Quantitative mass spectrometry in clinical chemistry

As has been demonstrated, mass spectrometry provides a powerful analytical tool for the accurate measurement of small amounts of substances in a complex biological matrix. In our laboratory this technique is used as a reference method for measuring the routine clinical chemical parameters creatinine, uric acid, cholesterol, total glycerol and the hormones cortisol, testosterone, oestradiol-17β, oestriol, progesterone, aldosterone and thyroxine in human serum. In general, the analytical procedure for measuring a substance by isotope dilution mass spectrometry (IDMS) consists of the following steps:

1. Addition of a certain amount of the isotopically labeled analyte to the serum sample.
2. Isolation and purification of the labeled and the non-labeled endogenous analyte from the biological matrix.
3. Derivative formation of the isolated and purified labeled and non-labeled compound.
4. Selected ion recording of characteristicm/z values of the labeled and non-labeled analyte using combined gas chromatography-mass spectrometry (GCMS).
5. Calculation of the concentration of the analyte from the isotope ratio measured by GCMS.

The methods described here are now routinely in use for the quality control scheme of the Deutsche Gesellschaft für Klinische Chemie for assessing target values in external quality control sera. The reference method values obtained by IDMS provide a reliable basis for evaluating and comparing the results of collaborative surveys.     

Einsatz der echten Integration in der Atomabsorptions-Spektroskopie

An improvement of the signal-to-noise ratio in atomic absorption spectroscopy may be obtained by each of the following three methods of signal averaging:

increase of the time constant

digital signal averaging

true integration of the signal

The most adequate of these methods appears to be true integration. Two examples are presented to demonstrate the improved signal-to-noise ratio due to the application of this method:

trace analysis of mercury in water by means of flameless AAS. The detection limit appears to be 0.2 ng which corresponds to a concentration of 2 · 10^?13 g/ml.

trace analysis of copper in human urine by means of flame AAS: 1,3 · 10^?8 g/ml copper could be detected without any solvent extraction process.

     

Applications of pattern recognition

This poster illustrates the lecture on Pattern Recognition and gives recently published and unpublished examples, mainly from the laboratory from the first author. The applications concern:

- the determination of metabolic pathways of branched chain fatty acids (by clustering),

- the development of a genetic classification of meteorites (by clustering),

- the classification of cholinergic agents according to their interaction with different receptors (by clustering),

- the structure of a data set consisting of gaschromatographic profiles in samples collected in pollution monitoring stations (by factor analysis and pattern recognition),

- factors determining GLC behaviour of solutes (by factor analysis and multiple regression),

- the classification of olive oils according to geographic origin (by principal components and pattern recognition),

- the diagnosis of thyroid status (by pattern recognition).

     

Zustandsdiagramme binärer Phospholipidmischungen

The transition curves were calculated on the basis of two types of ideal phase diagrams:

miscibility in the liquid phase and immiscibility in the solid phase;

complete miscibility in both liquid and solid phases.

A comparison was made of the theoretical curves with observed transition curves.     

Röntgenkristallstrukturanalyse und Organische Chemie

An introduction into X-ray crystal structure analysis as method to determine the structure of organic compounds is given. In this connection two new methods for phase determination of X-ray reflections of macromolecules are reported. A few specially selected samples of structures are given to demonstrate the efficiency of X-ray crystal structure analysis with regard to the elucidation of stereochemical problems in the field of organic chemistry. The examples of application are heterocyclic organic compounds (cycloaddition products of nitrile-ylides, a stable azomethinimine, a cyanine dyestuff) and biologically relevant molecules (aminoacids of the amatoxins, the main poison of the green deathcup toadstool). The mentioned molecular structures determined by X-ray crystal structure analysis are discussed under the following aspects.

1. Elucidation of constitution, configuration and conformation.
2. Identification and verification of chemical pathways for new classes of compounds.
3. Bond relationships, influence of ring strain and other intramolecular interactions upon bond lengths and angles.
4. The attempt to find general stereochemical principles among related molecules or molecular parts points to the fundamental aspects of these experiments. Generally, the 1,4-diaza-s-trans-1,3-butadiene system, the steric influence of a free electron pair at a double bonded nitrogen and the CF₃-group are studied.
5. Obtaining of initial parameters for quantum mechanical calculations and comparison with theoretical results.
6. With regard to the structure analysis of two amino acid fragments of the amatoxins the configuration of the hydroxylated isoleucines of the amatoxins and the configuration at the chiral SO center of the natural amatoxins and of the synthetic phalloidin sulphoxides are fixed. The configuration at the SO center is decisive for the poisonous effect.

     

Limits of cyclodextrin application in oral drug preparations

Cyclodextrins are applied to facilitate formulation problems, to improve stability and bioavailability. Following factors are determining whether or not cyclodextrins can be applied in oral pharmaceutical preparations:

- properties of the selected CD: solubility, price, specific catalytic properties,

- the drug to be complexed: molecular weight, polarity, solubility,

- drug dose

- solubility properties of the complex and the “super solubility” /temporary over-saturation/

- complex stability and possibility to shift the dissociation equilibrium toward the appropriate direction

- legislative procedures

     

Theoretical discussion of methods of elimination of matrix effects in non-dispersive X-ray analysis

Theoretical consideration concerning some possibilities for the elimination of matrix effects in non-dispersive X-ray fluorescence and absorption analysis are discussed. The theoretical treatment is concerned with the following methods:

1. double-channel absorption edge analysis,
2. concentration increase and dilution method in fluorescence analysis,
3. fluorescence-absorption method,
4. emission-transmission method,
5. fluorescence-Compton scattering method,
6. method of multicomponent analysis.

On the basis of the derived formulas, nomographic methods of interpretation of the data are given. Using these methods it is possible to determine unambiguously the concentration of the relevant element. The formulas are also convenient for numerical interpretation. The introduction of the concept of “generalized sensitivity” allows the comparison of various radiometric methods.     

Charakterisierung der Mischbarkeitseigenschaften binärer Monoschichten durch Messung der Gleichgewichtsspreitungsdrucke

1. The equilibrium spreading pressures were studied as a function of composition of the following binary systems by use of the Wilhelmy-method atT=295 K on aqueous substrate:

octadecyldiethylphosphine oxide/1-hexadecanol (I)

octadecyldiethylphosphine oxide/1-eicosanol (II)

octadecyldiethylphosphine oxide/stearic acid (III)

1. The equilibrium spreading pressures of the pure components of the systems I und II, respectively, decrease due to addition of the corresponding components of mixture. A minimum occurs at the mole fractionx=0,50. The equilibrium spreading pressures of mixtures of system III rise with increasing mole fraction of octadecyldiethylphosphine oxide.
2. The three spreading diagrams are based on one general type. The phase rule is applied and shows that the components of the 3 systems must be immiscible in the monolayer but miscible in the bulk phase.

     

Thermoanalytical control of paint-shop wastes prior to disposal

The paint-shop wastes under study originated from metallurgical factory painting houses. Displaying a high toxicity and flammability, they are classified as specifically hazardous to the natural environment. Paint-shop wastes can be disposed of in the following ways:

solidification and deposition in sanitary landfills;

storage on a special dumping ground;

incineration.

This paper presents the physicochemical composition and thermal analysis (DTA and TG) of paint-shop wastes and their mixtures with various components. Thermoanalytical measurements were carried out in a dynamic atmosphere of air. Enthalpies were calculated from the peak areas of the DTA curves. Thermoanalytical data were compared with calorimetric results obtained with an oxygen bomb.     

Drug targeting by drug entrapment into ultrafine compartments as carriers

The incorporation of drugs into vesicles is one of several technological methods for the optimization of targeted drug delivery and controlled drug targeting. The main problems will always remain

? To design inert auxiliary accompanying materials in order to overcome side reactions

? To use body-friendly and biodegradable macromolecular carrier materials for the therapeutic system

? To miniaturize the dosage form dramatically in the submicroscopic size range in order to eliminate foreign body irritations

? To develop ultrafine solid and amorphous vesicular compartments (nanocapsules, nanopellets, nanoparticles) to get stable systems with good tissue transfer and organ targeting properties

The actual stand of the incorporation of drugs and biologic active material into ultrafine colloidal solid capsules is reviewed here as for instance:

? Immunoactive material

? Fluorescent indicators in body fluids

? Controlled and sustained release systems

? Nonspecific drug targeting of the first-order (passage through endothelial tissues)

? Second-order targeting (a specific transparenchymal migration), and a highly specific targeting of the third-order (transcellular passage, especially lysosomal transports)

Examples for some of these applications are given. It can be shown that such ultrafine vesiculated capsules offer some advantages when applied parenterally, but also partly for oral application. In the future, still more studies are necessary finally to clarify the importance and practical use of such ultrafine targeting carriers.     

Parameter distortion by unappropriate nonisothermal treatment

The aim of this contribution is to draw attention to the following problems:

1. boundary conditions of kinetic models,
2. distortion of kinetic data by cutting-off the peak ends,
3. regression analysis involved in the evaluation of kinetic data.

     

Cellular and metabolic engineering

Metabolic engineering is defined as the purposeful modification of intermediary metabolism using recombinant DNA techniques. Cellular engineering, a more inclusive term, is defined as the purposeful modification of cell properties using the same techniques. Examples of cellular and metabolic engineering are divided into five categories:

1. Improved production of chemicals already produced by the host organism;
2. Extended substrate range for growth and product formation;
3. Addition of new catabolic activities for degradation of toxic chemicals;
4. Production of chemicals new to the host organism; and
5. Modification of cell properties.

Over 100 examples of cellular and metabolic engineering are summarized. Several molecular biological, analytical chemistry, and mathematical and computational tools of relevance to cellular and metabolic engineering are reviewed. The importance of host selection and gene selection is emphasized. Finally, some future directions and emerging areas are presented.     

Thermogravimetry of the NiO-Al2O3 system in a hydrogen flow

Thermogravimetry in a hydrogen flow is used to distinguish different forms of NiO-Al₂O₃ bonding at the temperatures of reduction of NiO. The influences of the temperature of preliminary treatment and the component ratio on the shapes of the TG and DTG curves of reduction were represented. By comparison of the results obtained with different weights and with different particle sizes, the influences of diffusion of water vapour in macropores on the characters of TG and DTG curves were studied. Different types of TG and DTG curves of reduction were observed:

1. Curves usual in thermal analysis, whose positions depend on the specimen weight and on the dispersity of the sample.
2. Curves whose position displays almost no change with variations of 1–2 orders the specimen weight.
3. Curves the number of reduction rate peaks in which depend on the experimental conditions.

     

Kinetics of immobilized pig heart fumarase

The kinetics of immobilized pig heart fumarase are described and compared with the properties of the enzyme free in solution.

1. An analogous pH dependence of initial activity is found for free and immobilized enzyme.
2. Immobilized and free fumarase deviate from classical Michaelis-Menten kinetics in the same way. The apparent K_m values are three to eight times higher for the immobilized (2 mg/g gel) enzyme.
3. The specific activity of immobilized fumarase is dependent on the final enzyme concentration on the gel; normal specific activities are observed when 50 ?g fumarase is immobilized per gram of gel, whereas the specific activity decreases with increasing enzyme concentration.
4. The activation energies for free and immobilized fumarase (50 ?g/g gel) were found to be identical between 22 and 32?C and with L-malate as substrate (E_a = 12,290 cal/mol at pH 7.9). Upon increasing the concentration of fumarase on the gel, the activation energy decreases.

Our results indicate that the true catalytic properties of fumarase are not affected by immobilization of this enzyme. The slight differences observed when fumarase is immobilized at concentrations higher than 50 ?g/g gel must be attributed to diffusional limitation at the surface of the Sepharose matrix.     

An experimental study of the surface rheology of adsorbed protein layers

Adsorbed gelatin layers have been characterized by measuring the surface equilibrium tension and surface rheological parameters of shear, found by surface creep experiments. After the determination of these parameters the solution below the adsorption layer (gelatin + phosphate buffer) was substituted by a gelatin-free phosphate-buffer-solution. The differences of the parameters calculated before and after the substitution of the bulk phase allowed us to prove some model assumptions of the protein adsorption and to characterize the physical contents of some of the surface rheological parameters determined. The parameters calculated and measured permit the distinction of three different concentration ranges of gelatin:

1. the range of reversible adsorption layer.
2. the ranges of saturated, irreversible adsorption layer, and
3. the range of multilayer formation.

     

Thermal analysis and microstructure of solids and solid state reactions

Thermoanalytical and microstructural measurement techniques allow the characterization of solids, their transformation, decomposition, specific surface and pore structure. These techniques are of importance for solids with high specific surface and/or porosity. Thermoanalytical techniques are applied to detect solid state reactions, to determine reaction temperatures and to monitor the progress of the reaction. They are useful to simulate thermal processes in the industry and to receive meaningful results with small samples in a short time. Specific surface, cumulative pore volume and pore volume distribution are obtained by intrusion and absorption techniques. Here selected applications of thermoanalytical and microstructure investigations in modern fields of materials research will be presented, as:

-pore analysis of sintering steps of ceramics,

-secondary structure of catalyst granules,

-template decomposition in zeolites,

-firing process in a composide ceramic tape,

-ignition of coke.

     

Mikrobestimmung von monosubstituierten Acetylenverbindungen durch Bestimmung des aktiven Wasserstoffs mit Alanat

1. The determination of monosubstituted acetylenic compounds was investigated at various temperatures (?60° to +95°C) by the alanate method described byMerz.
2. At 90°C all active hydrogen atoms were determined with sufficient accuracy and good precision, but at 0° C the active hydrogen atom of the ethinyl group in monosubstituted acetylenes did not react. This is the basis of a method for determining the acid hydrogen in the ethinyl group: the active hydrogen reactive at 0° C is first determined, then the temperature is raised to 90° C, when all the remaining active hydrogen atoms are determined.
3. The Merz method for determining active hydrogen is in our opinion the most suitable chemical method for determining ethinylcarbinols.
4. It has been shown that active hydrogen atoms react faster with alanate (sodium diethyldihydridoaluminate) than with Grignard reagent. The active hydrogen atoms in certain compounds do not react quantitatively with Grignard reagent within the normal tune. The alanate method is thus more generally applicable than the Grignard method.

     

Penetration von Polyethylenglycoldodecylethern in gespreitete Methylstearatmonoschichten

1. The penetration isotherms of octaethyleneglycoldodecyl ether and tetraethyleneglycoldodecyl ether in methyl stearate monolayers at different film areas A_M were measured at T=295 K.
2. The penetration isotherms of the system methyl stearate/octaethyleneglycoldodecyl ether depended only weakly on the area of methyl stearate. Also, the relative shift of the penetration isotherms, related to the adsorption isotherm, was small.
3. The general penetration behaviour of both the systems methyl stearate/tetraethyleneglycoldodecyl ether and methyl stearate/octaethyleneglycoldodecyl ether was nearly identical.
4. The thermodynamical analysis of the system methyl stearate/octaethyleneglycoldodecyl ether indicated that the components are only partially miscible.

     

The development of heat flow calorimetry as a tool for process optimization and process safety

Classical thermo-analytical micro methods (DTA, DSC) are still very useful for process work, but medium scale instruments based on heat flow measurement are attaining an increasingly important role in this domain. As in many areas, development of reaction calorimetry for industrial applications was driven by needs and by available means (technical capabilities). The needs have been fairly constant over the past decades. There are data needs:

Reaction rates

Heat release rates

Heat of desired reactions and decompositions

Heat capacities and heat transfer capacities

It took the specialists of calorimetry a long time to recognize and to accept the operational needs, namely:

Working under controlled temperature conditions (constant temperature, temperature ramps)

Adding components during runs (continuously or in portions)

Simulation of industrial mixing conditions

The main driving force for the development of process oriented calorimetric instruments was the evolution of electronic hardware which made the control of heat flow on a (non micro) laboratory scale easy. The paper gives an overview on the principles of heat flow control and reviews the developments of the fifties and sixties, when the matching of heat flow with heat release by reactions was the goal. With the advent of fast and powerful laptop computers, the focus has shifted. Now, the deduction of true heat release rates from signals which may be badly distorted, is the goal. Some recent developments are reviewed and the hope is expressed that calorimetric equipment, inexpensive enough to be affordable for every laboratory engaged in process work, will be available soon.     

Application of thermal analysis in solid industrial wastes treatment

The choice of an appropriate and safe disposal alternative should be based on the wide range of physicochemical examination thermal analysis in conjunction with other data enables identification of wastes, allows determination of weight losses at any stage of thermal decomposition and characterization of the combustible properties of wastes. In this paper the physicochemical composition of some industrial wastes, which create serious hazards to the natural environment is presented. The following waste materials were investigated:

tar wastes from several departments of the coking plant

paint-shop wastes from a metallurgical factory.

Thermoanalytic measurements were carried out in the dynamic atmosphere of air. Enthalpic values were calculated from the peak areas of the DTA curves. Thermoanalytic data were compared with calorimetric results obtained from an oxygen bomb. The disposal methods for above-mentioned wastes are proposed.