Particulates in a reconstituted solution: identifying the likely source — what changed since

Particulates in a reconstituted solution: identifying the likely source — what changed since — setting out what I have, and where I think it stops being reliable.

Posting the method first, because I know what the first three replies will otherwise be.

• Column: C8, 4.6 x 150 mm, 3.5 um
• Mobile phase: 0.1% TFA in water / 0.1% TFA in acetonitrile
• Gradient: 13% to 56% organic over 21 minutes
• Detection: 220 nm
• Injection: 14 uL
• Sample: retatrutide, reconstituted to 2.0 mg/mL, injected within an hour

The main peak integrates at 97.6% of total area. There is a small feature on the trailing edge that I cannot decide is a shoulder or a baseline artefact, and that is what I am actually asking about.

Related substances: compounds chemically related to the target peptide but not the target peptide itself. The standard method separates them and reports them as area percent. How related they can be before they exceed specification is a regulatory question.

On the opening post — agreed on the reasoning, with one qualification.

Two things before anyone answers the substance.

First, the context in the first post is clear and specific. Second, the question is framed so that an answer can actually address it. Both are the norm here and both matter more than they sound.