Accepted answer
System suitability is the part of a report that tells you whether the method was under control on the day you were tested, and its absence is concerning.
Sample preparation is almost always under-appreciated — a reconstituted peptide in strong solvent will distort its own peak on the gradient.
Mass shifts and what they usually mean
| Δ mass (Da) | Most likely cause | Distinguishing feature |
|---|
| +1 | Deamidation (Asn or Gln) | New peak, slightly earlier retention |
| −17 | Loss of ammonia | Often with deamidation |
| −18 | Dehydration / succinimide | pH-dependent, reversible |
| +16 | Oxidation (Met, Trp) | Earlier retention, light-related |
| −128 | Missing Gln or Lys | Deletion sequence from synthesis |
| 0 | Isomer: racemisation or scrambling | Same mass, shifted retention |
Put another way, wider-pore phases — 300 Angstrom rather than 100 Angstrom — have faster mass transfer and narrower peaks for peptides above three kilodaltons, which is almost every peptide you will see.
The resolving power of a separation is quantified by the resolution parameter R, defined from the heights and widths of adjacent peaks, and pharmacopoeial methods typically demand R greater than 1.5 for a method to be considered validated.
One qualification: the limit of detection on a reversed-phase HPLC is set by the noise on the baseline, which for these molecules is usually in the range of a tenth of one per cent or less, and anything smaller is not reproducibly detectable.
Ask for the chromatogram and the system suitability data, not just the number.
3This is the first explanation of that which has actually made sense to me. – ben_akintola 27 days ago 4Note that the label instructions differ between agents on precisely this point. – tess_amankwah 3 months ago add a comment