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Lactating ruminants require an adequate supply of absorbable amino acids for the synthesis of milk protein from two sources, that is crude protein (CP) synthesized microbially in the rumen and ruminally undegraded CP (RUP) from feed which can both be digested in the small intestine. Several chemical and physical methods have been identified as being effective in increasing the proportion of RUP of total CP of a feedstuff, yet there is a continuing need for developing and establishing methods which protect feed protein from ruminal degradation with acceptable expenditure of labour and other costs. The objective of this study was to identify and quantify effects of and interactions between chlorogenic acid and protein in solvent-extracted sunflower meal (SFM) as induced by alkali treatment. Response surface methodology was employed to investigate the influence of pH, reaction time and drying temperature on the resulting SFM and, subsequently, its protein value for ruminants estimated from laboratory values. For this purpose, alkali-treated SFM was subjected to a fractionation of feed CP according to the Cornell net carbohydrate and protein system as a basis for estimating RUP at different assumed ruminal passage rates (Kp ). To estimate the intestinal digestibility of the treated SFM and its RUP, a three-step enzymatic in vitro procedure was applied. Alkaline treatment of SFM increased RUP values with factors ranging from approximately 3 (Kp =.08/hr) to 12 (Kp =.02/hr). Furthermore, the intestinal digestibility of the alkali-treated SFM was enhanced by approximately 10% compared to untreated SFM. Increasing pH and reaction time led to both increasing RUP values and intestinal digestibility. In conclusion, a targeted alkaline treatment of naturally occurring compounds in feedstuffs might be a promising approach to provide high-RUP feeds for ruminants which, at the same time, have improved intestinal digestibility values.

Fast methods for the extraction and analysis of various secondary metabolites from cocoa products were developed and optimized regarding speed and separation efficiency. Extraction by pressurized liquid extraction is automated and the extracts are analyzed by rapid reversed-phase ultra high-performance liquid chromatography and normal-phase high-performance liquid chromatography methods. After extraction, no further sample treatment is required before chromatographic analysis. The analytes comprise monomeric and oligomeric flavanols, flavonols, methylxanthins, N-phenylpropenoyl amino acids, and phenolic acids. Polyphenols and N-phenylpropenoyl amino acids are separated in a single run of 33 min, procyanidins are analyzed by normal-phase high-performance liquid chromatography within 16 min, and methylxanthins require only 6 min total run time. A fourth method is suitable for phenolic acids, but only protocatechuic acid was found in relevant quantities. The optimized methods were validated and applied to 27 dark chocolates, one milk chocolate, two cocoa powders and two food supplements based on cocoa extract.

A fast isocratic liquid chromatography method was developed for the simultaneous quantification of eight xanthophylls (13-Z-lutein, 13'-Z-lutein, 13-Z-zeaxanthin, all-E-lutein, all-E-zeaxanthin, all-E-canthaxanthin, all-E-β-apo-8'-carotenoic acid ethyl ester and all-E-β-apo-8'-carotenal) within 12 min, compared to 90 min by the conventional high-performance liquid chromatography method. The separation was achieved on a YMC C30 reversed-phase column (100 mm x 2.0 mm; 3 μm) operated at 20°C using a methanol/tert-butyl methyl ether/water solvent system at a flow rate of 0.8 mL/min. The method was successfully applied to quantify lutein and zeaxanthin stereoisomers in egg yolk, raw and cooked spinach, and a dietary supplement. The method can be used for the rapid analysis of xanthophyll isomers in different food products and for quality control purposes.

Introduction Rhodiola rosea L. is a medicinal herb used for its adaptogenic properties. The main active components are the phenylpropanoids collectively referred to as rosavins. Objectives To develop an isolation method for phytochemicals present in Rhodiola rosea roots using high-speed counter-current chromatography (HSCCC). Methodology The roots of Rhodiola rosea were extracted with methanol and fractionated using liquid–liquid partition and polyamide column clean-up. The purified fraction (100 mg) was subjected to semi-preparative HSCCC using the two-phase solvent system ethyl acetate:butanol:water (3:2:5). The head-to-tail elution mode was employed with a flow rate of 1.5 mL/min and a rotary speed of 1000 rpm. Results The separation yielded six main fractions with four components more than 90% pure. The sixth fraction was further purified using semi-preparative HPLC with a Synergi-hydro RP C18-column to obtain rosin and geranyl 1-O-α-l-arabinopyranosyl(1 6)-β-d-glucopyranoside. The main components isolated were rosavin (3.4 mg, 97% purity), salidroside (0.5 mg, 90% purity), benzyl-O-β-d-glucopyranoside (1.2 mg, 85% purity), rosarin (1.3 mg, 99% purity), rosiridin (1.8 mg, 92% purity), rosin (1.2 mg, 95% purity) and geranyl 1-O-α-l-arabinopyranosyl(1 6)-β-d-glucopyranoside (6.5 mg, 97% purity). The identity and purity of these components were confirmed using ultrafast liquid chromatography–diode-array detector–MS/MS analysis, 1 H- and 13 C-NMR spectroscopy. Conclusion High-speed counter-current chromatography was successful in the isolation of several phytochemicals present in Rhodiola rosea roots, including two components that are not commercially available. Copyright © 2012 John Wiley & Sons, Ltd.

The aim of the study was to obtain information on the content of co-active compounds of a food supplement recommended as a weight reduction diet and on its short-term effectiveness and safety as a starter for lifestyle change. Eighty participants completed the protocol. The Sambucus nigra L. berry juice enriched with flower extract and tablets containing berry powder and flower extract provided a total of 1 mg anthocyanins, 370 mg flavonol glycosides and 150 mg hydroxycinnamates per day; the Asparagus officinalis L. powder tablets provided 19 mg saponins per day. After the diet, the mean weight, blood pressure, physical and emotional well-being and the quality of life had significantly improved (ITT analysis). The effectiveness and tolerability of the regimen were rated as very good or good by most of the completers. It remains to be established if any particular compounds contribute to the efficacy of the diet.

High-performance liquid chromatography (HPLC) coupled to electrospray ionization tandem mass spectrometry (ESI-MS(n)) was used to study the covalent interactions between chlorogenic acid (CQA) quinone and two amino acid derivatives, tert-butyloxycarbonyl-L-lysine and N-acetyl-L-cysteine. In a model system at pH 7.0, the formation of covalent addition products was demonstrated for both derivatives. The addition product of CQA dimer and tert-butyloxycarbonyl-L-lysine was characterized by LC/MS(n) as a benzacridine structure. For N-acetyl-L-cysteine, mono- and diaddition products at the thiol group with CQA quinone were found. In apple juice at pH 3.6, covalent interactions of CQA quinone were observed only with N-acetyl-L-cysteine. Taking together these results and those reported by other groups it can be concluded that covalent interactions of amino side chains with phenolic compounds could contribute to the reduction of the allergenic potential of certain food proteins.

5-Alkyl- and 5-alkenylresorcinols, as well as their hydroxylated derivatives, were extracted from mango (Mangiferaindica L.) peels, purified on polyamide and characterized by high-performance liquid chromatography/atmospheric pressure chemical ionization mass spectrometry (HPLC/APcI-MS) for the first time. Among the 15 compounds analyzed, 3 major and 12 minor C15-, C17-, and C19-substituted resorcinols and related analogues, showing varying degrees of unsaturation, were characterized by their specific fragmentation patterns in collision-induced dissociation experiments. This marks the first report on the occurrence of mono-, di-, and triunsaturated C15-homologues, saturated and triunsaturated C17-homologues, and mono- and diunsaturated C19-homologues in mango peels. Additionally, several hydroxylated C15- and C17-homologues, also not yet described in mango, and a C14-monoene, unique because of its even-numbered side chain, were tentatively identified on the basis of their fragmentation patterns. The results obtained in the present study indicate that HPLC-DAD-APcI-MSn, combined with the newly developed solid-phase extraction, is a powerful tool for the analysis of alk(en)ylresorcinols and could therefore be used for their determination in various matrices. Copyright © 2007 John Wiley & Sons, Ltd.

The Anacardiaceae is a plant family that includes numerous species of high economic importance, e.g. mango, cashew, pistachio, and pink pepper as well as plants that are of local importance. Members of this family have been known as polyphenol-rich, however, this early view was based mainly on the results obtained using relatively basic analytical methods. During the past decades, tremendous progress has been made in analytical chemistry, which has led to new knowledge about the polyphenol composition of Anacardiaceae species. This review, covering 160 references, summarizes the most important literature on phenolic compounds present in edible species of the Anacardiaceae family. Structures of selected phenolic compounds are presented and chemotaxonomic aspects are discussed.

Recent studies have revealed different acrylamide formation mechanisms, e. g. from carnosine (N-beta-alanyl-L-histidine) and aminopropionamide as additional precursors. The occurrence of acrylamide in food matrices devoid of common precursors such as meat supports an additional formation pathway. Gluten was recovered from wheat flour by water extraction. Starch, reducing sugars and amino acids were removed using alpha-amylase and NaCl solution and were completely absent in the purified gluten fraction. The gluten was dry heated at temperatures ranging from 160 to 240 degrees C for 8 to 12 min and analyzed for acrylamide and cinnamic amide using liquid chromatography-tandem mass spectrometry. Acrylamide could be detected up to 3997 microg/kg gluten dry weight. Cinnamic amide was detected and unambiguously identified in the gluten samples, thus confirming the proposed formation of acrylamide from proteins. After gluten addition to bread roll dough, protein pyrolysis to form acrylamide in the complex food matrix was assessed. Contents of asparagine and reducing sugars were diminished due to the addition of the gluten. In contrast to the expectation with respect to the well-established common formation mechanism of acrylamide, it increased from 53.4 to 63.9 microg/kg (+20%), which was in good correlation with the higher proportion of gluten. As demonstrated by the t-test, the increase in acrylamide was significant when comparing 0 and 15% gluten addition. Additionally, cinnamic amide could be found in crusts of bread rolls. Thus, evidence for pyrolytic formation of acrylamide from wheat gluten was provided.

Acrylamide levels of bakery products, e. g., bread and bread rolls, are usually below 100 microg/kg , often even below 50 microg/kg. Therefore, usual analytical methods which have an LOQ greater, not dbl equals 25 microg/kg are not sensitive enough for detailed investigations on acrylamide formation within these commodities. An improved method for trace level determination of acrylamide in bakery products was developed using ion trap LC-ESI-MS/MS. Samples were divided into crumbs and crusts to achieve an initial concentration by removing the crumbs since these are devoid of acrylamide. After sample extraction and clean-up using multimode SPE cartridges, further analyte enrichment was accomplished by solid-phase-supported liquid-liquid extraction with ethyl acetate prior to LC-MS/MS analysis. The method was evaluated using bread, bread rolls, alkali-baked bread rolls, and toast. LOQ was calculated from the confidence interval of the calibration curve and found to be 1.7 ng/mL, corresponding to 17 microg/kg of product. When crumbs and crusts were separated, an LOQ of 10.2 microg/kg of bakery product could be obtained. As demonstrated in preliminary comparative analyses, accuracy of the method met the requirements for determination of trace level acrylamide formation in bakery products. Mean recovery was 102.4% (CV 4.5%), intermediate reproducibility revealed a CV of 2.1%, and a repeatability of CV 6.0%.

Carotenoids and carotenoid esters were extracted from red pepper pods (Capsicum annuum L.) without saponification. Among the 42 compounds detected, 4 non-esterified, 11 mono- and 17 diesters were characterized based on their retention times, UV/Vis spectra and their fragmentation patterns in collision-induced dissociation experiments in atmospheric pressure chemical ionization mass spectrometry (APCI-MS). Positive and negative ion mode measurements were used for the characterization of major and minor carotenoids and their esters. Capsanthin esterified with lauric, palmitic and myristic acids represented the predominant compounds in the red pepper extracts. Additionally, three beta-cryptoxanthin and one zeaxanthin monoester were tentatively identified in red pepper pods for the first time. Furthermore, the specific fragmentation patterns of capsanthin-laurate-myristate and capsanthin-myristate-palmitate were used for the distinction of both regioisomers. The results obtained from LC-DAD-APCI-MSn experiments demonstrated that the carotenoid profile of red pepper pods is considerably more complex than considered hitherto.

Phenolic acids and flavonoids were extracted from a dandelion (Taraxacum officinale WEB. ex WIGG.) root and herb juice and characterized by high-performance liquid chromatography/electrospray ionization mass spectrometry. Among the 43 compounds detected, 5 mono- and dicaffeoylquinic acids, 5 tartaric acid derivatives, 8 flavone and 8 flavonol glycosides were characterized based on their UV spectra and their fragmentation patterns in collision-induced dissociation experiments. The predominant compound was chicoric acid (dicaffeoyltartaric acid). Furthermore, several caffeoylquinic acid isomers were distinguished in dandelion extracts for the first time by their specific mass spectral data. The present study reveals that even more quercetin glycosides were found in dandelion than hitherto assumed. The occurrence of di- and triglycosylated flavonoids in particular has not yet been described. This paper marks the first report on HPLC-DAD/ESI-MSn investigations of phenolic compounds in dandelion.

Polyphenolics were extracted from peels, pulp and kernels of mango fruits (Mangifera indica L. cv. 'Tommy Atkins') and characterized by high-performance liquid chromatography/electrospray ionization mass spectrometry. In the peel 18 gallotannins and five benzophenone derivatives were detected which were tentatively identified as galloylated maclurin and iriflophenone glucosides. Twenty-one and eight gallotannins were found in the kernels and pulp, respectively, whereas no evidence for the presence of benzophenone derivatives was obtained. Gallotannins quantified by the rhodanine assay amounted to 1.4 mg/g dm in the peels (expressed as gallic acid), while only small amounts (0.2 mg/g dm) were found in the pulp. In contrast, mango kernels contained 15.5 mg/g dm and thus proved to be a rich source of gallotannins.

Phenolic acids were extracted from black carrot roots (Daucus carota ssp. sativus var. atrorubens Alef.) and a black carrot juice concentrate, and characterized by high-performance liquid chromatography/electrospray ionization mass spectrometry. Most of the compounds detected were identified as depsides composed of p-coumaric, caffeic and ferulic acids. Additionally, three hydroxybenzoic acid derivatives and one quercetin glycoside were detected. 5-O-Caffeoylquinic acid (chlorogenic acid) represented the predominant compound amounting to 657 mg/kg in the roots and 5815 mg/kg in the concentrate. The specific fragmentation patterns of mono- and dihydroxycinnamoylquinic acids allowed the distinction of several stereoisomers. The presence of 4-O-caffeoylquinic acid and several further hydroxycinnamic acid esters, together with compounds not belonging to the depside type, is reported for the first time. The present study reveals that the phenolic profile of black carrots is even more complex than hitherto assumed and may contribute to pigment stability of extracts derived from the roots.

A stationary phase with hydrophilic endcapping was used for the separation of seven quercetin (Q) glycosides by high-performance liquid chromatography. The elution order was established as Q 3-rutinoside, Q 3-galactoside, Q 3-glucoside, Q 3-xyloside, Q 3-arabinopyranoside, Q 3-arabinofuranoside, and Q 3-rhamnoside. Since a recent study had revealed that a commercially available reference compound (Q 3-arap) was erroneously labeled as avicularin (Q 3-araf), standards of the two isomers of Q 3-ara were characterized by 1 D and 2D NMR spectroscopy, and by optical rotation. The predominant Q glycosides detected in apple pomace were Q 3-gal, Q 3-araf, Q 3-rha and Q 3-xyl, whereas considerably lower amounts of Q 3-glc and Q 3-arap were found. Total content of Q glycosides was approximately 900 mg/kg on a dry matter basis, indicating that apple pomace is a good source of natural antioxidants.

Amino acid (AA) enantiomers were determined as N(O)-pentafluoropropionyl-(2)-propyl esters by chiral gas chromatography-mass spectrometry (GC-MS) in 24 h samples of the urine of three healthy volunteers and in their blood sera. In urine the largest amounts were determined for D-Ser (64-199 micromol/day) and D-Ala (24-138 micromol/day). In blood sera, D-Ala (2.3-4.2 micromol/L) and D-Ser (1.0-2.9 micromol/L) were most abundant. Varying amounts of the D-enantiomers of Thr, Pro, Asx, Glx, Phe, Tyr, Orn and Lys were also found, albeit not in all urines and sera. Further, enantiomers were quantified in urine samples of two volunteers fasting for 115 h. Quantities of renally excreted D-AAs decreased in fasting, although amounts of D-Ser (69 and 77 micromol/L urine) as well as other D-AAs were still detectable. Time-dependent analyses of urine showed that D-AAs are continuously excreted.

Quantities of D-amino acids were determined in body fluids (urine, blood plasma and blood serum, milk) of mammals (hamster, horse, bovine, sheep, pig, and dog). Amino acids were isolated using a cation exchanger and converted into their N(O)-pentafluoropropionyl (or trifluoroacetyl) amino acid 2-propyl esters. Enantiomers were separated and quantified on a Chirasil-L-Val capillary column with mass spectrometric detection using selected ion monitoring. D-Enantiomers of most protein L-amino acids were detected. Largest absolute and relative amounts in most cases were determined for D-Ser and D-Ala in urine. Stereoisomers of 2,6-diaminopimelic acid were also measured in bovine, ovine, and porcine urine. Since D-amino acids were detected in all representative classes of the major orders of Mammalia, namely Artiodactyla, Perissodactyla, Rodentia, and Carnivora, and taking reports in the literature into account, it is postulated that D-amino acids occur in all mammals.

The amounts and the configuration of the stereoisomers of 2,6-diaminopimelic acid (Dap) and the enantiomeric content of other amino acids were determined in five individual species (Fibrobacter succinogenes, Streptococcus bovis, Selenomonas ruminantium, Prevotella ruminicola and Anaerovibrio lipolytica) of rumen bacteria, and in samples of mixed rumen bacteria isolated from sheep. The separation and quantification of the Dap stereoisomers was achieved by gas chromatography (GC) of trifluoroacetyl 2-propyl esters on a Chirasil-L-Val fused silica column, and detection was achieved by selected ion monitoring mass spectrometry (SIM-MS). No isomers of Dap were detected in S. bovis and P. ruminicola, two of the bacterial isolates. LL- and DD-Dap were not detected in any of the bacterial samples. As only the meso-isomer of Dap was detected in these microorganisms, it was quantified by adding LL-Dap as an internal standard before the bacteria were acid-hydrolyzed. Amounts of between 4.8 and 12.0 mg meso-Dap per gram of bacterial dry matter (DM) were determined. The presence in the rumen bacteria of free amino acid enantiomers, extractable with 70% aqueous ethanol, were determined by GC–SIM-MS; the D-amino acids were predominantly Ala, Asp and Glu, but there was considerable variation between the species. Copyright © 1999 John Wiley & Sons, Ltd.

Im Verlauf der Plasteinreaktion konzentrieren sich hydrophobe Peptide uberwiegend in den Aggregaten (Plasteinen), wahrend hydrophile Peptide in Losung (Uberstand) verbleiben. Liquidchromatographische und sequenzanalytische Untersuchungen der pankreatischen Casein-Plasteine haben ergeben, das die Aggregate uberwiegend aus den freien Aminosauren Tyrosin, Phenylalanin und Tryptophan bestehen. Daruber hinaus enthalten die Plasteine kurzkettige Peptide insbesondere vom C-terminalen Ende des β-Caseins. Die Charakterisierung der funktionellen Eigenschaften der Plasteine hat deutlich gemacht, das die Aggregation der kurzkettigen Peptide und freien Aminosauren auf nicht-kovalenten, hydrophoben und ionogenen Wechselwirkungen beruht. In den Uberstanden aus der Plasteinreaktion konnten Caseinophosphopeptidsequenzen insbesondere aus αs-Casein bestimmt werden. Characterization of pancreatic casein plasteins. In the course of the plastein reaction hydrophobic peptides concentrate mainly in the aggregates (plasteins), whilst hydrophilic peptides remain in solution (supernatant). Liquid chromatographic and sequence analytical studies of pancreatic casein plasteins have shown that the aggregates consist mainly of the free amino acids tyrosine, phenylalanine and tryptophan. Plasteins contain, in addition, short-chain peptides, particularly from the C-terminal of β-casein. Characterization of the functional properties of the plasteins has shown clearly that aggregation of the short-chain peptides and free amino acids is brought by non-covalent, hydrophobic and ionogenic interactions. In the supernatants resulting from the plastein reaction caseinophosphopeptide sequences, in particular from αs-casein, were determined.