What is Assay and How to Perform?

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Assay plays an important role in pharmaceutical development in managing the quality, safety and efficacy of the pharmaceuticals. The goal of an assay is to ensure that the drug meets the required standards of purity, strength, and quality before it reaches consumers.In this article you will know This article describes what assay is, how assay is performed and what precautions should be taken while doing assay

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• What is Pharmaceuticals Assay: Learn In 7 Minutes
• Potency, Purity and Assay in pharmaceuticals analysis

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 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.

Steps of HPLC and GC 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

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.

Advantages of Assay

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

Assay play a unique role in pharmaceutical development. I hope this article has helped you understand how to perform assay and its advantages. You may also want to check out other articles on my blog such as:

• GLP (Good Laboratory Practice) | How To Implement
• Analytical Method Development and Validation in Pharma

FAQs

References

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