Imre Boldizsár

Mass fragmentation study of the trimethylsilyl derivatives of arctiin, matairesinoside, arctigenin, phylligenin, matairesinol, pinoresinol and methylarctigenin: Their gas and liquid chromatographic analysis in plant extracts

The mass fragmentation patterns and the characteristic behavior of the trimethylsilyl (TMS) derivatives of the dibenzylbutyrolactone-type (arctiin, arctigenin, methylarctigenin, matairesinoside, matairesinol) and those of the diphenylperhydrofurotetrahydrofurane-type (phylligenin, pinoresinol) lignans, obtained by gas chromatography-mass spectrometry (GC-MS), were presented. It was shown that upon acidic hydrolysis the dibenzylbutyrolactone-type lignans are stable while the diphenylperhydrofurotetrahydrofurane-type ones decompose. As a novelty to the field we confirmed that the fragment species of the derivatized lignan glycosides, in the presence of excess hexamethyldisilazane, leaded to their in situ derivatization. Quantification of the selective fragment ions of the TMS derivatives by GC-MS, in respect of the ions found one by one, and concerning the selective fragment ions {SFI(s)} in total, provided acceptable reproducibilities, suitable for quantitation purposes: varying between 1.20% and 6.6% relative standard deviation percentages (RSD%). For characterization of the behavior of various type of lignans, analyses were performed with the untreated and with the trifluoroacetic acid hydrolyzed plant extracts, from the same sample, in parallel, both by GC-MS and by high performance liquid chromatography-mass spectrometry, working in the positive electron ionization mode (HPLC-ESPI-MS). The analysis of lignans in fruit and leaf extracts (obtained from the Arctium, Centaurea and Forsythia plants) was confirmed both by GC-MS and by HPLC-ESPI-MS. Our multicomponent system (including the identification and quantification of sugars, sugar alcohols, and several members of various homologous series of acids, anthraquinones and flavonoids) has been extended to the analysis of lignan glycosides and to the free lignans. Reproducibilities in the quantitation of lignans in plant matrices, as averages on GC and HPLC basis, varied between 0.9% and 11% (RSD). The distribution of the lignan constituents was presented for 5 Arctium, for 8 Centaurea and for 4 Forsythia plant extracts: the total of lignan contents varied between 0.42 and 87.9 mg/g, respectively.

Characteristic fragmentation patterns of trimethylsilyl and trimethylsilyl-oxime derivatives of plant disaccharides as obtained by gas chromatography coupled to ion-trap mass spectrometry

The characteristic fragmentation pattern of six reducing and two non reducing type disaccharides-(neohesperidose, acuminose, sambubiose, rutinose, vicianose, primverose, and two arabinosyl-inositols) has been described. These saccharides have not been previously identified by on-line chromatographic techniques. Unambiguous specific characteristics of the TMS (oxime)s such as mass distribution, syn/anti oximes ratios and elution order proved to be associated with their reducing or non reducing character, with their aldosyl property and with the position of their O-glycosidic linkages. The practical utility of the mass fragmentation study of these rare disaccharides was demonstrated, at the first time, by the simultaneous, on-line identification and quantification of the acuminose, vicianose, primverose and arabinosyl-inositol contents of tea leaves, from green and black tea blends of Indian and Chinese origin.

Simultaneous GC-MS quantitation of acids and sugars in the hydrolyzates of immunostimulant, water-soluble polysaccharides of basidiomycetes

SummaryThe simultaneous quantitation of acids and sugars as their trimethyl silyl (TMS) derivatives has been extended in order to identify and quantitate the simple acid and sugar constituents in the hydrolyzates of various immunostimulant, water-soluble polysaccharides obtained from various Basidiomycetes, such as Armillariella mellea, Auricularia auricula-judae, Coriolus versicolor, Flammulina velutipes, Fomes fomentarius, Ganoderma applanatum, Ganoderma lucidum, Pleurotus ostreatus, Schizophyllum commune, Trametes hirsuta. Optimum hydrolysis conditions, performed with 2 M trifluoroacetic acid (TFAA) for five hrs, proved the presence of several sugars and acids with maximum recovery. (i) the total sugar/sugar alcohol content of polysaccharides varied between 20- and 65% and consisted of arabitol (0.01–10.2%), arabinose (0.09–1.3%), ribose (0.2–1.8%), fucose (0.3–1.2%), mannitol (0.01–5.3%), sorbitol (0.01–0.05%), galactiol (0.04%), fructose (0.08–0.8%), galactose (0.9–29%), glucose (10–53%), uronic acids (0.14–3.7%), sucrose (0.03–2%), trehalose (0.2–1%), cellobiose (0.01–0.6%), maltose (0.2–1.9%), other disaccharides (0.2–8%). (ii) The total of acids varied from 1.5 to 30% including o-phosphoric (1.3–19%), malic (0.08–4.7%), citric (0.08–4.7%), isocitric; (3%) and C16−C18 fatty acids (1–6%).

Complementary fragmentation pattern analysis by gas chromatography-mass spectrometry and liquid chromatography tandem mass spectrometry confirmed the precious lignan content of Cirsium weeds

In this paper, as novelties to the field, it is confirmed at first, that the fruits of Cirsium species, regarded as injurious weeds, do contain lignans, two, different butyrolactone-type glycoside/aglycone pairs: the well known arctiin/arctigenin and the particularly rare tracheloside/trachelogenin species. These experiences were supported by gas chromatography-mass spectrometry (GC-MS), by liquid chromatography tandem mass spectrometry (LC-MS/(MS)) and by nuclear magnetic resonance (NMR) spectroscopy. The study reflects the powerful impact of the complementary chromatographic mass fragmentation evidences resulting in the identification and quantification, the extremely rare, with on line technique not yet identified and described, tracheloside/trachelogenin pair lignans, without authentic standard compounds. Fragmentation pattern analysis of the trimethylsilyl (TMS) derivative of trachelogenin, based on GC-MS, via two different fragmentation pathways confirmed the detailed structure of the trachelogenin molecule. The complementary chromatographic evidences have been unambiguously confirmed, by (1)H and (13)C NMR analysis of trachelogenin, isolated by preparative chromatography. Identification and quantification of the fruit extracts of four Cirsium (C.) species (C. arvense, C. canum, C. oleraceum, and C. palustre), revealed that (i) all four species do accumulate the tracheloside/trachelogenin or the arctiin/arctigenin butyrolactone-type glycoside/aglycone pairs, (ii) the overwhelming part of lignans are present as glycosides (tracheloside 9.1-14.5 mg/g, arctiin 28.6-39.3 mg/g, expressed on dry fruit basis), (iii) their acidic and enzymatic hydrolyses to the corresponding aglycones, to trachelogenin and arctigenin are fast and quantitative and (iv) the many-sided beneficial trachelogenin and arctigenin can be prepared separately, without impurities, excellent for medicinal purposes.

Optimum yields of dibenzylbutyrolactone-type lignans from cynareae fruits, during their ripening, germination and enzymatic hydrolysis processes, determined by on-line chromatographic methods

INTRODUCTION Dibenzylbutyrolactone-type lignans are the physiologically active constituents of the achene fruits of Cynareae. These lignans occur in glycoside/aglycone forms: in the highest quantity of the arctiin/arctigenin, matairesinoside/matairesinol and tracheloside/trachelogenin pairs found in the fruits of Arctium lappa L., Centaurea scabiosa L. and Cirsium arvense (L.) Scop. OBJECTIVE To optimise the extraction yield of the arctiin/arctigenin, matairesinoside/matairesinol and tracheloside/trachelogenin glycoside/aglycone pairs, from the fruits of Arctium lappa, Centaurea scabiosa and Cirsium arvense, under the ripening, germination and enzymatic hydrolysis processes of the fruits. METHODOLOGY Identification and quantification of lignans were performed with on-line gas chromatography-mass spectrometry (GC-MS) and with high performance liquid chromatography (HPLC), both with UV and mass selective detections (HPLC-UV/MS). RESULTS As novelties to the field it was confirmed that: (i) the unripe fruits provide a high amount of lignans, similar to the ripe fruit; (ii) the fruits of Arctium lappa and Cirsium arvense do have glycosidase activity to hydrolyse their lignan glycosides into free lignans; (iii) the glycosidase of Centaurea scabiosa fruit becomes activated under its germination process only; and (iv) the overwhelming part of the fruits lignan contents (80-94%) in all three species are accumulated in the embryo. CONCLUSION The best sources of (i) lignan aglycones are the enzyme-hydrolysed embryos, separating spontaneously during the germination process, and (ii) lignan glycosides are the unripe fruits.

Characterization of the endogenous enzymatic hydrolyses of Petroselinum crispum glycosides: Determined by chromatography upon their sugar and flavonoid products

The behavior of the flavonoid diglycosides, relevant constituents of parsley (Petroselinum crispum) fruit (PFr) and leaf (PLe) samples was characterized upon their enzymatic hydrolyses applying complementary liquid chromatography-ultraviolet (LC-UV) and gas chromatography mass selective (GC-MS) detections. Analyses were performed in quantitative manner, from the same extracts as a function of hydrolysis times. Both in fruit and leaf tissue extracts, in intact and in enzyme hydrolyzed ones, apigenin, chrysoeriol, their glycosides, sugars, sugar alcohols, carboxylic acids and phytosterols, in total 17 constituents were identified and quantified. Based primarily on the selective mass fragmentation properties of the trimethylsilyl (oxime) ether/ester derivatives of constituents, we confirmed several novelties to the field. (i) It was shown for the first time that in parsley tissues different types of glycosidase enzyme are active. In PFr samples, both the stepwise and disaccharide specific endogenous mechanisms were certified, quantifying simultaneously the continuous release of apigenin, chrysoeriol, 2-O-apiosyl-apiose, apiose and glucose. (ii) 2-O-Apiosyl-glucose was demonstrated as disaccharide due to its formation under derivatization conditions from parsley glycosides. (iii) Both in PFr and in PLe samples even the invertase enzyme activity was attainable: sucrose decomposition in both tissues was going on with the same intensity. Three different types of enzymatic glycosidase processes were followed with their specific hydrolysis products by means of HPLC-UV and GC-MS, simultaneously.

Endogenous enzyme-hydrolyzed fruit of Cirsium brachycephalum: Optimal source of the antiproliferative lignan trachelogenin regulating the Wnt/β-Catenin signaling pathway in the SW480 colon adenocarcinoma cell line

The molecular constituents of Cirsium brachycephalum fruits were identified, quantified and isolated for the first time. The lignan glycoside tracheloside was the main compound, which was transformed quantitatively into its aglycone trachelogenin by endogenous enzymatic treatment of the fruit. Following this transformation by high performance liquid chromatography (HPLC) hyphenated with UV and mass spectrometry (MS) detections on a quantitative basis, the enzyme-hydrolyzed fruit was found to be the richest raw material containing trachelogenin (17.2mg/g) reported to date. Thus, the enzyme-hydrolyzed fruit was used to isolate trachelogenin using preparative HPLC in order to (1) unambiguously confirm its identity by gas chromatography-MS, nuclear magnetic resonance spectroscopy and optical rotation, and (2) investigate its in vitro antiproliferative activities against the SW480 colon adenocarcinoma cell line. Trachelogenin significantly affected the phosphorylation of key proteins such as β-Catenin, c-Myc and GSK3 in the β-Catenin signaling pathway in a concentration-dependent manner. These changes account for the antiproliferative effects of trachelogenin.

The role of harmonized, gas and liquid chromatography mass spectrometry in the discovery of the neolignan balanophonin in the fruit wall of Cirsium vulgare

In order to identify and quantify fruit-lignans of Cirsium vulgare - authors introduced a special analysis system: with particular attention to the lignans enrichment/separation course. These synchronized, germination and enzymatic hydrolysis processes were followed by complementary gas and liquid chromatography, coupled with special mass selective detections (GC-MS, LC-MS/MS, LC-TOF/MS) and confirmed by nuclear magnetic resonance (NMR) spectroscopy. Mass fragmentations and NMR evidences, proved that the two main medicinal lignan constituents of the fruits of Cirsium vulgare are the neolignan-type, free balanophonin and the butyrolactone-type tracheloside. As novelty to the field, these two lignans of different chemical structures could be quantitatively extracted, separately from each others, without impurities. Balanophonin and tracheloside do accumulate in the fruits of C. vulgare, separately: balanophonin was found, in enormous high concentrations, in the fruit wall (23.2-24.9 mg/g), while in embryo part tracheloside was determined (20.3mg/g), exclusively. Consequently, the optimum source of balanophonin proved to be the fruit wall, while tracheloside, - providing trachelogenin upon enzymatic hydrolysis, - could be obtained from the embryo parts of fruits. As further novelties of the study balanophonin was identified and quantified at the first time with on-line chromatographic technique, in free form, without authentic standard compound.

Specific hydrolysis and accumulation of antiproliferative lignans in the fruit of Leuzea carthamoides (Willd.) DC.

Dibenzylbutyrolactone-type lignan glycosides (tracheloside and carthamoside), their aglycones (trachelogenin and carthamogenin) and feruloyl-serotonin isomers were determined in the fruits of Leuzea carthamoides by using LC–UV, LC–MS/MS and GC–MS techniques. The composition of the embryo and wall parts of the fruits was analysed before and after their hydrolysis. As a result of these studies, fruit part-specific accumulation of lignan glycosides and feruloyl-serotonins were confirmed, demonstrating that the embryo contains a high amount of lignan glycosides (tracheloside 32.9 mg/g, carthamoside 45.3 mg/g), while the wall part of the fruit accumulates feruloyl-serotonins (63.0 mg/g). Enzymatic hydrolysis of the embryo resulted in the quantitative transformation of lignan glycosides into their corresponding aglycones, allowing selective isolation of trachelogenin and carthamogenin. These aglycones were subjected to an antiproliferative study against the SW480 colon adenocarcinoma cell line. In this test, moderate activity of carthamogenin and a significant effect of trachelogenin were demonstrated in a concentration range of 22–185 μM.

Quantitative Silylation Speciations of Primary Phenylalkyl Amines, Including Amphetamine and 3,4-Methylenedioxyamphetamine Prior to Their Analysis by GC/MS

A novel, quantitative trimethylsilylation approach derivatizing 11 primary phenylalkyl amines (PPAAs), including amphetamine (A) and 3,4-methylenedioxyamphetamine (MDA), was noted. Triggering the fully derivatized ditrimethylsilyl (diTMS) species with the N-methyl-N-(trimethylsilyl)-trifluoroacetamide (MSTFA) reagent, a new principle was recognized followed by GC/MS. In the course of method optimization, the complementary impact of solvents (acetonitrile, ACN; ethyl acetate, ETAC; pyridine, PYR) and catalysts (trimethylchlorosilane, TMCS; trimethyliodosilane, TMIS) was studied: the role of solvent and catalyst proved to be equally crucial. Optimum, proportional, huge responses were obtained with the MSTFA/PYR = 2/1-9/1 (v/v) reagent applying catalysts; A and MDA needed the TMIS, while the rest of PPAAs provided the diTMS products also with TMCS. Similar to derivatives generated with hexamethyldisilazane and perfluorocarboxylic acid (HMDS and PFCA) ( Molnár et al. Anal. Chem. 2015 , 87 , 848 - 852 ), the fully silylated PPAAs offer several advantages. Both of our methods save time and cost by allowing for direct injection of analytes into the column; this is in stark contrast with the requirement to evaporate acid anhydrides by nitrogen prior to their injection. Efficiences of the novel catalyzed trimethylsilylation (MSTFA) and our recently introduced (now, for A and MDA extended) acylation principle were contrasted. Catalyzed trimethylsilylation led to diTMS derivatives resulting in on average a 1.7 times larger response compared to the corresponding acylated species. Catalyzed trimethylsilylation of PPAAs, A, and MDA were characterized with retention, mass fragmentation, and analytical performance properties (R(2), LOQ values). The practical utility of ditrimethylsilyation was shown by analyzing A in urine and mescaline (MSC) in cactus samples.