CEI-MS-01 — procedure

§4Procedure

The procedure below is written in the second person, which is the only place in the Institute's document set where that register is used. A procedure is an instruction and is written as one.

1. Equilibrate the column at the stated temperature with the initial mobile-phase composition for not fewer than 15 column volumes. Confirm baseline stability at 214 nm over 10 minutes with a drift not exceeding 0.5 mAU.
2. Prepare the diluent as mobile phase A. For hydrophobic acylated analogues, prepare the diluent as 20 % acetonitrile in mobile phase A to prevent on-column precipitation at injection.
3. Weigh the sample to give a nominal concentration of 0.5 mg/mL, correcting for stated peptide content if a content figure is available. Record whether a correction was applied.
4. Dissolve by gentle inversion. Do not sonicate an oxidation-labile peptide; sonication generates local heating and dissolved-gas cavitation, both of which accelerate methionine and tryptophan oxidation.
5. Inject the diluent blank. Confirm that no peak exceeding 0.05 % of the expected main-peak area appears within the integration window.
6. Inject the reference standard, where available, five times. Assess system suitability.
7. Inject the sample in duplicate. Where the two determinations differ by more than 0.3 percentage points absolute, investigate before reporting.
8. Integrate from the end of the void disturbance to the end of the gradient hold. Record the integration start time explicitly: for polar peptides eluting close to the void, the choice of integration start materially changes the reported purity.
9. Report purity as the main-peak area divided by the total integrated area, excluding blank peaks and the void disturbance, expressed to one decimal place.

References cited on this page

References are numbered in order of first citation in this document. Each superscript in the text links to its entry below.

1. United States Pharmacopeial Convention. General Chapter ⟨1225⟩ Validation of Compendial Procedures. United States Pharmacopeia — National Formulary (USP–NF) 2024;USP 2024 Issue 1. identifier not held by the Institute

Identifiers are reproduced only where the Institute holds them. Where a digital object identifier or PubMed identifier is not shown, the Institute has recorded the journal and year and has not constructed an identifier.