Identity by mass spectrometry: why a CoA needs two tests

Purity and identity are two different questions. HPLC answers what share of the UV signal belongs to the main peak. Mass spectrometry (MS) answers whether the substance has the mass its sequence predicts. A certificate of analysis that answers only one of these questions leaves the other open.

Two questions, two methods

Both tests belong to the same batch number. How to read the other fields of a certificate is covered in Reading a certificate of analysis.

Why HPLC does not settle identity

A chromatogram shows peaks and their areas. It does not show which substance sits in a peak. A high purity only means that one peak makes up most of the signal.

A research group at Ghent University that examined 98 ordered research peptides (Verbeke and others, 2015) checked what sat in the peaks for 15 of them. In 14 cases the main peak was the ordered peptide. For one peptide it was not. There the measured mass was 2.1 daltons below the expected one. The authors suggest that a disulfide bridge had formed between the two cysteines of the sequence, giving a ring-shaped molecule instead of the ordered linear one. They state plainly that the main peak of a chromatogram can be an impurity instead of the ordered peptide.

What mass spectrometry measures

MS determines the molecular mass of a substance. For a peptide, the expected mass can be calculated from the sequence. The certificate then compares two numbers: the expected and the measured mass. If they agree within the accuracy of the measurement, that points to the expected substance.

If the mass differs, the difference hints at what happened. Synthesis produces typical by-products, among them sequences with missing or extra amino acids, and oxidised forms. Two examples from the Ghent study:

• Minus 2.1 daltons: consistent with a disulfide bridge, as described above.
• Plus 14.04 daltons: consistent with an added methyl group.

Most impurities the group identified were sequences missing one or more amino acids.

Fragments for the sequence

A simple mass measurement shows the mass of the whole molecule. In tandem mass spectrometry (MS/MS) the instrument also breaks the molecule into fragments. Their masses allow conclusions about the order of the amino acids. The Ghent group confirmed the sequences of the impurities this way.

The limit of a mass measurement

Same mass does not mean same structure. Epimers, variants that differ only in the spatial arrangement at one position, have the same mass as the parent peptide. Mass spectrometry cannot tell them apart; that takes a chromatographic separation (Mazzoccanti and others, 2020).

In the other direction, a mass spectrum showing the expected mass does not tell you how large that substance's share of the sample is. HPLC measures that, with the limits described in HPLC purity explained. Whether it is the right substance, and how large its share is, only both measurements together can answer.

LC-MS: separation and mass in one run

In LC-MS the separation column is coupled directly to the mass spectrometer, so each peak in the chromatogram gets a mass. The Ghent group used this coupling to assign their impurities. A certificate based on an LC-MS measurement can therefore show that the expected mass sits in the main peak and not in a side peak.

What to look for on the certificate

• an identity test with method, expected and measured mass;
• a purity test with method, wavelength, specification and numerical result;
• the same batch number for both tests;
• ideally, the batch's mass spectrum and chromatogram attached.

At Novo Labo you can request the certificate of analysis for a batch by writing to hello@novolabo.com. All products are sold strictly for laboratory and research use.