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H L Makin

Publications and source records attributed to H L Makin.

At least 55 records · Page 3Linked to original sources
PubMed1989

Carrier gas flow-rate and injection system in capillary gas chromatography of unconjugated and glycine-conjugated bile acids.

The first description of successful capillary gas chromatography of intact glycine-conjugated bile acid derivatives used an automatic solid injection system and required very high carrier gas flow-rates (approximately 20 ml/min) to obtain satisfactory peak shape. Peak heights of unconjugated bile acid derivatives using this injection system and the low flow-rates (1-2 ml/min) usually used for such gas chromatographic analyses were very susceptible to small changes in flow-rate. This system has been re-examined in an attempt to explain this anomalous behaviour. An alternative injection system, the all-glass dropping needle, was also investigated. No explanation for the very high carrier gas flow-rates required when using the automatic solid injection system was found. The dropping needle injection system, however, produced excellent separation of bile acids and their glycine conjugates as dimethylethylsilyl ethers-methyl esters on non-polar wall-coated capillary columns using normal carrier gas flow-rates of 1-2 ml/min. It is clear that the automatic solid injection system originally used has some problem associated with it which can only be overcome, in the case of bile acids and their glycine conjugates, by increasing the carrier gas flow-rate to very high levels.

Bile Acids and Salts↗
PubMed1989

Measurement of 25-hydroxyvitamin D2, 25-hydroxyvitamin D3, 24,25-dihydroxyvitamin D2 and 25,26-dihydroxyvitamin D2 in a single plasma sample by mass fragmentography.

A specific and sensitive assay for the measurement of the concentration of 25-hydroxyvitamin D2, 25-hydroxyvitamin D3, 24,25-dihydroxyvitamin D2 and 25,26-dihydroxyvitamin D2 in a single plasma sample is described, using stable isotope dilution mass fragmentography. After addition of appropriate deuterium-labelled internal standards, plasma samples were treated with acetonitrile to precipitate protein, and vitamin D metabolites were extracted on prepacked microparticulate reverse-phase cartridges. Further purification was achieved using straight-phase cartridges and high-performance liquid chromatography. Gas chromatography-mass spectrometry was carried out after appropriate derivatisation of samples and standards. The method has been evaluated in terms of specificity, recovery of added standards, and reproducibility.

Calcifediol↗
PubMed1988

Isolation and identification of seven metabolites of 25-hydroxydihydrotachysterol3 formed in the isolated perfused rat kidney: a model for the study of side-chain metabolism of vitamin D.

The in vivo metabolism of dihydrotachysterol3, an analogue of vitamin D3 and a potent calcemic factor, has been studied in the rat. This in vivo metabolism is compared to the in vitro metabolism of 25-hydroxydihydrotachysterol3 in the perfused rat kidney. Using mass spectrometry and ultraviolet spectroscopy, we have identified seven novel metabolites derived from 25-hydroxydihydrotachysterol3. The seven compounds represent intermediates on two renal pathways (24-oxidation and 26,23-lactone formation) also observed for 25-hydroxyvitamin D3. No evidence was found for the renal synthesis of a 1-hydroxylated metabolite of 25-hydroxydihydrotachysterol3 analogous to the hormone 1,25-dihydroxyvitamin D3. Two of the compounds formed in vitro, 24,25-dihydroxydihydrotachysterol3 and 25-hydroxydihydrotachysterol 26,23-lactone, were also formed in vivo. In vivo studies also revealed the formation of two other unidentified metabolites which are presumed to be formed nonrenally and may be calcemic factors. This work shows that dihydrotachysterol3 metabolism is complex and probably utilizes the same side-chain enzymes as vitamin D3. In addition, our work also confirms that intermediates postulated to lie on pathways to 26,23-lactone in the vitamin D3 series are also formed for the side chain in dihydrotachysterol3.

Animals↗
PubMed1988

The measurement of vitamins D2 and D3 and seven major metabolites in a single sample of human plasma using gas chromatography/mass spectrometry.

Selected ion monitoring of vitamin D metabolites has previously been described but there has been only one detailed description of the measurement by gas chromatography/mass spectrometry (GC/MS) of a number of metabolites in a single plasma sample. We describe here a GC/MS method, using stable isotope labelled internal standards, which allows the estimation of vitamins D2 and D3, and their 25-hydroxy, 24,25-dihydroxy and 25,26-dihydroxy metabolites in a single 2 ml sample of plasma, although more is needed for the measurement of 1,25-dihydroxyvitamin D3. Plasma was extracted on Bond Elut C18 cartridges and initial fractionation carried out on Sep-Pak SIL. Straight-phase high-performance liquid chromatography was required for separation of polyhydroxylated metabolites prior to GC/MS using an LKB 2091 mass spectrometer with conventional packed columns. n-Butylboronate esters were formed across vicinal hydroxyls, followed by formation of trimethylsilyl ethers using trimethylsilylimidazole. The [M - 90 - 15]+ ion for each compound was monitored. Deuterated internal standards were not available for all metabolites and it was necessary to use (2H6)D3 and (2H6)25OHD3 as standards for the measurement of D2 and D3, and 25OHD3 and 25OHD2, respectively, and (2H6)24,25(OH)2D3 as a standard for 24,25(OH)2D3 and 25,26(OH)2D2. Although the [M - 90 - 15]+ ion of 24,25(OH)2D and 25,26(OH)2D has the same mass: charge ratio, derivatives of these compounds are completely separated in the GC system used. The intra-assay precision for all these assays is usually less than 5%.

Cholecalciferol↗
PubMed1988

Use of mass spectrometry in the identification of in vivo and in vitro metabolites of dihydrotachysterol3 in the rat.

The metabolism of dihydrotachysterol3 (DHT3), a vitamin D analogue, has been investigated in vivo in the rat after intraperitoneal injection, and the metabolism of the 25-hydroxylated metabolite of DHT3 was studied in vitro in the isolated perfused rat kidney. A large number of metabolites have been obtained and some have been identified. The rat plasma or kidney perfusate were extracted and the metabolites separated by high-performance liquid chromatography (HPLC) in straight- and reverse-phase systems and using cyano columns. Metabolites were identified, using a photodiode array assembly which monitored the HPLC eluate, by the characteristic ultraviolet spectrum of DHT compounds. Tentative structures were assigned to some of the metabolites obtained on the basis of their mobility in the various HPLC systems used in comparison to that of known metabolites of vitamin D. Gas chromatography/mass spectrometry (GC/MS) and direct probe mass spectrometry have been used to confirm the identity of seven metabolites formed in vitro, of which only two have been definitely shown also to be formed in vivo. GC/MS was carried out after derivatization forming trimethylsilyl ethers, n-butyl boronate cyclic esters, and N-O-methyl oximes before and after oxidation with sodium periodate and/or reduction with sodium borohydride. Molecular ions of these compounds are usually of low abundance and characteristic mass fragments at m/z 273, 255 and 121 are always seen with metabolites of DHT.

Animals↗
PubMed1988

High-performance liquid chromatography of steroid hormones.

Although a considerable amount of work has been carried out in the last ten years in developing methods for the separation of steroids by HPLC, it is still not widespread for the reasons discussed above. There is however no doubt that further developments in HPLC technology, in increasing sensitivity and/or specificity of detection systems, perhaps with microbore columns, may lead to an increase in the use of this powerful analytical procedure as an additional separation method to improve specificity of assay. Solution of the problem of simple interfacing of HPLC systems with mass spectrometers (discussed in another chapter by Games) should further increase the application of HPLC. HPLC is of particular value in providing a means of separating unstable compounds prior to assay by relatively nonspecific quantitation methods. Most steroids do not fall into this category, but the steroid vitamin D and its metabolites do and HPLC has proved in this area to be invaluable (see chapter by Jones & DeLuca). There are a multiplicity of different HPLC systems for the separation of steroids, varying in column type (and manufacturer), solvent composition and method of elution, temperature of elution, etc., and only a few attempts have been made to rationalise these data. It would therefore seem that a fruitful area of future study would be the investigation of computerised systems for the selection and optimisation of HPLC systems for particular steroid separations.

Adrenal Cortex Hormones↗
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↗