Development of an accurate and high-throughput methodology for structural comprehension of chlorophylls derivatives. (II) Dephytylated derivatives

Abstract

Dephytylated chlorophylls (chlorophyllides and pheophorbides) are the starting point of the chlorophyll catabolism in green tissues, components of the chlorophyll pattern in storage/processed food vegetables, as well as the favoured structural arrangement for chlorophyll absorption. In addition, dephytylated native chlorophylls are prone to several modifications of their structure yielding pyro-, 13(2)-hydroxy- and 15(1)-hydroxy-lactone derivatives. Despite of these outstanding remarks only few of them have been analysed by MS(n). Besides new protocols for obtaining standards, we have developed a new high throughput methodology able to determine the fragmentation pathway of 16 dephytylated chlorophyll derivatives, elucidating the structures of the new product ions and new mechanisms of fragmentation. The new methodology combines, by first time, high resolution time-of-flight mass spectrometry and powerful post-processing software. Native chlorophyllides and pheophorbides mainly exhibit product ions that involve the fragmentation of D ring, as well as additional exclusive product ions. The introduction of an oxygenated function at E ring enhances the progress of fragmentation reactions through the β-keto ester group, developing also exclusive product ions for 13(2)-hydroxy derivatives and for 15(1)-hydroxy-lactone ones. Consequently, while MS(2)-based reactions of phytylated chlorophyll derivatives point to fragmentations at the phytyl and propionic chains, dephytylated chlorophyll derivatives behave different as the absence of phytyl makes β-keto ester group and E ring more prone to fragmentation. Proposals of the key reaction mechanisms underlying the origin of new product ions have been made.

First‐Pass Metabolism of Chlorophylls in Mice

“…These products were identified according to their chromatographic behavior (retention time), UV‐Visible spectra, and MS high‐accuracy measurements, including mass error, isotopic pattern, and the featured product ions. These characteristic data, presented in Table S1, Supporting Information, were compared with those of pure standards and data available in the literature . However, the identification of phytyl‐chlorin e 6 was supported by the chromatographic behavior and features of the UV‐visible spectrum in comparison with its de‐esterified counterpart chlorin e 6 because this is the commercial standard available .…”

“…Standard of chlorophyll and pheophorbide a were purchased from Wako (Neuss, Germany) and chlorin e 6 from Frontier Scientific (Logan, USA). The other chlorophyll derivatives were obtained following previously detailed the protocols . Commercial Spirulina (NaturGreen) was purchased from a local store.…”

“…All procedures were performed under dimmed green light to avoid any photo‐oxidative process of the chlorophyll derivatives. Pellets (both standard and supplemented ones), livers, or feces were homogenized with 25 mL of DMF saturated with MgCO 3 and vacuum filtered. The solid was collected and extracted twice with the same conditions.…”

“…The liquid chromatograph was Dionex Ultimate 3000RS U‐HPLC (Thermo Fisher Scientific, Waltham, MA, USA). Chromatographic separation was performed as described before . A stainless steel column (200 × 4.6 mm, 3 μm particle size) packed with Mediterranea Sea18 (Teknokroma, Barcelona, Spain) was used.…”

“…The instrument control was performed using Bruker Daltonics HyStar 3.2. Data evaluation was performed with Bruker Daltonics DataAnalysis 4.1 as described previously . From the HPLC/qTOF–MS acquisition data, an automated peak detection on the EICs expected for the [M + H] + ion of each compound in the database was performed with Bruker Daltonics TargetAnalysis 1.2 software.…”