A pharmaceutical's purity, assay, and potency are terms commonly used when discussing drug quality testing. Due to its accuracy and efficiency, High-Performance Liquid Chromatography (HPLC) is commonly used to assess purity, assay, and potency. This blog is part of our primer series on separation science. It provides a simple overview of these key terms and explains how liquid chromatography can help measure them.
HPLC tracks the increase in percentage of purity and assay during the purification process using preparative HPLC. For instance, consider the purification of the peptide pharmaceutical glucagon-like peptide-1 (GLP-1) analogs, such as Liraglutide or Semaglutide. The initial crude product, obtained through solid-phase peptide synthesis, undergoes a series of purification cycles using reverse-phase liquid chromatography. Before the purification step, when you perform analytical HPLC on this crude sample, you may determine the purity to be 40% and the assay to be 28%.
After that, you load the crude sample into your preparative chromatography column and run it through the system. This process constitutes one purification cycle. As a result, the purity has increased by 80%, and the assay is at 70% (see this patent [1] as an example). But why are the purity values higher (40% and 80%) and the assay values lower (28% and 70%)?
Purity generally refers to chromatographic purity, the percentage ratio of a pharmaceutical's peak area to the total sum of all peak areas in an HPLC chromatogram.
For example, if you take 10 milligrams (mg) of crude GLP-1 analog, dissolve it in 10 milliliters (mL) of water, and inject it into the column. For simplicity, we have only one impurity peak (similar to Figure 1 ) and another peptide by-product (an impurity peak). Does purity of 80% mean 10 mg of the crude sample contains 8 milligrams of pure GLP-1 sample and 2 milligrams of the peptide by-product impurity?
The answer is no. The 10 mg crude sample also contains compounds present in the crude that may not be detected by the HPLC detector. Analytical HPLC methods do not necessarily identify all the components present. For instance, the UV detector commonly used in most HPLCs might not detect all compounds involved in solid-phase peptide synthesis. These undetected compounds could include:
Residual organic solvents and moisture. These trace amounts of organic solvents or water remain in the dried crude sample.
Residue on ignition (ROI). The ROI describes the total concentration of inorganic salts and trace metals that fail to ignite at 600 °C.
Counter ions. The charge on the pharmaceutical is paired with small molecules or ions of opposite charge to change important characteristics like solubility and stability.
In a sample weighing 10 mg of crude material, the composition can be outlined as follows:
- The amount of pure GLP-1 is represented by x mg.
- The amount of impurities, specifically peptide by-products, is represented by y mg.
- The amount of other compounds, which may include residual organic solvents, moisture, ignition residues, and counter ions, is represented by z mg.
Thus, the relationship can be expressed with the equation:
10 mg of crude material = x mg of pure GLP-1 + y mg of peptide impurities + z mg of other compounds.
The percentage purity is calculated as follows:
% Purity = (x mg of pure GLP-1 in the crude) / (x mg of GLP-1 in the crude + y mg of impurities (peptide by-products))
Therefore,
% Purity = (x / (10 - z)) x 100.
Assay generally is defined as the percentage ratio of a pharmaceutical's peak area to the total sum of all peak areas in an HPLC chromatogram and undetected compounds not detected by HPLC.
The percentage assay is calculated using the formula:
% Assay = (x mg of pure GLP-1 in the crude) / (x mg of pure GLP-1 + y mg of peptide impurities + z mg of other compounds (residual organic solvents, moisture, ignition residues, counter ions))
So,
% Assay = (x / 10) x 100.
Since purity is calculated by dividing x by an amount less than the total GLP-1 crude amount, and the assay is determined by dividing x by an amount equal to the total GLP-1 crude amount, the resultant values for the assay are always lower than those for purity.
How do you calculate purity with HPLC?
We use area normalization. This involves measuring the area of the GLP-1 peak (e.g., A1 in Figure 1) and dividing it by the total area of all the peaks (e.g., A1 + A2 in Figure 1) in the chromatogram (GLP-1 and other impurity peaks). The result is expressed as a percentage of purity.
Purity% = (Peak area of pharmaceutical /sum of all peak areas) x 100
How do you calculate %assay in the crude sample by HPLC?
You must know the drug's potency to calculate the % assay in a crude sample. The % assay can be determined by comparing the amount of the pharmaceutical in the crude sample to the amount in a reference standard. The amount of the pharmaceutical in the reference standard is referred to as the pharmaceutical's potency.
According to the U.S. Food and Drug Administration (FDA), a reference standard material is defined as a "highly purified compound that is well characterized." The U.S. Pharmacopeia (USP) describes reference standard materials as "highly characterized specimens of drug substances, excipients, reportable impurities, degradation products, compendial reagents, and performance calibrators."
Reference standards are essential for determining quantitative data (such as assay and impurity levels), qualitative data (such as identifying compounds), and equipment performance tests (like HPLC instrument calibration). Therefore, the quality and purity of the reference standards are critical.
Assay analysis relies on having a reference standard with accurately determined potency by mass balance, hinges on the precision and accuracy of the assay method used [2]. If the potency of the reference standard is not accurate, it will lead to errors in the assay results. Furthermore, if a drug substance is analyzed using different reference standards or different methods to determine the purity of the reference standards, this may result in differing assay outcomes for the same drug substance.
Let's assume you have a 10 mg GLP-1 reference standard sample dissolved in 10 mL. Additionally, let's say this standard sample contains 2% (or 0.2 mg) of compounds that are not detected by HPLC, such as moisture, residual organic solvents, residue from ignition, and counter ions. Therefore, you are injecting 9.8 mg (or 98%) of the actual GLP-1 drug sample. This 98% reflects the potency of the GLP-1 standard.
To determine the assay of a 10 mg GLP-1 crude sample, you need to compare the area of the GLP-1 peak from the crude sample (e.g., A1 in Figure 1) with that of the 10 mg reference standard. The formula for the % Assay is as follows:
%Assay = (Peak area of the pharmaceutical in the sample x concentration of standard) x 100
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(Peak area of the pharmaceutical in the standard x concentration of sample)
The peak area of the pharmaceutical in the standard/ concentration of standard is also called the response factor of the reference standard. Thus, one can write assay as:
%Assay = (Peak area of the pharmaceutical in the sample) x 100
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(concentration of sample) x Response factor of the reference standard
The Bottom Line
Purity, assay, and potency are essential terms in testing the quality of drugs. Understanding these terms can help avoid confusion when using HPLC methods for their calculations.
- Purity checks how much of the drug is in a specific sample. The calculation includes impurities detected by HPLC, but does not include any impurities that are not detected by HPLC.
- Assay measures how much of a drug is in a specific sample. The calculation includes impurities that are detected and undetected by HPLC.
- Potency shows how much of the drug is in a specific standard of that same drug. The calculation includes any impurities that are detected and undetected by HPLC.
For more information on how our NanoPak-C All Carbon media can address your peptide purification challenges or or to request samples, please email us at inquiry@millennialscientific.com, call us at 855 388 2800, or fill in our online form.
References
Kupiec, T. C., Skinner, R. & Lanier, L. Stability Versus Potency Testing: The Madness is in the Method. Int J Pharm Compd 12, 50-53 (2008).
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