Chapter 10 - Sources of Inaccuracy in Biochemical Genetics Testing

Abstract

Metabolic disease biomarker measurement offers a unique opportunity to identify potential analytical interferences because many of the tests used in this field provide positive identification of these compounds through mass spectrometric identification. The bulk of the clinical workload in biochemical genetics involves analyses of amino acids, organic acids, and acylcarnitine, and these analyses are the focus of this chapter. Most laboratories do not utilize mass spectrometry for amino acid analysis in body fluids but remain dependent on chromatographic separation with peak identification using ninhydrin, which specifically interacts with primary and secondary amines, and spectrophotometric detection as well as quantification. Because this process does not have positive peak identification through mass spectrometry, a number of interferences do occur from primary amines, particularly commonly used antibiotics of the penicillin family. Interference from other primary amines is particularly prevalent when analyzing urine amino acid profiles because multiple metabolites are generated from some interfering compounds. Issues related to interference are less problematic when separation technology is coupled to mass spectrometric detection such as in urine organic acid analysis. Interferences can usually be identified based on their fragmentation patterns and retention time. However, the presence of some exogenous compounds, including the commonly used anticonvulsant valproic acid, can cause interpretive difficulties due to interference with endogenous metabolism. When mass spectrometry is used without separation, as in flow injection multiple-reaction monitoring analysis of acylcarnitine or amino acids, the major source of interference derives from the presence of isobaric compounds such as leucine, isoleucine, alloisoleucine, and hydroxyproline, which cannot be separated and may mask the diagnosis of maple syrup urine disease.