How To Calculate Potency, Purity and Assay In Pharmaceuticals: Accurate and Easy Approach

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Potency, Purity, and Assay: Core Concepts in Pharmaceutical Analysis

Potency is the exact quantitative content of a pharmaceutical. It is applicable to standards and used for assay, purity and impurity profile calculation. It refers to the strength of the pharmaceutical. It is the absolute value.

Potency, purity, and assay are fundamental yet often misunderstood terms in pharmaceutical analysis. They play a critical role in drug development and quality control. Whether it’s qualitative analysis, such as pharmaceutical identification using techniques like HPLC, GC, UV, FTIR, GC-MS, or LC-MS, or quantitative analysis, including assays and content testing, these parameters are always at the forefront, with standards guiding every step.

Despite their widespread use, many chemists face confusion when it comes to correctly understanding and applying these concepts. That’s why I’ve decided to share my practical, experience-based insights into potency, purity, and assay.

In this article, I’ll discuss:

• What each term really means?
• The differences between potency, purity, and assay
• How to calculate each of them correctly
• Real-world case studies
• And answers to frequently asked questions (FAQs)

Whether you’re a student, analyst, or industry professional, this guide will help you gain clarity and confidence in handling these crucial pharmaceutical parameters.

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

Potency, Purity and Assay

Potency

Potency is the exact quantitative content of a pharmaceutical. It is only applicable to standards and used for assay, purity and impurity profile calculation. It refers to the strength of the pharmaceutical. It is the absolute value.

The highest pure material is used to calculate the potency. All types of impurities, like organic impurities, inorganic impurities, residual solvents, counter ions, loss on drying or water content, are taken into consideration for potency calculation.

Purity of Pharmaceuticals

Purity is the qualitative content of any pharmaceutical. It represents how free a pharmaceutical is from impurities.

• Example: If a compound is 99.5% pure, it contains 0.5% impurities.

Purity typically refers to chromatographic purity and is commonly expressed as peak area percentage (area%). It represents the proportion of the main pharmaceutical/analyte peak area relative to the total area of all detected peaks in a chromatogram, as determined by high-performance liquid chromatography (HPLC) or gas chromatography (GC)

It can also be calculated by other analytical techniques like chemicals and spectroscopic techniques. Chemicals and spectroscopic methods of calculating the purity are not specific and selective and that is the reason these methods are rarely used for calculating the purity. But chromatographic methods are specific and selective and that is the reason cinematographic methods are widely used for calculating the purity in Pharmaceutical industries.

Assay of Pharmaceuticals

The Assay is the quantitative content of a pharmaceutical. It is a relative value and it is calculated against its corresponding standard while using the chromatographic technique or spectroscopic technique. The potency of the standard is required in the assay calculation while using the chromatographic technique or spectroscopic technique.

The Assay can also be calculated using the chemical titration technique and in this technique, there is no need of any external standard. Since the chemical technique is not specific and that is why it is rarely used in the pharmaceutical industries.

Related Video:

How to Calculate Potency of pharmaceuticals?

The highest pure material is used to calculate the potency. All types of impurities, like organic impurities, inorganic impurities, residual solvents, counter ions, loss on drying or water content, are taken into consideration for potency calculation.

It is typically determined using a mass balance approach and is reported as the percentage ratio of the weight of the main pharmaceutical/analyte to the total weight of the drug substance (wt/wt %). This calculation accounts for all impurities present in the drug substance, including residual solvents, water content, related substances, and inorganic impurities, providing a true measure of the actual content and quality of the pharmaceutical.

Potency Calculation formula

Potency is calculated from purity in chromatography methods like HPLC and GC using the following formula-1 and formula -2:

Formula-1 and formula -2:

Note:

1. Formula-1 is widely used in the industry for potency calculation both by HPLC and GC methods
2. LOD (loss on drying or water test is decided based on the synthetic process or pharmaceutical test specification
3. When LOD is performed then there is no need to perform correction of residual solvents and water in potency calculation
4. Sulphated ash/residue on ignition test tells about content metal impurities

Case study: How to calculate potency from purity?

If a drug substance has an impurity A is 0.15%, LOD is 1.0%, and Sulphated ash is 0.1%. Calculate the potency

Using Formula-1 (purity: 99.85%, Sulphated ash 0.10% and LOD 1.0%)

• Potency = 99.85 x {100-(LOD + Sulphated ash)}/100 = 99.85 x {100 – (1 + 0.1)}/100 = 98.35%

Potency of drug substances that exist in salt form

For drug substances that exist in salt form, potency can be reported in two ways:

1. Freebase potency – which reflects only the mass of the active moiety (API) and excludes the mass of the counter-ion. For example, Ranitidine HCl, Rosuvastatin calcium etc.
2. Salt form potency – which includes the mass of the counte-rion, representing the total mass of the salt.

The choice between these depends on regulatory requirements and nature of the pharmaceutical.

Analytical Methods for Potency Calculation

The following analytical techniques are widely used in the pharmaceutical industries in for potency calculations:

1. Potency Calculation by HPLC Method
2. Potency Calculation by GC Method
3. Potency Calculation by Titrimetric Method

Procedure for Potency Calculation using HPLC Method

1. Take the purest pharmaceutical and make it ultra pure using different purification methods
2. Use existing validated related substances/purity HPLC method.
3. If existing method is not available then develop a new method and validate the same prior to potency calculation
4. Prepare the sample at assay concentration. If assay concentration is not suitable then pharmaceutical concentration can be increased
5. Prepare system suitability solution, QL/sensitivity solution and sample solution
6. Equlibriate the HPLC system as pert STP(standard test procedure)/Monograph
7. Inject the blank/diluent, system suitability test (SST), QL/sensitivity solution and sample solution
8. Check the system suitability acceptance criteria
9. Calculate the purity (using for Formula-3 or Formula-4)
10. Calculate the potency (using for Formula-1 or Formula-2)
11. Report the potency
12. Prepare the certificate of analysis (COA) and take approval of all concerned persons

Procedure for Potency Calculation by GC Method

1. Take the purest pharmaceutical and make it ultra pure using different purification methods
2. Use existing validated related substances/purity GC method.
3. If existing method is not available then develop a new method and validate the same prior to potency calculation
4. Prepare the sample at assay concentration. If assay concentration is not suitable then pharmaceutical concentration can be increased
5. Prepare system suitability solution, QL/sensitivity solution and sample solution
6. Condition the GC system as pert STP (standard test procedure)/Monograph
7. Inject the blank/diluent, system suitability test (SST), QL/sensitivity solution and sample solution
8. Check the system suitability acceptance criteria
9. Calculate the purity (using for Formula-3 or Formula-4)
10. Calculate the potency (using for Formula-1 or Formula-2)
11. Report the potency
12. Prepare the certificate of analysis and take approval of all concerned persons

Procedure for Potency Calculation by Titrimetric Method

• Take the purest pharmaceutical and make it ultra pure using different purification methods
• Perform the assay using validated method
• Assay on as is basis may be considered as a potency in Titrimetric Method

Note: Titrimetric Method is not specific and selective and that is the reason it is rarely used for potency calculation

How to Calculate Purity of Pharmaceuticals?

Purity can be calculated using chromatographic techniques like high-pressure liquid chromatography , Gas chromatograph etc as per method requirements. The following methods are used in the industries for calculating the purity:

1. Area normalisation method and
2. External standard method

Purity calculation formula: Area normalisation method or area% method

Calculate the percentage purity by the following formula (Formula-3) by area normalisation method or area% method:

Formula-3:

Note: Impurities peaks having value ≥QL( Quantitation limit) are considered in purity calculation

Purity calculation formula: External standard method

In this method first each impurity is calculated against either diluted standard (see external standard assay calculation formula-4 in assay section) of main analyte or against their corresponding impurity (as per method) and then purity is calculated using following formula:

Formula-4

Purity= (100 – sum of all impurities* present in the chromatogram)

Note*:

• Only those impurities having value ≥ Quantitation limit or QL are considered in purity calculation
• Purity is applicable for both standard and samples

Case study: purity Calculation

If a drug substance has an impurity A is 0.15%. calculate purity

Using Formula-3

• Purity = 100-0.15=99.85%

Using Formula-2

If the area response of impurity A of a drug substance is 7500 mAU and the area response of the main analyte is 50000 mAU; then calculate the purity

% Purity = Area response of main analyte/Sum of area of Impurity I and Main Analyte = 500000/50750 = 99.85%

How to Calculate Assay?

Assay by chromatographic techniques (HPLC and GC) is a widely used for quantifying the amount of main pharmaceutical/Analyte in a pharmaceutical. The measurement is made by comparing the main analyte/pharmaceutical peak area response in the sample to that of a reference standard with a known potency. The assay result is calculated using the sample response factor and standard response factor and potency.

Assay results can be reported in two ways:

• As-is basis: The result reflects the API content without correcting for the presence of water and residual solvents.
• Anhydrous and solvent-free basis: The result is adjusted by subtracting the mass of water and volatile impurities, providing a more accurate representation of the pure API content.

The External standard method and Internal standard method are used for assay calculation by chromatographic methods like HPLC and GC

Assay Calculation Formula: External standard and Internal standard methods

Formula-4:

Difference between Purity, Potency and Assay

Difference between Potency and Assay: Difference between Potency and purity

Note:

• Potency is more about the effectiveness or strength.
• Purity focuses on how clean or uncontaminated the compound is.
• Assay is about measuring what’s actually there in a defined amount.

Factors Affecting Potency

The following factors affect the potency:

1. Chromatographic Purity: Potency is directly proportional to chromatographic purity. Higher the chromatographic purity better (higher) is the potency.
2. Impurities: Potency is inversely proportional to impurities . Higher the impurities lower is the potency
3. LOD (Loss on drying): Potency is inversely proportional to LOD.
4. Water content: Potency is inversely proportional to Water content. Higher is the water content lower is the potency.
5. Residual solvents: Potency is inversely proportional to Residual solvents. Higher is the Residual solvents lower is the potency.
6. Sulphated ash: Potency is inversely proportional to Sulphated ash. Higher is the Sulphated ash lower is the potency.
7. Storage condition: Temperature, humidity etc may affect the drug standard and hence the standard should be maintained as recommended

Impact of potency on pharmaceutical analysis

Pharmaceutical analysis like Assay, Related substances by external method, Content test and Residual solvent tests depend on the use of a reference standard with accurately assigned potency, typically determined through a mass balance approach. The reliability of these tests are closely tied to the precision and accuracy of the analytical method employed. If the potency of the reference standard is inaccurate, this error will directly impact the accuracy of the assay results. Additionally, using different reference standards or employing varying methods to determine the purity of the reference standard can lead to inconsistent assay results, even when analyzing the same drug substance.

Potency plays a vital role in:

• Ensuring Product Safety and Efficacy
• Fulfilling the Regulatory Standards
• Supporting Pharmaceutical development
• Ensuring Batch-to-Batch Consistency

Precautions required while calculating the potency

• Use the validated analytical method for potency determination
• Sample concentration must be within the linear range, and each impurity should be detected
• Temperature, humidity etc may affect the drug standard and hence the standard should be maintained as recommended. Secondly standard must be re-qualified before the retest period.

How to calculate potency from Assay?

Assay on as is basis of highest pure material can be considered as a potency

Applications

• Purity is applicable for both standard and sample and it is used to calculate the potency of the standard.
• Potency is related to only standard and it is used to calculate assay, related substances, content test and residual solvent tests
• The assay is the relative value and it is calculated using the potency of the standard

Regulatory guidelines related to Potency, Purity and Assay

Regulatory guidelines for potency, purity, and assay are critical in pharmaceutical development and manufacturing. These parameters ensure that a drug product is safe, effective, and of consistent quality.

Unit of Potency, Purity and assay

The following units are widely used for potency:

1. %W/W (Weight/Weight

• It is widely used for APIs (Active pharmaceutical ingredients) and their stages
• Example: Potency of Paracetamol standard 99.7%w/w

2.0 Milligrams (mg), Micrograms (mcg/µg), or Grams (g)

• Most common for small molecule drugs.

3.0 International Units (IU)

• Used for vitamins, hormones, vaccines, and biologics.
• Represents biological activity rather than mass.
• Example: Insulin 100 IU/mL or Vitamin D 1000 IU

4.0 Units (U)

• Used for enzymes, heparin, some antibiotics (e.g., penicillin).
• Represents activity based on a standard reference.
• Example: Heparin 5000 units/mL

5.0 Activity Units (AU)

• Often used with enzymes or allergens in allergy extracts.
• Similar to IU but not always internationally standardized.
• Example: Lactase enzyme 9000 FCC units

6.0 Equivalents (Eq, mEq, µEq)

• Used for electrolytes or ion-exchange resins.
• Based on chemical activity (molar equivalents).
• Example: Potassium chloride 10 mEq

Note:

• Purity is represented as area % or %w/w
• Assay is represented %w/w, Micrograms and International Units (IU)

Conclusion

Purity, potency, and assay are closely interconnected and fundamental concepts in the pharmaceutical industry. They play a crucial role in ensuring the safety, efficacy, and quality of drug products. Understanding these parameters is essential for compliance with regulatory standards and for maintaining product integrity throughout its life-cycle.

If you have any questions or need further clarification on any of these topics, feel free to leave a comment below. I’ll be happy to respond and assist you as a priority!

You may also want to check out other articles on my blog, such as:

1. How to control Pharmaceutical Impurities
2. How To Calculate Solubility BY HPLC: Learn Easily in 11 Minutes
3. Biopharmaceutics Classification System (BCS): How Helpful in Drug Development
4. ppm, mcg and percentage (%) In Pharmaceutical Analysis: 5 Minutes Quick Learning
5. How to Calculate pKa: Top 3 Simple Techniques
6. Specified and Unspecified Impurities In APIs: How To Set Limits
7. Control of Nitrosamine Impurities in Pharmaceuticals

FAQs: Interview questions on potency, purity and assay

How to calculate potency from an assay?

Potency is not calculated from assay, but the assay is calculated from potency. Potency is calculated from purity.

What does purity and potency mean?

Both purity and potency tell about the exactness of the pharmaceutical. Purity is the qualitative value whereas potency is the quantitative value. Secondly, potency is only applicable for standard whereas purity is applicable for both standard and sample.

What are the differences between purity, potency and assay?

Purity, potency and assay tell about the exactness of the pharmaceutical. Purity is the qualitative value whereas potency and assay are the quantitative value. Secondly, potency is only applicable to standard whereas purity is applicable to both standard and sample. Assay is applicable to sample.

What is the formula for assay calculation?

Assay (%w/w) = (Test area ÷ Standard area) × (Standard weight. ÷Test weight) × (Test Volume ÷ volume) x Potency

What is the formula for percentage purity?

Percentage purity is calculated by normalisation method and external standard method. The following formulae is used to calculate the purity:

Purity= (100 – sum of all impurities* present in the chromatogram)

What is the potency pharmaceutical analysis?

Potency is the exact quantitative content of a drug substance or its stages and It is only applicable to standard.

How to calculate purity from potency?

Potency = Purity – (Sulphated ash/residue of ignition + Loss on drying/water content/residual solvent + counter ion) etc..) and hence Purity = Potency + (Sulphated ash/residue of ignition + Loss on drying/water content/residual solvent + counter ion) etc..)

What is the difference between assay by HPLC and purity by HPLC?

HPLC purity is the qualitative value whereas the HPLC assay is the quantitative value. Secondly, HPLC purity is the absolute value whereas HPLC assay is the relative value.

How assay is calculated?

Assay (%w/w) = (Test area ÷ Standard area) × (Standard weight. ÷Test weight) × (Test Volume ÷ volume) x Potency

How potency is related to Purity?

Potency = Purity – (Sulphated ash/residue of ignition + Loss on drying/water content/residual solvent + counter ion) etc..)

How assay is related to Potency?

Assay (%w/w) = (Test area ÷ Standard area) × (Standard weight. ÷Test weight) × (Test Volume ÷ volume) x Potency

Can a compound be 100% pure and still have low potency?

Yes. A sample might be pure but contain less than the expected API amount.

Is potency the same as assay?

Not exactly. Potency is more related to strength (often from formulation), while assay is a lab measurement of API content.

Why is purity important if the drug is potent?

Impurities may be toxic or reduce the drug’s shelf life, even if the API is effective.

Abbreviations

• ROI: Residue on ignition
• LOD: Loss on drying
• GC: Gas chromatography
• HPLC: High pressure liquid chromatography
• MS: Mass spectroscopy
• UV: Ultra violet spectroscopy
• FTIR: Fourier transform interferometer
• QL: Quantitation limit

Further Reading:

• 21 CFR 211.165(a) (FDA)
• ICH Q6A: Recommends establishing potency as a critical quality attribute (CQA) for biologics and chemical drugs.
• USP <1032>, <1033>, <1034>: Provide detailed methods and validation criteria for biological assays.
• ICH Q3A/B/C: Define thresholds and identification requirements for impurities in new drug substances and products
• Q3A: Impurities in drug substances (organic/inorganic).
• Q3B: Impurities in drug products.
• Q3C: Residual solvents..
• 21 CFR 211.160 and 211.165
• USP <1086>: Provides guidance on impurities in pharmaceutical
• ICH Q2(R2)
• What is difference between potency, purity and assay?