Total Ion Current Chromatograms Research Articles

Systematic toxicological analysis: computer-assisted identification of poisons in biological materials

A new software was developed to improve the chances for identification of a “general unknown” in complex biological materials. To achieve this goal, the total ion current chromatogram was simplified by filtering the acquired mass spectra via an automated subtraction procedure, which removed mass spectra originating from the sample matrix, as well as interfering substances from the extraction procedure. It could be shown that this tool emphasizes mass spectra of exceptional compounds, and therefore provides the forensic toxicologist with further evidence—even in cases where mass spectral data of the unknown compound are not available in “standard” spectral libraries.

Analysis of rabbit tear proteins by high-pressure liquid chromatography/electrospray ionization mass spectrometry.

The aim of this study was to develop a fast and reliable analytical procedure for the display of the protein components of tears that can be used to differentiate the status of the ocular surface. Using this new procedure, we analyzed the tear protein components following a corneal wound in the rabbit. Calibrated 10-microL glass, fire-polished capillary micropipettes were used to collect tears from New Zealand White rabbits prior to and daily for 9 days following a unilateral 6-mm diameter centrally placed anterior keratectomy. Tear proteins were eluted by a reversed-phase high-performance liquid chromatography (RP-HPLC) column and the tear protein profile was monitored by electrospray ionization (ESI) mass spectrometry positive total ion current (TIC) chromatography. Tear proteins were reliably separated into 17 peaks, each of which contained one or a number of protein components. The molecular weight of each protein component was determined by on-line ESI. Major tear protein components, lactoferrin, lysozyme (minimally detectable in rabbit tears), albumin, lipocalin, lipophilin and beta2-microglobulin, were tentatively identified by this method. Based on the mass spectrometric data, beta2-microglobulin was found to be glycosylated with N-acetylhexosamine. ESI-positive TIC chromatograms and mass spectra revealed comparative differences in the tear protein spectra after corneal wounding. One day after wounding, rabbit lysozyme with a molecular weight of 14,717 Da was found to be 8-fold higher in the tears of wounded eyes when compared with tears from unwounded eyes. It dropped back to normal 3 days after wounding. The expression of an unidentified tear protein with the molecular weight of 16,060 Da was also elevated after corneal wounding and returned to normal level by day 5. In this study, LC/ESI-MS was developed as a fast, reproducible and simple method for the identification and analysis of many of the protein components of the tears. Importantly, this technique also allows quantification of each component resolved in the chromatogram. This method is very suitable for mapping peptides and proteins (<80 kDa) in tears.

Simultaneous analysis of caffeic acid derivatives and alkamides in roots and extracts of Echinacea purpurea by high-performance liquid chromatography–photodiode array detection–electrospray mass spectrometry

High-performance liquid chromatography (HPLC) coupled with UV photodiode-array detection and electrospray ionization mass spectrometry was developed for the simultaneous analysis of caffeic acid derivatives and alkamides in the roots and extracts of Echinacea purpurea. Caffeic acid derivatives and alkamides produced very abundant peaks in the total ion current chromatogram during negative and positive cone voltage switching. Cichoric acid and the isomer pair, dodeca-2 E,4 E,8 Z,10 E/ Z-tetraenoic acid isobutylamide, were used as a standard for quantification of caffeic acid derivatives and alkamides in E. purpurea. This novel method surpasses previously published ones in product quality control and providing the HPLC chromatographic fingerprints of biological active components in E. purpurea.

Supercritical fluid chromatography coupled to electrospray mass spectrometry: a powerful tool for the analysis of chiral mixtures

Supercritical fluid chromatography coupled to a hybrid mass spectrometer (Q-Tof2) equipped with electrospray ion source has been used to separate and characterise a wide range of pharmaceutical racemates. We have chosen diverse molecular structures to demonstrate the potential of such experimental arrangement for high throughput analyses. The use of three different chiral stationary phases and different pressure/temperature working conditions provided clear indications on how such a high throughput method can be developed. The use of mass spectrometry was found to be essential for an unambiguous assignment of the eluting components particularly in the case of complex mixtures. The direct coupling of both systems without the need for a special interface resulted in similar peak shapes and peak widths in the UV and total ion current (TIC) chromatograms.

Comparison between liquid chromatography–time-of-flight mass spectrometry and selected reaction monitoring liquid chromatography–mass spectrometry for quantitative determination of idoxifene in human plasma

This study compares HPLC electrospray time-of-flight mass spectrometry and selected reaction monitoring (SRM) LC–MS for high throughput quantitative determination of a small molecule drug in biological samples. A high throughput LC–MS method was developed for quantitatative determination of idoxifene in human plasma and the evaluation was accomplished with the cross-validation of the developed LC–MS method between the time-of-flight mass spectrometer, and a triple quadrupole mass spectrometer operated in the SRM mode. A simple one-step semi-automated 96-well liquid–liquid extraction procedure was used to prepare 96 samples in approximately 30 min and a rapid gradient was used to shorten the LC run time. Time-of-flight mass spectrometry provides acquisition of full-scan mass spectra and extracted ion current chromatograms, which may be extracted from the total ion current chromatogram for peak area determination. The limit of quantitation for idoxifene in human plasma obtained with the time-of-flight mass spectrometer was 5 ng/ml based on 100-μl aliquots of human plasma, and the linear dynamic range was from 5 ng/ml to 2000 ng/ml. The quantitative LC–MS results from the time-of-flight mass spectrometer demonstrated that precision did not exceed 7.1% and accuracy did not exceed 1.7% with reference to quality control samples at three concentration levels in replicates of six. In contrast, the limit of quantitation for idoxifene in human plasma using a tandem triple quadrupole mass spectrometer was 0.5 ng/ml with a linear dynamic range to 1000 ng/ml. The results from the triple quadrupole instrument show that the precision did not exceed 2.2% and accuracy did not exceed 2.9%. The overall results suggest time-of-flight mass spectrometry may be a viable technique for high throughput bioanalytical work for the quantitative determination of a representative small molecule drug in the low ng/ml range in human plasma.

Simultaneous determination of acidic and non-acidic pesticides in natural waters by liquid chromatography–mass spectrometry

There is increasing interest and demand for real multi-residue methods able to simultaneously determine pesticides with a broad spectrum of chemical characteristics in environmental and biological matrices. A method based on solid-phase extraction with a Carbograph 4 cartridge and liquid chromatography with electrospray mass spectrometry (LC–ES-MS) enabling simultaneous determination of non-acidic and acidic pesticides in real water samples is described. On repeatedly ( n=5) extracting 4 l of drinking water (spike level 50 ng/l), 2 l of ground water (spike level 100 ng/l) and 1 l of river water (spike level 200 ng/l), recovery of 26 base/neutral pesticides and 13 acidic pesticides were equal to or better than 80%, except for carbendazim (67%), butocarboxim (73%), aldicarb (75%) and molinate (77%). Relative standard deviations ranged between 4 and 15%. Final extracts containing acidic and non-acidic pesticides were analyzed in a single chromatographic run while the ES-MS system was operated in both positive and negative ion modes. With the aim of finding the best operating conditions, in terms of sensitivity, the pH of the LC eluent was varied in the 2.9–8.4 range. Altogether, the best results were obtained by using an LC eluent containing 1 mmol/l formic acid. Over the entire pH range considered, well shaped peaks for both basic and acidic analytes were achieved by the use of a new generation LC column. By extracting selected ion current profiles from the total ion current mass chromatogram relative to analysis of 4 l of drinking water spiked with 50 ng/l of each of the 39 analytes, estimated limits of detection ranged between 0.05 and 1.5 ng/l, except for propyzamide (8 ng/l) and 2,4-DB (3 ng/l).

Iron(III)-Immobilized Metal Ion Affinity Chromatography and Mass Spectrometry for the Purification and Characterization of Synthetic Phosphopeptides

A method based upon immobilized metal ion affinity chromatography (IMAC) is described for purification of phosphopeptides from the crude preparations of solid-phase peptide synthesis step. Affinity chromatography consists of iron(III) immobilized on iminodiacetate–agarose gel. The method was applied for purification of seven synthetic enkephalin-related phosphorylated peptides. The effectiveness of the method was evaluated by analyzing the IMAC-retained and -nonretained components using reversed-phase (RP) high-performance liquid chromatography (HPLC) and an on-line combination of RP-HPLC and electrospray ionization mass spectrometry. The UV and total ion current chromatograms demonstrated that the phosphopeptides were effectively separated and purified.

ANALYSIS OF PHOSPHOENKEPHALINS BY COMBINED HIGH PERFORMANCE LIQUID CHROMATOGRAPHY AND ELECTROSPRAY IONIZATION MASS SPECTROMETRY

A method based upon a combination of high performance liquid chromatography (HPLC) and electrospray ionization (ESI) mass spectrometry (MS) for the analysis of phosphorylated peptides in a complex mixture of 14 phosphoenkephalins and their nonphosphorylated analogues is described. A narrow-bore column is directly connected to the ESI interface without the need of a solvent flow-splitter. A full-scan mass analysis in the positive-ion mode is used to provide the total ion current chromatogram and the molecular mass of each eluting component. Selective detection of phosphorylated peptides is achieved by selected-ion recording of the phosphate group marker ion (PO3 −) in the negative-ion mode. These two scans are acquired simultaneously on a single injection of the sample. The influence of water clusters on background ion current is also discussed.

Analysis of alkamides in roots and achenes of Echinacea purpurea by liquid chromatography–electrospray mass spectrometry

High-performance liquid chromatography paired with UV photodiode-array and electrospray mass spectrometry was investigated as a method for the analysis of alkamides in the roots and achenes of the popular herb Echinacea purpurea (L.) Moench. All alkamides showed very abundant peaks in the reconstructed total ion current chromatogram. Nine alkamides were identified in the root of E. purpurea, similar to the fingerprint reported by Bauer and Remiger [Planta Med., 55 (1989) 367]. Several minor alkamides, not identifiable by previously published techniques, have been also tentatively identified. Two major peaks were identified in E. purpurea achenes as undeca-2 E,4 Z-diene-8, 10-diynoic acid isobutylamide ( 1) and dodeca-2 E,4 E,8 E,10 E/Z tetraenoic acid isobutylamide ( 8/ 9). The isomer pair, tetraene 8/ 9, was purified as a standard for quantification of alkamide content in E. purpurea achenes and roots, as well as for the achenes of E. angustifolia DC and E. pallida (Nutt.) Nutt.

Particle beam LC/MS screening of polar compounds of contaminated ground water samples from a former gas plant

A commercial particle beam interface coupled to a single quadrupole mass spectrometer has been used for a nontarget screening of highly contaminated water samples from a former gas plant. A liquid chromatographic system has been used to separate hydrophilic compounds with a high percentage water gradient without any loss of efficiency in the MS interface. The ground water showed a high pollution burden in the total ion current chromatogram. Full scan EI mass spectra have been obtained for identification of unknown compounds which could be assigned to oxidation products of heterocyclic aromatic compounds, some known constituents of coal tar. The identification of 6(5H)-phenanthridinone has been confirmed by comparison with a corresponding standard. The difficulties of an unequivocal identification of isoquinolinone and methyl derivatives of quinolinone based on their EI mass spectra are discussed.

Protein digest analysis by pressurized capillary electrochromatography using an ion trap storage/reflectron time-of-flight mass detector.

Pressurized capillary electrochromatography (pCEC) has been coupled to an ion trap storage/reflectron time-of-flight mass spectrometer for the analysis of peptide mixtures and protein digests. Taking advantage of the electroosmotic flow, high separation efficiency has been achieved in pCEC due to a relatively flat flow profile and the use of smaller packing materials. A supplementary pressure was used in these experiments which suppressed bubble formation and also allowed the tuning of the elution of peptides using the electrical field. In this work, a fast separation of a six-peptide mixture has been successfully performed. Using columns only 6 cm long, a tryptic digest of bovine cytochrome c was fully separated in around 14 min by properly tuning the applied voltage and the supplementary pressure. In addition, relatively complex protein digests, such as a tryptic digest of chicken ovalbumin, were analyzed using this pCEC/MS system, and more than 20 peaks were resolved in the total ion current chromatogram within 17 min. The use of an ion trap storage/reflectron time-of-flight mass spectrometer as an on-line detector further increased the resolving power of the pCEC by unambiguously identifying coeluting components. The nonscanning property of the time-of flight mass analyzer and the ion signal integration capability of the ion trap were successfully combined to provide rapid and sensitive full-mass range detection in these experiments.

Structural analysis of the CHO‐derived interleukin‐4 by liquid‐chromatography/electrospray ionization mass spectrometry

AbstractElectrospray (ES) ionization mass spectrometric analysis of CHO‐derived recombinant interleukin‐4 (IL‐4) before and after deglycosylation with peptide: N‐glycosidase F is described. That proved useful not only in deriving the size of the attached carbohydrate components but also in identifying the major glycoforms as the mono‐and disialylated complex‐type N‐linked oligosaccharides. Additional signals arising from glycoforms containing carbohydrate components with more extended or higher branching were also detected. Further mapping of CHO IL‐4 was carried out by combining proteolytic digestion and chromatographic separation of the resulting peptide mixture with on‐line ES mass spectrometric detection. Comparative analysis of the V8 protease digests of CHO IL‐4 and its deglycosylated product revealed the Asn38 N‐glycosylation site. Glycopeptide‐containing fractions were identified by searching the resulting raw ES data for signal pairs whose m/z values differ by the mass of various carbohydrate units adjusted for the signal's charge state (e.g. 97 u difference for a triply charged ES signal of a glycopeptide containing NeuAc units). Furthermore, ES mass spectrometric analysis at elevated orifice potentials allowed the rapid location of the glycopeptides in the total ion current chromatogram by monitoring several carbohydrate‐specific fragment ions. This high orifice‐induced fragmentation is a highly sensitive method for generating sugar diagnostic ions in chromatographically separated components, even when glycopeptide and peptide fragments are co‐eluting.

Thermal degradation of polymers in the melt, 1. Characterization of volatile oligomers formed by thermal degradation of polyisobutylene

AbstractChemical structures of a number of components of volatile oligomers including (n+2)‐mers (n ≧ 0) produced by thermal degradation of polyisobutylene were systematically determined by high‐resolution capillary gas chromatogaphy/mass spectrometry (GC/MS). The total ion current (TIC) chromatogram consists of about 100 peaks ranging from dimers (2‐mers, n = 0) to dodecamers (12‐mers, n = 10) and most of the main peaks are classified into four types of terminal monoolefins: a trisubstituted olefin with a tert‐butyl end, a vinylidene olefin with a tert‐butyl end, a trisubstituted olefin with an isopropyl end, and a vinylidene olefin with an isopropyl end. The formation of these monoolefins is reasonably interpreted by intramolecular hydrogen abstractions (back‐biting) of primary and tertiary terminal macroradicals and subsequent β; scissions at the inner position of the main chain. In all the chromatograms of each (n+2)‐mer (n ≧ 1), the retention times of terminal trisubstituted types of monoolefins were shorter than those of terminal vinylidene types of monoolefins, in contrast to the elution order of dimers. The relative intensities between the interesting peaks of each (n+2)‐mer (n ≧ 1) clearly represent that back‐biting more predominantly occurs at the methylene hydrogen rather than at the methyl group consistent with the difference in the bond dissociation energy of the HC bond of interest, as opposed to the steric hindrance mechanism5,7.

Characterization of neutral metabolites of benzo[a]pyrene in urine from germfree rats.

[14C]Benzo[a]pyrene (BP) was administered to male germfree rats. Urinary metabolites, constituting 9% of the administered radioactivity, were fractionated by lipophilic ion exchange chromatography. More than 80% of the urinary metabolites were conjugated, while neutral metabolites constituted 13-18%. The latter group was characterized by reversed-phase HPLC, ultraviolet spectrometry (UV) and gas chromatography/mass spectrometry (GC/MS). Relative quantities of BP metabolites were estimated from the distribution of radioactivity upon HPLC fractionation. Some coeluted peaks were further quantitated from the total ion current chromatograms obtained in the GC/MS analysis. Two 7,8,9,10-tetrols which might be produced from the ultimate carcinogen r-7-t-8-dihydroxy-t-9,10-oxy-7,8,9,10-tetrahydro-BP (anti-7,8-diol-9,10-epoxide) were detected at trace levels and indicated to be the r-7-t-8,9-c-10-(7,10/8,9-) and r-7-t-8,9,10-(7/8,9,10-) isomers. A trans-11,12-dihydrodiol was characterized as a major metabolite, while a trans-7,8-dihydrodiol was present at trace levels. Three additional quantitatively important metabolites were identified as isomeric trihydroxy-BPs. Two metabolites coeluting with BP quinones on HPLC were detected with relatively high abundance and tentatively identified as carboxylic methyl ester derivatives of BP quinones. Three quinones were detected with 1,6-, 3,6- and 6,12-substitutions. The 6,12- and 11,12-dihydroxy-BPs were also found at trace levels. A group of quinone-like metabolites were tentatively identified as trioxo-BPs. No monohydroxy-BP was detected in the neutral fraction of the urine extract. The time course of excretion was also studied and found to differ between individual metabolites.

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.

Ion spray mass spectrometric detection for liquid chromatography: A concentration- or a mass-flow-sensitive device?

As the mass spectrometer becomes more accepted as a detector for HPLC, its characteristics should become better understood by those performing routine LC-MS experiments. In particular, the ion current response for quantitative analysis studies involving significant dynamic range in concentration for target analytes must be determined as well as other factors that affect MS response. This work describes the concentration-sensitive response for the ion spray (pneumatically-assisted electrospray) LC-MS interface from the chromatographer's perspective. A comparison of LC-MS ion current response in the isocratic mode resulting from studies of a synthetic mixture containing alkyl benzoates is presented. LC-MS total ion current chromatograms from three different column sizes (1 mm I.D., 2.1 mm I.D. and 4.6 mm I.D.) with and without a post-column split, and high-flow ion spray LC-MS without a post-column split illustrates that the former behaves as a concentration-sensitive detector whereas the latter behaves as a mass-flow-sensitive detector. The flexibility of ion spray to high-flow applications allows the use of HPLC eluent flow ranging from 0.001-2.0 ml/min. The use of solvent—buffer post-column addition also allows optimization for improved analyte ion current response.

Arson investigation using the ion trap detector

A technique of fire debris analysis for traces of accelerant in investigations of suspected arson is described which uses a relatively inexpensive mass spectrometer of recent design. Thermal elution of the volatiles from the debris is followed by adsorption of the vapour onto a porous polymer using headspace sampling. A concentrated extract of the adsorbate is analysed on a capillary column gas chromatograph coupled to an ion trap detector which generates a total ion current chromatogram of the sample. The presence of accelerant in the adsorbate can readily be determined by inspection of a series of mass chromatograms, replotted from the total ion current chromatograms, and corresponding to ions of specific masses which are characteristic of the accelerant used. The mass chromatograms collectively define the accelerant profile. Such profiles can usually be distinguished from the accompanying background contributions even in samples having significant matrix interferences.

Ion‐trap detector electron impact mass spectrometry: Practical considerations

AbstractAn investigation of the quality of mass spectra obtainable by the Finnigan ion‐trap detector (ITD) and on how this quality may depend on sample size is described. For compounds which are not very thermolabile or too prone to fragmentation, near‐normal spectra can be expected if the sample sizes do not exceed amounts of the order of 100 ng. Analyte loads which generate acceptable total ion current chromatograms with narrow‐bore capillary columns (about 500 ng) could still produce ITD mass spectra having enhanced quasi‐molecular ions, [M + 1]+, abnormal distributions of peak intensities and unusual fragments. Even under optimum conditions, certain analytes, e.g. acyclic alkanes, n‐alkanols and pyridines, will manifest spectral characteristics which are strictly typical of the ITD. In favourable cases, the formation of quasi‐molecular ions can be exploited for relative molecular mass determination. Even under conditions of self‐chemical ionization, the ITD spectra of most analytes generally retain enough electron impact mass spectral character to be readily recognizable by inspection or by library algorithms.

Complex mixture analysis based on gas chromatography-mass spectrometry with time array detection using a beam deflection time-of-flight mass spectrometer

A beam deflection time-of-flight mass spectrometer was developed in conjunction with an integrating transient recorder to provide time array detection, permitting high mass spectral scan file acquisition rates for complex mixture analysis by capillary gas chromatography-mass spectrometry (GC-MS). Results are presented for the analysis of a urinary organic acid mixture by GC-MS at a scan file acquisition rate of 10 scan files per second (sf/s), showing the advantages of such data collection in the deconvolution of partially resolved components. The reconstructed total ion current (RTIC) chromatogram available from data acquired at this scan file generation rate is shown to be comparable to the profile obtained from a flame ionization detector in representing the chromatography performed under identical experimental parameters. The RTIC chromatogram available from the database obtained at 10 sf/s is compared with that available from a database obtained at 1 sf/s, the latter representing that scan rate typically used with most GC-MS instruments. The advantages of the higher scan file acquisition rate in representing the chromatographic profile and in allowing mass spectral data to be obtained for components in the complex mixture that are unresolved chromatographically are discussed.

Structural characterization of bacitracin components by Frit-fast atom bombardment (FAB) liquid chromatography/mass spectrometry (LC/MS).

The structural characterization of minor components of bacitracin (BC) complex was carried out using a technique of liquid chromatography/mass spectrometry (LC/MS). Satisfactory total ion current chromatogram of BC complex and excellent mass spectra of many components were given by Frit-fast atom bombardment (FAB) LC/MS analytical system, and the structures of 13 minor components could be proposed. The 13 minor components were classified into two groups, bacitracin A (BC-A) related components and bacitracin F (BC-F) related components depending on their common N-terminal moieties. The structures of BC-A related components and BC-F related components were the same as those of BC-A and BC-F, respectively, except that one to three of isoleucine and leucine residues are replaced by valines. The BC-F related components were degradation products of BC-A related components through the same degradation process as that of BC-A.