Relative Response Factor (RRF) and its Calculation in HPLC

Table of Contents

• Introduction and Background
•  Method for calculating impurities
• Result of impurities using different methods
• Method for calculating the RRF
• Relative response factor calculation by Response ratio method
• RRF calculation by slope method
• Factors affecting RRF
• RRF of Isomeric impurities
• Can RRF of unspecified impurities be calculated ?
• Conditions for RRF Considerations
• Advantages of RRF
• Conclusion
• FAQs

Introduction and Background; Relative Response Factor (RRF) and its Calculation in HPLC and GC Analysis

Relative Response Factor (RRF) plays a unique role in pharmaceutical analysis. The quality of Active Pharmaceutical Ingredients (APIs) or their stages depends on their impurity profile or in other words, we can say purity. Impurity standards are required to test the impurity profile of APIs or pharmaceutical products or their stages. However, given the cost, analysis time, and availability of impurities standards, it is impossible for any pharmaceutical company to manage each impurity and use each impurity during routine analysis. Now the question is what is the solution? The only solution is the use of Relative Response Factor (RRF) and that is why I decided to share my skill-based knowledge on “Relative Response Factor (RRF) and its calculation in HPLC and GC analysis“.

In this post, you will learn about RRF (Relative response factor), Response factor, need for response factor, method of calculating impurities, case studies, procedure of calculating response factor and FAQs. This article will clarify all your doubts related to RRF

 Method for calculating impurities

The following techniques are used to calculate the impurities in the impurity profile or related substances test:

• Area normalisation method or area % method
• Against a diluted sample of the main analyte
  • Against corresponding impurity standard  or external standard method

Area normalisation method or area % method

The following formula is used to calculate the value of impurity:

% Impurity = (Area of impurity in the sample chromatogram÷Sum of area of all peaks in the chromatogram)x100

Against diluted sample of the main analyte:

In this method diluted  Analyte standard is used to calculate the impurities

Against corresponding impurity standard/external standard method:

In this method, each impurity is calculated against its corresponding impurity standard.

Result of impurities using different methods

In figure-1, sample analyte A contain two impurities B and C. Now let us calculate each impurity B and C using all three techniques.

Figure- 1 (Value of impurities calculated using different techniques

Result of impurities B and C calculated using different methods

Challenges with the different methods for calculating the impurities

It is clear from the above (figure -1 and table-1) that the results obtained by the area normalization method and the diluted standard methods are almost identical for impurity “C” while the results obtained by the external standard method are different for impurity B.
Now the question is which method is correct out of area normalization, dilute analyte method and external standard method. In other words, which of the above results is correct?
The answer is result obtained by external standard is correct using all detectors. The result obtained by the Area normalisation method and diluted analyte method only be correct with the universal detectors like ELSD, LC-MS. GC-Ms and not by the UV detector.

For UV detector:

• The result depends upon concentration as well as absorbance of the molecules
• The absorbance of a molecule depends upon its structure as well as the wavelength used

As each impurity may have different wavelength maxima and hence, the result obtained by the Area normalisation method and the diluted analyte method is not correct.

The result obtained by the external standard method is correct.
But now the question is, can an external standard method be possible to use every time?

The answer is no because of the following reasons:

• The external standard method needs each individual impurity standard while performing the analysis.
• Most impurity standards are not commercially available.
• Impurities preparation/isolation and characterization is costly as well as time-consuming.
• It increases the cost of the project many folds.
• If the impurity is highly hygroscopic or unstable in that case, it is impossible to preserve and use as an external standard.

Now the question is what is the solution?

To get rid of the above challenges related to calculation by different methods, the Relative Response Factor (RRF) has been introduced. It is defined as the ratio between the Response Factor of impurity and the Response Factor of the main analyte standard :

RRF= Response factor of Impurity/Response factor of the Main analyte

And the following formula is used for the calculation of impurities:

% Impurity = (Area of impurity in the sample chromatogram/Sum of area of all peaks in the chromatogram)x100

RF approach is very helpful and acceptable by all regulatory agencies:

• USP uses F = RRF. It is used in the denominator for the calculation
• PHEur uses CF, where CF = 1/RRF, It is used in the numerator for calculation
• Generally, RRF is used in the denominator for the calculation

RRF Calculation Method

The following methods are widely used in the Pharmaceutical industries for calculating Relative response factor (RRF):

• Response ratio method
• RRF calculation by slope method

Relative response factor calculation by Response ratio method

This is a tentative method for calculating RRF and is generally used during the development stage. Impurity and Analyte standards are prepared at the same concentration and injected into a chromatographic system to get the chromatograms. RRF is calculated by the following formula:

RRF= Response factor of Impurity/Response factor of the Main analyte

RRF calculation by slope method

The relative response factor will be determined by dividing the slope of specified impurities by the slope of the reference substance

Procedure for calculating RRF:

• Prepare at least 5 concentrations from two different stock solutions for each impurity as well as for the main analyte at the concentration range QL (quantitation limit) to ≥ 150%
• Generate the chromatograms for each concentration for each impurity as well as for the main analyte using the test method
• Calculate the slope using  using Excel for each impurity and API/main analyte.
• Calculate the RRF

RRF Calculation Case Study-1:

Let us consider an API which contains impurity “I” with a limit of NMT 0.15%. QL of the method is 0.04%. The sample analysis concentration is 400mcg/ml (See figure – 2)

Now let us discuss the procedure for calculating the RRF of impurity “I“.
Prepare at least 5 concentrations from two stock solutions for each I as well as for the A at the concentration range QL (quantitation limit) to ≥ 150% and calculate RRF (See below table – 2)

RRF for I is 0.034 and it will be used while calculating this impurity in routine analysis.

RRF calculation case study-2

Let us consider an API which contains impurity “A” with a limit of NMT 0.075%. QL of the method is 0.0075%. The sample analysis concentration is 3000mcg/ml (3mg/ml).

Procedure

• Prepare at least solutions from two different stock solutions for each impurity “A” as well as for the API between QL to 150%
• Generate the chromatograms for each concentration for each impurity as well as for the main analyte using the test method
• Calculate the slope using  using Excel for each impurity and API/main analyte.
• Calculate the RRF

Impurity A and API solutions preparation

• The sample concentration is 3000 mcg and hence impurity concentration at the specification level will be 3000 x 0.075/100 = 2.25mcg/ml.
• It means 2.25mcg/ml solution is equal to 0.075%. Now other solutions can be prepared in the following ways:
  • Impurity A concentration QL (10%) will be 2.25 x 10/100 =0.169 mcg/ml
  • Impurity A concentration 25% will be 2.25 x 25/100 =0.56 mcg/ml
  • Impurity A concentration 50% will be 2.25 x 50/100 =1.125mcg/ml
  • Impurity A concentration 75% will be 2.25 x 75/100 =1.69 mcg/ml
  • Impurity A concentration 125% will be 2.25 x 125/100 =2.81 mcg/ml
  • Impurity A concentration 150% will be 2.25 x 150/100 =3,38 mcg/ml

Note: Similarly API solution can also be prepared

RRF of A = Slope of A /Slope of B = (9675.245121/19224.64333) = 0.50

RRF for A is 0.54 and this will be used while calculating this impurity in routine analysis.

Factors affecting RRF

Relative response factor or RRF may change due to a change of:

• Wavelength)
• Column brand
• Column stationary phase particle Size
• Detector ( UV and PDA)
• Solvent grade
• Buffer concentration
• Column temperature
• pH variation
• Structure of the molecule

RRF of Isomeric impurities

RRF of Isomeric impurities may or may not be same.

Case study : ortho Benzaldehyde, meta hydroxy Benzaldehyde and para hydroxy Benzaldehyde are not identical RRF or RRF equal to 1

Can RRF of unspecified impurities be calculated ?

If unspecified impurity is known then RRF must be calculated and considered in impurity calculation. IF unspecified impurity is unknown then RRF can not be calculated.

Conditions for RRF Considerations

RRF between 0.8 and 1.2 should be considered as 1.0 and may not be used for calculation

• The RRF below 0.8 should be expressed with two significant numbers
• The RRF of less than 0.2 or more than 5 should not be used calculation

Advantages of RRF

• Helping in giving fast and exact results of impurities in drug substances, drug intermediates and drug product
• Universally acceptable e.g., by guidelines, by Pharmacopeia
• One-time evaluation job and lifetime calmness
• Avoid the stability/storage/management issues with standards
• Reduce the analysis/project development cost drastically
• Accepted by all Regulatory agencies

Conclusion

Relative response factor or RRF play a vital role in calculating the impurities by chromatographic techniques. and hence clear understanding is required for effective method development. This is all about this article. I hope now all your doubts related to RRF have been cleared and you will be able to use this technique in chromatographic analytical method development and routine analysis. Any question related to this article write in the comment section and i will answer on priority basis.

FAQs

What is the relative response factor?

Relative response factor (RRF) is used to control the impurities by chromatographic methods like HPLC and GC. In this method impurity standard is not used during the analysis and in place of impurity a RRF is used in the calculation.

How to calculate response factor?

There are several methods for calculating the response factor (RRF) in which the slope method is widely used in the industries. To calculate the RRF by this method, slope of impurity is divided by the slope of the main analyte.

RRF = Slope of an impurity/Slope of the API

What is response factor?

Response factor is the 1/RRF

What is RF and what is RRF?

RF is the response factor whereas RRF is the relative response factor.

What is the response ratio method?

In the response ratio method, Impurity and Analyte standards are prepared at the same concentration and injected into a chromatographic system to get the chromatograms. The area response of impurity is divided with the area response of main analyte to calculate the response factor.

What are the challenges in calculating the impurities by different techniques?

Different methods like the area normalisation method, external standard method and internal standard methods are used to calculate the impurities. In most of the cases, each method gives different result and that is why RRF method is used for impurity profile calculation.

What is the difference between Relative response factor and Response factor?

Response factor = 1/RRF

Why Relative response factor and Response factor is required in calculating the impurities?

Different methods like the area normalisation method, external standard method and internal standard methods are used to calculate the impurities. In most of the case each method gives different result and that is why RRF method is used for impurity profile calculation.

What are the factors affecting the Relative response factor?

Several factors like wavelength, buffer concentration, column temperature, column brand and detectors are affecting the RRF.

What are conditions for using the Relative response factor?

RRF between 0.8 and 1.2 should be considered as 1.0 and may not be used for calculation

What are the limitations of Relative response factor?

The RRF of less than 0.2 or more than 5 should not be used calculation

Can isomeric impurities having same RRF?

May or may not be same depending upon the nature of the molecule.

Can RRF of unspecified impurities be calculated?

If structure is known in that case can be calculated and if structure is not known in that case can not be calculated.

What is the difference between RRF and CF?

RRF is the Relative response factor whereas CF is the correlation factor.

CF = 1/RRF

Abbreviations:

• HPLC: High performance liquid chromatography
• GC: Gas chromatography
• RF: Response factor
• RRF: Relative response factor
• MS: Mass spectrophotometer
• ELSD: Electrospray detector
• QL: Quantification limit

References

• USP
• EP
• WHO guideline

Note:

• Some of the data have been designed to explain the topic