Isotopic fractionation of organic compounds in chromatography

Filer, Crist N.

doi:10.1002/(sici)1099-1344(199902)42:2<169::aid-jlcr178>3.3.co;2-s

Cited by 12 publications

(14 citation statements)

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“…Deuterated compounds, in general, are expected to elute earlier because their interactions with the non-polar stationary phase are not as strong as the protonated compounds [10]. It has been shown that chromatographic separation of isotopes is related to the number of deuterium atoms for structurally similar molecules [16].…”

Section: Discussionmentioning

confidence: 99%

“…Unfortunately, the chromatographic separation of isotopically labelled peptide pairs is commonly observed, especially for deuterated compounds. In this case, the heavy isotope generally elutes in advance of its lighter counterpart [10]. This phenomenon has influenced current practices of quantitative proteomics.…”

Section: Introductionmentioning

confidence: 99%

“…In contrast, it has been observed that labels incorporating isotopes other than deuterium generally lead to a much smaller separation of isotopic pairs. This is not surprising given that there is a doubling of mass for D vs H, while the change for 13 C over 12 C, for example, is only about 8% [10]. A general consequence of this observation is that labels employing isotopes other than deuterium, though more costly, have become the preferred choice in proteomics studies [11].…”

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Chromatographic behaviour of peptides following dimethylation with H2/D2-formaldehyde: Implications for comparative proteomics

Boutilier

Warden

Doucette

et al. 2012

Journal of Chromatography B

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Chromatographic behaviour of peptides following dimethylation with H2/D2-formaldehyde: Implications for comparative proteomics

Boutilier

Warden

Doucette

et al. 2012

Journal of Chromatography B

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“…Most probably, this is due to the enhanced clean up as for the older method a weak correlation was found between the CIR of both steroids and the concentration ratio of 5bDIOL/5aDIOL emerging from incomplete separation of both steroids on the HPLC. Small portions of 5bDIOL could contribute to the fraction collected for 5aDIOL depleting the CIR of this steroid according to chromatographic isotopic fractionation on a reversed-phase column [30,31]. This effect was circumvented by the modified HPLC conditions and differences in fraction collection as performed with the enhanced method.…”

Section: Reference Population-based Valuesmentioning

confidence: 99%

Combination of carbon isotope ratio with hydrogen isotope ratio determinations in sports drug testing

et al. 2013

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Carbon isotope ratio (CIR) analysis has been routinely and successfully applied to doping control analysis for many years to uncover the misuse of endogenous steroids such as testosterone. Over the years, several challenges and limitations of this approach became apparent, e.g., the influence of inadequate chromatographic separation on CIR values or the emergence of steroid preparations comprising identical CIRs as endogenous steroids. While the latter has been addressed recently by the implementation of hydrogen isotope ratios (HIR), an improved sample preparation for CIR avoiding co-eluting compounds is presented herein together with newly established reference values of those endogenous steroids being relevant for doping controls. From the fraction of glucuronidated steroids 5β-pregnane-3α,20α-diol, 5α-androst-16-en-3α-ol, 3α-Hydroxy-5β-androstane-11,17-dione, 3α-hydroxy-5α-androstan-17-one (ANDRO), 3α-hydroxy-5β-androstan-17-one (ETIO), 3β-hydroxy-androst-5-en-17-one (DHEA), 5α- and 5β-androstane-3α,17β-diol (5aDIOL and 5bDIOL), 17β-hydroxy-androst-4-en-3-one and 17α-hydroxy-androst-4-en-3-one were included. In addition, sulfate conjugates of ANDRO, ETIO, DHEA, 3β-hydroxy-5α-androstan-17-one plus 17α- and androst-5-ene-3β,17β-diol were considered and analyzed after acidic solvolysis. The results obtained for the reference population encompassing n = 67 males and females confirmed earlier findings regarding factors influencing endogenous CIR. Variations in sample preparation influenced CIR measurements especially for 5aDIOL and 5bDIOL, the most valuable steroidal analytes for the detection of testosterone misuse. Earlier investigations on the HIR of the same reference population enabled the evaluation of combined measurements of CIR and HIR and its usefulness regarding both steroid metabolism studies and doping control analysis. The combination of both stable isotopes would allow for lower reference limits providing the same statistical power and certainty to distinguish between the endo- or exogenous origin of a urinary steroid.

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“…The need of using 13 C containing amino acids is mostly due to differences in retention time displayed by the two isotopomers under reversed phase (RP) high performance liquid chromatography (HPLC). It is welldocumented that deuterated analogues can be chromatographically resolved varying the number of isotopes incorporated and the gradient conditions used in the LC run [21,22]. An additional advantage of site-specific metabolic incorporation of isotopes is the possibility to increase confidence in protein identification.…”

Section: In Vivo Stable Isotopic Labellingmentioning

confidence: 99%

Mass Spectrometry Based Strategies in Quantitative Proteomics

Brancia¹

2006

CAC

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Quantification of gene expression is the sine qua non in understanding how the components of a biological cell or an organism change and interact following external or internal perturbations. Analytical techniques that combine stable isotopic protein and peptides labelling and mass spectrometric analysis are becoming increasingly popular in determining protein abundance. The common motif, which recurs in these bio-analytical methods, is the parallel labelling of the protein samples with stable isotopes. The ratio of the differently labelled peptide ions observed in the mass spectrum reflects the differences in concentration between the species in the combined mixture. Isotopes can be introduced using labelled nutrients prior to proteolytic digestion or by chemical derivatisation of the peptide functional groups after proteolysis. Although metabolic incorporation of labelled isotopes provides a more global quantification strategy, chemical derivatisation based approaches are also suitable for those laboratories with basic proteomics facilities. Advantages and limitations of these techniques are reviewed, highlighting examples of specific biological applications.

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Isotopic fractionation of organic compounds in chromatography

Cited by 12 publications

References 0 publications

Chromatographic behaviour of peptides following dimethylation with H2/D2-formaldehyde: Implications for comparative proteomics

Chromatographic behaviour of peptides following dimethylation with H2/D2-formaldehyde: Implications for comparative proteomics

Combination of carbon isotope ratio with hydrogen isotope ratio determinations in sports drug testing

Mass Spectrometry Based Strategies in Quantitative Proteomics

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