Chromatographia (2015) 78:251–258
DOI 10.1007/s10337-014-2842-2
ORIGINAL
Variations in GC–MS Response Between Analytes
and Deuterated Analogs
Muhammed Alzweiri · Mohammad Khanfar ·
Yusuf Al‑Hiari Received: 24 November 2014 / Revised: 18 December 2014 / Accepted: 30 December 2014 / Published online: 20 January 2015
© Springer-Verlag Berlin Heidelberg 2015
Abstract Isotopic analogs are commonly used as appropriate internal standards. It was observed that analytes usually have higher mass responses than their equimolar deuterated analogs leading to quantification discrepancy. The
standard addition method was adopted on dimethyl azelate (DMA) and d6-dimethyl azelate (d6-DMA) to investigate possible reasons for this behavior. Cross contribution
of mass responses, intermolecular deuterium–hydrogen
exchange during chromatographic separation, and deviation in mass ionization response of C–H against C–D bonds
were studied. GC–MS analysis revealed that neither cross
contribution of ions nor H2/H exchange was responsible for
the difference in responses between DMA and d6-DMA.
On the other hand, a study of carbon nucleus relaxation
conducted by 13C-NMR showed that relaxation rate of carbonyl carbon in d6-DMA is faster than DMA (9 and 3 s−1,
respectively). This indicates rapid interaction between spin
of deuterium nucleus with spin of unpaired electrons in the
structure. Accordingly, DMA has more electrons in triplet/
singlet state (i.e., unpaired electrons) in each time moment
promoting propagation of radicalization reactions inside
the electron impact chamber. In conclusion, this should
increase the response of total ion current (TIC) and generate overestimated results for equimolar ratios of analytes
such as dimethyl azelate, dimethyl adipate and dimethyl
phthalate against their deuterated counterparts.
Keywords Gas chromatography · Deuterated analog ·
Mass response · Carbon relaxation
M. Alzweiri (*) · M. Khanfar · Y. Al‑Hiari Department of Pharmaceutical Sciences, Faculty of Pharmacy,
The University of Jordan, Amman 11942, Jordan
e-mail: [email protected]
Introduction
Isotopic analogs are frequently used in chromatography
as appropriate internal standards for tracing the analyte of
interest in complex mixtures, such as bio-fluids [1, 2]. One
of the commonly used isotopes is the deuterium-labeled
standard [3]. It has the advantage of compensating losses
of native analytes during sample preparation, including
extraction, filtration and dilution procedures [4, 5]. Additionally, deuterated analogs (DAs) of native analytes enable
correction of ion response due to change in mass detector
sensitivity resulted from matrix effect, either ion suppression or enhancement [2, 6, 7]. Similarity in physiochemical
properties of analytes and their 2H counterparts presumes
equivalent ion response for each pair [8].
However, LC–MS analysis carried by Wang et al. [8]
revealed that analyte and its DA have different responses.
They assumed that analyte and its DA encountered different matrix effects [8]. Additionally, Lee and co-authors
observed the same difference and referred it to a minute
change in chromatographic retention time [9]. This change
might be occurred owing to alteration in lipophilicity
between deuterium and hydrogen bonds (isotope effect)
[10, 11]. Response variation in analytes/DAs system was
also reported in GC–MS [12, 13]. Chang et al. reported
inconsistency in response to barbiturates and their DAs that
varied with the deviation in programming of column temperature [14, 15]. Cross contribution of analyte’s intense
ions shared with its DA might lead to non-linear mass
response [16–18]. Cross contribution might have resulted
from impurities of native analyte in DA (or vice versa)
and similar degradation products in both analytes and DAs
[18]. Deterioration of chromatographic separation between
analyte and its DA is also a possible reason for cross contribution [19, 20]. Moreover, low number of deuterium
13
252
atoms (i.e., <3) in DA increases the chance of cross contribution between ions of analytes and its DA [15]. On the
other hand, potential exchange of deuterium and hydrogen
atoms between analyte/pair is another possible reason for
unexpected mass response [21]. Presumably, high temperature in the GC port and in the column may accelerate deuterium–hydrogen exchange [22].
To obtain better insight into understanding the response
variation between analytes and their DAs, “standard addition” method was adopted in this study. This method
achieves better control for dissimilarities in chromatographic systems [14, 23] more than “direct measurement”,
in which analytes and their DAs analyzed separately. Further investigation of mass ionization was carried out by
studying carbon atom relaxation and energy transfer among
atoms of dimethyl azelate (DMA) and d6-dimethyl azelate
(d6-DMA). Carbon relaxation was studied by 13C-NMR
because its rate of relaxation is attributed to the relaxation
of hydrogen/deuterium atoms. The structure, which contains atoms with slow rate of relaxation, cannot dissipate
the energy quickly. This implies that it has radicals with
higher tendency for further radicalization than those with
a tendency for recombination. Consequently, this generates
higher number of fragments and better mass response in
mass ion chamber.
Materials and Methods
Reagents and Chemicals
Azelaic acid 98 % and methanol HPLC grade were purchased from Acros Organics (NJ, USA). Sulfuric acid 98 %
was purchased from S D Fine-Chem (Mumbai, India).
Sodium hydroxide was purchased from Lonover (London,
UK) whereas n-hexane 95 % GC grade was purchased from
Tedia (OH, USA) and used in sample preparation. Nonane
GC grade was purchased from Fluka (Geneva, Switzerland). Deuterated methanol (CD3OD) and deuterated chloroform (CDCl3) 99.8 % containing 1 % v/v trimethylsilane
(TMS) were purchased from Sigma–Aldrich (St. Louis,
USA).
Adipic acid was purchased from Merck (Darmstadt,
Germany), whereas Phthalic acid was purchased from Riedel–de Haen (Seelze, Germany).
Methyl Derivatization of Acids
DMA was synthesized as described by Alzweiri et al.
[24] and modified to suit this work. Briefly, azelaic acid
(1 g, 5.3 mmoL) was dissolved in 5 mL methanol and
0.5 mL sulfuric acid 98 % in a sealed vial. The obtained
solution was heated for 90 min at 70 °C. After cooling to
13
M. Alzweiri et al.
room temperature, 4.5 mL of hexane and 0.5 mL of 0.1 M
sodium hydroxide were added and then vortex centrifuged
for 2 min, 2,000 rpm. Hexane layer was then completely
dried by a rotavapor. Using the same procedure, d6-DMA
was prepared from d4-methanol. A series of DMA concentrations were prepared against a constant concentration of
d6-DMA (1 mM) and another series were prepared from
variable concentrations of d6-DMA and 1 mM of DMA.
The same synthetic procedure was also applied for adipic acid and phthalic acid to produce dimethyl adipate and
dimethyl phthalate, respectively from methanol. d6-dimethyl adipate and d6-dimethyl phthalate were produced from
d4-methanol. Subsequently, 25 mg from dimethyl adipate
was diluted to 25 mL with hexane. And also equimolar
amounts from each of the other products were diluted to
25 mL with hexane. 2 mL from each sample was mixed
with the same amount from the other products and the
final volume adjusted to 25 mL using hexane to make them
ready for GC–MS analysis. Therefore, the amount of dimethyl adipate injected into the system, taking in consideration the split ratio, was 0.8 ng.
Gas Chromatographic Conditions
1.0 μl aliquots (n = 2) of each sample were injected into a
TRACE GC 2000 SERIES (ThermoQuest CE Instruments,
Austin, TX, USA) gas chromatograph equipped with a
split–splitless injector (split ratio, 1:100). The column was
Rtx®-5MS-fused silica capillary column (30 m × 0.25 mm
i.d., 0.25 μm film thickness), consisting of Crossbond®
(5 % diphenyl 95 % dimethyl polysiloxane). Helium was
the carrier gas at a flow rate of 1.0 mL/min. The GC was
interfaced with a GCQ plus (ThermoQuest-Finnigan) mass
detector operating in EI mode (70 eV). The mass spectra
were recorded over 35–650 amu full-scan mode. A linear
temperature program was applied from 45 to 210 °C at
3 °C/min. The run time of analysis for each sample was
55 min. The temperatures of the injector base and ion
source were maintained at 250 and 180 °C, respectively.
Peak identification was based on comparison of mass
spectra of the product with standard mass charts included
in GC–MS database libraries, namely, Mainlib, Wiley and
Replib.
Nuclear Magnetic Resonance Conditions
Nuclear magnetic resonance (NMR) was recorded on
Bruker Avance III NMR spectrometer (1H 500.13, 13C
125.76 MHz) controlled by Topspin 3.1 software. Samples were dissolved in d-chloroform prior to the analysis. Four transients of pulse sequence Zg30 were used to
acquire 1H spectra and 99 transients of Zgpg30 pulse were
used for acquiring 13C data. Temperature of the probe was
253
Variations in GC–MS Response
maintained at 27 °C during the data acquisition. Chemical
shifts were reported in ppm related to tetramethylsilane
(TMS) as internal standard.
DMA and d6‑DMA Identity
The identity and the purity of DMA and d6-DMA were confirmed by in-source mass fragmentation as described in our
previous work [24]. Additionally, 1H-NMR was used to confirm the identity and to test the possibility of cross contamination between DMA and d6-DMA which might have resulted
from methanol impurity in d4-methanol reagent. The integration of 1H-NMR signals is equivalent to 20 hydrogen atoms
which are exactly equal to the number of hydrogens in DMA
(Fig. 1). Signals from 1–2.5 ppm belong to azelaic acid methylene backbone and the signal at 3.7 ppm belongs to the hydrogens of two equivalent methoxy groups. Methoxy signal was
disappeared from the 1H-NMR spectrum of d6-DMA (Fig. 1)
due to replacement of hydrogens with deuterium atoms. This
ensures the high purity of d6-DMA from any DMA traces
which might be produced from the reaction of azelaic acid
with trace contaminant of methanol in d4-methanol.
Results and Discussion
Based on the assumption of similar responses, isotopic
analog is routinely used as a chromatographic equivalent
to the tested analyte and as an appropriate internal standard [8, 9]. However, it has been revealed that they may
not necessarily have similar mass responses [8]. Accordingly, this study will investigate the reasons behind such
differences using DMA/d6–DMA pair as a study model.
These compounds were synthesized from highly pure
starting materials and used shortly after preparation
to guarantee high stability and purity of the DMA and
d6-DMA.
1' & 9'
0.9
TMS
0.8
Normalized Intensity
0.7
0.6
0.5
0.4
O
H3 C
O
9
9'
O
7
5
CH3
1 O 1'
3
0.3
4, 5 & 6
2&8
0.2
3&7
0.1
0
5.86
7
6
5
4
4.00
3
4.00 6.03
2
1
0
-1
Chemical Shift (ppm)
TMS
0.9
0.8
Normalized Intensity
Fig. 1 1H NMR spectrum of
dimethyl azelate (DMA) and
deuterated dimethyl azelate
(d6-DMA). Appearance of
methoxy hydrogens at 3.7 ppm
with correct integration confirms the methyl ester derivatization of azelaic acid and
its disappearance confirms the
purity of d6-DMA from DMA
0.7
0.6
0.5
0.4
O
0.3
O 9
9' CD 3
0.2
7
5
4, 5 & 6
2&8
O
1 O
1' CD 3
3
3&7
0.1
0
4.00
7
6
5
4
3
4.05 6.16
2
1
0
-1
Chemical Shift (ppm)
13
254
M. Alzweiri et al.
2.0E+07
1.8E+07
DMA
RoD
1.6E+07
Adjusted area under the curve
(relave to internal standard)
Response Factor Variation Between DMA and d6‑DMA
and Other Dicarboxylate Esters
d6-DMA
1.4E+07
1.2E+07
1.0E+07
8.0E+06
6.0E+06
4.0E+06
2.0E+06
0.0E+00
0
5
10
15
20
Concentraon (mM)
25
Fig. 2 Mass chromatographic responses of DMA and d6-DMA
adjusted to internal standard response (Nonane) according to standard
addition method procedure. RoD (ratio of difference) was calculated
by subtracting the response of d6-DMA from DMA at each concentration point and then dividing the yield with its corresponding concentration value
By implementing standard addition method, GC–MS
analysis of DMA and d6-DMA revealed a significant difference in response (Fig. 2). It is clear that d6-DMA has a
coefficient of response less than the corresponding analyte
(DMA).
Similarly, this pattern of deviation between DMA and
d6-DMA is apparent in an example of aliphatic molecules
(dimethyl adipate) and of aromatic molecules (dimethyl
phthalate). Equimolar amounts of analyte and its corresponding deuterated counterparts exhibited different
response factors as depicted in Fig. 3. Obviously, deuterated analogs preceded their hydrogenated counterparts and
also exhibited lower response factors.
Identities of dimethyl adipate, d6-dimethyl adipate,
dimethyl phthalate and d6-dimethyl phthalate were confirmed by mass spectrometry. Molecular ion of dimethyl
adipate was observed and the degradation pattern resembles the library best hit. Methoxy loss and Fred McLafferty rearrangement products were observed as shown in
Fig. 3a. d6-Dimethyl adipate revealed almost identical
mass fragmentation pattern of its 1H-counterpart. Mass
Fred McLafferty ( )
-Cleavage
-Cleavage
O
AH
AD
BH
BD
H 3C
O
59
O
143
100
O
CH 3 [M] +. =174
114
Fred McLafferty ( )
a
AD
77
O
105, 92
O
O
BD
CH 3
[M]
+.
=194
CH 3
Tropylium
O
133
163
b
Fig. 3 GC–MS chromatogram of a mixture consisted of dimethyl adipate (AH) and its deuterated analog (AD), dimethyl phthalate
(BH) and its deuterated counterpart (BD) as well as their mass charts.
13
Masses were generated from different fragmentation reactions including (γ) and (δ) Fred McLafferty rearrangements, α-cleavage and tropylium formation
255
Variations in GC–MS Response
shifts were also confirmed the structural identity where
6 amu shift was noticed between molecular ion of dimethyl adipate and its deuterated analog. Deuterated demethoxy product at 146 m/z has a shift of 3 amu and also
the same shift was recorded for masses: 117, 113 and
104 m/z.
Again dimethyl phthalate identity was confirmed by
the best match within the mass libraries. The products of
α-cleavage reaction and tropylium fragments (benzylium
and benzoylium) confirm the molecular identify as shown
in Fig. 3b. Mass shifts between dimethyl phthalate and
its counterpart were noticed particularly on the base peak
which represents demethoxy products.
Explanation of Response Variation Between DMA
and d6‑DMA
The hypotheses that might explain the response variation
between analytes and their DAs are summarized as follows:
(1) the possibility of cross contribution between analyte
and its DA, (2) the chance of deuterium/hydrogen exchange
between co-migrated analyte/DA pair, and (3) a difference
in ionization energy between carbon–hydrogen bond and
carbon–deuterium bond.
Possibility of Cross Contribution
The cross contribution between masses of DMA and d6DMA is significantly affected by chromatographic resolution between their peaks [25]. Several concentrations
of DMA were added to single concentration of d6-DMA
(and vice versa) according to standard addition procedure. This method was adopted to remove any possibilities of response differences between analyte and its DA
resulted from variations in chromatographic system such
as those generated from fluctuation in temperature programming [14, 15]. Increase in the concentration of DMA
against its DA is associated with a decrease in the chromatographic resolution (Fig. 4). The excessive addition of
DMA can almost overwhelm the peak of d6-DMA. However, the ratios of differences in response between DMA
and d6-DMA to their corresponding concentrations were
almost constant (RoD, Fig. 2). This explicit behavior indicates that the molecular ions of DMA and d6-DMA do not
affect the response of each other, even if their chromatographic resolution is minimal. However, cross contribution of molecular ions may take place if the mass weights
between analyte and its DA are trivial (˂3 amu) [15]. This
is unlikely in our model since the difference in masses
(between DMA and d6-DMA) is 6 amu. Therefore, cross
contribution can be excluded as a possible reason that
could explain the difference in response between DMA
and d6-DMA.
Fig. 4 Examples of chromatograms obtained for DMA and d6-DMA
mixtures carried out by standard addition method using constant concentration of d6-DMA (1 mM) and variable concentration of DMA,
namely, 0, 1, 3 and 9 mM
Possibility of Deuterium/Hydrogen Exchange
and Simultaneous Transesterification
Deuterium/hydrogen exchange was also investigated as a
possible reason for the difference in mass response between
DMA and d6-DMA through exchange of methoxy/d3-methoxy groups occurred by transesterification process. Standard
addition method was used to give enough time for nearly
co-migrated molecules (DMA and d6-DMA) to react during
the time of separation. Mixtures of DMA and d6-DMA were
analyzed by total ion current procedure (TIC) to measure the
content of d3-DMA (azelaic acid esterified by one equivalent of methanol and one equivalent of deuterated methanol).
As depicted in Fig. 5, the extracted chromatogram (EC) at
216 m/z signal represents the content of DMA in this mixture, whereas 222 m/z signal corresponds to d6-DMA content.
Negligible response was found for d3-DMA at 219 m/z (the
content of d3-DMA to the total TIC response was 0.007 %).
Furthermore, the expected possibility of simultaneous transesterification of two methoxy units by two d3-methoxy groups
(or vice versa) is extremely low. Again D/H exchange can
be excluded as a possible reason for the difference in mass
response between DMA and d6-DMA. Moreover, other transfers between deuterium/hydrogen atoms are unlikely since no
other responses were detected in the range between 216 and
222 m/z. Chang et al. [15] reported the same observation in a
series of tested barbiturates and their DAs.
Possibility of Mass Ionization Variation Between C–H
and C–D bonds
Next, the theory was investigated based on the assumption that variation in the degree of mass ionization between
13
256
M. Alzweiri et al.
24.84
34.03
28.86
76.78
77.03
77.28
DMA
174.24
13
24.85
28.86
76.78
77.28
d6-DMA
77.03
28.91
34.03
174.21
Fig. 6 13C-NMR spectra of
DMA and d6-DMA. Signal of
methoxy carbon (at a chemical
shift of 51 ppm) was almost
disappeared
51.42
Fig.  5 a Chromatogram of equivalent molar ratio of DMA and
d6-DMA mixture analyzed by total ion current (TIC) mode, b, c are
extracted chromatograms (EC) of DMA and d6-DMA, respectively.
EC of d represents the area of azelaic acid esterified by one equivalent of methanol and one equivalent of deuterated methanol
C–H and C–D bonds may explain the deviation of mass
response in analyte/DA pair. This possibility was experimentally investigated using 13C-NMR relaxation. The
carbon signal of methoxy group in d6-DMA disappeared,
whereas its counterpart carbon in DMA appeared at a
chemical shift of 51 ppm (Fig. 6). This phenomenon can
be attributed to quick relaxation of spin 1/2 nucleus which
leads to signal broadening or even to signal fading [26, 27].
The rapid energy dissipation from the carbon nuclei toward
the terminal deuterium atoms should reduce the data accumulation of the carbon signal during the scanning time.
Consequently, this will minimize the signal-to-noise ratio
of carbon intensities. The adjacent carbonyl carbon was
also affected by relaxation to deuterium nuclei, however,
to a lesser extent. As depicted in Fig. 7, the signal of carbonyl carbon of d6-DMA (174.24 ppm) becomes broader
and less intense than the corresponding equimolar carbon of DMA (174.21 ppm). This might be justified by the
fact that nuclear relaxation is diffused and accelerated by
spinning of unpaired electrons (S = 1 in triplet state) [28,
29]. Unpaired electrons should promote the initiation and
propagation radicalization reactions occurring inside the
257
DMA & d 6-DMA (1:1)
0.9
174.4
34.03
174.1
174.21
174.24
174.2
d 6-DMA
174.4
180
28.91
28.86
24.84
174.3
51.42
174.4
0.4
0.1
174.0
DMA
0.5
0.2
174.1
174.21
0.6
0.3
174.2
174.3
0.7
174.23
Normalized Intensity
0.8
TMS
77.28
77.03
76.78
Fig. 7 13C-NMR of DMA:
d6-DMA equimolar ratio
mixture and the chemical shift
of their carbonyl groups using
highly pure DMA and d6-DMA
samples
174.23
174.21
Variations in GC–MS Response
160
174.3
140
174.2
120
174.1 174.0
100
80
60
40
20
0
Chemical Shift (ppm)
chamber of electron impact ionizer, resembling the mechanism by which photoreactions are enhanced by electrons in
triplet state [30, 31]. Hence, rapid relaxation of C–D bond
and next attached bonds in comparison with C–H bond
implies a rapid diffusivity of energy to molecular electron
structure in deuterated analogs. Consequently, this reduces
the population of triplet/singlet electrons in C–D when
compared to C–H in each time moment. According to the
Lindemann hypothesis, breakage of bonds in unimolecular
gaseous systems is mediated by activation state preceded
the decomposition step of bonds and the rate is quite sensitive to the applied pressure [32, 33]. Because the analytes
are in gaseous state inside the EI chamber [32, 34], we
assume that the population of unpaired electrons in some
bonds promotes the mass decompositions and the response
from total ion current (TIC). It is worth to mention that the
mass response should be increased by increasing the generation of ionized fragments when TIC is used [35]. Furthermore, it was also described in the literature that C–D bond
has less zero point energy than C–H bond, 3 and 4.15 kcal/
mole [36]. This will make the kinetics of the reactions in
deuterated molecules, relying on C–D bond dissociation,
slower than hydrogenated counterparts (i.e., kinetic isotope effect) [37]. Additionally, the substitution of hydrogen
with deuterium might show slight shielding effect on next
carbon as described by Hansen if deuterium atoms are not
allowed to rotate [38]. However, free rotation of X–D bond
makes the shielding effect unpredictable [39, 40].
The relaxation rate of carbonyl carbon was calculated
from the following formula [41]:
R=
1
= πw h
2
T2
(1)
where R is the rate of relaxation, T2 is spin–spin relaxation
time and wh/2 is the width of the signal at half height.
The wh/2 of carbonyl signal in d6-DMA is equivalent to
the distance difference between 21909.40 and 21912.23 Hz,
whereas wh/2 of carbonyl signal in DMA is the difference
between 21908.05 and 21909.04 Hz. Accordingly, the rate
of carbonyl carbon relaxation in d6-DMA and DMA is 9
and 3 s−1, respectively. Three times difference in energy
relaxation between the two carbonyl carbons is quite significant, particularly for carbons which are not directly
attached to the deuterium atoms.
Conclusion
In this work, we present a GC–MS experimental
approach to explain quantification problems resulted
from variation in mass responses between analytes and
their DAs using DMA and d6-DMA as a model. It was
concluded that neither cross contribution of ions nor
H/2H exchange was responsible for such variation. However, the difference in energy transfer between C–H and
C–D bonds, confirmed by carbon nucleus relaxation, can
strongly explain the difference in mass response between
DMA and d6-DMA.
Conclusively, the study showed that DA cannot substitute the standard material in quantifying the analyte, particularly at high concentrations. Additionally,
the study introduced a novel use of NMR relaxation
rate in understanding some principles of mass ionization. This might open the door for more NMR applications in studying other time-dependant physiochemical
processes.
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Acknowledgments We wish to thank The University of Jordan represented by the Deanship of Academic Research for supporting and
funding the project.
Conflict of interest The authors declare that there are no conflicts
of interest.
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