Identification of flavonoids and their glycosides by high-performance liquid chromatography with electrospray ionization mass spectrometry and with diode array ultraviolet detection

Identification of flavonoids and flavonoid glycosides was carried out on Psidium guajava Linn leaves by means of high-performance liquid chromatography ultraviolet (HPLC-UV) analysis and HPLC mass spectrometry. By using HPLC-UV, two known phenolics (gallic acid and quercetin) and five newly reported ones (procatechuic acid, chlorogenic acid, caffeic acid, kaempferol and ferulic acid) were identified in alcohol guava leaf extract. Structural information about the compounds was obtained from the retention times, the UV spectra and mass spectra without the need to isolate the individual compounds. Two flavonoids (quercetin and kaempferol) and four flavonoid glycosides (three known components, quercetin 3-O-alpha-L-arabinoside, quercetin 3-O-beta-D-glucoside and quercetin 3-O-beta-D-galactoside, along with one novel compound, kaempferol-glycoside) and three other unknown compounds have been identified in the fractions.     

A method for the analysis of ginsenosides, malonyl ginsenosides, and hydrolyzed ginsenosides using high-performance liquid chromatography with ultraviolet and positive mode electrospray ionization mass spectrometric detection

A high-performance liquid chromatographic separation coupled to diode array absorbance and positive mode electrospray mass spectrometric detection has been developed for the analysis of ginsenosides, malonyl ginsenosides, and hydrolyzed ginsenosides in extracts of Asian ginseng (Panax ginseng) and American ginseng (P. quinquefolius). The method is capable of separating, identifying, and quantifying the predominant ginsenosides found in heated alcoholic extracts of Asian and American ginseng roots routinely sold as nutraceuticals. It also separates and identifies the malonyl ginsenosides often found in cold alcoholic extracts of ginseng root and has the potential to quantify these compounds if pure standards are available. Furthermore, it can separate and identify ginsenoside hydrolysis products such as those readily produced in situations mimicking gastric situations, including those used for dissolution studies (i.e., 0.1 N HCl, 37 degrees C).     

Development of a liquid chromatography/electrospray ionization tandem mass spectrometric method for the determination of hydroxyl radical

A rapid method combining liquid chromatography with electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) was developed for the determination of the hydroxyl radical (.OH). .OH generated via Fenton reaction was spin-trapped by 5,5-dimethyl-1-pyrroline N-oxide (DMPO) and then analyzed by LC/ESI-MS/MS in multiple reaction monitoring (MRM) mode, using N-methyl-2-pyrrolidone (NMP) as the internal standard. The peak area ratio of DMPO-OH to NMP positively correlated with the concentration of .OH. The relative standard deviation (RSD) of the method was 1.13% (n = 8). The present method was successfully applied to evaluate the .OH scavenging capacity of several phenolic acids.     

Fesoterodine stress degradation behavior by liquid chromatography coupled to ultraviolet detection and electrospray ionization mass spectrometry

In the present study, a rapid validated stability-indicating LC method was established and comprehensive stress testing of fesoterodine was carried out according to ICH guidelines. Fesoterodine was subjected to stress conditions of acid and basic hydrolysis, oxidation, photolysis and thermal decomposition. The degradation products formed under stress conditions were investigated by LC-UV and LC-ESI-MS. Successful separation of the drug from its degradation products was achieved on a monolithic C₁₈ column (100 mm × 4.6 mm i.d.) maintained at 45 °C using acetonitrile-methanol-0.03 mol L⁻¹ ammonium acetate (pH 3.8) (30:15:55, v/v/v) as the mobile phase. The flow rate was 2.4 mL min⁻¹ and the detection wavelength was 208 nm. Validation parameters such as specificity, linearity, precision, accuracy, and robustness were evaluated. Chromatographic separation was obtained within 2.5 min and it was suitable for high-throughput analysis. Fragmentation patterns of degradation products formed under different stress conditions were studied and characterized through LC-ESI-MS fragmentation. Based on the results, a drug degradation pathway was proposed, and the validated LC method was successfully applied to the quantitative analysis of fesoterodine in tablet dosage forms, helping to improve quality control and to assure therapeutic efficacy.     

Identification and analysis of alkaloids in cortex Phellodendron amurense by high‐performance liquid chromatography with electrospray ionization mass spectrometry coupled with photodiode array detection

Alkaloids from Cortex Phellodendron amurense Rupr. were identified to determine the material basis for the bioactivity of this herb. HPLC–ESI‐MS with photodiode array detection coupled to XCharge C₁₈ column was applied to analyze the alkaloids qualitatively and quantitatively. A total of 37 alkaloids were identified and tentatively characterized from the ethanol extract by online ESI‐MSⁿ fragmentation and UV spectral analysis. A total of ten alkaloids, including four novel natural products, were tentatively identified for the first time in P. amurense. The fragmentation pathways for certain compounds were analyzed. The contents of a pair of isomers (columbamine and jatrorrhizine) and four main alkaloids (phellodendrine, magnoflorine, berberine, and palmatine) were simultaneously quantified using the aforementioned method. Results showed that the newly discovered and known components of P. amurense were helpful in determining the material basis for the bioactivity of the herb. The application of the XCharge C₁₈ column is a suitable and practical method for the isolation of alkaloids in plants.     

Ultrafast liquid chromatography/ultraviolet and liquid chromatography/tandem mass spectrometric analysis

Optimal liquid chromatography/mass spectrometric [LC/MS(/MS)] analysis depends on both the LC selectivity and the electrospray efficiency. Here, we outline a simple and comprehensive LC/MS/MS strategy for the rapid analysis of a wide range of pharmaceutical compounds. To achieve ultrafast LC separation with little sacrifice in peak capacity, one needs to start with a column that provides a good peak capacity at short gradient run times; secondly, it is important to use high flow rates to achieve a good gradient peak capacity. Following this strategy, it was possible to baseline-resolve a mixture (containing acidic, neutral, and basic pharmaceutical analytes) in seconds. By coupling the selectivity provided by fast LC separation with the specificity of MS/MS detection, it is possible to separate and identify a wide range of analytes in 1-min gradient analyses. Also, the impact of mobile phase pH on both the chromatographic selectivity and the MS/MS sensitivity is demonstrated.     

Capillary supercritical fluid chromatography with simultaneous flame ionization and mass spectrometric detection

A simple interface between a capillary supercritical fluid chromatograph and an Extranuclear Simulscan mass spectrometer is described. The SFC column is directly inserted into the ion source through the existing GC-interface. The system is equipped with a splitting device which allows simultaneous EI/MS and flame ionization detection when CO₂ is used as the supercritical phase. The effect of source temperature and pressure on CO₂ clustering was studied for optimization of source conditions. The performance of the system was evaluated with a series of model compounds and standard mixtures.     

Studies of iridoid glycosides using liquid chromatography/electrospray ionization tandem mass spectrometry

Fragmentation pathways of five iridoid glycosides have been studied by using electrospray ionization multi-stage tandem mass spectrometry (ESI-MS(n)). The first-stage MS data of the five iridoid glycosides were compared. The MS spectra showed that the adduct ions of iridoid glycosides and the formate anion were diagnostic ions to distinguish iridoid glycosides with a carboxyl group at the C-4 position or an ester group at the C-4 position. The MS fragmentation pathways of the five iridoid glycosides were also studied. Analyzing the product ion spectra of iridoid glycosides, some neutral losses were observed, such as H(2)O, CO(2) and glucose residues, which were very useful for the identification of the functional groups in the structures of iridoid glycosides. Furthermore, specific loss of one molecule of methyl 3-oxopropanoate or 3-oxopropanic acid was firstly discussed, which corresponded to the isomerization of the hemiacetal group in the structure of iridoid aglycone. According to the fragmentation mechanisms and HPLC/MS(n) data, the structures of five iridoid glycosides in a crude extract of Gardenia jasminoisdes fruit have been identified. Three compounds were compared with standards and the other two were identified as shanzhiside and genipin gentibioside by their MS(n) data without standard compounds. In order to further validate the veracity of the deduction, genipin gentiobioside was isolated from the extract of Gardenia jasminoisdes fruit using Purification Factory and was further identified by C- and H-NMR.     

Evaluation of major active components in St. John's Wort dietary supplements by high-performance liquid chromatography with photodiode array detection and electrospray mass spectrometric confirmation

A RP-HPLC method with photodiode array detection and LC-electrospray ionization (ESI) MS confirmation was established for the determination of major active components in St. John's Wort dietary supplement capsules. The samples alternatively were extracted with ethanol-acetone (2:3) using a 55 degrees C water-bath shaker or an ambient temperature ultrasonic bath. Extracts were separated by RP-C18 chromatography using a 95-min water-methanol-acetonitrile-trifluoroacetic acid gradient. The major components were identified by photodiode array detection and then confirmed by LC-ESI-MS. The quantification of components was performed using an internal standard (luteolin). This method may serve as a valuable tool for the quality evaluation of St. John's Wort dietary supplement products.     

Identification and determination of phase II nabumetone metabolites by high-performance liquid chromatography with photodiode array and mass spectrometric detection

Chromatographic analyses play an important role in the identification and determination of phase I and phase II drug metabolites. While the chemical standards of phase I metabolites are usually available from commercial sources or by various synthetic, degradation or isolation methods, the phase II drug metabolites have usually more complicated structures, their standards are in general inaccessible and their identification and determination require a comprehensive analytical approach involving the use of xenobiochemical methods and the employment of hyphenated analytical techniques. In this work, various high-performance liquid chromatography (HPLC) methods were employed in the evaluation of xenobiochemical experiments leading to the identification and determination of phase II nabumetone metabolites. Optimal conditions for the quantitative enzymatic deconjugation of phase II metabolites were found for the samples of minipig bile, small intestine contents and urine. Comparative HPLC analyses of the samples of above-mentioned biomatrices and of the same biomatrices after their enzymatic treatment using beta-glucuronidase and arylsulfatase afforded the qualitative and quantitative information about phase II nabumetone metabolites. Hereby, three principal phase II nabumetone metabolites (ether glucuronides) were discovered in minipig's body fluids and their structures were confirmed using liquid chromatography (LC)-electrospray ionization mass spectrometric (MS) analyses.     

Determination of sulphonated azo dyes in food by ion-pair liquid chromatography with photodiode array and electrospray mass spectrometry detection

An ion-pair liquid chromatography method with on-line photodiode-array and electrospray mass spectrometry detection was developed to determine 10 commonly used sulphonated azo dyes (Tartrazine, Amaranth, New Coccine, Sunset Yellow FCF, Allura Red AC, Ponceau R, Ponceau 3R, Orange I, Orange II and Metanil Yellow) in food. A reversed phase C(18) column with gradient elution was utilized to separate these compounds. Triethylamine was added in the mobile phase as an ion-pair reagent for chromatographic separation. Photodiode-array detection was employed for quantitative determination and electrospray mass spectrometry was used for identification. Good linearity (0.05-10 ppm, r(2)=0.999) and detection limit (<0.01 ppm) were determined with 5 mul injection. In addition, precision and accuracy associated with this newly developed method will be presented. A liquid extraction method was also developed to extract these dyes from different foodstuffs. The application of this method was demonstrated by analyzing sulphonated azo dyes in soft drinks, fruit jam, and salted vegetables.     

Direct and simultaneous profiling of epoxyeicosatrienoic acid enantiomers by capillary tandem column chiral-phase liquid chromatography with dual online photodiode array and tandem mass spectrometric detection

Despite first evidence for the cytochrome P450-mediated enantioselective biosynthesis and activity of cis-epoxyeicosatrienoic acids (EETs), as yet little is known about the stereospecifity of EET generation and physiology, because the existing chiral methods are time consuming, labor intensive, and not sensitive enough. We present a method for highly sensitive, direct, and simultaneous chiral analysis of all eight EET enantiomers consisting of (i) solid-phase extraction, (ii) reversed-phase high-performance liquid chromatographic purification followed by (iii) consecutive regio- and enantiomeric separation of the four underivatized EET regioisomers within one chromatographic run employing capillary tandem column chiral-phase liquid chromatography with (iv) reliable dual online photodiode array and gentle electrospray ionization tandem mass spectrometric identification and quantitation of the eluting optical antipodes. This one-step, simple, expeditious, and highly sensitive measurement allows profiling of all eight EET enantiomers at once, thus avoiding substance loss and enabling high sample throughput. Limits of quantification in the low picogram range were achieved by the use of capillary columns with typical high quantitative sensitivity instead of conventional columns with low chromatographic signal intensity employed by previous methods. Application to tissue homogenates demonstrated the suitability of this approach for routine and reliable “enantioprofiling” of free endogenous EETs, i.e., EETs not esterified into cellular membrane phospholipids, typically occurring at very low concentrations. The technique can readily be employed for preparative purification of enantiomers in the microgram range using large-inner-diameter columns. Figure Direct and simultaneous enantioprofiling of the four free endogenous epoxyeicosatrienoic acids (EETs) from a complex biological matrix, like the cardiopulmonary system, within one chromatographic run by highly sensitive, one-step capillary tandem column chiral-phase liquid chromatography with dual online photodiode array and tandem mass spectrometric detection (CapTC-CP-LC-PDAD-ESI-MS²) enables accurate, systematic, and routine correlation between the absolute configuration of EETs and their physiological actions Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Formation of iron complexes from trifluoroacetic acid based liquid chromatography mobile phases as interference ions in liquid chromatography/electrospray ionization mass spectrometric analysis

Two unexpected singly charged ions at m/z 1103 and 944 have been observed in mass spectra obtained from electrospray ionization mass spectrometric analysis of liquid chromatography effluents with mobile phases containing trifluoroacetic acid (TFA) that severely interfered with sample analysis. Accurate mass measurement and tandem mass spectrometry studies revealed that these two ions are composed of three components; clusters of trifluoroacetic acid, clusters of mass 159 and iron. Formation of these ions is inhibited by removing TFA from the mobile phases and using formic acid in its place, replacing the stainless steel union with a titanium union or by adding a small blank fused-silica capillary column between the chromatography column and the electrospray tip via a stainless steel union without any adverse effects to chromatographic separation, peak broadening or peptide identifications.     

Characterization of dielectric barrier electrospray ionization for mass spectrometric detection

A novel electrospray interface is presented which induces an electric field by dielectric polarization through a non-conductive barrier. Therefore, a square-wave high-voltage signal is applied. This technique allows mass spectrometric measurements in the positive as well as in the negative mass spectrometry mode without changing the polarity of the potential applied, and it decreases the risk of undesired discharges, induced by high electric currents. The applicability of this technique is demonstrated by mass spectrometric determination of reserpine.     

Characterization of fifty-one flavonoids in a Chinese herbal prescription Longdan Xiegan Decoction by high-performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry and photodiode array detection

High-performance liquid chromatography coupled to electrospray ionization (ESI) tandem mass spectrometry and photodiode array detection (HPLC-DAD-ESI-MS(n)) was developed to identify and characterize the flavonoids in a Chinese formulated preparation, Longdan Xiegan Decoction (LXD). In total, fifty-one flavonoids (27 flavones, 10 flavanones, 7 chalcones, 5 flavonols and 2 isoflavones) were characterized. Eighteen compounds among them including a newly detected flavonoid, naringin, from the ingredient herbs, were unambiguously determined by comparing the retention times (t(R)), UV spectral data and mass fragmentation behaviors with those of the reference compounds. Another thirty-three compounds were tentatively identified by referencing to the reported data of their UV and MS spectra. The ESI-MS/MS fragmentation behavior of flavones (OMe-substituted, O-glycosides, C-glycosides), chalcones, flavonols and their appropriate characteristic pathways were proposed. In negative ion ESI-MS all the flavonoids yielded prominent [M--H](-) ions in the first order mass spectra. Fragmentation with a loss of mass of 15 Da (CH(3)), 18 Da (H(2)O), 28 Da (CO), 44 Da (CO(2)), 56 Da (2CO) and the residues of glucose and glucuronic acid observed in the MS/MS spectra were useful for aiding the structural identification of the flavonoids investigated.     

Separation and determination of alkylglycosides by liquid chromatography with electrospray mass spectrometric detection

The separation of alkylpolyglycosides by liquid chromatography with electrospray mass spectrometric detection, using either an alkylamide or a cyanopropyl column, and acetonitrile/water mixtures as mobile phases, was developed. Using the alkylamide column and isocratic elution, the α- and β-epimers and ring isomers (pyranosides and furanosides) of the alkylmonoglycosides were resolved. The ring isomers were also resolved in a much shorter time using the cyanopropyl column with gradient elution. Using these columns, the isomers of the alkyldiglycosides and alkyltriglycosides were also partially resolved. The equilibration time was much shorter with the cyanopropyl column, which was selected to perform quantitation studies. The response factors increased more than an order of magnitude with the length of the alkyl chain, from the methyl to the decylmonoglycoside, and decrease largely for the dodecyl and tetradecylmonoglycoside. The limits of detection were of ca. 25 μM from the hexyl up to the dodecylmonoglycoside. The procedures were applied to the characterisation and determination of alkylmonoglycosides in toiletries.     

High-throughput liquid chromatography ultraviolet/mass spectrometric analysis of combinatorial libraries using an eight-channel multiplexed electrospray time-of-flight mass spectrometer

We report a high-throughput liquid chromatography/mass spectrometry (LC/MS) protocol for analyzing large combinatorial libraries using an eight-channel parallel LC/UV/MS (MUX-LCT) system. System configuration, linear response range in UV absorbance, LC column selection, and flow rate were optimized for 24 h/7 day unattended operations. Combinatorial libraries were analyzed on this system at a rate of 3200 compounds per day for a 3.5 min cycle time per injection. This parallel system is compared with a single-channel system in terms of performance and operation.     

Optimization of electrospray interface and quadrupole ion trap mass spectrometer parameters in pesticide liquid chromatography/electrospray ionization mass spectrometry analysis

Optimization of both the ionization process and ion transportation in the mass spectrometer is of crucial importance in order to achieve high sensitivity and low detection limits and acceptable accuracy in liquid chromatography/electrospray ionization mass spectrometry (LC/ESI‐MS) analysis. In this paper four optimization procedures of electrospray interface and quadrupole ion‐trap mass spectrometer parameters (ESI‐MS) (nebulizer gas and drying gas flow rate, end plate voltage, capillary voltage, skimmer voltage, octopoles direct current and radio frequency, trap drive and lens voltages) were studied on three pesticides – thiabendazole, aldicarb and imazalil. The results demonstrate that the methodology of optimization strongly influences the effectiveness of finding true optima of the operating parameters. Both eluent flow rate and composition during optimization have to mimic the situation during real analysis as closely as possible in order to achieve parameters giving the highest sensitivity. Therefore, post‐column addition of analyte to the mobile phase identical in composition to the one in which analyte elutes during real analysis combined with software‐based optimization was found to be the most effective and fastest method for achieving intensity maxima. The parameters most strongly affecting ion formation and transportation, hence sensitivity, were capillary voltage, direct current of the first octopole, trap drive and the second lens for all pesticides under study. In addition to sensitivity and detection limit matrix effect was considered in the optimization process. It was found that the matrix effect can be reduced but not eliminated by adjusting the ESI and MS parameters. The optimal parameters from the point of view of the matrix effect can only be found with factorial design. Parameters giving higher sensitivity tended to be more affected by matrix effect causing higher ionization suppression by co‐eluting compounds. Copyright © 2010 John Wiley & Sons, Ltd.     

Determination and quantitation of sulfonylurea and urea herbicides in water samples using liquid chromatography with electrospray ionization mass spectrometric detection

A multianalyte method has been developed for the confirmation and quantitation of five sulfonylureas, bensulfuron-methyl, imazosulfuron, pyrazosulfuron-ethyl, flazasulfuron and halosulfuron-methyl, and for three ureas, siduron, dymron (daimuron) and diuron (DCMU) in water. Samples were extracted from water by off-line solid-phase extraction (SPE) with a polystyrene polymer cartridge (PS2), an ODS C₁₈-bonded silica cartridge (C₁₈) and an N-vinylpyrrolidone polymer cartridge (Oasis). Analyte determination and quantitation were performed by liquid chromatography with mass spectrometry (LC-MS). Extraction efficiency experiments demonstrated the ability of this method to extract sulfonylureas and ureas from water samples. Confirmatory analysis was carried out by LC-electrospray mass spectrometry (LC-ESI-MS) instrumentation equipped with a single-quadrupole mass filter. MS data acquisition was performed by a single or two-ion selected ion monitoring (SIM) program. It is required for confirmation that LC-MS retention times of the analytes are within 1% of the retention times of the standards, and that the molecular ion or characteristic fragment ion is present for each analyte. Fragment ions from distinctive structures must be obtained to identify and characterize specific herbicide molecules. These were obtained by controlled decomposition of sulfonylurea and urea adduct ions after suitably adjusting the electrical field in the desolvation chamber. The eight herbicides were also measured in fortified pure water (water purified by a milli-Q system), tap water and river water. Average recoveries of the eight analytes from water samples were in the range of 70-120% with relative standard deviations (R.S.D.s) of <20%. The limit of quantitation (LOQ) for each of the eight herbicides was between 10 and 100 ng l⁻¹.     

Determination of polycyclic aromatic compounds by high-performance liquid chromatography with simultaneous mass spectrometry and ultraviolet diode array detection

A major problem in the determination of polycyclic aromatic compounds (PACs) in environmental samples is the extreme complexity of the extracts, even after extensive fractionation. The combination of high-performance liquid chromatography (HPLC) with simultaneous mass spectrometry (MS) and ultraviolet diode array detection (DAD) is a powerful tool for the identification and quantitation of such species with a high degree of confidence. HPLC allows the selective separation of a wide variety of PACs, including thermally labile and high molecular weight compounds. Electron ionization MS with the moving belt interface provides high sensitivity and selectivity, as well as structural information such as molecular weight, functional groups, and elemental composition. The diode array detector helps to differentiate isomeric structures and confirm compound identity.