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Application of screening experimental designs to assess chromatographic isotope effect upon isotope-coded derivatization for quantitative liquid chromatography-mass spectrometry.

Szarka S, Prokai-Tatrai K, Prokai L - Anal. Chem. (2014)

Bottom Line: Together with a simultaneous matrix effect, this could lead to unacceptable accuracy in quantitative liquid chromatography-mass spectrometry assays, especially when electrospray ionization is used.Results confirmed that the most effective approach to avoid chromatographic isotope effect is the use of (15)N or (13)C labeling instead of deuterium labeling, while chromatographic parameters had no general influence.On the basis of our results, we recommend the modification of the AIDA protocol by replacing d3-2,4-dinitrophenylhydrazine with (15)N- or (13)C-labeled derivatizing reagent to avoid possible unfavorable consequences of chromatographic isotope effects.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Neuroscience, and ‡Department of Pharmaceutical Sciences, UNT System College of Pharmacy, University of North Texas Health Science Center , 3500 Camp Bowie Boulevard, Fort Worth, Texas 76107-2699, United States.

ABSTRACT
Isotope effect may cause partial chromatographic separation of labeled (heavy) and unlabeled (light) isotopologue pairs. Together with a simultaneous matrix effect, this could lead to unacceptable accuracy in quantitative liquid chromatography-mass spectrometry assays, especially when electrospray ionization is used. Four biologically relevant reactive aldehydes (acrolein, malondialdehyde, 4-hydroxy-2-nonenal, and 4-oxo-2-nonenal) were derivatized with light or heavy (d3-, (13)C6-, (15)N2-, or (15)N4-labeled) 2,4-dinitrophenylhydrazine and used as model compounds to evaluate chromatographic isotope effects. For comprehensive assessment of retention time differences between light/heavy pairs under various gradient reversed-phase liquid chromatography conditions, major chromatographic parameters (stationary phase, mobile phase pH, temperature, organic solvent, and gradient slope) and different isotope labelings were addressed by multiple-factor screening using experimental designs that included both asymmetrical (Addelman) and Plackett-Burman schemes followed by statistical evaluations. Results confirmed that the most effective approach to avoid chromatographic isotope effect is the use of (15)N or (13)C labeling instead of deuterium labeling, while chromatographic parameters had no general influence. Comparison of the alternate isotope-coded derivatization assay (AIDA) using deuterium versus (15)N labeling gave unacceptable differences (>15%) upon quantifying some of the model aldehydes from biological matrixes. On the basis of our results, we recommend the modification of the AIDA protocol by replacing d3-2,4-dinitrophenylhydrazine with (15)N- or (13)C-labeled derivatizing reagent to avoid possible unfavorable consequences of chromatographic isotope effects.

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Bland–Altman plots of quantities of the selectedaldehydesmeasured in fortified mouse tissue extracts by AIDA using d3 vs 15N4 labeling. Boldsolid lines show the measurement bias between the two methods, dottedlines represent the 95% confidence limits of the bias, and the dashedlines indicate the limits of agreement values.
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fig5: Bland–Altman plots of quantities of the selectedaldehydesmeasured in fortified mouse tissue extracts by AIDA using d3 vs 15N4 labeling. Boldsolid lines show the measurement bias between the two methods, dottedlines represent the 95% confidence limits of the bias, and the dashedlines indicate the limits of agreement values.

Mentions: The resultsobtained by the two labeling approaches that reliedon d3-DNPH24 and [15N4]DNPH,14 respectively, as reagents are visualized on scatterplots in FigureS-6 (Supporting Information). The statisticallysignificant differences in the slopes of Deming regression lines fromthe lines of equality indicate that a proportional error exists betweenthe data generated by the use of d3-DNPHversus [15N4]DNPH. The confidence intervalsfor the biases and limits of agreement (±1.96 SD) could be estimated,because the percentile differences followed normal distribution (datanot shown). These data are included in the Bland–Altman plotsshown in Figure 5. The measurement bias valueswere calculated as 3.0%, −1.3%, and 0.4% for MDA, ACR, andHNE, respectively. The 95% confidence intervals for the bias of thesealdehydes also include zero; therefore, measurement biases were notsignificant. The limits of agreement show whether 95% of the differenceswould lie between the limits, if the differences were normally distributed.These intervals ranged from −13% to 19%, −23% to 21%,and −9% to 10% for MDA, ACR, and HNE, respectively (Figure 5). Accordingly, the two methods in both matrixesafforded similar quantitative results only for HNE. For the quantificationof MDA and ACR, the limit of agreement values exceeded the maximumtolerable inaccuracy (±15%).38,39 Therefore,these methods may not be considered equivalent or interchangeablefor the quantitative analysis of these aldehydes by AIDA from thegiven matrixes.


Application of screening experimental designs to assess chromatographic isotope effect upon isotope-coded derivatization for quantitative liquid chromatography-mass spectrometry.

Szarka S, Prokai-Tatrai K, Prokai L - Anal. Chem. (2014)

Bland–Altman plots of quantities of the selectedaldehydesmeasured in fortified mouse tissue extracts by AIDA using d3 vs 15N4 labeling. Boldsolid lines show the measurement bias between the two methods, dottedlines represent the 95% confidence limits of the bias, and the dashedlines indicate the limits of agreement values.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4215859&req=5

fig5: Bland–Altman plots of quantities of the selectedaldehydesmeasured in fortified mouse tissue extracts by AIDA using d3 vs 15N4 labeling. Boldsolid lines show the measurement bias between the two methods, dottedlines represent the 95% confidence limits of the bias, and the dashedlines indicate the limits of agreement values.
Mentions: The resultsobtained by the two labeling approaches that reliedon d3-DNPH24 and [15N4]DNPH,14 respectively, as reagents are visualized on scatterplots in FigureS-6 (Supporting Information). The statisticallysignificant differences in the slopes of Deming regression lines fromthe lines of equality indicate that a proportional error exists betweenthe data generated by the use of d3-DNPHversus [15N4]DNPH. The confidence intervalsfor the biases and limits of agreement (±1.96 SD) could be estimated,because the percentile differences followed normal distribution (datanot shown). These data are included in the Bland–Altman plotsshown in Figure 5. The measurement bias valueswere calculated as 3.0%, −1.3%, and 0.4% for MDA, ACR, andHNE, respectively. The 95% confidence intervals for the bias of thesealdehydes also include zero; therefore, measurement biases were notsignificant. The limits of agreement show whether 95% of the differenceswould lie between the limits, if the differences were normally distributed.These intervals ranged from −13% to 19%, −23% to 21%,and −9% to 10% for MDA, ACR, and HNE, respectively (Figure 5). Accordingly, the two methods in both matrixesafforded similar quantitative results only for HNE. For the quantificationof MDA and ACR, the limit of agreement values exceeded the maximumtolerable inaccuracy (±15%).38,39 Therefore,these methods may not be considered equivalent or interchangeablefor the quantitative analysis of these aldehydes by AIDA from thegiven matrixes.

Bottom Line: Together with a simultaneous matrix effect, this could lead to unacceptable accuracy in quantitative liquid chromatography-mass spectrometry assays, especially when electrospray ionization is used.Results confirmed that the most effective approach to avoid chromatographic isotope effect is the use of (15)N or (13)C labeling instead of deuterium labeling, while chromatographic parameters had no general influence.On the basis of our results, we recommend the modification of the AIDA protocol by replacing d3-2,4-dinitrophenylhydrazine with (15)N- or (13)C-labeled derivatizing reagent to avoid possible unfavorable consequences of chromatographic isotope effects.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Neuroscience, and ‡Department of Pharmaceutical Sciences, UNT System College of Pharmacy, University of North Texas Health Science Center , 3500 Camp Bowie Boulevard, Fort Worth, Texas 76107-2699, United States.

ABSTRACT
Isotope effect may cause partial chromatographic separation of labeled (heavy) and unlabeled (light) isotopologue pairs. Together with a simultaneous matrix effect, this could lead to unacceptable accuracy in quantitative liquid chromatography-mass spectrometry assays, especially when electrospray ionization is used. Four biologically relevant reactive aldehydes (acrolein, malondialdehyde, 4-hydroxy-2-nonenal, and 4-oxo-2-nonenal) were derivatized with light or heavy (d3-, (13)C6-, (15)N2-, or (15)N4-labeled) 2,4-dinitrophenylhydrazine and used as model compounds to evaluate chromatographic isotope effects. For comprehensive assessment of retention time differences between light/heavy pairs under various gradient reversed-phase liquid chromatography conditions, major chromatographic parameters (stationary phase, mobile phase pH, temperature, organic solvent, and gradient slope) and different isotope labelings were addressed by multiple-factor screening using experimental designs that included both asymmetrical (Addelman) and Plackett-Burman schemes followed by statistical evaluations. Results confirmed that the most effective approach to avoid chromatographic isotope effect is the use of (15)N or (13)C labeling instead of deuterium labeling, while chromatographic parameters had no general influence. Comparison of the alternate isotope-coded derivatization assay (AIDA) using deuterium versus (15)N labeling gave unacceptable differences (>15%) upon quantifying some of the model aldehydes from biological matrixes. On the basis of our results, we recommend the modification of the AIDA protocol by replacing d3-2,4-dinitrophenylhydrazine with (15)N- or (13)C-labeled derivatizing reagent to avoid possible unfavorable consequences of chromatographic isotope effects.

Show MeSH
Related in: MedlinePlus