TLC-MALDI-TOF-MS-based identification of flavonoid compounds using an inorganic matrix.

INTRODUCTION: Thin layer chromatography (TLC) is frequently used to obtain the fingerprint of a plant extract. Although the retardation factor and the response to visualisation give primary information about compound identification, the direct TLC-mass spectrometry (MS) coupling allows a more detailed characterisation of samples. OBJECTIVES: To demonstrate the potential for the flavonoid dereplication using an inorganic matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF-MS) method with and without TLC separation. MATERIAL AND METHODS: Samples derived from wine, apple or rose were deposited on an aluminium-backed silica gel TLC sheet compatible with the MS adapter. Unlike the wine sample, for apple and rose samples compound derivatisation was necessary. These two samples were deposited twice and the plate was cut in two parts. One half was oversprayed with Neu-Peg reagent to visualise flavonoids while the inorganic matrix was deposited on each flavonoid zone on the second half for MS ionisation. RESULTS: Mass spectra obtained for samples without plate development showed numerous ions corresponding to glycosylated flavonoids. The lower m/z observed could be due either to aglycone flavonoids or to in-source fragment ions. After plate development, a separation of many spots was observed and each spot was analysed separately leading to a deeper identification of the present flavonoids. Moreover, isobaric flavonoids with different hRf values could be differentiated. CONCLUSION: TLC-MALDI-TOF-MS using an inorganic matrix enabled the analysis of anthocyanins in positive mode and of flavonols, flavanols, dihydrochalcones and phenolic acids in negative mode, reducing adduct, aggregate forms giving thus simple and reliable spectra for the dereplication approach of flavonoids in complex samples.

Molecular Fingerprint Comparison of Closely Related Rose Varieties based on UHPLC-HRMS Analysis and Chemometrics.

INTRODUCTION: The "Jardin de Granville" modern rose variety not only combines the morphological properties of its two parental cultivars, but also possesses better agronomic characteristics (abundant blooms, strong growth and vitality, high resistance to common rose diseases). In addition, it shows remarkable biological properties such as a high ability to decrease inflammatory and oxidative stress on skin cells. That is why Parfums Christian Dior selected this rose variety to be an active ingredient in luxury cosmetics. OBJECTIVES: To identify the characteristic molecular signature of "Jardin de Granville" compared with its parents "Annapurna" and "John Clare", by the mean of a non-targeted metabolomic comparison. MATERIAL AND METHODS: Wood, flower and leaf hydro-alcoholic extracts were analysed by UHPLC-ESI-HRMS. The fingerprints were then submitted to unsupervised multivariate analyses involving principal component analysis (PCA) and hierarchical ascendant classification (HAC). Analysis of variance (ANOVA) was finally performed to highlight the significant differences in each group of organs. RESULTS: The extracts were composed of phenolic compounds such as hydrolysable and condensed tannins and flavonol derivatives. Three groups of extracts were clustered as a function of the variety. The compounds overexpressed in "Jardin de Granville" variety were highlighted thanks to ANOVA test. Flower was the most discriminative organ with 15 overexpressed molecules. Auto MS/MS analyses led to their tentative identifications. CONCLUSION: The non-targeted metabolomic approach revealed the importance of tannins to discriminate close rose varieties. The overexpressed hydrolysable tannins characteristic of "Jardin de Granville" can be responsible for the antioxidant and anti-inflammatory properties of the rose cosmetic ingredients. Copyright © 2016 John Wiley & Sons, Ltd.

Development of a gas chromatography-mass spectrometry method to monitor in a single run, mono- to triterpenoid compounds distribution in resinous plant materials.

A new procedure based on gas chromatography coupled to mass spectrometry (GC-MS) was developed for the simultaneous determination of mono- to triterpenoid compounds in resinous materials. Given the difference of volatility and polarity of the studied compounds some critical steps in this methodology had to be identified and investigated. The recovery of volatile compounds after sample extraction was studied. A recovery range from 30% to 100% from the more volatile monoterpene to the least one was observed. Then the mandatory derivatization step for the analysis of pentacyclic triterpenes bearing hydroxyl and carboxyl groups was optimized. Results showed that derivatization using N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) and trimethylchlorosilane (TMCS) in pyridine (22:13:65 v/v/v) for 2h at 30 °C was the most efficient method of derivatizing all the hydroxyl and carboxylic acid groups contained in the triterpene structures. After choosing the best injection parameters for these compounds, the selectivity of the GC column towards the separation of these terpenoids was investigated using statistical tools (principal component analysis and desirability functions). A separation with a good resolution was achieved on an HP-5ms column using a programmed temperature vaporizing injector (PTV). The method was pre-validated in terms of detection limits (LOD from 100 µg L(-1) to 200 µg L(-1) depending on the compound), linearity and repeatability using seven compounds representative of mono- and triterpenoid classes. An exhaustive characterization of various types of resins (di-, triterpenic and oleo-gum resins) was achieved.

Optimization of the derivatization protocol of pentacyclic triterpenes prior to their gas chromatography-mass spectrometry analysis in plant extracts.

This paper focuses on the application of a two-level full factorial design to optimize the key derivatization step before the GC-FID and GC-MS analysis of pentacyclic triterpenes. The derivatization reaction was screened for influential factors and statistically significant parameters with a p value less than 0.05. A multi-response optimization based on a desirability function was then applied, while simultaneously considering overall detection enhancement of compounds. Results showed that derivatization using N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) and trimethylchlorosilane (TMCS) in pyridine (22:13:65v/v/v) for 2h at 30°C was the most efficient method of derivatizing all the hydroxyl and carboxylic acid groups contained in the triterpene structures. The validity of the method was demonstrated using GC-MS analyzes of a mixture containing eleven standards (ß-amyrin, α-amyrin, lupeol, erythrodiol, uvaol, betulin, oleanolic acid, betulinic acid, ursolic acid, maslinic acid and corosolic acid). These compounds are representative of different classes of terpene compounds bearing different functional groups such as alcohols, diols, and carboxylic acids. The derivatization procedure was then tested on four plant extracts: apple pomace, salvia sclarea (dried leaves and flowers), sea buckthorn (Hyppophae rhammnoides L.) berries, and B. serrata resin. The identification of triterpenes was based on the comparison of their retention time and mass spectra to those of standards. The presence of compounds already identified in the literature was confirmed and new ones such as maslinic and corosolic acids were identified in apples, sea buckthorn and salvia sclarea.

Treatment with KLEPTOSE® CRYSMEB reduces mouse atherogenesis by impacting on lipid profile and Th1 lymphocyte response.

The ability of pharmacological agents to target both "classical" risk factors and inflammation may be key for successful outcomes in the prevention and treatment of atherogenesis. Among the promising drugs interfering with cholesterol metabolism, we investigated whether methyl beta-cyclodextrin (KLEPTOSE® CRYSMEB) could positively impact on atherogenesis, lipid profile and atherosclerotic plaque inflammation in ApoE-/- mice. Eleven-week old ApoE-/- mice were fed either a normal diet (N.D.) or a high-cholesterol diet (H.D.), resulting in different levels of hypercholesterolemia. KLEPTOSE® CRYSMEB (40mg/kg) or vehicle was intraperitoneally administrated 3 times per week in the last 16weeks before euthanasia in mice under N.D. and in the last 11weeks under H.D. Treatment with KLEPTOSE® CRYSMEB reduced triglyceride serum levels in both atherogenesis mouse models. In H.D. mice, treatment with KLEPTOSE® CRYSMEB increased HDL-cholesterol levels and reduced free fatty acids and spleen weight. In both mouse models, treatment with KLEPTOSE® CRYSMEB reduced atherosclerotic plaque size in thoraco-abdominal aortas and intraplaque T lymphocyte content, but did not induce relevant improvements in other histological parameters of vulnerability (macrophage, neutrophil, MMP-9 and collagen content). Conversely and more markedly in H.D. mice, treatment with KLEPTOSE® CRYSMEB was associated with a reduction in genetic markers of Th1-mediated immune response. In vitro, KLEPTOSE® CRYSMEB dose-dependently abrogated Th1 proliferation and IFNγ release. In conclusion, treatment with KLEPTOSE® CRYSMEB reduced atherosclerotic plaque size by improving triglyceride serum levels and Th1-mediated response. These results indicate this drug as a potential tool for blocking atheroprogression associated with different severity degrees of hypercholesterolemia.

Non-targeted molecular characterisation of a rose flower ethyl acetate extract using Ultra-HPLC with atmospheric pressure photoionisation and quadrupole time-of-flight MS/MS.

INTRODUCTION: A non-targeted approach to characterise the phytochemical composition of the flower organ of an original rose cultivar 'Jardin de Granville'® was developed. Particular attention was paid to the less documented molecular families of intermediate polarity, compared with the polyphenol family (anthocyanins, flavonoids, tannins) and volatile compounds. OBJECTIVE: To develop a molecular fingerprinting method for the rapid qualitative phytochemical characterisation of the rose flower ethyl acetate extract. MATERIAL AND METHODS: An ultra-HPLC with atmospheric pressure photoionisation (APPI) and quadrupole time-of-flight (QTOF) MS/MS combined with microwave-assisted extraction was carried out for ethyl acetate extracts as an intermediate polarity extraction solvent in order to obtain the most exhaustive extract containing a large range of molecular families. An optimised methodology based on the coupling of the UHPLC and APPI source with a QTOF analyser was developed to characterise the extracted molecules. RESULTS: Sixty-one compounds were identified in the extract, covering eight molecular families of intermediate polarity ranging from polyphenols to triglycerides. The presence of flavonoids with anti-oxidant properties and of triterpenoids with potential anti-inflammatory activity was evidenced and cell-wall constituents such as fatty acids, glycolipids, sphingolipids and acylated sterol glycosides were characterised. Some chlorophyll derivatives were also detected. CONCLUSION: The method developed is appropriate for fast phytochemical evaluation of rose ethyl acetate extract. It produced accurate mass and MS/MS spectra, which permitted identification of a wide range of compounds of intermediate polarity.

Computer-assisted management of unconsumed drugs as a cost-containment strategy in oncology.

BACKGROUND: Cost-containment strategies are required to deal with rising drug expenditure, also in oncology. Drug wastage related to the preparation of chemotherapy drugs for patients is costly, but solutions exist for optimizing the use of unconsumed anticancer drugs. OBJECTIVE: Our pharmacy department makes use of a computerized drug storage bank, which records stability data and the amounts of unconsumed drugs available, and is connected to prescription software via an interface. We aimed to evaluate the real cost savings generated by this system. METHOD: We assessed the cost savings achieved with this system, for 37 different anticancer drugs, over a 1-year period. French drug pricing and the amounts of drugs from the storage bank potentially re-used were assessed. RESULTS: The re-use of unconsumed anticancer drugs generated substantial cost savings, for nine drugs in particular: azacitidine, bevacizumab, bortezomib, cetuximab, docetaxel, liposomal doxorubicin, rituximab, topotecan and trastuzumab. Overall cost savings accounted for about 5 % of total anticancer drug expenditure at our hospital (8.5 M). CONCLUSION: In medical hematology-oncology, drug wastage reduction and a computerized physician order entry system could be applied in routine practice at centralized drug-processing units, with significant financial benefits.

Phytochemical analysis of Rosa hybrida cv. 'Jardin de Granville' by HPTLC, HPLC-DAD and HPLC-ESI-HRMS: polyphenolic fingerprints of six plant organs.

The Rosa hybrida cultivar 'Jardin de Granville', a delicate clear pink flower, is here investigated through a progressive analytical strategy using complementary phytochemical screening methods in order to characterize the polyphenol content of several parts of the plant. The microwave hydro-ethanolic extract analysis of six different parts of the plant, carried out by High Performance Thin Layer Chromatography (HPTLC) and High Performance Liquid Chromatography coupled with a Diode Array Detector (HPLC-DAD) enabled initial identification of the polar molecular families present in each organ, namely tannins and flavonoids (quercetin and kaempferol derivatives). The HPLC fingerprints displayed different profiles for each organ, attesting to the original composition and potential valuation of the different plant parts. More detailed analyses of the extracts were then carried out by High Performance Liquid Chromatography coupled with electrospray ionization (ESI) mass spectrometry with a Q-TOF analyzer (ESI-HR-Q-TOF). Around 60 compounds were identified, mainly gallo-tannins, ellagi-tannins, catechin derivatives and glycoside derivatives of quercetin and kaempferol. Some compounds such as hyperoside or ellagic acid appeared to be ubiquitous and were found in abundance in each plant part. Woods were the richest organ in catechin and proanthocyanidin derivatives while kaempferol derivatives were more numerous and abundant in bud and flower parts.

New advances in countercurrent chromatography and centrifugal partition chromatography: focus on coupling strategy.

Countercurrent chromatography (CCC) is an attractive separation method because the analytes are partitioned between two immiscible liquid phases avoiding problems related to solid stationary phase. In recent years, this technique has made great progress in separation power and detection potential. This review describes coupling strategies involving high speed CCC (HSCCC) or centrifugal partition chromatography (CPC). It includes on-line extraction-isolation, hyphenation with mass spectrometry (MS) and nuclear magnetic resonance (NMR) detectors, multidimensional CCC (MDCCC), two-dimensional CCC (2D-CCC), on-line coupling with liquid chromatography (LC), and biological tests, and innovative off-line developments. The basic principles of each method are presented and applications are summarized.

Optimization of the isolation and quantitation of kahweol and cafestol in green coffee oil.

Kahweol and cafestol are two diterpenes that exist mainly as esters of fatty acids in green coffee oil. To recover them under their free form they have to be either saponified or trans-esterified. These two compounds are well known to be sensitive to heat, and reagents, therefore experimental conditions used in the transesterification reaction are critical. In this paper, a Doehlert experimental design plan is used to optimize the transesterification conditions using some key variables such as the temperature of the reaction, the reagent base concentration and the duration of the reaction. Therefore, the optimal parameters determined from the Doehlert design are equal to 70 °C, temperature of the reaction; 1.25 mol L(-1) concentration of the reagent base; and 60 min reaction time. The contour plots show that the extracted quantity of kahweol and cafestol can depend greatly from the experimental conditions. After transesterification, the free form of the diterpernes is extracted from the lipid fraction using liquid-liquid extraction and analyzed using GC-FID without prior derivatization. The amount of kahweol and cafestol obtained from green coffee oil obtained by cold mechanical press of Catuai coffee bean is equal to 33.2±2.2 and 24.3±2.4 g kg(-1)oil, respectively. In an attempt to streamline the process, the transesterification reaction is performed in an in-flow chemistry reactor using the optimal conditions obtained with the Doehlert experimental design. The amount of kahweol and cafestol obtained from the same green coffee oil is equal to 43.5 and 30.072 g kg(-1)oil, respectively. Results are slightly higher compared to the ones obtained with the batch procedure. This can be explained by a better mixing of the coffee oil with the reagents and a faster transesterification reaction.

Development of a LC-MS/MS method to monitor palmitoyl peptides content in anti-wrinkle cosmetics.

Palmitoyl peptides are anti-aging agents widely used in cosmetics. This article describes the development of a LC-MS/MS analytical procedure that allows, after a liquid-liquid extraction procedure, their unambiguous detection in cosmetic formulation. MS/MS detection is shown to be specific regarding placebo formulations. Limits of quantification, linearity, accuracy and precision of the method were estimated. The results presented show that palmitoyl peptides can be thus reliably assayed. The palmitoylated pentapeptide palmitoyl-lysyl-threonyl-threonyl-lysyl-serine (pal-KTTKS) was assayed in anti-wrinkle creams using palmitoyl-glycyl-histidyl-lysine (pal-GHK) as internal standard. From the results obtained, the influence of the formulation on pal-KTTKS availability is evidenced.

Liquid chromatography analysis of monosubstituted sulfobutyl ether-beta-cyclodextrin isomers on porous graphitic carbon.

The retention behaviour of the three positional isomers of monosubstituted sulfobutyl ether-beta-cyclodextrin was investigated on a porous graphitic carbon (PGC) column. The influence of the mobile phase composition (nature and concentration of organic and electronic modifiers) was studied as well as the effect of column temperature. These hydrophilic and anionic analytes were highly retained on the PGC stationary phase compared to octadecyl bonded phases. The retention is mainly governed by a reversed-phase mechanism with electronic interaction playing a secondary role. An increase in solute retention and efficiency with temperature was observed. Successful isocratic separation with satisfactory baseline resolution of the three isomers of monosubstituted sulfobutyl ether-beta-cyclodextrin was achieved at 75 degrees C on a Hypercarb column by using ammonium acetate as electronic modifier in water-acetonitrile (83:17). The chromatographic methodology developed can be easily used for relative quantification of each isomer within a mixture and can be applied for semi-preparative purification of each one. The evaporative light scattering detector allows the detection of these non UV-visible absorbing molecules.

Study of selenium distribution in the protein fractions of the Brazil nut, Bertholletia excelsa.

The high selenium content of the Brazil nut, Bertholletia excelsa, makes this seed a healthy food qualified as an antiradical protector. The studied nut contained 126 ppm of selenium. Selenium was found to be distributed in the nut protein fractions. The water-extracted fraction, which represented 17.7% of the cake protein, was the richest in selenium with 153 ppm. Analysis by HPLC-MS showed that selenium was linked by a covalent bond to two amino acids to form selenomethionine and selenocystine. The selenomethionine represented a little less than 1% of the total amount of methionine.

A comparative study of commercial liquid chromatographic detectors for the analysis of underivatized amino acids.

This study compares the main commercial detectors that can detect amino acids in their underivatized form. The detectors tested are: the chemiluminescent nitrogen detector (CLND), the evaporative light scattering detector (ELSD), the nuclear magnetic resonance spectrometer, conductivity detector, refractive index, UV, and electrospray quadrupole mass spectrometry (in simple and tandem MS mode). As ELSD, CLND and MS require a volatile mobile phase, an ion-pair reversed-phase liquid chromatographic system was selected, consisting of an octadecyl column and an aqueous mobile phase containing pentadecafluorooctanoic acid as volatile ion-pairing reagent. Underivatized taurine, hypotaurine, aspartic acid, hydroxyproline, asparagine, serine, glycine, glutamine, cysteine, glutamic acid, threonine and alanine were simultaneously analysed with each detector. In order to test the applicability of these detectors to "real world" samples, the amino acid stoichiometry of the tetrapeptide Gly-Gly-Asp-Ala was determined with each detector after acid hydrolysis. The detectors were compared in terms of linearity, limit of detection, advantages and disadvantages as well as special features (capacity to provide structural information, specificity, quantification with single calibration curve, etc.).