![]() Deuterated EDTA is available, but this is quite expensive. ![]() However, the use of EDTA introduces a number of 1H NMR signals for the free form as well as the Ca- and Mg-bound chelate forms. Chelation of the divalent metals with EDTA has been used with promising results (Asiago et al. A potential problem with this approach however may be the degradation of pH sensitive metabolites through hydrolysis or redox reactions.Ī number of approaches have been proposed to remove the divalent metal ions from urine samples and therefore remove their influence on metabolite peak chemical shifts. 2011 Lehnert and Hunkler 1986 Sze and Jardetzky 1994 Wevers et al. An alternative approach to minimise pH-dependent shifts is to move the sample pH to extreme values away from metabolite p K a values and towards the acid/base chemical shift limits (Beneduci et al. In addition, very salty samples reduce NMR sensitivity, especially for cryogenically cooled probes. While the use of a strong buffer may limit many metabolite peaks chemical shifts, a small number of metabolites, such as citrate and histidine, still exhibit strong inter-sample chemical shift variations (Lindon et al. ( 2009) recommended using 1.5 M buffer solutions in a 1:10 volume ratio of urine. 2007) recommended a minimum final concentration of 0.3 M for normal urine, and 1 M for concentrated urine samples. Buffers are commonly added to urine samples to control the pH variation of different samples. 2008).Ī number of studies have investigated different sample preparation techniques in order to limit the pH and metal ion dependent NMR peak position variation. The main metal ions present in urine are sodium, potassium, and the divalent ions calcium and magnesium, however it is the divalent ions that are the main contributors (after pH) to peak chemical shift variability (Ackerman et al. However, differences in its other components (such as urea, salts and other ions) are common, and result in often large variations in metabolite peak chemical shifts (Lindon et al. Urine is a popular biofluid used for metabolomic investigations, as it consists of various metabolites that can provide insight into a number of metabolic processes and disease states (Lindon et al. However, metabolite chemical shifts are sensitive to the chemical environment and subtle matrix effects, such as differences in pH and ionic strength, generally lead to inter-sample peak position variation (Weljie et al. 1H NMR is widely used for the metabolomic analysis of biofluids, as it provides quantitative, structural information on a wide range of metabolites, in a non-destructive and highly reproducible manner (Nicholson and Wilson 1989 Wishart 2008 Zhang et al.
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