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Biomedical subjects

R F Venn

Publications and source records attributed to R F Venn.

At least 19 recordsLinked to original sources
PubMed2004

Molecularly imprinted polymers for the determination of a pharmaceutical development compound in plasma using 96-well MISPE technology.

The use of molecularly imprinted polymers (MIPs) as sorbents for the solid phase extraction (SPE) of a pharmaceutical compound in development, prior to quantitative analysis was investigated. Three MIPs were synthesised using a structural analogue as the template molecule. Each polymer was prepared with different monomers and porogens. The MIPs were then tested for their performance both in organic and aqueous environments, the final aim being to load plasma directly onto the polymers. At an early development stage, there is a limited amount of compound available. Due to this limitation, reducing the amount of template required for imprinting was investigated. A MIP capable of extracting the analyte directly from plasma was produced. The specificity of the polymer allowed the method to be validated at a lower sensitivity than a more conventional SPE assay. For the first time, MIPs were packed into 96-well blocks enabling high throughput analysis. The analytical method was fully validated for imprecision and inaccuracy down to 4 ng/ml in plasma.

Drug Design↗
PubMed2000

Fluorescence determination of sulphobutylether-beta-cyclodextrin in human plasma by size exclusion chromatography with inclusion complex formation.

A selective method for the determination of sulphobutylether-beta-cyclodextrin (SBECD) in human plasma has been developed and validated over the range 4-200 microg ml(-1). SBECD is extracted from plasma using end-capped cyclohexyl solid phase extraction cartridges. This is followed by high performance size exclusion chromatography with a mobile phase consisting of 1-naphthol (0.1 mM) in methanol-potassium nitrate (0.2 M) (1:9 v/v), 1 ml min(-1). The high aqueous content of the mobile phase quenches the fluorescence of 1-naphthol. However, the naphthol forms an inclusion complex with SBECD. The non-polar 'bucket' environment of the inclusion region restores the fluorescence, which is measured at excitation and emission wavelengths of 290 and 360 nm, respectively, when SBECD elutes from the column.

Chromatography, Gel↗
PubMed1998

Clinical analysis of sampatrilat, a combined renal endopeptidase and angiotensin-converting enzyme inhibitor I: assay in plasma of human volunteers by atmospheric-pressure ionisation mass-spectrometry following derivatisation with BF3-methanol.

Sampatrilat is a dual inhibitor of angiotensin converting enzyme (ACE) and neutral endopeptidase (NEP) under development for the treatment of hypertension and congestive heart failure. In order to support the early clinical development (with oral administration and an expected low bioavailability), a sensitive and selective assay was required. A method for plasma was developed and validated employing HPLC APCI MS-MS. The plasma samples were extracted on solid-phase extraction cartridges, derivatised with BF3-methanol, diluted, extracted again and then subjected to HPLC APCI-MS-MS. Derivatisation was necessary because the two carboxyl group in the molecule prevented efficient ionisation in the heated nebuliser source. The calibration range was from 0.5 to 20 ng ml(-1) and the lower limit of quantification was 0.5 ng ml(-1). Imprecision and inaccuracy were determined on three separate occasions at three concentrations (0.5, 5 and 20 ng ml[-1]) and shown to be lower than 10% in every case.

Angiotensin-Converting Enzyme Inhibitors↗
PubMed1998

Clinical analysis of sampatrilat, a combined renal endopeptidase and angiotensin-converting enzyme inhibitor II: assay in the plasma and urine of human volunteers by dissociation enhanced lanthanide fluorescence immunoassay (DELFIA).

Sampatrilat is a dual inhibitor of angiotensin converting enzyme (ACE) and neutral endopeptidase (NEP) under development for the treatment of hypertension and congestive heart failure. In order to support the early clinical development (with oral administration and an expected low bioavailability), a sensitive and selective assay was required. An HPLC-atmospheric-pressure chemical ionisation mass-spectrometric (HPLC-APCI-MS-MS) assay had been already validated (R.F. Venn et al., J. Pharm. Biomed. Anal., in press), but due to its low throughput an alternative method was sought. As the molecule is peptide-like and not metabolised, we believed the immunoassay approach was appropriate. Thus we developed an immunoassay for the compound using time-resolved fluorescence as an end-point (DELFIA) with lower limits of quantification of 0.2 ng ml(-1) for the plasma assay and 5 ng ml(-1) for the assay in urine. This assay is a 96-well plate based competitive immunoassay; the end-point is the determination of a (non-radioactive) europium label by time-resolved fluorimetry. Sampatrilat is labelled with chelated europium via isothiocyanate chemistry. The advantage of this assay is its extremely high throughput, allowing rapid analysis of many thousands of samples. The DELFIA method was successfully cross-validated with the HPLC-APCI-MS-MS method.

Angiotensin-Converting Enzyme Inhibitors↗
PubMed1996

Rapid, solid phase extraction technique for the high-throughput assay of darifenacin in human plasma.

A novel method has been developed for the rapid solid phase extraction of drugs and metabolites from biological fluids, prior to further analysis. The newly designed, 96-tube micropreparation block facilitates high throughput by enabling the extraction of 96 samples simultaneously. The system is described, linked to HPLC/APCI-MS/MS, for the determination of darifenacin in human plasma. The resulting procedure, using deuterated darifenacin as internal standard, is validated over the concentration range 25-2000 pg/mliter; accuracy (0.6-4.6%) and precision (3.6-18.8%) are considered acceptable and overall recovery was determined to be approximately 50%.

Benzofurans↗
PubMed1993

Analysis of human beta-endorphin 28-31 (melanotropin potentiating factor) and analogues by high-performance liquid chromatography of their 9-fluorenylmethoxycarbonyl derivatives.

A method is described for the determination of sub-picomole amounts of Lys-Lys-Gly-Glu [the C-terminal tetrapeptide sequence of human beta-endorphin, referred to as melanotropin potentiating factor (MPF)], a putative neurotrophic agent. Attempts to raise antibodies to the peptide were not successful and we have therefore developed a method based on the fluorescence of its 9-fluorenylmethyl chloroformate (FMOC) derivative which provides a sensitivity comparable to that of radioimmunoassay. Standard solutions, cerebrospinal fluid or central nervous tissue extracts are first treated with FMOC-Cl. The resulting mixture of FMOC-peptides is then subjected to high-performance liquid chromatography (HPLC) and quantified using a fluorescence monitor. By this procedure, MPF and related peptides can be analysed from one sample in a single HPLC run. The method was also applied to determine the rate of release into a phosphate-buffered saline medium of a metabolically stable analogue of MPF from a slow-release formulation of the compound.

Amino Acid Sequence↗
PubMed1992

CSF enkephalins in diabetic neuropathy.

CSF methionine and leucine enkephalins were measured by high performance liquid chromatography and radioimmunoassay in diabetic patients with painful neuropathy (n = 22) and painless neuropathy (n = 5), and non-diabetic subjects with low back pain (n = 11). Wide variations in CSF enkephalin levels were found and they were often below the limit of detection (less than 0.1 pmol/l) in the diabetic and non-diabetic groups. The origin of CSF enkephalins is unknown and CSF levels may not reflect tissue concentrations. In conclusion, CSF enkephalin levels are difficult to interpret and do not provide useful information on the function of enkephalinergic pathways.

Adult↗
PubMed1990

Fast reliable assay for morphine and its metabolites using high-performance liquid chromatography and native fluorescence detection.

A method for the fast analysis of morphine (M), normorphine (NM), morphine-3- and -6-glucuronides (M3G and M6G) and codeine (C) is described which has the advantages of sensitivity, speed and specificity. Dihydrocodeine and heroin can also be assayed. The method is based on extraction of the opiates from serum, plasma and cerebrospinal fluid using reversed-phase solid-phase extraction columns, followed by reversed-phase high-performance liquid chromatography with native fluorescence detection. The extraction step provides greater than 95% recovery, and the response of the detection system is linear from 0.5 to beyond 750 ng. The method allows analysis of M, NM, M3G, M6G and C. No other drugs have been found to interfere with the assay. The assay offers a quick, cheap and reliable method of specifically determining morphine and its metabolites, including the potent M6G, from a small sample volume; this will be of advantage to both clinician and basic scientist.

Chromatography, High Pressure Liquid↗
PubMed1987

Combined high-performance liquid chromatographic-radioimmunoassay method for the analysis of endorphins, enkephalins and other neurotransmitter peptides.

Assays for beta-endorphin (BE) and its precursors such as beta-lipotropin (LPH) in cerebrospinal fluid (CSF), plasma and some tissues have been difficult because of their low concentrations in limited sample volumes, the non-specificity of most antisera. These problems are compounded by the lack of suitable separation methods. Similar problems exist for the enkephalins, tachykinins and dynorphins, among others. This study reports a high-performance liquid chromatographic (HPLC) separation method in which BE and LPH are well separated from each other and which also separates other neuropeptides of interest. The method uses volatile solvents which do not interfere with radioimmunoassay (RIA). Thus by combining HPLC with RIA the method offers, for the first time, a specific assay method for the endorphin, enkephalin and dynorphin families of peptides which does not suffer from the uncertainties in RIA due to cross-reactivities of antisera. Peptide concentrations obtained from the CSF of a small group of chronic pain patients are also presented.

Buffers↗
PubMed1986

A specific radioimmunoassay for [Leu] and [Met]enkephalin applied to a HPLC separation of human CSF.

Highly specific antisera have been raised to [Leu]enkephalin and applied in a radioimmunoassay that is sufficiently sensitive and precise for the measurement of enkephalin pentapeptides in body fluids. Extraction of CSF by Sep-pak cartridges, followed by reverse-phase HPLC to separate [Leu] and [Met]enkephalin prior to assay, has assessed their concentrations as 59-170 pmol/l and 0.5-30 pmol/l, respectively, in three patients with chronic pain. The reproducibility of the HPLC separation step was checked by adding to the sample a synthetic analogue of very low cross-reactivity in UV detectable quantities. The assay procedure described is generally applicable to the assessment of [Leu] and [Met]enkephalin concentrations in CSF.

Chromatography, High Pressure Liquid↗
PubMed1984

Opiate binding to subcellular fractions from guinea pig ileum.

Binding of 3H-etorphine and 3H-D-Ala2-D-Leu5-enkephalin to opiate receptors in synaptosomal and microsomal fractions prepared from guinea pig ileum homogenates has been studied. It is found that the dissociation constants for etorphine from all fractions are the same. The binding capacity for etorphine for the purified synaptosomal fraction is greater than for other fractions by a factor of 5. For the enkephalin derivative binding to the microsomal fraction the dissociation constant is greater than for etorphine while the binding capacity is a factor of 3 lower. These results are in contrast to the case for binding to central nervous system subcellular fractions.

Animals↗
PubMed1983

A rapid binding assay for immunoglobulins which may be used as an alternative to ammonium sulfate precipitation methods.

This paper describes the conditions under which a short column packed with Sephadex G-25 may be eluted centrifugally to separate labeled ligands from binding immunoglobulins in a manner which can yield quantitative binding information. This method may be used as an alternative to the classical binding assay involving ammonium sulfate precipitation. The advantage is a large saving in total assay time over that procedure. The column assay is applicable to antisera and purified polyclonal and monoclonal immunoglobulins. The restrictions are that the labeled ligands must be of molecular weight less than approximately 5000 Da.

Ammonium Sulfate↗
PubMed1983

Properties of murine anti-morphine antibodies.

Four lines of high affinity monoclonal antibodies directed against morphine have been isolated and affinity purified. Some of their properties, including cross-reactivities to a large set of selected opiate agonists and antagonists are described. Importantly, none of the immunoglobulins cross-react with D-Ala2-D-Leu5-enkephalin.

Animals↗
PubMed1981

A potent peptide affinity reagent for the opiate receptor.

The synthesis and characterization of a novel enkephalin analogue, Tyr-D-Ala-Gly-Phe-Leu-chloromethyl ketone, is described. The biological potency of the compound in various assays has been determined to be very high. The compound is an alkylating affinity reagent and irreversibly inactivates a defined population of enkephalin receptors in rat brain membrane preparations, as well as irreversibly inhibiting electrically stimulated contractions in the mouse vas deferens tissue preparation.

Affinity Labels↗
PubMed1979

The preparation of stable enzyme-coenzyme complexes with endogenous catalytic activity.

N6-(6-Aminohexylcarbamoylmethyl)-NAD+ was coupled to lactate dehydrogenase by using glutaraldehyde to form an active complex. The stability of this complex could be considerably improved by reduction with KBH4, although this treatment caused a partial decrease in specific activity. NAD+ was also coupled directly to the enzyme by this method. All of these complexes exhibited an intrinsic activity in the absence of exogenous NAD+.

Borohydrides↗