Academic literature on the topic 'Hyphenated techniques'

In metabolomics, the analytical challenge is to capture the chemical diversity of the metabolome. With the current technologies only a portion of the metabolome can be analysed. As a result there is a drive to direct significant analytical efforts towards capturing the metabolome or changes in the metabolome reliably and reproducibly in biological systems. Apart from analytical challenges, the challenges also include development of appropriate methodologies to quench and extract metabolites which is a crucial parameter in sample preparation and is required to achieve an accurate representation of phenotype. This thesis focuses on addressing both the challenges in mammalian and microalgal metabolomics. Metabolomics in cancer research is gaining momentum as a tool to understand the molecular mechanism of disease progression and for the identification of specific biomarkers which may assist distinguishing between normal, benign and metastatic cancer states. In our first investigation we developed GC-MS based modified direct cell scraping, bead harvesting and LN2 methods for harvesting three adherently grown mammalian cell lines (two breast cancer cell lines MDA-MB 436, MCF7 and an endothelial cell line HMEC1) which provided rapid and reliable route with three fold improved metabolome coverage and reduced the artifacts due to metabolome leakage compared to conventional methods. Later optimized treatments were employed and the influence of various washing and quenching solvents (buffered/unbuffered) on metabolite leakage was investigated for metastatic cancer cell line MDA-MB-231. This identified one washing step with PBS followed by quenching with 60% methanol (buffered with HEPES) as the best washing and quenching solvents. Further validation and comparison of proposed workflows for metabolomic study of two metastatic TNBC cell lines (MDA-MB-231 and MDA-MB-436) resulted in recovery of 154 unique metabolites and demonstrated the robustness and reliability of these methods in pathway based analysis in cancer. In case of GC-MS based microalgal metabolomics, with comprehensive evaluation of selected quenching and extraction methods in model microalga C. reinhardtii, we have successfully demonstrated that the choice of quenching and extraction solvents have significant impact on recovery of different classes of metabolites. Our results clearly indicate that 60% methanol (buffered with HEPES) and 25 % aqueous methanol are the best suited quenching and extraction solvent respectively for untargeted metabolomic analysis of C. reinhardtii, as the highest number of metabolites belonging to various chemical classes were recovered with good intensities and reproducibilities with this miniaturized proposed method compared to other evaluated methods. Later impact of various stages involved in biodiesel production workflow from microalga on recovery of biodiesel was assessed in three microalgal species namely C. reinhardtii, D. salina and N. salina. Within which we have developed an optimized GC-FID method and miniaturized direct TE method for quantification of fatty acids, which can be applied to a small amount of biomass and saves tremendous amounts of time, solvents and reagents required, is less expensive and uses environment friendly solvents making it more suitable for sustainable large scale production. In our final investigation, we directed our efforts towards preliminary optimization and comparative analysis of HILIC and IP-RP-HPLC based separation for the retention and separation of specific metabolites classes. This identified HILIC as the best available column till date for untargeted metabolomic studies. The descriptive understanding gained from each of these investigations provides greater insight into biology of mammalian and algal systems by improving the metabolome coverage for various metabolite classes. These insights illustrating the underlying molecular pathways involved in respective biology's, will help scientific communities in identifying as-of-yet-missing reactions in the metabolic network. In addition these insights will surely help in generating many hypothesis based investigations in microalgal and cancer community.

To improve detection of the misuse of erythropoietin (EPO) for performance enhancing purposes, this PhD examined ways to improve recovery and preanalysis concentration of EPO from urine. It also looked at ways to enhance the signal in liquid chromatography tandem mass spectrometry of the acidic glycopeptides from digested EPO, and at distinguishing between recombinant EPO and human urinary EPO based on differences in their glycosylation. Due to a shortage of supply of available analytical standards, model glycoproteins were frequently used in place of endogenous EPO. Immunoextraction with magnetic beads effectively recovered EPO from urine which had been filtered to remove large proteins, but was unsuccessful from unfiltered urine, suggesting more research into the right choice of antibody was needed. The specific and reversible binding of boronic acids to cis-diol groups found in the glycan groups of glycoproteins was investigated as a device for the selective binding of EPO. Attempts were made to functionalise mesoporous silica for use as a column packing material. Although there was evidence that at least one method of functionalisation was successful, the use of this silica to extract glycoproteins and glycopeptides was not. Signal enhancement through the introduction of ‘superchargers’ into LC solvents was investigated. This was effective with small molecules, and also improved detection of sialylated glycopeptides. The results do not fit entirely with current models of how superchargers exert their effect, suggesting they are incomplete. Finally, the cleavage, digestion and derivatisation of N-glycans to identify bisected and non-bisected structures as a way to discriminate between rEPO and huEPO was examined. Samples were analysed using LC-MS and CE-LIF, and although much of the work was carried out using a model glycoprotein, there is some evidence that the approach may be capable of discriminating between artificial and endogenous EPO at the levels found in anti-doping samples.

Hops (Humulus lupulus L.) are an indispensable component of beer, with the essential oil responsible for imparting distinctive odour and aroma characteristics to beer. However, not all character-impact odorants in hop essential oil have been identified and hop aroma in beer is still not completely understood. The composition of hop essential oil is very complex with 485 compounds currently identified in the literature, and recent research suggests that up to 1000 compounds may actually be present. Only a certain number will be present at concentrations above threshold and make a direct contribution to the odour of the oil. In addition, many important odorants are only present at trace concentrations. Gas chromatography-olfactometry (GC-O) using human assessors is the best way to locate potent odorants and facilitate their identification. A novel methodology was developed to identify the character-impact odorants in hop essential oil samples using hyphenated techniques in gas chromatography. GC-O was used to locate odour active compounds and determine relative importance using CharmAnalysis[TM] according to the odour potency principle. Due to the chemical complexity of the samples, considerable co-elution of peaks occurs during single column gas chromatography (1DGC), making the detection and identification of character-impact odorants challenging. Therefore, comprehensive two-dimensional gas chromatography (GCxGC) combined with time-of-flight mass spectrometry (TOFMS) was used to resolve and identify compounds eluting in the odour active regions. The methodology was developed in a case study on coriander (Coriandrum sativum) and wild coriander (Eryngium foetidum) leaf essential oils. During GC-O analysis of these samples it was recognised that odour active regions frequently coincide with several co-eluting compounds. To address this, a heart-cut multidimensional gas chromatography-olfactometry (MDGC-O) instrument was developed to resolve these 'co-eluting odour clusters' and determine the compound(s) responsible for the odour perception. The 'spicy' character of hops is considered to be a desirable attribute in beer associated with 'noble hop aroma'. However, the compounds responsible have yet to be adequately elucidated. This character was investigated using a commercial 'Spicy' fraction of hop essential oil, selectively enriched for monoterpene and sesquiterpene alcohols. The odour active compounds in (i) the spicy fractions and (ii) the whole essential oils of four different hop varieties were compared using the presented methodology. A compound with an intense 'woody, cedarwood' odour was determined to be a potent, character-impact odorant in all samples. This odour coincided with a complex region of the chromatogram where up to thirteen compounds were co-eluting. The peak responsible for this odour was determined by (i) correlation of peak areas with odour potency (Charm) values and (ii) MDGC-O. The compound was tentatively identified by GCxGC-TOFMS as 14-hydroxy-β-caryophyllene, which has not previously been reported as an odorant in hop essential oil. It was concluded that this compound and other 'woody, cedarwood' odorants contributed to the 'spicy' character of the investigated hop samples. Compounds previously associated with noble hop aroma, notably caryophyllene oxide, humulene epoxides I and II, and humulenol II, did not contribute to the odour character of the hop samples. Other potent odorants that were identified in the whole essential oil and spicy fractions of hops were: geraniol, linalool, β-ionone, eugenol, isovaleric acid, and β-damascenone. While myrcene was a moderately potent odorant in the whole hop essential oil samples, the abundant sesquiterpene hydrocarbons α-humulene, β-caryophyllene and β-farnesene did not significantly contribute to the odour character.

<p>This thesis is based on studies in which the suitability of various gas chromatography (GC) injection techniques was examined for the determination of polybrominated diphenyl ethers (PBDEs) and carbazoles, two groups of compounds that are thermally labile and/or have high boiling-points. For such substances, it is essential to introduce the samples into the GC system in an appropriate way to avoid degradation and other potential problems. In addition, different types of gas chromatographic column system and mass spectrometric detectors were evaluated for the determination of PBDEs.</p><p>Conventional injectors, such as splitless, on-column and programmed temperature vaporizing (PTV) injectors were evaluated and optimized for determination of PBDEs. The results show on-column injection to be the best option, providing low discrimination and high precision. The splitless injector is commonly used for “dirty” samples. However, it is not suitable for determination of the high molecular weight congeners, since it tends to discriminate against them and promote their degradation, leading to poor precision and accuracy. The PTV injector appears to be a more suitable alternative. The use of liners reduces problems associated with potential interferents such as polar compounds and lipids and compared to the hot splitless injector, it provides gentler solvent evaporation, due to its temperature programming feature, leading to low discrimination and variance.</p><p>Increasing the injection volume from the conventional 1-3 µL to >50 µL offers two main benefits. Firstly, the overall detection and quantification limits are decreased, since the entire sample extract can be injected into the GC system. Secondly, large volume injections enable hyphenation of preceding techniques such as liquid chromatography (LC), solid phase extraction and other kinds of extraction. Large-volume injections were utilized and optimized in the studies included in this thesis.</p><p>With a loop-type injector/interface large sample volumes can be injected on-column providing low risk of discrimination against compounds with low volatility. This injector was used for the determination of PBDEs in air and as an interface for the determination of carbazoles by LC-GC. Peak distortion is a frequently encountered problem associated with this type of injector that was addressed and solved during the work underlying this thesis.</p><p>The PTV can be used as a large volume injector, in so-called solvent vent mode. This technique was evaluated for the determination of PBDEs and as an interface for coupling dynamic sonication-assisted solvent extraction online to GC. The results show that careful optimization of the injection parameters is required, but also that the PTV is robust and yields reproducible results.</p><p>PBDEs are commonly detected using mass spectrometry in electron capture negative ionization (ECNI) mode, monitoring bromine ions (m/z 79 and 81). The mass spectrometric properties of the fully brominated diphenyl ether, BDE-209, have been investigated. A high molecular weight fragment at m/z 486/488 enables the use of 13C-labeled BDE-209 as an internal surrogate standard.</p>