GLP-3 purity: what a purity percentage does and doesn't tell you

Purity is the number most people look at first on a peptide certificate, and the one most often misread. This article explains what an HPLC purity percentage measures, how it differs from peptide content and net content, where impurities in synthetic peptides come from, and how to compare purity results between lots or suppliers. GLP-3 RT lot data is used throughout.

Three different numbers

Keeping these apart avoids most purity misunderstandings. A sample can score very high on one and still differ on another.

Purity is relative

HPLC purity is the main peak's area divided by the total area of all detected peaks.¹ It describes the composition of what the detector sees. Water, residual salts and counterions such as acetate or trifluoroacetate produce little or no signal at the detection wavelength and are not counted. A sample can therefore be very pure by HPLC and still contain a meaningful fraction of non-peptide mass.

Purity is also method-relative. It reflects one column, one gradient and one detection wavelength. A different validated method might resolve an impurity that this method merges into the main peak, or count a peak this method doesn't integrate.²

Where impurities in synthetic peptides come from

Synthetic peptides are built one residue at a time. Each coupling step is highly efficient but not perfect, and the impurities HPLC is designed to find usually reflect that process:¹

• Deletion sequences: one residue missing because a coupling step didn't complete.
• Truncated sequences: chains that stopped growing early.
• Incompletely deprotected sequences: a protecting group left on a side chain.
• Diastereomers: one residue switched to its mirror-image configuration during synthesis.
• Modification variants: for modified peptides (for example lipidated, amidated or cyclized sequences), forms with an incomplete or altered modification.

After synthesis, storage can add degradation products such as deamidated, oxidized or aggregated forms.³ Many of these differ only slightly from the target and elute close to the main peak, which is why method specificity matters.

Purity and identity are separate questions

A purity figure says the sample is homogeneous. It does not say what the main component is. A sample of the wrong peptide can still show a single clean peak. That is why every GLP-3 RT lot also has a separate identity test (LC-MS), and why a certificate showing purity without identity is incomplete. See LC-MS analysis of GLP-3.

GLP-3 RT purity by lot

GLP3 Peptide Co.’s stated release specification is ≥95% purity by HPLC-UV with identity confirmed by LC-MS. Current lots exceed it by more than four percentage points.

Net content and label size

The current certificates report measured net content above each labeled vial size:

Net content applies to the vial the laboratory tested. Individual vials within a lot can vary.

Comparing purity between lots

The current lots report 99.98% (KB89232), 99.67% (KB48721) and 99.93% (KB3011). Two cautions apply when comparing numbers like these:

• Reporting precision is not accuracy. A result printed to two decimal places is not necessarily reliable to two decimal places. Every validated method has a characterized precision.²
• Small differences may be method noise. Differences within a few tenths of a percent can reflect normal variation in sample preparation and integration rather than a real difference in the material.

Comparing purity claims between suppliers

Purity figures from different sources are directly comparable only when the method is comparable. Before comparing, check:

1. Is the figure tied to a specific lot, or is it a general product claim?
2. Is the method named (HPLC-UV, area percent), and is the chromatogram available?
3. Was the testing performed by an identified independent laboratory?
4. Is identity confirmed by a separate method on the same lot?
5. Is the testing date shown?

A general statement such as "99% pure" with no lot, method or laboratory cannot be evaluated. A lot-specific result with its chromatogram, method and laboratory can.

What a purity figure does not tell you

• Which compound the main peak is.
• How much peptide is in the vial.
• Whether non-UV-absorbing material (water, salts, counterions) is present.
• Whether the material has changed since the test date.