An on-line heart-cutting two-dimensional liquid chromatography method for intracellular 2-hydroxyglutarate enantiomers

Ohtawa, Takuma; Tsunoda, Makoto

doi:10.1039/d3ay00263b

Cited by 2 publications

(1 citation statement)

References 20 publications

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“…2D-LC can increase the resolution of the peaks and enhance the specificity of the method as compared with one-dimensional separation [27][28][29][30]. As mentioned before, this method was chosen because the separation of the NBD-urea cycle amino acids from the 18 NBD-proteinogenic amino acids on an ODS column was difficult.…”

Section: Retention Of Nbd-urea Cycle Amino Acids On Ion-exchange Columnmentioning

confidence: 99%

Development of a Two-Dimensional Liquid Chromatographic Method for Analysis of Urea Cycle Amino Acids

Sumida,

Tsunoda

2024

Molecules

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The urea cycle has been found to be closely associated with certain types of cancers and other diseases such as cardiovascular disease and chronic kidney disease. An analytical method for the precise quantification of urea cycle amino acids (arginine, ornithine, citrulline, and argininosuccinate) by off-line two-dimensional liquid chromatography (2D-LC) combined with fluorescence-based detection was developed. Before analysis, the amino acids were derivatised with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) to obtain NBD-amino acids. The first dimension involved the reversed-phase separation, in which NBD derivatives of urea cycle amino acids were completely separated from each other and mostly separated from the 18 NBD-proteinogenic amino acids. The samples were eluted with stepwise gradient using 0.02% trifluoroacetic acid in water–acetonitrile as the mobile phase. In the second dimension, an amino column was used for the separation of NBD-ornithine, -citrulline, and -argininosuccinate, while a sulfonic acid column was used to separate NBD-arginine. The developed 2D-LC system was used to analyse human plasma samples. The fractions of NBD-urea cycle amino acids obtained in the first dimension were collected manually and introduced into the second dimension. By choosing appropriate mobile phases for the second dimension, each NBD-urea cycle amino acid eluted in the first dimension was well separated from the other proteinogenic amino acids and interference from endogenous substance. This could not be achieved in the first dimension. The urea cycle amino acids in human plasma sample were quantified, and the method was well validated. The calibration curves for each NBD-urea cycle amino acid showed good linearity from 3 (ASA) or 15 (Orn, Cit, and Arg) to 600 nM, with correlation coefficients higher than 0.9969. The intraday and interday precisions were less than 7.9% and 15%, respectively. The 2D-LC system is expected to be useful for understanding the involvement of the urea cycle in disease progression.

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Section: Retention Of Nbd-urea Cycle Amino Acids On Ion-exchange Columnmentioning

confidence: 99%

Development of a Two-Dimensional Liquid Chromatographic Method for Analysis of Urea Cycle Amino Acids

Sumida,

Tsunoda

2024

Molecules

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Simultaneous Determination of D and L Enantiomers of 2-Hydroxyglutarate by UHPLC–MS/MS Method in Human Biological Fluids and its Clinical Application

Li,

Bo,

et al. 2024

Journal of Chromatographic Science

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2-hydroxyglutarate has limited abundance in normal tissues but a high level under certain pathologic conditions. To clarify the diagnostic value of two chiral isomers of 2-hydroxyglutarate in plasma and urine of Chinese cancer patients, an ultra-high performance liquid chromatography-tandem mass spectrometric method was developed for simultaneous quantification of D-/L-2-hydroxyglutarate. The selected D-/L-2-hydroxyglutarate-d5 as internal standards were added to samples before the SPE on Waters Oasis® MAX 96-Well plate (30 μm, 60 mg). A derivatization step with (+)-O,O′-diacetyl-L-tartaric anhydride permitted the chromatography separation of D-/L-2-hydroxyglutarate on an ACQUITY UPLC-HSS T3 column (50 × 2.1 mm, i.d. 1.8 μm) with acetonitrile and water (containing 0.1% formic acid and 10 mmol ammonium acetate) as the mobile phase. The calibration curves showed good linearity (R ≥ 0.99) over the concentration ranges of 200–5,000 ng/mL and 500–20,000 ng/mL for analysis of D-/L-2-hydroxyglutarate in plasma and urine samples, respectively. Intra- and inter-run precision were ≤ 12.33%, and the accuracy was within the range of −10.44 to 13.90%. This method was further successfully applied to clinical sample analysis in isocitrate dehydrogenase 1/2 mutated Chinese cancer patients.

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An on-line heart-cutting two-dimensional liquid chromatography method for intracellular 2-hydroxyglutarate enantiomers

Abstract: An analytical method for 2-hydroxyglutarate 2-HG enantiomers was developed using on-line heart-cutting two-dimensional liquid chromatography (2D-LC) with fluorescence detection.

Cited by 2 publications

References 20 publications

Development of a Two-Dimensional Liquid Chromatographic Method for Analysis of Urea Cycle Amino Acids

Development of a Two-Dimensional Liquid Chromatographic Method for Analysis of Urea Cycle Amino Acids

Simultaneous Determination of D and L Enantiomers of 2-Hydroxyglutarate by UHPLC–MS/MS Method in Human Biological Fluids and its Clinical Application

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