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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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Chemical structure ofselected reactive aldehydes and their DNPHderivatives.
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fig1: Chemical structure ofselected reactive aldehydes and their DNPHderivatives.

Mentions: Despiteearlier reports,5−9,13,20 the influence of operating parameters (e.g., stationary phase, mobilephase pH, temperature, organic solvent, and gradient slope) relevantto most gradient LC–MS-based bioassays on chromatographic isotopeeffects has not been assessed and understood. Non-deuterium-labeledspecies also have not been studied extensively in this context, especiallywhen the most widely applied analytical gradient reversed-phase HPLC(RPLC) conditions are considered.21 Therefore,we evaluated the chromatographic isotope effect brought about by stableisotope labeling on different atoms in the framework of isotope-codedderivatization (ICD) that represents an emerging method for quantitativemetabolite profiling.22,23 We focused on its implementationknown as alternate isotope-coded derivatization assay (AIDA) and appliedit to the LC–MS/MS analysis of biologically relevant aldehydes.24 AIDA uses 2,4-dinitro-3,5,6-trideuterophenylhydrazine(d3-DNPH) for isotope-coded “heavy”labeling, and has been validated for malondialdehyde (MDA) and 4-hydroxy-2-nonenal(HNE) quantifications. Here we also included 15N- and 13C-labeled DNPH reagents, and extended our study to acrolein(ACR) and 4-oxo-2-nonenal (ONE), as additional lipid peroxidation(LPO) end-products (Figure 1).


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)

Chemical structure ofselected reactive aldehydes and their DNPHderivatives.
© Copyright Policy
Related In: Results  -  Collection

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

fig1: Chemical structure ofselected reactive aldehydes and their DNPHderivatives.
Mentions: Despiteearlier reports,5−9,13,20 the influence of operating parameters (e.g., stationary phase, mobilephase pH, temperature, organic solvent, and gradient slope) relevantto most gradient LC–MS-based bioassays on chromatographic isotopeeffects has not been assessed and understood. Non-deuterium-labeledspecies also have not been studied extensively in this context, especiallywhen the most widely applied analytical gradient reversed-phase HPLC(RPLC) conditions are considered.21 Therefore,we evaluated the chromatographic isotope effect brought about by stableisotope labeling on different atoms in the framework of isotope-codedderivatization (ICD) that represents an emerging method for quantitativemetabolite profiling.22,23 We focused on its implementationknown as alternate isotope-coded derivatization assay (AIDA) and appliedit to the LC–MS/MS analysis of biologically relevant aldehydes.24 AIDA uses 2,4-dinitro-3,5,6-trideuterophenylhydrazine(d3-DNPH) for isotope-coded “heavy”labeling, and has been validated for malondialdehyde (MDA) and 4-hydroxy-2-nonenal(HNE) quantifications. Here we also included 15N- and 13C-labeled DNPH reagents, and extended our study to acrolein(ACR) and 4-oxo-2-nonenal (ONE), as additional lipid peroxidation(LPO) end-products (Figure 1).

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