Application of high-performance liquid chromatography with isotope-ratio mass spectrometry for measuring low levels of enrichment of underivatized materials

Abramson, Fred P.; Black, Gavin E.; Lecchi, Paolo

doi:10.1016/s0021-9673(00)01032-3

Cited by 26 publications

(21 citation statements)

References 15 publications

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“…Several attempts have been made over the last decades to couple LC with IRMS, including a chemical reaction interface [32] and a moving wire [33][34][35]. The first commercially available instrument was introduced in 2004 and is based on wet chemical oxidation [36].…”

Section: Liquid Chromatography-irms (Lc-irms)mentioning

confidence: 99%

Current challenges in compound-specific stable isotope analysis of environmental organic contaminants

et al. 2012

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Compound-specific stable-isotope analysis (CSIA) has greatly facilitated assessment of sources and transformation processes of organic pollutants. Multielement isotope analysis is one of the most promising applications of CSIA because it even enables distinction of different transformation pathways. This review introduces the essential features of continuous-flow isotope-ratio mass spectrometry (IRMS) and highlights current challenges in environmental analysis as exemplified for the isotopes of nitrogen, hydrogen, chlorine, and oxygen. Strategies and recent advances to enable isotopic measurements of polar contaminants, for example pesticides or pharmaceuticals, are discussed with special emphasis on possible solutions for analysis of low concentrations of contaminants in environmental matrices. Finally, we discuss different levels of calibration and referencing and point out the urgent need for compound-specific isotope standards for gas chromatography-isotope-ratio mass spectrometry (GC-IRMS) of organic pollutants.

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Section: Liquid Chromatography-irms (Lc-irms)mentioning

confidence: 99%

Current challenges in compound-specific stable isotope analysis of environmental organic contaminants

et al. 2012

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“…To determine the carbon isotope ratios, a reactant or product mixture is delivered by high-pressure liquid chromatography (HPLC) to a chemical reaction interface (CRI), where it is converted by pyrolysis to C⌷ 2 , and injected into the IRMS. This analytical system is ideally suited to measure isotope ratios in nonvolatile samples (33)(34)(35). We have used these techniques, along with a suite of specifically labeled 18 O-labeled GTP substrates ( Fig.…”

mentioning

confidence: 99%

Kinetic isotope effects in Ras-catalyzed GTP hydrolysis: Evidence for a loose transition state

Black

Lecchi

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

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A remote labeling method has been developed to determine 18 O kinetic isotope effects (KIEs) in Ras-catalyzed GTP hydrolysis. Substrate mixtures consist of 13 C-depleted GTP and [ 18 O, 13 C]GTP that contains 18 O at phosphoryl positions of mechanistic interest and 13 C at all carbon positions of the guanosine moiety. Isotope ratios of the nonvolatile substrates and products are measured by using a chemical reaction interface͞isotope ratio mass spectrometer. The isotope effects are 1.0012 (0.0026) in the ␥ nonbridge oxygens, 1.0194 (0.0025) in the leaving group oxygens (the ␤-␥ oxygen and the two ␤ nonbridge oxygens), and 1.0105 (0.0016) in the two ␤ nonbridge oxygens. The KIE in the ␤-␥ bridge oxygen was computed to be 1.0116 or 1.0088 by two different methods. The significant KIE in the leaving group reveals that chemistry is largely rate-limiting whereas the KIEs in the ␥ nonbridge oxygens and the leaving group indicate a loose transition state that approaches a metaphosphate. The KIE in the two ␤ nonbridge oxygens is roughly equal to that in the ␤-␥ bridge oxygen. This indicates that, in the transition state, Ras shifts one-half of the negative charge that arises from P ␥-O␤-␥ fission from the ␤-␥ bridge oxygen to the two ␤ nonbridge oxygens. The KIE effects, interpreted in light of structural and spectroscopic data, suggest that Ras promotes a loose transition state by stabilizing negative charge in the ␤-␥ bridge and ␤ nonbridge oxygens of GTP. R as is the prototypical member of the family of small G proteins, which along with G␣ subunits of heterotrimeric G proteins, constitute a class of GTP hydrolases that regulate diverse signaling pathways in eukaryotes (1). Ras orchestrates multiple signaling pathways and regulates cell differentiation, proliferation, and apoptosis (2-4). The GTP-bound forms of G proteins are functionally active: that is, they bind to ''effector'' molecules and regulate their activities or location within the cell. Hydrolysis of GTP results in deactivation and effector release (5). In the absence of other factors, the duration of the active signaling state depends on the intrinsic hydrolytic rate of the G protein, which is typically very slow. However, Ras and other G proteins are subject to specific regulation by GTPase-activating proteins (GAPs), which accelerate intrinsic hydrolytic rates by factors ranging from 10 to 10 5 . In particular, RasGAP increases the GTPase rate of Ras by a factor of 10 5 , from 10 Ϫ4 s Ϫ1 to 10 s Ϫ1 (6). Mutations that impair either intrinsic or GAP-facilitated GTPase activity leave Ras in a prolonged state of activation, which is responsible for its role in oncogenic diseases (7).Ras catalyzes the in-line attack of water on the ␥ phosphate of GTP with inversion of configuration (8). However, the nature of the transition state and the rate-limiting step of Ras-catalyzed GTP hydrolysis remain unclear (9-16). A phosphoryl transfer reaction may either proceed through a metaphosphate or a phosphorane intermediate, or by a concerted pathway (Fig. 1) (17, 18...

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“…4). The LC/IRMS device became commercially available in 2004 (Krummen et al, 2004) after experiments conducted over the last two decades were able to link (or hyphenate) LC to IRMS (Caimi & Brenna, 1993;Teffera et al, 1993;McLean et al, 1996;Brenna et al, 1997;Abramson, Black, & Lecchi, 2001).…”

Section: E Lc/irmsmentioning

confidence: 99%

High‐precision mass spectrometric analysis using stable isotopes in studies of children

Schierbeek

Akker

Fay

et al. 2011

Mass Spectrometry Reviews

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The use of stable isotopes combined with mass spectrometry (MS) provides insight into metabolic processes within the body. Herein, an overview on the relevance of stable isotope methodology in pediatric research is presented. Applications for the use of stable isotopes with MS cover carbohydrate, fat, and amino acid metabolism as well as body composition, energy expenditure, and the synthesis of specific peptides and proteins, such as glutathione and albumin. The main focus of these studies is on the interactions between nutrients and the endogenous metabolism within the body and how these factors affect the health of a growing infant. Considering that the early imprinting of metabolic processes hugely impacts metabolism (and thus functional outcome) later in life, research in this area is important and is advancing rapidly. The major fluxes on a metabolic level are the synthesis and breakdown rates. They can be quantified using kinetic tracer analysis and mathematical modeling. Organic MS and isotope ratio mass spectrometry (IRMS) are the two most mature techniques for the isotopic analysis of compounds. Introduction of the samples is usually done by coupling gas chromatography (GC) to either IRMS or MS because it is the most robust technique for specific isotopic analysis of volatile compounds. In addition, liquid chromatography (LC) is now being used more often as a tool for sample introduction of both volatile and non-volatile compounds into IRMS or MS for (13)C isotopic analyses at natural abundances and for (13)C-labeled enriched compounds. The availability of samples is often limited in pediatric patients. Therefore, sample size restriction is important when developing new methods. Also, the availability of stable isotope-labeled substrates is necessary for measurements of the kinetics and concentrations in metabolic studies, which can be a limiting factor. During the last decade, the availability of these substrates has increased. Furthermore, improvements in the accuracy, precision, and sensitivity of existing techniques (such as GC/IRMS) and the development of new techniques (such as LC/IRMS) have opened up new avenues for tackling these limitations.

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Application of high-performance liquid chromatography with isotope-ratio mass spectrometry for measuring low levels of enrichment of underivatized materials

Cited by 26 publications

References 15 publications

Current challenges in compound-specific stable isotope analysis of environmental organic contaminants

Current challenges in compound-specific stable isotope analysis of environmental organic contaminants

Kinetic isotope effects in Ras-catalyzed GTP hydrolysis: Evidence for a loose transition state

High‐precision mass spectrometric analysis using stable isotopes in studies of children

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