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

Article abstract of DOI:10.1021/ac501309s

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-, ¹³C₆-, ¹⁵N₂-, or ¹⁵N₄-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 ¹⁵N or ¹³C labeling instead of deuterium labeling, while chromatographic parameters had no general influence. Comparison of the alternate isotope-coded derivatization assay (AIDA) using deuterium versus ¹⁵N 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 d ₃-2,4-dinitrophenylhydrazine with ¹⁵N- or ¹³C-labeled derivatizing reagent to avoid possible unfavorable consequences of chromatographic isotope effects.

Full text of DOI:10.1021/ac501309s

Article
pubs.acs.org/ac
Application of Screening Experimental Designs to Assess
Chromatographic Isotope Effect upon Isotope-Coded Derivatization
for Quantitative Liquid Chromatography−Mass Spectrometry
Szabolcs Szarka,† Katalin Prokai-Tatrai,†,‡ and Laszlo Prokai*,†
†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
S
* Supporting Information
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, malondialde-
hyde, 4-hydroxy-2-nonenal, and 4-oxo-2-nonenal) were derivatized
with light or heavy (d3-, 13C6-, 15N2-, or 15N4-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 15N or 13C labeling instead of deuterium labeling, while
chromatographic parameters had no general influence. Comparison of the alternate isotope-coded derivatization assay (AIDA)
using deuterium versus 15N 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 15N- or 13C-labeled derivatizing reagent to avoid possible unfavorable consequences of
chromatographic isotope effects.
igh-performance liquid chromatography coupled with
Htandem mass spectrometric (LC−MS/MS) detection is a
frequently used technique for the identification and quantifi-
cation of a wide range of chemical species in biological matrixes.
The superior specificity of MS allows the simplification of
sample preparation, and the application of fast gradient
methods, where a baseline separation of all components is
not required. However, coeluting matrix components can
significantly affect the accuracy and precision of a method.1−4
The use of a stable-isotope-labeled internal standard (IS),
presumed to have identical physicochemical properties to those
of the corresponding unlabeled analyte, is considered to correct
for the variability arising from sample preparations and
instrumental analyses. Generally, deuterium (d-) labeled ISs
are available commercially; however, they may elute at different
time points than their analytes due to chromatographic isotope
effect.5−7 The shifts in the retention times can be large enough
to cause differential matrix effect in LC−MS, especially when
electrospray ionization (ESI) is used.2 Therefore, the change in
the analyte to IS peak area ratios may be significant enough to
influence the accuracy of quantitation.8−13
Several approaches have been recommended to overcome
caveats associated with the use of d-labeled ISs.12−17 Additional
sample preparation steps, the use of shallow gradient profiles to
achieve adequate separation of analytes from major matrix
components eluting with the solvent front, application of
atmospheric pressure chemical ionization (APCI) which is less
prone to matrix effect than ESI,2 or using other than d- (i.e.,
13C-, 15N-, or 18O-) labeled ISs are examples of these efforts.
Unfortunately, all of these practices have limitations; for
instance, multiple sample preparation steps and longer
chromatography using shallow gradients decrease sample
throughput without actually eliminating problems related to
matrix effect.4 In many cases, the APCI technique results in
sensitivity loss compared to ESI.18,19 It has also been reported
that data obtained through the use of the “minimal labeling
approach” require corrections by an isotope pattern deconvo-
Received: April 1, 2014
Accepted: June 12, 2014
© XXXX American Chemical Society
A
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Article
lution algorithm.16,17 On the other hand, the limited availability
and/or high price of other than d-labeled analogues may be a
practical hurdle that prevents their widespread applications.
Despite earlier reports,5−9,13,20 the influence of operating
parameters (e.g., stationary phase, mobile phase pH, temper-
ature, organic solvent, and gradient slope) relevant to most
gradient LC−MS-based bioassays on chromatographic isotope
effects has not been assessed and understood. Non-deuterium-
labeled species also have not been studied extensively in this
context, especially when the most widely applied analytical
gradient reversed-phase HPLC (RPLC) conditions are
considered.21 Therefore, we evaluated the chromatographic
isotope effect brought about by stable isotope labeling on
different atoms in the framework of isotope-coded derivatiza-
tion (ICD) that represents an emerging method for
quantitative metabolite profiling.22,23 We focused on its
implementation known as alternate isotope-coded derivatiza-
tion assay (AIDA) and applied it 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).
relevant to most gradient LC−MS-based bioassays and stable
isotope labeling to the chromatographic isotope effect that
could compromise the accuracy and precision of quantitation
by RPLC−ESI-MS. These multivariate approaches allow for
predicting the effect of a given factor at several levels of
combinations with other factors.
EXPERIMENTAL SECTION
■
Chemicals and Reagents. Butylated hydroxytoluene
(BHT), ACR, DNPH, MDA-tetrabutylammonium salt,
[15N]KNO3 (98% isotope purity), [15N2]hydrazine sulfate
(98% isotope purity), and d5-chlorobenzene (99% isotope
purity) were obtained from Sigma-Aldrich (St. Louis, MO,
U.S.A.). [13C6]Chlorobenzene (99% isotope purity) was from
Cambridge Isotope Laboratories, Inc. (Tewksbury, MA,
U.S.A.). HNE (10 mg/mL in ethanol) and ONE (5 mg/mL
in methyl acetate) were purchased from Cayman Chemicals
(Ann Arbor, MI, U.S.A.). Solvents used for LC−MS/MS
measurements were of HPLC grade and supplied by Fisher
Scientific (Atlanta, GA, U.S.A.). Preparative TLC plates (silica
gel G, 20 cm × 20 cm, UNIPLATE-T taper plate) were from
Analtech Inc. (Newark, DE, U.S.A.), and analytical TLC silica
gel 60 F254 plates were from AMD Millipore (Billerica, MA,
U.S.A.). Charcoal-stripped human serum was ordered from
Innovative Research (Novi, MI, U.S.A.).
Synthesis of Stable Isotope Labeled DNPH Ana-
logues. The isotope-labeled “heavy” hydrazines, d3-DNPH,
[13C6]DNPH, [15N4]DNPH, and [15N2]DNPH, were synthe-
sized according to routine procedures based on the nucleophile
substitution reaction between hydrazine and 2,4-dinitrochlor-
obenzene, as reported before.14 First, the appropriately labeled
2,4-dinitrochlorobenzenes were synthesized; d3-2,4-dinitro-
chlorobenzene and [13C6]2,4-dinitrochlorobenzene were pre-
pared from d5-chlorobenzene and [13C6]chlorobenzene,
respectively, in a mixture of KNO3/H2SO4. The 15N-labeled
2,4-dinitrochlorobenzene was obtained from chlorobenzene in
a mixture of [15N]KNO3/H2SO4. Then, the individual 2,4-
dinitrochlorobenzenes were reacted with hydrazine in acetic
acid, with the exception of the synthesis leading to
[15N4]DNPH where [15N2]hydrazine sulfate was used. Each
product was then purified on preparative TLC using hexane/
ethyl acetate (6:1, v/v). On analytical TLC, we detected only
one UV−vis spot, with Rf = 0.63, using hexane/ethyl acetate
(6:1, v/v).
Overall, we aspired to a comprehensive assessment of the
subject through experimental screening designs25,26 for the first
time to address the contributions of operating parameters
For the derivatization of the carbonyls, individual DNPH
reagents (light and heavy: d3-, [15N2]-, [15N4]-, or [13C6]-
labeled) were dissolved in formic acid/acetonitrile 1:50 (v/v)
to obtain 1 mg/mL of derivatizing stock solutions.
Sample Solutions for the Evaluation of Isotope Effect.
Stock solutions of MDA, ACR, HNE, and ONE, respectively,
were prepared freshly in acetonitrile at 1 mg/mL concentration
for each aldehyde. Aliquots of 10 μL of each aldehyde were
added into 160 μL of derivatizing solution containing the light
or individual heavy (d3-, [15N2]-, [15N4]-, or [13C6]-labeled)
DNPH, respectively. The samples were incubated at 40 °C for
2 h in the dark. Then, equal aliquots (10 μL) of the light
hydrazones’ mixture and each of their heavy hydrazones’
mixture were brought together, resulting in four heavy−light
labeled solutions of the derivatized aldehydes. These samples
were then diluted with 50% (v/v) acetonitrile to obtain 0.1 and
1.0 μg/mL solutions (expressed in terms of free carbonyl
content), respectively, for isotope effect screenings.
Figure 1. Chemical structure of selected reactive aldehydes and their
DNPH derivatives.
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Table 1. Gradient RPLC Conditions Selected for the Evaluation of Chromatographic Isotope Effects
column
PhenHex
C18
Phenomenex Kinetex phenyl-hexyl (50 mm × 2.1 mm i.d., 5 μm particles) with SecurityGuard (2 mm × 2.1 mm i.d.)
Phenomenex Kinetex C18 (50 mm × 2.1 mm i.d., 5 μm particles) with SecurityGuard (2 mm × 2.1 mm i.d.)
mobile phase
eluent A1
0.1% (v/v) acetic acid in water at pH 3.3
eluent A2
5 mM ammonium acetate in water at pH 8.2
eluent B1
ACN
eluent B2
MeOH
gradient profile
50% to 100% B
gradient time (min)
a
tg1
2 (k* = 3.5 )
a
tg2
4 (k* = 7 )
a
tg3
8 (k* = 14 )
flow rate (mL/min)
0.4
column oven temperature (°C)
T1
25
40
50
5
T2
T3
injection volume (μL)
a
k* denotes gradient retention factor.
Evaluation of Isotope Effect Using Screening Exper-
imental Designs. A Surveyor LC system (Thermo Electron
Corporation, Trace Chemical Analysis, Austin, TX, U.S.A.) was
employed throughout the studies. A Phenomenex Kinetex
phenyl-hexyl silica column (50 mm × 2.1 mm i.d., 5 μm
particles) protected by a Phenomenex 2 mm × 2.1 mm i.d.
SecurityGuard phenyl-hexyl silica guard column (PhenHex)
and a Phenomenex Kinetex C18 column (50 mm × 2.1 mm
i.d., 5 μm particles) protected by a Phenomenex 2 mm × 2.1
mm i.d. SecurityGuard C18 guard column (C18) were used to
assess the isotope effect. RPLC conditions, including mobile
phase pH, column temperature, and gradient time, are
summarized in Table 1. The k* gradient retention factors
were within the recommended optimal range (0.5 < k* < 20)
for analytical HPLC.21 The selected seven factors to study their
effects on the chromatographic isotope effects are shown in
Table S-1 (Supporting Information). An asymmetrical
experimental design (Table S-2 in the Supporting Information)
was constructed from Addelman’s basic plan using a template
proposed by Hund et al.25 The experiments were sorted
according to RP analytical columns and mobile phases used,
from practical reasons. The Plackett−Burman experimental
design (Table S-3 in the Supporting Information) was set up
according to Vander Heyden et al.26 To investigate the
influence of uncontrolled factors such as time effects due to
response drifts, we repeated selected experiments, e.g.,
experiment 1 in Supporting Information Table S-2 and
experiment 12 in Supporting Information Table S-3, before
and after executing the sequences specified in these tables.
Statistical and graphical interpretations of data included visual
inspection of half-normal probability plots and estimation of
the critical effects such as negligible factor effects (Ecritical), and
margin of error (ME), and simultaneous margin of error
(SME).26
Then, the column was flushed with 100% B for 1 min and,
finally, was re-equilibrated with 40% B for 3 min. Flow rate was
maintained constant at 0.4 mL/min, and the injection volume
was 5 μL.
Mass Spectrometry. LC−MS/MS analysis of the light and
heavy DNPH derivatives was performed on a TSQ Quantum
Ultra (Thermo Electron Corporation) mass spectrometer
equipped with heated ESI (H-ESI) probe. With the exception
of DNPH−MDA, negative ion mode was applied. H-ESI spray
voltage, H-ESI temperature, and capillary temperature were
maintained at 3.5 and −3.0 kV (positive and negative
ionization, respectively), 350, and 325 °C, respectively.
Nitrogen sheath gas and auxiliary gas flow rates were 30 and
25 arbitrary units (corresponding to approximately 0.45 and 7.5
L/min according to the manufacturer’s specification), respec-
tively. Collision-induced dissociation was performed with argon
at 1.5 mTorr pressure. Selected reaction monitoring (SRM)
with unit mass resolution for the precursor and product ions
was used for quantification (Table S-4 in the Supporting
Information). Tube lens voltage was optimized for MDA−
DNPH and HNE−DNPH in positive and negative mode,
respectively. The optimal collision energy determined for each
hydrazone was obtained by direct infusion experiments. Data
acquisition and processing were carried out by the Xcalibur
software (version 2.1).
Direct infusion and isocratic (acetonitrile/water/acetic acid,
50:50:0.1, v/v; all other LC parameters are specified above in
the previous section) RPLC−MS scan and MS/MS product ion
scan (Figure S-1 in the Supporting Information) analysis was
applied for the confirmation of identity and for assessment of
purity of the labeled DNPHs. We estimated that the purity was
>98% after preparative TLC purification for each reagent.
Animals and Tissue Collection. Ovariectomized CD-1
mice were purchased from Harlan Laboratories (Indianapolis,
IN, U.S.A.). All procedures were reviewed and approved by the
Institutional Animal Care and Use Committee at the UNT
Health Science Center. Animals were euthanized by intra-
peritoneal administration of 60 mg/kg ketamine and 10 mg/kg
xylazine. Brain and liver were collected and processed
immediately.
RPLC Conditions for the Quantification of Hydra-
zones. Analyses were done using the same PhenHex column
(50 mm × 2.1 mm i.d., 5 μm particles) as specified above with
gradient elution at 30 °C. The mobile phase was a mixture of
(A1) 0.1% (v/v) acetic acid in water and (B1) acetonitrile (see
Table 1), with linear gradient from 40% B to 100% B in 4 min.
C
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Sample Preparation for Quantification. Tissue extracts
were prepared in a BeadBug microtube homogenizer D1030
(Benchmark Scientific, Edison, NJ, U.S.A.). Brain and liver
(100 mg) were mixed with 400 μL of acetonitrile/water (3:1,
v/v) containing 25 μg/mL BHT, a strong phenolic antioxidant,
in a 2 mL homogenizer tube prefilled with zirconium beads (1.5
mm i.d.) and homogenized for 3 min at 3300 rpm. The tissue
homogenate was centrifuged (5 min at 14 500 rpm), and the
clean supernatant was separated. It was then spiked with
different amounts (ranging from 20 to 2000 ng/g fresh tissue)
of MDA, ACR, and HNE to obtain quality control (QC)
samples for quantification according to the AIDA protocol.24
Briefly, to a 100 μL aliquot of a QC sample 100 μL of light or
heavy derivatizing stock solution was added. The reaction was
carried out as specified above. ICD standards (i.e., heavy- and
light-labeled DNPH−aldehyde mixtures) were prepared in 100
μL of charcoal stripped human serum precipitated with 300 μL
acetonitrile containing BHT (25 μg/mL). After centrifugation,
the clear supernatant was spiked with 200 ng of MDA, 100 ng
of ACR, and 100 ng of HNE and derivatized as specified above.
After derivatization, the samples were diluted with 800 μL of
acetonitrile. An amount of 200 μL of heavy-labeled ICD
standard was then added to 200 μL of light-labeled QC sample,
and 200 μL of light-labeled ICD standard was added to 200 μL
of heavy-labeled QC sample. Next, the resultant mixtures were
diluted with 1.6 mL water and subjected to solid-phase
extraction (SPE). Briefly, Supelclean LC-18 SPE cartridges (1
mL, 100 mg; Sigma-Aldrich, St. Louis, MO, U.S.A.) were
conditioned with 1 mL of acetonitrile, then with 1 mL of 0.1%
formic acid in water. After sample loading, the cartridges were
washed with 2 × 1 mL of water/acetonitrile/formic acid
(70:30:0.1, v/v), and the hydrazones were then eluted with 2 ×
200 μL of acetonitrile. The effluents were dried at room
temperature in an Eppendorf Vacufuge (Hauppauge, NY,
U.S.A.) and reconstituted in 50 μL of 50% acetonitrile for
subsequent LC−MS/MS analysis. All samples were analyzed
within 3 days.24 No aldehydes were detected in the unspiked
blank QC samples; therefore, blank correction24 was not
applied in AIDA calculations.
First, we employed asymmetrical screening experimental
design (Supporting Information Table S-2) using factor nos.
1−6 from Supporting Information Table S-1. The levels were
chosen to cover a wide range of conditions applied 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 eluting compound,
retention of ACR−DNPH was intermediate, while the
derivatized HNE and ONE were late-eluting compounds
upon simultaneously analyzing these model aldehydes by
gradient RPLC. As shown in Table 2 and Figure 2, both
Table 2. Absolute Effects of Various Factors Calculated from
the Retention Time Shift between Light and Heavy Pairs of
DNPH Derivatives Obtained from the Asymmetrical
a
Experimental Design
factor
MDA
ACR
HNE
ONE
isotope labeling
d3-15N4
b,
b,
b,
c
c
c
,
,
,
d
d
d
b
b
b
,
,
,
c
c
c
,
,
,
d
d
d
b,
b,
b,
c
c
c
,
,
,
d
d
d
0.539
0.619
0.659
0.081
0.120
0.039
1.099
1.000
1.025
0.098
0.074
0.024
0.697
0.746
0.669
0.049
0.028
0.077
0.947
1.044
1.020
0.098
0.073
0.025
d3-15N2
d3-13C6
15N4-15N2
15N4-15C6
15N2-13C6
temperature
25−50 °C
25−40 °C
40−50 °C
gradient time
2−4 min
2−8 min
4−8 min
stationary phase
pH
0.479
0.550
0.071
0.344
0.382
0.039
0.149
0.188
0.039
0.248
0.284
0.036
0.010
0.480
0.470
0.443
0.143
0.290
0.137
0.396
0.260
0.062
0.112
0.186
0.087
0.302
0.215
0.114
0.063
0.086
0.086
0.395
0.309
0.112
0.209
0.185
e
organic solvent
dummies
dummy 1
dummy 2
dummy 3
critical effects
Ecritical
Statistical Analysis. Statistical evaluations were carried out
with the XLSTAT (version 2014.1.01) and the Primer of
Biostatistics (version 5.0; McGraw-Hill, New York, NY, U.S.A.)
software. Statistical differences between means of data sets were
determined with one-way ANOVA at p < 0.05. Statistical
interpretation of the method comparison test results was
achieved following the guidelines proposed in previous
reports.27−29 Linear regression lines were obtained by using
the Deming regression model.28 Percentile differences were
visualized in Bland−Altman plots.27 Normal distribution of the
data was confirmed with the Shapiro−Wilk test.
0.191
0.250
0.190
0.135
0.087
0.187
0.086
0.189
0.135
0.136
0.136
0.186
0.675
0.791
1.300
0.452
0.439
0.715
0.455
0.301
0.490
0.491
0.419
0.683
ME
SME
a
Critical effects were estimated according to Vander Heyden et al. (ref
b
c
26). Significant effect compared to Ecritical
compared to simultaneous margin of error (SME). Significant effects
from the half-probability normal plot. Possibly significant effect
.
Significant effect
d
e
compared to margin of error (ME).
RESULTS AND DISCUSSION
■
Multivariate approaches employed by screening experimental
designs25,26 have several advantages over the classically applied
one-variable-at-a-time methodologies, where only one factor at
a time is changed. Specifically, screening experimental designs
calculate the influence of a given factor at several levels through
combinations with other factors, which permits general
conclusions.25,26,30 Moreover, they permit the evaluation of
relatively large number of factors in a relatively small number of
experiments, and thus, the strategy can be used in high-
throughput assays.
statistical and graphical data interpretation revealed that only
deuterium isotope substitution 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
chromatographic conditions applied. The calculated absolute
effect (0.302) was slightly larger than the margin of error (ME
= 0.301) for HNE, when the 2 and 8 min gradients were
compared. At the same time, it was smaller than the
simultaneous margin of error (SME = 0.490). Since there is
D
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Figure 2. Half-normal probability plots of the absolute effects on the retention time difference measured between light and heavy labeled hydrazones
in the asymmetrical design with identification of margin of error (ME) and simultaneous margin of error (SME) as critical effects. A, B, and C factors
denote stable isotope labeling, column temperature, and gradient time, respectively.
an increased chance for false positive decisions using ME and
significance was not confirmed by other statistical and graphical
interpretation methods, 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 confound the main effects, and therefore, individual RPLC
factors affecting chromatographic deuterium isotope effect may
be underestimated by the approach. To overcome this potential
problem, the asymmetric design was deconstructed and a
Plackett−Burman design was adopted using only light- and d3-
DNPH-derived hydrazones. Results of this method are listed in
Table 3 and plotted in Figures 3 and 4.
In agreement with findings concluded from the asymmetric
experimental plan (Table 2), no significant effects were
identified for MDA−DNPH (Table 3) with the Plackett−
Burman approach. For ACR, we found only one factor (the
organic modifier in the mobile phase) that had significant effect
on the retention time difference between the ACR−DNPH and
d3-ACR−DNPH upon comparing the absolute effect (0.560)
with critical effect derived from negligible factor effects (Ecritical
= 0.513). On the other hand, the gradient time had significant
effect for the two late-eluting analytes (HNE−DNPH and
ONE−DNPH). All three data interpretation methods (i.e.,
comparison to Ecritical and SME, as well as inspection of the half-
normal probability plot) confirmed this finding for the HNE
derivative (Table 3), while for ONE−DNPH only “possibly
significant” effect can be concluded in terms of SME. As such,
the isotope effect can be significantly reduced for these
compounds by applying a steeper gradient slope (data not
shown). It is interesting to note that the analyte’s concentration
also had significant effect, when the absolute effects (0.228 and
0.287) were compared to the corresponding Ecritical values
(0.146 and 0.247) for HNE−DNPH and ONE−DNPH,
Table 3. Absolute Effects of Various Factors on the
Retention Time Difference between Light and d3-Labeled
DNPH Derivatives Obtained from the Plackett−Burman
a
Design
factor
MDA
ACR
HNE
ONE
temperature
gradient time
stationary phase
pH
0.376
0.106
0.589
0.494
0.392
0.249
0.366
0.436
0.098
0.032
0.131
0.464
0.167
0.162
0.065
0.228
0.189
0.655
0.153
0.224
0.189
0.287
b
b
b
,
c
,
d
b
,d,e
b
organic solvent
analyte concn
dummies
dummy 1
dummy 2
dummy 3
dummy 4
dummy 5
critical effects
Ecritical
0.560
0.299
b
b
0.310
0.380
0.036
0.450
0.319
0.161
0.300
0.168
0.168
0.164
0.067
0.030
0.073
0.066
0.032
0.009
0.022
0.189
0.048
0.087
0.849
0.758
1.214
0.513
0.642
1.032
0.146
0.267
0.427
0.247
0.556
0.894
ME
SME
a
Critical effects were estimated according to Vander Heyden et al. (ref
b
c
26). Significant effect compared to Ecritical
compared to simultaneous margin of error (SME). Significant effects
from the half-probability normal plot. Possibly significant effect
.
Significant effect
d
e
compared to margin of error (ME).
respectively. Although the significance of analyte concentration
was not confirmed by alternative interpretation methods, such
as the considering SME or the visual inspection of the half-
normal probability plots (Figure 3), this finding may need to be
considered in terms of accuracy and precision of quantitation.
Additionally, small but significant effects of the stationary phase
and the pH of the mobile phase were also revealed for HNE−
DNPH by one of interpretation methods we employed (Table
3).
E
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Analytical Chemistry
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Figure 3. Half-normal probability plots of the absolute effects on the retention time difference measured between light and d3-labeled aldehyde
hydrazones in the Plackett−Burman experimental design with identification of margin of error (ME) and simultaneous margin of error (SME) as
critical effects. C factor denotes gradient time.
To validate findings summarized in Table 3, we repeated the
Plackett−Burman design using [15N4]DNPH. This setup,
however, did not yield any statistically significant main effects
on chromatographic isotope effect (Table S-5 and Figure S-2 in
the Supporting Information). As expected, the use of
[15N4]DNPH brought about a statistically significant decrease
in percentile single isotope effect (%IE, Figure 4A) compared
to d3 labeling, along with a significant decrease in the retention
time difference (ΔtR) for all aldehydes except for MDA (Figure
4B). The observed large standard deviation for the d3-labeled
MDA derivative suggests that this compound is more sensitive
to changes in RPLC conditions than the later-eluting
only by 2 Da, and such a small difference can cause significant
cross-talk effect if these peaks overlap. As shown in Figure S-4
in the Supporting Information, we achieved acceptable
resolution (Rs ≥ 2)21 by using acetonitrile as organic mobile
phase, while the pH of the aqueous phase and the stationary
phase chemistry had only little effect. On the other hand,
acceptable separation was not obtained at all when methanol
was used. Moreover, acetonitrile considerably decreased the
analysis time, consequently increasing assay throughput. The
detector sensitivity was dramatically influenced by the mobile
phase pH. As expected, MDA−DNPH response significantly
decreased upon high pH (5 mM ammonium acetate at pH 8.2)
chromatography, when positive ion formation is not favored.
The other aldehyde derivatives produced abundant negative
ions during ESI; thus, negative ionization mode is preferred for
the monitoring of these species.24,31,32 Contrary to our
expectation, high pH decreased the signal even in the negative
ion mode. This unique phenomenon, which is known as the
“wrong-way-round ionization,”18,19 is actually advantageous,
since efficient ionization can be obtained using both polarity
modes within one assay.
On the basis of the results obtained by the asymmetric
screening experiment, we propose an LC−MS/MS method for
quantification of MDA, ACR, HNE, and ONE by ICD. We
suggest gradient separation to be performed using PhenHex
column with low pH (0.1% acetic acid at pH 3.3) aqueous
phase and acetonitrile as the organic modifier. When the
organic content of the initial mobile phase is reduced to 40%,
adequate retention (kapp ≥ 3) for the first-eluting MDA−
DNPH can be achieved. Gradient time of 4 min and column
temperature set at 30 °C also allow for the excellent separation
of the critical peak pair (RsHNE−DNPH/ONE−DNPH = 3.3) within
only 3.2 min (Figure S-5 in the Supporting Information).
hydrazones; we found a strong linear correlation (R2
=
0.9178) between kapp and ΔtR (Figure S-3 in the Supporting
Information). Accordingly, negligible ΔtR can only be achieved
for MDA−DNPH when kapp < 3, while kapp ≥ 3 is
recommended for bioanalytical LC−MS applications.21
Taken together, the only way to eliminate universally the
retention time difference between light and heavy isotopo-
logues is when ICD applies reagents labeled on heavy atoms
(C, N, O, etc.). 15N4-labeled DNPH could be safely used for
correcting LC−ESI-MS detector response under a wide range
of RPLC conditions without the risk of manifesting significant
ΔtR. Therefore, we propose the modification of the AIDA
protocol24 to replace d3-DNPH with 15N4- or 13C6-DNPH for
the derivatization of carbonyls.
Results, such as critical resolution, analysis time, and detector
response obtained by the asymmetrical design experiment, were
also used to select the optimal RPLC conditions21 for the
reliable and fast simultaneous analysis of the chosen model
aldehydes in mixtures. Satisfactory separation of HNE−DNPH
and ONE−DNPH was a very important aspect of method
optimization. The molecular masses of these hydrazones differ
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Analytical Chemistry
Article
them to well-documented LPO-related pathology34−36 and
thereby putting AIDA in the context of application to high-
relevance biological studies, ideal as complex matrixes for a
comparative evaluation of the previously developed method
and the modification we propose herein. Because we did not
detect ONE upon screening the chosen mouse tissue
homogenates by LC−MS/MS using DNPH derivatization,
therefore, we conducted this evaluation with MDA, ACR and
HNE as analytes. Leaving out ONE also avoided addressing its
potential conversion to E-1-hydroxynon-2-en-4-one in bio-
logical samples,37 which was beyond the scope of our work
primarily focusing on chromatographic isotope effects and its
potential impact on bioanalyses using ICD followed by LC−
MS/MS.
The results obtained by the two labeling approaches that
relied on d3-DNPH24 and [15N4]DNPH,14 respectively, as
reagents are visualized on scatterplots in Figure S-6
(Supporting Information). The statistically significant differ-
ences in the slopes of Deming regression lines from the lines of
equality indicate that a proportional error exists between the
data generated by the use of d3-DNPH versus [15N4]DNPH.
The confidence intervals for the biases and limits of agreement
( 1.96 SD) could be estimated, because the percentile
differences followed normal distribution (data not shown).
These data are included in the Bland−Altman plots shown in
Figure 5. The measurement bias values were calculated as 3.0%,
−1.3%, and 0.4% for MDA, ACR, and HNE, respectively. The
95% confidence intervals for the bias of these aldehydes also
include zero; therefore, measurement biases were not
significant. The limits of agreement show whether 95% of the
differences would 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 matrixes afforded similar quantitative results only for
HNE. For the quantification of MDA and ACR, the limit of
agreement values exceeded the maximum tolerable inaccuracy
( 15%).38,39 Therefore, these methods may not be considered
equivalent or interchangeable for the quantitative analysis of
these aldehydes by AIDA from the given matrixes.
Figure 4. Mean (A) percentile single isotope effect (%IE) and (B)
absolute retention time differences (|ΔtR|) obtained by the Plackett−
Burman experimental designs. Error bars represent standard deviation;
asterisks (∗) indicate statistically significant difference determined by
one-way ANOVA (p < 0.05).
In order to reveal potential accuracy differences, a series of
sample pairs were quantified based on this optimized LC−MS/
MS assay using both the previously used d3-DNPH24 and our
own [15N4]DNPH reagent14 in AIDA. To compare the
proposed modification to the original method,24 we used
mouse brain and liver homogenates as biological matrixes to
generate QC samples focusing on the quantification of selected
LPO end-products. The accumulation of these compounds in
various human organs and body fluids has been linked to major
tissue and cellular dysfunction believed to play a role in aging
and most age- and oxidative stress-related diseases.33 However,
high lipid content make the brain and liver, besides exposing
CONCLUSIONS
■
The synthesis of DNPH is relatively simple. Isotope labeling
can be customized, because several different stable isotope
atoms (such as d, 13C, 15N, and/or 18O) can be incorporated in
various numbers, even at specific positions, into the structure of
this carbonyl-specific reagent. Therefore, it provided a
Figure 5. Bland−Altman plots of quantities of the selected aldehydes measured in fortified mouse tissue extracts by AIDA using d3 vs 15N4 labeling.
Bold solid lines show the measurement bias between the two methods, dotted lines represent the 95% confidence limits of the bias, and the dashed
lines indicate the limits of agreement values.
G
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isotope effects, is that the most effective approach to avoid a
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deuterium-labeled reagents in AIDA. When AIDA relies on
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ASSOCIATED CONTENT
* Supporting Information
Additional information as noted in text. This material is
available free of charge via the Internet at http://pubs.acs.org.
■
S
AUTHOR INFORMATION
Corresponding Author
*Phone: +1-817-735-2206. Fax: +1-817-735-2118. E-mail:
Laszlo.Prokai@unthsc.edu.
■
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Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Dr. Vien Nguyen and Mr. Kurt Kamrowski for their
excellent technical assistance. This work was supported in part
by the National Institutes of Health (Grant No. AG025384)
and the Robert A. Welch Foundation (endowment BK-0031 to
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