M.F. Lefevere

Isotope-dilution mass spectrometry in clinical chemistry

Publisher Summary This chapter discusses isotope dilution mass spectrometry in clinical chemistry. Mass spectrometry (MS) in clinical chemistry is increasingly used as an analytical tool to solve complex problems in biochemistry and medicine. As a matter of fact, there are five ways that mass spectrometry can be applied to problems in these areas: (1) confirmation of identity, (2) identification of unknowns, (3) elucidation of the structure of complex molecules, (4) quantitation of selected constituents present in ultratrace quantities, and (5) dynamic analysis of major constituents in vivo. In quantitative mass spectrometry, the signal intensity depends not only on the amount of sample but also on a number of other variables such as the ionization yield, focusing of the ion beam, and the amplification factor of the detector. As it is very difficult to keep these parameters constant over the whole period of analysis, nearly all quantitative applications of MS are based on a comparison of the ion current obtained from the component of interest with the ion current obtained from a standard. In quantitative analysis this can be accomplished either by the continuous admission of a reference sample at a constant rate concurrently with the sample under investigation or by the use of an internal standard (IS) that is added to the sample prior to MS analysis. The complexity and cost of these techniques, however, prevent a more generalized use in the near future. Meanwhile, there is no doubt that isotope dilution in combination with “classical” MS techniques has a great future in clinical chemistry.

High-performance liquid chromatographic assay of vitamin K in human serum.

A sensitive combined adsorption and reversed-phase high-performance liquid chromatographic procedure is described that permits the determination of endogenous vitamin K1 levels in serum. The separation of vitamin K1 (cis- + trans-isomers), vitamin K1 epoxide and the menaquinones MK-2, MK-4 and MK-9 by adsorption, reversed-phase and cyano-bonded phase chromatography is discussed. The methodology was further developed for the quantitative measurement of vitamin K1 in human serum. Concentrations as low as 500 pg/ml in serum could be detected. The identity of the vitamin K1 peak was confirmed by UV absorption spectrophotometry and re-chromatography. The method has been applied to the determination of serum levels in normal healthy individuals and patients treated with vitamin K1.

Selectivity effects in non-aqueous reversed-phase and normal-phase chromatography of geometric canthaxanthin isomers

Abstract Non-aqueous reversed-phase and normal-phase systems are reported for the separation of geometric isomers of the carotenoid canthaxanthin. On Zorbax ODS

Chromatographic determination of N-acetyl-DL-tryptophan and octanoic acid in human albumin solutions.

Chromatographic procedures have been developed for determination of the stabilizers N-acetyl-DL-tryptophan and octanoic acid in human albumin solutions. N-Acetyl-DL-tryptophan and the internal standard, N-formyl-DL-tryptophan, were separated by liquid chromatography on a reversed-phase column with UV detection at 280 nm. Deproteinization and extraction were carried out with methanol. The extraction recovery at the level of 4.9 mM was 92.5 +/- 2.5% (S.D.) (n = 10), and the average coefficient of variation (C.V.) for replicate analyses of albumin solutions (mean = 2.57, 10.44 and 17.10 mM) was 1.10% (n = 27). Octanoic acid was determined gas chromatographically as its methyl ester, with nonanoic acid as the internal standard. The sample pretreatment included acidification, extraction with hexane and derivatization with methanol-sulphuric acid. The relative recovery from albumin solutions was 89.7 +/- 5.8% (S.D.) (n = 6), and replicate determinations of the compound yielded a C.V. of 5.5% (mean = 14.82 mM, n = 9).

Bioanalytical mass spectrometry as applied to organic acid profiling and steroid reference methodology.

Gas chromatography-mass spectrometry (GC-MS) is a versatile and powerful diagnostic tool in clinical chemistry. Instrumental aspects of the technique are considered. The use of GC-MS for identification purposes is exemplified by the metabolic profiling of organic acids for the diagnosis of inborn errors of metabolism. Quantitative GC-MS is discussed with particular emphasis on the choice of the internal standard. For highest precision and accuracy, isotope dilution GC-MS is the most suitable technique; the application of ID-GC-MS to reference methods and reference materials is discussed in relation to the analysis of steroid hormones. The principles involved in devising calibration procedures and measurement protocols in quantitative GC-MS are outlined.