[Paper memo] Oxidation of sample by UV of UV detector on HPLC-UV-MS
Treatise memo
In HPLC-UV-MS analysis, it is reported that the ultraviolet rays emitted from the UV detector oxidize the sample and are detected on the MS spectrum as artifacts.
Target literature
HPLC–UV–MS Analysis: A Source for Severe Oxidation Artifacts
Author (Affiliation): Fritz Schweikart and Gustaf Hulthe (AstraZeneca)
Journal (published date): Analytical Chemistry (2019/01)
https://pubs.acs.org/doi/10.1021/acs.analchem.8b05845
Original abstract
HPLC coupled to both UV and MS is an established setup for purity assessments in many areas. With evolving technology, instrument sensitivity increases, calling for lower sample concentration, while light flux in a commercial UV detector cell is considerably higher than earlier. This evolution has In this work we show several examples from pharmaceutical development where UV degradation in the UV detector leads to severely misleading mass spectra in typical day to day samples.
(Full translation)
The HPLC-UV-MS system, which connects a UV / Vis (ultraviolet-visible spectroscopic) detector and a mass spectrometer (MS) to high performance liquid chromatography (HPLC), is widely used in many analysis sites.With technological innovation, it has become possible to measure and detect samples with lower concentrations, while the luminous flux emitted to the UV detector cell is also higher than before.As a result, a relatively large amount of radicals were generated by UV compared to the analysis target, and artifacts were generated on the MS spectrum.In this study, taking drug development as an example, we show that oxidation by UV detectors results in a misleading mass spectrum in ordinary daily analytical samples.
Overview
【method】
The authors have demonstrated oxidation of the analytical sample during measurement with a UV detector using more than XNUMX different samples.Measurement targets include amino acids, peptides, proteins, nucleic acids, low molecular weight compounds, etc.
【result】
The effect of UV was greater in areas of low sample concentration in the liquid delivery route.Certainly, the oxidant peak appears more strongly at the start point (XNUMX) and end point (XNUMX) of the peak than at the peak top (XNUMX).Even +XNUMX to +XNUMX oxidants are detected at the end of the peak.
Figure 1. Mass spectra of Bombesin (Pyr-QRLG-NQWAVGHLM-NH2) in the (1) early, (2) main, and (3) late eluting fraction of the peak (Agilent / Bruker system). For comparison, the MS spectrum with the UV lamp switched off is shown as “no UV.”.
CultureIn the donation, known oxidative decomposition products and monooxidants to trioxides (+10 to +1) are measured for various samples (Bombesin, Cytochrome C, peptides of about 3 residues, Trp, Met, ketoprofen, etc.). However, in some cases, each of the 16-48 oxide peaks was detected with a relative intensity of about 1% of that of the unchanged peak.
Furthermore, the authors have shown that the appearance of such oxidant peaks can be prevented in two ways:
① Turn off UV light
② Add BHT during the mobile phase
Oxidation of the sample is observed in both methanol and acetonitrile as the organic solvent of phase B, but it seems that acetonitrile produced more oxides.Also, since the flow velocity is directly related to the irradiation time, the faster it was, the less oxidized it was.
Impressions
I knew it was possible that the UV light from the UV / vis detector would cause oxidation of the sample, but I didn't think it would actually be this far.
Is the content of this document a problem when measuring unknown substances?When measuring a very small amount of unknown metabolites with metabolomics, oxidants are mistakenly counted as different substances, and relative quantification is likely to cause an error.
The cause is the reaction with reactive oxygen species (ROS: Reactive Oxygen Species) such as hydroxyl radicals generated by UV, and the excitation of compounds with an absorption wavelength near 254 nm for decomposition or indirect photodecomposition.
It seems that you should be careful when measuring samples that are easily oxidized.In particular, amino acids such as methionine (Met), tryptophan (Trp), cysteine (Cys), and tyrosine (Tyr), peptide proteins containing these, and compounds classified as photosensitizers, that is, aromatic rings and carbonyl groups. Compounds with.
Well, honestly, I don't often refer to the UV spectrum firmly in MS analysis, so it may be an ant even if it is OFF.
In general quantitative analysis using LC-UV-MS, internal and external standard substances are used, and these should be oxidized in the same way, so I feel that they do not affect the analysis so much (extreme). Oxidation of the sample at low concentration levels may raise the lower limit of quantification ...).