Kinetic and molecular orbital analyses of dicarboxylic acylcarnitine methylesterification show that derivatization may affect the screening of newborns by tandem mass spectrometry

Article abstract of DOI:10.1016/j.bmcl.2015.11.016

Newborns are routinely screened for organic acidemias by acylcarnitine analysis. We previously reported the partial catalytic methylesterification of dicarboxylic acylcarnitines by benzenesulfonic acid moiety in the solid extraction cartridge during extraction from serum. Since the diagnosis of organic acidemias by tandem mass spectrometry is affected by the higher molecular weight of these derivatized acylcarnitines, we investigated the methylesterification conditions. The kinetic constants for the methylesterification of carboxyl groups on the acyl and carnitine sides of carnitine were 2.5 and 0.24 h^-1, respectively. The physical basis underlying this difference in methylesterification rates was clarified theoretically, illustrating that methylesterification during extraction proceeds easily and must be prevented.

Full text of DOI:10.1016/j.bmcl.2015.11.016

Bioorganic & Medicinal Chemistry Letters 26 (2016) 121–125
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Kinetic and molecular orbital analyses of dicarboxylic acylcarnitine
methylesterification show that derivatization may affect the
screening of newborns by tandem mass spectrometry
b
a
a
c
d
Yasuhiro Maeda a, , Yoko Nakajima , Kana Gotoh , Yuji Hotta , Tomoya Kataoka , Naruji Sugiyama ,
⇑
Naohiro Shirai a, Tetsuya Ito b, Kazunori Kimura a,c
a Graduate School of Pharmaceutical Sciences, Nagoya City University, 3-1 Tanabe-dori, Mizuho-ku, Nagoya 467-8603, Japan
b School of Medicine, Fujita Health University, 1-98 Dengakugakubo, Kutsukake-cho, Toyoake 470-1192, Japan
c Graduate School of Medical Sciences, Nagoya City University, 1 Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya 467-8601, Japan
d Aichi-Gakuin University, School of Pharmacy, 2-11 Suemori-dori, Chikusa-ku, Nagoya 464-8651, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Newborns are routinely screened for organic acidemias by acylcarnitine analysis. We previously reported
the partial catalytic methylesterification of dicarboxylic acylcarnitines by benzenesulfonic acid moiety in
the solid extraction cartridge during extraction from serum. Since the diagnosis of organic acidemias by
tandem mass spectrometry is affected by the higher molecular weight of these derivatized acylcarnitines,
we investigated the methylesterification conditions. The kinetic constants for the methylesterification of
carboxyl groups on the acyl and carnitine sides of carnitine were 2.5 and 0.24 hꢀ1, respectively. The phys-
ical basis underlying this difference in methylesterification rates was clarified theoretically, illustrating
that methylesterification during extraction proceeds easily and must be prevented.
Received 10 September 2015
Revised 2 November 2015
Accepted 6 November 2015
Available online 10 November 2015
Keywords:
Carnitine
Acylcarnitine
Methylesterification
Newborn screening
Tandem mass spectrometry
Ó 2015 Elsevier Ltd. All rights reserved.
Screening newborns for organic acidemias and fatty acid
oxidation defects using tandem mass spectrometry (MS/MS)
typically requires analysis of acylcarnitines, which are extracted
using methanol from a spot of blood dried on filter paper.1,2 We
previously reported an HPLC-MS/MS method for determining the
concentrations of acylcarnitine containing dicarboxylic acylcarniti-
nes such as glutarylcarnitine (1a), succinylcarnitine (1b), malonyl-
carnitine (1c) and methylmalonylcarnitine (1d) in serum and urine
(see Scheme 1).3,4
had been catalytically methylesterified by the benzenesulfonic
acid moiety in the SPE cartridge.7 Under these conditions, acylcar-
nitines with no carboxyl group in the acyl residue were not
methylesterified. Generally, a change in the molecular weight of
an analyte during pre-treatment can adversely affect the MS/MS
diagnosis of diseases such as inherited metabolic diseases.
Thus, it is important to prevent esterification to allow for
accurate screening. It was therefore necessary to investigate the
methylesterification of dicarboxylic acylcarnitines. An understand-
ing of the rate of this reaction should allow us to prevent esterifi-
cation from taking place.
Compounds 1–4 were analyzed by MS/MS using multiple reac-
tion monitoring (MRM) in positive ion mode. Generally, MS/MS
analysis of acylcarnitine gives a product ion of m/z 85, whereas
acylcarnitine methyl ester gives a product ion of m/z 99, as the lat-
ter contains the additional ester moiety.8 Although the precursor
ions of compounds 2 and 3 are the same, the product ion of 2 is
m/z 85, while that of 3 is m/z 99 (Fig. 1), thus allowing products
1–4 to be distinguished. The MRM transformations are shown in
Table 1.
Compound 1a is a biomarker of glutaric aciduria type I,5 while
1d is an indicator of methylmalonic acidemia.3,6 These acylcarniti-
nes were extracted using methanol from serum immobilized on a
cation-exchange solid phase extraction (SPE) cartridge (Oasis
MCX; Waters, Milford, MA, USA), and the extract was analyzed
by HPLC-MS/MS. A peak with a mass 14 Da higher than the
expected m/z value of the dicarboxylic acylcarnitines was
observed, suggesting that the carboxyl group in the acyl residue
Abbreviations: MS/MS, tandem mass spectrometry; SPE, solid phase extraction;
MRM, multiple reaction monitoring.
Since the sensitivity of MS to carboxyl groups is heightened
following their methylesterification, the sensitivity of MS to the
⇑
Corresponding author. Tel./fax: +81 52 836 3413.
E-mail address: maeda@phar.nagoya-cu.ac.jp (Y. Maeda).
http://dx.doi.org/10.1016/j.bmcl.2015.11.016
0960-894X/Ó 2015 Elsevier Ltd. All rights reserved.

122
Y. Maeda et al. / Bioorg. Med. Chem. Lett. 26 (2016) 121–125
Table 1
The MS/MS transitions of dicarboxylic acylcarnitines (1–4)
Products
Transition (m/z)
1c
248 > 85
262 > 85
262 > 99
276 > 85
276 > 99
290 > 85
290 > 99
304 > 99
1b, 1d, 2c
3c
1a, 1e, 2b, 2d
3b, 3d, 4c
2a, 2e
3a, 3e, 4b, 4d
4a, 4e
at regular time intervals and evaporated under a flow of nitrogen
gas. The residue was dissolved in 50% aqueous acetonitrile and
injected into the HPLC-MS/MS. Only the substrates and reaction
products were detected in the analysis. The concentrations of
1–4, 5–6, and 7–8 were determined from the peak area of each
compound. Reactions with
ing to the same procedure.
x
-amino acids were conducted accord-
The time course of the methylesterification of 1 (1
lmol/L) in 1%
HCl/methanol solution is shown in Figure 2. The concentrations of
1–4 were calculated by correcting for the MS area ratios of 1–4,
noted above. In the reaction of 1a, the carboxyl group on the glu-
taric acid side was easily methylesterified, and the product, 2a,
reacted slowly to produce 4a (Fig. 2A). Although a trace amount
of 3a was produced, this was below the limit of quantification.
Although the formation of 2b and 2c from 1b and 1c was not as
pronounced as that of 2a from 1a (Fig. 2B and C), products 3b and
3c were indeed formed. Thus, the methylesterification rate of the
carboxyl on the acyl group decreases with decreasing acyl group
length. Thus, it is apparent that the two carboxyl groups compete
during the methylesterification reaction.
Compound 1d accumulates in patients with methylmalonic
acidemia.3 Methylesterification of 1d gave 3d preferentially,
although a small amount of 2d was also formed (Fig. 2D). The pro-
duction rate of 4d was significantly slower than that of 4a–c, likely
due to steric hindrance from the methyl group. Furthermore the
methylesterification of dimethylmalonylcarnitine (1e) produced
primarily 3e, although a trace amount of 2e was also formed,
Scheme 1. The methylesterification reaction schemes for 1, 5, and 7.
detection of 1–4 was examined using identical concentrations of
each compound. A mixture containing 1 mol/L of 1–4 provided
peak area ratios of 1:2:3:4 = 1.0:1.5:4.3:8.2.
An HCl/methanol solution (5% (v/w), 200
l
l
L, Nacalai Tesque,
L of 1, carnitine (5), or propionyl-
mol/L) and vortexed. The solution
L aliquots were removed
Kyoto, Japan) was added to 800
l
carnitine (7) in methanol (1.25
l
was stored at 23.5 0.5 °C, and then 50
l
Figure 1. Product ion MS/MS spectra of: (A) glutarylcarnitine (1a), (B) glutarylcarnitine monomethyl ester on the acyl side (2a), (C) glutarylcarnitine monomethyl ester on
the carnitine side (3a), (D) glutarylcarnitine dimethyl ester (4a).

Y. Maeda et al. / Bioorg. Med. Chem. Lett. 26 (2016) 121–125
123
perhaps because the dimethyl group of 1e is bulkier than the
methyl in the acyl group of 1d (Fig. 2E). Thus the formation of
dimethyl ester product 4e was particularly slow.
First-order plots for the decrease in 1a at three initial
concentrations are shown in Figure 3.
All plots are linear, allowing the pseudo-first-order rate
constants, kobs, to be determined according to Eq. 1:
logðCt=C0Þ ¼ ꢀðkobs=2:303Þ ꢁ t
ð1Þ
where Ct and C0 are the concentrations of 1a at time = t and
time = zero, respectively. The kobs values obtained are listed in
Table 2, and are independent of the initial concentrations of 1a.
The kobs values for the reaction of 1a in 2% and 3% HCl/methanol
solution are also listed in Table 2.
The values of kobs are proportional to the HCl concentration, giv-
ing the relationship kobs = 2.4 ꢁ [HCl]%, where [HCl]% represents
the w/v percentage of HCl in methanol. Although acid catalyzed
methylesterification is a second-order reaction of the carboxylic
acid with methanol, the reactions in this study can be treated as
pseudo-first-order, as methanol was used in large excess compared
to compound 1a, and thus the semi-log plots of the concentration
of 1a versus time are linear, and the kobs values obtained are
independent of the initial concentration of 1a.
The time courses of the methylesterification reactions of 1a, 2a,
and 4a shown in Figure 2A suggest the reaction path represented in
Scheme 1 for the overall methylesterification of 1a. In this study,
k13 is assumed to be significantly smaller than k12, since the
amount of 3a was significantly smaller than that of 1a, 2a, and
4a. According to the reaction scheme (consecutive reactions or ser-
ies reactions) shown in Scheme 1, the concentrations of 1a, 2a and
4a (C1, C2, and C4) can be represented by Eqs. 2, 4 and 5,
respectively.
C1=C0 ¼ expðꢀkT ꢁ tÞ
ð2Þ
ð3Þ
ð4Þ
kT ¼ k12 þ k13ðk12 >> k13
Þ
C2=C0 ¼ ðk12=ðk24 ꢀ kT ÞÞðexpðꢀkT ꢁ tÞ ꢀ expðꢀk24 ꢁ tÞÞ
C4=C0 ¼ 1 þ ð1=ðk24 ꢀ kT ÞÞðk24 expðꢀkT ꢁ tÞ ꢀ k12 expðꢀk24 ꢁ tÞÞ
ð5Þ
In Eq. 3, the sum of k12 and k13 is represented as kT. The individual
rate constants (kT (ꢂk12) and k24) were determined by applying non-
linear least-squares analysis to Eqs. 2, 4 and 5, and the constants
derived are listed in Table 2. The k12 and k24 values derived from
Figure 3. Pseudo-first-order plot for the decrease in glutarylcarnitine (1a) concen-
Figure 2. Methylation reaction curves of acylcarnitines in 1% HCl/MeOH. (A)
Glutarylcarnitine, (B) succinylcarnitine, (C) malonylcarnitine, (D) methylmalonyl-
carnitine, (E) dimethylmalonylcarnitine.
tration. The starting concentrations of 1a were: N; 1
mol/L.
lmol/L, d; 2 lmol/L, ꢁ;
3
l

124
Y. Maeda et al. / Bioorg. Med. Chem. Lett. 26 (2016) 121–125
Table 2
Table 4
Kinetic constants for the esterification of glutarylcarnitine (1a) and (4-methoxycar-
bonyl)butyrylcarnitine (2a)
Kinetic constants for the esterification of decanoic acid, x-aminoacids, carnitine (5),
and propionylcarnitines (7) under the condition of 1% HCl
Substrate Concentration of
Concentration of
HCl (%)
Kinetic
Substrate
Kinetic constantsa (hꢀ1
)
substrate (
lmol/L)
constantsa (hꢀ1
)
kobs
k56
k78
kobs
k12 k24
Decanoic acid
3.4
—
—
1a
1a
1a
1a
1a
2a
1
2
3
1
1
1
1
1
1
2
3
1
2.8
3.2
2.6
5.9
7.4
0.25
2.5 0.24
3.5 0.26
2.7 0.25
5.2 0.37
7.0 0.63
4-Dimethylaminobutylic acid
6-Dimethylaminohexanoic acid
8-Dimethylaminooctanoic acid
5
7
1.3
3.5
4.7
0.45
0.37
—
—
—
0.43
—
—
—
—
—
0.37
—
0.26
a
The kinetic constant kij represents the rate constant for the reaction of substrate
a
The kinetic constant kij represents the rate constant for the reaction of substrate
i to product j. The kij values were obtained from non-liner least-squares analysis.
i to product j. The kij values were obtained from non-liner least squares analysis. The
kobs values were obtained from Eq. 1.
The kobs values were obtained from Eq. 1.
The kinetic constants (kobs) of the methylesterification of
x
-amino acids (4-dimethylaminobutylic acid, 6-dimethylamino-
Table 3
Kinetic constants for the esterification of dicarboxylic acylcarnitines (1b–e) in the
presence of 1% HCl
hexanoic acid and 8-dimethylaminooctanoic acid), which differ
only in the length of their carbon chains, are 1.3, 3.5 and 4.7 hꢀ1
respectively (Table 4).
,
Substrate
Kinetic constantsa (hꢀ1
)
The longer the carbon chain, the larger the kinetic constant,
thus indicating that the distance between the ammonium nitrogen
and the carbonyl oxygen influences the methylesterification rate
under acidic conditions.
The methylesterifications of 5 (k56) and 7 (k78) were also exam-
ined (Scheme 1), and their rate constants listed in Table 4. Compar-
ison of these rate constants indicates that the methylesterification
rate of the carboxyl group on the carnitine side is not significantly
influenced by differences in acyl chain length, as observed also for
the reactions of 1b–e (k13 and k24).
k12
k13
k24
k34
1b
1c
1d
1e
0.76
0.70
0.051
0.022
0.29
0.44
0.17
0.23
0.32
0.31
0.28
0.31
0.38
0.81
0.080
NDb
a
The kinetic constant kij represents the rate constant for the reaction of substrate
i to product j. The kij values were obtained from non-liner least-squares analysis.
The kobs values were obtained from Eq. 1.
b
Not determined.
Eq. 4 are identical to those from Eq. 5. In addition, the k12 values
derived from Eqs. 4 and 5 are in good agreement with kobs and k12
from Eqs. 1 and 2, respectively. To confirm that the described ana-
lytical method is valid, the reaction rate of independently synthe-
sized 2a in 1% HCl/methanol solution was measured, giving a kobs
value of 0.25 (Table 2). This value is comparable to the k24 value cal-
culated from Eqs. 4 and 5 for the consecutive reaction of 1a, thus
strongly supporting the validity of this analytical method.
The kinetic constants of the methylesterification of 1b–e are
listed in Table 3.
Since the k12 value of 1a is significantly larger than those of 1b–
e, the methylesterification of the carboxyl group on the glutaric
acid side of 1a proceeds easily. The kinetic constants k13 and k24
represent the methylesterification of the carboxyl group on the
carnitine side. These values are comparable, thus suggesting that
methylesterification of the carboxyl group is not influenced by acyl
group length.
Methylesterification of glutamic acid in acidic methanol results
in preferential methylesterification of the carboxyl group furthest
from the amino group.9,10 The difference in reactivity between
the two carboxyl groups in glutarylcarnitine (3a) is therefore likely
due to the distance between the carboxyl and amino groups. When
decanoic acid, which lacks an amino group, is dissolved in 1% HCl/
methanol at 23.5 0.5 °C, the kinetic constant kobs is 3.4 hꢀ1
(Table 4).
This indicates that the methylesterification of the carboxyl
group on the carnitine side is particularly slow, and that the
shorter the acyl moiety, the slower the methylesterification of
the carboxyl group of the acyl moiety. This effect is likely due to
the influence of the quaternary ammonium moiety. The first step
in the methylesterification under acidic methanol conditions is
the addition of a proton to the carbonyl oxygen atom. Since the
quaternary ammonium group accepts an electron from the
carbonyl oxygen, the carbonyl oxygen is electron poor when the
carbonyl and ammonium groups are in close proximity, and so
the addition of a proton to the carbonyl oxygen is inhibited.
Interestingly, the distance between the ammonium nitrogen
and the carbonyl oxygen is equal in 4-dimethylaminobutylic acid,
5 and 7. However, the kobs of 5 and 7 is lower than that of dimethy-
laminobutylic acid. This is likely due to the electron density of the
carbonyl oxygens on 5 and 7 being lower than that of 4-dimethy-
laminobutylic acid because of the electron withdrawing oxygen
atom at the 3-position.
The difference in reactivity of the two carboxylic groups (the
carnitine side and the acyl side) in 1 is likely due to the difference
in rate of addition of a proton to the carbonyl oxygen atom. This
can be explained by molecular orbital analysis. Thus, optimized
structures and electronic states of 1 were calculated by the density
functional theory (DFT) approach using B3LYP/6-31+G⁄⁄ 11–14
For
.
compounds 1a–e, the electron density of the highest occupied
molecular orbital (HOMO) is localized on the carbonyl oxygen lone
pair on the acyl side in dicarboxylic acylcarnitine (Fig. 4).
In addition, the electron on the carbonyl oxygen on the car-
nitine side in 1a is found three levels below the HOMO, while
the carbonyl oxygen electron on the carnitine side in 1b is found
two levels below the HOMO. Therefore, methylesterification of 1a
and 1b proceeds preferentially on the acyl side. The electron on
the carbonyl oxygen on the carnitine side in 1c–e is only one level
below the HOMO, and the energy gap between the HOMO and one
level below ranges from 0.011 eV to 0.017 eV in 1c–e. These values
are less than those of 1a (0.042 eV) and 1b (0.049 eV). Thus, the
proton is able to access the carbonyl oxygen atom from both sides
in 1c–e because of the low energy gap, resulting in poor reaction
selectivity of 1c for 2c and 3c. However, the selectivity of 1d for
3d, or of 1e for 3e, is better than that of 1c, since the neighboring
methyl groups in 1d and 1e blocks the addition of a proton to the
carbonyl oxygen on the acyl side.
In summary, acylcarnitines are extracted with methanol for
MS/MS screening of newborns for organic acidemias and fatty acid
oxidation defects. Methylesterification of the carboxyl group on
the acyl side of dicarboxylic acylcarnitines in acidic methanol pro-
ceeds easily. However, methylesterification of the carboxyl group

Y. Maeda et al. / Bioorg. Med. Chem. Lett. 26 (2016) 121–125
125
Figure 4. Carbonyl oxygen lone-pair orbitals of acylcarnitines (1a–e).
4. Maeda, Y.; Ito, T.; Ohmi, H.; Yokoi, K.; Nakajima, Y.; Ueta, A.; Kurono, Y.; Togari,
H.; Sugiyama, N. J. Chromatogr., B Analyt. Technol. Biomed. Life Sci. 2008, 870, 154.
5. Baric, I.; Zschocke, J.; Christensen, E.; Duran, M.; Goodman, S. I.; Leonard, J.;
Müller, V. E.; Morton, D. H.; Superti-Furga, A.; Hoffmann, G. F. J. Inherit. Metab.
Dis. 1998, 21, 326.
6. Deodato, F.; Boenzi, S.; Santorelli, F. M.; Dionisi-Vici, C. Am. J. Med. Genet. C,
Semin. Med. Genet. 2006, 142C, 104.
7. The extraction with acetonitrile from the SPE cartridge avoided the
methylesterification without lowering the recovery (our previous data).3
8. Millington, D. S.; Kodo, N.; Terada, N.; Roe, D.; Chace, D. H. Int. J. Mass Spectrom.
Ion Processes 1991, 111, 211.
on the carnitine was inhibited by the electron acceptance effect of
the quaternary ammonium nitrogen atom. This explanation was
supported by molecular orbital calculations. Since the carboxyl
group in glutarylcarnitine is distant from the ammonium nitrogen,
the methylesterification rate is faster than for other acylcarnitines.
It is important to prevent the methylesterification of glutarylcar-
nitine during extraction procedures for MS-based diagnosis of glu-
taric acidemia type I.
9. Cox, R. J.; Wang, P. S. H. J. Chem. Soc., Perkin Trans. 1 2001, 2006.
10. Baldwin, J. E.; North, M.; Flinn, A.; Moloney, M. G. J. Chem. Soc., Chem. Commun.
1988, 828.
Acknowledgement
11. Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.;
Cheeseman, J. R.; Montgomery, J. A., Jr.; Vreven, T.; Kudin, K. N.; Burant, J. C.;
Millam, J. M.; Iyengar, S. S.; Tomasi, J.; Barone, V.; Mennucci, B.; Cossi, M.;
Scalmani, G.; Rega, N.; Petersson, G. A.; Nakatsuji, H.; Hada, M.; Ehara, M.;
Toyota, K.; Fukuda, R.; Hasegawa, J.; Ishida, M.; Nakajima, T.; Honda, Y.; Kitao,
O.; Nakai, H.; Klene, M.; Li, X.; Knox, J. E.; Hratchian, H. P.; Cross, J. B.; Bakken,
V.; Adamo, C.; Jaramillo, J.; Gomperts, R.; Stratmann, R. E.; Yazyev, O.; Austin, A.
J.; Cammi, R.; Pomelli, C.; Ochterski, J. W.; Ayala, P. Y.; Morokuma, K.; Voth, G.
A.; Salvador, P.; Dannenberg, J. J.; Zakrzewski, V. G.; Dapprich, S.; Daniels, A. D.;
Strain, M. C.; Farkas, O.; Malick, D. K.; Rabuck, A. D.; Raghavachari, K.;
Foresman, J. B.; Ortiz, J. V.; Cui, Q.; Baboul, A. G.; Clifford, S.; Cioslowski, J.;
Stefanov, B. B.; Liu, G.; Liashenko, A.; Piskorz, P.; Komaromi, I.; Martin, R. L.;
Fox, D. J.; Keith, T.; Al-Laham, M. A.; Peng, C. Y.; Nanayakkara, A.; Challacombe,
M.; Gill, P. M. W.; Johnson, B.; Chen, W.; Wong, M. W.; Gonzalez, C.; Pople, J. A.
Gaussian 03, Revision E.01; Gaussian: Wallingford CT, 2004. http://
www.gaussian.co.
This research was supported by a Grant-in-Aid for Research
from Nagoya City University.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2015.11.
016.
References and notes
1. Rashed, M. S.; Ozand, P. T.; Bucknall, M. P.; Little, D. Pediatr. Res. 1995, 38, 324.
2. Chace, D. H.; Kalas, T. A.; Naylor, E. W. Clin. Chem. 2003, 49, 1797.
3. Maeda, Y.; Ito, T.; Suzuki, A.; Kurono, Y.; Ueta, A.; Yokoi, K.; Sumi, S.; Togari, H.;
Sugiyama, N. Rapid Commun. Mass Spectrom. 2007, 21, 799.
12. Lee, C. T.; Yang, W. T.; Parr, R. G. Phys. Rev. B 1988, 37, 785.
13. Miehlich, B.; Savin, A.; Stoll, H.; Preuss, H. Chem. Phys. Lett. 1989, 157, 200.
14. Becke, A. D. J. Chem. Phys. 1993, 98, 5648.