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D J Trafford

Publications and source records attributed to D J Trafford.

At least 37 records · Page 2Linked to original sources
PubMed1988

A semi-automated high-performance liquid chromatographic system for the determination of 25-hydroxyvitamin D in human plasma: elimination of interference by barbiturates and use of photodiode array detection.

A semi-automated high-performance liquid chromatographic (HPLC) method for the measurement of 25-hydroxyvitamins D(2) and D(3) is described. Plasma was extracted using acetonitrile and a Bond-Elut C(18) cartridge system, eluted with methanol and fractionated on Sep-Pak SIL. After formation of isotachysterol isomers straight-phase HPLC was carried out monitoring the mobile phase with a photodiode array detection system. Two internal standards have been used, namely [(3)H]25-hydroxyvitamin D(3) and 25-hydroxydihydrotachysterol(3) both of which are shown to give satisfactory results. The use of the extraction system described eliminates interference from barbiturates which had previously been reported to interfere. Photodiode array detection allows confirmation of the identity of the analytes.

Journal Article↗
PubMed1986

Capillary gas-liquid chromatography of glycine-conjugated bile acids without prior hydrolysis.

A method is described for the gas-liquid chromatographic (GLC) analysis of intact glycine conjugates of the major bile acids present in human plasma. It is, therefore, now possible to analyze glycine-conjugated and unconjugated bile acids together on a single GLC column without the necessity for a hydrolytic step. A large number of derivatives of bile acid glycine conjugates were examined, but only acetate- and silyl ether-derivatives of carboxylic acid methyl esters were found initially to be suitable. It was not possible to make acetates consistently, and trimethylsilyl ethers did not allow resolution of the glycine conjugates of cholic and chenodeoxycholic acids. Dimethylethylsilyl ether methyl ester derivatives were subsequently found to give the best results. Chromatographic conditions for successful analysis of these derivatives were examined and it was found to be necessary to use wall-coated capillary columns of thin film thickness (0.12 micron) and very high carrier gas flow rates (ca. 20 ml/min hydrogen). Using acetonitrile and Bond Elut extraction, fractionation on Sep-Pak SIL cartridges, and derivatization as dimethylethylsilyl ether methyl esters, the capillary gas-liquid chromatography of intact glycine-conjugated bile acids from human plasma was demonstrated for the first time.

Bile Acids and Salts↗
PubMed1985

Extraction and fractionation of bile acids and their conjugates using pre-packed microparticulate silica cartridges (Sep-Pak Sil and Bond-Elut C18).

A new method for the extraction of bile acids from human plasma using acetonitrile precipitation of plasma protein and subsequent use of Bond-Elut C18 cartridges is described. After extraction the bile acids can be separated into three fractions: unconjugated, glycine-, and taurine-conjugated, using Sep-Pak SIL cartridges at 4 degrees C, eluting with ethanol--chloroform--water--glacial acetic acid mixtures. These extraction and fractionation procedures were evaluated in terms of recovery, reproducibility and resolution between the fractions. All these parameters were found to be satisfactory. Although the reproducibility of fractionation on Sep-Pak SIL cartridges was found to vary between batches, this did not give rise to significant difficulties. Plasmas from normals and patients with hepatobiliary disease were analysed by capillary gas-liquid chromatography after extraction and fractionation using the procedure described.

Bile Acids and Salts↗
PubMed1985

Specific mass fragmentographic assay for 25,26-dihydroxyvitamin D in human plasma using a deuterated internal standard.

A specific mass fragmentographic assay for the measurement of 25,26-dihydroxyvitamin D3 [25,26(OH)2D3] in human plasma, using a stable isotope labelled internal standard ([26,27-2H5]25,26(OH)2D3), is described. Plasma samples (5 ml) were extracted with acetonitrile and applied to a C18 Sep-Pak cartridge, from which the vitamin D metabolites were eluted with methanol. The metabolites were then applied to a Sep-Pak SIL cartridge and three fractions were collected. The most polar fraction, containing the polyhydroxylated metabolites, was further purified by high-performance liquid chromatography on Zorbax SIL. The eluent containing 25,26(OH)2D3 was collected, and the 25,26-n-butylboronate cyclic ester 3-trimethylsilyl ether derivative was formed. Gas chromatography-mass spectrometry was carried out, monitoring the intensities of the ions at m/z 449 and m/z 454 (for the internal standard). These ions represent the loss of a methyl group and the 3-silanol group, (M-90-15)+. The minimum limit of detection of the assay was estimated to be approximately 0.05 microgram/l. Inter-assay (3.7%) and intra-assay (8.0%) precision was acceptable and added 25,26(OH)2D3, over the concentration range 0.5-1.5 microgram/l, was recovered quantitatively. The plasma 25,26(OH)2D3 level was estimated in 26 healthy volunteers and ranged from 0.05 to 1.30 microgram/l, with a mean value of 0.54 microgram/l.

24,25-Dihydroxyvitamin D 3↗
PubMed1985

Vitamin D metabolites in idiopathic infantile hypercalcaemia.

Metabolites of vitamin D were measured in plasma from 83 patients with idiopathic infantile hypercalcaemia syndrome who were mentally handicapped but had normal calcium values at the time of the study. No significant difference was detected in the mean plasma concentrations of 25-hydroxyvitamin D2, 1,25-dihydroxyvitamin D, 24,25-dihydroxyvitamin D3, or 25,26-dihydroxyvitamin D3 between patients and age matched controls. The mean plasma concentration of 25-hydroxyvitamin D3 was significantly lower in patients than controls but this may be a secondary phenomenon related to less sunlight exposure. In addition, two hypercalcaemic patients with this syndrome were studied during the first year of life, and were found to have normal concentrations of vitamin D metabolites. These findings do not support a role for abnormal vitamin D metabolism in the pathogenesis of this syndrome.

24,25-Dihydroxyvitamin D 3↗
PubMed1984

Specific estimation of 24,25-dihydroxyvitamin D in plasma by gas chromatography-mass spectrometry.

This paper describes a specific mass-fragmentographic method, involving a stable-isotope-labeled internal standard, for measurement of 24,25-dihydroxyvitamin D in human plasma. Vitamin D metabolites were rapidly extracted from plasma by using Sep-Pak C18 cartridges and separated into fractions on Sep-Pak SIL cartridges. The polar fraction, containing the dihydroxylated metabolites, was further purified by "high-performance" liquid chromatography on Zorbax SIL. The fraction containing 24,25-dihydroxyvitamin D was collected, evaporated, and converted to the 24:25-cyclic n-butyl boronate-3-trimethylsilyl ether derivative before analysis by gas chromatography-mass spectrometry. The intensity of the mass fragment (m/z 449, m/z 455 for the hexadeuterated internal standard) arising from the loss of one of the angular methyls and the 3-silanol group [( M-90-15]+) was monitored. The minimum limit of detection for this method is about 0.1 microgram/L. Inter- and intra-assay reproducibility was acceptable, and analytical recovery of added 24,25-dihydroxyvitamin D3 over the concentration range 1.0 to 5.0 micrograms/L was quantitative. Concentrations of 24,25-dihydroxyvitamin D3 in plasma of 21 apparently healthy volunteers were between 0.55 and 5.39 micrograms/L, higher values being obtained after prolonged exposure to the sun. No 24,25-dihydroxyvitamin D2 could be detected in any plasma sample examined.

24,25-Dihydroxyvitamin D 3↗
PubMed1983

Studies on the presence of 25-hydroxyvitamin D in human saliva.

Saliva collected from volunteers of both sexes at various times throughout the day was examined for the presence of 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3. The method used was a specific high-performance liquid chromatographic procedure with a minimum detectable limit for 25-hydroxyvitamin D of 0.05 microgram/1, (130 pmol/1 for 25-hydroxyvitamin D3) using 20 ml of saliva. No 25-hydroxyvitamin D could be detected in any of the samples. Examination of larger quantities of saliva (100 ml) by a combination of high-performance liquid chromatography and gas chromatography-mass spectrometry showed the presence of a peak possibly derived from 25-hydroxyvitamin D3, at a concentration not in excess of 65 pmol/l. Using known binding characteristics of 25-hydroxyvitamin D and plasma vitamin D binding protein, and comparing them with similar characteristics for cortisol and plasma cortisol binding globulin and the known salivary concentration of cortisol, simple calculations indicated that a likely concentration of 25-hydroxyvitamin D in saliva would be around 8 pmol/l. Previously reported values ranging from 130-4160 pmol/l (50-1600 micrograms/l) using a simple competitive protein binding assay could not be confirmed.

25-Hydroxyvitamin D 2↗
PubMed1983

An evaluation of the use of Sep-Pak C18 cartridges for the extraction of vitamin D3 and some of its metabolites from plasma and urine.

The use of Sep-Pak C18 cartridges for the extraction of vitamin D and some of its metabolites from plasma and urine has been evaluated by studying the recovery of added tritiated secosteroids. The preparation of the cartridges, recoveries, extraction and elution with a number of solvents, effect of varying flow rates for application and elution, and the effect of increasing volumes of plasma and urine have been investigated. Two methods for the application of secosteroids present in plasma to Sep-Pak C18 cartridges have been examined, using methyl cyanide extracts removing precipitated protein by centrifugation, and using acidified methanolic plasma. Methyl cyanide extracts applied to Sep-Pak C18 cartridges and eluted with methanol or methyl cyanide gave the cleanest extracts suitable for direct HPLC. Acidified methanolic plasma, applied to Sep-Pak C18 cartridges and eluted with methanol or methyl cyanide gave extracts which could not be applied directly to an HPLC--further fractionation using Sep-Pak SIL cartridges was necessary. Recoveries of added tritiated secosteroids using both methods were greater than 80% with the exception of vitamin D itself which was poorly recovered--methyl cyanide extraction giving only 30% recovery and use of acidified methanolic plasma giving 66% recovery.

Cholecalciferol↗
PubMed1983

The quantitative estimation of bile acids and their conjugates in human biological fluids.

This review attempts to provide a concise and critical summary of modern methods for the analysis of bile acids and their conjugates in human biological fluids. Most emphasis is given to more up-to-date procedures that have been applied to the study of human disease and attention is drawn to previous reviews in areas that have not been covered here. An increasing awareness of the possibility that bile acids may be involved in the etiology of a number of disorders, or that such disorders may give rise to changes in bile acid concentration, has stimulated the study of bile acid methodology. Although many procedures have been described using, for example, high-pressure liquid chromatography (HPLC), gas-liquid chromatography (GLC), gas-liquid chromatography--mass spectrometry (GLC-MS), and radioimmunoassay (RIA), no simple but comprehensive procedure for the estimation of bile acids and their conjugates has yet been published. Further study in this area is still required in order to establish the role of bile acid estimations in the routine diagnosis and treatment of disease.

Bile Acids and Salts↗
PubMed1982

A rapid and simple method for the measurement of plasma 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 using Sep-Pak C18 cartridges and a single high-performance liquid chromatographic step.

A simple one step high-performance liquid chromatographic (HPLC) procedure for the analysis of plasma concentrations of 25-hydroxyvitamin D3 (25-OHD3) and 25-hydroxyvitamin D2 (25-OHD2) is described. Plasma (2-4 ml) was extracted with methyl cyanide which was passed through a Sep-Pak C18 cartridge, washed with methanol:water (70:30, v/v) and the 25-OHD fraction eluted with methyl cyanide. After isomerisation to their isotachysterol derivatives, the secosteroids were estimated using a straight-phase HPLC system, monitoring the eluent at 301 nm. Radioactive 25-OHD3, added to plasma at the start of the procedure, was used to correct for losses. Recovery of added 25-OHD3 was quantitative and values obtained using this method were similar to those obtained on the same plasma samples using a mass fragmentographic technique. Normal ranges were similar to those described by other workers and within- (5.8% for 25-OHD3) and between-(8.0% for 25-OHD3) batch reproducibilities were satisfactory.

25-Hydroxyvitamin D 2↗
PubMed1982

A semi-automated computerised gas-liquid chromatographic system for urinary steroid profile analysis.

A simple gas-liquid chromatographic method for the measurement of some of the major steroids in urine is described. The system uses conventional packed columns, automatic injection and is linked to an integrator. Calculations are all carried out by computer. The system is recommended as a simple screening procedure, producing a urinary steroid 'profile', which can process large numbers of urine samples cheaply and quickly, enabling patients worthy of more intensive investigation to be identified.

17-Hydroxycorticosteroids↗
PubMed1981

High-performance liquid chromatography of 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 in human plasma. Use of isotachysterols and a comparison with gas chromatography--mass spectrometry.

A high-performance liquid chromatographic (HPLC) method for estimating plasma 25-hydroxyvitamin D2 (25-OHD2) and 25-hydroxyvitamin D3 (25-OHD3) is described. The method involves plasma extraction, Lipidex 5000 chromatography and HPLC on straight-phase Zorbax-SIL, collecting the 25-OHD2 + 25-OHD3 fractions. These secosteroids are isomerised to their isotachysterol derivatives and re-run in the same HPLC system, monitoring at 290 nm. 3H-Labelled 25-OHD3 is used as an internal standard. The method was evaluated in terms of reproducibility, and recovery of added secosteroids was quantitative. Values obtained using this method were in close agreement with those values obtained on the same plasma sample using gas chromatography--mass spectrometry.

25-Hydroxyvitamin D 2↗
PubMed1981

A rapid and simple method for the esterification of fatty acids and steroid carboxylic acids prior to gas-liquid chromatography.

A simple procedure for the esterification of steroid carboxylic acid groups using an acetyl chloride/alcohol reagent has been carefully evaluated. The ease with which these esters can be formed using this reagent depends on the acid and the ester required. C24 bile acids form methyl esters using acetyl chloride/methanol (1:20, v/v) almost instantaneously at 50 degrees C, whereas the C20 aetienic acid studied requires 2 h at 50 degrees C. The formation of isopropyl esters of 5 beta-cholanic acid takes longer (4 h at 50 degrees C) than the formation of n-propyl esters (1 h at 50 degrees C). The identity of various esters fo cholanic acids formed using this reagent have been checked by gas chromatography-mass spectrometry and various other procedures used for the methylation of fatty acids have been examined to establish whether they can be used for the quantitative esterification of bile acids.

Carboxylic Acids↗
PubMed1981

The in vivo metabolism of Althesin (alphaxalone + alphadolone acetate) in man.

Gas chromatograph-mass spectrometry has been used to study the metabolism of Althesin (alphaxalone and alphadolone acetate) in man. Two metabolites, 20 alpha-reduced alphaxalone and alphadolone, as well as the two parent steroids, have been detected in the plasma during and after an infusion of Althesin. The main urinary metabolites were alphaxalone and 20 alpha-reduced alphaxalone, with smaller amounts of alphadolone, all of which were excreted mainly as the glucuronide conjugates. No alphadolone acetate was detected in the urine. In 3 patients in whom bile was collected over the 1st 24 h post-operation from an indwelling T-tube, it has not been possible using the methods described to detect in bile the presence of the parent steroids, or any of their probable metabolites.

Adult↗