Reversed‐phase HPLC‐ESI/MS analysis of birch leaf proanthocyanidins after their acidic degradation in the presence of nucleophiles

Abstract

Mountain birch leaves contain large amounts of structurally variable polymeric proanthocyanidins. Their isolation procedure was enhanced by the addition of liquid-liquid extractions prior to column chromatography over Sephadex LH-20. Isolated polymeric proanthocyanidins were depolymerised by acid-catalysis in the presence of benzyl mercaptan or phloroglucinol in order to study their composition. The resulting degradation products, flavan-3-ols and flavan-3-ol adducts, were analysed with reversed-phase high-performance liquid chromatography using UV photodiode array detection for quantification and electrospray ionisation mass spectrometry for identification. The results showed that polymeric proanthocyanidins contained (epi)gallocatechins and (epi)catechins as the extension units and, mainly, (+)-catechin as the terminal unit. The mean degree of polymerisation was found to be 26 based on thiolysis and 31 based on phloroglucinol degradation.

Thiolysis-HPLC analysis of proanthocyanidins in health foods and their materials

“…Then, the extract was evaporated to 1 mL and was repurified by using a SinoChrom SiO 2 column (250 mm  20 mm id, 10 μm), and the mobile phase of the ethyl acetate: petroleum ether was 60∶40 v/v. Both compounds were confirmed on the basis of spectra data mainly MS, 1 H, and 13 C-NMR after comparison with published data (Gu et al, 2002;Vidal et al, 2004;Karonen et al, 2007) which confirmed EC-S and ECG-S.…”

“…This reaction can be quantitatively completed and thus would be able to make a very complex mixture of PC produce only a limited number of degradation products after the thiolysis reaction. Recently, a number of HPLC analyses upon this reaction have been developed (Labarbe et al,1999;Guyot et al, 2001;Kennedy and Jones, 2001;Gu et al, 2002;Dueñas et al, 2003;Karonen et al, 2007). However, these methods do not use the thiolysis derivatives as the reference substance, and the samples to be analyzed are often single plant materials.…”

Deconvolution of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry isotope patterns to determine ratios of A-type to B-type interflavan bonds in cranberry proanthocyanidins

“…Thiolysis and pholoroglucinolysis depends on cleavage through auto-oxidation of the interflavan bond in the presence of strong acid and subsequent reaction of extension units with a nucleophile (Karonen et al, 2007). Thiolysis operates under the assumptions that the cleavage of the interflavan bond is complete, the reaction follows a 1:1 stoichiometry (Karonen et al, 2007), and flavan-3-ol benzylthioethers have the same molar absorptivities as their respective flavan-3-ol monomers (Zhou, Lin, Wei, & Tam, 2011). Therefore the application of thiolysis and pholoroglucinolysis for estimating DP and presence of A-type interflavan bonds may be inaccurate when these assumptions are incorrect.…”

Analysis of proanthocyanidins

“…Koupai-Abyazani et al [95] developed a similar method using the acid degradation of procyanidins in the presence of the nucleophilic agent phloroglucinol and subsequent RP-HPLC separation. More recently, Karonen et al [84] reported a limitation of the thiolysis method as some polymeric PAC are resistant to degradation with thiols (notably when PAC originate from aged tissues, e.g., outer bark). Therefore, the method using a solution of 0.1 M HCl in methanol containing 50 mg/mL phloroglucinol and 10 mg/mL ascorbic acid seems to be more suitable [84].…”

“…More recently, Karonen et al [84] reported a limitation of the thiolysis method as some polymeric PAC are resistant to degradation with thiols (notably when PAC originate from aged tissues, e.g., outer bark). Therefore, the method using a solution of 0.1 M HCl in methanol containing 50 mg/mL phloroglucinol and 10 mg/mL ascorbic acid seems to be more suitable [84]. During this reaction, the extension units of the PAC are transformed into benzylthiol or phloroglucinol adducts, respectively (Fig.…”