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Made available in DSpace on 2022-04-28T17:23:35Z (GMT). No. of bitstreams: 0 Previous issue date: 2022-04-07 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) We analyzed the effectiveness of manual cleaning protocols performed on high-speed dental handpieces, using protein identification by fluorescence. Although one protocol was able to able to lower the amount of protein, >40% of the handpieces showed amounts of residual protein at unacceptable levels. Univ Fed Pernambuco, Dept Prosthodont & Oral Facial Surg, Recife, PE, Brazil Univ Sao Paulo, Med Surg Nursing Dept, Escola Enfermagem, Sao Paulo, Brazil Fed Govt, Natl Hlth Surveillance Agcy, Brasilia, DF, Brazil State Univ Sao Paulo, Sch Pharm, Dept Biol Sci, Araraquara, SP, Brazil Queen Mary Univ London, William Harvey Res Inst, London, England State Univ Sao Paulo, Sch Pharm, Dept Biol Sci, Araraquara, SP, Brazil CAPES: 001

Traveling in cognito, Henry Ford and his wife, Clara, spent April 1928 in England where guided by Herbert Morton, an engineer with Fords Manchester plant they traveled around looking for old steam ...

This paper is a personal review of the role developments in separation science over the last four decades have played in the diagnosis and understanding of purine and pyridine metabolism particularly in man. In 1967 the separation of nucleotides was used to demonstrate a new chromatography technique. This technique became known as HPLC and which continues to dominate the analysis of purines etc. The resolution and quantitation offered by even the earliest HPLC systems completely changed our understanding of matters such as nucleotide instability in cells and tissues, diagnosis of in born errors, etc. Capillary Electrophoresis also enabled high resolution as well as the quantitation of usual analytes such allantoin. Now LC-MS dominates the diagnostic field. This paper is based on the Anne Simmonds Lecture given by the author at PP17 in Gdansk in 2017.

Paracetamol is arguably the most commonly used analgesic and antipyretic drug worldwide, however its mechanism of action is still not fully established. It has been shown to exert effects through multiple pathways, some actions suggested to be mediated via N-arachidonoylphenolamine (AM404). AM404, formed through conjugation of paracetamol-derived p-aminophenol with arachidonic acid in the brain, is an activator of the capsaicin receptor, TRPV1, and inhibits the reuptake of the endocannabinoid, anandamide, into postsynaptic neurons, as well as inhibiting synthesis of PGE2 by COX-2. However, the presence of AM404 in the central nervous system following administration of paracetamol has not yet been demonstrated in humans. Cerebrospinal fluid (CSF) and blood were collected from 26 adult male patients between 10 and 211 minutes following intravenous administration of 1 g of paracetamol. Paracetamol was measured by high-performance liquid chromatography with UV detection. AM404 was measured by liquid chromatography-tandem mass spectrometry. AM404 was detected in 17 of the 26 evaluable CSF samples at 5–40 nmol⋅L−1. Paracetamol was measurable in CSF within 10 minutes, with a maximum measured concentration of 60 μmol⋅L−1 at 206 minutes. This study is the first to report on the presence of AM404 in human CSF following paracetamol administration. This may represent an important finding in our understanding of paracetamol’s mechanism of action, although measured concentrations were far below the previously documented IC50 for this metabolite., Video abstract

Topically applied tetracaine is a local anaesthetic. A novel HPLC method for the rapid and sensitive analysis of tetracaine was developed and compared with a short end direction capillary zone electrophoresis (CZE) method. The method was developed and validated for the separation and quantification of tetracaine in skin samples removed by ‘tape-stripping’. Tetracaine was extracted from tape with 100% methanol, which was then diluted to 50% with water for injection. Tetracaine and the internal standard, procaine, were separated on a reversed-phase Luna PFP(2), 3 µm, 150 × 4.6 mm column at ambient temperature using isocratic elution with KH2PO4 buffer (pH 2.5) and methanol (35:65, v/v). The flow rate was 1 mL/min, with detection at 312 nm. The limit of quantification for tetracaine was 0.03 µg/mL. Calibration lines were linear with r2 values >0.99. The within- and between-assay imprecision and the percentage of inaccuracy for the QC samples including lower and upper limits of quantitation were 92%. Compared with CZE, the mean percentage error and the absolute mean percentage error were 0.62 and 6.29, respectively. The two methods were compared in a number of pharmacokinetic studies. Copyright © 2014 John Wiley & Sons, Ltd.

An understanding of the strength of materials is essential for modern mechanical and structural engineering. The paper discusses whether Kirkaldy's Machine, the foundation to the modern system of materials testing should be saved.

Ciclosporin is used as an immunosuppressant in post-organ transplantation. Recently, many questions have been raised about using generic substitutes, especially with narrow therapeutic index drugs (NTIDs). In this study, a simple high-performance liquid chromatography (HPLC) method was developed, validated and applied to detection ciclosporin in dissolution testing. Seven ciclosporin products (gelatin capsules) were included in this study, obtained from Columbia (C), Egypt (E), India (I), Jordan (J), Pakistan (P), Saudi Arabia (S), and Turkey (T). The dissolution test was done for all capsules. The Conditions were as follows: 500 ml deionized water as the medium in apparatus 2 (Pharmatest, Germany), temperature 37.5 ± 0.5°C; 50 rev/min, sampling times were 5, 10, 15, 30, 60 and 90 min with 5 ml for each sample. HPLC separation was done by a C18 column, 5 μm, (4.6 × 250 mm, ACE 5) held at 50 ± 0.3°C. Analytes were isocratically eluted at 0.7 ml/min with acetonitrile and water (70+30%) and 0.03% trifluoroacetic acid, over the 25-min run time. The intra-day and inter-day imprecision for ciclosporin across the standard range was

The Sailing Stone project animates a stone sculpture such that a traditional, large scale, normally static installation physically interacts with the public. The Sailing Stone moves so as to disrupt the normal motion of visitors to the space so as to encourage changes in the nature of the interaction between visitors and the sculpture itself. The public gallery space is monitored through a network of video cameras, and this information is used to develop a model of human motion through the gallery space. Based on this model of visitor motion, a motion plan is developed and executed for the sculpture so as to maximally disrupt the motion paths of visitors.

Background HIF-1 deficiency has marked effects on tumour glycolysis and growth. We therefore investigated the consequences of HIF-1 deficiency in mice, using the well established Hepa-1 wild-type (WT) and HIF-1β-deficient (c4) model. These mechanisms could be clinically relevant, since HIF-1 is now a therapeutic target. Methods Hepa-1 WT and c4 tumours grown in vivo were analysed by 18FDG-PET and 19FDG Magnetic Resonance Spectroscopy for glucose uptake; by HPLC for adenine nucleotides; by immunohistochemistry for GLUTs; by immunoblotting and by DIGE followed by tandem mass spectrometry for protein expression; and by classical enzymatic methods for enzyme activity. Results HIF-1β deficient Hepa-1 c4 tumours grew significantly more slowly than WT tumours, and (as expected) showed significantly lower expression of many glycolytic enzymes. However, HIF-1β deficiency caused no significant change in the rate of glucose uptake in c4 tumours compared to WT when assessed in vivo by measuring fluoro-deoxyglucose (FDG) uptake. Immunohistochemistry demonstrated less GLUT-1 in c4 tumours, whereas GLUT-2 (liver type) was similar to WT. Factors that might upregulate glucose uptake independently of HIF-1 (phospho-Akt, c-Myc) were shown to have either lower or similar expression in c4 compared to WT tumours. However the AMP/ATP ratio was 4.5 fold higher (p < 0.01) in c4 tumours, and phosphofructokinase-1 (PFK-1) activity, measured at prevailing cellular ATP and AMP concentrations, was up to two-fold higher in homogenates of the deficient c4 cells and tumours compared to WT (p < 0.001), suggesting that allosteric PFK activation could explain their normal level of glycolysis. Phospho AMP-Kinase was also higher in the c4 tumours. Conclusions Despite their defective HIF-1 and consequent down-regulation of glycolytic enzyme expression, Hepa-1 c4 tumours maintain glucose uptake and glycolysis because the resulting low [ATP] high [AMP] allosterically activate PFK-1. This mechanism of resistance would keep glycolysis functioning and also result in activation of AMP-Kinase and growth inhibition; it may have major implications for the therapeutic activity of HIF inhibitors in vivo. Interestingly, this control mechanism does not involve transcriptional control or proteomics, but rather the classical activation and inhibition mechanisms of glycolytic enzymes.

IntroductionDissolution is the amount of substance that goes into solution per unit time under standardised conditions of liquid/solid interface, solvent composition and temperature. Dissolution is one of the most important tools to predict the in-vivo bioavailability and in some cases to determine bioequivalence and assure interchangeability.AimTo compare the differences in dissolution behaviour of solid dosage forms between innovators (reference products) and their generic counterparts (tested products).MethodsFour replicates for each batch of 37 tested medicines was carried out using A PT-DT70 dissolution tester from Pharma Test. A total of 13 branded medicines and 24 generic counterparts were obtained locally and internationally to detect any differences in their dissolution behaviour. They were tested according to the British Pharmacopeia, European Pharmacopeia and the US Pharmacopeia with the rate of dissolution determined by ultra-violet Spectrophotometery.ResultsMost tested medicines complied with the pharmacopoeial specifications and achieved 85% dissolution in 60min. However, some generic medicines showed significant differences in dissolution rate at 60 and 120min. Many generic medicines showed a slower dissolution rate than their branded counterparts such as the generic forms of omeprazole 20mg. Some showed an incomplete dissolution such as the generic form of nifedipine 10mg. Other generics showed faster dissolution rate than their branded counterpart such as the generic forms of meloxicam 15mg. Moreover, some generics from different batches of the same manufacturer showed significant differences in their dissolution rate such as the generic forms of meloxicam 7.5mg. Nevertheless, some generic medicines violated the EMA and the FDA guidelines for industry when they failed to achieve 85% dissolution at 60min, such as the generic form of diclofenac sodium 50mg.ConclusionMost medicines in this study complied with the pharmacopeial limits. However, some generics dissolved differently than their branded counterparts. This can clearly question the interchangeability between the branded and its generic counterpart or even among generics.

Aims The aims of this study were (A) to investigate whether the number of years of forensic experience affected the accuracy with which forensic experts (FEs) were able to age bruises and (B) to identify the properties and colours of a bruise that were utilized by FEs in their assessment of bruise age. The study then investigated the possibility of using a more objective technique. It was decided to use readily available digital photography and software to objectively assess changes in bruise colouration and to investigate if this can be used to age bruises. Methods Twenty-three FEs were shown 25 photographs of bruises of varying but known ages and asked to estimate the ages. In part two of the study, bruises were inflicted on volunteers using a vacuum pump and photographs taken of the bruise daily from infliction to resolution. The images were analysed using Adobe Photoshop®. Red, green and blue (RGB) values were recorded for each bruise and analyses carried out comparing the values over time between subjects and within subjects. Results This study both enhanced and supported a previous conclusion that visual assessment of photographs is an unreliable method for ageing bruises. Additionally, it found that the degree of forensic experience had no effect on accuracy. It also identified that colour (particularly yellow, red and purple) and intensity of colour were the most commonly used properties of a bruise in the assessment of its age. The RGB method proved to be a reliable technique with which to measure bruise colour, but its validity in the assessment of bruise age was poor. Conclusions Visual assessment of bruises is unreliable and the accuracy of ageing was not improved by the degree of forensic experience. The RGB method gave highly reproducible results, but did not accurately assess bruise age. However, results within subjects suggested that there may be individual variation in haemoglobin metabolism.

The aim of this study was to investigate the involvement of autoimmune reactions to native and post-translationally modified extracellular matrix components in the pathogenesis of periodontitis. Sera from individuals with aggressive periodontitis (AgP, n = 25), chronic periodontitis (CP, n = 14), and gingivitis (G, n = 18) were tested for the presence of autoantibodies against: (a) native collagen type I (CI) and collagen type III (CIII); (b) CI and CIII post-translationally modified by reactive oxygen species (ROS) of the type present during inflammation; and (c) citrullinated filaggrin-derived peptides (CCP). Autoantibodies to native and ROS-modified CI and CIII as well as autoantibodies to CCP were observed exclusively in patients with AgP and not in those with CP or G. In conclusion, autoimmune reactions to native and post-translationally modified self-antigens may play a role specifically in the pathogenesis of AgP.

This study investigated the biochemical relationship between human follicular/oocyte maturity and the levels of follicular fluid purines. Intrafollicular levels of purine metabolites and creatinine are associated with oocyte presence, and the presence of such high levels of adenosine indicates a privileged site with no adenosine deaminase activity. Subgrouping according to oocyte recovery and fertilization revealed differences in correlation between the purine metabolites: Only where an oocyte was recovered and subsequently fertilized did follicular fluid adenosine, adenine, and hypoxanthine levels correlate with each other. Significantly, purines' correlation with levels of the terminal degradation product, uric acid, could only be seen in failed fertilization samples. Given the established metabolic pathways for adenosine triphosphate/adenosine diphosphate/adenosine monophosphate degradation, the results indicate maximization of 2 purine salvage pathways (from adenine and hypoxanthine) that pivot on the presence of high adenosine levels. Such optimized recovery may be necessary to build a store of salvaged adenosine phosphate for oocyte survival.

Although Anne was not able to attend the international symposium commemorated in this volume, she was a solid supporter of the international series. Anne took part in every meeting on Purine and Py...

Some of the major serum proteins that are also found in follicular fluid, including transferrin, alpha-macroglobulin and albumin, are thought to play a role in oocyte maturation. This study set out to identify proteins in human follicular fluid by capillary zone electrophoresis and to investigate their relationship to follicular/oocyte maturity and fertility outcome. 176 individual follicular fluid samples, from 30 women undertaking in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI), were run using an optimized capillary zone electrophoresis method that gave a good separation of sixteen peaks in most samples. Nine of the peaks were identified and quantified but seven remain unknown and require further proteomic identification. Of the identified protein peaks, levels of each were corrected for follicular volume and total content calculated. No significant difference in protein levels was found with regard to oocyte recovery and fertilization. Protein concentrations tended to decrease as the follicular sphere increased whilst total content in follicular fluid increased in proportion to size. This is consistent with simple transudation across a sphere surface area which does not increase in proportion to the follicular fluid. This is not true of the concentration and content pattern of other proteins/biomolecules which are produced by follicular cells locally. In conclusion, neither concentration nor absolute levels of nine major proteins identified in follicular fluids correlated with oocyte presence and fertility outcome. Future work to remove more concentrated proteins (e.g. albumin) would enhance separation of smaller peaks and identification of the unknown molecules.

Study Objective To characterize the insoluble particulate matter generated during hysteroscopic monopolar and bipolar electrosurgery. Design In vitro comparative study (Canadian Task Force Classification II-1). Setting Teaching hospital. Patients Eight patients undergoing hysterectomy with benign indications. Intervention Tissue blocks were divided, and 1 g of tissue was vaporized using a monopolar and a bipolar vaporizing electrode. Measurements and Main Results The particles were examined at light microscopy to study their morphologic features. Particle analysis was carried out using a laser diffraction particle analyser. Monopolar uterine tissue vaporization generated particles of a smaller median diameter than did bipolar vaporization (0.13 μm and 0.25 μm, respectively). The distribution by particle surface area in both instruments seems to be complex, with peaks at 0.35 and 4.0 μm for monopolar-generated particles and 0.5 and 5.0 μm for bipolar-generated particles. Another smaller peak with larger particles produced by both instruments was identified at 30 μm. During monopolar uterine tissue vaporization, 9.96% of particles generated were smaller than 5 μm, whereas during bipolar vaporization, 3.81% of particles were smaller than 5 μm (p = .01). Conclusion Particulate matter generated during hysteroscopic electrosurgical vaporization using monopolar and bipolar instruments is heterodisperse and complex in shape. Monopolar vaporization results in particles of a smaller median diameter compared with bipolar vaporization. Both instruments generate 2 distinct populations of particles by surface area. The distribution of particles by volume during hysteroscopic electrosurgery is complex.

A rapid and simple short-end (reverse) capillary zone electrophoresis method was developed and validated for the separation and quantification of tetracaine in skin using tape samples. The separation was performed in a 485 mm (400 mm to window) x 50 microm internal diameter fused silica capillary using a background electrolyte of phosphoric acid-Tris pH2.5 at -25 kV. The extraction of tetracaine from tape samples was achieved using methanol diluted to 50% with water before injection. Procaine was the internal standard. The migration times for procaine and tetracaine were 1.25 and 1.36 min, respectively. The limit of quantification for tetracaine was 50 microg, with a signal-to-noise ratio greater than 10. The calibration curve was linear from 50 to 1200 microg with r(2) greater than 0.99. The CV for both within- and between-assay imprecision and the percentage inaccuracy for the quality control samples including lower and upper limits of quantitation were12.1% and11%, respectively. The absolute mean recovery of tetracaine was97%. The accuracy and selectivity of this method allowed the rapid measurement of tetracaine in tape samples obtained from a skin tape stripping study of local anaesthetics in healthy subjects.

(2008). I. K. Brunel in London. Transactions of the Newcomen Society: Vol. 78, No. 1, pp. 47-52.

Insulin is the most specific beta cell antigen and a potential primary autoantigen in type 1 diabetes. Insulin autoantibodies (IAAs) are the earliest marker of beta cell autoimmunity; however, only slightly more than 50% of children and even fewer adults newly diagnosed with type 1 diabetes are IAA positive. The aim of this investigation was to determine if oxidative post-translational modification (oxPTM) of insulin by reactive oxidants associated with islet inflammation generates neoepitopes that stimulate an immune response in individuals with type 1 diabetes. oxPTM of insulin was generated using ribose and various reactive oxygen species. Modifications were analysed by SDS-PAGE, three-dimensional fluorescence and MS. Autoreactivity to oxPTM insulin (oxPTM-INS) was observed by ELISA and western blotting, using sera from participants with type 1 or type 2 diabetes and healthy controls as probes. IAA was measured using the gold-standard radiobinding assay (RBA). MS of oxPTM-INS identified chlorination of Tyr16 and Tyr26; oxidation of His5, Cys7 and Phe24; and glycation of Lys29 and Phe1 in chain B. Significantly higher binding to oxPTM-INS vs native insulin was observed in participants with type 1 diabetes, with 84% sensitivity compared with 61% sensitivity for RBA. oxPTM-INS autoantibodies and IAA co-existed in 50% of those with type 1 diabetes. Importantly 34% of those with diabetes who were IAA negative were oxPTM-INS positive. Altogether, 95% of participants with type 1 diabetes presented with autoimmunity to insulin by RBA, oxPTM-INS or both. Binding to oxPTM-INS was directed towards oxPTM-INS fragments with slower mobility than native insulin. These data suggest that oxPTM-INS is a potential autoantigen in individuals with new-onset type 1 diabetes.

'Multi-dimensional' liquid separations have a history almost as long as chromatography. In multi-dimensional chromatography the sample is subjected to more than one separation mechanism; each mechanism is considered an independent separation dimension. The separations can be carried out either offline via fraction collection, or directly coupled online. Early multi-dimensional separations using combinations of paper chromatography, electrophoresis and gels, in both planar and columnar modes are reviewed. Developments in HPLC have increased the number of measurable analytes in ever more complex matrices, and this has led to the concept of 'global metabolite profiling'. This review focuses on the theory and practice of modern 'comprehensive' multi-dimensional liquid chromatography when applied to biomedical and pharmaceutical analysis.

Objective Collagen-induced arthritis is a commonly accepted model of rheumatoid arthritis (RA). However, it has been difficult to substantiate the involvement of autoimmunity to type II collagen (CII) in the pathogenesis of RA. The aim of this investigation was to determine if CII, modified by reactive oxidant species present within the inflamed joint, could generate neoantigenic epitopes. Methods Oxidants that play a role in acute and chronic inflammation and are present in the rheumatoid joint (hydroxyl radical, hypochlorous acid, and peroxynitrite) were used for modification of native CII. In addition, CII was glycated with ribose, since nonenzymatic oxidative reactions by glycation are evident in RA. Modifications were analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and 3-dimensional fluorescence followed by enzyme-linked immunosorbent assay (ELISA) and Western blotting, using, as probes, sera from patients with RA and from patients with other inflammatory and noninflammatory joint diseases. Results Only 1 RA serum sample showed strong binding to native CII. In contrast, binding to modified CII was increased in 14 of 31 RA sera, of which 7 were strong binders and 7 were moderate binders. Among the non-RA serum samples, only 1 yielded a strong reaction to modified CII and 5 of 41 were moderate binders. Samples that showed the strongest binding to modified CII in ELISA also showed strong binding to various fragmented or aggregated forms of CII in Western blots, as well as strong binding to fragmented CII present in RA synovial fluid. Conclusion When modified by conditions found within the inflamed joint, CII acts as an autoantigen in RA.

Separations of human urine by microemulsion electrokinetic chromatography (MEEKC) and micellar electrokinetic capillary chromatography (MEKC) with respect to resolution, migration times and efficiencies were optimized and compared. The optimised MEEKC and MEKC methods were simple and fast, both of which are excellent characteristics for the complex separations required in clinical and biomedical studies. However, resolution in MEKC was significantly greater than in MEEKC although migration times were 30% faster for the optimised MEEKC method. In addition, a faster analysis method (short-end injection) specifically for routine screening purposes was also investigated. With both MEEKC and MEKC modes, this provided short separations (less than 4 min for urine) with no major compromise in resolution. In conclusion, we found that MEEKC offered no real advantage over MEKC for urine analysis.

In our experience the efficient design of molecularly imprinted polymer (MIPs) for novel templates has proved difficult. Following commonly used imprinting protocols, MIPs designed against one template show both a lack of capacity and poor specificity for rebinding either the template or structurally similar analytes. Optimisation methods that involve changing one factor at a time can be laborious. A novel approach for the optimisation of MIPs using chemometrics is described. Sulfonamides, common drug residues in foodstuffs, were used as the model analytes with a methacrylic acid/ethylene glycol dimethacrylate MIP. To avoid the inaccuracies in measurement caused by template bleed a multi-analyte competition rebind assay was developed to select suitable sulfonamides to be used as the template for the MIP, and for the rebind analyte in the chemometric optimisation study. The rebinding efficiencies were monitored by HPLC. The template sulfonamide was selected as sulfamethazine (SMZ), and the rebind analyte as sulfadimethoxine (SDIM). The template:monomer:cross-linker (T:M:X) ratio of the SMZ block MIP was then optimised using a three-level full factorial design to predict a MIP with the highest rebind capacity. On synthesis this was 38.8% for SDIM in a solid phase extraction (SPE) application agreeing with the predication. The factorial design was further utilised to predict an optimum T:M:X ratio for the production of a class specific MIP, capable of binding a range of sulfonamides simultaneously. The predicted optimum T:M:X ratios of (1:10:55) and (1:10:10) were found to be different to commonly used ratios from the MIP literature.

The aim of this study was to isolate and identify antigenic peptides associated with HLA class I molecules on a bladder tumour cell line. HLA-A1 moelcules were purified using an immunobead-purification technique and following elution of nonapeptides associated with the complex, their HPLC profile was determined by Tandem mass spectrometry (MS/MS). Three peptides were identified namely: (1) P991 (VTDPGNLLY); (2) P1041.5 (LTDLGFLVY), and (3) P1057.7 (LTDPHLLSY); these sequences matched elements of hepatitis B, MAGE1-A1 and herpes simplex viruses. These antigens had half-lives of approximately 120 min which is within the theoretical range of such short peptides. These results indicate that identification of MHC-associated peptides is possible without using an assay for cytotoxic T cells. Although this approach was applied on a relatively small scale, broader applications can be foreseen.

Advanced oxidation protein products (AOPP) are reportedly elevated in the plasma of patients with a number of diseases, including diabetes mellitus, that involve oxidative stress. However, the accurate measurement of AOPP in human plasma is hampered by the formation of a precipitate following the addition of potassium iodide and glacial acetic acid according to the published assay procedure. Here we describe a modification of the AOPP assay which eliminates interference by precipitation and provides a robust, reliable, and reproducible protocol for the measurement of iodide oxidising capacity in plasma samples (intra-assay CV 1.7–5.3%, interassay CV 5.3–10.5%). The improved method revealed a significant association of AOPP levels with age (p<0.05) and hypertension (p=0.01) in EDTA-anticoagulated plasma samples from 52 patients with diabetes and 38 nondiabetic control subjects, suggesting a possible link between plasma oxidising capacity and endothelial and/or vascular dysfunction. There was no significant difference between AOPP concentrations in diabetic (74.8 ± 7.2 μM chloramine T equivalents) and nondiabetic (75.5 ± 7.0 μM chloramine T equivalents) individuals.

On a very wet evening in November 2014 Barnsley Metropolitan Council held a spectacular light show to mark the Official opening of the Elsecar Atmospheric engine after a period restoration to worki...

Topically applied tetracaine is a local anaesthetic. A novel HPLC method for the rapid and sensitive analysis of tetracaine was developed and compared with a short end direction capillary zone electrophoresis (CZE) method. The method was developed and validated for the separation and quantification of tetracaine in skin samples removed by 'tape-stripping'. Tetracaine was extracted from tape with 100% methanol, which was then diluted to 50% with water for injection. Tetracaine and the internal standard, procaine, were separated on a reversed-phase Luna PFP(2), 3 µm, 150 × 4.6 mm column at ambient temperature using isocratic elution with KH2 PO4 buffer (pH 2.5) and methanol (35:65, v/v). The flow rate was 1 mL/min, with detection at 312 nm. The limit of quantification for tetracaine was 0.03 µg/mL. Calibration lines were linear with r(2) values0.99. The within- and between-assay imprecision and the percentage of inaccuracy for the QC samples including lower and upper limits of quantitation were6 and10%. The absolute mean recovery of tetracaine was92%. Compared with CZE, the mean percentage error and the absolute mean percentage error were 0.62 and 6.29, respectively. The two methods were compared in a number of pharmacokinetic studies.

This paper introduces a novel improvement to high pressure slurry packing of CEC capillaries. Slurry homogeneity is maintained by constantly stirring in a ‘balanced density’ solvent system in a vessel with a configuration that prevents silica agglomerates from blocking the capillary inlet. Up to nine, one metre length capillaries may be packed simultaneously and repeatably in under 3 hours. All capillaries produced peak efficiencies greater than 100,000 plates/metre with 5 μm Spherisorb ODS1 and showed capillary to capillary variability of less than 10% in terms of peak symmetry and peak efficiency. The data has been presented in part, at the 23rd International Symposium in Chromatography 2000.

The major histocompatibility complex (MHC) class I molecules present processed self and non-self peptides to T lymphocytes. Given that the class I peptide complex plays a critical role in cell-mediated immunity, it is important to identify the nature of class I-associated peptides unique to malignant cells as a prelude to the development of vaccines. The aim of this study was to combine immuno-bead purification (using anti-class I antibody W6/32) technique, sequential ultra-filtration and high performance liquid chromatography (HPLC) to isolate class I antigens and associated peptides from an in-house established bladder tumour cell line (Fen) whose missing class I antigens had been restored by beta2-microglobulin (beta2-m) gene transfaction. The results were as follows: (a) class I antigens could be separated from tumour cell lysate but only from the class I positive Fen cells; (b) treatment of CNBr-W6/32 beads pre-exposed to class I positive Fen lysate and eluted with dissociation agent (mild acid) resulted in the release of more than 20 peptides at an approximate molecular weight of between 700 and 3000 Da based on SDS-PAGE and silver staining analysis; (c) purified and eluted peptides from class I antigens showed distinct peaks when analysed by HPLC. The data presented in this investigation demonstrated the feasibility of isolating class I antigens and associated peptides from a bladder tumour cell line. The extension of these approaches to isolate peptides from tissue tumour biopsies may help the future of vaccine therapy in cancer patients.

HPLC was used in combination with immuno-bead separation technique for identification of an individual protein from a pool of proteins. This was carried out using an in-house monoclonal antibody (ATC2) specific for placental alkaline phosphatase (PLAP) as a primary antibody for conjugation to CNBr beads. The phosphatase activity (ALP) of PLAP was measured by colorimetric assay (MEDC). The data from this study has so far indicated that: 1. HPLC analysis of molecules following isolation with ATC2-conjugated beads showed high degree of purity. This could be achieved using protein mixtures prepared from lysates of tumour cell lines or tumour fragments. 2. HPLC-isolated PLAP maintained phosphatase activity. 3. Out of the four dissociation reagents used, diethyl amine (DEA) was found to be the best reagent for dissociation of antigen, ie PLAP, but not mAb from CNBr beads. 4. The profile of ALP activity was different for samples prepared from testis and kidney fragments, both in terms of the HPLC peak profile as well as the sensitivity. These data confirmed that the immuno-bead separation technique in conjunction with HPLC were powerful tools for identifying an individual protein from a pool of proteins. These approaches are being used for the identification of PLAP molecules, as a tumour marker in patients suspected of testicular malignancies with equivocal ultrasound.

A novel triple column capillary electrophoresis system is described. Design specifications facilitate method development and analyses by providing on-line, selective, pre-concentration and clean-up of both high (ml) and low (μl) volumes of specific analytes in two dimensions and separation via an additional third dimension. The system described additionally provides four distinct detection capabilities via both contactless conductivity and UV. The addition of a third dimension to the previously reported “coupled-column” systems, and further modifications made, has allowed for optimal identification, separation, and quantitation of micro-components in complex mixtures. The ability to perform both capillary zone electrophoretic and isotachophoretic separations on-line and in any combination enhances the scope for rapid analytical method development and analysis of complex or trace sample components.

Experimental colitis is associated with anorexia that is attenuated by treatment with an interleukin (IL)-1 receptor antagonist. Serotonin (5-hydroxytryptamine [5-HT]) is a potent inhibitor of feeding, and its release from the hypothalamus is stimulated by IL-1. We have tested the hypotheses that anorexia associated with experimental colitis results from increased activity of hypothalamic 5-HT neurons and that the increase in activity occurs secondary to an increase in availability of tryptophan, the precursor of 5-HT.In vivo 5-HT release and regional hypothalamic 5-HT and tryptophan concentrations were measured in rats with 2,4,6,-trinitrobenzene sulfonic acid (TNBS)-induced colitis, healthy controls, and animals pair-fed to match the food intake of the colitic group. Food intake in the colitic group was assessed after depletion of brain 5-HT by p-chlorophenylalanine (PCPA).In the colitic group, release of 5-HT from the hypothalamic paraventricular nucleus (PVN) was 3-fold (P = 0.01) and 14-fold (P0.001) higher than in control and pair-fed groups, respectively. Concentrations of tryptophan were similar in each group in all hypothalamic regions. Food intake was significantly increased in the colitic group after PCPA treatment but was not restored to control values.In animals with TNBS-induced colitis, 5-HT release from the PVN is increased. The increase in food intake after depletion of brain 5-HT suggests that hypothalamic 5-HT contributes to anorexia but is not the only mediator. Increased 5-HT release in the colitic group was not driven by increased precursor availability.

In this preliminary study we have optimised micellar electrokinetic chromatography (MEKC, a form of capillary electrophoresis) to enable the chromatographic and spectral characteristics of human ouabainlike compound (OLC) to be investigated. Sera from fifty patients were combined to form a pool (100ml) whilst urine (92.5ml) was obtained from a normal healthy volunteer. Both samples were initially concentrated and partially purified by solid phase extraction before further purification by sequential HPLC separations. Final volumes for both extracts were 100μl. MEKC was performed on a HP3D CE instrument with voltage set at 20KV, capillary temperature at 20°C, injection time 4s, sample volume 10nl, with detection by photodiode array. A compound was found in both serum and urine that had similar elution and spectral characteristics to authentic ouabain. We conclude that MEKC is potentially a useful tool for the analysis of human OLC. (Hypertens Res 2000; 23 Suppl: S29-S32)

A simple but rapid capillary electrophoresis method was developed for the measurement of nitrite and nitrate in human extracellular fluids and other aqueous solutions. The capabilities of the method were demonstrated by the measurement of endogenous nitrite and nitrate in plasma and serum samples from healthy volunteers, and serum and synovial fluid samples from rheumatoid arthritis patients. Furthermore, this method was used to simultaneously measure nicotinamide adenine dinucleotide, reduced (NADH), nicotinamide adenine dinucleotide (NAD+), nitrite, and nitrate, when studying the nitrite reductase activity of xanthine oxidase. The stability of nitrite was also investigated and it was found that when whole blood was spiked with nitrite and then processed, the nitrite was more stable in the plasma than in the serum. Our findings may help to explain the variations in basal nitrite concentrations reported in the literature.

Within the clinical chemistry community, electrophoretic separations are traditionally considered to be of low resolving power, nonquantitative and best suited to high molecular weight species, such as proteins. The instrumentation is simple, relatively inexpensive and cheap to run, but even at their best such electrophoretic methods are only semi-quantitative. High-performance liquid chromatography (HPLC), on the other hand, is considered to be precise, useful for small molecular weight species, especially drugs, and reproducible, but requires skilled operators. Capillary electrophoresis (CE) combines many of the advantages of HPLC with, I would argue, many of the most attractive features of electrophoresis with respect to biomolecules.

Advances in separation science are leading to more sensitive and more specific analysis of the complex mixtures of metabolites, vitamins and nutrients found in biofluids. Recent developments include capillary electrokinetic separations such as capillary electrophoresis, and the linking of separation methods to new forms of mass spectrometry.

A micellar electrokinetic chromatographic method is described for the determination and quantitation of allantoin, an end-product of purine metabolism in mammals that is applicable to biofluids of different mammal species and man. The method was optimised following a study on the effect of pH and sample preparation procedure. Final conditions were 30 mM sodium tetraborate, pH 9.5, 75 mM sodium dodecyl sulphate, 20 kV and 20 degrees C. Allantoin was well resolved from endogenous compounds and could be determined in horse, dog, mouse and rabbit urine. No allantoin could be found in man. No complicated sample treatment was necessary, thus the developed method was rapid (5 min), sensitive (5 microM) and simple. Results from this work will permit the determination of allantoin in man as a measure of free radical generation reactions as well as its presence in the plasma and other biofluids with modification of the sample preparation procedures.

We describe a capillary electrophoresis procedure, using uridine as an internal standard, for the analysis of urinary pseudouridine following solid-phase extraction. This method retains the advantages of existing chromatographic techniques but has superior resolving power and is technically less demanding. The standard curves were linear and reproducible with a detection limit of 60 fmol; chromatographic analysis was complete in under 10min. Injection variability was

Capillary electrochromatography (CEC) with gradient elution was used to separate mixtures of corticosteroids (adrenosterone, hydrocortisone, dexamethasone, fluocortolone) in extracts of equine urine and plasma. Urine samples were first purified using solid phase extraction. Two purification steps were necessary to prevent contamination of the CEC column. Plasma was purified using automated dialysis. A laboratory-built CEC interface, connected to a gradient HPLC system, delivered samples and mobile phase to the CEC column. CEC was performed in fused-silica capillaries of 50 μm i.d., 24 cm total length, and 16 cm effective length packed with Apex ODS, 3 μm particle size. The mobile phase was ammonium acetate (5 mM) in water/acetonitrile. Acetonitrile in the mobile phase was varied from 9 to 80% (v/v) using the gradient HPLC system. Detection was by UV absorbance at 240 nm. Samples, 10-250 μL, were injected into the mobile phase stream and loaded onto the CEC column under an applied field of 1.04 kV cm(-1) and a CEC column head pressure of 12 bar. Mobile phase flow rate through the sampling interface was 100 μL min(-1). The system was reproducible and could be left in unattended operation for long periods. After injection of 200 urine extracts, a broadening of peaks was observed but the CEC column was still serviceable.

Widespread evidence supports the existence of an endogenous digitalis-like compound in mammals. We report here the development of a novel enzyme immunoassay for ouabain that, in conjunction with a detailed HPLC study, identifies a ouabain-like compound (OLC) in extracted human plasma. The assay is sensitive—minimum detection limit for OLC 37 pmol/L (11 pmol/L in plasma)—and has a working range (between-assay CV

There are many high-performance liquid chromatographic assays for the determination of sulphonamides, dihydrofolate reductase inhibitors and β-lactam antibiotics individually in food products. Recently, some capillary electrophoresis (CE) assays for these drugs have appeared. Sulphonamides plus dihydrofolate reductase inhibitors are readily separated by capillary zone electrophoresis (CZE) and micellar electrokinetic capillary chromatography (MECC) can separate β-lactams. However, if CE is to prove its value over HPLC techniques in practice then its intrinsic high resolution must be exploited fully. A combination of a sulphonamide-dihydrofolate reductase inhibitors separation by CZE with a MECC assay of β-lactams would be advantageous in the screening of food products for veterinary drug residues, saving both time and effort. The analysis of all three classes of drugs, in a single run, was tested using our established β-lactam MECC conditions, but this gave only a partial separation, with a very crowded region. A Box-Behnken factorial design was used to optimise this separation. It was carried out parallel with the “traditional trial and error procedure”, based on our knowledge of the individual CE separations. Both methods arrived at a remarkably similar set of conditions. The final separation produced 25 peaks, within which, were 30 compounds, the separation taking 25 min.

This paper demonstrates the use of micellar electrokinetic capillary chromatography (MECC) to monitor enzyme reaction conditions. The hydrolysis reactions of model conjugated substrates (morphine and reduced flunixin glucuronides, napthyl sulfate), by proprietary beta-glucuronidase preparations, were studied under varied experimental conditions. Reactions were carried out in autosampler vials with incubation in a thermostatted CE autosampler tray. MECC was performed using borax buffer (17.5 mM, pH 9.3) modified with sodium dodecyl sulfate (70 mM). Repetitive injections were made from the sample vial throughout the course of the reactions at a frequency of up to 10 h-1. MECC provided a rapid and reproducible assay for the model substrates. Baseline interference from the enzymes prevented measurement of product increase, therefore substrate decrease was measured from the peak areas. Monitoring of reactions in this way has proved valuable in the optimisation of hydrolysis conditions used in sample preparation for drug analysis. beta-Glucuronidase preparations from Helix pomatia were found to give the best performance of those evaluated in terms of deconjugation efficiency.