Relative Response Factor (RRF) in Pharmaceutical Analysis: Learn In 5 Steps

Understanding Relative Response Factor (RRF) in Pharmaceutical Analysis: A Comprehensive Guide

In pharmaceutical analysis, ensuring the quality and purity of Active Pharmaceutical Ingredients (APIs) is of paramount importance. This quality is often determined by analyzing the impurity profile, which requires precise testing methods. However, managing and using impurity standards for every possible contaminant during routine analysis can be costly, time-consuming, and impractical for pharmaceutical companies. So, what is the solution?

The answer lies in the Relative Response Factor (RRF), a crucial tool that allows for the effective calculation of impurities without needing to reference every individual impurity standard. This approach not only saves costs and time but also enhances the reliability and efficiency of pharmaceutical testing.

In this blog post, we will dive into everything you need to know about RRF, its significance in High-Performance Liquid Chromatography (HPLC) and Gas Chromatography (GC) analysis, and how it can be applied in impurity testing. We’ll explore topics such as:

1. What is RRF and how it differs from Response Factor?
2. The importance of Response Factor in pharmaceutical analysis
3. Step-by-step methods for calculating impurities using RRF
4. Real-world case studies and examples
5. Commonly asked questions (FAQs) about RRF and its practical application

By the end of this post, you’ll have a solid understanding of RRF and its vital role in pharmaceutical quality control. Whether you’re new to this concept or looking to refine your skills, this guide will help clarify all your questions and provide valuable insights into how to leverage RRF for more efficient and accurate pharmaceutical analysis.

Relative Response Factor (RRF)

The Relative Response Factor (RRF) is defined as the ratio between the Response Factor of the impurity and the Response Factor of the main analyte standard. It can also be defined as

It can also be written as: RRF is the ratio of the slope of the specified impurities to the slope of the main analyte

RRF is used when comparing the responses of different analytes to a standard. It is useful for calculating the concentration of an impurity in a sample when the impurity and the standard have different detector responses.

Note: See Figure 2 to know more about the Response factor

Related topic: How To Control Impurities In Pharmaceuticals: Get Mastery In ..

Pharmacopeias’ Definition of RRF

RRF Definition as per United States Pharmacopoeia (USP)

The Relative response factor (RRF) is the ratio of the responses of equal amounts of the Impurities and the drug substance. USP refers RRF as Correction factor (CF) or Response factor or Relative response factor. It is denoted as F

RRF Definition as per European Pharmacopoeia (Ph.Eur)

Response Factor, expresses the sensitivity of a detector for a given substance relative to a standard substance. The correction factor is reciprocal of the response factor. Ph.Eur refers RRF as Correction factor or Response factor.

RRF Definition as per British Pharmacopoeia (BP

The Response Factor is a relative term, being the response of equal weights of one substance relative to that of another in the conditions described in the test. BP refers RRF as Response factor.

Response Factor (RF)

The Response Factor is a measure of the sensitivity of a detector to a specific analyte (compound) relative to its concentration. It is calculated by dividing the detector’s response (area response ) by the amount or concentration of the analyte.

RF helps in quantifying the amount of a specific analyte based on the detector’s response. It is generally used when analyzing a single compound and determining its concentration in a sample.

Formula for calculating Response Factor (RF)

Difference Between RF (Response Factor) and RRF (Relative Response Factor) in HPLC

The following are the main differences between RF and RRF

• Basis of Calculation: RF is calculated for a single analyte, whereas RRF compares the RF of two analytes (usually an unknown analyte to a standard).
• Usage: RF is used to quantify the concentration of a single compound, while RRF is used for comparisons between different compounds (analyte vs. standard) to account for differences in detector response.
• Context: RF is typically used when you analyze one specific compound at a time, while RRF is important in multi-compound analysis, where it helps in adjusting for differences in detector sensitivities.
• RF gives the absolute response per unit of analyte, while RRF is a relative comparison, often used to simplify quantification when using an internal standard or comparing multiple analytes.

Example:

• Response Factor (RF): If you are analyzing a single drug in a pharmaceutical, RF will be used to quantify the drug based on its peak area.
• Relative Response Factor (RRF): If you are analyzing the drug and comparing it to an internal standard (say, a known compound with a similar structure), RRF will be used to adjust for the difference in detector responses between the drug and the standard.

Different Methods for Calculating Impurities in Pharmaceuticals

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

1. Area normalization method or area % method
2. Against a diluted sample of the main analyte
3. Against the corresponding impurity standard  or the external standard method
4. Impurity Calculation using RRF

Area normalization 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-3, sample analyte A contain two impurities B and C. Now let us calculate each impurity B and C using all the above three 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 -3 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 that the result obtained by the external standard is correct using all detectors. The result obtained by the Area normalization method and diluted analyte method can only be correct with 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

RRF 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

Impurity Calculation using RRF

The following formula is used to calculate impurity using RRF:

Different Methods for Calculating RRF

The following methods are widely used in the pharmaceutical industries in calculating the RRF:

1. Response Factor (RF) Method or Direct Method
2. Slope Method
3. RRF Calculation using standards (impurity and main analyte) with multiple concentrations
4. Standard addition Method

Note: Methods 1 and 2 are widely used in the industry for RRF calculation

RRF calculation by Response Factor (RF) Method or Direct Method

Steps for Calculation:

• Prepare Impurity Standard Solution: Prepare the concerned impurity standard solution and inject it into the HPLC or GC and generate the chromatogram
• Prepare Analyte Standard Solution: Prepare the Analyte Standard Solution and inject it into the HPLC or GC and generate the chromatogram
• Calculate the RRF (using the following formulae):

Case Study-1:

Let us consider we have to determine the RRF of impurity B against drug substance A:

• Peak area for B = 500 units
• Peak area for A = 2000 units
• Concentration of Impurity B = 20 µg/mL
• Concentration of main analyte A = 10 µg/mL
  • RRF B = (500 ÷ 1000) × (10 ÷ 20) = 0.25

Thus RRF impurity B is 0.25 against Drug substance A .

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

• 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

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 for RRF of impurity A

• Prepare at least 5 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 1500 mcg and hence impurity concentration at the specification level will be 1500 x 0.15/100 = 2.25mcg/ml.
• It means 2.25mcg/ml solution is equal to 0.15%. 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

(Table-2)

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.

Case Study 3

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 – )

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 -3)

(Table 3)

RRF for I is 0.514 and it will be used while calculating this impurity in routine analysis.

RRF Calculation using standard solutions (impurity and main analyte) with multiple concentrations

This method requires preparing a series of standard solutions with known concentrations of the impurity and main analyte. The response factors are determined using these standards. It is similar to the direct method but uses multiple data points to improve accuracy.

RRF Calculation using Standard Addition Method

This is a less common method, and it is widely used in GC analysis. In the standard addition method, a known amount of reference compound is added to the sample, and the response is measured. The RRF is determined by comparing the response from the sample and the added reference.

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.

Relative Response Factor (RRF) Round Off Procedure

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

Note: RRF round off rage may be modified (between 0.9 to 1.1 or between 0.95 to 1.05) depending upon the nature of the impurity and process requirement.

Numerical presentation of RRF

• Q3A (R) guideline indicates that the impurity results should be reported to one decimal place if they are at or above 1.0 and to two decimal places if they are below 1.0.
• 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

Advantages of Relative Response Factor (RRF)

The Relative Response Factor (RRF) is crucial in pharmaceutical analysis to ensure accurate and consistent quantification of impurities in complex pharmaceuticals containing multiple impurities. Its importance can be outlined in several key aspects:

• Accurate Quantification of Impurities: The RRF helps to correct for variations in the detector’s response to different analytes. Since different analytes may not respond equally to the same analytical detector (e.g., UV, HPLC, LC-MS or GC-MS), using an RRF ensures that the response for each compound is normalized. This results in more accurate measurements of the analytes’ concentrations.
• Ensuring Consistency Across Batches: During the manufacturing process or stability testing, RRFs allow for the comparison of results from different batches or formulations. This helps ensure consistency and quality control, making it easier to monitor the stability and effectiveness of the drug over time
• Application in different detection methods: The RRF is crucial when different detection methods or systems are used. For instance, in high-performance liquid chromatography (HPLC), if two analytes are being detected but have different UV absorbances, their RRFs will allow accurate comparison and quantification.
• Regulatory Compliance: Regulatory agencies like the FDA, KFDA, PMDA and EMA often require that pharmaceuticals meet quality control standards. Using RRFs ensures that pharmaceuticals are analyzed with precision, meeting regulatory guidelines for drug quality, safety, and efficacy.

• 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 and lifetime calmness
• Avoid the stability/storage/management issues with standards
• Reduce the analysis/project development cost drastically
• Accepted by all Regulatory agencies

Conclusion

In conclusion, the Relative Response Factor (RRF) is an essential parameter in chromatographic techniques, particularly when calculating impurities in pharmaceutical analysis. A thorough understanding of RRF is crucial for the effective development of analytical methods and ensuring the accuracy of routine analyses. By grasping the role of RRF, analysts can better compare the responses of different compounds, leading to more reliable impurity quantification. I hope this article has provided you with a clear and comprehensive understanding of RRF, and you can now apply this knowledge in your chromatographic method development and routine testing.

If you have any further questions or need clarification on any aspect of this topic, please feel free to leave a comment, and I will address your queries as a priority.

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 the 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 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.

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 normalization 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.

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

References

• (PDF) The use of Relative Response Factors to determine …
• Q 2 (R1) Validation of Analytical Procedures
• USP Guideline for Submitting Requests for Revision to …