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Planar Chromatography
Published in Joseph Chamberlain, The Analysis of Drugs in Biological Fluids, 2018
TLC is also well suited to two-dimensional chromatography. A single sample is spotted near the corner of a square plate which is developed in the usual way, dried, turned through 90°C, and developed with a different solvent. If the two solvent systems are complementary to each other, this can be a powerful method of identification, because the detected position will be very characteristic of the individual components. Alternatively, the first channel of development can be reacted with a derivatizing agent, for example by acetylation of hydroxyl groups, and the same solvent system used for the second development. Figure 5.3 shows the characterization of organic alcohols using such an approach.
Homologous Edman-Type Reagents in Microsequence Analysis of Polypeptides
Published in Ajit S. Bhown, Protein/Peptide Sequence Analysis: Current Methodologies, 1988
B. Wittmann-Liebold, J. Shan-Wei, J. Salnikow
The thin-layer identification, however, is obscured to some extent (above the origin and in the area of the basic derivatives) by additional fluorescing spots caused by side reactions of the degradation and obviously even more by traces of fluorescence-positive contaminants of the sheets. It was found that the quality of the thin-layer chromatogram can largely be improved by prechromatography of the sheets prior to sample application in the second-dimension solvent mixture and in the direction of the second dimension and then followed by application of the DNSA-derivatives and performing the two-dimensional chromatography. In this manner, clear spots of individual degradation cycles can be obtained, thus rendering this technique useful for manual microsequence analysis. When performing the DNSAPITC degradations in shortened “dansyl glass” tubes, the method can be applied for 100 to 500 pmol; simultaneously, up to 30 samples can be degraded.47
Toxicokinetics of Nerve Agents
Published in Brian J. Lukey, James A. Romano, Salem Harry, Chemical Warfare Agents, 2019
Marcel J. van der Schans, Hendrik P. Benschop, Christopher E. Whalley
Toxicokinetic studies are only interesting if the agents can be determined at levels that are toxicologically relevant. The relevant levels should be related to the binding constant of the agent with AChE. Since nerve agents inhibit AChE with rates up to 108/M/min, it can be derived from this that blood levels down to a few picograms per milliliter can still cause a significant inhibition over a period of hours. Only the most sophisticated gas chromatographic techniques combined with sensitive detection methods such as NPD, FPD, or mass spectrometry (MS) are sensitive enough to fulfill this task. In the period of time when the toxicokinetic studies mentioned in this chapter were performed, only the NPD detector was available (Benschop et al., 1985; Spruit et al., 2001). Nowadays, organophosphofluoridates are analyzed by means of mass spectrometry with chemical ionization using ammonia as a reaction gas (Degenhardt et al., 2004; Jakubowski et al., 2004). This ionization mode is efficient for ionization of organophosphofluoridates but also more selective than electron impact ionization, since it is a softer ionization mode. As mentioned in the previous section, it is also essential that the isomers can be distinguished from each other, in view of the extreme difference in toxicity of the isomers. The combination of chirally sensitive analysis was realized by two-dimensional gas chromatography in combination with large volume sample introduction (up to 400 µl) by means of thermal desorption. This allows the detection of low concentrations of nerve agent, that is, in the picograms per milliliter range. The two-dimensional chromatography used the heart cutting technique. The configuration comprises two GC-columns, a pre-column and a second analytical chiral column, which are connected in series. The sample is introduced on the pre-column. After separation on the pre-column, a small fraction of the effluent is injected on the second analytical chiral column. The reason for using two-dimensional chromatography is twofold. First, the combination of two stationary phases creates additional selectivity, which is required for the detection of nerve agents at trace levels in biological samples. Second, the configuration preserves the chiral column, which has a fragile stationary phase that deteriorates with the formation of liquid phases on the column wall and the elution of interfering matrix components. With this configuration, it was possible to detect amounts of soman and sarin down to 1–5 pg while separating all isomers of sarin and soman (Benschop and De Jong, 1987, 1990, 1993).
Breathing new life into clinical testing and diagnostics: perspectives on volatile biomarkers from breath
Published in Critical Reviews in Clinical Laboratory Sciences, 2022
Jordan J. Haworth, Charlotte K. Pitcher, Giuseppe Ferrandino, Anthony R. Hobson, Kirk L. Pappan, Jonathan L. D. Lawson
As the gold standard for breath sample analysis, gas chromatography mass spectrometry (GC-MS) offers the opportunity to separate and identify a wide range of compounds from breath and to perform detailed analysis and quantifications of each of those compounds. GC has been the leading choice to separate compounds since the era of modern breath research was ushered in by Pauling and colleagues [20]. Advances in mass spectrometry technologies have further increased our ability to distinguish and identify molecules on breath, first with two-dimensional chromatography methods such as GCxGC-MS and more recently with high resolution accurate mass (HRAM) GC-MS. GC-MS offers the ability to identify compounds by matching to reference standards or existing compound libraries, and the confidence in naming compounds allows potential biomarkers to be associated with specific biological pathways and disease processes. Demonstrating the metabolic origin for biomarkers helps demonstrate their disease relevance, establish their clinical validity, and inform further development of targeted sampling and analysis methods that are more suitable in clinical contexts. GC-MS approaches also show the highest sensitivity for low abundance biomarkers, as low as parts per trillion (ppt), and have a dynamic range of up to six orders of magnitude, which is necessary to reliably cover the various VOC abundances seen on breath.
Relevance of animal studies in the toxicological assessment of oil and wax hydrocarbons. Solving the puzzle for a new outlook in risk assessment
Published in Critical Reviews in Toxicology, 2021
Juan-Carlos Carrillo, Dirk Danneels, Jan Woldhuis
To round up and confirming the observations of Boitnott and Margolis on the type of hydrocarbons retained in human livers (Boitnott and Margolis 1970), the hydrocarbon composition of the tissues collected by Barp et al. 2014 was characterized using comprehensive two-dimensional chromatography (GCxGC-FID), presently the best technique for characterizing the composition of saturated hydrocarbons (Biedermann et al. 2015). The absence of n-alkanes (both those from vegetable origin and mineral oil derived products) was noted in the liver. Furthermore, all hydrocarbon components forming distinct signals are essentially removed including lightly branched, pristane and phytane, which are clearly present in the fat. What is left behind in the liver is an unresolved “gray cloud” of hydrocarbons mainly represented by highly isomerized and polycyclic compounds, as shown in Figure 9.
Native size-exclusion chromatography-mass spectrometry: suitability for antibody–drug conjugate drug-to-antibody ratio quantitation across a range of chemotypes and drug-loading levels
Published in mAbs, 2020
Jay Jones, Laura Pack, Joshua H. Hunter, John F. Valliere-Douglass
We have deliberately emphasized the quantitative strengths of nSEC-MS for tracking ADC DAR and drug distribution, but the approach has the added benefit of providing an experimental mass for all species that are quantitated. Obviously, mass verification is important for guaranteeing that ADC forms are quantitated correctly, but the intact mass can also provide additional insight. For example, Figure 5 demonstrates that nSEC-MS and HIC quantitation of even-load vcMMAE ADCs agree quite well across the DAR range over which method accuracy was assessed. However, the odd-load vcMMAE ADCs do not line up nearly as well. While these species are at low levels to begin with, nSEC-MS results indicate that DAR1 and DAR3 species are, on average, observed at considerably lower levels than they are by HIC (Figure 4). Conversely, DAR5 and DAR7 species were not detected at all by HIC, but were detected by nSEC-MS at levels higher than the QL and were definitively identified on the basis of experimental mass. Previous work on the characterization of ADC separations by HIC provides some insight into why nSEC-MS and HIC do not align on detection and quantitation of odd-loaded species.35 Gilroy et al. used two-dimensional chromatography to show that putative DAR3 species isolated from HIC separation actually consisted predominantly of DAR4 ADC with 1 aglycosylated HC and/or a Man-5 N-glycan and that DAR3 was likely a minor component. This observation suggests that HIC over-represents DAR3, which aligns with our finding that true DAR3 is not as abundant as HIC results would indicate. Regarding the higher odd-loaded ADCs, the HIC chromatogram is not as well resolving in the post 4-load region and the peaks are relatively broad, which suggests that DAR5 and DAR7 are not sufficiently resolved to allow detection and quantitation of these species. In principle, HIC is a hydrophobicity-based separation, but molecule attributes such as glycosylation status and composition may have an impact on hydrophobicity, and therefore HIC retention,36 potentially leading to the conflation of these species with drug-load variants. It is important to note that our findings may depend on the specifics of the HIC method and the glycosylation status of the ADCs used in this study. Nevertheless, our findings highlight a key point, which is that off-line chromatographic methods used for quantitation of DAR may be influenced by unexpected coelution of odd-loaded species with even-loaded ADC post-translational variants. While HIC is well suited for measuring DAR and is stability-indicating, MS approaches may offer a key advantage, namely: the quantitated species are unambiguously identified by mass.