When a peptide is synthesized, the final product is never 100% pure. Synthesis is a chemical process with inherent inefficiencies — incomplete couplings, side reactions, and residual reagents all contribute to impurities. The question is never "is this pure?" — it's "how pure is this, and are the impurities at levels that won't affect my research?"

Purity testing answers this question with analytical data rather than assumptions. Two complementary techniques form the standard: HPLC for purity and LC-MS for identity.

What HPLC Does

HPLC stands for High-Performance Liquid Chromatography. It separates the components of a sample based on their chemical properties — how they interact with a stationary phase (the column) and a mobile phase (the solvent flowing through the column).

Here's the process in plain terms:

1. A small amount of the peptide sample is dissolved and injected into the HPLC system
2. A solvent (mobile phase) carries the sample through a packed column (stationary phase) at high pressure
3. Different molecules interact with the column differently — some pass through quickly, others are retained longer
4. As each component exits the column, a UV detector measures its absorbance
5. The detector output is plotted as a chromatogram: signal intensity (y-axis) versus time (x-axis)

Each component appears as a peak at a specific retention time. The target peptide should appear as a single dominant peak. Impurities appear as smaller peaks at different retention times.

Reading a Chromatogram

The main peak: This is your target compound. It should be:

• Tall and sharp (indicating a concentrated, well-resolved compound)
• Symmetric (not tailing or fronting, which can indicate column issues or compound degradation)
• At the expected retention time for that compound under those conditions

The baseline: The flat line between peaks. A clean, flat baseline indicates good separation and low background noise. A noisy or rising baseline can obscure small impurity peaks.

Secondary peaks: Any peaks besides the main peak represent impurities. Their size relative to the main peak determines the purity calculation. Small peaks near the baseline are normal and expected. Large secondary peaks warrant investigation.

Purity calculation: The area under the main peak divided by the total area under all peaks, expressed as a percentage. If the main peak area is 99.2 and total peak area is 100.0, purity = 99.2%.

What HPLC Cannot Tell You

HPLC measures purity but not identity. A perfectly pure sample of the wrong compound would show a single peak at 100% purity — and the HPLC would not flag the error. This is why mass spectrometry is the essential complement to HPLC.

HPLC also cannot detect impurities that co-elute with the target compound (elute at exactly the same retention time). Different HPLC methods (different columns, solvents, gradients) can resolve co-eluting peaks that appear as a single peak under other conditions. This is why method selection matters and why purity numbers aren't directly comparable across different HPLC methods.

What LC-MS Does

LC-MS (Liquid Chromatography–Mass Spectrometry) combines HPLC separation with mass spectrometry detection. After the HPLC column separates the components, each component enters a mass spectrometer that measures its molecular weight (more precisely, its mass-to-charge ratio, m/z).

This solves the identity problem: the mass spectrometer confirms that the molecule exiting the column at the expected retention time has the correct molecular weight for your target peptide.

Reading Mass Spec Data

Expected vs Observed Molecular Weight:

• The COA reports the theoretical molecular weight (calculated from the amino acid sequence) and the observed molecular weight (measured by the instrument)
• A match within ±1-2 Da confirms identity
• Example: BPC-157 expected MW = 1419.53 Da, observed MW = 1419.51 Da — this is a positive identity confirmation

Mass Spectrum: The mass spectrum shows peaks at different m/z values. The dominant peak should correspond to the target compound. Common ionization states include [M+H]+ (molecular weight + 1 proton), [M+2H]2+ (doubly charged), and [M+Na]+ (sodium adduct). The COA should note which ionization state was observed so you can verify the calculation.

What LC-MS Cannot Tell You

LC-MS confirms molecular weight but not purity — a sample contaminated with 20% of a different compound would show two mass peaks, but the relative amounts are better quantified by HPLC. LC-MS also cannot distinguish between structural isomers that have the same molecular weight but different amino acid sequences or configurations (e.g., D-amino acid substitutions).

Neither technique alone provides complete characterization. Together, they answer both essential questions:

A credible COA should include results from both techniques. If a vendor provides only HPLC purity without mass spec confirmation, or mass spec without purity data, the characterization is incomplete.

Some research applications require additional characterization beyond standard HPLC + LC-MS:

Amino Acid Analysis (AAA): Hydrolyzes the peptide into individual amino acids and quantifies each. Confirms amino acid composition. The most definitive sequence verification short of Edman degradation or tandem MS sequencing.

Endotoxin Testing (LAL): Detects bacterial endotoxin contamination. Critical for cell culture and in vivo applications where endotoxins can activate immune responses and confound results.

Counterion Analysis: Identifies the salt form of the peptide (acetate, TFA, hydrochloride). The counterion affects peptide content calculations and may matter for certain research applications.

Circular Dichroism (CD): Measures secondary structure (alpha-helix, beta-sheet, random coil). Relevant for peptides where conformation affects activity.

These tests are not routinely included in standard COAs but may be available on request for specific research applications.

Every batch of every OSYRIS product undergoes:

1. HPLC purity analysis — confirming ≥98% purity (typical: 99.0-99.5%)
2. LC-MS identity confirmation — confirming correct molecular weight within ±1-2 Da
3. Third-party independent testing — performed by a laboratory with no financial relationship to OSYRIS or the manufacturer

Results are published as a downloadable COA on every product page. When a new batch is tested, the COA is updated.