Determination of adenosine nucleotides in cultured cells by ion-pairing liquid chromatography–electrospray ionization mass spectrometry

Qian, Tianxiu; Cai, Zongwei; Yang, M. S.

doi:10.1016/j.ab.2003.10.028

Cited by 70 publications

(79 citation statements)

References 36 publications

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“…N,N-dimethylhexylamine (N,N-DMHA) has also caused the formation of adducts (Tuytten et al, 2002). These adducts have been used as quantifying ions by some (Cai, Song, & Yang, 2002;Qian, Cai, & Yang, 2004), whereas others, using 1,5-DMHA, found that the product ions of these adducts were not sufficiently specific because only the adduct was lost upon fragmentation . Unless the intensity of the non-adduct is unacceptable, the use of adducts should be avoided in quantitative bioanalysis.…”

Section: B Ionization Suppressionmentioning

confidence: 97%

Mass spectrometry in the quantitative analysis of therapeutic intracellular nucleotide analogs

Jansen

Rosing

Schellens

et al. 2011

Mass Spectrometry Reviews

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Nucleoside analogs are widely used in anti-cancer, anti-(retro)viral, and immunosuppressive therapy. Nucleosides are prodrugs that require intracellular activation to mono-, di-, and finally triphosphates. Monitoring of these intracellular nucleotides is important to understand their pharmacology. The relatively involatile salts and ion-pairing agents traditionally used for the separation of these ionic analytes limit the applicability of mass spectrometry (MS) for detection. Both indirect and direct methods have been developed to circumvent this apparent incompatibility. Indirect methods consist of de-phosphorylation of the nucleotides into nucleosides before the actual analysis. Various direct approaches have been developed, ranging from the use of relatively volatile or very low levels of regular ion-pairing agents, hydrophilic interaction chromatography (HILIC), weak anion-exchange, or porous graphitic carbon columns to capillary electrophoresis and matrix-assisted light desorption--time of flight (MALDI-TOF) MS. In this review we present an overview of the publications describing the quantitative analysis of therapeutic intracellular nucleotide analogs using MS. The focus is on the different approaches for their direct analysis. We conclude that despite the technical hurdles, several useful MS-compatible chromatographic approaches have been developed, enabling the use of the excellent selectivity and sensitivity of MS for the quantitative analysis of intracellular nucleotides.

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Section: B Ionization Suppressionmentioning

confidence: 97%

Mass spectrometry in the quantitative analysis of therapeutic intracellular nucleotide analogs

Jansen

Rosing

Schellens

et al. 2011

Mass Spectrometry Reviews

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“…IEC using high-salt concentrations was first described in the 1950s [12]. Long run times [11,13] and incompatibility with mass spectrometry, however, have limited its application for nucleotide quantification mostly to in vitro studies [14].…”

Section: Introductionmentioning

confidence: 99%

Quantitation of intracellular purine intermediates in different Corynebacteria using electrospray LC-MS/MS

et al. 2012

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Intermediates of the purine biosynthesis pathway play key roles in cellular metabolism including nucleic acid synthesis and signal mediation. In addition, they are also of major interest to the biotechnological industry as several intermediates either possess flavor-enhancing characteristics or are applied in medical therapy. In this study, we have developed an analytical method for quantitation of 12 intermediates from the purine biosynthesis pathway including important nucleotides and their corresponding nucleosides and nucleobases. The approach comprised a single-step acidic extraction/quenching procedure, followed by quantitative electrospray LC-MS/MS analysis. The assay was validated in terms of accuracy, precision, reproducibility, and applicability for complex biological matrices. The method was subsequently applied for determination of free intracellular pool sizes of purine biosynthetic pathway intermediates in the two Gram-positive bacteria Corynebacterium glutamicum and Corynebacterium ammoniagenes. Importantly, no ion pair reagents were applied in this approach as usually required for liquid chromatography analysis of large classes of diverse metabolites.

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“…In particular, separation of ATP and adenosine 5'-diphosphate (ADP), that differ by a single negative charge, could be achieved by using conventional reversedphase columns under isocratic conditions [1][2][3][4], so that, for example, tenths of a percent of contaminating ADP in commercial ATP preparations (and vice versa) could be rapidly determined. This is also the practical limit of the method, and lower amounts of ADP in ADP/ATP mixtures cannot be detected by the above procedures.…”

Section: Introductionmentioning

confidence: 99%

In situ monitoring of kinetics of metabolic conversion of ATP to ADP catalyzed by MgATPases of muscle Gastrocnemius skinned fibers using micellar electrokinetic chromatography

Kulp¹,

Kaljurand²,

Käämbre

et al. 2004

Electrophoresis

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A method for the in situ measurement of the kinetics of ATP metabolic transformation using capillary electrophoresis (CE) has been developed. The depletion of ATP and formation of ADP were monitored in situ by using saponin-permeabilized muscle fibers. The method of micellar electrokinetic chromatography, employing reversed electroosmotic flow by cationic surfactant and reversed-polarity mode, provided an efficient and reproducible separation of nucleotides and enabled kinetic analysis of the reaction to be performed in a large range of nucleotide concentrations that approaches physiological concentrations of ATP in the muscle cells, without the need for precipitation of proteins prior to sample application. The analytes were detected at a nM level with a reproducibility of about 7%. This reproducibility enabled the comparison of different competing kinetic models of ATP conversion to ADP and the results show that the MgATPase activity in the fast-twitch gastrocnemius muscle followed biphasic kinetics that corresponds to the allosteric character of regulation of the enzyme(s) activity at physiological ATP concentrations. The results also confirmed that the combination of minimal sample volume requirements, rapid measurement and reproducibility makes the micellar CE a valuable tool for the analysis of biological fluids and understanding the processes of biological interest.

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Determination of adenosine nucleotides in cultured cells by ion-pairing liquid chromatography–electrospray ionization mass spectrometry

Cited by 70 publications

References 36 publications

Mass spectrometry in the quantitative analysis of therapeutic intracellular nucleotide analogs

Mass spectrometry in the quantitative analysis of therapeutic intracellular nucleotide analogs

Quantitation of intracellular purine intermediates in different Corynebacteria using electrospray LC-MS/MS

In situ monitoring of kinetics of metabolic conversion of ATP to ADP catalyzed by MgATPases of muscle Gastrocnemius skinned fibers using micellar electrokinetic chromatography

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