Dissertation Thomas Bachmann

Synthesis of isotopically labeled standards of vitamin B6 and their application in the development of an LC-MS/MS method for quantification of B6 in food

ISBN:  978-3-945762-07-3

231 Seiten, 29 Abbildungen, 6 Tabellen, Format DIN A5

Vitamin B6 repräsentiert einen der wichtigsten am menschlichen Stoffwechsel beteiligten Mikronährstoffe. Trotz seines reichlichen Vorkommens in Nahrungsmitteln stellt die genaue Quantifizierung, insbesondere in Bezug auf individuelle Vitamere, bis heute eine fortlaufende Herausforderung dar. Das Ziel dieser Arbeit war die Entwicklung einer auf LC-MS/MS und SIDA basierenden analytischen Methode zur gleichzeitigen Quantifizierung von B6-Vitameren in Lebensmittelproben. Zunächst wurden ausgehend von [13C3]-Propionsäure isotopenmarkierte interne Standards chemisch synthetisiert. Die Strategie beinhaltete eine Oxazolbildung und eine DIELS-ALDER-Reaktion als Schlüsselschritte. Darüber hinaus wurden Strategien für die Herstellung glykosylierter Derivate adressiert. Die synthetisierten Substrate wurden anschließend als interne Standards bei der Entwicklung einer auf LC-MS/MS und SIDA basierenden analytischen Methode zur Bestimmung der individuellen Vitamere in Lebensmitteln eingesetzt.

Publikationen aus der Dissertation:

Bachmann T, Rychlik M (2018) Synthesis of 13C3-B6 vitamers starting from 13C3-propionic acid, Molecules, 23, 2117; doi:10.3390/molecules23092117 (open access)

Bachmann T , Schnurr C , Zainer L, Rychlik M (2020) Chemical synthesis of 5’-β-glycoconjugates of vitamin B6 , Carbohydrate Research 489: 107940, https://doi.org/10.1016/j.carres.2020.107940

Bachmann T, Rychlik M (2020) Chemical glucosylation of pyridoxine, Carbohydrate Research 489: 107929, https://doi.org/10.1016/j.carres.2020.107929

Bachmann T, Maurer A, Rychlik M (2020) Development of a LC-MS/MS method using stable isotope dilution for the quantification of individual B6 vitamers in fruits, vegetables and cereals, Analytical and Bioanalytical Chemistry, 412:7237-7252, https://doi.org/10.1007/s00216-020-02857-5

Ausführliche Zusammenfassung:

With the increasing technological progress in the analytical field opening the doors for analytical surveys, a manifold of methods was established for the analysis of vitamin B6 with the focus oriented towards food and biological samples due to the grave importance of the vitamers in the human metabolism. Step-wise incorporation of improving aspects such as deproteinizing agents for milder sample processing or increased analyte separation and selectivity via application of HPLC/fluorescence detection methods over time contributed their fraction to a more detailed painting. Today however, the research revolving around this vitamin resembles a slumbering giant, relaxing from the eruptive outburst of creativity in research during the first decades. Modern methods such as LC-MS/MS and SIDA bring the innovation to disrupt the slumber and inherit the potential for further development in the field of vitamin B6. Despite the stagnating research, this group’s importance did not forfeit any value when discussions revolving around nutritional or medical standpoints, e.g. food processing and bioavailability, arise. Those topics remain contemporary and their influx even increases with ongoing time, feeding the need for improved analytical methods regarding this set of molecules. The current study aimed to establish a fundament for following projects through exploration of the chemical and analytical aspects surrounding B6 by investigating new chemical strategies for the synthesis of the vitamers and the development of an analytical method for their individual quantification based on LC-MS/MS and SIDA.

The three branches growing towards chemical strategies for the synthesis of isotopically labeled vitamers bore fruitful results.

First investigations centered around the preparation of pyridoxine, which included key reactions such as the formation of an oxazole and a Diels-Alder reaction, revealed the need for a cheaper alternative to the known preparations with respect to the synthesis of isotopically labeled substrates. Although alanine marks a cheap and well available starting material for the synthesis of vitamin B6, isotopologues of the amino acid proof to be an expensive investment particularly when the need for multiple isotopes in the molecule for application in the analytical field arises. By introduction of a five step sequence with the goal to prepare alanine, the expanded chemical route offers the access to vitamers of B6 containing multiple isotopic labels in the core-structure starting from a cheaper material. After extensive optimization, the chemical route was applied in the synthesis of the free vitamers [13C3]-PN, [13C3]-PL and [13C3]-PM starting from isotopically labeled [13C3]-propionic acid.

The next glance was cast upon the preparation of pyridoxine-5’-β-glucoside, a derivative of the B6 group, whose important footprint in nature has yet to be explored and fully understood. Most preparations of this molecule rely on the utilization of biotechnological processes, whereas chemical preparations remain scarce and non-optimized. By exploring the extensive field of glycosylation reactions, an improved chemical strategy involving the application of 2,3,4,6-tetra-O-acetyl-d-glucopyranosyl fluoride and boron trifluoride diethyl etherate (2.0 eq.) as promoter at 0 °C with subsequent stepwise deprotection using potassium/sodium hydroxide in acetonitrile/water followed by acid hydrolysis with formic acid was found for the preparation of PNG, which is capable to compete with biotechnological processes in aspects such as yield and working effort.

With the glucoside of PN contributing a major part to the overall vitamin B6 content in plants, light does rarely fall on other glycosides of the B6 group, casting their potential impact in darkness. The third project, therefore, focused on synthesizing new glycoconjugates. Utilizing improved Koenigs-Knorr conditions, a variety of β-glycosides, i.e. the mannoside, galactoside, glucuronide, maltoside, cellobioside, xyloside and arabinoside, was synthesized and characterized. Furthermore, two chemical strategies for the preparation of pyridoxal glucoside were successfully applied starting from either PL·HCl or twofold protected PNG and the synthesis of isotopically labeled [13C6]-5’-β-PNG using 2,3,4,6-tetra-O-acetyl-[13C6]-α-d-glucopyranosyl bromide and α4,3-O-isopropylidene pyridoxine was accomplished. Lastly, the synthesized standards were applied in the development of an LC-MS/MS method based on SIDA. Hereby, the vitamers PN, PL, PM, PMP and – for the first time – PNG were analyzed using [13C3]-PN, [13C3]-PL and [13C6]-PNG as internal standards. After its successful validation the method was applied for the quantification of B6 vitamers in 14 plant-based food samples, where the obtained quantities aligned well with results from the literature.