Comparative chemical profiling of three TCM drugs in the Paeoniaceae family by UPLC-MS/MS combined with chemometric methods

Paeoniae Radix Rubra (Chi-Shao in Chinese) and Paeoniae Radix Alba (Bai-Shao in Chinese) are two valuable traditional Chinese medicine (TCM) drugs, usually indicated for menstrual disorders and viral infections. Paeonia anomala subsp. anomala (Xinjiang-Shaoyao in Chinese) is taken as Chi-Shao substitute in Xinjiang, China. Due to the diverse growing conditions, there are some differences in chemical compositions of three TCM drugs. An UPLC fingerprint analysis with chemometric methods, including similarity analysis and hierarchical clustering analysis, was applied in the study. By virtue of UPLC-QTOF-MS, 29 components including 18 monoterpene glycosides, 5 galloyl glucoses and 6 phenolic compounds were simultaneously identified. It could be concluded that the UPLC-QTOF-MS combined with chemometric methods could efficiently identify the three TCM drugs, and was a powerful approach for the chemical profiling of three TCM drugs. The developed method provide an available approach for quality control of three TCM drugs.     

Identification and quantification of galloyl derivatives,flavonoid glycosides and anthocyanins in leaves of Pistacia lentiscus L

Separation, identification and quantification of polyphenols was carried out on leaves of Pistacia lentiscus L., an evergreen member of the family Anacardiaceae, using semi-preparative HPLC, HPLC-photodiode array detection and HPLC-MS analysis, together with 1H- and 13C NMR. Three major classes of secondary metabolites were detected: (i) gallic acid and galloyl derivatives of both glucose and quinic acid; (ii) flavonol glycosides, i.e. myricetin and quercetin glycosides; and (iii) anthocyanins, namely delphinidin 3-O-glucoside and cyanidin 3-O-glucoside. Low amounts of catechin were also detected. The concentration of galloyl derivatives was extremely high, representing 5.3% of the leaf dry weight, and appreciable amounts of myricetin derivatives were also detected (1.5% on a dry weight basis). These findings may be useful in establishing a relationship between the chemical composition of the leaf extract and the previously reported biological activity of P. lentiscus, and may also assign a new potential role of P. lentiscus tissue extracts in human health care.     

Chemotaxonomy and flavonoid profiling of Torilis species by HPLC/ESI/MS2

The flavonoid profiles of Turkish Torilis Gaertn. (Apiaceae) species were studied by TLC, HPLC-UV and HPLC/ESI/MS² (negative mode). O-glycosides of luteolin, apigenin and chrysoeriol were identified from crude extracts with the help of mass spectra in different MS/MS modes, such as full scan, precursor ion scan and product ion scan. Luteolin-7-O-glucoside and luteolin-7-O-rutinoside were common to all species. Flavonoid profiles usually differ from one species to another and can be put to use for a genus such as Torilis which has been little studied. By the help of different flavonoid profiles, it is concluded that, the plants, which are recognised as less rayed subspecies of Torilis arvensis (Huds.) Link. in various floras including Turkish one, must be classified in species category as Torilis chrysocarpa and Torilis purpurea. Flavonoid profiles seem to be in relation with evolutionary biogeography of the species. Because the most isolated species of the genus, endemic Torilis triradiata, has the most different flavonoid pattern. Moreover, geographically isolated species, T. triradiata and Torilis leptocarpa, do not share any flavonoid except for the two which are common to all species.     

Differentiation of isomeric malonylated flavonoid glyconjugates in plant extracts with UPLC-ESI/MS/MS

Introduction: Glycosylation at different hydroxyl groups of flavonoids and acylation of sugar moieties are ubiquitous modifications observed in plants. These modifications give rise to simultaneous presence of numerous isomeric and isobaric compounds in tissues and extracts thereof. Objective: To develop UPLC‐MS method capable for resolution of isomeric malonylated glycoconjugates of flavonoids and recognition of structural differences. Methodology: Flavonoid glycoconjugates were extracted from leaves of blue lupin (Lupinus angustifolius L.) plants with 80% methanol. Extracts were analysed using ultraperformance liquid chromatography (UPLC) combined with tandem (quadrupole–time of flight, QToF) mass spectrometry. Results: Differentiation of malonylated glycosides of isoflavones and flavones is demonstrated in this paper. The use of UPLC‐MS/MS enabled 38 flavonoid conjugates to be distinguished, including the discrimination of five different isomers of a single 3′‐O‐methylluteolin glycoside. Additionally, pseudo MS³ experiments (CID spectra registered at high cone voltages) enabled confirmation of the aglycone structures by comparison of their spectra with those obtained from aglycone standards. Conclusions: Application of UPLC‐MS/MS allows separation and identification numerous positional isomers of malonylated glycosides of flavonoids and isoflavonoids in plant material. Provided there is strict control of the MS ionisation parameters, this method may be useful for preparation of a flavonoids spectra database, enabling the inter‐laboratory comparison of analytical results. Copyright © 2008 John Wiley & Sons, Ltd.     

Flavonoid glycosides from the leaves of Aphananthe aspera (Thunb.) Planch. (Cannabaceae) and their chemotaxonomic significance

From the leaves of Aphananthe aspera (Thunb.) Planch. (Family: Cannabaceae), six flavonol glycosides, such as quercetin 3-O-β-glucopyranoside (1), kaempferol 3-O-β-glucopyranoside (2), quercetin 3-O-rutinoside (3), kaempferol 3-O-rutinoside (4), quercetin 3-O-neohesperidoside (5) and kaempferol 3-O-neohesperidoside (6) were isolated and identified. Structure elucidation of these compounds was performed on the basis of NMR spectral data. All these compounds were isolated for the first time from the genus Aphananthe. Chemotaxonomic significance and distribution of these flavonoid derivatives among the genera of Cannabaceae are explained in detail.     

The flavonoid chemistry of the genusTrillium

Four flavonol glycosides (Fig.1) were isolated from the leaves ofTrillium tschonoskii Maxim. By means of UV, NMR, and mass spectral analyses, they were identified to be acetylated kaempferol 3-O-arabinosylgalactoside (TK-1), kaempferol 3-O-arabinosylgalactoside (TK-2), acetylated quercetin 3-O-arabinosylgalactoside (TQ-1) and quercetin 3-O-arabinosylgalactoside (TQ-2). High performance liquid chromatography (HPLC) profiles of 172 specimens ofT. tschonoskii collected from nine different places in Japan were grouped into three different types based on the flavonoid components: type I and type II containing TK-1 and TQ-1, and TK-2 and TQ-2, respectively, as main component, and type III containing all of four flavonol glycosides. Those results show that the intraspecific variation ofT. tschonoskii with different geographical distribution has not only been found by the analysis of karyotype, but also that of flavonoid components.     

MS/MS library facilitated MRM quantification of native peptides prepared by denaturing ultrafiltration

ABSTRACT: Naturally occurring native peptides provide important information about physiological states of an organism and its changes in disease conditions but protocols and methods for assessing their abundance are not well-developed. In this paper, we describe a simple procedure for the quantification of non-tryptic peptides in body fluids. The workflow includes an enrichment step followed by two-dimensional fractionation of native peptides and MS/MS data management facilitating the design and validation of LC- MRM MS assays. The added value of the workflow is demonstrated in the development of a triplex LC-MRM MS assay used for quantification of peptides potentially associated with the progression of liver disease to hepatocellular carcinoma.     

Methylmalonic acid quantification in low serum volumes by UPLC-MS/MS

Methylmalonic acid (MMA) is a metabolic intermediate transformed to succinic acid (SA) by a vitamin B(12)-dependent catalytic step, and is broadly used as a clinical biomarker of functional vitamin B12 status. However, reported methods use between 100 and 1000 μL of serum or plasma making them sub-optimal for sample-limited studies, including those with neonates and infants. LC-MS/MS based protocols to measure MMA as n-butyl esters in the presence of tri-deuterated MMA (MMA-d(3)) were modified for use with 25 μL of human serum by scaling down sample processing volumes and analysis by UPLC-MS/MS. Plasma-based calibration solutions were found to be unnecessary, and chromatographic resolution and peak shape of SA and MMA was optimized in <4 min with isocratic 53:47 methanol/1.67 mM (pH 6.5) ammonium formate. Additionally, 1-cyclohexyl-urido-3-dodecanoic acid (CUDA) was included as internal standard allowing direct assessment of MMA recovery. Sample concentrations in the low normal range produced a signal:noise of >100:1. MMA intra- and inter-assay variability was under 10%. MMA-d(3) surrogate recovery averaged 93±14%. MMA stability exceeded three years in frozen samples and was unaffected by up to five freeze/thaw cycles. In conclusion, we report that methylmalonic acid can be measured with 25 μL of serum using water based standards. The assay signal:noise per concentration indicates that the method could perform as implemented with as little as 5 μL of serum. The reported method is applicable for studies of functional B12 status in sample limited experiments including investigations of nutritional status in neonates and in studies where low normal MMA levels are expected.     

Individual variation of flavonoid glycosides in Chaerophyllum aureum

Individuals (45) of Chaerophyllum aureum collected from six stations in a 1500 m perimeter of the Ubaye Valley (southern Alps, France) were surveyed for their flavonoid glycoside content. Factor analysis of correspondence applied to the distribution of these compounds showed—as noted before—luteolin-7 diglucoside is a discriminating factor of specimens from tall grass prairies (Adenostylion) against those from meadows (Triseto-Polygonion). In addition, populations of Triseto-Polygonion and Adenostylion differ in their chemical homogeneity: those from meadows display a far greater heterogeneity than those growing in the tall grass prairies. This can be interpreted as the result of a stronger selective pressure in the meadows, from human activities.     

Sensitive quantification of sirolimus and everolimus by LC–MS/MS with online sample cleanup

Sirolimus and its derivative everolimus are widely used today as immunosuppressive agents for example in the transplantation medicine. The problematic pharmacokinetic behavior of those substances makes therapeutic drug monitoring mandatory. Therefore, a fast, simple and sensitive high-throughput procedure using online extraction with turbulent flow chromatography for the concurrent measurement of sirolimus and everolimus has been developed. 200 μl of whole blood was mixed with internal standard (23-desmethoxyrapamycin) and the precipitation solution and centrifuged. An aliquot of the supernatant was transferred into autosampler vials. 50 μl of the supernatant was injected into the LC system, where the analytes were extracted using turbulent flow chromatography and thereafter analyzed using reversed phase chromatography. Detection was done by atmospherical pressure chemical ionization (APCI) mass spectrometry in the negative ionization mode. The method has been fully validated and compared to a previously used method. The method was shown to be linear over the entire calibration range (2.2–43.7 μg/l for everolimus and 2.9–51.2 μg/l for sirolimus). The lower limit of quantification was 0.5 μg/l for both compounds. For within-day and between-day analysis, the CV's were <7.6% for everolimus and <8.7% for sirolimus, respectively. The accuracy was between 92.1% and 105% for everolimus and 96.1% and 106% for sirolimus. Recovery ranged between 46.3% and 50.6% for everolimus and 51.2% and 57.2% for sirolimus. The method was demonstrated to be free of matrix effects and comparable to the previously used method. The presented LC–MS/MS method, using turbulent flow chromatography online extraction, allows a fast, simple and reliable determination of everolimus and sirolimus.     

Characterization of pertussis toxin by LC-MS/MS

Liquid chromatography-mass spectrometry (LC-MS) with a dual spray electrospray ionization source has been used to measure the molecular weights of pertussis toxin (PT) subunits. Measurement accuracy better than 0.4 Da was achieved for all PT subunits in the molecular weight range of 11,000 to 27,000 Da. At this mass assignment accuracy level, the sequences of the PT subunits investigated in this study are easily determined based on molecular weight alone. The subunits 1, 2, and 5 of PT were observed to undergo oxidation under normal storage conditions as ammonium sulfate suspension at 2 to 8 degrees C. These oxidized subunits can be separated completely or partially by reverse-phase high-performance liquid chromatography (HPLC) from their native counterparts. For the determination of oxidation sites, the oxidized subunits and their nonoxidized counterparts were fraction collected, trypsin digested, and mapped by LC-MS. The oxidized peptides and their nonoxidized counterparts were further studied by liquid chromatography-tandem mass spectrometry (LC-MS/MS) to confirm their identities. The methionines at position 212 of subunit 1, at position 89 of subunit 2, and at position 40 of subunit 5 were found to be the primary sites of oxidation.     

Identification and quantification of components in extracts of Uncaria tomentosa by HPLC-ES/MS

The two main classes of secondary metabolites, alkaloids and quinovic acid glycosides, of Uncaria tomentosa (Willd.) DC. (Rubiaceae), a Peruvian plant commonly known as ‘uña de gato’, have been analysed. Separation of the alkaloidal fraction was achieved using a solid phase extraction method based on cationic exchange, and an analytical method employing HPLC‐ES/MS has been developed. Quantitative data for commercial wild bark, cultivated bark and leaves are reported. The analysis of quinovic acid glycosides was performed directly on the crude extract using both a fast analytical method based on ?ow injection ES/MS, and a more complete analytical technique using HPLC‐MS. Copyright © 2004 John Wiley & Sons, Ltd.     

Characterization of the human tear metabolome by LC-MS/MS

The tear film overlying the epithelial cells of the eye's surface is vital to visual function, and its composition is reflective of ocular surface health. The ultrasmall volume of tears poses challenges in its analysis, contributing to the limited number of reports on the tear metabolome. In addition, using a standard clinical method of tear collection posed some confounding factors in metabonomic analysis. We sought to establish an analytical platform for the global characterization of human tear metabolites. Following information dependent acquisition (IDA) directed liquid chromatography-tandem mass spectrometry (LC-MS/MS), isotope pattern matched peak mining was performed using Extracted Ion Chromatogram (XIC) manager within the PeakView software. Sixty metabolites representing diverse compound classes were identified in human tears, most of which have not been previously reported. Selected metabolites were verified using pure standards. Unsupervised chemometric analysis showed good separation between tear samples and blanks (PC1 = 87%, R(2) = 0.91, Q(2) = 0.87). The results demonstrated the potential of our platform for untargeted metabonomic studies of eye diseases.