Practical recommendations for the reduction of memory effects in compound‐specific ¹⁵N/¹⁴N‐ratio analysis of enriched amino acids by gas chromatography/combustion/isotope ratio mass spectrometry

Abstract

Gas chromatography/combustion/isotope ratio mass spectrometry (GC-C-IRMS) is a highly sensitive approach which allows the analysis of the (13)C/(12)C and (15)N/(14)N isotope composition of amino acids in the range of natural abundance or in slightly (13)C- and (15)N-enriched samples. However, the accuracy of measurements remains a permanent challenge. Here we show the effect of the presence of slightly (15)N-enriched compounds in physiological samples on the accuracy and reproducibility of (15)N-abundances of amino acids within or between analytical runs. We spiked several individual amino acids with the respective (15)N-labelled isotopomer and measured the (15)N/(14)N ratios of other amino acids in the same sample or in the following analytical runs. Intra- and inter-run memory effects can be observed in (15)N/(14)N ratios of amino acids. Sample throughput is reduced when cleaning runs using standard mixtures are required to restore initial conditions after runs of samples with (15)N-enriched analytes. Possible reasons for the observed phenomenon and its implications for work in the lower (15)N-enrichment range (<0.5 APE) are discussed and include different aspects of gas chromatography, derivatisation, and hot catalytic metal surface effects. Results need to be interpreted with caution if complex physiological samples contain (15)N-enriched amino acids beyond 500‰ δ(15)N (~0.18 APE).

The dietary protein paradox and threonine 15N‐depletion: Pyridoxal‐5'‐phosphate enzyme activity as a mechanism for the δ15N trophic level effect

“…The hydrolysates were dried and derivatized to form N ‐pivaloyl‐ i ‐propyl esters (NPP) of AAs as described in detail in Metges et al . and Petzke and Metges . The 15 N/ 14 N ratios were determined by gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS) using a Delta S isotope ratio mass spectrometer (Thermo Scientific, Bremen, Germany).…”

“…) that frequently used Glu or Asp are biochemically inaccurate, and the correct designations are Glx or Asx, which are used here and recommended for all future GC/C/IRMS studies. The nitrogen stable isotope composition was reported using the delta (δ) per mil (‰) notation as δ 15 N values relative to the international standard AIR‐N 2 (Ambient Inhalable Nitrogen, δ 15 N = 0.0‰, [ 15 N]/[ 14 N] = 0.0036765) . Differences between the δ 15 N values are expressed as Δ 15 N values as indicated.…”

“…[50,51] The hydrolysates were dried and derivatized to form N-pivaloyl-i-propyl esters (NPP) of AAs as described in detail in Metges et al [52] and Petzke and Metges. [53] The 15 N/ 14 N ratios were determined by gas chromatography/ combustion/isotope ratio mass spectrometry (GC/C/IRMS) using a Delta S isotope ratio mass spectrometer (Thermo Scientific, Bremen, Germany). Based on retention times the following AAs were detected: alanine (Ala), glycine (Gly), valine (Val), leucine (Leu), isoleucine (Ile), proline (Pro), aspartic acid/asparagine (Asx), threonine (Thr), serine (Ser), methionine (Met), glutamic acid/glutamine (Glx), phenylalanine (Phe), lysine (Lys), histidine (His), and tyrosine (Tyr).…”

“…The nitrogen stable isotope composition was reported using the delta (δ) per mil (‰) notation as δ 15 N values relative to the international standard AIR-N 2 (Ambient Inhalable Nitrogen, δ 15 N = 0.0‰, [ 15 N]/[ 14 N] = 0.0036765). [52,53] Differences between the δ 15 N values are expressed as Δ 15 N values as indicated. The plasma AAs from each individual rat were derivatized once and measured five times by GC/C/IRMS.…”

Compound-specific &lt;sup&gt;15&lt;/sup&gt;N stable isotope probing of N assimilation by the soil microbial biomass: a new methodological paradigm in soil N cycling

“…The technique uses 15 N stable isotope tracers and compound-specific 15 N analysis, but unlike previous 15 N stable isotope tracer work, uses amino acids (AAs; representing biochemical products) rather than ammonium (NH + 4 ) and nitrate (NO − 3 ) to directly investigate the flow of N into the biologically active soil organic N pool. Our 15 N compound-specific stable isotope probing ( 15 N-SIP) approach exploits the high selectivity and sensitivity of gas chromatographycombustion-isotope ratio mass spectrometry (GC-C-IRMS;Meier-Augenstein, 1999;Evershed et al, 2006;Maxfield et al, 2012;Petzke and Metges, 2012) ensuring highly "diluted" metabolic products of the 15 N tracer are readily detectable in the incubated soils. Furthermore, by using only "low" tracer doses perturbations to native soil conditions are minimised.…”