Acetonitrile-derived cyanohydrin (ketone-bearing peptides)

Trace HCN in acetonitrile (residual from acrylonitrile manufacture, or from MeCN pyrolysis under acid) attacks ketone carbonyls in modified peptides → cyanohydrin. +27 Da.

Why it happens (mechanism)

CH₃-C≡N decomposes under acid (or heat) to release HCN. HCN ⇌ CN⁻ + H⁺. CN⁻ adds to ketone C=O → R₂C(OH)(CN), a cyanohydrin. +27 Da on the ketone-bearing residue.

When it strikes (triggers)

Aqueous MeCN (TFA buffer) for HPLC of ketone-modified peptides. Aged MeCN. Hot MeCN. Basic pH adds significantly. Mostly affects peptides with ketone groups (semicarbazide, levulinyl, oxime ligation precursors).

How to spot it (MS signature)

+27.0 Da (CN). On ketone-modified peptides only — natural amino acids don't have ketone carbonyls.

How to prevent it

• Use methanol/H₂O/TFA instead of acetonitrile/H₂O/TFA for HPLC of ketone-bearing peptides (caveat: methanol has its own issues with esterification on Asp/Glu).
• Use freshly distilled, peptide-grade MeCN.
• Keep purification at low pH (acid suppresses CN⁻ ionization).

If it already happened (salvage)

• Cyanohydrin is reversible: mild base (pH 9-10, RT) ionizes it back to ketone + CN⁻.

Source

Yi Yang, Side Reactions in Peptide Synthesis (Elsevier, 2016), Chapter 14, §14.4.