/mL as follows.

Ws × DFs × Ps × 1000

Cs = ---------------------------------------
100
Where,

Ws = Weight of the drug substance or working standard or reference standard in mg.

DFs = Dilution factor at each level of drug substance standard solution.

Ps = Potency of drug substance standard.

Plot a graph of concentration versus response for impurity and standard solution. Take concentration of
standard solution on “X” axis and response on “Y” axis. Do not include zero" in linearity plot. Determine
the slope of individual linearity plots. Calculate the relative response factor for all the known impurities
using following formula.
Slope of impurity solution curve

RRF of Impurity = ------------------------------------------------------

Slope of standard solutions curve

Verification of RRF by Recovery Study: Conduct recovery studies to prove that the established RRF's are
correct. Prepare an unspiked test preparation, inject into the chromatographic system and record the
chromatograms. Prepare spiked test solutions by spiking the test preparation with impurities preferably
at 0.3% and 0.6% level.

I × Cs × 100

Impurity found in % = --------------------------------

 S × Ct × RRF

OR

I × Cs × 1000

Impurity found in µg =

S × DFt × RRF

Where,

Area of individual known impurity in unspiked/ spiked test preparation.

Area of diluted standard solution of drug substance.

Concentration of diluted standard solution of drug substance.

Concentration of drug substance in unspiked / spiked test preparation.

Dilution factor for test

Calculate the % of impurity added as follows.

Ci × 100

impurity added in % =

Cst

OR

impurity added in µg = Ci × Spiked volume (mL) x 1000

Where,
 Ci = Concentration of impurity added in spiked test preparation.

Cst = Concentration of drug substance in spiked test preparation.

Calculate the % recovery of individual known impurities by using the following formula.
(Rst – Rust) X 100

% of Recovery =

R add

Where,

Rst = % of impurity/µg of impurity found in spiked test preparation.

Rust = % of impurity/µg of impurity found in unspiked test preparation.

R add = % of impurity/µg of impurity added in spiked test preparation.

% Recovery shall be within the range of 90 % to 110 % for each trail. If not, investigate and repeat the
experiment again after omitting the possible errors. If the % recovery is within the range of 90 % to
110%, then RRF's shall be incorporated in calculation of % of known individual impurities.