What is Pharmaceuticals Assay: Learn In 7 Minutes

Assay is the cornerstone of pharmaceutical development, playing a crucial role in ensuring the quality, safety, and efficacy of pharmaceuticals. The primary objective of an assay is to verify that pharmaceuticals meet the necessary standards of purity, strength, and overall quality before they are made available to consumers. In this article, we’ll explore what an assay is, the steps involved in performing an assay, and the essential precautions that must be taken to ensure accurate and reliable results. Whether you’re new to the topic or looking to deepen your knowledge, this guide will provide valuable insights into the significance of assays in the pharmaceutical industry

Pharmaceuticals Assay

The process of determining the actual concentration of pharmaceuticals (such as APIs, raw materials and finished products) using analytical techniques is called assay.

Analytical techniques used for Assay

The following analytical techniques are widely used to perform assay:

• High-performance liquid chromatography (HPLC): Used to separate, identify, and quantify Pharmaceuticals and their related impurities. This is one of the most used techniques for assay determination in industries.
• UV-visible spectrophotometry: Measures the absorbance of light by a sample to determine concentration.
• Titration: A method where a reagent of known concentration is added to a sample until a reaction is complete, allowing the concentration of the substance to be calculated.
• Gas chromatography (GC): Used for assay determination of the volatile compounds.

Related Topic: How To Control Impurities In Pharmaceuticals: Get Mastery In 

Types of Pharmaceuticals Assay

There are several types of pharmaceutical assays, each serving a different purpose depending on the objective. The following types of assay are widely used in pharmaceuticals development:

1. Titrimetric assay: Different types of titrimetric methods are used to perform such assays
2. Chromatographic assay: Techniques like HPLC and GC are used to perform such assay
3. Mass spectroscopic assay: Techniques like HPLCMS and GC-MS are used to perform such assay
4. Spectroscopic assay: Techniques like FTIR and UV-Visible spectrophotometer are used to perform such assay
5. Biological Assays: Determine the biological activity or potency of a substance using living organisms or cells.
6. Immunoassays: Detect and quantify substances using antigen-antibody reactions
7. Dissolution Testing: Measure the rate at which a drug dissolves in a specific medium

How to Perform Assay?

Performing an assay test by High-Performance Liquid Chromatography (HPLC) and Gas Chromatography (GC) involves multiple steps such as:

• Instruments (such as HPLC and GC)
• Preparation of system suitability solution
• Preparation of Samples and Standards
• Selection of Chromatographic Conditions
• Standard and Sample Injection
• Chromatographic Separation
• Evaluation of System Suitability test acceptance criteria test

Preparation of Samples and Standards

• Sample Preparation:
  • Prepare the sample solution according to the method specification (dissolving in an appropriate solvent or diluting).
  • Ensure the sample is filtered, if necessary, to remove particulates.
• Preparation of Standard Solutions:
  • These standards should cover the concentration range expected in the samples.

Selection of Chromatographic Conditions

• Column Selection:
  • Choose an appropriate HPLC column based on the chemical nature of the compound (e.g., reversed-phase, normal phase, ion-exchange).
• Mobile Phase Selection:
  • Select a suitable mobile phase (solvents) that will provide good separation of the analyte from other components in the sample.
  • The composition, pH, and flow rate of the mobile phase are adjusted to optimize separation.
• Detector Selection:
  • Choose the appropriate detector for the analyte (commonly UV-Vis, fluorescence, or refractive index detectors).
• System Setup:
  • Set the temperature of the column, if necessary.
  • Program the pump for the required flow rate and gradient, if applicable.

Standard and Sample Injection

• Inject the prepared sample and standard solution into the HPLC system and generate the chromatogram. Note down the area response of main analyte from standard and sample chromatograms.

Chromatographic Separation

• Allow the sample to pass through the column with the mobile phase.
• The analyte will separate based on its interaction with the stationary phase and the mobile phase.
• The detector records the separated components as a chromatogram (peaks).

Procedure

• Inject blank, System suitability test solution, standard solution and sample solution. Calculate the assay using the following formula:
• Assay Calculation Formulae:
  • The assay is typically calculated by comparing the area of the sample peak to the area of the standard peak. The following formulae are widely used for assay calculation:

Note: Formulae-1 is used for assay calculation by HPLC method and formulae-2 is used for assay calculation by GC method

Assay Calculation Formula

Evaluation of System Suitability test acceptance criteria test

• Ensure system suitability (check parameters like resolution, tailing factor, etc.) must pass as defined in the STP or Monograph.

11 Characteristics of a Good Pharmaceutical Assay Method

A good assay method should be accurate, precise, sensitive, specific, and practical to use while being cost-effective, robust, and compliant with relevant standards. The following are the 11 key characteristics of any assay method to ensure accuracy, reliability, and practicality:

1. Regulatory Compliance: It must meet relevant regulatory requirements and standards (such as those from the FDA, EMA, or ISO).
2. Ease of Use: A good assay should be user-friendly and not require highly specialized skills or equipment. It should be straightforward to perform, interpret, and analyze.
3. Speed: The method should provide results in a reasonable amount of time, especially in scenarios where quick results are critical, such as clinical diagnostics or quality control.
4. Cost-effectiveness: The assay should be affordable in terms of reagents, equipment, and time. The cost should not be prohibitively high for the intended use, especially when scaling up for larger samples or routine use.
5. Stability: The reagents and the assay itself should be stable over time, ensuring that results remain consistent even after long storage periods, provided the conditions are appropriate.
6. Precision: .It should give precise results under the same conditions and over time
7. Accuracy: The assay method should produce results that are close to the true or accepted value for the substance being measured. It should give correct results consistently
8. Sensitivity: The assay method should be sensitive enough to detect detect analyte as mentioned in the method
9. Specificity: The assay should be selective for the analyte being measured and not be significantly affected by other substances (impurities). This ensures that the results are truly representative of the target analyte.
10. Linearity and Range: A good assay should produce results that are linearly related to the concentration of the analyte within a certain range. This allows for accurate quantification over a wide range of concentrations.
11. Robustness: The assay should remain reliable and consistent even when small changes are made to experimental conditions, such as temperature or reagent lot. This is important for ensuring the method is practical in real-world scenarios.

Advantages

Assays are important for the following reasons:

1. Quality Control: Assays help verify that a pharmaceutical product contains the correct amount of the active ingredient, ensuring consistency between different batches of a drug.
2. Regulatory Compliance: Regulatory bodies, like the FDA (U.S. Food and Drug Administration) or EMA (European Medicines Agency), require pharmaceutical companies to perform assays to confirm that their products meet required specifications.
3. Formulation Development: Assays are also used in the development and optimization of drug formulations to ensure efficacy and safety.

Conclusion

In conclusion, pharmaceutical assays are vital in ensuring the quality, safety, and efficacy of drugs throughout their development and distribution. By rigorously testing for purity, strength, and overall quality, assays serve as the cornerstone for regulatory compliance and consumer safety. As pharmaceutical technologies continue to evolve, so too does the importance of reliable and precise assays. Ultimately, these testing methods not only safeguard public health but also maintain the integrity of the pharmaceutical industry. With this understanding, you can now appreciate the critical role assays play in shaping the medicines we rely on daily.

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

• Analytical Method Development and Validation in Pharma

FAQs

References

Overview of USP General Chapters <476> and <1086>