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L Siekmann

Publications and source records attributed to L Siekmann.

At least 37 records · Page 2Linked to original sources
PubMed1987

Measurement of thyroxine in human serum by isotope dilution mass spectrometry. Definitive methods in clinical chemistry, V.

The determination of thyroid hormones is widely used for the diagnosis and therapy control of thyroid disorders. In particular, thyroxine in serum is one of the most frequently determined endocrine parameters. Unfortunately, the results obtained by the use of different commercial test kits vary significantly, and until now there has been no means to decide whether a particular enzyme or radioimmunoassay kit yields accurate results or not. It seemed, therefore, necessary to develop definitive or reference methods for the measurement of thyroxine and to apply this technique for the assessment of target values in control sera for internal and external quality control. In the present investigation, an analytical protocol using the isotope dilution mass spectrometry technique is described which is herewith proposed as a definitive method for the measurement of thyroxine in human serum. The procedure consists of the following steps. (i) Equilibration of endogenous thyroxine in a serum sample with 100 ng [13C2]thyroxine. (ii) Isolation of the thyroid hormones by using a cation exchange resin. (iii) Formation of the methyl ester by reaction with methanolic hydrochloric acid. (iv) Purification of the methyl ester by column chromatography on Sephadex LH-20. (v) Formation of the N,O-bistrifluoroacetyl derivative with trifluoracetic anhydride. (vi) Selected ion monitoring of fragment ions of the thyroxine and the [13C2]thyroxine derivatives at m/z 870 and 872 using a magnetic sector field mass spectrometer with electron impact ionization combined with a capillary gas chromatography column. This method is now used to assign target values in a German quality control scheme. The precision of the method is of the order of 1-2% (coefficient of variation).

Chromatography, Gas↗
PubMed1985

Determination of uric acid in human serum by isotope dilution-mass spectrometry. Definitive methods in clinical chemistry, III.

A method for the measurement of uric acid in human serum by isotope dilution-mass spectrometry is described. The analytical procedure consists of the following steps: Addition of [1,3-15N2]uric acid to the serum sample; ion exchange chromatography on AG1-X2; formation of the trimethylsilyl derivative; gas liquid chromatography-mass spectrometry (GC-MS), selected ion monitoring (SIM) at m/z-values 456 and 458; calculation of the amount of uric acid in the serum sample from the isotope ratio, as measured by GC-MS. The accuracy of the method is obtained by the use of the highly specific technique of selected ion recording in combination with the exact control of recovery as performed by the isotope dilution procedure. On the basis of the high accuracy of the isotope dilution-mass spectrometry technique, the method presented here may be proposed as a definitive method in clinical chemistry. The imprecision of the method was estimated by measuring replicates in 18 lyophilised serum pools on different occasions. The coefficient of variation proved to be between 0.6 and 1.1% in the concentration range of 200 to 500 mumol/l. The lower limit of detection (ratio of signal to noise 3:1) of SIM was about 10 ng uric acid per sample.

Chromatography, Ion Exchange↗
PubMed1985

Determination of creatinine in human serum by isotope dilution-mass spectrometry. Definitive methods in clinical chemistry, IV.

A method for the determination of creatinine in human serum by isotope dilution-mass spectrometry is described. The analytical procedure comprises the following steps: Addition of [13C,15N2 )creatinine to the serum sample; ion exchange chromatography on the cation exchange resin AG 50W-X2; formation of the trimethylsilyl derivative; gas liquid chromatography-mass spectrometry (GC-MS); selected ion monitoring (SIM) at the m/z-values 329 and 332; calculation of the amount of creatinine in the serum sample from the isotope ratio, as measured by GC-MS. [13C,15N2]Creatinine was prepared by chemical synthesis. The substance is then used as internal standard for the measurement of creatinine in serum samples. The imprecision of the method was in the range from 0.35 to 1.05% (coefficient of variation) as determined by repetitive measurements of creatinine in 13 different control sera on different occasions. The lower limit of detection of the mass spectrometer in the selected ion monitoring mode is about 0.5 ng creatinine with a signal to noise ratio of 3:1 The accuracy of the method is achieved by the use of the isotope dilution principle in combination with GC-MS. In view of the high specificity and exact control of recovery, the procedure for the measurement of creatinine in human serum, as described here, may be considered as a definitive method in clinical chemistry.

Chromatography, High Pressure Liquid↗
PubMed1984

Determination of oestradiol-17 beta in human serum by isotope dilution-mass spectrometry. Definitive methods in clinical chemistry, II.

A definitive method is described for the measurement of oestradiol-17 beta in human serum. The method is based on the principle of isotope dilution-mass spectrometry. The analytical procedure consists of the following steps: addition of [4-14 C]oestradiol-17 beta to the plasma sample; extraction of the 14C-labelled and the non-labelled oestradiol-17 beta with dichloromethane; purification of the oestradiol-17 beta fraction by column chromatography on Sephadex LH-20; formation of the 3,17 beta-diheptafluorobutyric ester of oestradiol-17 beta; selected ion monitoring at m/z-values 664 and 666 by gas liquid chromatography-mass spectrometry. The accuracy of the method is based on the high specificity of mass spectrometry and on the exact control of recovery employing the principle of isotope dilution. Coefficients of variation from day to day varied between 1.0% and 2.5% in the range of 0.319 to 3.53 nmol/l. The lower limit of detection (ratio of signal to noise 3:1) is 5 pg oestradiol-17 beta per sample.

Estradiol↗
PubMed1983

[Collaborative tests of steroid hormone determinations: accuracy and precision of analytic results].

In the years 1977 to 1981, 14 quality-control surveys for the determination of steroid hormones were performed in cooperation with the Deutsche Gesellschaft für Klinische Chemie. Hereby the laboratories participating could in each case analyze the following steroids: aldosterone, cortisol, oestradiol-17 beta, oestriol, progesterone, and testosterone. In the light of the results an investigation was made as to whether, in the course of time, an improvement in the accuracy or in the precision of the determinations had been attained, and to what extent the determinations depend on the qualities of the test material. A clear improvement in the accuracy of the results of the analyses could only be ascertained for oestradiol-17 beta. For aldosterone and cortisol, values were found in pool-plasma whose medians were significantly above the definitive values. An improvement in precision could be noted especially with oestradiol-17 beta and to lesser degrees with cortisol and oestriol. The kind of test material--plasma which contained only the hormones to be analyzed on the one hand, and, on the other hand, pool-plasma, which also contained all endogenous hormones--had no influence on the precision of the results from various laboratories. Low concentrations of the individual steroids led--on the basis of the methodological principle of radioimmunoassays--in almost all cases to a reduced interlaboratory precision in regard to values. The accuracy of the analysis values was considerably impaired only with aldosterone and oestradiol-17 beta by low concentrations: the medians here were in part twice as high as the definitive values.

Aldosterone↗
PubMed1983

[Candidate selected method for the enzymatic determination of total cholesterol in serum].

An enzymatic method is described for the determination of total cholesterol in serum using a single aqueous reagent which can easily be prepared from commercial substrates and enzymes. The determination is carried out manually, the resulting stable chromogen is measured at a wavelength of 500-550 nm. The cholesterol concentration may be calculated either using a primary cholesterol standard or a constant factor for a given wavelength. The reliability of the method is reported: Data for the imprecision are given on the basis of a survey in 3 laboratories, the accuracy is established by comparison with a definitive and a reference method. Analytical and biological interferances are briefly discussed and results with this enzymatic method are reported concerning the reference values for serum cholesterol.

Cholesterol↗
PubMed1982

Determination of cortisol in human plasma by isotope dilution-mass spectrometry. Definitive methods in clinical chemistry, I.

A definitive method is described for the determination of cortisol in human plasma. The method is based on the principle of isotope dilution-mass spectrometry. The analytical procedure comprises the following steps: (1) Addition of [4-14C]cortisol to the plasma sample; (2) extraction of the 14C-labelled and of the non-labelled cortisol by dichloromethane; (3) purification of the cortisol fraction by column chromatography on Sephadex LH-20; (4) formation of the trimethylsilyl ether of cortisol methoxime; (5) selected ion monitoring at m/z-values 605, 607, 636 and 638 during gas liquid chromatography. The accuracy of the method is based on the high specificity of mass spectrometry and on the exact control of recovery employing the principle of isotope dilution. The precision of the method was evaluated by calculation of the coefficient of variation from day to day; this varied from 0.7-2.3% in the range of 256-845 nmol/l. The lower limit of detection (ratio of signal to noise 3:1) is 200 pg cortisol per sample.

Carbon Radioisotopes↗
PubMed1980

Measurement by isotope dilution mass spectrometry of 17 alpha-ethynyloestradiol-17 beta and norethisterone in serum of women taking oral contraceptives.

The highly specific and accurate technique of isotope dilution mass spectrometry has been used for the measurement of 17 alpha-ethynyloestradiol-17 beta and norethisterone in serum. Serum samples were obtained from female volunteers who received 2.5 mg lynestrenol and 50 micrograms 17 alpha-ethynyloestradiol-17 beta in two different galenical preparations. The determination of total 17 alpha-ethynyloestradiol-17 beta (conjugated and non-conjugated) was carried out by the following procedure: (1) adsorption of the steroids from 1 ml serum to Amberlite XAD-2; (2) enzyme hydrolysis of the conjugated steroid; (3) addition of 1 ng [6,7-3H2] 17 alpha-ethynyloestradiol-17 beta as internal standard; (4) column chromatography on Sephadex LH 20; (5) derivative formation with heptafluorobutyric anhydride; (6) isotope dilution mass spectrometry at m/z 474 and 478 using a glass capillary gas-liquid chromatography column. For the measurement of norethisterone, which is the major metabolite of lynestrenol, 1 ng [7-3H]norethisterone was added to 0.5 ml serum. The labelled and the non-labelled steroids were extracted and purified by column chromatography on Sephadex LH 20. The norethisterone was reacted to form the 3-enol-17 beta-trimethylsilyl ether of norethisterone and [3H]norethisterone. For isotope dilution mass spectrometry the derivative was injected into the glass capillary column which was coupled to the mass spectrometer. The instrument was adjusted to m/z 442 and 444, corresponding to the molecular ions of the ether derivatives of norethisterone and [7-3H]norethisterone. Accuracy was achieved by the use of highly specific technique of mass spectrometry and the exact control of recovery using the isotope dilution principle. The precision was 4.5% (CV) for the determination of 17 alpha-ethynyloestradiol-17 beta and 2.5% (CV) for norethisterone. The lower limit of detection was at 20 pg ml-1 for both methods.

Contraceptives, Oral↗
PubMed1980

Biosynthesis of 16 alpha, 17 alpha-epoxy-oestratrienol in rat liver microsomes.

After incubation of 1,3,5(10),16-oestratetraenol, a 16-dehydrosteroid, with rat liver microsomes, 16 alpha, 17 alpha-epoxy-oestratrienol was isolated as metabolite. The compound was detected by the use of mass fragmentography after purification of the incubation extract by thin-layer chromatography. Since the epoxide is rapidly hydrolysed by a hepatic epoxide hydratase, only very small concentrations of this metabolite were present in the incubation extract. When styrene oxide was added to the incubation mixture as inhibitor of the epoxide hydratase, the yield of the steroid epoxide increased considerably. Final identification of the oestrogen epoxide was performed by recording mass spectra and by comparison with authentic reference material.

Animals↗
PubMed1980

Biosynthesis of 16 alpha, 17 alpha-epoxy-4-androsten-3-one in rat liver microsomes.

4,16-Androstadien-3-one was incubated with the microsomal fraction of male rat liver in the presence of a NADPH generating system and oxygen. The metabolites formed were extracted from the incubation medium and purified by thin-layer chromatography (tlc). Final identification was performed by combined gas liquid chromatography-mass spectrometry. Incubation of 4,16-androstadien-3-one resulted in the formation of a non-polar metabolite which proved to be 16 alpha, 17 alpha-epoxy-4-androsten-3-one. This epoxide is a shortlived intermediate which is rapidly hydrolysed by the microsomal epoxide hydratase to 16 beta, 17 alpha-dihydroxy-4-androsten-3-one. In order to increase the amounts of epoxide in the incubation mixtures, styrene oxide which is a potent inhibitor of the epoxide hydratase was added. Under these conditions, up to 8% of the 16-dehydro-steroid incubated was transferred to the 16 alpha, 17 alpha-epoxy-compound.

Androstane-3,17-diol↗