Rapid screening and identification of antioxidants in the leaves of Malus hupehensis using off‐line two‐dimensional HPLC–UV–MS/MS coupled with a 1,1′‐diphenyl‐2‐picrylhydrazyl assay

The leaves of Malus hupehensis have a strong antioxidant activity and are commonly consumed as a healthy tea. However, detailed information about its antioxidants is incomplete. Herein, we developed an effective strategy based on combining off‐line two‐dimensional high‐performance liquid chromatography with ultraviolet and tandem mass spectrometry detection with a 1,1′‐diphenyl‐2‐picrylhydrazyl assay to rapidly screen and identify the antioxidants from the leaves of M. hupehensis. In the orthogonal two‐dimensional liquid chromatography system, a Venusil HILIC column was used for the first dimension, while a Universil XB‐C₁₈ column was installed in the second dimension. As a result, 32 antioxidants, including ten dihydrochalcones, two flavanones, nine flavonols, four flavones, and seven phenolic acids were tentatively identified, out of which 23 compounds, as far as we know, were isolated and characterized from the leaves of M. hupehensis for the first time. To the best of our knowledge, this is the first systematic investigation of the antioxidants from the leaves of M. hupehensis. The results indicated that the proposed method is an efficient technique to rapidly investigate antioxidants, especially for coeluted and minor compounds in a complex system.     

Homoisoflavonoids profiling of Ophiopogon japonicus by off‐line coupling high‐speed countercurrent chromatography with high‐performance liquid chromatography–diode array detector‒quadrupole time‐of‐flight tandem mass spectrometry

Roots of Ophiopogon japonicus have been used as a functional food ingredient and traditional Chinese medicine for a long time in China. Homoisoflavonoids are one of the major kinds of bioactive compounds in O. japonicus; however, literature data about its homoisoflavonoids profile are scarce because of the complex ingredients with low abundance. Here, homoisoflavonoid fraction was prepared by petroleum ether extraction. Then, a high‐speed countercurrent chromatography off‐line coupling with high‐performance liquid chromatography–diode array detector?quadrupole time‐of‐flight tandem mass spectrometry was developed for systematic identification of homoisoflavonoids. After that, 39 homoisoflavonoids, including 29 homoisoflavanone and 10 homoisoflavone, were unambiguously or tentatively identified, while 12 of them were reported in O. japonicus for the first time. Finally, eight available homoisoflavonoids were sensitively, precisely, and accurately determined by standard calibration curves, with limit of detection and limit of quantification in the range of 0.05–0.30 μg/mL and 0.12–0.66 μg/mL, relative standard deviation less than 7.3% for intra‐ and interday variations, and recovery at 94.5–105.2%. Collectively, our developed method is efficient, reliable, and valuable to profile chemical components of complex natural products.     

Identification of metabolites in WZS‐miniature pig urine after oral administration of Danshen decoction by HPLC coupled with diode array detection with electrospray ionization tandem ion trap and time‐of‐flight mass spectrometry

Danshen (DS) is a widely used traditional Chinese medicine for treating cardiovascular and cerebrovascular diseases. A simple, rapid and sensitive method was developed for identification of the in vivo metabolites in urine of WZS‐miniature pigs after oral administration of DS decoction by HPLC coupled with diode array detection with electrospray ionization tandem ion trap and time‐of‐flight mass spectrometry. This method has been successfully applied to simultaneous identification of 50 compounds (including 11 new ones) in pig urine. In addition, one new compound, (3‐hydroxyphenyl) crylic acid glycine methyl ester (C1), along with eight known ones were first isolated by column chromatography and identified by spectroscopic means, including 1D/2DNMR and mass spectrometry, as reference substances. Ten phenolic compounds (protocatechuic aldehyde, protocatechuic acid, caffeic acid, danshensu, ferulic acid, isoferulic acid, rosmarinic acid and salvianolic acid A/B/D) were found to be the main absorbed original constituents of DS decoction, which underwent the metabolic reactions of glucuronidation, sulfation, methylation, hydrogenation and glycine conjugation in vivo. In conclusion, the developed method is applicable to the analysis and identification of constituents in biological matrices after administration of DS decoction. Copyright © 2012 John Wiley & Sons, Ltd.     

In vivo metabolite profiles of isoimperatorin and phellopterin in rats analyzed using HPLC coupled with diode array detector and electrospray ionization ion trap time‐of‐flight mass spectrometry technique

Isoimperatorin (IP) and phellopterin (PP) are two furocoumarins existing in Angelicae Dahuricae Radix. There is an isopentenyloxyl substituted at C‐5 in IP, and an isopentenyloxyl and a methoxyl substituted at C‐8 and C‐5, respectively, in PP. To elucidate the in vivo metabolic characteristics of PP and IP, HPLC coupled with diode array detector and electrospray ionization ion trap time‐of‐flight mass spectrometry technique was used. In total, 111 metabolites, including 53 new ones, were identified from the urine and plasma samples of rats after oral administration of IP and PP, respectively. The metabolites were formed through eight reactions on IP and PP: oxidation, hydroxylation–hydrogenation, carboxylation on the isopentenyloxyl, O‐dealkylation, hydroxylation on the furocoumarin nucleus, ring‐opening reaction on the furan ring and reduction or ring‐opening reaction on the lactone ring. Among these, hydroxylation on the furocoumarin nucleus was found for the first time for in vivo metabolites of PP and IP, and the ring‐opening reaction on the furan ring or lactone ring was found for the first time for in vivo metabolites of isopentenyloxyl furocoumarins. The research gave us a new insight into the in vivo metabolic profiles of IP and PP, which could help us better understand their important roles as two active constituents of Angelicae Dahuricae Radix.     

Characterization and simultaneous quantification of biological aporphine alkaloids in Litsea cubeba by HPLC with hybrid ion trap time‐of‐flight mass spectrometry and HPLC with diode array detection

The root and rhizome of Litsea cubeba (Lour) Pers., named ‘Dou‐chi‐jiang’ in Chinese, has been traditionally used for treatment of cardiovascular and cerebrovascular diseases, rheumatic arthralgia, and other diseases in China. Aporphine alkaloids are its characteristic ingredients and responsible for its bioactivities, especially anti‐inflammatory and analgesic effects. A sensitive and reliable high‐performance liquid chromatography with diode array detection‐tandem mass spectrometry method was developed for characterization and simultaneous determination of biological aporphine alkaloids in ‘Dou‐chi‐jiang’. The optimized chromatographic conditions were performed on an Eclipse XDB C₁₈ column with a gradient of acetonitrile/water containing 0.1% formic acid as the mass spectrometry mobile phase and acetonitrile/water containing 0.2% diethylamine (pH 3.10, adjusted by acetic acid) as the liquid chromatography mobile phase. The fragmentation pathways by loss of CO, ·CH₃, ·NH₃, and ·NH₂CH₃ were detected as characteristic for aporphine alkaloids. Based on these characteristics, total 12 analogues were identified. The quantification method was validated in terms of linearity, precision, and accuracy for six major aporphine alkaloids, which was successfully applied for simultaneous determination in ten batches of samples. The established method is simple, rapid, and specific for characterization and quantitation of aporphine alkaloids in ‘Dou‐chi‐jiang’ and other traditional Chinese medicines rich in this kind of ingredient.     

Lipid profiling of cyanobacteria Synechococcus sp. PCC 7002 using two‐dimensional liquid chromatography with quadrupole time‐of‐flight mass spectrometry

Glycerolipid is a main component of membranes in oxygenic photosynthetic organisms. Up to now, the majority of publication in this area has focused on the physiological functions of glycerolipids and lipoprotein complexes in photosynthesis, but the study on the separation and identification of glycerolipids in thylakoid membrane in cyanobacteria is relatively rare. Here we report a new method to separate and identify five photosynthetic glycerolipid classes, including monoglucosyl diacylglycerol, monogalactosyl diacylglycerol, digalactosyl diacylglycerol, sulfoquinovosyl diacylglycerol, and phosphatidylglycerol, in cyanobacteria Synechococcus sp. PCC 7002 by two‐dimensional (normal‐ and reversed‐phase) liquid chromatography online coupled to quadrupole time‐of‐flight mass spectrometry. Over twice as many lipid species were detected by our method compared to the previously reported methods. Ten new odd‐chain fatty acid glycerolipids were discovered for the first time. Moreover, complete separation of isomers of monogalactosyl diacylglycerol and monoglucosyl diacylglycerol was achieved. According to the tandem mass spectrometry results, we found that the head group of monoglucosyl diacylglycerols was not as stable as that of monogalactosyl diacylglycerols, which might explain why the organism chose monogalactosyl diacylglycerols and digalactosyl diacylglycerols instead of monoglucosyl diacylglycerols as the main content of the photosynthetic membranes in the history of evolution. This work will benefit further research on the physiological function of glycerolipids.     

Simultaneous determination of niacin and pyridoxine at trace levels by using diode array high‐performance liquid chromatography and liquid chromatography with quadrupole time‐of‐flight tandem mass spectrometry

A highly sensitive and simple diode‐array high‐performance liquid chromatography and liquid chromatography with quadrupole time‐of‐flight tandem mass spectrometry method was developed for the simultaneous determination of niacin and pyridoxine in pharmaceutical drugs, tap water, and wastewater samples. To determine the in vivo behavior of niacin and pyridoxine, analytes were subjected to simulated gastric conditions. The calibration plots of the diode‐array high‐performance liquid chromatography and liquid chromatography with quadrupole time‐of‐flight tandem mass spectrometry method showed good linearity over a wide concentration range with close to 1.0 correlation coefficients for both analytes. The limit of detection/limit of quantitation values for liquid chromatography quadrupole time‐of‐flight tandem mass spectrometry analysis were 1.98/6.59 and 1.3/4.4 μg/L for niacin and pyridoxine, respectively, while limit of detection/limit of quantitation values for niacin and pyridoxine in high‐performance liquid chromatography analysis were 3.7/12.3 and 5.7/18.9 μg/L, respectively. Recovery studies were also performed to show the applicability of the developed methods, and percentage recovery values were found to be 90–105% in tap water and 94–97% in wastewater for both analytes. The method was also successfully applied for the qualitative and quantitative determination of niacin and pyridoxine in drug samples.     

Determination and comparison of agarwood from different origins by comprehensive two‐dimensional gas chromatography–quadrupole time‐of‐flight mass spectrometry

Agarwood, a species of resinous heartwood, is a precious medicinal plant and a type of rare natural spice, which is widely used in medicine, cosmetics, religious activities, and other fields. In this study, agarwood samples from eight different regions across four countries were analyzed by comprehensive two‐dimensional gas chromatography?quadrupole time‐of‐flight mass spectrometry. A total of 232 species were identified (the match factors of these compounds were above 750). The main compounds of agarwood are oxygenated sesquiterpenes and chromones. The compositions of India1 and Malaysia2 were significantly different from those of other samples, which might be attributed to the different production processes of agarwood. For further investigation, factor analysis was conducted for six agarwood samples. The results showed that the data classification possessed a regional characteristic; according to the retention time and relative content, characteristic compositions were determined by factor scores. Finally, the differences of characteristic compositions were simply analyzed, and the reasons were speculated.     

Chemometrics comparison of gas chromatography with mass spectrometry and comprehensive two‐dimensional gas chromatography with time‐of‐flight mass spectrometry Daphnia magna metabolic profiles exposed to salinity

The performances of gas chromatography with mass spectrometry and of comprehensive two‐dimensional gas chromatography with time‐of‐flight mass spectrometry are examined through the comparison of Daphnia magna metabolic profiles. Gas chromatography with mass spectrometry and comprehensive two‐dimensional gas chromatography with mass spectrometry were used to compare the concentration changes of metabolites under saline conditions. In this regard, a chemometric strategy based on wavelet compression and multivariate curve resolution–alternating least squares is used to compare the performances of gas chromatography with mass spectrometry and comprehensive two‐dimensional gas chromatography with time‐of‐flight mass spectrometry for the untargeted metabolic profiling of Daphnia magna in control and salinity‐exposed samples. Examination of the results confirmed the outperformance of comprehensive two‐dimensional gas chromatography with time‐of‐flight mass spectrometry over gas chromatography with mass spectrometry for the detection of metabolites in D. magna samples. The peak areas of multivariate curve resolution–alternating least squares resolved elution profiles in every sample analyzed by comprehensive two‐dimensional gas chromatography with time‐of‐flight mass spectrometry were arranged in a new data matrix that was then modeled by partial least squares discriminant analysis. The control and salt‐exposed daphnids samples were discriminated and the most relevant metabolites were estimated using variable importance in projection and selectivity ratio values. Salinity de‐regulated 18 metabolites from metabolic pathways involved in protein translation, transmembrane cell transport, carbon metabolism, secondary metabolism, glycolysis, and osmoregulation.     

A strategy for screening and identifying mycotoxins in herbal medicine using ultra‐performance liquid chromatography with tandem quadrupole time‐of‐flight mass spectrometry

The objective of this study was to develop an effective strategy for screening and identifying mycotoxins in herbal medicine (HM). Here, Imperatae Rhizoma, a commonly used Chinese herb, was selected as a model HM. A crude drug contaminated with fungi was analyzed by comparing with uncontaminated ones. Ultra‐performance LC coupled to tandem quadrupole TOF‐MS (UPLC–Q‐TOF‐MS) with collision energy function was applied to analyze different samples from Imperatae Rhizoma. Then, MarkerLynx^TM software was employed to screen the excess components in analytes, compared with control samples, and those selected markers were likely to be the metabolites of fungi. Furthermore, each of the accurate masses of the markers obtained from MarkerLynx^TM was then searched in a mycotoxins/fungal metabolites database established in advance. The molecular formulas with relative mass error between the measured and theoretical mass within 5 ppm were chosen and then applied to MassFragment^TM analysis for further confirmation of their structures. With the use of this approach, five mycotoxins that have never been reported in HM were identified in contaminated Imperatae Rhizoma. The results demonstrate the potential of UPLC–Q‐TOF‐MS coupled with the MarkerLynx^TM software and MassFragment^TM tool as an efficient and convenient method to screen and identify mycotoxins in herbal materials and aid in the quality control of HM.     

Ultra high performance liquid chromatography with photodiode array detector and quadrupole time‐of‐flight tandem mass spectrometry coupled with discriminant analysis to evaluate Angelicae pubescentis radix from different regions

A rapid and effective method was developed for the qualitative and quantitative analysis of the major chemical constituents in Angelicae pubescentis radix by ultra high performance liquid chromatography with photodiode array detection and quadrupole time‐of‐flight tandem mass spectrometry. The chromatographic separation was achieved on an ACQUITY UHPLC BEH C₁₈ column (2.1 × 100 mm, 1.7 μm). Nine phenolic acids, 30 coumarins, bisabolangelone, and adenosine were identified by quadrupole time‐of‐flight tandem mass spectrometry. All calibration curves exhibited good linearity (r > 0.9996) within the linear ranges. The relative standard deviation calculated for intraday and interday precision, stability, and accuracy were <5%. The mean recovery ranged from 95.8 to 106%. The overall limits of detection and quantification were 0.025–0.160 and 0.100–0.560 μg/mL, respectively. Discriminant analysis was investigated as a method for evaluating the quality of the samples with 100% correction in their classification. The results demonstrated that the developed method could successfully be used to differentiate samples from different regions and could be a helpful tool for detection and confirmation of the quality of traditional Chinese medicines.     

Serum metabolomics of laryngeal cancer based on liquid chromatography coupled with quadrupole time‐of‐flight mass spectrometry

The discovery of new laryngeal cancer‐related metabolite biomarkers could help to facilitate early diagnosis. A serum metabolomics study from laryngeal cancer patients and healthy individuals was conducted using liquid chromatography coupled with quadrupole time‐of‐flight mass spectrometry. Univariate and multivariate statistics were used to discriminate laryngeal cancer patients and healthy individuals. 1‐Palmitoyl‐sn‐glycero‐3‐phosphocholine (LysoPC 16:0), 1‐o‐hexadecyl‐2‐acetyl‐sn‐glycero‐3‐phosphocholine (PAF) and 1,2‐dipalmitoyl‐sn‐glycero‐3‐phosphocholine were found to be significantly different between the laryngeal cancer group and the healthy group. They are mainly involved in phospholipids catabolism, linoleic acid metabolism, α‐linoleic acid metabolism and arachidonic acid metabolism. The area under the curve of the biomarker combined by two metabolites (LysoPC 16:0 and PAF) was 0.935, the sensitivity was 0.962 and the specificity was 0.825. LysoPC 16:0 and PAF may show diagnostic potential for laryngeal carcinoma.     

Analysis of volatile organic compounds in indoor environments using thermal desorption with comprehensive two‐dimensional gas chromatography and high‐resolution time‐of‐flight mass spectrometry

Building‐related health effects are frequently observed. Several factors have been listed as possible causes including temperature, humidity, light conditions, presence of particulate matter, and microorganisms or volatile organic compounds. To be able to link exposure to specific volatile organic compounds to building‐related health effects, powerful and comprehensive analytical methods are required. For this purpose, we developed an active air sampling method that utilizes dual‐bed tubes loaded with TENAX‐TA and Carboxen‐1000 adsorbents to sample two parallel air samples of 4 L each. For the comprehensive volatile organic compounds analysis, an automated thermal desorption comprehensive two‐dimensional gas chromatography high‐resolution time‐of‐flight mass spectrometry method was developed and used. It allowed targeted analysis of approximately 90 known volatile organic compounds with relative standard deviations below 25% for the vast majority of target volatile organic compounds. It also allowed semiquantification (no matching standards) of numerous nontarget air contaminants using the same data set. The nontarget analysis workflow included peak finding, background elimination, feature alignment, detection frequency filtering, and tentative identification. Application of the workflow to air samples from 68 indoor environments at a large hospital complex resulted in a comprehensive volatile organic compound characterization, including 178 single compounds and 13 hydrocarbon groups.     

Identification and metabolite profiling of alkaloids in aerial parts of Papaver rhoeas by liquid chromatography coupled with quadrupole time‐of‐flight tandem mass spectrometry

Papaver plants can produce diverse bioactive alkaloids. Papaver rhoeas Linnaeus (common poppy or corn poppy) is an annual flowering medicinal plant used for treating cough, sleep disorder, and as a sedative, pain reliever, and food. It contains various powerful alkaloids like rhoeadine, benzylisoquinoline, and proaporphine. To investigate and identify alkaloids in the aerial parts of P. rhoeas, samples were collected at different growth stages and analyzed using liquid chromatography coupled with quadrupole time‐of‐flight tandem mass spectrometry. A liquid chromatography with mass spectrometry method was developed for the identification and metabolite profiling of alkaloids for P. rhoeas by comparing with Papaver somniferum. Eighteen alkaloids involved in benzylisoquinoline alkaloid biosynthesis were used to optimize the liquid chromatography gradient and mass spectrometry conditions. Fifty‐five alkaloids, including protoberberine, benzylisoquinoline, aporphine, benzophenanthridine, and rhoeadine‐type alkaloids, were identified authentically or tentatively by liquid chromatography coupled with quadrupole time‐of‐flight tandem mass spectrometry in samples taken during various growth stages. Rhoeadine alkaloids were observed only in P. rhoeas samples, and codeine and morphine were tentatively identified in P. somniferum. The liquid chromatography coupled with quadrupole time‐of‐flight tandem mass spectrometry method can be a powerful tool for the identification of diverse metabolites in the genus Papaver. These results may help understand the biosynthesis of alkaloids in P. rhoeas and evaluate the quality of this plant for possible medicinal applications.