How Sapho Bio Tests for Peptide Potency

We correct our reference standards to the Net Peptide Content of every peptide API — so your potency result reflects exactly how much peptide is in the vial.

Potency testing answers a deceptively simple question: How much active pharmaceutical ingredient (API) is really in a compounded preparation?

A pharmacy sends us a vial labeled 10 mg/mL of a peptide API. We measure it by HPLC-UV (USP <621>) against our internal reference standards and report whether it meets its label claim (90.0–110.0%). That result is only as trustworthy as the standard behind it, which is why we correct our standards to the Net Peptide Content of the API.

About Sapho Bio: Sapho Bio is a precision measurement company offering a comprehensive suite of lot release and stability tests for compounding pharmacies, including USP validated methods for rapid sterility, endotoxin and potency.

The Problem We Kept Seeing

A common error when compounding peptides is improperly correcting the mass of lyophilized powder using data from the Certificate of Analysis (COA). A batch of lyophilized peptide does not only contain the peptide of interest: counterion salts (such as TFA or acetate) and water can be a substantial fraction of the powder. "HPLC Purity" is usually the highest number on the COA and the easiest to reach for. But it doesn't accurately describe all the impurities in a batch; correcting to HPLC Purity alone can miss a massive shortfall. The result is a preparation that is subpotent, even when the label was followed exactly, and a patient who is under-dosed. We design our testing to detect this problem before it reaches a patient.

What a Certificate of Analysis Tells You

A complete COA reports many values that carry different information. Three matter, and each measures something different:

No single value is the whole story; each holds valuable information about what is actually in your product. For that reason, all of these values need to be incorporated into your corrections.

How We Determine Net Peptide Content

Here at Sapho Bio, when we prepare standards from lyophilized powder, we measure what we call the Net Peptide Content. The Net Peptide Content for a peptide API is defined as:

Net Peptide Content = Measured Mass × (Peptide Content %) × (HPLC Purity %) × (Assay %)

The Net Peptide Content represents the actual amount of peptide API in a formulation and accounts for all of the shortfalls described in a COA.

Other Benefits of using Net Peptide Content:

• Simplicity - one standard, one sample, one measurement. Allows us to focus on accuracy and rapid results

• Consistency - we don't have to account for different formulations, APIs, preparations, salts, etc. Our test measures everything to the same specification.

• Clarity - we measure what is on the label, nothing more, nothing less. Pharmacies and patients don't have to wonder what our number represents or perform additional math.

Example Calculation: A peptide standard measured at 10 mg, with an 85% Peptide Content, 98% HPLC Purity, and 99% Assay.

Net Peptide Content = 10 mg × (0.85) × (0.98) × (0.99) = 8.25 mg

That is a 17.5% gap from the label — greater than the difference between a preparation that passes and one that fails.

How Our Potency Testing Works

1. You send us the vial, its label claim (e.g. 10 mg/mL), and the Certificate of Analysis of your current lot.

2. Using validated HPLC-UV methods, we compare your preparation to corrected reference standards.

3. We report the Net Peptide Content in your formulation and issue a pass or fail against label claim (90.0–110.0%)

By correcting our standards to measure the Net Peptide Content, we ensure our result reflects the actual amount of API in your formulation. During onboarding we ask how you correct based on your COAs. If your approach differs from ours, we will tell you and, at your request, describe any correction we apply and document it on our certificate.

Frequently Asked Questions

Why isn't HPLC Purity enough?

HPLC Purity only compares the correct molecule to other molecules the HPLC-UV can detect. It does not account for the water and salt that make up a real share of many powders, and it ignores content shortfalls reported elsewhere on the COA. A material can be highly "pure" on an HPLC and still be well below its labeled concentration.

My COA lists HPLC Purity and Assay, but no separate "Peptide Content" — is that a problem?

Not necessarily. Suppliers differ in how they report information in a COA, and many report the Peptide Content shortfall under "Assay." When this is the case, we apply all the values that are present. When in doubt, it is always best to reach out to your supplier.

My COA reports percentages of acetate, water, and other different substances. Do I need to also account for these values?

No. The Peptide Content value already accounts for all non-peptide substances, including acetate and water. The exception is when your API forms a complex with a metal ion (for example, the Copper ion in GHK-Cu). In this case, the Peptide Content can be replaced with the sum of the percentage of peptide and metal percentages to measure the total mass of the Peptide-Metal complex.

My peptide API is a salt or a hydrate — how is that handled?

The counterion and water are not active product, so they are not measured. We work from the COA's quantitative assay on the basis that matches your dose, just as we remove a peptide's counterion and water.

Further reading. Sigma-Aldrich, Peptide Sample Amount Determination; Bachem, Quality Control of Amino Acids & Peptides; AmbioPharm, What is Net Peptide Content?; Iris Biotech, Net Content and Purity.

This article is educational and does not replace your facility's quality program or professional judgment. Compounded-preparation potency expectations follow USP <795> / <797>.