Academic literature on the topic 'Portable liquid chromatography'

With the explosive growth of the dietary supplements industry, new demands have emerged that cannot be faced with the sophisticated instrumentation available in well-equipped laboratories. In particular, there is a demand for simplified and easy-to-use instruments, capable of providing results in short times of analysis. In this study, a hand-portable miniaturized liquid chromatograph (portable LC) has been tested for the determination of chlorogenic acids (CGAs) in products intended to supplement the diet and elaborated with green coffee extracts. CGAs offer several health benefits due to their antioxidant properties, and an increasing number of dietary supplements are marketed with claimed high contents of these compounds. The results obtained with the proposed portable LC approach have been compared with those obtained with two other miniaturized benchtop liquid chromatography instruments, namely, a capillary liquid chromatograph (capLC) and a nano liquid chromatograph (nanoLC). Although compared with the methods that used the benchtop instruments, the sensitivity attainable was lower, the portable LC instrument provided a comparable analytical performance for the quantification of the main GCAs at low mg g−1 levels, and it was clearly superior in terms of speed. The proposed portable LC-based method can be applied to assess the content and distribution profile of the predominant CGAs in this kind of dietary supplement. It can be also used to estimate the antioxidant power due to CGAs, as well as their preservation state.

Abstract There is a growing need for chemical analyses to be performed in the field, at the point of need. Tools and techniques often found in analytical chemistry laboratories are necessary in performing these analyses, yet have, historically, been unable to do so owing to their size, cost and complexity. Technical advances in miniaturisation and liquid chromatography are enabling the translation of these techniques out of the laboratory, and into the field. Here we examine the advances that are enabling portable liquid chromatography (LC). We explore the evolution of portable instrumentation from its inception to the most recent advances, highlighting the trends in the field and discussing the necessary criteria for developing in-field solutions. While instrumentation is becoming more capable it has yet to find adoption outside of research.

This dissertation focuses on the development of hand-portable capillary liquid chromatography (LC) instrumentation. In this work, battery-operable nano-flow pumping systems (isocratic and gradient) were developed and integrated with portable UV-absorption detectors for capillary LC. The systems were reduced in size to acceptable weights and power usage for field operation. A major advantage of the pumps is that they do not employ a splitter, since they were specifically designed for capillary column use, thereby greatly reducing solvent consumption and waste generation. UV-absorption detectors were specifically designed and optimized for on-column detection to minimize extra-column band broadening. Initially, an isocratic nano-flow pumping system with a stop-flow injector was integrated with an on-column UV-absorption detector (254 nm). The pumping system gave excellent flow rate accuracy (<99.94%) and low percent injection carry-over (RSD 0.31%) suitable for quantitative analysis. Using sodium anthraquinone-2-sulfonate, the detector gave an LOD (S/N = 3) of 0.13 µM, which was 12 times lower than a commercial UV-absorption detector. Reversed-phase separations of a homologous series of alkyl benzenes was demonstrated. Further miniaturization of UV-absorption detection was accomplished using a 260 nm deep UV LED. The detector was small in size and weighed only 85 g (without electronics). No optical reference was included due to the low drift in the signal. Two ball lenses, one of which was integrated with the LED, were used to increase light throughput through the capillary column. Stray light was minimized by the use of a band-pass filter and an adjustable slit. Signals down to the ppb level (nM) were easily detected with a short-term noise level of 4.4 µAU, confirming a low limit of detection and low noise. The detection limit for adenosine-5'-monophosphate was 230 times lower than any previously reported values. Isocratic separations of phenolic compounds were performed using a poly(ethylene glycol) diacrylate monolithic capillary column. Finally, a novel nano-flow gradient generator integrated with a stop-flow injector was developed. Gradient performance was found to be excellent for gradient step accuracy (RSD < 1.2%, n = 4) and linear gradient reproducibility (RSD < 1.42%, n = 4). Separations of five phenols were demonstrated using the nano-flow gradient system. Efforts to develop a 405 nm laser diode-based UV-absorption detector for hemoglobin analysis were described.

Le contrôle qualité des médicaments est une étape essentielle de la chaine de distribution des médicaments. Il garantit leurs fiabilités avant leurs utilisations et contribue à la lutte contre les médicaments falsifiés ou de qualité inférieure. Il est conventionnellement réalisé selon les méthodes des monographies des pharmacopées. Ces méthodes se caractérisent souvent par la complexité de leur mise en œuvre et des temps longs consacrés à leurs réalisations. Surtout, ces méthodes nécessitent souvent l’utilisation de réactifs et solvants toxiques pour le personnel de laboratoire et l’environnement.L’objectif de ce travail est de contribuer à l’amélioration du contrôle qualité des médicaments à travers le développement de méthodes d’analyses peu polluantes et de mise en œuvre facile et faisable dans les laboratoires dont ceux à ressources limitées. Il s’est agi en particulier de développer des méthodes de chromatographie liquide haute performance à polarité de phases inversée avec des phases mobiles à base d’éthanol qui est l’un des solvants les plus verts. Pour faire la preuve de la pertinence de cette approche, des formulations d’antipaludiques à base d’artésunate et d’amodiaquine d’une part, et d’artéméther et de luméfantrine d’autre part, ont été choisis.La première partie du travail a commencé par une étude de criblage utilisant l’éthanol comme solvant organique afin d’évaluer l’effet de différents paramètres critiques tels que le pH et la nature de la phase stationnaire sur le comportement chromatographique des molécules en termes de symétrie de pic, de rétention et de détection. L’association artésunate/amodiaquine a été choisie comme preuve de concept car il s’agit d’un modèle intéressant impliquant une molécule à caractère acide avec peu de groupements chromophores et une molécule à caractère basique. Les résultats du criblage ont permis d’optimiser différentes méthodes en phase inverse vertes à base d’éthanol permettant d’analyser l’artésunate, l’amodiaquine et leurs impuretés. L’approche « Quality by Design » recommandée par les autorités règlementaires pharmaceutiques et permettant d’obtenir des méthodes plus flexibles et robustes, a été choisi comme stratégie de développement. Les méthodes ont été ensuite validées selon le guide ICH Q2 (R1), en utilisant la stratégie du profil d’exactitude.La deuxième partie du travail a consisté à démontrer le potentiel d’un spectromètre proche infrarouge portable et à bas prix, en association avec les outils chimiométriques, dans la détection de médicaments falsifiés. L’analyse a porté sur l’association artémether/luméfantrine qui est l’un des médicaments antipaludiques les plus falsifiés. Malgré sa zone spectrale réduite et sa faible résolution comparativement aux appareils classiques, le spectromètre portable a permis de détecter des médicaments falsifiés ne contenant aucune substance active et d’identifier spécifiquement les différentes marques de comprimés. Les résultats obtenus ont démontré le grand potentiel de ces nouveaux équipements proche infrarouge innovants et à bas prix pour leur utilisation en première ligne dans la détection et la lutte contre les faux médicaments. Afin de mieux interpréter les résultats de l’analyse proche infrarouge, une méthode de chromatographie liquide en phase inverse, verte et rapide a été aussi développée pour doser l’artéméther et la luméfantrine dans les comprimés.Ces différentes approches ont montré qu’il est possible de mettre en œuvre le concept de chimie analytique verte pour le contrôle qualité sans nécessité l’acquisition de nouveaux équipements pour obtenir des méthodes performantes qui respectent la règlementation pharmaceutique
The quality control of pharmaceutical products is a key issue in the medicine supply chain. It guarantees the reliability before consumption and contribute to fight against substandard and falsified drugs. It is conventionally performed according to pharmacopeias in which methods are most often long and use harmful reagents for the technical staff, health, and environment.The objective of this work was to improve the quality control of drugs through the development green analytical methods easy to implement in laboratories including those with limited resources. It was consisted, particularly, to develop reversed phase-high performance liquid chromatography methods with mobile phases based on ethanol which is one the best green solvents. Analyzes were applied to the antimalarial combination therapies such as artesunate/amodiaquine and artemether/lumefantrine.The first part of the study started by a screening phase where impacts of critical parameters, such as mobile phase pH and stationary phase, on compound peak symmetry, retention and detection were investigated. Artesunate/amodiaquine combination therapy, which includes an acidic compound with few chromophores and a basic compound was chosen as proof of concept. Several pH modifiers selected for their ecofriendly character and stationary phases were screened. Based on the screening results, different green RP-HPLC methods using ethanol as organic solvent et allowing to analyze artesunate, amodiaquine and their related impurities were developed. Quality by Design approach, recommended by the pharmaceutical regulatory authorities and allowing to obtain robust methods, was chosen as development strategy. Developed methods were validated according ICH Q2 (R1) guideline by using accuracy profile methodology.The second part of the study consisted in investigating the qualitative performance of a low-cost handheld near infrared spectrophotometer associated to chemometric methods as screening tool in the identification of falsified drugs. Analysis was performed on artemether/lumefantrine tablets which are one of the most falsified drugs, Despite its limited spectral range and low resolution compared to bench top devices, the handheld device allowed to detect falsified drugs with no active ingredients and to identify specifically a tablet brand name. This innovated low-cost handheld near infrared spectrophotometer offers a promising performance and could be used as a first line screening tool in the detection and fight against falsified drugs. For a better interpretation of the near infrared results, a green and fast HPLC method was also developed, validated, and used to analyze artemether and lumefantrine in the tablets.These different approaches demonstrated that green analytical methods could be implemented in the pharmaceutical quality control field without the need of new equipment and with analytical performances in accordance with pharmaceutical requirements

Capillary electrophoresis was used for determination of 6 fractions of caseins. Those fractions were measured in 144 samples of cow’s milk originated from the feeding experiment focused on explanation the influence of the feeding onto casein productions. In this work were separated 6 fraction of caseins first time with total resolution of the peaks. Capillary electrophoresis was applied for determination of short-chain organic acids during fermentation of wine must. It was compared the fermentation of must fermented by different yeast. The difference of profile short-chain organic acids during fermentation were not statistically significant. The once difference was in the utilisation of the malic acid and production of the lactic acid. A portable miniaturized system for medium pressure liquid chromatography was developed. The components were tested and system was used for the isocratic and gradient elution of various analytes (food dyes, parabens). New line of electroluminescent diodes (LEDs) for deep-UV areas of wavelength based on a different materials substrate was characterised. The new line was compared with old line LEDs. The new line LEDs was incorporated in deep-UV absorbance detectors. Detectors were characterised and tested for a detection various analytes in modes flow injection analysis and chromatography separation. First time was characterised this new line of the LEDs and the origin of the parasitic emission band produced by deep-UV LEDs light sources was explained. This origin is given by disturbances of a materials substrates. This work is a contribution for an advance of low-cost and portable systems and detection devices in the field of analytical chemistry.

Aims The method for the confirmation of drugs of abuse for addiction testing within King’s College Hospital prior to 2008 was a labour intensive thin layer chromatography method. To replace this with a faster method more suited to future requirements, the laboratory bought a liquid chromatography system with ion trap mass spectrometric detection. The development of the routine analytical method and the implementation of this method within the laboratory using on-line solid phase extraction and Turboflow® sample preparation will allow the laboratory to operate successfully in the field of clinical drugs of abuse testing in the future. Method Analyses are performed on an ion trap mass spectrometer with an electrospray ion source following reversed phase liquid chromatography, initially using on-line solid phase extraction with a Jasco XLC® series autosampler and pump and later a Thermo Turboflow® on-line extraction method with a CTC Combi-Pal® autosampler and Agilent 1100 series liquid chromatography system. Elution of drugs and metabolites is performed with a multi-step gradient of ammonium formate buffer and acetonitrile, followed by regeneration of the extraction and analytical columns to starting conditions. Detection is achieved with a Thermo LCQ Fleet ion trap mass spectrometer with a combination of full spectrum survey scans, dedicated product ion scans, neutral loss scans and data dependent product ion scans in two analysis segments. Total run time is only 20 minutes, allowing a throughput of around 65 samples per day. Results The methods include the novel combination of the elimination of any hydrolysis step, on-line SPE or Turboflow extraction, detection of multiple drug groups, full spectrum analysis and library matching, the use of data dependent scans and ion trap mass spectrometry using MS3 and neutral loss scans. The methods developed were validated using a departmental method validation protocol and accepted for routine use. Simultaneous detection of over fifty analytes has been found possible in a range of clinically relevant drug groups, including opiates, amphetamines, methadone, propoxyphene, cocaine, ketamine and their metabolites. The use of neutral loss scans and product ion scans of phase 2 drug metabolites permits the addition of previously unidentified drugs and metabolites to the method, allowing the laboratory’s services to develop in line with requirements of the service. Quality is maintained through the use of standard operating procedures, staff training, quality control samples and external quality assessment. Conclusion Drugs of abuse testing is key for treatment and monitoring of drug addiction. The introduction of modern mass spectrometry techniques has reduced the turnaround time of routine analysis for a range of drugs and metabolites and increased the range of drugs that can be analysed. The methods introduced have revolutionised testing at King’s College Hospital and produced a method which is capable of evolving with the needs of the service to keep abreast of future requirements of the service.

Substance misuse remains problematic with current concerns being the rise in acute poisoning deaths, particularly opioid-associated, and the ever-widening range of drugs available. Strategies for tackling opioid addiction and opioid related-deaths include researching alternative routes of therapeutic agent administration. Initial urine screening for substance misuse has traditionally employed immunoassays, with confirmation of specific analytes by chromatographic methods. Liquid chromatography-high resolution mass spectrometry (LC-HRMS) offers untargeted analysis without compromising selectivity, and enables users to ascertain putative elemental compositions of an analyte, retrospectively interrogate data, and to incorporate novel analytes easily. These features enable screening and confirmation of drugs in a single method, and may be advantageous for detecting novel psychoactive substances (NPS). This thesis aims to investigate the role of LC-HRMS in drug analysis in the clinical setting. A simple system was developed that is capable of detecting a wide range of commonly-encountered drugs and metabolites. Non-selective sample preparation was used to enable detection of as many compounds as possible, but significant matrix effects were observed. Additional information regarding selected NPS was ascertained through retrospective identification of mephedrone metabolites in patient urines, and through later incorporation of ethylphenidate, methylphenidate, and ritalinic acid, into the method. A separate quantitative LC-HRMS method was developed to facilitate pharmacokinetic studies of naloxone and naltrexone administered through alternative routes. The method was also applied to urine samples, with naloxone-3-glucuronide identified as a potential marker to differentiate between Subutex and Suboxone use. LC-HRMS has advantages in drug detection, particularly in regard to NPS, and in method development. However, application in the clinical setting is restricted by requirements for high throughput, timely results, and operation to accepted ‘cutoff’ values that introduce awkward compromises in system operation. LC-HRMS may have greater application in the forensic setting where more time is available for the analysis of a single sample.

Cancer cells have been known for some time to have very different metabolismas compared to that of normal non proliferating cells. As metabolism is involvedin almost every aspect of cell function, there has been a recent resurgence ofinterest in inhibiting cancer metabolism as a therapeutic strategy. Inhibitors thatspecifically target altered metabolic components in cancer cells are being developedas antiproliferative agents. However, many such inhibitors have not progressedinto the clinic due to limited efficacy either in vitro or in vivo. In this study weexplore the hypothesis that this is often due to the robustness of the metabolicnetwork and the differences between individual cancer cell lines in their metaboliccharacteristics. We take a systems biology approach. We investigate the cellular bioenergetic profiles of a panel of five non-small celllung cancer cell lines before and after treatment with a novel inhibitor of theglutaminase-1 (GLS1) enzyme. Additionally, we explore the effects of this inhibitoron intracellular metabolism of these cell lines as well as on the uptake and secretionof glucose, lactate and amino acids. To be able to do the latter robustly, wehad to modify the experimental assay considerably from procedures that seemto be standard in the literature; using these earlier procedures the metabolicenvironment of the cells was highly variable, leading to misleading results onthe metabolic effects of the inhibitor. We reduced cell density, altered mediumvolume and changed the time-window of the assay. This led to the cells growingexponentially, appearing indifferent to the few remaining changes. In this newassay, the metabolic effects of the glutaminase inhibitor became robust. One of the most significant results of this study is the metabolic heterogeneitydisplayed across the cell line panel under basal conditions. Differences in themetabolic functioning of the cell lines were observed in terms of both theirbioenergetic and metabolic profile. The amount of respiration attributed tooxidative phosphorylation differed between cell lines and respiratory capacity wasattenuated in most cells. However, the rate of glycolysis was similar betweencell lines in this assay. These results suggest that the Warburg effect arisesthrough a greater diversity of mechanisms than traditionally assumed, involvingvarious combinations of changes in the expression of glycolytic and mitochondrialmetabolic enzymes. The effects of GLS1 inhibition on cellular bioenergetics and metabolism alsodiffered between cell lines, even between resistant cell lines, indicating that theremay also be a diversity of resistance mechanisms. The metabolomic response ofcell lines to treatment suggests potential resistance mechanisms through metabolicadaptation or through the prior differences in the metabolic function of resistantcell lines. Part of the metabolome response to GLS1 inhibition was quite specificfor sensitive cells, with high concentrations of IMP as the strongest marker. Our results suggest that the metabolome is a significant player in what determinesthe response of cells to metabolic inhibitors, that its responses differ between cancercells, that responses are not beyond systems understanding, and that thereforethe metabolome should be taken into account in the design of and therapy withanti-cancer drugs.

Metabolism of xenobiotic drug molecules can result in the formation of metabolites which are more chemically reactive than the parent drug from which they are derived. These reactive species have the potential to covalently modify biological macromolecules if they are not detoxified. The formation of drug-protein adducts carries a potential risk of clinical toxicities and idiosyncratic adverse drug reactions which can, in severe cases, result in hospitalisation and even death. Current methods for the evaluation of the risk for a drug to cause adverse drug reactions due to drug-protein binding rely on risk factors such as quantitative covalent binding value, structure, dose etc. The objective of this project was to develop methods for the detection of reactive metabolites directly bound to proteins, which could be used in future evaluations of the mechanisms of binding of candidates in drug development. Three compounds known to produce reactive metabolites, acetaminophen, SB-648969 and amodiaquine, were used as tool substrates. In vitro incubations with human liver microsomes and individual cytochrome P450 enzymes (as Supersomes ) were used to produce reactive metabolite species and binding with the incubation proteins evaluated. Analysis of the intact proteins, peptides generated via trypsin digestion of the incubation protein, and amino acids generated via digestion with pronase were evaluated using a combination of LC/MS and LC-MS/MS. Reactive metabolite trapping experiments with glutathione were used to provide information about the likely structure of the bound species and the specificity of binding, and were useful in the development of sensitive targeted precursor ion scanning and multiple reaction monitoring methods. [14C] radiolabelled acetaminophen and SB-649868 were used to assess the quantitative levels of binding (<5% modification of protein in both cases). Radiodetection using accelerator mass spectrometry (AMS) was used to evaluate the stoichiometry of binding and aid the identification of adducted peptides through retention time comparison. Chemical and electrochemical methods were utilised to produce stable solutions of N-acetyl-p-benzoquinone imine (NAPQI) and amodiaquine quinone imine (AQQI), reactive metabolites of acetaminophen and amodiaquine, respectively, which were bound to selected proteins and used as chromatographic and mass spectrometric standards. These methods were used to successfully identify an acetaminophen-modified peptide (T56) of cytochrome P450 CYP2E1. No modified proteins were observed for the SB-649868 incubations, however, examination of the AMS chromatograms for the incubations with acetaminophen and SB-649868 revealed a difference in the stoichiometry of binding, with one modified peptide observed with acetaminophen, and several for the incubations with SB-649868. The detection and identification of drug-protein adducts remains extremely challenging due to the low levels of any adducts observed, which can be exacerbated by binding on multiple sites of a protein; however this project has demonstrated that sensitive and selective LC/MS methods can be successfully developed to identify drug-protein adducts.

Hepcidin-25 is regarded as the master regulator of iron homeostasis. Three N-truncated isoforms of hepcidin-25 have been identified in human serum; hepcidin-20, -22, and -24, although information is scant as to the serum concentrations of these isoforms. A liquid chromatography-high resolution-mass spectrometry (LC-HR-MS) assay was developed for the simultaneous quantitation of hepcidin isoforms in human serum. Serum (200 μL) was mixed with aqueous formic acid (600 μL), and the supernatant loaded onto a 96-well- SPE-plate. Eluted sample (70 μL) was diluted with deionised water (60 μL) and analysed using LC–HR–MS. Samples previously analysed by a published LC-MS/MS assay were analysed for method comparison. All hepcidin isoforms were quantified in samples from healthy volunteers as controls, and patients with hereditary haemochromatosis (HH), non-alcoholic fatty liver disease (NAFLD), iron deficient anaemia (IDA), anaemia of chronic disease (ACD), and sickle cell anaemia (SCA). Samples were also analysed from individuals with chronic kidney disease (CKD) not requiring haemodialysis, and those pre- and post-haemodialysis. Intra-/inter-assay accuracy and precision were acceptable, calibration was linear (R2 > 0.90, all analytes), and the LLoQ was 1 μg/L (all analytes). There was a good correlation for hepcidin-25 to a published LC-MS/MS assay (y = 0.85x -3.2, R2 = 0.96). Median (range) hepcidin-25 concentrations in controls, and individuals with IDA, SCA, HH, ACD and sepsis were: 8 (1–31), <1 (< 1–2), < 1 (< 1–10), 2 (< 1–15), 60 (10–213), and 92 (11–216) μg/L, respectively. Hepcidin-20, -22, or -24 were not detected in any control sample, but were detected in 30–100 % of all samples at 10–20 % of the hepcidin-25 concentration. Following haemodialysis, all hepcidin isoforms declined by some 35–50 %. Hepcidin-25 was most strongly correlated to hepcidin-24, and less so to hepcidin-22 and -20, in all disease states. The developed method was applicable for clinical use. However, further controlled studies are required to fully evaluate the role of hepcidin-20, -22, and -24 measurement in a clinical setting.

High performance liquid chromatography (HPLC) is a dominant laboratory-based analytical technique, but despite its dominant position within analytical laboratories, HPLC has not seen significant application to-date in many important field-based applications. For example, a portable and field-deployable liquid chromatographyultraviolet/ mass spectrometric system (LC-UV/MS) would be of considerable value to facilitate on-site analysis for the pharmaceutical manufacturing sector, areas of industrial process monitoring, threat analysis, and environmental monitoring. However, there are major challenges associated with development of fully functional and field-deployable portable LC-UV/MS systems, particularly for the pharmaceutical manufacturing sector and related on-site industrial applications. Therefore the main aims of this research have been to; (1) Develop and demonstrate the analytical performance of miniaturized UV absorbance detectors with suitability for application within portable capillary LC systems; (2) Undertake a configuration and compatibility investigation into the coupling of a portable briefcase-sized capillary LC-UV platform with new portable mass spectrometric (MS) detector for point-of-need LC-UV-MS analysis; (3) Integrate and demonstrate the performance of the portable LC with industry-standard auto-sampling devices for automated process analysis within pharmaceutical laboratories; and (4) Undertake an in-field demonstration of the coupled portable LC/MS system for environmental analysis. Light-emitting diodes (LEDs) are small in size, have low power consumption requirements, short warm-up times, and deliver long lifetimes. These make LEDs attractive light sources for the miniaturization of absorbance-based detectors. However, LEDs are quasi-monochromatic with narrow emission spectra. This significantly limits the potential of LEDs for applications that require multi-wavelength detection capability. A number of limitations and improvements in low-UV LED-based absorbance detectors were investigated within the first phase of this research. A new miniature deep multi- LED UV absorbance detector was developed using low-cost LEDs, which could be operated in both individual wavelength (240, 255, and 275 nm) and scanning (230 – 300 nm) detection modes. The detector was mostly composed of off-the-shelf components, such as the LEDs, a trifurcated fiber assembly (TFFA), a commercial capillary Z-type flow cell, and the detector photodiodes. The detector was subsequently incorporated within a portable ‘briefcase-sized’ capillary LC system. The detector was characterized in terms of performance and was further benchmarked against a commercial variable wavelength capillary LC detector (Dionex UltiMate 3000RS). The developed detector showed low levels of stray light (<0.4%), delivered an effective path length of up to 99.0% of the detection flow cell, gave a wide dynamic range (0.5 to 200 μg/mL for sulfamethazine, carbamazepine, and flavone), and exhibited low noise levels (at 300 μAU level). Further to the above, in the second phase of this research, the design and performance demonstration of a compact, modular and broadband (D\(_2\) lamp-based) UV-Vis absorbance detector was investigated, as an alternative to the LED-based detectors. The detector was composed of a miniature D\(_2\) light source, an end-capillary extended optical pathlength (10 mm) flow-cell, and a miniature spectrometer. The assembled detector showed low levels of stray light (<0.2%), utilization of up to 91.0% of the effective path length of the Z-type flow-cell, a linearity limit of ~ 3.0 AU, and low noise levels (58.0×10\(^{-5}\) AU). Further, the detector was again integrated and demonstrated with the above portable capillary LC and was then also benchmarked against a commercial capillary LC detector. The detector provided full UV-Vis scanning spectral capability for analytes when used with the portable capillary HPLC. Limits of detection (LODs) for five test pharmaceuticals ranged from 190 to 602 μg L\(^{−1}\). The system was then demonstrated for applications within the pharmaceutical manufacturing sector. Integrating a portable LC system as a process analytical technology (PAT) within industrial settings is a significant technical challenge. This typically includes several steps, including automated sampling, automated sample preparation, the actual chromatographic analysis, and subsequent data processing. In the third phase of this research, the portable capillary LC with the above miniaturized detection options was deployed as a PAT system within a commercial pharmaceutical laboratory. The system was integrated with an industry standard automated sampling device and used for studying the kinetics of a synthetic reaction and benchmarked against a commercial process laboratory full ultra-high performance LC system. The deployed system gave low detection limits (3 μgL\(^{−1}\) and delivered a wide dynamic range (up to 200 μg mL\(^{−1}\). The on-site demonstration confirmed robust performance in automated process analysis, with less than 5.0% relative standard deviation (RSD) on sample-to-sample reproducibility, and less than 2% carry-over between samples. The system has shown potential to significantly increase sample throughput by providing near real-time analysis and to improve understanding of synthetic processes. Further to the above industrial application, it should be noted that there are very few reports to-date of field-deployable LC-based systems which can be successfully employed for on-site trace analysis. However, portable and field-deployable LC-MS systems for in-field analysis of complex mixtures and highly challenging samples is a definite future need. In the final phase of this research work, the portable and field-deployable capillary LC-MS system was configured for in-field analysis of per- and polyfluoroalkyl substances (PFAS) in soils. Thus, a portable ultrasound-assisted extraction (UAE) methodology was developed, optimized, and applied with the portable capillary LC/MS system for on-site analysis of PFAS in real soil samples. The critical parameters for the integration of the capillary LC with MS detection, and on the separation and performance of the coupled capillary LC/MS were studied and optimized. The UAE method were also studied and optimized using response surface methodology (RSM) based on a central composite design (CCD). Spiked soil samples with known concentrations of 50 μg/L or 100 μg/L were used for method validation. The mean recovery for eleven PFAS ranged between 70 –110%, with relative standard deviations (RSD) ranging from 3 to 12%. When deployed to a field site, method reporting limit for 12 PFAS ranged from 0.6 to 0.1 ng g\(^{−1}\), with wide dynamic ranges (1 - 600 ng g\(^{−1}\) and excellent linearities (R\(^2\) ˃ 0.991). The in-field portable system was again benchmarked against a commercial lab-based LC/MS for the analysis of PFAS in real soil samples, with comparison results showing good agreement. Twelve PFAS were detected and identified in real soil samples at concentrations ranging from 8.1 ng g\(^{−1}\) (for perfluorooctanesulfonic acid, PFOS) to 2.9 μg g\(^{−1}\) (perfluorohexanesulphonic acid, PFHxS). The system demonstrated significant potential and robustness, and it is anticipated the system could help to deliver sensitive and fast analysis for rapid on-site environmental monitoring in the near future.

A state-of-the-art miniaturised and field-portable liquid chromatography (LC) system has been developed for use in the pharmaceutical sector. Developments on the instrumentation included (1) a small-footprint high-pressure pumping system that allows fast LC separation, (2) integration of an auto-sampling interface that enables automatic injection and cleaning, (3) utilisation of high-efficiency LC columns for fast separation, (4) adoption of robust and versatile detection approaches with superior sensitivity. The first phase of development involved the integration and performance testing of a fully automated hand-portable capillary LC system with a total weight of ~2.7 kg. This portable LC integrated multiple small footprint LC components within a 3D-printed enclosure, including a modular microfluidic system with automatic injection, column heater, and low-UV light emitting diode (LED) based Z-cell absorbance detector. This portable LC when used with a 5 µm C18column (100 mm × 300 µm I.D.) delivered highly reproducible chromatograms with retention time and peak area relative standard deviations (RSDs) of <0.7% and <3.3% in isocratic mode (n = 10), as well as of <0.1% and <2.3% in gradient mode (n = 10), respectively, with sub-4 min run times. Limits of detection (LODs) for four test small molecule pharmaceuticals ranged from 65 to 101 µgL\(^{-1}\) based on a 316 nL injection volume, with separation efficiencies between 18,000 and 29,700 Nm\(^{-1}\). In addition, the portable LC was coupled to a small footprint mass spectrometer (MS) to demonstrate compatibility and ‘point-of-need’ LC-MS capability. The limitations and improvements of low-UV LED and Z-cell based absorbance detection were addressed in the second phase. Commercial low-UV LEDs produced limited spectral outputs with low external quantum efficiencies (EQEs), which affected the detection sensitivity, especially when light was transmitted through narrow capillary-format optical paths. In order to achieve higher emission intensity, the LED was operated at high input currents. However, this led to a stressed LED as a result of excessive heat generation. A solution proposed and investigated herein was to apply active cooling for effective heat dissipation, which recovered the performance of the affected LED. Hence, a simple 3D-printed liquid cooling interface was developed for low-UV LED based absorbance detection. This active cooling interface improved LED performance by reducing noise and LOD by a factor of 2 as well as shortening equilibration time by 6-fold. Another solution was to use a high-power UV-LED in conjunction with collimating lenses to increase photon throughput. A novel surface mount device (SMD) LED emitting at 235 nm with improved output power and EQEs, was used in capillary-scale UV detection. This SMD had extremely low light transmission (~0.001%) through the 100 × 100 µm channel of the Z-cell detector, so the optical alignment was optimised by the simulation and integration of focusing lenses into the optical path. A 9-fold increase of light transmission with a dual-lens setup yielded an enhancement of detector sensitivity by a factor of 2.2. In the third phase, an upgraded portable LC with higher pressure capability was deployed as process analytical technology (PAT) for use within an industrial pharmaceutical setting, namely reaction monitoring prototype (RMP). For at-line analysis, a self-packed 3.5 µm XBridge C18 capillary column was used for separation in the RMP system. The difference in quantitation of the starting compound between the RMP and a full-sized benchtop LC was minimal, which is not statistically significant. To cope with the sample degradation issue within a highly aqueous environment, a normal-phase LC was attempted using a 1.7 µm BEH HILIC packed capillary column on the RMP system. A successful attempt at developing an on-line coupling of the RMP system with a sample dilution cart demonstrated its high potential and flexibility as a robust PAT approach.

This chapter deals with separation methods from the perspective of green analytical chemistry. Gas chromatography, the oldest and most familiar method of separation, is inherently green; however, even this method can be improved by eliminating sample preparation procedures whenever possible. High-performance liquid chromatography (HPLC) employs organic solvents, especially acetonitrile, as eluents. If the laboratory operates many liquid chromatographs, the amount of spent eluent generated is several litres per day – a situation that makes the greening of liquid chromatography pressing. Several possibilities for greening liquid chromatography are described: microscale HPLC, temperature elevation and programming in HPLC, application of green eluent modifiers, supercritical fluid chromatography, and ultra performance liquid chromatography. Capillary electrophoresis (CE) is another liquid-phase separation technique that uses much less eluent, and is therefore an inherently green separation method. The CE method is also a basis for miniaturising separation methods. Miniaturisation is a promising route to greening analytical methods since it significantly reduces energy consumption. It also facilitates the development of portable analytical instruments that can be taken wherever needed, eliminating the time and energy required to transport samples to the laboratory. The emergence of microfluidics as a new paradigm in chemical measurement science is the result of miniaturising analytical instrumentation.

The antithrombotic effect of aspirin might be enhanced if platelet cyclooxygenase could be inhibited in the portal ciculation while sparing cyclooxygenase in the systemic vascular endothelium. This might be achieved by modifying the dose and formulation to maximise presystemic aspirin clearance by the liver. To test this hypothesis low dose enteric coated aspirin (Astrix, 50mg single dose, lOOmg single dose and lOOmg daily for 1 week) was orally administered to pigs with permanent indwelling arterial and portal vein catheters. Plasma aspirin concentrations were measured by high performance liquid chromatography in blood obtained simultaneously from the artery and portal vein for 6 hours after dosage. Platelet aggregation and thromboxane generation were measured in 4 pigs before and after the lOOmg chronic dosage regimen. Aortic prostacyclin production was measured in aspirin treated (lOOmg daily for 1 week) and untreated pigs after sacrifice. After the 50mg single dose the arterial:portal areas under the plasma concentration versus time curve (AUC) ratio was 0.63±0.09 (n=6). In 3 pigs which received all 3 dosage regimens the arterial:portal AUC ratios were 0.48±0.05 after 50mg single dose, 0.52±0.02 after lOOmg single dose and 0.47+0.03 after lOOmg daily for 1 week. Platelet aggregation in response to sodium arachidonate (1.65mM) was completely abolished after chronic aspirin administration. Thromboxane production (pg/106 platelets) by this stimulus decreased from 536±117 before aspirin to 57±14 after aspirin (n=4; p=0.017). Aortic prostacyclin synthesis (ng/disc after 10 min incubation) was 1.66±0.28 (n=4) in untreated pigs and 0.97±0.25 (n=4) in treated pigs (p=0.06).With this slow release aspirin formulation there was substantial but incomplete clearance of aspirin by the liver. This may not be sufficient to spare cyclooxygenase in the systemic vessels from the effect of aspirin.

Commercially available handheld chemical analyzers for forensic applications have been available for over a decade. Portable systems from multiple vendors can perform X-ray fluorescence (XRF) spectroscopy, Raman spectroscopy, Fourier transform infrared(FTIR) spectroscopy, and recently laser-induced breakdown spectroscopy (LIBS). Together, we have been exploring the development and potential applications of a multisensor system consisting of XRF, Raman, and LIBS for environmental characterization with a focus on soils from military ranges. Handheld sensors offer the potential to substantially increase sample throughput through the elimination of transport of samples back to the laboratory and labor-intensive sample preparation procedures. Further, these technologies have the capability for extremely rapid analysis, on the order of tens of seconds or less. We have compared and evaluated results from the analysis of several hundred soil samples using conventional laboratory bench top inductively coupled plasma atomic emission spectroscopy (ICP-AES) for metals evaluation and high-performance liquid chromatography (HPLC) and Raman spectroscopy for detection and characterization of energetic materials against handheld XRF, LIBS, and Raman analyzers. The soil samples contained antimony, copper, lead, tungsten, and zinc as well as energetic compounds such as 2,4,6-trinitrotoluene(TNT), hexahydro-1,3,5-triazine (RDX), nitroglycerine (NG), and dinitrotoluene isomers (DNT). Precision, accuracy, and sensitivity of the handheld field sensor technologies were compared against conventional laboratory instrumentation to determine their suitability for field characterization leading to decisional outcomes.