Multianalytical Approach for Deciphering the Specific MS/MS Transition and Overcoming the Challenge of the Separation of a Transient Intermediate, Quinonoid Dihydrobiopterin

Abstract

Despite recent technological developments in analytical chemistry, separation and direct characterization of transient intermediates remain an analytical challenge. Among these, separation and direct characterization of quinonoid dihydrobiopterin (qH2Bip), a transient intermediate of tetrahydrobiopterin (H4Bip)-dependent hydroxylation reactions, essential in living organisms, with important and varied human pathophysiological impacts, are a clear illustration. H4Bip regeneration may be impaired by competitive nonenzymatic autoxidation reactions, such as isomerization of qH2Bip into a more stable 7,8-H2Bip (H2Bip) isomer, and subsequent nonenzymatic oxidation reactions. The quinonoid qH2Bip intermediate thus plays a key role in H4Bip-dependent hydroxylation reactions. However, only a few experimental results have indirectly confirmed this finding while revealing the difficulty of isolating qH2Bip from H4Bip-containing solutions. As a result, no current H4Bip assay method allows this isomer to be quantified even by liquid chromatography–tandem mass spectrometry (MS/MS). Here, we report isolation, structural characterization, and abundance of qH2Bip formed upon H4Bip autoxidation using three methods integrated into MS/MS. First, we characterized the structure of the two observed H2B isomers using IR photodissociation spectroscopy in conjunction with quantum chemical calculations. Then, we used differential ion mobility spectrometry to fully separate all oxidized forms of H4Bip including qH2Bip. These data are consistent and show that qH2Bip can also be unambiguously identified thanks to its specific MS/MS transition. This finding paves the way for the quantification of qH2Bip with MS/MS methods. Most importantly, the half-life value of this intermediate is nearly equivalent to that of H4Bip (tens of minutes), suggesting that an accurate method of H4Bip analysis should include the quantification of qH2Bip.

Autoxidation Kinetics of Tetrahydrobiopterin—Giving Quinonoid Dihydrobiopterin the Consideration It Deserves

“…Except for qH2Bip, we used two MRM transitions for each pterin, the first one corresponding to the most intense fragment for identification, and the second one, less intense, for confirmation. For qH2Bip, we only used the previously deciphered characteristic MRM transition ( m/z , 240 > 166) [ 37 ].…”

“…However, all these data were obtained at neutral pH and were only based on the spectrophotometric detection of H4Bip autoxidation products. Moreover, the data related to H4Bip stability and qH2Bip half-life remain scarce and conflicting [ 23 , 24 , 37 ]. Thus, we used the proposed LC-MS/MS method to further investigate this issue.…”

“…Very recently, by using a multi-analytical approach including differential ion mobility spectrometry (DMS), we have demonstrated that H2Bip isomers can be distinguished and identified based on their MS/MS transition ions [ 37 ]. We also reported that the half-life of qH2Bip is longer than it was previously believed under our experimental conditions [ 37 ]. However, we did not check the effects of pH and the type of buffer on qH2Bip’s stability.…”

“…Considering the recent findings on the specific detection of qH2Bip [ 37 ] based on its characteristic MS/MS transition, it becomes evident that LC-MS/MS may be used to specifically detect this pterin with the multiple reaction monitoring (MRM) mode of detection. Hence, here we developed an HPLC-MS/MS method able to separate all the autoxidation products of H4Bip, including qH2Bip.…”

Insights into Molecular Structure of Pterins Suitable for Biomedical Applications

“…Detection of quinonoid 6,7-dihydrobiopterin (qH 2 Bip) is a challenging task since it quickly transforms to a more stable 7,8-dihydrobiopterin (H 2 Bip). Thus, integration of an infrared photodissociation spectroscopy along with liquid chromatography–tandem mass spectrometry is required [ 116 ].…”