Preparation and characterization of long methacrylate monolithic column for capillary liquid chromatography

Long methacrylate monolithic columns (100 cm x 320 mum i.d.) were prepared from silanized fused-silica capillaries of 320 mum i.d. by in situ copolymerization of butyl methacrylate (BMA) with ethylene dimethacrylate (EDMA) in the presence of a suitable porogen. The separation performance and selectivity of the column were evaluated and compared with a 25 cm x 320 mum i.d. column prepared in the same way by capillary high-performance liquid chromatography (mu-HPLC) The results showed that the 1 m long monolithic column can generate 33 x 10(3) plate number and exhibited good permeability, higher sample loadability, and separation capability. (C) 2004 Elsevier B.V. All rights reserved.

Analysis on Iridoid Glycosides in Crude and Processed Extracts from Cornus Officinals by Liquid Chromatography-electrospray Ionization Mass Spectrometry

The iridoid glycosides in crude and processed extracts from cornus officinals have been analyzed by high performance liquid chromatography-electrospray ionization mass spectrometry. Samples were analyzed by a reversed-phase C18 column using a binary eluent under gradient conditions. Seven iridoid glycosides could be separated and detected. The [M-H](-) ions of iridoid glycosides in the negative ion mode were observed, which reflect their molecule mass information. An in-source collision induced dissociation (in-source CID) experiment was carried out in order to identify the structures and to measure the contents of iridoid glycosides. The epimers were discovered in the experiment for the first time, namely 7 alpha-O-ethyl-morroniside and 7 beta-O-ethylmorroniside.

DETERMINATION OF AMINOPYRINE AND ITS METABOLITE IN BIOLOGICAL FLUID BY LIQUID-CHROMATOGRAPHY ELECTROCHEMISTRY WITH A GLASSY-CARBON ELECTRODE DISPERSED WITH ALPHA-ALUMINA PARTICLES

A sensitive high-performance liquid chromatographic method has been developed for the quantitative determination of aminopyrine (AM) and its metabolite 4-aminoantipyrine (AAN). The method utilizes reverse-phase chromatography/amperometric detection with a glassy carbon electrode dispersed with alpha-arumina particles as the working electrode, on which the oxidation of AM and AAN was greatly improved compared with that on a bare glassy carbon electrode. As a result, the detection limit was as low as 1.4 ng for AM and 0.8 ng for AAN, and the calibration plots for the above compounds have wide linear ranges from 100 ng/mL to 100 mu g/mL and 60 ng/mL to 80 mu g/mL (for AM and AAN, respectively). The above method was applied for the detection of these materials in human urine with satisfactory results.

Purification of human tissue prokallikrein excreted from insect cells by liquid chromatography

Tissue kallikrein, generally existing in living bodies as prokallikrein, is a serine proteinase that has proven of great significance to treat hypertension, cardiopathy and nephropathy. Although the extraction of tissue kallikrein from human urine is the most commonly used method to obtain such a protein, not only the yield is very little, but also the procedure is rather complex. Furthermore, the biological safety is uncertain. Therefore, the preparation of such a protein by genetic engineering method, including gene expression, cell culture, separation and purification, is very important. In this paper, a new method to obtain purified tissue prokallikrein excreted from insect cells by liquid chromatography has been proposed. In contrast to the previously published papers, the purification procedure is simplified to only three steps with the final yield of 57% and the purity of 95%, which is not only convenient, but also low-cost and suitable for the large-scale preparation of such a protein. The purified protein is further validated as prokallikrein by high performance liquid chromatography-mass spectrometry and amino acid sequencing. (c) 2005 Elsevier B.V. All rights reserved.

A high resolution study of the chlorophyll and bacteriochlorophyll pigment distributions in a calcite/gypsum microbial mat

Application of a high resolution high performance liquid chromatography-mass spectrometry method to the study of a microbial mat system has permitted the identification of a greater number of pigments derived from green bacteria than reported in a previous study. Although the green bacteria found in the mat were identified as Chloroflexus-like, bacteriochlorophylls and bacteriophaeophytins c that can be attributed to Chloroflexaceae on the basis of literature reports account for less than 10% of the pigments derived from green bacteria in the mat. Analysis of the bacteriochlorophylls and bacteriophaeophytins c and d using atmospheric pressure chemical ionisation-liquid chromatography-tandem mass spectrometry reveals complex depth profiles, signalling inputs from a number of organisms. The pigment compositions provide evidence for green bacteria living in close proximity to the living cyanobacterial mat. Depth profiles of pigments derived from green, purple and cyanobacteria indicate that the remnants of mats present in the deeper part of the section contain a record dominated by signatures from anoxygenic photoautotrophs.

Dried blood spot liquid chromatography assay for therapeutic drug monitoring of metformin

The use of blood spot collection cards is a simple way to obtain specimens for analysis of drugs for the purpose of therapeutic drug monitoring, assessing adherence to medications and preventing toxicity in routine clinical setting. We describe the development and validation of a microanalytical technique for the determination of metformin from dried blood spots. The method is based on reversed phase high-performance liquid chromatography with ultraviolet detection. Drug recovery in the developed method was found to be more than 84%. The limits of detection and quantification were calculated to be to be 90 and 150 ng/ml, respectively. The intraday and interday precision (measured by CV%) was always less than 9%. The accuracy (measured by relative error, %) was always less than 12%. Stability analysis showed that metformin is stable for at least 2 months when stored at -70 degrees C. The small volume of blood required (10 mu L), combined with the simplicity of the analytical technique makes this a useful procedure for monitoring metformin concentrations in routine clinical settings. The method is currently being applied to the analysis of blood spots taken from diabetic patients to assess adherence to medications and relationship between metformin level and metabolic control of diabetes. (c) 2006 Elsevier B.V. All rights reserved.

Towards Surface Plasmon Resonance biosensing combined with bioaffinity-assisted nano HILIC Liquid Chromatography Time-of-flight Mass Spectrometry identification of Paralytic Shellfish Poisons

The potential for coupling technologies to deliver new, improved forms of bioanalysis is still in its infancy. We review a number of examples in which coupling has been successful, with special emphasis on combining surface-plasmon-resonance biosensors with mass spectrometry. We give an overview of current progress towards combining biosensor-based bioanalysis with chemical analysis for confirmation of paralytic shellfish poisons that are marine toxins. This comprehensive approach could be an alternative to the official methods currently used (e.g., animal testing and high-performance liquid chromatography with fluorescence detection) and could serve as a model for many more such applications. (C) 2009 Elsevier Ltd. All rights reserved.

Gas liquid equilibria at high pressures

Este trabalho tem como objectivo a medição da solubilidade de gases em líquidos a altas pressões. Para a realização experimental foi usada uma célula de altas pressões de volume variável e um método de observação directa para a detecção das transições de fase. As condições usadas neste trabalho experimental foram temperaturas entre 276.00 e 373.15 K e pressões ate 100 MPa. Numa primeira fase foram efectuadas medições de solubilidade de metano em anilina pura e para uma solução aquosa de composição mássica igual a 5% em anilina. A anilina é fundamentalmente usada em processos como a produção de borracha, de poliuretanos, pigmentos e tintas, fármacos, herbicidas e fungicidas. É usualmente produzida através de processos de redução do nitrobenzeno por reacção com o HCl. De facto, esta síntese é um processo químico complexo onde um grande número de processos compete entre si. Alterações nas condições do processo com a possível formação de produtos intermediários podem afectar a eficiência do processo. Para a sua melhoria foi sugerido que o hidrogénio usado fosse simultaneamente produzido e gasto no reactor principal. Neste caso e para a produção do mesmo, era necessário adicionar metano e água ao reactor. Tendo por base a ideia de que as reacções onde o hidrogénio é reagente e produto ocorrem em simultâneo, torna-se clara a importância do estudo da solubilidade do metano em anilina pura e em soluções aquosas desta. Numa segunda fase foi estudada a solubilidade do dióxido de carbono em soluções aquosas de tri-iso-butil(metil)fosfónio tosilato, com composições molares são de 4, 8 e 12% em líquido iónico. Este pertencente à família dos fosfónios. Possui uma viscosidade e densidade elevadas, é térmica e quimicamente estável e ainda possui uma elevada polaridade. Apresenta uma miscibilidade completa em água e nos solventes mais usuais, como o diclorometano e tolueno, não sendo no entanto míscivel em hexano. O tri-isobutil( metil)fosfónium tosilato é usado como solvente nos processos de hidroformilação de olefinas e ainda em processos de captura e conversão de dióxido de carbono. Neste trabalho experimental, a temperatura e a pressão foram inicialmente aumentadas até o sistema atingir o equilíbrio. A pressão é diminuída lentamente até se verificar o aparecimento/desaparecimento da última bolha de gás. A pressão à qual a última bolha de gás desaparece representa a pressão de equilíbrio para aquela temperatura. Este procedimento foi efectuado para vários sistemas e várias temperaturas.

High temperature vapour-liquid equilibria of water-polyalcohol mixtures

Tese de doutoramento, Química (Química Tecnológica), Universidade de Lisboa, Faculdade de Ciências, 2014

Liquid chromatography-tandem mass spectrometry (LC/APCI-MS/MS) methods for the quantification of captan and folpet phthalimide metabolites in human plasma and urine.

Captan and folpet are fungicides largely used in agriculture. They have similar chemical structures, except that folpet has an aromatic ring unlike captan. Their half-lives in blood are very short, given that they are readily broken down to tetrahydrophthalimide (THPI) and phthalimide (PI), respectively. Few authors measured these biomarkers in plasma or urine, and analysis was conducted either by gas chromatography coupled to mass spectrometry or liquid chromatography with UV detection. The objective of this study was thus to develop simple, sensitive and specific liquid chromatography-atmospheric pressure chemical ionization-tandem mass spectrometry (LC/APCI-MS/MS) methods to quantify both THPI and PI in human plasma and urine. Briefly, deuterated THPI was added as an internal standard and purification was performed by solid-phase extraction followed by LC/APCI-MS/MS analysis in negative ion mode for both compounds. Validation of the methods was conducted using spiked blank plasma and urine samples at concentrations ranging from 1 to 250 μg/L and 1 to 50 μg/L, respectively, along with samples of volunteers and workers exposed to captan or folpet. The methods showed a good linearity (R (2) > 0.99), recovery (on average 90% for THPI and 75% for PI), intra- and inter-day precision (RSD, <15%) and accuracy (<20%), and stability. The limit of detection was 0.58 μg/L in urine and 1.47 μg/L in plasma for THPI and 1.14 and 2.17 μg/L, respectively, for PI. The described methods proved to be accurate and suitable to determine the toxicokinetics of both metabolites in human plasma and urine.

Liquid Chromatography-Electrospray Linear Ion Trap Mass Spectrometry Analysis of Targeted Neuropeptides in Tac1-/- Mouse Spinal Cords Reveal Significant Lower Concentration of Opioid Peptides.

Tachykinin and opioid peptides play a central role in pain transmission, modulation and inhibition. The treatment of pain is very important in medicine and many studies using NK1 receptor antagonists failed to show significant analgesic effects in humans. Recent investigations suggest that both pronociceptive tachykinins and the analgesic opioid systems are important for normal pain sensation. The analysis of opioid peptides in Tac1-/- spinal cord tissues offers a great opportunity to verify the influence of the tachykinin system on specific opioid peptides. The objectives of this study were to develop a HPLC–MS/MRM assay to quantify targeted peptides in spinal cord tissues. Secondly, we wanted to verify if the Tac1-/- mouse endogenous opioid system is hampered and therefore affect significantly the pain modulatory pathways. Targeted neuropeptides were analyzed by high performance liquid chromatography linear ion trap mass spectrometry. Our results reveal that EM-2, Leu-Enk and Dyn A were down-regulated in Tac1-/- spinal cord tissues. Interestingly, Dyn A was almost 3 fold down-regulated (p < 0.0001). No significant concentration differences were observed in mouse Tac1-/- spinal cords for Met-Enk and CGRP. The analysis of Tac1-/- mouse spinal cords revealed noteworthy decreases of EM-2, Leu-Enk and Dyn A concentrations which strongly suggest a significant impact on the endogenous pain-relieving mechanisms. These observations may have insightful impact on future analgesic drug developments and therapeutic strategies.

Coupling liquid MALDI MS to liquid chromatography

Matrix-assisted laser desorption/ionisation (MALDI) coupled with time-of-flight (TOF) mass spectrometry (MS) is a powerful tool for the analysis of biological samples, and nanoflow high-performance liquid chromatography (nanoHPLC) is a useful separation technique for the analysis of complex proteomics samples. The off-line combination of MALDI and nanoHPLC has been extensively investigated and straightforward techniques have been developed, focussing particularly on automated MALDI sample preparation that yields sensitive and reproducible spectra. Normally conventional solid MALDI matrices such as α-cyano-4-hydroxycinnamic acid (CHCA) are used for sample preparation. However, they have limited usefulness in quantitative measurements and automated data acquisition because of the formation of heterogeneous crystals, resulting in highly variable ion yields and desorption/ ionization characteristics. Glycerol-based liquid support matrices (LSM) have been proposed as an alternative to the traditional solid matrices as they provide increased shot-to-shot reproducibility, leading to prolonged and stable ion signals and therefore better results. This chapter focuses on the integration of the liquid LSM MALDI matrices into the LC-MALDI MS/MS approach in identifying complex and large proteomes. The interface between LC and MALDI consists of a robotic spotter, which fractionates the eluent from the LC column into nanoliter volumes, and co-spots simultaneously the liquid matrix with the eluent fractions onto a MALDI target plate via sheath flow. The efficiency of this method is demonstrated through the analysis of trypsin digests of both bovine serum albumin (BSA) and Lactobacillus plantarum WCFS1 proteins.

Detection and Characterization of Cholesterol-Oxidized Products Using HPLC Coupled to Dopant Assisted Atmospheric Pressure Photoionization Tandem Mass Spectrometry

Oxidation of cholesterol (Ch) by a variety of reactive oxygen species gives rise mainly to hydroperoxides and aldehydes. Despite the growing interest in Ch-oxidized products, the detection and characterization of these products is still a matter of concern. In this work, the main Ch-oxidized products, namely, 3 beta-hydroxycholest-5-ene-7 alpha-hydroperoxide (7 alpha-OOH), 3 beta-5 alpha-cholest-6-ene-5-hydroperoxide (5 alpha-OOH), 3 beta-hydroxycholest-4-ene-6 alpha-hydroperoxide (6 alpha-OOH), 3 beta-hydroxycholest-4-ene-6 beta-hydroperoxide (6 beta-OOH), and 3 beta-hydroxy-5 beta-hydroxy-B-norcholestane-6 beta-carboxaldehyde (ChAld), were detected in the same analysis using high-performance liquid chromatography (HPLC) coupled to dopant assisted atmospheric pressure photoionization tandem mass spectrometry. The use of selected reaction monitoring mode (SRM) allowed a sensitive detection of each oxidized product, while the enhanced product ion mode (EPI) helped to improve the confidence of the analyses. Isotopic labeling experiments enabled one to elucidate mechanistic features during fragmentation processes. The characteristic fragmentation pattern of Ch-oxidized products is the consecutive loss of 1120 molecules, yielding cationic fragments at m/z 401, 383, and 365. Homolytic scissions of the peroxide bond are also seen. With (18)O-labeling approach, it was possible to establish a fragmentation order for each isomer. The SRM transitions ratio along with EPI and (18)O-labeled experiments give detailed information about differences for water elimination, allowing a proper discrimination between the isomers:Phis is of special interest considering the emerging role of Ch-oxidized products in the development of diseases.

Ultrasensitive Simultaneous Quantification of 1,N(2)-Etheno-2 `-deoxyguanosine and 1,N(2-)Propano-2 `-deoxyguanosine in DNA by an Online Liquid Chromatography-Electrospray Tandem Mass Spectrometry Assay

Exocyclic DNA adducts produced by exogenous and endogenous compounds are emerging as potential tools to study a variety of human diseases and air pollution exposure. A highly sensitive method involving online reverse-phase high performance liquid chromatography with electrospray tandem mass spectrometry detection in the multiple reaction monitoring mode and employing stable isotope-labeled internal standards was developed for the simultaneous quantification of 1,N(2)-etheno-2`-deoxyguanosine (1,N(2)-epsilon dGuo) and 1,N(2)-propano-2`-deoxyguanosine (1,N(2)-propanodGuo) in DNA. This methodology permits direct online quantification of 2`-deoxyguanosine and ca. 500 amol of adducts in 100 mu g of hydrolyzed DNA M the same analysis. Using the newly developed technique, accurate determinations of 1,N(2)-etheno-2`-deoxyguanosine and 1,N2-propano-2`-deoxyguanosine levels in DNA extracts of human cultured cells (4.01 +/- 0.32 1,N(2)-epsilon dGuo/10(8) dGuo and 3.43 +/- 0.33 1,N(2)-propanodGuo/10(8) dGuo) and rat tissue (liver, 2.47 +/- 0.61 1,N(2)-epsilon dGuo/10(8) dGuo and 4.61 +/- 0.69 1,N(2)-propanodGuo/108 dGuo; brain, 2.96 +/- 1.43,N(2)-epsilon dGuo/10(8) dGuo and 5.66 +/- 3.70 1,N(2)-propanoclGuo/10(8) dGuo; and lung, 0,87 +/- 0.34 1,N(2)-edGuo/ 10(8) dGuo and 2.25 +/- 1.72 1,N(2)-propanodGuo/10(8) dGuo) were performed. The method described herein can be used to study the biological significance of exocyclic DNA adducts through the quantification of different adducts in humans and experimental an with pathological conditions and after air pollution exposure.

Determination of lomefloxacin in tablet preparations by liquid chromatography

A sensitive, precise, and specific high-performance liquid chromatography (HPLC) method was developed for the assay of lomefloxacin (LFLX) in raw material and tablet preparations. The method validation parameters yielded good results and included the range, linearity, precision, accuracy, specificity, and recovery. It was also found that the excipients in the commercial tablet preparation did not interfere with the assay. The HPLC separation was performed on a reversed-phase Phenomenex C18 column (150 x 4.6 mm id, 5 pm particle size) with a mobile phase composed of 1% acetic acid-acetonitrile-methanol (70 + 15 + 15, v/v/v), pumped isocratically at a flow rate of 1.0 mL/min. The effluent was monitored at 280 nm. The calibration graph for LFLX was linear from 2.0 to 7.0 mg/mL. The interday and intraday precisions (relative standard deviation) were less than 1.0%. The method was applied for the quality control of commercial LFLX tablets to quantitate the drug.