David Perrett

Methodological considerations in the determination of plasma catecholamines by high-performance liquid chromatography with electrochemical detection.

We report on a systematic investigation into some frequently encountered problems in the estimation of catecholamines in plasma and biological fluids using high-performance liquid chromatography with electrochemical detection. The kinetics of adsorption and desorption of catecholamines from plasma on to alumina has been studied using laboratory-prepared and some commercial aluminas with various acids. Chromatographic conditions yielding optimal resolution and sensitivity have been characterised. Some possible interfering peaks have been identified. The stability of catecholamines in plasma has been studied.

APPROACHES TO THE RATIONAL DESIGN OF MOLECULARLY IMPRINTED POLYMERS

Abstract 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.

Analysis of Corticosteroids in Biofluids by Capillary Electrochromatography with Gradient Elution

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.

Optimized separation of purine bases and nucleosides in human cord plasma by capillary zone electrophoresis

An optimized separation of the main purine compounds of human serum by capillary zone electrophoresis is presented. Separations were performed in an uncoated silica capillary (44 cm x 75 microns I.D., 37 cm to window) on a SpectraPhoresis 1000 system with UV detection. The separation of adenine (Ade), adenosine (Ado), guanine (Gua), guanosine (Guo), hypoxanthine (Hyp), inosine (Ino), xanthine (Xan) and uric acid (UA) was optimized with respect to pH, temperature, applied potential and hydrodynamic injection time. Optimum conditions were 20 mM borate buffer (pH 9.4), 37 degrees C, 20 kV and 9 s load and detection at 260 nm. Linearity extended from 1 to 125 microM. The sensitivity of the method was 0.5 microM, which is adequate for measuring Ade, Gua, Hyp and UA in plasma samples. Plasma samples from newborns were precipitated with an equal volume of perchloric acid (7%, v/v), the supernatant was adjusted to neutral pH with potassium carbonate and, before injection, the sample was alkalized with sodium hydroxide. The method presented here allows the determination of Ade, Guo, Hyp and UA. The levels of the determined purines were compared in samples from control newborns, preterm babies and newborns with asphyxia or acidic serum pH values.

Capillary electrophoresis in clinical chemistry.

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.

Involvement of Autoimmunity in the Pathogenesis of Aggressive Periodontitis

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.

Rapid assay for hard tissue collagen cross-links using isocratic ion-pair reversed-phase liquid chromatography.

Following a detailed study, a rapid and sensitive assay for the naturally fluorescent collagen cross-links pyridinoline and deoxypyridinoline has been developed using ion-pair reversed-phase high-performance liquid chromatography in the presence of 1-octanesulphonic acid (OSA). Pyridinoline and deoxypyridinoline were separated on an Exsil 100 ODS, 5-microns column (100 mm X 4.6 mm I.D.) using 25 mM sodium formate, 5 mM OSA and 1 mM ethylenediaminetetraacetic acid adjusted to pH 3.25, containing 20% (v/v) methanol. The mobile phase flow-rate was 1.5 ml/min. Compounds were detected by their natural fluorescence (xenon lamp; excitation wavelength 290 nm, emission wavelength 400 nm). Peak areas were linear to 25 pmol injected for pyridinoline and 20 pmol injected for deoxypyridinoline (r = 0.99). Intra-assay coefficients of variation for urinary extracts were 7.65 and 9.07% (n = 10), respectively. Limit of detection (signal-to-noise ratio = 5) was 200 fmol injected. Quantification of the cross-links in acid hydrolysates and human urine samples was possible in under 15 min.

Studies on d-penicillamine metabolism in cystinuria and rheumatoid arthritis: Isolation of S-methyl-D-penicillamine

Abstract An unknown metabolite of D -penicillamine found in the urine of patients receiving the drug has been isolated by ion-exchange chromatography. It was identified as S-methyl- D -penicillamine by mass spectrometry and its structure was confirmed by synthesis. In subjects receiving D -penicillamine for treatment of cystinuria and rheumatoid arthritis 4 and 8 per cent respectively of the dose was methylated. The total percentage of the D -penicillamine dose excreted (as cysteine-penicillamine plus penicillamine disulphide plus S-methyl- D -penicillamine) was 40 and 34 per cent in cystinuria and rheumatoid arthritis respectively. The findings in two cases of Wilsons Disease were similar to those in rheumatoid arthritis. In studies on four cystinuria patients an average of 12 per cent of the administered D -penicillamine was recovered in the faeces mainly as penicillamine disulphide. However, only between 42 and 53 per cent of the dose was identified in the urine and faeces, so that approximately 50 per cent of the administered drug was unaccounted for. D -Penicillamine caused a 32 per cent reduction in the urinary excretion of cysteine residues in cystinuria but a 400 per cent increase in their excretion in rheumatoid arthritis.

Rapid determination of drugs in biofluids by capillary electrophoresis. Measurement of antipyrine in saliva for pharmacokinetic studies

A micellar electrokinetic capillary chromatography method was developed that permitted the resolution of antipyrine from endogenous compounds and its quantitation in neat saliva in as little as 1 min. Final conditions were: SpectraPhoresis 1000, 30(23) cm x 50 microns silica capillary, 50 mM sodium phosphate pH 9.6, 50 mM SDS, 10 s hydrodynamic load, detection scanning 200-300 nm or 260 nm, run 25 kV. To overcome the effects of Joule heating the capillary was cooled to 15 degrees C. Sensitivity was < 10 microM and linearity extended to 350 microM. Comparison with an HPLC assay demonstrated that hydrodynamic injection gave a loading bias unless samples and standards were of equal viscosity. For 75 samples from five subjects the correlation of CE vs. HPLC was then r = 0.99.

Simultaneous analysis of nitrite, nitrate and the nicotinamide nucleotides by capillary electrophoresis: Application to biochemical studies and human extracellular fluids

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.