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Measurement traceability is probably the most important tool for achievement of comparability in clinical chemistry. As stipulated by the In Vitro Diagnostica Directive of the European Union and several ISO standards, values assigned to calibrators and control materials must be traceable to reference materials and/or reference procedures of higher order. In the German proficiency testing system, statutory use of reference measurement procedures for several measurands has been in force since 1988. As a result, reference procedures are now regularly applied for the setting up of target values in the control samples of internal and external quality assessment and for assigning values to the manufacturer's calibrator and control materials. Noticeably, the comparability of results obtained by different diagnostic tests has greatly improved for the measurement of many metabolites and substrates, e.g. creatinine, cholesterol, uric acid, total glycerol and urea. For many measurands in laboratory medicine the implementation of the concept of traceability proves to be much more difficult; this mainly concerns the measurement of proteins, in particular enzymes, proteo-hormones, tumour markers and cardiac markers. For such measurands the analyte must first of all be distinctively defined before a reference system can be established which comprises reference procedures, reference materials and networks of reference laboratories.

The new version of ISO Guide 34 requires producers of certified reference materials (CRMs) to include contributions of possible instability to the overall CRM uncertainty, to obtain a value for the uncertainty in compliance with the Guide to the Expression of the Uncertainty in Measurement (GUM). A pragmatic approach to estimating the uncertainty of stability is presented. It relies on regression analysis of stability data with subsequent testing of the slope of the regression line for significance. If the slope is found to be statistically insignificant, a shelf life is chosen and the uncertainty connected with this time is estimated via the standard deviation of the slope. This uncertainty is included in the overall uncertainty of the CRM. This approach is explained with examples showing its applicability to matrix CRMs.

Uncertainties of four enzyme-CRMs that have recently been certified in a co-operation between the IRMM and the International Federation for Clinical Chemistry were estimated. Estimation was based on the sum of the uncertainties of characterization, homogeneity and stability. Data from the certification collaborative study were used to estimate laboratory uncertainties, which form the basis for the uncertainty of characterization. Estimations for the uncertainty of homogeneity were derived from classical homogeneity studies. The estimations of uncertainty of stability caused the most difficulties. Realistic uncertainties fitting the needs of customers while being derived from measurement data based on theoretical considerations were obtained.

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: The methods described here are now routinely in use for the quality control scheme of the Deutsche Gesellschaft fur 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.

Untersuchungen der letzten Jahre haben gezeigt, das Oestrogene in der Leber zu wasserloslichen Metaboliten umgewandelt werden. Unter diesen Verbindungen konnten die 1- und 4-Glutathionthioather der 2-Hydroxyoestrogene identifiziert werden. In der vorliegenden Arbeit werden Verfahren zur Isolierung, Trennung und Identifizierung der Thioather aus biologischem Material beschrieben. Die Verfahren bestehen aus Saulen-Chromatographie an Sephadex, Hochspannungspapierelektrophorese und Massenspektrometrie. Es werden verschiedene Verfahren zur Darstellung geeigneter Derivate fur die Massenspektrometrie diskutiert. Als am besten geeignet erwiesen sich die Trimethylsilylderivate, deren Fragmentierung an Hand von Beispielen erlautert wird. Mit Hilfe der Massenspektrometrie konnen die 1- und 4-Thioather der 2-Hydroxyoestrogene einwandfrei voneinander unterschieden werden. Der Substanzbedarf fur die Aufnahme gut auswertbarer Massenspektren liegt zwischen 1 und 5 μg.

Die quantitative Bestimmung geringer Steroidkonzentrationen in Korperflussigkeiten erfordert mit den heute bekannten physikalisch-chemischen Verfahren (Gas-Chromatographie, Doppelisotopenderivatverdunnungsmethoden) einen erheblichen methodischen und zeitlichen Aufwand. Eine wesentliche Vereinfachung bei hoher Empfindlichkeit und Spezifitat bietet der Einsatz des Gas-Chromatographen-Massenspektrometers. Hierbei wird das Massenspektrometer als hochspezifischer Detektor fur den Gas-Chromatographen benutzt; dies erfolgt durch Einstellung eines geeignetenm/e-Wertes. Mit Hilfe des Multiple Ion Detectors ist es auch moglich, mehrere Massen gleichzeitig aufzuzeichnen. Die Brauchbarkeit dieses Verfahrens wurde am Beispiel folgender Methoden zur Bestimmung von Steroidhormonen demonstriert: Testosteron im Plasma, Aldosteron im Urin, Oestradiol-17β und Oestron im Plasma. Als besonders vorteilhaft erwies sich bei der quantitativen Bestimmung der Oestrogene die Verwendung der entsprechenden Dideuteroverbindungen als Standardsubstanzen. Die vorlaufigen Nachweisgrenzen der hier angegebenen Verfahren liegen fur Aldosteron und Testosteron bei 1 ng, fur Oestron und Oestradiol-17β bei 0,05 ng.

A method for quantitative of ethinyl estradiol, using isotopic-dilution mass spectrometry, is described. In addition, the equation for calculating the analytical results of the assay is provided. The method's sensitivity is 25 pg/ml at its lower limit, and its specificity and precision are based on a coefficient of variation of only 2.5%, as calculated from duplicate determinations. This method requires, ideally, an isotopically labeled conjugate for ethinyl estradiol; however, none is commercially available. Therefore, it is not possible to measure the accurate recovery of product during the complete analytical procedures, but only from the step after enzyme hydrolysis (complete instructions for assay performance are the bulk of the text). Nevertheless, it is argued that this technique for determining ethinyl estradiol in human plasma will be a valuable tool for comparing bioavailability of different oral contraceptive drugs.