Biomedical subjects

D M Goodall

Publications and source records attributed to D M Goodall.

At least 19 recordsLinked to original sources

PubMed2005

Differential glycosylation of polyclonal IgG, IgG-Fc and IgG-Fab isolated from the sera of patients with ANCA-associated systemic vasculitis.

Post-translational modifications (PTMs) of proteins produced in vivo may be tissue, developmentally and/or disease specific. PTMs impact on the stability and function of proteins and offer a challenge to the commercial production of protein biotherapeutics. We have previously reported a marked deficit in galactosylation of oligosaccharides released from polyclonal IgG isolated from sera of patients with the anti-neutrophil cytoplasmic antibodies (ANCA) associated vasculitides; Wegener's granulomatosis (WG) and microscopic polyangiitis (MPA). Whilst normal polyclonal IgG molecules are glycosylated within the IgG-Fc region, approximately 20% of molecules also bear oligosaccharides attached to the variable regions of the light or heavy chain IgG-Fab. It is of interest, therefore to compare profiles of oligosaccharides released from the IgG-Fc and IgG-Fab of normal IgG with that isolated from the sera of patients with WG or MPA. This study shows that whilst the oligosaccharides released from ANCA IgG-Fc are hypogalactosylated those released from IgG-Fab are galactosylated and sialylated. These results show that hypogalactosylation of IgG-Fc is not due to a defect in the glycosylation or processing machinery. It rather suggests a subtle change in IgG-Fc conformation that influences the addition of galactose. Remarkably, this influence is exerted on all plasma cells. Interestingly, a licensed monoclonal antibody therapeutic, produced in Sp2/0 cells, is also shown to be hypogalactosylated in its IgG-Fc but fully galactosylated in its IgG-Fab.

Antibodies, Antineutrophil Cytoplasmic↗

PubMed2004

Evaluation of large volume-difficult matrix introduction-gas chromatography-time of flight-mass spectrometry (LV-DMI-GC-TOF-MS) for the determination of pesticides in fruit-based baby foods.

The European Union Baby Food Directive (1999/39/EC), which came into force on 1 July 2002, set legal maximum residue levels at 0.01 mg kg(-1) for all pesticides in baby foods. The combination of large volume-difficult matrix introduction (LV-DMI) with gas chromatography-time of flight-mass spectrometry (GC-TOF-MS), described herein, provides the analyst with a simple but rapid alternative GC-MS technique for the multiresidue analysis of pesticides in fruit-based baby foods. Samples were extracted with ethyl acetate in the presence of Na2SO4 and NaHCO3 and the crude extracts were analysed directly using LV-DMI-GC-TOF-MS. The best overall results (98 pesticides quantified satisfactorily at a spiking level of 0.01 mg kg(-1)) were obtained by analysis of concentrated extracts (2.5 g crop ml(-1)) using a 30-m column, with a chromatographic run time of 25 min. A good signal-to-noise ratio was obtained at the lowest calibrated level (0.0125 microg ml(-1)), with excellent linearity achieved over the range 0.0125-0.25 microg ml(-1) (equivalent to 0.005-0.1 mg kg(-1)). Average recoveries for the analysis of five replicate determinations at a spiking level of 0.01 mg kg(-1) were between 79 and 114% with relative standard derivations generally less than 20%.

Calibration↗

PubMed2001

High-performance liquid chromatography applications of optical rotation detection with compensation for scattering and absorbance at the laser wavelength.

Use of instrumentation developed to enable simultaneous monitoring of optical rotation (OR) and transmittance allows OR measurements to be made in the presence of high levels of absorbance, scattering or other effects that change the intensity of the plane-polarised light at the photodiode detector. This extends the application of OR detection to areas where it was previously difficult. Examples of the application of high-performance liquid chromatography (HPLC) with the improved OR detector include (i) the analytical scale separation of fructose and sucrose and (ii) the semi-preparative separation of enantiomers of warfarin and Trögers base. A signal-to-noise improvement of up to 150% is found when comparing signals with and without correction for transmittance changes. The improved OR detector has been used in series with a UV detector and the system shown to be suitable for on-line measurement of peak purity in separations using a chiral column under overload conditions.

Chromatography, High Pressure Liquid↗

PubMed2001

Predicting peak shape in capillary zone electrophoresis: a generic approach to parametrizing peaks using the Haarhoff-Van der Linde (HVL) function.

We have found that the Haarhoff-Van der Linde (HVL) peak function provides excellent fitting to the shapes of CZE peaks. Initially designed for overloaded peaks in gas chromatography, this function describes a Gaussian peak when there is no peak distortion, and a triangular peak when there is no diffusional peak broadening. As such, it is ideal for CZE peaks distorted by electromigration dispersion (EMD). Fitting peaks with this function gives four parameters: three of them can be related to the Gaussian peak that would have been obtained in case of no EMD; the last one is a measure of the peak distortion. Using moving boundary theory, this peak distortion parameter may readily be expressed in terms of analyte and background electrolyte mobilities and concentrations, electric field, and sample injection length. The variance of an HVL peak is shown to be described by a universal function, and a master equation is presented. The region where EMD adds less than 10% to the Gaussian variance is shown to be very narrowly spread around the mobility matching condition. Under typical CZE operating conditions with an analyte at 1% of the BGE concentration, significant peak distortion is always present. Because the total peak variance is not an addition of the Gaussian and triangular contributions, the HVL model and the methodology introduced here should always be used to correctly combine variances.

Journal Article↗

PubMed2001

Trace analysis of gamma-cyclodextrin in a sample of beta-cyclodextrin by capillary electrophoresis.

A new capillary electrophoretic method for trace analysis of gamma-cyclodextrin, gamma-CD, in a sample of beta-CD has been developed, building on our recent work in which the tetraphenylborate ion, Ph4B-, was found to bind to gamma-CD three orders of magnitude more strongly than to beta-CD. The method involves measurement of the change of net electrophoretic mobility of Ph4B- and its CD complexes in a background electrolyte containing a fixed concentration of beta-CD. Good linearity was found between 1/deltamu and 1/Cgamma where deltamu is the difference in the mobility of Ph4B- in the beta-CD solution at a given and at zero concentration of gamma-CD, and Cgamma the gamma-CD concentration. The limit of detection for gamma-CD in a beta-CD sample was found to be 0.020% (w/w), and high precision and accuracy were obtained.

Calibration↗

PubMed2001

Comparison of binding of tetraphenylborate and tetraphenylphosphonium ions to cyclodextrins studied by capillary electrophoresis.

Binding constants for tetraphenylborate and tetraphenylphosphonium ions (Ph4B- and Ph4P+) to cyclodextrins (CDs) to give 1:1 host-guest complexes have been measured using capillary electrophoresis. Mobilities of the ions as a function of gamma-CD concentration give binding constants, K, of 1.08 x 10(5) M-1 for Ph4B- and 0.6 x 10(1) M-1 for Ph4P+. This dramatic difference of four orders of magnitude in binding constants is not seen with beta-CD (K = 7.7 x 10(1) M-1 for Ph4B- and 3.7 x 10(1) M-1 for Ph4P+) or dimethyl (DM)-beta-CD (K = 46 x 10(1) M-1 for Ph4B-1 and 7.7 x 10(1) M-1 for Ph4P+). The crystal and hydrodynamic radii of the ions, the latter calculated from their absolute mobilities, indicate that Ph4B- is smaller than the gamma-CD cavity, whereas Ph4P+ is approximately the gamma-CD cavity size. Results suggest that Ph4B- fits exactly into a gamma-CD cavity, with hydrophobic contacts involving several of the phenyl rings, whereas Ph4P+ is too large to enable these multiple contacts to be made. When only a single phenyl ring can fit into the CD cavity, binding strengths are in the order DM-beta-CD > beta-CD > gamma-CD and Ph4B- > Ph4P+. Measurement of electrophoretic mobilities of the complexes shows that their hydrodynamic radii are in the order gamma-CD < beta-CD approximately DM-beta-CD for Ph4B- and gamma-CD > beta-CD approximately DM-beta-CD for Ph4P+.

Cyclodextrins↗

PubMed2001

Separation of iota-, kappa and lambda-carrageenans by capillary electrophoresis.

The present study reports a novel method for the separation of the high-molecular-weight anionic polysaccharides, iota, kappa, and lambda carrageenans, in capillary electrophoresis (CE). Carrageenan samples are first derivatised with 9-aminopyrene-1,4,6-trisulfonic acid (APTS), separated in an ammonium acetate background electrolyte (BGE) and detected with laser-induced fluorescence (LIF). The effects of changes of instrumental parameters (temperature, injection mode, field strength) and the composition of the BGE (concentration and pH) are reported, and are explained in terms of the physical chemistry of the BGE and the biopolymers. Optimal separation conditions for kappa, iota, and lambda carrageenans, including an APTS internal standard, were found in a polyvinyl alcohol coated capillary with an ammonium acetate BGE of low concentration (25 mM) and moderate pH (8.0). This BGE gave the best reproducibility in tests on iota/kappa mixtures, with relative standard deviations (RSDs) in migration times and normalised peak areas (relative to the APTS internal standard) of less than 0.1% and 1%, respectively. Using this BGE at 50 degrees C and a voltage of 30 kV, all three carrageenan subtypes were separated in a run time of 3 min.

Carrageenan↗

PubMed2001

Tandem mass spectrometric analysis of quaternary ammonium pesticides.

A detailed MS(n) study on an ion trap instrument of the quaternary ammonium pesticides paraquat, diquat, difenzoquat, mepiquat and chlormequat reveals a number of ions not reported previously, and has allowed examination of the fragmentation pathways. A number of transitions that are highly specific to each quat have been identified. Optimal ion trap operating conditions determined using Simplex optimisation can promote either detection of a particular fragmentation transition or a range of MS/MS product ions with a high overall signal response. Thus, fragmentation conditions were optimised to enhance the specificity or sensitivity of MS/MS methods.

Chromatography, Liquid↗

PubMed2001

Formation of gas-phase clusters monitored during electrospray mass spectrometry: a study of quaternary ammonium pesticides.

Gas-phase cluster formation between the quaternary ammonium pesticides paraquat, diquat, difenzoquat, chlormequat and mepiquat, and chloride and acetate anions present in a liquid chromatography (LC) mobile phase, has been studied using electrospray mass spectrometry. The clusters of paraquat, mepiquat and chlormequat were revealed over the entire m/z range of the mass spectrometer, and their formation is dependent on the concentrations of both the cationic and the anionic species. Mepiquat and chlormequat form clusters of the type [2M(q)(+) + A(-)](+), where M(q)(+) is the quaternary ammonium cation and A(-) is the anion. Paraquat forms a cluster species with ammonia and also an ion-pair complex with chloride anions. Diquat and difenzoquat did not form observable ion-pair complexes or clusters with any of the anions studied. Competitive binding of acetate and chloride anions reflects the higher charge density of chloride, which forms the dominant clusters with mepiquat and chlormequat. The formation of cluster species has implications for the quantification of quaternary ammonium pesticides and may have an influence on the linearity of calibrations.

Acetates↗

PubMed2001

Characterization of peptides formed during fermentation of cocoa bean.

Analysis by SDS-PAGE and GPC-MS of fermented cocoa extracts shows changes in the amount and composition of the major proteins, accompanied by formation of complex distributions of peptides. MS/MS studies and application of SEQUEST sequencing software have allowed identification of two related peptides, a hexapeptide and a nonapeptide, formed from vicilin, one of the cocoa storage proteins. Time course studies of the two peptides show different abundance profiles and indicate, in part, production of the hexapeptide from the nonapeptide.

Cacao↗

PubMed2000

Improved sensitivity in detection of chlormequat by liquid chromatography-mass spectrometry.

Two published separations, using electrospray mass spectrometry (ES-MS), exhibit significant differences in limits of detection (LODs) for chlormequat cation in pear. Separation on ODS1, confirmed to result from ion-exchange, gives shorter analysis times and calibration over a wider concentration range than on an SCX cation-exchange column. The superior LOD using ODS1 (0.04 ng ml(-1) vs. 1.0 ng ml(-1)) results mainly from better chromatographic peak shape. Separation on ODS1 combined with optimised ES-MS detection allows direct quantification of chlormequat on an ion trap instrument at levels lower than those required for residue analysis in foods and also in drinking water.

Chlormequat↗

PubMed2000

Optimisation of ion trap parameters for the quantification of chlormequat by liquid chromatography/mass spectrometry and the application in the analysis of pear extracts.

Optimisation of the activation parameters for ion trap mass spectrometric analysis of the chlormequat cation using simplex optimisation enabled the product ion (m/z 58) response to be improved 1000-fold. A comparison of the sensitivity of the optimised ion trap mass spectrometer with that of a triple quadrupole mass spectrometer for liquid chromatography/tandem mass spectrometry (LC/MS/MS) showed that similar limits of detection (LODs) could be achieved. For the MS/MS transition of the (35)Cl precursor to the most abundant product, LODs were 0.8 ng cation mL(-1) (0.004 mg cation kg(-1) pear equivalent) and 1.0 ng cation mL(-1) (0.005 mg cation kg(-1) pear equivalent) on the triple quadrupole and ion trap instrument, respectively.

Chlormequat↗

PubMed1997

Analysis of biological particles using dielectrophoresis and impedance measurement.

A novel electrical technique for detecting the collection of particles by dielectrophoresis (DEP) is described. The method is based on the impedance changes resulting from this collection in a microfabricated, integrated dual-channel electrode structure. The results show good agreement with measurements of DEP collection by optical method for suspensions of bacterial species B. subtilis, E. coli and abiotic latex beads, but with substantially reduced experimental uncertainties. The technique overcomes the restriction on particle size of optical techniques and can potentially be used to investigate highly sub-micron sized particles like viruses and DNA fragments. The dual-channel electrode cells can be integrated with other structures for analysing sub-micron scale particles, for example chip-based capillary electrophoresis.

Bacteria↗

PubMed1996

Self-consistent framework for standardising mobilities in free solution capillary electrophoresis: applications to oligoglycines and oligoalanines.

A theoretical analysis of deviations from ideality in ionic transport is presented to correct mobilities, mu, measured in free solution capillary electrophoresis (CE) to mobility at infinite dilution, mu degree (limiting mobility). Non-ideality is treated at the same level of approximation as in equilibrium, using a correction factor for the sum of the analyte and counter-ion radius originally suggested by Robinson and Stokes (Electrolyte Solutions, 1961). Unlike previous corrections using Debye-Hückel-Onsager theory, which are strictly applicable only at very low ionic strengths, this treatment is expected to be valid for univalent ions migrating in a uni-univalent background electrolyte for ionic strengths up to 0.075 mol kg-1, a range typical of CE experiments. The analysis is applied to the determination of mu degree in acidic and basic buffers for oligoalanines and oligoglycines with degree of polymerisation 2 to 6. Limiting mobilities for the fully protonated and deprotonated peptides are found to be numerically equal but opposite in sign, consistent with a change in charge from +1 to -1. In all uni-univalent buffers studied (borate, citrate, low pH lithium phosphate and sodium phosphate) mu degree values established using data over a range of pH and ionic strength are found to be identical and in excellent agreement with previous values from isotachophoresis. Values of mu degree in high pH sodium phosphate buffer are systematically 0.2.10(-8) m2 V-1 s-1 higher than those in other buffers; this may be attributed to limitations of the model for a buffer with 1+:2- and 1+:3- ions. This self-consistent framework for standardising mobilities in free solution CE is expected to be widely applicable to univalent analytes migrating in a 1:1 background electrolyte.

Chemical Phenomena↗

PubMed1994

Capillary electrophoresis with chiral selectors: optimization of separation and determination of thermodynamic parameters for binding of tioconazole enantiomers to cyclodextrins.

A systematic approach is outlined for optimization of enantiomeric separations in free solution capillary electrophoresis using chiral mobile-phase additives. Maximum electrophoretic mobility difference between the enantiomers occurs when the concentration of free selector is equal to the reciprocal of the average binding constant. General equations and data analysis methods are presented to relate mobilities to equilibrium constants in simple and competitive binding equilibria and used to determine thermodynamic parameters for host-guest complexation of tioconazole enantiomers with a range of cyclodextrin selectors. Selectivities are found to be in the reverse order of binding constants in the series dimethyl-beta-cyclodextrin (K1 = 6.9 x 10(3) M-1, alpha = 1.10) to hydroxypropyl-beta-cyclodextrin (K1 = 0.72 x 10(3) M-1, alpha = 1.29). For beta-cyclodextrin (K1 = 1.32 x 10(3)M-1, alpha = 1.20), delta H zero provides the dominant contribution to binding but delta delta H zero and T delta delta S zero terms give comparable contributions to the selectivity. Addition of alcohol does not affect the selectivity, but allows displacement of the optimum separation conditions to higher cyclodextrin concentration through either competitive binding (with cyclohexanol) or preferential solvation of reactants (with methanol).

Antifungal Agents↗

PubMed1994

Enantiomeric separation of salbutamol and related impurities using capillary electrophoresis.

The enantiomeric resolution of salbutamol and its chiral and achiral related impurities is investigated using capillary electrophoresis. The effects of nine varieties of cyclodextrin, cyclodextrin concentration and organic modifier concentration were studied in an attempt to resolve all possible analytes in a complex mixture of salbutamol-related solutes. Eleven components including three enantiomeric pairs were baseline resolved using 112 mM dimethyl-beta-cyclodextrin at pH 2.5 in a citric acid/phosphate buffer. Both methanol and iso-propyl alcohol at up to 20% were found to have a deleterious effect on the separation. Binding constants and mobility values for the free and complexed forms for each solute were determined. The results are interpreted by considering the physical properties of the molecules under the conditions employed and a rationale proposed for the underlying basis for chiral and achiral selectivity.

Albuterol↗

PubMed1994

Quantitative applications and validation of the resolution of enantiomers by capillary electrophoresis.

The separation and quantitation of enantiomers is an important application of capillary electrophoresis. This paper reviews the performance and validation of capillary electrophoretic methods for this application. Limits of detection of < 0.1% are shown for determinations of minor enantiomer levels. Application areas include enantiomeric purity testing of pharmaceuticals and herbicides, reaction rate monitoring, stability testing, and clinical analysis. It is concluded that the number of quantitative application areas will undoubtedly expand as capillary electrophoresis becomes more widely established as an alternative and complement to high-performance liquid chromatography.

Artifacts↗

PubMed1994

Two enzyme linked immunosorbent assays for detecting antibodies against meningococcal capsular polysaccharides A and C.

AIMS: To evaluate two of the recent methods of coating microtitre plates in the enzyme linked immunosorbent assay (ELISA) for detecting human antibodies against meningococcal capsular polysaccharides A and C with a view to validating a specific meningococcal antibody assay for routine clinical use. METHODS: Two four-layer ELISA protocols were standardised: one method utilised meningococcal polysaccharides conjugated to poly-L-lysine polypeptide for coating the microtitre plates; another used polysaccharides mixed with methylated human serum albumin (mHSA). Titration curves were plotted for the ELISAs and the squared Pearson correlation coefficient (R2) was used to determine the degree of accuracy of fit of the curves. Specificity tests were performed by inhibition and adsorption studies. RESULTS: Both methods gave good titration curves with a high R2 of > 0.98, indicating a high degree of accuracy in forming the curves. The titration end point after vaccination, obtained by the mHSA method, was 20 times higher, however, than that obtained by the poly-L-lysine method. Specificity tests showed that in the ELISA using polysaccharide/poly-L-lysine, antibody activity of a pre-vaccination serum sample was inhibited by 37%, and of post-vaccination serum by 50% with 1000-fold excess antigen. Antibody activity (post-vaccination) was reduced by 51% and 59%, respectively, by adsorption with antigen-coated Sepharose beads or adsorption with suspensions of killed meningococci. In contrast, antibody activity of a pre-vaccination serum was inhibited by 60% and a post-vaccination serum by 90% in ELISA employing polysaccharides mixed with mHSA. Reproducibility was better with the use of methylated human serum albumin than with poly-L-lysine; the former showed intrabatch and interbatch coefficients of variation of 4% and 2%, respectively, compared with 43% (intrabatch) and 16% (interbatch) obtained with the poly-L-lysine. CONCLUSION: It is concluded that the antibody assay using meningococcal polysaccharides groups A and C mixed with mHSA is much better than that using polysaccharides coupled with poly-L-lysine.

Antibodies, Bacterial↗