Determination of catechins and caffeine in proposed green tea standard reference materials by liquid chromatography-particle beam/electron ionization mass spectrometry (LC-PB/EIMS)

Presented here is the quantitative analysis of green tea NIST standard reference materials (SRMs) via liquid chromatography-particle beam/electron ionization mass spectrometry (LC-PB/EIMS). Three different NIST green tea standard reference materials (SRM 3254 Camellia sinesis Leaves, SRM 3255 C. sinesis Extract and SRM 3256 Green Tea-containing Oral Dosage Form) are characterized for the content of caffeine and a series of catechin species (gallic acid, catechin, epicatechin, epigallocatechin, epicatechin gallate and epigallocatechin gallate (EGCG)). The absolute limits of detection for caffeine and the catechin species were determined to be on the nanogram level. A reversed-phase chromatographic separation of the green tea reference materials was carried out on a commercial C₁₈ column using a gradient of water (containing 0.1% TFA) and 2:1 methanol:acetonitrile (containing 0.1%TFA) at 0.9 mL min⁻¹ and an analysis time of 50 min. Quantification of caffeine and the catechin species was carried out using the standard addition and internal standard methods, with the latter providing appreciable improvements in precision and recovery.     

Investigation of pyrrolizidine alkaloids and their N-oxides in commercial comfrey-containing products and botanical materials by liquid chromatography electrospray ionization mass spectrometry

Pyrrolizidine alkaloids (PAs) and their N-oxides are found in several plant families throughout the world. PAs are potentially toxic to the liver and/or lungs in humans and may cause acute liver failure, cirrhosis, pneumonitis, or pulmonary hypertension. PAs are also carcinogenic to animals, and they have been linked to the development of hepatocellular and skin squamous cell carcinomas as well as liver angiosarcomas. According to experimental studies, the quantity of PAs in some herbal teas and dietary supplements is sufficient to be carcinogenic in exposed individuals. A method for the extraction and identification of PAs and their N-oxides in botanical materials and commercial comfrey-containing products has been developed using liquid chromatography electrospray ionization mass spectrometry. Following optimization of the extraction procedure and the chromatographic conditions, the method was applied to the analysis of 10 herbal remedies. All of the products that were labeled to contain comfrey were found to contain measurable quantities of PAs.     

Development of botanical and fish oil standard reference materials for fatty acids

As part of a collaboration with the National Institutes of Health’s Office of Dietary Supplements and the Food and Drug Administration’s Center for Drug Evaluation and Research, the National Institute of Standards and Technology has developed Standard Reference Material (SRM) 3274 Botanical Oils Containing Omega-3 and Omega-6 Fatty Acids and SRM 3275 Omega-3 and Omega-6 Fatty Acids in Fish Oil. SRM 3274 consists of one ampoule of each of four seed oils (3274-1 Borage (Borago officinalis), 3274-2 Evening Primrose (Oenothera biennis), 3274-3 Flax (Linium usitatissimum), and 3274-4 Perilla (Perilla frutescens)), and SRM 3275 consists of two ampoules of each of three fish oils (3275-1 a concentrate high in docosahexaenoic acid, 3275-2 an anchovy oil high in docosahexaenoic acid and eicosapentaenoic acid, and 3275-3 a concentrate containing 60 % long-chain omega-3 fatty acids). Each oil has certified and reference mass fraction values for up to 20 fatty acids. The fatty acid mass fraction values are based on results from analyses using gas chromatography with flame ionization detection (GC-FID) and mass spectrometry (GC/MS). These SRMs will complement other reference materials currently available with mass fractions for similar analytes and are part of a series of SRMs being developed for dietary supplements.

Application of liquid chromatography-particle beam mass spectrometry and gas chromatography-mass spectrometry for the identification of metabolites of polycyclic aromatic hydrocarbons

Chromatographia - Two liquid chromatography-particle beam mass spectrometry (LC-PBMS) systems have been used for analysis of polycyclic aromatic hydrocarbon metabolites in water samples from a...     

Capillary electrophoresis-diode array detection--electrospray mass spectrometry for the analysis of selected tropane alkaloids in plant extracts

Capillary zone electrophoresis, coupled to UV and interfaced with electrospray ionization mass spectrometry (ESI-MS), is described for the simultaneous analysis of hyoscyamine and scopolamine. On-line UV detection occurred at 22 cm from the inlet of the capillary and ESI-MS monitoring was performed along the entire length of the capillary (85 cm). An alkaline solution of 40 mM ammonium acetate at pH 8.5 was suitable for the analysis of the alkaloids under consideration. Under the optimized conditions, including CE and ESI-MS parameters, the two alkaloids were resolved within a short time and with very high sensitivity. The differentiation of hyoscyamine and its positional isomer littorine, commonly encountered in plant material, is also presented using up-front collision-induced dissociation. Finally, the developed method was applied to the analysis of these alkaloids in Belladonna leaf extract and in Datura candida x D. aurea hairy root extract.     

New liquid chromatography/electron ionization mass spectrometry methods in water analysis

A method to extract and analyze organic compounds from water is presented. A solid phase micro-trap (micro-SPE) directly connected to the micro-analytical column is used. Sensibility and specificity needed for trace analysis are guaranteed by mass spectrometric electron ionization (EI) detection. A new micro-HPLC/EI-MS interface called Capillary-EI (Cap-EI) is described. The ultimate evolution of this interface is also presented: in this extremely simplified interface the analytes are nebulized, vaporized and ionized in the small volume of the ion source. This interface, called Direct-EI, exploits nano- and micro-HPLC columns with a mobile phase flow rates ranging from 0.3 to 1.5 microL/min. Contemporary use of micro SPE, Cap-EI or Direct-EI gives us a powerful technique to identify and quantify organic pollutants at part per billion level (ppb).     

Parameter optimization of electrothermal vaporization inductively coupled plasma mass spectrometry for oligoelement determination in standard reference materials

The operational parameters of the graphite furnace for electrothermal vaporization inductively coupled plasma mass spectrometry (ETV-ICP-MS), i.e. the internal carrier gas flow rate, the total carrier gas flow rate, the sample pretreatment temperature and the volatilization temperature, are optimized for oligoelement determinations (⁷⁵As, ⁹Be, ¹¹²Cd, ⁵⁰Cr, ⁶⁵Cu, ¹⁰³Rh, ¹²³Sb). The volatilization temperatures of As and Cr are compared to those obtained by graphite furnace atomic absorption spectrometry (GFAAS). Several modifiers Mg(NO₃)₂, Pd(NO₃)₂, Mg(NO₃)₂/Pd(NO₃)₂, Ni(NO₃)₂, KI, (NH₄)H₂PO₄ have been tested using the concentrations recommended for GFAAS. The concentration of Mg(NO₃)₂ alone and in combination with NaCl has been varied to find the optimal modifier conditions. ETV-ICP-MS signal enhancements by a factor of 10 to 130 respective to those of conventional nebulization have been obtained. The optimized parameters are evaluated by analyzing the water standard reference NIST 1643c and the aqua regia solution of the lake sediment reference material BCR 280.     

Parameter optimization of electrothermal vaporization inductively coupled plasma mass spectrometry for oligoelement determination in standard reference materials

The operational parameters of the graphite furnace for electrothermal vaporization inductively coupled plasma mass spectrometry (ETV-ICP-MS), i.e. the internal carrier gas flow rate, the total carrier gas flow rate, the sample pretreatment temperature and the volatilization temperature, are optimized for oligoelement determinations (⁷⁵As, ⁹Be, ¹¹²Cd, ⁵⁰Cr, ⁶⁵Cu, ¹⁰³Rh, ¹²³Sb). The volatilization temperatures of As and Cr are compared to those obtained by graphite furnace atomic absorption spectrometry (GFAAS). Several modifiers Mg(NO₃)₂, Pd(NO₃)₂, Mg(NO₃)₂/Pd(NO₃)₂, Ni(NO₃)₂, KI, (NH₄)H₂PO₄ have been tested using the concentrations recommended for GFAAS. The concentration of Mg(NO₃)₂ alone and in combination with NaCl has been varied to find the optimal modifier conditions. ETV-ICP-MS signal enhancements by a factor of 10 to 130 respective to those of conventional nebulization have been obtained. The optimized parameters are evaluated by analyzing the water standard reference NIST 1643c and the aqua regia solution of the lake sediment reference material BCR 280.     

Comparison of amino acid sequence analysis by electron ionization and negative-ion chemical ionization mass spectrometry

The positive electron ionization and negative chemical ionization mass spectra of 15 different derivatives of the tripeptide Phe-Ala-Leu have been compared. Total ion currents and ion currents of sequence-characterizing ions have been measured and compared. The negative-ion spectra, using 10% carbon dioxide in argon as moderator gas, proved to be simpler and contained more abundant sequence ions than the positive electron ionization spectra.     

Identification of caffeine and its metabolites in human urine extracts by electron impact ionization tandem mass spectrometry

Electron impact ionized purine bases such as caffeine and its metabolites (theopbylline, theobromine, paraxanthine, 1-methylxanthine, 3-metnybumthine and 7-methyIxanthine) were characterized by tandem mass spectrometry. The direct analysts of human urinary extracts by tandem mass spectrometry allows the unambiguous identification of caffeine and its metabolites present simultaneously in the urine.     

A general soft ionization method for mass spectrometry: Resonance-enhanced multi-photon ionization of biomolecules

We show here that ‘double-barrelled’ laser desorption/resonance-enhanced multi-photon ionization mass spectrometry has many advantages over traditional single-step desorption/ionization techniques, particularly if combined with Reflectron-Time-of-Flight mass analysers. We demonstrate the effectiveness of this technique for large labile biomolecules, namely native chlorophylls, porphyrins and a peptide, as examples of structural and mass analysis.     

Determination of pyrrolizidine alkaloids in comfrey by liquid chromatography-electrospray ionization mass spectrometry

Symphytum officinale L. (comfrey) is a medicinal plant commonly used in decoctions and aliments. Besides therapeutic bioactive compounds present in the herb, it is found to contain hepatotoxic pyrrolizidine alkaloids (PAs), such as lycopsamine and others. In the present study, PAs such as lycopsamine, echimidine and lasiocarpine were determined using electrospray liquid chromatography-mass spectrometry (LC-MS) with the method precision (relative standard deviation, RSD) <10%. Detection of lycopsamine, symviridine and their N-oxides could be confirmed with a newly developed method based on HPLC ion-trap and orbitrap MS with electrospray ionization interface. With LC-MS, quantitative analysis of lycopsamine in the botanical extract was carried out. The effect of extraction solvent was optimized by sonication and methanol: H₂O (50:50) was selected. Then a rapid method based on pressurized hot water extraction (PHWE) was employed for the extraction of lycopsamine from comfrey followed by the comparison with heating under reflux with the RSD ranging from 2.49% to 19.32%. Our results showed a higher extraction efficiency for heating under reflux compared with PHWE. It was proposed that the lower extraction efficiency for PHWE was attributable to dissolved nitrogen from air which caused the reduction in the solubility of lycopsamine in the compressed hot solvent. In this study, quantitative analysis of PAs in comfrey was demonstrated. In addition, it was found that the use of subcritical water for extractions depended on the physical properties of the dissolved solutes and their tendency to degrade under the chosen extraction conditions.     

Isotope-ratio measurements of lead in NIST standard reference materials by multiple-collector inductively coupled plasma mass spectrometry

The capability of a second-generation Nu Instruments multiple collector inductively coupled plasma mass spectrometer (MC-ICP-MS) has been evaluated for precise and accurate isotope-ratio determinations of lead. Essentially the mass spectrometer is a double-focusing instrument of Nier-Johnson analyzer geometry equipped with a newly designed variable-dispersion ion optical device, enabling the measured ion beams to be focused into a fixed array of Faraday collectors and an ion-counting assembly. NIST SRM Pb 981, 982, and 983 isotopic standards were used. Addition of thallium to the lead standards and subsequent simultaneous measurement of the thallium and lead isotopes enabled correction for mass discrimination, by use of the exponential correction law and 205Tl/203Tl = 2.3875. Six measurements of SRM Pb-982 furnished the results 206Pb/204Pb = 36.7326(68), 207Pb/204Pb = 17.1543(30), 208Pb/204Pb = 36.7249(69), 207Pb/206Pb = 0.46700(1), and 208Pb/206Pb = 0.99979(2); the NIST-certified values were 36.738(37), 17.159(25), 36.744(50), 0.46707(20), and 1.00016(36), respectively. Direct isotope lead analysis in silicates can be performed without any chemical separation. NIST SRM 610 glass was dissolved and introduced into the MC-ICP-MS by means of a micro concentric nebulizer. The ratios observed were in excellent agreement with previously reported data obtained by TIMS and laser ablation MC-ICP-MS, despite the high Ca/Pb concentration ratio (200/1) and the presence of many other elements at levels comparable with that of lead. Approximately 0.2 microg lead are sufficient for isotope analysis with ratio uncertainties between 240 and 530 ppm.     

Screening for pyrrolizidine alkaloids in plant materials by electron ionization RP-HPLC-MS with thermabeam interface

Plant samples from leaves of Cerinthe minor, Cynoglossum clandestinum, Echium tuberculatum (as well roots), Eritrichium rupestre, Lithospermum purpureo-coerulem, Nonnea lutea, Nonnea setosa, Onosma stellulatum and Cynoglossum amabile were screened for toxic pyrrolizidine alkaloids (PAs) with a newly elaborated procedure comprising gradient HPLC with diode array (DAD) and thermabeam electron impact mass spectrometry (EI-MS). Dried plant material was extracted with boiling 1% tartaric acid in methanol for 2 h on an electric basket and crude extracts purified with cation-exchange solid phase extraction (CE-SPE). Purified extracts containing alkaloids were separated on Zorbax SB RP18 stationary phase in gradient of 0.1% formic acid in methanol. The flow rate was 0.25 mL/min and was suitable both for DAD and EI MS detections. Applied gradient procedure permitted quite sufficient separation of PAs in various plant extracts. On the basis of EI MS spectra, toxic PAs with unsaturated 1,2-double bond in the necine moiety were found in all plant materials and in nine of them (excluding only Cynoglossum amabile) for the fi rst time. They included the following types of structures: 9- and 7-viridifloryl-retronecine monoesters, 9-angeloyl-7-viridifloryl-retronecine, 9-angeloyl-retronecine diester, 9-viridifloryl-retronecine saturated ester, 7-angeloyl-9-viridifloryl-retronecine, 7-angeloyl-9-echimidinyl-retronecine, trachelanthamine and others. Selected ion monitoring (SIM) chromatograms at m/z 119, 120 and 136 together with analysis of UV spectra from DAD detector can be applied in rapid screening for toxic PAs in new plant extracts but to obtain detailed structural information (molecular weight and stereochemistry) more expensive hyphenation is required. Consumption of all analysed plants should be avoided as carcinogenic and hepatotoxic properties of the alkaloids detected are expected.     

Atmospheric-pressure laser ionization: a novel ionization method for liquid chromatography/mass spectrometry

We report on the development of a new laser-ionization (LI) source operating at atmospheric pressure (AP) for liquid chromatography/mass spectrometry (LC/MS) applications. APLI is introduced as a powerful addition to existing AP ionization techniques, in particular atmospheric-pressure chemical ionization (APCI), electrospray ionization (ESI), and atmospheric pressure photoionization (APPI). Replacing the one-step VUV approach in APPI with step-wise two-photon ionization strongly enhances the selectivity of the ionization process. Furthermore, the photon flux during an ionization event is drastically increased over that of APPI, leading to very low detection limits. In addition, the APLI mechanism generally operates primarily directly on the analyte. This allows for very efficient ionization even of non-polar compounds such as polycyclic aromatic hydrocarbons (PAHs). The APLI source was characterized with a MicroMass Q-Tof Ultima II analyzer. Both the effluent of an HPLC column containing a number of PAHs (benzo[a]pyrene, fluoranthene, anthracene, fluorene) and samples from direct syringe injection were analyzed with respect to selectivity and sensitivity of the overall system. The liquid phase was vaporized by a conventional APCI inlet (AP probe) with the corona needle removed. Ionization was performed through selective resonance-enhanced multi-photon ionization schemes using a high-repetition-rate fixed-frequency excimer laser operating at 248 nm. Detection limits well within the low-fmol regime are readily obtained for various aromatic hydrocarbons that exhibit long-lived electronic states at the energy level of the first photon. Only molecular ions are generated at the low laser fluxes employed ( approximately 1 MW/cm(2)). The design and performance of the laser-ionization source are presented along with results of the analysis of aromatic hydrocarbons.     

Determination of nitrated polycyclic aromatic hydrocarbons in diesel particulate-related standard reference materials by using gas chromatography/mass spectrometry with negative ion chemical ionization

Gas chromatography/mass spectrometry (GC/MS) with negative ion chemical ionization (NICI) detection was utilized for quantitative determination of nitrated polycyclic aromatic hydrocarbons (nitro-PAHs) in diesel particulate-related standard reference materials (SRMs). Prior to GC/MS analysis, isolation of the nitro-PAHs from the complex diesel particulate extract was accomplished using solid phase extraction (SPE) and normal-phase liquid chromatographic (LC) fractionation using an amino/cyano stationary phase. Concentrations of eight to ten mononitro-PAHs and three dinitropyrenes were determined in three diesel particulate-related SRMs: SRM 1650a Diesel Particulate Matter, SRM 1975 Diesel Particulate Extract, and SRM 2975 Diesel Particulate Matter (Industrial Forklift). The results from GC/MS NICI using two different columns (5% phenyl methylpolysiloxane and 50% phenyl methylpolysiloxane) were compared to each other and to results from two other laboratories for selected nitro-PAHs. 1-Nitropyrene was the most abundant nitro-PAHs in each of the diesel particulate SRMs (19.8+/-1.1 micro g g(-1) particle in SRM 1650a and 33.1+/-0.6 micro g g(-1) particle in SRM 2975). Three dinitropyrene isomers were measured in SRM 1975 at 0.5-1.4 micro g g(-1) extract and in SRM 2975 at 1-3 micro g g(-1) particle.     

Liquid chromatography/electrospray ionization mass spectrometry method for the quantification of valproic acid in human plasma

A simple, sensitive and rapid liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS) method was developed and validated for the quantification of valproic acid, an antiepileptic drug, in human plasma using benzoic acid as internal standard (IS). Following solid-phase extraction, the analytes were separated using an isocratic mobile phase on a reversed-phase C18 column and analyzed by MS in the single ion monitoring mode using the respective [M-H]- ions, m/z 143 for valproic acid and m/z 121 for the IS. The assay exhibited a linear dynamic range of 0.5-60 microg/mL for valproic acid in human plasma. The lower limit of quantification was 500 ng/mL with a relative standard deviation of less than 10%. Acceptable precision and accuracy were obtained for concentrations over the standard curve range. The average absolute recoveries of valproic acid and the IS from spiked plasma samples were 96.1+/-4.2 and 95.6+/-2.7%, respectively. A run time of 4.5 min for each sample made it possible to analyze more than 250 human plasma samples per day. The validated method has been successfully used to analyze human plasma samples for application in pharmacokinetic, bioavailability and bioequivalence studies.     

Flow-injection electrospray ionization mass spectrometry of crude cell extracts for high-throughput bacterial identification

Flow-injection electrospray ionization mass spectrometry (FI-ESI-MS) of unfractionated cell-free extracts obtained from bacterial cells suspended in a solvent mixture was investigated as a rapid analytical method for reproducible, high-throughput bacterial identification. Five bacterial strains (two Escherichia coli, two Bacillus spp. and one Brevibacillus laterosporus) were studied in this investigation. Axenically grown bacterial cells were suspended in an acidic organic solvent and the cell-free extract was sequentially injected into a solvent flow stream that was sprayed into the ionization chamber of the ESI-MS. The spectra produced contained reproducible information, which was useful for discriminating between the bacteria. Tandem mass spectrometry was used to characterize further the peaks, and at least three classes of macromolecules, namely phospholipids, glycolipids, and proteins, were found to contribute most to the spectral information. Bacterial extracts stored under different conditions gave very similar mass spectra for each of the five bacterial strains, indicating that the extracts were stable even at room temperature for up to 24 h, with no loss of information content, which has obvious implications for automated high-throughput analysis. An analysis of the components of the extracting solvent mixture and their effects on the spectral information showed that acetonitrile contributes most significantly to the extraction process and hence to the information content of the spectra.