Identification of the Major Components of Buddleja officinalis Extract and Their Metabolites in Rat Urine by UHPLC‐LTQ‐Orbitrap

Buddleja officinalis Maxim, one of the most popular herbal medicines in China, is widely prescribed for curing eye diseases for centuries. In this study, the major components of B. officinalis extract (BOE) and their metabolites in rat urine were detected and identified by ultra‐high‐pressure liquid chromatography coupled with linear ion trap‐orbitrap tandem mass spectrometry (UHPLC‐LTQ‐Orbitrap). A total of 19 compounds, including 8 flavonoids and 11 phenylethanoid glycosides, were confirmed or tentatively identified from BOE. In vivo, 33 components, including 3 prototypes and 30 metabolies, were confirmed or tentatively identified in rat urine samples. The metabolic pathways of different types of compounds were also proposed. This study would effectively narrow the range of potentially bioactive constituents of BOE and shed light to its action mechanism.     

Fermentation dynamics of Lactobacillus helveticus H9 revealed by ultra‐performance liquid chromatography quadrupole time‐of‐flight mass spectrometry

Lactobacillus (L.) helveticus H9 is a probiotic strain that can produce antihypertensive peptides during milk fermentation. This study analysed the dynamics of skim milk fermentation by L. helveticus H9 by ultra‐performance liquid chromatography coupled to quadrupole time‐of‐flight mass spectrometry (UPLC/Q‐TOF MS). A total of 1992 metabolites were detected from all of the fermented samples in the LC‐MS analysis by multivariate statistical analysis. Metabolites with variable importance in projection (VIP) values ≥2 were considered differentially abundant among samples and were responsible for the unique taste and nutritional and functional qualities of fermented milk. Valine, threonine, l ‐methionine, tyrosine, asparagine and leucine were the predominant amino acids produced during fermentation, and their quantities changed remarkably during the fermentation process. Citric acid and uric acid were the major, and only detectable, organic acids. Some intermediate metabolites, such as N‐acryloylglycine and nicotinamide‐N‐oxide, were also detected. Moreover, certain oligopeptides such as Val‐Leu, Lys‐Gly, Ala‐Glu, Asp‐Ser, Leu‐Pro and Val‐Phe‐Ala were not detected until the middle and late fermentation periods. This study demonstrated dynamic metabolic changes, providing a strong foundation for deciphering the physiological and biochemical mechanisms of the fermentation process.     

Comparison of ginsenosides in radix and rhizome of wild Panax species using LC‐ELSD and LC‐Q‐TOF‐MS

High‐performance liquid chromatography (HPLC) with evaporative light scattering detector (ELSD) and quadrupole time‐of‐flight mass spectrometry (Q‐TOF‐MS) were used for qualitative and quantitative analysis of wild Panax species, especially wild P. vietnamensis, which is an important medicinal plant. We determined the types and concentrations of ginsenosides in radix and rhizome of wild P. vietnamensis. Identification of ginsenosides was achieved using Q‐TOF‐MS; concentrations were determined by ELSD. The most abundant ginsenosides in wild Vietnamese ginseng were of the ocotillol type, accounting for more than 50% of the total. Compared to the rhizome, the radix had 31% higher ginsenoside content due to variation in protopanaxatriol‐type ginsenoside contents. We also found an unusual difference in the chromatograms of the two parts of wild P. vietnamensis. This difference did not appear in other wild species such as P. ginseng and P. quinquefolius. Our study provides an opportunity for further in‐depth study of the distinctive characteristics of P. vietnamensis.     

Comparison and Characterization of Compounds with Antioxidant Activity in Lycium barbarum Using High‐Performance Thin Layer Chromatography Coupled with DPPH Bioautography and Tandem Mass Spectrometry

Methanol extracts from 50 batches of Lycium barbarum (L. barbarum, wolfberry) in China were compared and characterized using high‐performance thin‐layer chromatography coupled with 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH) bioautography (HPTLC‐DPPH) and electrospray ionization quadrupole time‐of‐flight tandem mass spectrometry (ESI‐Q‐TOF‐MS/MS), respectively. Results showed that similar components occupying the major antioxidant activity existed in L. barbarum collected from different origins. However, the average antioxidant capacities of methanol extracts of L. barbarum collected in Ningxia were significantly higher than those of Qinghai, Xinjiang, Inner Mongolia, and Gansu, which may contribute to rational use of L. barbarum in China. Furthermore, the chemical structure of compound with the highest antioxidant capacity was tentatively identified as 2‐O‐β‐d ‐glucopyranosyl‐l ‐ascorbic acid using ESI‐Q‐TOF‐MS/MS analysis, which possessed high potentials to be used as an antioxidant biomarker for the quality control of L. barbarum. Results are helpful for the bioactivity‐based quality control of L. barbarum, and beneficial for the improvement of their performance in functional/health foods area, suggesting that HPTLC‐DPPH bioautography with ESI‐Q‐TOF‐MS/MS could be used as a routine approach for quality control of antioxidant components in L. barbarum.     

Bioavailability study of a polyphenol‐enriched extract from Hibiscus sabdariffa in rats and associated antioxidant status

The aqueous extracts of Hibiscus sabdariffa have been commonly used in folk medicine. Nevertheless, the compounds or metabolites responsible for its healthy effects have not yet been identified. The major metabolites present in rat plasma after acute ingestion of a polyphenol‐enriched Hibiscus sabdariffa extract were characterized and quantified in order to study their bioavailability. The antioxidant status of the plasma samples was also measured through several complementary antioxidant techniques. High‐performance liquid chromatography coupled to time‐of‐flight mass spectrometry (HPLC‐ESI‐TOF‐MS) was used for the bioavailability study. The antioxidant status was measured by ferric reducing ability of plasma method, thiobarbituric acid reactive substances assay, and superoxide dismutase activity assay. Seventeen polyphenols and metabolites have been detected and quantified. Eleven of these compounds were metabolites. Although phenolic acids were found in plasma without any modification in their structures, most flavonols were found as quercetin or kaempferol glucuronide conjugates. Flavonol glucuronide conjugates, which show longer half‐life elimination values, are proposed to contribute to the observed lipid peroxidation inhibitory activity in the cellular membranes. By contrast, phenolic acids appear to exert their antioxidant activity through ferric ion reduction and superoxide scavenging at shorter times. We propose that flavonol‐conjugated forms (quercetin and kaempferol) may be the compounds responsible for the observed antioxidant effects and contribute to the healthy effects of H. sabdariffa polyphenolic extract.     

The Cannizzaro‐like metabolites of secoiridoid glucosides in some olive cultivars

Oleuropein (the most abundant bitter principle) and its analogue ligstroside, both secoiridoid biophenols, were extracted from Hojiblanca black olives and analysed by preparative flash chromatography and HPLC. During olive growth, maturation and processing, these compounds undergo biological and technological degradation to various metabolites, by enzymatic degradation through esterases, phenoloxidases and β‐glucosidases. An enzymatic system with regiospecific Cannizzaro‐like activity could be responsible for two novel metabolites, isolated and characterised by ¹H‐ NMR, ¹²C NMR and FAB‐MS as hydroxycarboxylic and lactone derivatives of terpenoid origin from the precursor oleoside‐11‐methylester, formed from both secoiridoid biophenols. The occurrence of these two secoiridoid metabolites requires a thermodynamically controlled sequential process, via hydride rearrangements, through the complex metabolic pathway. The possible reaction sequence was interpreted by molecular calculations performed on competitive isomers. Copyright © 2004 Society of Chemical Industry     

Absorption and twenty‐four‐hour metabolism time‐course of quercetin‐3‐O‐glucoside in rats,in vivo

Rats were meal‐fed a semi‐synthetic diet, with or without quercetin 3‐O‐glucoside (Q3G; 100 mg per meal) and groups of three were killed either fasting, or at 2, 5 and 24 post‐feeding. Flavonoids and their metabolites in the diet, stomach contents, small intestinal lumen and mucosa, caecal contents and plasma were determined by LC/MS. Q3G was not hydrolysed in the stomach, but deglycosylation and further metabolism occurred in the small intestinal mucosa. At least 17 flavonoid glucuronides were identified in the lumen and mucosa, with evidence of time‐dependent changes such as de‐ and re‐glucuronidation. Quercetin mono‐sulphate was also detected in the small intestinal contents. Metabolites were still present in tissue and plasma 24 h after feeding. There was also evidence of complex microbial processing of Q3G in the caecal lumen with the appearance of at least one methylquercetin‐mono‐glucuronide, mono‐sulphate unique to this site in the gut, together with phenolic acid derivatives. Intestinal flavonoid metabolism is thus a very complex process in mammals, involving both enterocytes and bacteria. Copyright © 2004 Society of Chemical Industry     

Metabolomics reveals consistency of the shoot system in Medicago truncatula by HPLC‐UV‐ESI‐MS/MS

Flavonoids not only play crucial roles in plant development and resistance, but also provide one of the major natural sources in human nutrition. To investigate the distribution of flavonoids in the shoot system of Medicago truncatula, a high‐performance liquid chromatography coupled with electrospray ionisation tandem mass spectrometer (HPLC‐ESI‐MS/MS) method was established and then applied to determine the quantitation of flavonoids in different parts of the plant. There were twenty‐two, fifteen and eleven different kinds of flavonoids identified from the flower, leaf and stem of M. truncatula, respectively. The identified constituents were either aglycone or glycosides of the typical flavonoid backbones, such as myricetin, quercetin, luteolin, kaempferol, tricin, apigenin and laricitrin. It was found that the shoot system of M. truncatula can be differentiated by flavonoids in terms of structures and contents. Our results provide instruction to utilise the shoot system of legume crops as fodder and herb medicine in the future.     

Quantification of 1,8‐cineole and of its metabolites in humans using stable isotope dilution assays

The metabolism of 1,8‐cineole after ingestion of sage tea was studied. After application of the tea, the metabolites 2‐hydroxy‐1,8‐cineole, 3‐hydroxy‐1,8‐cineole, 9‐hydroxy‐1,8‐cineole and, for the first time in humans, 7‐hydroxy‐1,8‐cineole were identified in plasma and urine of one volunteer. For quantitation of these metabolites and the parent compound, stable isotope dilution assays were developed after synthesis of [²H₃]‐1,8‐cineole, [9/10‐²H₃]‐2‐hydroxy‐1,8‐cineole and [¹³C,²H₂]‐9‐hydroxy‐1,8‐cineole as internal standards. Using these standards, we quantified 1,8‐cineole by solid phase microextraction GC‐MS and the hydroxyl‐1,8‐cineoles by LC‐MS/MS after deconjugation in blood and urine of the volunteer. After consumption of 1.02 mg 1,8‐cineole (19 μg/kg bw), the hydroxycineoles along with their parent compound were detectable in the blood plasma of the volunteer under study after liberation from their glucuronides with 2‐hydroxycineole being the predominant metabolite at a maximum plasma concentration of 86 nmol/L followed by the 9‐hydroxy isomer at a maximum plasma concentration of 33 nmol/L. The parent compound 1,8‐cineole showed a low maximum plasma concentration of 19 nmol/L. In urine, 2‐hydroxycineole also showed highest contents followed by its 9‐isomer. Summing up the urinary excretion over 10 h, 2‐hydroxycineole, the 9‐isomer, the 3‐isomer and the 7‐isomer accounted for 20.9, 17.2, 10.6 and 3.8% of the cineole dose, respectively.     

Determination of purine alkaloids and catechins in different parts of Camellia assamica var. kucha by HPLC‐DAD/ESI‐MS/MS

BACKGROUND: Kucha (Camellia assamica var. kucha) is a novel wild tea resource grown in China and a tea plant containing a sizable amount of theacrine (1,3,7,9‐tetramethyluric acid). High‐performance liquid chromatography (HPLC) analysis of purine alkaloids and catechins in young leaves of Kucha has been reported previously. However, the compositions of purine alkaloids and catechins in other parts of the plant remain unknown, and more information about the chemical constituents of Kucha is also necessary for further research and development of this new tea resource. RESULTS: Using HPLC with diode array detection coupled with electrospray ionisation tandem mass spectrometry (HPLC‐DAD/ESI‐MS/MS), three purine alkaloids, seven catechins and four non‐catechin phenolic compounds were identified or tentatively identified in Kucha. Purine alkaloids and catechins in leaves at different developmental stages, flowers, stems, pericarps and seeds of the plant were also quantified for the first time by the HPLC method, which was fully validated. Recoveries of the quantified compounds ranged from 96.67 to 104.33%. CONCLUSION: The results showed that the total contents of purine alkaloids and catechins were highest in young leaves of Kucha. Theacrine was detected in all parts of the plant and found to be most abundant in pericarps. Copyright © 2009 Society of Chemical Industry     

The C‐glycosyl flavonoid,aspalathin, is absorbed,methylated and glucuronidated intact in humans

Human bioavailability of the flavonoid dihydrochalcones is little understood, and no evidence exists for C‐glycosyl flavonoid absorption in humans. The present study uses catechol‐O‐methyltransferase to generate methylated metabolites of aspalathin (a C‐glycosyl dihydrochalcone from rooibos tea). One of the methylated forms, both with and without glucuronidation, was detected using LC‐MS/MS in the urine of human subjects (n = 6), demonstrating that deglycosylation is not a prerequisite for C‐glycosyl flavonoid absorption. Methylation is catalysed by both intestine and liver cytosolic extracts. The results show that flavonoid C‐glycosides are methylated and glucuronidated in vivo in an intact form in humans.     

Flavan‐3‐ol C‐glycosides – Preparation and model experiments mimicking their human intestinal transit

In order to study the human intestinal transit of flavan‐3‐ol C‐glycosides, several C‐glycosyl derivatives were prepared by non‐enzymatic reaction of (+)‐catechin with α‐D ‐glucose, α‐D ‐galactose and α‐D ‐rhamnose, respectively. In contrast to literature data, we propose that the reaction mechanism proceeds in analogy to the rearrangement of flavan‐3‐ols during epimerization under alkaline conditions. Four of the 12 synthesized flavan‐3‐ol C‐glycosides were incubated under aerobic conditions at 37°C using saliva (2 min) and simulated gastric juice (3 h). To simulate human intestine, the C‐glycosides were also incubated under anaerobic conditions at 37°C both in human ileostomy fluid (10 h) and colostomy fluid (24 h), respectively. The flavan‐3‐ol C‐glycosides under study, i.e. (+)‐epicatechin 8‐C‐β‐D ‐glucopyranoside (1a), (+)‐epicatechin 6‐C‐β‐D ‐glucopyranoside (1d), (+)‐catechin 6‐C‐β‐D ‐galactopyranoside (2b), (+)‐catechin 6‐C‐β‐D ‐rhamnopyranoside (3b) were analyzed in the incubation samples by HPLC‐DAD and HPLC‐DAD‐MS/MS. They were found to be stable in the course of incubation in saliva, simulated gastric juice and ileostomy fluid and underwent degradation in colostomy fluid. While the 6‐C‐β‐D ‐glucopyranoside 1d was completely metabolized between 2 and 4 h, decomposition of the 6‐C‐β‐D ‐galactopyranoside 2b reached only 16±2% within 4 h of incubation. Linear degradation rates of 1d and 2b in colostomy fluid differed significantly. As microbial metabolism of flavan‐3‐ols is known not to be influenced by the stereochemistry of the aglycon, varying degradation rates are ascribed to the effect of the sugar moiety. Based on these results we assume that flavan‐3‐ol C‐glycosides pass through the upper gastrointestinal tract (oral cavity, stomach and small intestine) unmodified and are then metabolized by the colonic microflora.     

Absorption,metabolism, and excretion of green tea flavan‐3‐ols in humans with an ileostomy

Green tea containing 634 μmol of flavan‐3‐ols was ingested by human subjects with an ileostomy. Ileal fluid, plasma, and urine collected 0–24 h after ingestion were analysed by HPLC‐MS. The ileal fluid contained 70% of the ingested flavan‐3‐ols in the form of parent compounds (33%) and 23 metabolites (37%). The main metabolites effluxed back into the lumen of the small intestine were O‐linked sulphates and methyl‐sulphates of (epi)catechin and (epi)gallocatechin. Thus, in subjects with a functioning colon substantial quantities of flavan‐3‐ols would pass from the small to the large intestine. Plasma contained 16 metabolites, principally methylated, sulphated, and glucuronidated conjugates of (epi)catechin and (epi)gallocatechin, exhibiting 101–256 nM peak plasma concentration and the time to reach peak plasma concentration ranging from 0.8 to 2.2 h. Plasma pharmacokinetic profiles were similar to those obtained with healthy subjects, indicating that flavan‐3‐ol absorption occurs in the small intestine. Ileostomists had earlier plasma time to reach peak plasma concentration values than subjects with an intact colon, indicating the absence of an ileal brake. Urine contained 18 metabolites of (epi)catechin and (epi)gallocatechin in amounts corresponding to 6.8±0.6% of total flavan‐3‐ol intake. However, excretion of (epi)catechin metabolites was equivalent to 27% of the ingested (?)‐epicatechin and (+)‐catechin.     

Profiling the Ginsenosides of Three Ginseng Products by Lc‐Q‐Tof/Ms

Ginseng is a well‐known herbal medicine that has been gaining increasingly popularity as a potential chemopreventive agent. In traditional Chinese medicine practice, white ginseng (WG), red ginseng (RG), and dali ginseng (DG) are 3 different ginseng‐processed products used for different purposes. Although the morphological appearance and some constituents contained in these ginseng products are similar, their pharmacological activities are significantly different due to the varied types and quantity of ginsenosides in each product. In the present study, a practical method based on rapid liquid chromatography coupled with quadrupole time of flight mass spectrometry (LC‐Q‐TOF/MS) was developed to identify the chemical profiles of ginsenosides in these 3 ginseng products. The results demonstrated that a total of 55, 53, and 43 compounds were unambiguously assigned or tentatively identified in DG, WG, and RG samples, respectively. The featured compounds are mainly malonyl ginsenosides in WG, and decarboxyl products of mal‐ginsenosides and the dehydrated compounds from polar ginsenosides were characteristic in RG, while DG contain some characteristic components present both in WG and RG. We presume that heating processing is the major factor affecting the chemical profile of ginseng products. The difference of chemical information revealed by LC‐Q‐TOF/MS could be used to discriminate the WG, RG, and DG samples.     

LC‐MS identification of anthocyanins in boysenberry extract and anthocyanin metabolites in human urine following dosing

The anthocyanin composition of boysenberry (Rubusloganbaccus × baileyanus Britt) extract was determined by LC‐ESI‐MS. Four anthocyanins were identified, all comprising a cyanidin‐anthocyanidin‐type skeleton. The two major components were identified as the disaccharide cyanidin‐3‐O‐sophoroside and the monosaccharide cyanidin‐3‐O‐glucoside. The two less abundant components were identified as the rutinosides, cyanidin‐3‐O‐2^G‐glucosylrutinoside and cyanidin‐3‐O‐rutinoside, respectively. These same four anthocyanins were also detected in human urine following a dosing study with boysenberry extract indicating that glycosylated anthocyanins can be absorbed from the gut and excreted intact in the urine. Several anthocyanin metabolites were also detected in the urine and were identified by LC‐ESI‐MS as monoglucuronides of peonidin, cyanidin and pelargonidin. The results suggest that anthocyanins consumed as part of a diet are bioavailable and are present as intact or metabolized forms in the body. Copyright © 2004 Society of Chemical Industry     

Isolation,purification and characterisation of angiotensin I‐converting enzyme–inhibitory peptides derived from catfish (Clarias batrachus) muscle protein thermolysin hydrolysates

The angiotensin I‐converting enzyme (ACE)‐inhibitory activities of catfish (Clarias batrachus) muscle protein hydrolysates were investigated. Thermolytic digests of C. batrachus sarcoplasmic and myofibrillar proteins exhibited inhibitory activity towards ACE and were purified with the aim of ultrafiltration, gel filtration and reversed‐phase high‐performance liquid chromatography (RP‐HPLC). The amino acid sequences of hydrolysates with the highest ACE‐inhibitory activities were determined using electrospray quadrupole time‐of‐flight tandem mass spectrometry (ESI‐TOFQ MS/MS). The sequences of GPPP (IC₅₀ = 0.86 μm ) and IEKPP (IC₅₀ = 1.2 μm ) corresponding to the fragments 986–989 and 441–445 of myosin‐I heavy chain were identified for the sarcoplasmic and myofibrillar protein hydrolysates, respectively. Peptide GPPP exhibited a mixed‐type inhibition whereas peptide IEKPP could only bind to the active sites of ACE. The results demonstrate that hydrolysates of C. batrachus muscle proteins obtained by thermolysin may contain bioactive peptides.     

Determination of antioxidant activity and phenolic compounds of Muntingia calabura Linn. peel by HPLC‐DAD and UPLC‐ESI‐MS/MS

Antioxidant activity in Muntingia calabura Linn. peel was evaluated by DPPH radical, ORAC, ABTS cation radical, FRAP assays and total phenolic contents by different extraction conditions. In addition, a method for determination of phenolic compounds in calabura peel samples harvested in Brazil using methanol:water and magnetic stirring as the extraction method, HPLC‐DAD and UPLC‐ESI‐MS/MS analysis were developed. Calabura peel showed antioxidant activity for all extraction conditions and assays evaluated, the most polar solvents being more effective. The developed HPLC‐DAD method allowed the accurate determination of phenolic compounds, with recoveries in the range of 72–107% and precision values ≤4%, with exception for chlorogenic acid. Gallic acid was determined at the highest concentration levels, followed by myricetin, ferulic acid and vanillic acid. However, all the five proposed phenolic compounds were identified in calabura peel samples by UPLC‐ESI‐MS/MS. Thus, calabura peel, an uncommon edible fruit part, can be appointed as a rich source of phenolic compounds.     

Preparation of Methylated Products of A‐type Procyanidin Trimers in Cinnamon Bark and Their Protective Effects on Pancreatic β‐Cell

Polyphenols are partial metabolized to methylated conjugations in vivo, and then could modify bioavailability and bioactivity related to the uptake of parent compounds. Our previous studies have found that the antidiabetic effects of cinnamon barks are mainly related to polyphenol components, particularly A‐type procyanidin trimer cinnamtannin‐1 (CT1). It is necessary to understand the antidiabetic activity of methylations of CT1, nevertheless, sufficient amounts of methylated CT1 are difficult to obtain from metabolites in vivo. In this study, O‐methyl derivatives of CT1 were prepared through one‐pot methyl iodide reaction and isolation via column chromatography and RP‐HPLC semipreparation. The structures of O‐methyl substituents were determined through NMR (Nuclear Magnetic Resonance) and HPLC‐ESI‐MS (High‐performance liquid chromatography‐electrospray ionization‐mass spectrometry). Five purified O‐methyl substituents and 2 isomers of CT1 were obtained. Their protective effects on a palmitic acid‐induced pancreatic β‐cell apoptosis model were then evaluated. Results showed that the protective effects on pancreatic β‐cell of O‐methyl substituents were weaker than those of CT1. The results suggested that the methylation of catechol groups could be a relevant factor contributing to the decline of protective effects on pancreatic β‐cell of CT1 via obstructing quinone intermediate formation and affecting antioxidant abilities. The antidiabetic effects of O‐methyl derivatives of CT1 should be further determined by other antidiabetic models.     

Improved extraction of disulphide‐rich bioactive proteins from soya hulls: characterisation of a novel aspartic proteinase

Soya hull, an underutilised coproduct of soya processing, was investigated as a source of disulphide‐rich bioactive proteins. A Viscozyme L‐assisted extraction method was developed to improve the yield of extracted proteins. The extracted proteins were identified by MALDI TOF–TOF MS, and the most abundant disulphide‐rich protein among identified proteins was purified and the enzymatic properties were evaluated. The results indicated that the Viscozyme L‐assisted extraction method extracted significantly (P < 0.05) more proteins (39.01%) than did the aqueous method (4.52%). The yield of the purified aspartic proteinase nepenthesin‐1‐like [Glycine max] (GmAPN1K) (the most abundant disulphide‐rich protein) is 570 mg Kg^?1. The specific activity of GmAPN1K was 62.1 U mg^?1 at pH 3.0 and 37 °C. The enzyme was optimally active at pH 3.0 and 55 °C. It was stable within pH range 3.0–10.0 and up to 55 °C, respectively, and was specifically inhibited by pepstatin A.     

Liquid Chromatography‐Diode Array and Electrospray High‐Accuracy Mass Spectrometry of Iso‐α‐Acids in DCHA‐Iso Standard and Beer

Excellent liquid chromatographic (LC) separation of cis/trans stereoisomers of iso‐α‐acids has been achieved with reversed‐phase sorbent XTerra MS C18. An isocratic alkaline mobile phase, consisting of a mixture of 5 mmol l^?1 ammonium acetate (pH 8)‐acetonitrile‐methanol, 62:21:17 (v/v/v), was used. In the DCHA‐Iso international calibration standard trans‐isoposthumulone was identified combining photo diode array (PDA) spectra and electrospray high‐accuracy mass spectrometric (MS) data. Moreover, the molecular mass of two degradation products resulting from the in‐solution storage of the DCHA‐Iso standard was determined. The presence of trans and cis isomers of isoposthumulone, isocohumulone, isoadhumulone and iso‐n‐humulone in beer samples was confirmed. The trans isomers of iso‐α‐acids showed characteristic and reproducibly slightly different ultraviolet absorbance spectra with respect to cis isomers.