Total Ion Current Traces Research Articles

DETECTION AND QUANTIFICATION OF PHYTOCHEMICAL MARKERS OFIlex paraguariensisBY LIQUID CHROMATOGRAPHY

Ilex paraguariensis (yerba-mate) is used as a beverage, and its extract requires adequate quality control methods in order to guarantee quality and safe use. Strategies to develop and optimize a chromatographic method to quantify theobromine, caffeine, and chlorogenic acid in I. paraguariensis extracts were evaluated by applying a quality by design (QbD) model and ultra high-performance liquid chromatography (UHPLC). The presence of these three phytochemical markers in the extracts was evaluated using UHPLC-MS and was confirmed by the chromatographic bands in the total ion current traces (m/z of 181.1 [M+H]+, 195.0 [M+H]+, and 353.0 [M−H]−, respectively). The developed method was then transferred to a high-performance liquid chromatography (HPLC) platform, and the three phytochemical markers were used as external standards in the validation of a method for analyses of these compounds in extracts using a diode array detector (DAD). The validated method was applied to quantify the chlorogenic acid, caffeine, and theobromine in the samples. HPLC-DAD chromatographic fingerprinting was also used in a multivariate approach to process the entire data and to separate the I. paraguariensis extracts into two groups. The developed method is very useful for qualifying and quantifying I. paraguariensis extracts.

Isobaric metabolite interferences and the requirement for close examination of raw data in addition to stringent chromatographic separations in liquid chromatography/tandem mass spectrometric analysis of drugs in biological matrix

In addition to matrix effects, common interferences observed in liquid chromatography/tandem mass spectrometry (LC/MS/MS) analyses can be caused by the response of drug-related metabolites to the multiple reaction monitoring (MRM) channel of a given drug, as a result of in-source reactions or decomposition of either phase I or II metabolites. However, it has been largely ignored that, for some drugs, metabolism can lead to the formation of isobaric or isomeric metabolites that exhibit the same MRM transitions as parent drugs. The present study describes two examples demonstrating that interference caused by isobaric or isomeric metabolites is a practical issue in analyzing biological samples by LC/MS/MS. In the first case, two sequential metabolic reactions, demethylation followed by oxidation of a primary alcohol moiety to a carboxylic acid, produced an isobaric metabolite that exhibits a MRM transition identical to the parent drug. Because the drug compound was rapidly metabolized in rats and completely disappeared in plasma samples, the isobaric metabolite appeared as a single peak in the total ion current (TIC) trace and could easily be quantified as the drug since it was eluted at a retention time very close to that of the drug in a 12-min LC run. In the second example, metabolism via the ring-opening of a substituted isoxazole moiety led to the formation of an isomeric product that showed an almost identical collision-induced dissociation (CID) MS spectrum as the original drug. Because two components were co-eluted, the isomeric product could be mistakenly quantified and reported by data processing software as the parent drug if the TIC trace was not carefully inspected. Nowadays, all LC/MS data are processed by computer software in a highly automated fashion, and some analysts may spend much less time to visually examine raw TIC traces than they used to do. Two examples described in this article remind us that quality data require both adequate chromatographic separations and close examination of raw data in LC/MS/MS analyses of drugs in biological matrix.

A validated higher-performance liquid chromatography method for quantification of cinchonain Ib in bark and phytopharmaceuticals of Trichilia catigua used as Catuaba

The hydroalcoholic extract, prepared from authentic chopped barks of Trichilia catigua, was evaluated by high-performance liquid chromatography using a diode array detector (200–400 mn). The crude extract was purified by rotation locular counter-current chromatography and the chloroform fraction obtained was clean-up by solid-phase extraction. With the aim of getting preliminary structure information on-line, the methanol fraction thus obtained was analyzed by gradient elution using the diode array detector coupled to a mass spectrometer. The presence of flavalignan in this extract was inferred by the chromatographic band, in the total ion current trace, that had an [M–H] − = 451. With this information, cinchonain Ib was isolated as a pure compound from the crude hydroalcoholic extract using a solid-phase extraction procedure for the sample clean-up followed by a semi-preparative separation using the reverse mode of elution. The isolated compound, after complete characterization, was used as an external standard for the development and validation of a method for the analysis of this compound in herbal medicines using the ultraviolet as the detector. The validated method has been successfully applied for quantification of cinchonain Ib in commercialized herbal medicines sold as Catuaba in Brazil and also in standard chopped barks of T. catigua.

Porous polymer monolith assisted electrospray from a glass microdevice

The coupling of a lab-on-a-chip microfluidic device to a nanoelectrospray ionization mass spectrometer has the potential to automate many routine analytical procedures and produce a powerful analytical tool. However, past coupling strategies have relied on complex manufacturing steps including drilling and etching the device to attach a capillary or building a nanospray emitter directly into the device. This study shows that a nanospray emitter can be easily fabricated using a porous polymer monolith (PPM) at the end of a glass microdevice. These devices are able to obtain a stable electrospray at a variety of flow rates (50-500 nL/min) but optimal results are obtained at lower flow rates (50-100 nL/min) compatible with electroosmotic flow processes. The PPM is photo-patterned so that it can be placed in any position within the channel of the device with no dead volume. The porous character and the hydrophobic nature of the PPM both aid in development of a stable electrospray process. Total ion current traces for the constant infusion of leucine-enkephalin and PPG show relative standard errors as low as 4%, and produce mass spectra with good signal-to-noise (S/N 43) from only 2 fmol of material. In addition, multiple experiments in a given day show good repeatability with variability as low as 13%, and the multiple flow paths inherent in the PPM limit sprayer clogging.

Porous Polymer Monolith Assisted Electrospray

Coupling low-flow analytical separation instrumentation such as capillary electrophoresis, capillary electrochromatography, nano-HPLC, and microfluidic-based devices with electrospray ionization mass spectrometry has yielded powerful analytical tools. However, conventional coupling methodologies such as nanospray suffer from limitations including poor conductive coating robustness, constant clogging, complicated fabrication processes, and incompatibility with large flow rate regimes. This study demonstrates that robust nanospray emitters can be fabricated through the formation and utilization of a porous polymer monolith (PPM) at the end of a fused-silica capillary. Stable electrosprays can be produced from capillaries (75-100-microm i.d.) at a variety of flow rates (50-1000 nL/min) without the need to taper the capillaries by etching or pulling. The PPM is photopatterned to be present only near the capillary exit aperture using conditions that generate pore sizes similar to those seen with nanospray tips. The porous nature of the PPM aids in developing a stable electrospray generating a single clearly visible Taylor cone at relatively high flow rates while at low flow rates (<100 nL/min) a mist, presumably from multiple small Taylor cones, develops. The hydrophobic nature of the PPM should limit problems with band broadening associated with droplet spreading at the capillary exit, while the multiple flow paths inherent in the PPM minimize clogging problems associated with conventional nanospray emitters. Total ion current traces for a constant infusion of standard PPG and cytochrome c solutions are very stable with deviations ranging from only 3 to 8%. The PPM-assisted electrospray produces mass spectra with excellent signal-to-noise ratios from only a few femtomoles of material.

Polar organic pollutants in the Elbe river Liquid chromatographic-mass spectrometric and flow-injection analysis-mass spectrometric analyses demonstrating changes in quality and concentration during the unification process in Germany

During the unification process of the two German states from 1989 to 1994, water extracts from the Elbe river were analyzed by gas chromatography-mass spectrometry (GC-MS) and by high-performance liquid chromatography (HPLC) combined with ultraviolet (UV) and/or mass spectrometric detection (MS) in order to monitor the pollutants in one of the most contaminated European rivers. After column chromatography (LC) or flow-injection analysis (FIA) bypassing the analytical column, ionization was performed by a thermospray interface (TSP). Semiquantitative estimations of the pollution of the Elbe were made form the total-ion current traces (TIC) of the extracts. Determination of the total dissolved organic carbon (DOC) indicated a reduction of more than 55% of pollutants in the water phase. The pollutants were identified by tandem mass spectrometry (MS-MS), generating daughter-ion spectra by collision-induced dissociation (CID) using either column chromatography or direct mixture analysis. Compound-specific analyses indicated a reduction in the amount of pollutants as well as a change in their composition. This could be explained by reduced industrial production and by intensified construction of biological sewage treatment plants.

Use of liquid chromatography—thermospray mass spectrometry in phytochemical analysis of crude plant extracts

Qualitative analyses of selected compounds by high-performance liquid chromatography of crude plant extracts are not always straightforward owing to the lack of a suitable chromophore for UV detection or because insufficient material is present. In both instances, however, coupling liquid chromatography with thermospray mass spectrometry has proved to be ideal for the direct detection of these compounds. Different examples of analyses of bioactive constituents (including ginkgolides, sesquiterpene lactones, acetogenins and saponins) in crude plant extracts are described. Total ion current traces and mass chromatograms allow the easy detection of these compounds in the extracts. Depending on the ionization pattern, [M + H] + or [M + NH 4] + ions are used for selected ion monitoring studies.

Studies on the metabolism of omeprazole in the rat using liquid chromatography/ionspray mass spectrometry and the isotope cluster technique with [34S]omeprazole.

This paper describes the application of the ionspray (pneumatically-assisted electrospray) interface for liquid chromatography (LC) and atmospheric-pressure ionization mass spectrometry (API MS) to samples obtained in a study on the metabolism of omeprazole. In this study [34S]omeprazole was utilized for the stable isotope cluster technique. Over forty metabolites in a sample of partially purified rat urine were resolved by gradient elution LC with ionspray API MS detection, and each of them produced molecular ion 1:1 clusters (MH+ and [MH + 2]+). The chromatographic fidelity of the total-ion current (TIC) was excellent. The endogenous matrix of the sample was quite low, allowing a background-subtracted averaged mass spectrum of the entire TIC trace to produce a 'metabolite mass profile' depicting all the molecular ion 1:1 clusters in the sample. From this mass profile, it was possible to obtain direct information concerning oxygenation and conjugation reactions of the parent compound.

Identification of Endogenous Gibberellins in the Winter Annual Weed Thlaspi arvense L

Eleven endogenous gibberellins (GAs) were identified by combined gas chromatography-mass spectrometry in purified extracts from shoots of field pennycress (Thlaspi arvense L.): GA(1,9,12,15,19,20,24,29,44,51,53). Traces of GA(8) and GA(25) were tentatively indicated by combined gas chromatography-mass spectrometry-selected ion monitoring. Comparison of the total ion current traces indicated that GA(19) and GA(44) were most abundant, while GA(12,15,20,24,29,53) occurred in lesser amounts. Only small amounts of GA(1,9,51) were present. The levels of GA(8) and GA(25) were barely detectable. Consideration of hydroxylation patterns of the ent-gibberellane ring structure indicates two families of GAs: one with a C-13 hydroxyl group (GA(1,8,19,20,29,44,53)) and another whose members are either nonhydroxylated (GA(9,12,15,24,25)) or lack a C-13 hydroxyl group (GA(51)). This suggests that in field pennycress there are two parallel pathways for GA metabolism with an early branch point from GA(12): an early C-13 hydroxylation pathway, leading ultimately to GA(1) and GA(8) and a C-13 deoxy pathway culminating in the formation of GA(9) and GA(51).

Selected-ion monitoring analyses of environmental samples using improved software

Computer programs have been written which significantly increase the storage capabilities of a low-cost gas chromatography—mass spectrometry (GC-MS)-calculator system. These programs convert raw selected ion monitoring (SIM) data to a more compact form and allow the storage of fourteen 60-min GC-MS analyses on disks which were previously limited to four analyses. SIM data can be displayed by means of an interactive program which uses an x- y plotter. The total ion current and mass chromatograms generated during operation of the GC-MS system in scanning mode can be displayed on the same plot as SIM analyses with this versatile program. In addition, the sum of SIM ion abundances, analogous to a scan run total ion current trace can be displayed or integrated. The software was used in a GC-MS analysis of organic compounds extracted from a ground water leachate sample taken at a municipal dumpsite.

Combined liquid chromatography mass spectrometry of glycosides, glucuronides, sugars and nucleosides

On-line liquid chromatographic mass spectrometric studies of glycosides, glucuronides, mono- and disaccharides and nucleosides are reported. Using ammonia chemical ionization good total ion current traces were obtained together with mass spectra which enabled characterization of the compounds studied.

The characterization of closely related polymeric materials by thermogravimetry—mass spectrometry

The potential of a new technique in forensic science, thermogravimetry—mass spectrometry, is illustrated by its application to the examination of acrylic fibres and white alkyd gloss paints, small samples of which are difficult to differentiate even by a combination of current techniques. A simple interface allowing the coupling of a magnetic sector mass spectrometer to a thermobalance is described. This combination allows up to five experimental parameters to be determined simultaneously either for classification of the material under investigation or for the detailed direct comparison of samples. The thermobalance allows the temperatures at which weight losses occur and their relative proportions to be determined; the basic parameter obtained from the mass spectrometer is the total ion current trace. From this, single qualitative mass spectra may be chosen for examination and ions, whose presence or absence may be diagnostic or which vary characteristically throughout the analysis, can be selected. The variations of these ions may then be examined by computer-generated mass thermograms (analagous to mass chromatograms in g.c.—m.s.) or, if computer facilities are not available, by multiple ion detection. The residue from the analysis can be used for trace element analysis, e.g. by energy-dispersive x-ray fluorescence spectrometry.