Isolation and characterization of methoxylated flavones in the flowers of Primula veris by liquid chromatography and mass spectrometry

Characterization of six flavones, which were named substances G1, G2, G3, G4, G5 and G6 according to their R_F values in normal-phase thin-layer chromatography, is reported. The pure flavones were purified after maceration with methanol by normal-phase solid-phase extraction, normal-phase medium-pressure liquid chromatography, normal-phase preparative thin-layer chromatography and preparative reversed-phase high-performance liquid chromatography (RP-HPLC). The collected fractions of several isolation steps were analyzed by normal-phase (NP) and RP-HPLC. Detection and identification of the substances G was accomplished by UV detection at 213–216 nm, diode array UV detection, or fluorescence detection (λ_ex=330 nm; λ_em=440 nm). The molecular mass, the elementary composition, and the structure of the six components was determined by electron-impact high-resolution mass spectrometry (EI-HRMS). Substance G4 was identified as 3′,4′,5′-trimethoxyflavone. The substances G1–G6 were shown to be mono-, di- tri- and pentamethoxyflavones. HPLC–electrospray ionization tandem mass spectrometry (ESI-MS–MS) of the flavones was carried out employing a 150×2 mm I.D. column packed with a 3 μm/100 Å octadecylsilica stationary phase and a mobile phase comprising 1.0% acetic acid in water–acetonitrile (50:50). Comparative RP-HPLC–ESI-MS of the raw methanol extract and the isolated substances G1–G6 proved that the isolated compounds were pure and were not artifacts. Finally, RP-HPLC–ESI-MS–MS was used to identify substances G1–G6 in phytopharmaceutical drugs.     

Identification and quantification of eight flavones in root and shoot tissues of the medicinal plant huang-qin (Scutellaria baicalensis Georgi) using high-performance liquid chromatography with diode array and mass spectrometric detection

A method of analysis of eight flavones using high-performance liquid chromatography (HPLC)-diode array detection (DAD)-mass spectrometry (MS) in root and aerial tissues of the medicinal plant Scutellaria baicalensis was developed. The identity of the analytes was confirmed using retention time, UV-vis and mass spectral comparisons to commercial standards. Both UV-vis and mass spectral patterns were characterized for glycosylated flavones. Two additional flavone glycosides were tentatively identified as chrysin-7-glucuronide and wogonoside, but not quantified. Greenhouse and in vitro-grown tissues were analyzed with flavone concentrations ranges of 0.14-150 and 0.030-1.7 microg/mg for greenhouse root and shoot tissue, respectively, and 0.0068-6.4 and 0.082-1.5 microg/mg for in vitro-grown roots and shoots, respectively.     

Preparative separation of five flavones from flowers of Polygonum cuspidatum by high‐speed countercurrent chromatography

A preparative high‐speed countercurrent chromatography method was successfully used for the isolation of five minor flavones from Polygonum cuspidatum flowers. Among them, three compounds were obtained from P. cuspidatum for the first time. A twin two‐phase solvent system composed of n‐hexane/ethyl acetate/ethanol/water (1:6:3:6, v/v/v/v) and petroleum ether/ethyl acetate/methanol/water (2:4:3:3, v/v/v/v) was developed. Compounds were obtained from the fraction B and fraction C prepurified by silica gel column chromatography. Five minor compositions, 6.8 mg of hesperidin, 11.2 mg of phloridzin, 4.9 mg of luteolin, 5.3 mg of hyperin, and 3.7 mg of luteoloside were obtained from 140 mg of the fraction B and 110 mg of fraction C with a purity of 95.3, 96.4, 98.0, 96.8, and 95.3%, respectively, as determined by high‐performance liquid chromatography. The structures of these compounds were identified by ¹H and ¹³C NMR spectroscopy.     

Study on the photostability of guaiazulene by high-performance liquid chromatography/mass spectrometry and gas chromatography/mass spectrometry

The photostability of guaiazulene (1,4-dimethyl-7-isopropylazulene; GA), a natural azulenic compound used in cosmetic and health-care products, as well as in pharmaceutical preparations, was investigated in solution (methanol, ethanol, acetonitrile), by different techniques: gas chromatography/mass spectrometry (GC/MS) and high-performance liquid chromatography combined with atmospheric pressure chemical ionization mass spectrometry and UV detection (LC/APCI-MS and HPLC/UV). A solar simulator (xenon-arc lamp) was used as UV-A radiation source. The study involved: monitoring compound decomposition, identifying products of photodegradation (PPs), assessing the role of oxygen and evaluating the kinetics of the process. Minor PPs are volatile compounds and were characterized by GC/MS, while oligomeric polyoxygenated compounds, tentatively characterized on the basis of MS and MS/MS spectra, were found to be the main photoproducts. The photodegradation was found to be enhanced by the presence of oxygen; nevertheless, determination of the singlet oxygen quantum yield for GA gave a lower value than that for the reference standard Rose Bengal. The obtained results and the developed stability-indicating methods (GC/MS and LC/MS) are of interest for stability studies and/or quality control purposes of GA as raw material or cosmetic products.     

Monitoring stevioside in soju by high-performance liquid chromatography and liquid chromatography/mass spectrometry

A method using high-performance liquid chromatography (HPLC) with UV absorption detection was developed to monitor stevioside in soju, a distilled spirits product that is commercially available. The method uses a single-step dilution for sample preparation. It completely eliminates the time-consuming process of solid-phase extraction. A method using HPLC/mass spectrometry was optimized to confirm the identities of stevioside and other related impurities, including rebaudioside A, rebaudioside C, and dulcoside. The method was validated. The validation parameters included range (10.1-1007.3 ppm), precision, linearity, accuracy, robustness, system suitability, and intermediate precision. Stevioside standard solutions at 6 concentration levels were prepared for the validation work, including the tests for precision, linearity, and accuracy. The solutions were prepared in triplicate for each concentration. The relative standard deviation for the precision test was <3% for all 6 concentration levels. The correlation coefficient for the linearity within the concentration range was determined to be > 0.999. The average recovery ranged from 95.7 to 101.1% for the soju samples spiked with stevioside standard. The detection limit for stevioside was estimated at 75 ppb. The method was used to screen several soju samples; no detectable stevioside was found in the samples.     

Determination of the metabolites of gestrinone in human urine by high performance liquid chromatography, liquid chromatography/mass spectrometry and gas chromatography/mass spectrometry

Gestrinone was studied by high performance liquid chromatography (HPLC) for screening and by gas chromatography/mass spectrometry (GC/MS) for confirmation. When the chromatograms of blank, spiked urine and dosed urine were compared by HPLC, two unknown metabolites were found and these were excreted as the conjugated forms. Metabolites 1 and 2 were tested by LC/MS and LC/MS/MS and both had parent ions at m/z 325. The fragment ion of metabolite 1 was at m/z 263 and ions for metabolite 2 were m/z 307 [MH - H(2)O](+), 289, 279 and 241. LC/MS/MS of m/z 263 as the parent ion of metabolite 1 gave fragment ions at m/z 245 and 217, which were assumed to be [263 - H(2)O](+) and [235 - H(2)O](+), respectively. The trimethylsilyl (TMS)-enol-TMS ether derivative of gestrinone displayed three peaks in its GC/MS chromatogram, formed by tautomerism.     

Determination of steroids by liquid chromatography/mass spectrometry

On-line atmospheric pressure chemical ionization (APCI) and electrospray ionization (ESI) liquid chromatography/mass spectrometry (LC/MS) were evaluated for the analysis of a variety of steroids. Steroids were classified into three major groups based on the spectra and the sensitivities observed: (I) those containing a 3-one, 4-ene functional group, (II) those containing at least one ketone group without conjugation, and (III) those containing hydroxy group(s) only. In the APCI mode, the best sensitivity and the lowest detection limit for all three groups were obtained by using a mobile phase consisting of methanol and 1%–2% acetic acid in water. The APCI spectra were characterized by MH⁺, MH⁺-H₂O, MH⁺-2H₂O, etc., with the degree of H₂O loss being compound dependent: group I steroids produced stable MH⁺ and group III steroids showed extensive water loss. In the electrospray mode the best sensitivity and the lowest detection limit for the first two groups were obtained when pure methanol and water were used as the mobile phase. This condition produced abundant stable MNa⁺ due to ubiquitous sodium. Detection limits in the 5–15 pg range can be easily achieved using ESI LC/MS. Addition of ammonium acetate or use of acetonitrile in the mobile phase, common in the LC/MS analysis of steroids, decreased the sensitivity for the group I and II steroids and thus should be avoided. For group III steroids, the detection limit can be improved by the addition of acetic acid to the mobile phase.     

Determination of chlormequat and mepiquat in foods by liquid chromatography/mass spectrometry or liquid chromatography/tandem mass spectrometry: interlaboratory study

Interlaboratory validation studies have been performed on 2 methods for the determination of chlormequat (CLQ) and mepiquat (MPQ). Both methods used identical extraction procedures and stable isotope internal standardization but differed in the use of liquid chromatography/mass spectrometry (LC/MS) or LC/tandem mass spectrometry (LC/MS/MS) for the determination, the amount of internal standard used, and the expected limit of detection. After addition of deuterated internal standards, CLQ and MPQ were extracted with methanol-water and determined by LC//MS or LC/MS/MS with positive electrospray ionization. Eight European laboratories participated in the LC/MS method study, analyzing mushroom, pear, wheat flour, and fruit puree with residues of CLQ in the range 0.040-1.19 mg/kg and of MPQ in the range 0.041-0.39 mg/kg. For CLQ, the Horwitz ratio (HoRat) values for individual test materials/levels were in the range 0.85-1.13 with a mean of 1.00, showing good method performance. For MPQ, the Ho values for mushroom, pear (both levels), and wheat flour were in the range 0.83-0.94, again indicating good method performance. For the determination of MPQ in infant food (fruit puree) at 0.041 mg/kg, the Ho was 1.7 when a value of 0 reported by one participant was excluded. In the LC/MS/MS study, in which 11 laboratories participated, a separate sample set was analyzed with residues of CLQ in the range 0.007-1.03 mg/kg and of MPQ in the range 0.008-0.72 mg/kg. Ho values for CLQ were in the range 0.27-1.36 and for MPQ in the range 0.51-2.10, all corresponding to acceptable method performance.     

Poly(amic acid) and polyimide characterization using gas chromatography/mass spectrometry and particle beam liquid chromatography/mass spectrometry

A number of polymers were hydrolyzed in NH₄OH and studied using gas chromatography/ mass spectrometry (GC/MS) and particle beam liquid chromatography/mass spectrometry (particle beam LC/MS) techniques. The polymers studied in this report were as follows: BPDA-PDA, BPDA-PDA-ODA, BPDA-PDA-GFDA, PMDA-ODA, and BTDA-APB. Some of the polymer samples were hydrolyzed in both their acid and imide forms to see if any mass spectrometric differences could be detected. ln all cases, the acid and imide spectra looked the same. GC/MS was unable to determine either the amine or acid portion of these polymers via a direct injection of the sample, but when the samples were first extracted with diethyl ether and this ether extract was injected into the chromatograph, the amine portion of the polymers was readily detected. The acid portion was, again, not detected in either the sample or the ether extract. The particle beam was able to detect both the amine and acid monomeric units in the nonextracted sample.     

Characterization of explosives by liquid chromatography/mass spectrometry and liquid chromatography/tandem mass spectrometry using electrospray ionization and parent-ion scanning techniques

Analytical techniques for the detection of small amounts of explosives (in the picogram range) are now involved in various application. Some of them concern soil, water and air monitoring in order to face environmental problems related to improper handling procedures either in stocking or in wasting of the explosive products. Other areas are strictly related to forensic analysis of samples coming either from explosion areas where the matrix is various (metal, glass, wood, scraps), or from explosives transportation related to international terrorism. Generally speaking, for these applications the bulk of the matrix seriously interferes in the detection of the explosive analyte, which is usually present at trace levels. Unfortunately, despite some improvements, analytical techniques developed up today in this domain are still faced to two main constraints: the introduction of new products with unanticipated chemico-physical properties and the requirement of a routine and fast analytical method which can handle any matrix with a minimal clean-up and performing a sensitivity compatible either with the ever-decreasing demanded detection limit and with the ever-decreasing available specimen amount. These requirements can be fulfilled now by the new LC-MS and LC-MSMS techniques: mass spectrometry (MS) is likely an universal detector but even specific, especially when implemented in tandem MS (MSMS); LC is by far the most suitable technique to handle such a kind of compounds. Moreover, of a particular concern are some explosives which are reported to be thermally stable but difficult to dissolve. Some of the experiments on characterization of explosives [Octagen (HMX), Ethyleneglycol dinitrate (EGDN), Exogen (RDX), Propanetriol trinitrate (NG), Trinitrotoluene (TNT), N-Methyl-N-tetranitrobenzenamine (TETRYL), Dintrotoluene (DNT), Bis-(nitrooxy-methyl) propanediol dinitrate (PETN), Hexanitrostilbene (HNS), Triazido-trinitrobenzene (TNTAB), Tetranitro-acridone (TENAC), Hexa-nitrodiphenylamine (HEXYL), Nitroguanidine (NQ)] by LC-MS and LC-MSMS with the API-IonSpray source and using the Parent-Scan technique are presented.     

Radical identification by liquid chromatography/thermospray mass spectrometry

Radical adducts of 5,5-dimethyl-1-pyrroline N-oxide (DMPO) with hydroxyl, methanol-derived, and ethanol-derived radicals were detected by a combination of liquid chromatography with either electron paramagnetic resonance or thermospray mass spectrometry (LC/EPR or LC/TSP-MS) in the Fenton system (with methanol or ethanol). One radical adduct was observed in the reaction of DMPO with the hydroxyl radical or the methanol-derived radical, while two adducts were detected in the reaction of DMPO with ethanol-derived radicals. The LC/TSP-MS spectra showed quasi-molecular ions [M + H]+ at m/z 146 and m/z 160 for the methanol-derived and ethanol-derived radical adducts, respectively, and an apparent molecular ion M+ at m/z 130 for the hydroxyl radical adduct. Use of methyl-D3 alcohol (CD3OH) and ethyl-D5 alcohol (CD3CD2OH) indicated that carbon-centered radicals are formed. Experiments with partially deuterated ethanol (CD3CH2OH and CH3CD2OH) indicated that the two adducts observed in the reaction of DMPO with ethanol-derived radicals correspond to the two diastereomeric adducts of DMPO with the alpha-hydroxyethyl free radical.     

Characterization of C-glycosyl flavones O-glycosylated by liquid chromatography-tandem mass spectrometry

Fifty three O-glycosyl-C-glycosyl flavones with O-glycosylation on phenolic hydroxyl or on the C-glycosyl residue or combination of both forms have been studied by liquid chromatography-UV diode array detection-electrospray ionisation mass spectrometry ion trap in the negative mode. The study of the relative abundance of the main ions from the MS preferential fragmentation on -MS2 and/or -MS3 events allows the differentiation of the position of the O-glycosylation, either on phenolic hydroxyl or on the sugar moiety of C-glycosylation. In addition, it is possible to discriminate between O-glycosylation at 2' and at 6' positions. The occurrence of an abundant ion Y(0)(-) ([(M-H)-132/-146/-162](-), mono-O-pentosyl/rhamnosyl/hexosyl-C-glycosyl derivatives) after -MS2 fragmentation characterizes the O-glycosylation on phenolic hydroxyls. The preferential fragmentation leading to a relevant Z(1)(-) ([Y(1)-18](-)) fragment is characteristic of 2'-O-glycosyl-C-glycosyl derivatives. The 6'-O-glycosyl-C-glycosyl derivatives are characterized by (0,2)X(0)(-), which is generated by a global loss of the sugar moiety from the O-glycosylation at 6' and the glycosidic fraction that involves the carbons 6'-3' of the C-glycosyl residue ([(M-H)-162-120](-), in the case of 6'-O-hexosyl-C-hexosyl derivatives). Regarding the combined O-glycosylated compounds (both on phenolic hydroxyl and on sugar moiety at C-glycosylation), the main fragmentation on -MS2 events produces a Y(0)(-) characterizing the O-glycosylation on the phenolic hydroxyl, and the -MS3[(M-H)-->Y(0)](-) fragmentation of the O-glycosylation on the C-glycosyl residue.     

Polymer characterization by combining liquid chromatography with MALDI and ESI mass spectrometry

High-performance polymers are complex mixtures of materials of different size and chemical composition and with different end groups and architecture. To determine the molecular heterogeneity of such systems, hyphenation of several techniques is required. The value of coupling mass spectrometry (MS) with separation techniques has already been recognized - such methods have proved to be among the most powerful for molecular characterization of complex polymer systems.The review focuses on matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) MS coupled with liquid chromatography (LC). Such hyphenation has been used for most polymer analysis by mass spectrometry coupled with separation techniques. The advantages and/or limitations of these techniques for polymer characterization are discussed. Future prospects are briefly outlined.     

Analysis of cannabinoids by liquid chromatography— Thermospray mass spectrometry and liquid chromatography— Tandem mass spectrometry

Summary A rapid method based on liquid chromatography and thermospray mass spectrometry without any derivatization or pre-purification steps has been developed for the identification and quantification of cannabinoids in drugs from cannabis plants. The extracts were separated on a C₁₈ reversed-phase column with an acidic acetonitrile-water gradient. Liquid chromatographymass spectrometry was performed with a thermospray interface and protonated molecular ions were obtained from the cannabinoids of interest. Liquid chromatography-tandem mass spectrometry experiments on the molecular ions gave additional structural information online. The sensitivity and selectivity of the method was sufficient to enable the detection of 100 pg of the cannabinoids.     

Quantitative liquid chromatography/time-of-flight mass spectrometry

Over the last decade, time-of-flight (TOF) instruments have increasingly been used as quantitation tools. In addition, because of their high resolving power, they can be used for verification of empirical formulas. Historically, TOF instruments have had limited quantitation capabilities because of their narrow dynamic range. However, recent advances have improved these limitations. This review covers the rationale for using TOF for LC detection, and describes the many methods currently in the literature for the quantitation of pharmaceuticals, environmental pollutants, explosives and many phytochemicals.     

Protein open-access liquid chromatography/mass spectrometry

Each year increasing numbers of proteins are submitted for routine characterization by liquid chromatography/mass spectrometry (LC/MS). This paper reports a solution that transforms routine LC/MS analysis of proteins into a fully automated process that significantly reduces analyst intervention. The solution developed, protein open-access (OA) LC/MS, consists of web-enabled sample submission and registration, automated data processing, data interpretation, and report generation. Sample submissions and results are recorded in a LIMS that utilizes an Oracle database. The protein sequence is captured during the sample submission process, stored in the database, and utilized to determine the theoretical protein molecular weight. This calculated mass is used to set the parameters for transformation of the mass-to-charge spectra to the mass domain and evaluate the presence or absence of the desired protein. Three protein OA-LC/MS instruments have been deployed in our facility to support protein characterization, purification, and modification efforts.     

Determination of histamine in tomatoes by liquid chromatography/mass spectrometry

The histamine level in tomato fruits and pastes was determined by 2 orthogonal techniques as a means of comparing accuracy. Close statistical agreement was found between assays for free histamine by capillary electrophoresis (with UV absorbance detection), and for the dansyl derivative by reversed-phase liquid chromatography (LC). Both techniques have adequate sensitivity for the analysis of endogenous histamine in tomatoes, but LC/electrospray tandem mass spectrometry was more sensitive by at least an order of magnitude, down to a level of 0.05 mg/kg.     

Two-dimensional liquid chromatography/mass spectrometry/mass spectrometry separation of water-soluble metabolites

Off-line two-dimensional liquid chromatography with tandem mass spectrometry detection (2D-LC/MS-MS) was used to separate a set of metabolomic species. Water-soluble metabolites were extracted from Escherichia coli and Saccharomyces cerevisae cultures and were immediately analyzed using strong cation exchange (SCX)-hydrophilic interaction chromatography (HILIC). Metabolite mixtures are well-suited for multidimensional chromatography as the range of components varies widely with respect to polarity and chemical makeup. Some currently used methods employ two different separations for the detection of positively and negatively ionized metabolites by mass spectrometry. Here we developed a single set of chromatographic conditions for both ionization modes and were able to detect a total of 141 extracted metabolite species, with an overall peak capacity of ca. 2500. We show that a single two-dimensional separation method is sufficient and practical when a pair or more of unidimensional separations are used in metabolomics.