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P N Shaw

Publications and source records attributed to P N Shaw.

At least 19 recordsLinked to original sources
PubMed2006

Shortcomings of protein removal prior to high performance liquid chromatographic analysis-A case study using method development for BAY 11-7082.

During the analytical method development for BAY 11-7082 ((E)-3-[4-methylphenylsulfonyl]-2-propenenitrile), using HPLC-MS-MS and HPLC-UV, we observed that the protein removal process (both ultrafiltration and precipitation method using organic solvents) prior to HPLC brought about a significant reduction in the concentration of this compound. The use of a structurally similar internal standard, BAY 11-7085 ((E)-3-[4-t-butylphenylsulfonyl]-2-propenenitrile), was not effective in compensating for the loss of analyte as the extent of reduction was different to that of the analyte. We present here a systematic investigation of this problem and a new validated method for the determination of BAY 11-7082.

Chromatography, High Pressure Liquid↗
PubMed2006

Preparation of immuno-stimulating complexes (ISCOMs) by ether injection.

This study investigated the application of the solvent dispersion technique, specifically ether injection, which has been successfully used in the preparation of liposomes, as a new, continuous and potentially scaleable method for the preparation of ISCOMs. Phosphatidylcholine (PC) and cholesterol (Chol) were dissolved in ether, which was injected into an aqueous solution, maintained at 55 degrees C, containing Quil A. The influences of the following variables on ISCOM formation were investigated: ratio of PC:Quil A:Chol used, pumping rate, total lipid mass and concentration of buffer salts and Quil A in the aqueous phase. All samples were characterized by negative stain transmission electron microscopy, photon correlation spectroscopy and sucrose ultracentrifugation gradient. It was demonstrated that ISCOMs could be produced by this method but the homogeneity of the preparation was influenced by the conditions used. Homogeneous ISCOM preparations were consistently produced only when the weight ratio of PC:Quil A:Chol was 5:3:2 with a total lipid mass of 20 mg, the Quil A dissolved in a 0.01 M phosphate buffer at a concentration of 6 mg in 4 ml, and the ether solution injected into the warmed buffer solution at a rate of 0.2 ml/min. Changing any of these variables resulted in more heterogeneous preparations in which ISCOMs typically co-existed with other colloidal structures such as worm-like and helical micelles, liposomes, lamellae and lipidic particles.

Adjuvants, Immunologic↗
PubMed2005

Enhancing the ratio of molecular ions to non-covalent compounds in the electrospray interface of LC-MS in quantitative analysis.

A common problem encountered during the development of MS methods for the quantitation of small organic molecules by LC-MS is the formation of non-covalently bound species or adducts in the electrospray interface. Often the population of the molecular ion is insignificant compared to those of all other forms of the analyte produced in the electrospray, making it difficult to obtain the sensitivity required for accurate quantitation. We have investigated the effects of the following variables: orifice potential, nebulizer gas flow, temperature, solvent composition and the sample pH on the relative distributions of ions of the types MH+, MNa+, MNH4+, and 2MNa+, where M represents a small organic molecule: BAY 11-7082 ((E)-3-[4-methylphenylsulfonyl]-2-propenenitrile). Orifice potential, solvent composition and the sample pH had the greatest influence on the relative distributions of these ions, making these parameters the most useful for optimizing methods for the quantitation of small molecules.

Chromatography, High Pressure Liquid↗
PubMed2004

Selectivity of 7-alkoxycoumarins as probe substrates for rat hepatic cytochrome P450 forms is influenced by the substitution pattern on the coumarin nucleus.

1. The O-dealkylation of 7-alkoxycoumarins is widely used as an assay to characterize cytochrome P450 (CYP) activity. These substrates can also undergo oxidative attack at additional sites on the coumarin nucleus, which may influence their apparent selectivity for particular CYP forms. 2. Accordingly, the effect of blockade of these additional sites was investigated on the selectivity towards rat hepatic CYP forms, with emphasis on the CYP1A and 2B forms. 3. Blockade of the 3-/4- and 6-positions resulted in substrates for which the CYP1A1/2 selectivity of the unsubstituted 7-alkoxycoumarins was altered to a CYP2B selectivity; this was achieved with little overall change in the molecular dimensions of the substrate. Limited analysis of other inducible CYP forms indicated at most only small effects of structure modification on activity. 4. The findings suggest that the sensitivity of probe substrates for CYP forms may be limited by the occurrence of competing side reactions of the substrate, and that better probes may be derived by blocking the sites of these side reactions.

7-Alkoxycoumarin O-Dealkylase↗
PubMed2003

Quantification of clarithromycin, its 14-hydroxy and decladinose metabolites in rat plasma, gastric juice and gastric tissue using high-performance liquid chromatography with electrochemical detection.

A rapid, selective and sensitive HPLC assay has been developed for the simultaneous analysis of clarithromycin, its 14-hydroxy-clarithromycin metabolite, and its decladinose acid degradation product, in small volumes of rat gastric juice aspirate, plasma and gastric tissue. Sample were extracted with n-hexane/2-butanol (4:1) and the internal standard was roxithromycin. A Kromasil ODS 5 micrometer(75x4.6 mm I.D.) column was used with a mobile phase consisting of acetonitrile/aqueous phosphate buffer (pH 7, 0.086 M) (45:55 v/v). The column temperature was 30 degrees C and coulometric detection was used at 850 mV using a screen voltage of 600 mV. The analysis time was less than 8 min. The limits of quantitation for clarithromycin, 14-OH clarithromycin and decladinose clarithromycin were 0.15 microgram ml(-1) or lower in plasma (0.05 ml); 0.16 microgram ml(-1) or lower in gastric juice (0.2 ml); and 0.51 microgram g(-1) or lower for gastric tissue (0.25 g). The method was linear up to at least 20.3, 15.4 and 12.5 microgram ml(-1) for clarithromycin, 14-OH-clarithromycin and decladinose, respectively, in gastric juice aspirate and plasma and up to 40.6, 30.9 and 25.0 microgram g(-1) in gastric tissue. The assay was applied to the measurement of clarithromycin, 14-OH-clarithromycin and, for the first time, decladinose clarithromycin in pharmacokinetic studies of gastric transfer of clarithromycin in individual rats.

Animals↗
PubMed2002

Measurement of amoxicillin in plasma and gastric samples using high-performance liquid chromatography with fluorimetric detection.

A rapid, selective and sensitive HPLC assay has been developed for the routine analysis of amoxicillin in rat plasma, gastric juice aspirate and gastric tissue which is applicable to low concentrations of amoxicillin (<1 microg mL(-1)) or small sample volumes. Amoxicillin was converted, via an internal rearrangement, to form a fluorescent product which was subsequently recovered using liquid-liquid extraction. A Kromasil ODS 3 microm (150 x 3.2 mm I.D.) column was maintained at 40 degrees C and used with a mobile phase consisting of methanol-water (55:45, v/v). Fluorimetric detection was at an lambda(ex) of 365 nm and an lambda(em) of 445 nm. The limits of quantitation for amoxicillin were 0.1 microg mL(-1) for gastric juice aspirate (500 microL), 0.5 microg mL(-1) for plasma (50 microL) and 0.075 microg g(-1) for gastric tissue (250 mg). The method was linear up to at least 15 microg mL(-1) in gastric juice aspirate, up to 200 microg mL(-1) in plasma and up to 100 microg g(-1) in gastric tissue, with inter- and intra-day RSDs being less than 19%. The assay has been applied to the measurement of amoxicillin in rat plasma, gastric juice aspirate and gastric tissue for pharmacokinetic studies in individual rats.

Amoxicillin↗
PubMed2002

Impact of acid secretion, gastritis, and mucus thickness on gastric transfer of antibiotics in rats.

BACKGROUND AND AIMS: The success of Helicobacter pylori eradication regimens depends on gastric pH, inflammation, and mucus thickness. Our aim was to investigate the effects of acid secretion, inflammation, and mucolysis on gastric antibiotic transfer. SUBJECTS AND METHODS: A total of 134 anaesthetised rats were given metronidazole, amoxicillin, or clarithromycin intravenously and gastric contents were aspirated via an indwelling cannula. Acid secretion was controlled by either omeprazole or pentagastrin while gastritis was induced by infection with H pylori or dosing with iodoacetamide. Mucolysis was achieved by instilling pronase into the gastric lumen. RESULTS: Metronidazole transfer increased with acid secretion and fell with omeprazole, independently of gastric pH. Clarithromycin was also transferred with acid but was then rapidly degraded. Omeprazole prevented this degradation, raising gastric luminal concentrations. Omeprazole did not alter amoxicillin transfer. Gastritis induced by H pylori did not alter gastric transfer of metronidazole and amoxicillin but that of clarithromycin was increased by 23%. However, gastritis induced by iodoacetamide reduced clarithromycin transfer without any effect on metronidazole or amoxicillin transfer. Pronase treatment increased amoxicillin transfer fourfold and metronidazole by 66% but reduced clarithromycin transfer by 35%. CONCLUSIONS: Metronidazole and clarithromycin are predominantly transferred with gastric acid rather than by an acid trapping mechanism. Pronase increases the appearance of amoxicillin and metronidazole in gastric secretions.

Amoxicillin↗
PubMed2001

Resistance to nonspecific protein adsorption by poly(vinyl alcohol) thin films adsorbed to a poly(styrene) support matrix studied using surface plasmon resonance.

Thin films of poly(vinyl alcohol) (PVA) polymer were prepared on a flat, nonporous, poly(styrene) support matrix by adsorption from aqueous solution and were characterized in order to investigate the nonspecific adsorption of proteins to a chromatographically relevant surface. The integrity and surface coverage of the PVA thin films were established by surface analysis and atomic force microscopy imaging. The adsorption of the PVA polymers to the poly(styrene) substrate and the nonspecific adsorption of proteins to the PVA-coated surface were monitored using surface plasmon resonance. PVA was strongly bound to the poly(styrene) surface, but the surface density of the adsorbed PVA polymers was affected substantially by the concentration, molecular weight, and degree of hydrolysis of PVA polymers used. There was evidence of increasing degrees of unfolding of the PVA polymer onto the poly(styrene) surface as the concentration of the the PVA coating solution increased. Complete PVA coverage of the poly(styrene) surface was observed at PVA concentrations of 0.1 mg/mL or greater but with significant influence of both molecular weight and degree of hydrolysis of the PVA polymers. Resistance of the PVA-coated poly(styrene) surface to the nonspecific adsorption of human serum albumin (HSA) correlated with the degree of surface coverage of the PVA. The use of anti-HSA as a probe for adsorbed HSA suggested that HSA was displacing PVA from the poly(styrene) surface at the lower PVA surface coverage. A complete barrier to nonspecific protein adsorption was observed with a PVA coating solution concentration of greater than 0.1 mg/ mL with a degree of hydrolysis of <88%.

Adsorption↗
PubMed2001

Quantification of metronidazole in small-volume biological samples using narrow-bore high-performance liquid chromatography.

A rapid, selective and sensitive HPLC assay has been developed for the routine analysis of metronidazole in small volumes of rat plasma, gastric aspirate and gastric tissue. The extraction procedure involves liquid-liquid extraction and a protein precipitation step. A microbore Hypersil ODS 3 microm (150 x 2.1 mm I.D.) column was used with a mobile phase consisting of acetonitrile-aqueous 0.05 M potassium phosphate buffer (pH 7) containing 0.1% triethylamine (10:90). The column temperature was at 25 degrees C and the detection was by UV absorbance at 317 nm. The limit of detection was 0.015 microg ml(-1) for gastric juice aspirate and plasma and 0.010 microg g(-1) for gastric tissue (equivalent to 0.75 ng on-column). The method was linear up to a concentration of 200 microg ml(-1) for plasma and gastric juice aspirate and up to 40 microg g(-1) for tissue, with inter- and intra-day relative standard deviations less than 14%. The measured recovery was at least 78% in all sample matrices. The method proved robust and reliable when applied to the measurement of metronidazole in rat plasma, gastric juice aspirate and gastric tissue for pharmacokinetic studies in individual rats.

Animals↗
PubMed2001

Effects of acid treatment on the trace metal content of chromatographic silica: bulk analysis, surface analysis and chromatographic performance of bonded phases.

A series of studies has been carried out on the effect of refluxing silica chromatography particles for 0.5 h and 18 h in water, dilute hydrochloric acid and dilute hydrofluoric acid. The bulk and surface trace metal concentrations were measured by inductively-coupled plasma atomic emission spectroscopy, static secondary ion mass spectrometry (SSIMS) and X-ray photoelectron spectroscopy. Diffuse reflectance Fourier transform infrared spectroscopy was used to determine changes in 'isolated" and "bonded" silanol groups. The chromatographic behaviour of a series of weakly basic analytes was investigated on C8 and C18 bonded phases manufactured from the acid-treated silicas. The different reflux treatments all resulted in a reduction in the numbers of isolated silanols compared with the untreated silica and SSIMS analysis suggested that the HF-treated silicas had undergone a more efficient surface rehydroxylation. Bulk trace metals were removed most effectively by the HF treatment, with the multivalent elements (Ti and Al) being the most difficult to remove. Surface specific analysis suggested that trace metals were removed more rapidly from the surface of the silica compared to the bulk matrix and that the acid treatments resulted in halide contamination of the silica surface. Evidence is presented to suggest that the bulk metal content of the silica is not representative of the concentration of metals at the chromatographic surface. The chromatographic investigations showed that the HF-treated silica gave substantially better performance towards weak bases than the HCl-treated silicas.

Acids↗
PubMed2001

Electrophoretic behaviour of oligonucleotides and mono-, di- and triphosphate nucleotides by capillary zone electrophoresis.

A systematic investigation has been made into the mechanisms of the capillary zone electrophoresis (CZE) separation of 12 common nucleotides (mono-, di- and triphosphorylated) and polydeoxythymidylic acid oligonucleotides (pd(T)5-18) using electrophoretic mobility values calculated from migration time data. Relationships between electrophoretic mobility and the physicochemical characteristics of the analytes (charge, dissociation constants, charge-to-mass ratio) and the background electrolyte conditions (buffer strength, percentage organic modifier and buffer pH) were characterised. Nucleotide migration was dominated by the negatively charged phosphate groups. Additionally, there were important contributions to migration behaviour from the ionised amide groups of the nucleobases guanine and uracil at higher buffer pH values or with the presence of methanol in the electrolyte. Calculated electrophoretic mobility values for the nucleotides showed a substantially improved (5-fold) inter-run repeatability compared with migration time data. These studies show the value of representing nucleotide migration data as electrophoretic mobility in CZE for obtaining a more thorough analysis of separation mechanisms and to compensate for variation in migration time data caused by small changes in electrosmotic flow. Oligonucleotides pd(T)5-11 could be adequately resolved from their nearest neighbour, but the limit of single-base separation was pd(T)10 from pd(T)11 under the conditions used. It was calculated that a difference in charge-to-mass ratio of 2.64 x 10(-5) was required for resolution under the CZE conditions used.

Adenine Nucleotides↗
PubMed2001

Systemic levels of glyceryl trinitrate following topical application to the anoderm do not correlate with the measured reduction in anal pressure.

BACKGROUND: Topical 0.2 per cent glyceryl trinitrate (GTN) lowers resting anal pressure (RAP) and heals two-thirds of chronic anal fissures. Over 50 per cent of patients experience headache, presumably through systemic absorption, but the pharmacokinetics of GTN ointment are unknown. This study evaluated the systemic absorption profile of GTN, and correlation between plasma GTN levels, RAP, haemodynamic variables and side-effects. METHODS: Thirty healthy volunteers were recruited with local medical school ethics committee approval. Continuous static anal manometry was performed for 10 min before and 2 h after application of 0.2 per cent GTN (0.5 g) to the anoderm. Blood samples were taken from an intravenous cannula, and pulse and blood pressure were measured before application of GTN, and 10, 20, 30, 40, 50, 60, 90, 120 and 180 min thereafter. Details of side-effects were recorded. RESULTS: GTN was detected in the plasma 10 min to 3 h after topical application. RAP was significantly reduced after 10 min, but had returned to pretreatment values by 120 min. Pulse was statistically unchanged during the study; systolic blood pressure was significantly lower 20-90 min after GTN application, and diastolic pressure was decreased throughout the study. Headaches were experienced by 14 of 30 volunteers after a median (range) of 41 (4-120) min, persisting for 74 (30-176) min, with an intensity score of 19 (5-30) mm represented on a 100-mm linear visual analogue scale. There was no correlation between plasma GTN concentration, RAP, and the onset, duration or intensity of headaches. CONCLUSION: Topical GTN acts locally on the internal anal sphincter; systemic levels do not contribute significantly to the reduction in RAP. There is marked interindividual variation in plasma GTN levels, and no correlation with haemodynamic variables and side-effects.

Absorption↗
PubMed1999

Antitumor benzothiazoles. 7. Synthesis of 2-(4-acylaminophenyl)benzothiazoles and investigations into the role of acetylation in the antitumor activities of the parent amines.

2-(4-Aminophenyl)benzothiazoles display potent and selective antitumor activity against inter alia breast, ovarian, colon, and renal cell lines, but their mechanism of action, though yet to be defined, may be novel. Metabolism is suspected to play a central role in the mode of action of these benzothiazoles since drug uptake and biotransformation were observed in sensitive cell lines (e.g., breast MCF-7 and MDA 468 cells) in vitro, whereas insensitive cell lines (e.g., prostate PC 3 cells) showed negligible uptake and biotransformation. N-Acyl derivatives of the arylamines have been synthesized, and in vitro studies confirm N-acetylation and oxidation as the main metabolic transformations of 2-(4-aminophenyl)benzothiazoles, with the predominant process being dictated by the nature of the 3'-substituent. The prototype amine 3 underwent mainly N-acetylation in vitro, while 3'-substituted analogues 4 and 5 were primarily oxidized. N-Acetylation of 4 to 11 exerts a drastic dyschemotherapeutic effect in vitro, but acetylation of the halogeno congeners 5-7 gave acetylamines 12-14 which substantially retain selective antitumor activity. In vivo pharmacokinetic studies in rats confirmed rapid and exclusive N-acetylation of the 3'-methyl analogue 4, but less acetylation with the 3'-chloro analogue 5. Distinct expression patterns of N-acetyltransferase NAT1 and NAT2 have been demonstrated in our panel of cell lines.

Acetylation↗
PubMed1998

Reversed-phase high-performance liquid chromatographic assay methods for the analysis of a range of penicillins in in vitro permeation studies.

Simple, rapid and selective HPLC methods have been developed for the analysis of amoxycillin, ampicillin, epicillin, mecillinam and propicillin. A Hypersil ODS 5 microm (150x4.6 mm I.D.) column was used with mobile phases containing aqueous phosphate buffers, pH 3-4.6 and either methanol or acetonitrile as the organic modifier. Samples were detected by their optimal UV absorption (210-230 nm). The lower limits of quantitation of the compounds (100 microl injection volume) were 0.1 microg/ml. The assays were linear in the range of 0.1-100 microg/ml with r2 values greater than 0.99. The methods have been applied successfully for the measurement of the flux of the compounds across Caco-2 cells monolayers.

Caco-2 Cells↗
PubMed1998

The effect of protein binding and lipophilicity of penicillins on their in-vitro flux across gastric mucosa.

Delivery of amoxycillin across the human gastric mucosa to Helicobacter pylori is poor compared with that of metronidazole and clarithromycin, limiting the clinical effectiveness of this penicillin. To investigate the physicochemical properties of penicillins that influence their flux across gastric mucosa, the fluxes of metronidazole and eight penicillins were measured in vitro across rat gastric mucosa. The lipophilicity of these drugs was also measured using potentiometric titration. The mean fluxes of monobasic penicillins (range 0.66-0.89 nmol/cm2/h) were significantly lower than those of the aminopenicillins (range 1.94-2.80 nmol/cm2/h) (P < 0.005). Penicillin flux was not significantly correlated with lipophilicity as measured, but was significantly correlated with published protein binding data (rs = 0.9048, P < 0.002). Metronidazole flux was significantly higher than that of any penicillin at 22.6 (+/-0.9) nmol/cm2/h (P < 0.001). Therefore, the in-vitro gastric delivery of penicillins can be predicted from protein binding which may in turn predict activity against H. pylori in vivo.

Animals↗
PubMed1997

The effect of omeprazole on the pharmacokinetics of metronidazole and hydroxymetronidazole in human plasma, saliva and gastric juice.

AIMS: To evaluate the effect of omeprazole on the pharmacokinetics of metronidazole and hydroxymetronidazole in plasma, gastric juice and saliva following intravenous infusion or oral dosing of metronidazole. METHODS: Eight volunteers received single doses of metronidazole (400 mg) intravenously and orally, whilst taking placebo or omeprazole (40 mg, twice daily for 5 days) in a randomized 4-way crossover study. Metronidazole and hydroxymetronidazole concentrations in plasma, saliva and gastric juice samples were determined by h.p.l.c. Pharmacokinetic parameters for metronidazole and hydroxymetronidazole were calculated, and the significance of the mean differences in parameters between omeprazole and placebo co-administration was assessed using a two-tailed, paired t-test. RESULTS: There were no significant differences (P < 0.05) in any of the plasma or saliva pharmacokinetic parameter values for metronidazole between volunteers receiving omeprazole or placebo when metronidazole was administered either as an intravenous infusion or orally. Following intravenous administration of metronidazole to the placebo group and omeprazole treated group respectively, the gastric transfer of metronidazole was significantly reduced from 15.5 +/- 10.4% to 2.6 +/- 1.0% of the dose (P = 0.007; 95% CI of difference 4.8 to 21.0) with concomitant changes in the metronidazole AUC (from 77.5 +/- 18.0 mumol l-1 h to 352.6 +/- 182.1 mumol l-1 h; P = 0.0003; 95% CI of difference 127.6 to 422.7), Cmax (from 61.4 +/- 26.5 mumol l-1 to 271.8 +/- 104.3 mumol l-1; p = 0.0001; 95% CI of difference 118.6 to 302.1). Similarly, the gastric juice AUC of hydroxymetronidazole was significantly reduced from 3.2 +/- 1.9 mumol l-1 h to 1.5 +/- 0.8 mumol l-1 h of the dose (P = 0.0043; 95% CI of difference 0.4 to 3.0) with a concomitant change in Cmax (from 5.0 +/- 2.5 mumol l-1 to 3.0 +/- 1.2 mumol l-1; P = 0.0007; 95% CI of difference 0.7 to 3.4). CONCLUSIONS: Omeprazole had little effect on the plasma and salivary pharmacokinetics of metronidazole (or its hydroxymetabolite) after intravenous or oral administration, but it did have a substantial effect on the pharmacokinetics of metronidazole and hydroxymetronidazole in gastric juice.

Adult↗
PubMed1997

The stability of amoxycillin, clarithromycin and metronidazole in gastric juice: relevance to the treatment of Helicobacter pylori infection.

Although omeprazole is an important component in anti-Helicobacter pylori therapeutic regimes using clarithromycin, amoxycillin and metronidazole, the mechanism by which it enhances antimicrobial action is unknown. One potential explanation for this effect is increased antibiotic chemical stability resulting from gastric pH changes induced by co-administration of omeprazole. The chemical stability of clarithromycin, amoxycillin and metronidazole was investigated in aqueous solutions and in human gastric juice collected before and after a 7-day course of omeprazole. Amoxycillin, clarithromycin and metronidazole were prepared in buffered aqueous solutions of pH 1.0 to 8.0 and in gastric juice of pH 2.0 and 7.0. The gastric juice samples were obtained from fasted H. pylori-negative volunteers before and after they had received a 7-day course of omeprazole. All the samples were incubated at 37 degrees C and analysed at intervals by HPLC. Amoxycillin, clarithromycin and metronidazole were stable in aqueous solutions of pH 4.0-7.0, pH 5.0-8.0 and pH 2.0-7.0, respectively. At pH 2.0, the degradation half-lives were 19.0 +/- 0.2 h, 1.3 +/- 0.05 h and 2200 +/- 1100 h, respectively. In gastric juice samples of pH 2.0, the degradation half-lives were 15.2 +/- 0.3 h, 1.0 +/- 0.04 h and > or = 800 h, respectively. The half-lives of the drugs in the gastric juice samples of pH 7.0 were all > 68 h. The co-administration of omeprazole with amoxycillin or clarithromycin is likely to increase the chemical stability of amoxycillin and clarithromycin in gastric juice. Clarithromycin degrades rapidly at normal gastric pH (1.0-2.0) but amoxycillin and metronidazole are sufficiently stable at this pH to maintain an antibacterial concentration in the stomach.

Amoxicillin↗