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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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Mean (A) percentile singleisotope effect (%IE) and (B) absoluteretention time differences (/ΔtR/) obtained by the Plackett–Burman experimental designs. Errorbars represent standard deviation; asterisks (∗) indicate statisticallysignificant difference determined by one-way ANOVA (p < 0.05).
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fig4: Mean (A) percentile singleisotope effect (%IE) and (B) absoluteretention time differences (/ΔtR/) obtained by the Plackett–Burman experimental designs. Errorbars represent standard deviation; asterisks (∗) indicate statisticallysignificant difference determined by one-way ANOVA (p < 0.05).

Mentions: First, we employed asymmetrical screening experimental design (Supporting Information Table S-2) using factornos. 1–6 from Supporting Information Table S-1. The levels were chosen to cover a wide range of conditionsapplied in practical gradient RPLC separations.21 Four biologically relevant reactive aldehydes (ACR, MDA,HNE, and ONE) were selected as model compounds for these studies,and ICD involved conversion to hydrazones with unlabeled (“light”)and various heavy (d3-, 15N2-, 15N4-, or 13C6-) labeled DNPH. The derivatized MDA represented an early elutingcompound, retention of ACR–DNPH was intermediate, while thederivatized HNE and ONE were late-eluting compounds upon simultaneouslyanalyzing these model aldehydes by gradient RPLC. As shown in Table 2 and Figure 2, both statisticaland graphical data interpretation revealed that only deuterium isotopesubstitution had a significant effect on the retention time shifts,with the exception of MDA. The latter may be due to the poor retention(apparent retention factor, kapp <4) of the first-eluting MDA–DNPH under the chromatographicconditions applied. The calculated absolute effect (0.302) was slightlylarger than the margin of error (ME = 0.301) for HNE, when the 2 and8 min gradients were compared. At the same time, it was smaller thanthe simultaneous margin of error (SME = 0.490). Since there is anincreased chance for false positive decisions using ME and significancewas not confirmed by other statistical and graphical interpretationmethods, this effect may only be considered as “possibly significant”according to Vander Heyden et al.26 Nevertheless,the results obtained by our asymmetrical design experiment (Table 2) pointed out that neither 13C nor 15N labeling had a significant effect on retention time difference;only deuterium isotope substitution ended up as a significant factor.However, two-factor or higher-order interaction effects can confoundthe main effects, and therefore, individual RPLC factors affectingchromatographic deuterium isotope effect may be underestimated bythe approach. To overcome this potential problem, the asymmetric designwas deconstructed and a Plackett–Burman design was adoptedusing only light- and d3-DNPH-derivedhydrazones. Results of this method are listed in Table 3 and plotted in Figures 3 and 4.


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)

Mean (A) percentile singleisotope effect (%IE) and (B) absoluteretention time differences (/ΔtR/) obtained by the Plackett–Burman experimental designs. Errorbars represent standard deviation; asterisks (∗) indicate statisticallysignificant difference determined by one-way ANOVA (p < 0.05).
© Copyright Policy
Related In: Results  -  Collection

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

fig4: Mean (A) percentile singleisotope effect (%IE) and (B) absoluteretention time differences (/ΔtR/) obtained by the Plackett–Burman experimental designs. Errorbars represent standard deviation; asterisks (∗) indicate statisticallysignificant difference determined by one-way ANOVA (p < 0.05).
Mentions: First, we employed asymmetrical screening experimental design (Supporting Information Table S-2) using factornos. 1–6 from Supporting Information Table S-1. The levels were chosen to cover a wide range of conditionsapplied in practical gradient RPLC separations.21 Four biologically relevant reactive aldehydes (ACR, MDA,HNE, and ONE) were selected as model compounds for these studies,and ICD involved conversion to hydrazones with unlabeled (“light”)and various heavy (d3-, 15N2-, 15N4-, or 13C6-) labeled DNPH. The derivatized MDA represented an early elutingcompound, retention of ACR–DNPH was intermediate, while thederivatized HNE and ONE were late-eluting compounds upon simultaneouslyanalyzing these model aldehydes by gradient RPLC. As shown in Table 2 and Figure 2, both statisticaland graphical data interpretation revealed that only deuterium isotopesubstitution had a significant effect on the retention time shifts,with the exception of MDA. The latter may be due to the poor retention(apparent retention factor, kapp <4) of the first-eluting MDA–DNPH under the chromatographicconditions applied. The calculated absolute effect (0.302) was slightlylarger than the margin of error (ME = 0.301) for HNE, when the 2 and8 min gradients were compared. At the same time, it was smaller thanthe simultaneous margin of error (SME = 0.490). Since there is anincreased chance for false positive decisions using ME and significancewas not confirmed by other statistical and graphical interpretationmethods, this effect may only be considered as “possibly significant”according to Vander Heyden et al.26 Nevertheless,the results obtained by our asymmetrical design experiment (Table 2) pointed out that neither 13C nor 15N labeling had a significant effect on retention time difference;only deuterium isotope substitution ended up as a significant factor.However, two-factor or higher-order interaction effects can confoundthe main effects, and therefore, individual RPLC factors affectingchromatographic deuterium isotope effect may be underestimated bythe approach. To overcome this potential problem, the asymmetric designwas deconstructed and a Plackett–Burman design was adoptedusing only light- and d3-DNPH-derivedhydrazones. Results of this method are listed in Table 3 and plotted in Figures 3 and 4.

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