What LC-MS Tells You
HPLC tells you how pure a sample is. LC-MS tells you what that sample is. The two are complementary — a batch with 99% purity by HPLC tells you 99% of the material is one compound, but it doesn’t confirm the identity of that compound. LC-MS bridges this gap.
How LC-MS Works
The chromatographic step separates the sample as in standard HPLC. As compounds elute from the column, they enter the mass spectrometer where they are ionized — typically via electrospray ionization (ESI). The ionized molecules are accelerated through an electromagnetic field, and the mass-to-charge ratio (m/z) of each is measured.
Reading the Mass Spectrum
The output is a 2D plot with m/z on the x-axis and signal intensity on the y-axis. Each peak represents an ion of a specific mass. For peptides, you’ll typically see multiple peaks corresponding to different charge states — for example, [M+H]+ (singly protonated), [M+2H]2+ (doubly protonated), [M+3H]3+ (triply protonated). The molecular weight of the peptide is calculated by deconvoluting these multi-charge signals.
Acceptance Criteria
The standard for identity confirmation is observed mass within ±0.5 Da of theoretical mass. Theoretical mass is calculated directly from the amino acid sequence — each amino acid contributes a known monoisotopic mass, and the peptide bond formation loses one water molecule per bond. For a 15-amino-acid peptide like BPC-157, the theoretical mass is approximately 1419.53 Da — confirmable against the PubChem reference. Any deviation beyond ±0.5 Da indicates the molecule is not the intended sequence.
Common Causes of Mass Discrepancies
If the observed mass differs from theoretical, the cause is usually one of: oxidation (+16 Da per oxidized methionine or cysteine), deletion sequence (peptide missing one amino acid during synthesis), truncation (peptide cut short during cleavage), or incomplete deprotection (protecting groups still attached). Each has a characteristic mass signature that experienced analysts recognize.
VialPepLab COA Format
Every batch in our catalog has a COA documenting: HPLC chromatogram with calculated purity, LC-MS spectrum with observed molecular weight, theoretical mass calculation, mass deviation in Da, batch number, and synthesis date. Browse the COA Library to see live examples from current inventory. View available compounds or contact our QA team for batch-specific questions.