Analytica Chimica Acta 694 (2011) 177–180

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Analytica Chimica Acta

journal homepage: www.elsevier.com/locate/aca

Response to “Comments on the uncertainties in isotope patterns of molecules” by

J. Meija and Z. Mester (doi:10.1016/j.aca.2010.09.029)
J.I. Garcia Alonso ∗ , P. Rodríguez-González
Department of Physical and Analytical Chemistry, Faculty of Chemistry, University of Oviedo, Julián Clavería 8, 33006 Oviedo, Spain

a r t i c l e i n f o a b s t r a c t

Article history: This manuscript presents a response to a recently published article in Analytica Chimica Acta by J. Meija
Received 4 November 2010 and Z. Mester (doi:10.1016/j.aca.2010.09.029) in which the authors comment on some aspects of our
Received in revised form 19 January 2011 previous work entitled “Determination of the uncertainties in the theoretical mass isotopomer distribu-
Accepted 2 March 2011
tion of molecules” Anal. Chim. Acta 664 (2010) 68–76. We present here new arguments to support our
Available online 8 March 2011
previous findings and to clarify some aspects regarding to the calculation of the uncertainty of isotope
patterns. The uncertainty calculations proposed by Meija and Mester for carbon-only clusters have been
Keywords:
compared with our previously developed first-order model based on the spreadsheet approach described
Mass isotopomer distributions
Error propagation
by Kragten (J. Kragten, Analyst 119 (1994) 2161). The results obtained in this comparison demonstrate
Natural variations that the Kragten procedure can be applied safely for the calculation of the uncertainties in the mass
Mass spectrometry isotopomer distribution of molecules when the u average value is calculated. In addition the procedure
provides good results even when the first-order error propagation law failed for some particular cases
(C93 and C186 clusters).
© 2011 Elsevier B.V. All rights reserved.

1. Introduction
The uncertainties in the isotope distribution of molecules due
to the natural variability in the isotope composition of their con-
stituting elements were approached for the first time in a recent
paper from our laboratory [1]. In our approach, we used a first-order
error propagation model [2] taking into account the correlation
coefficients between the isotope abundances of the different ele-
ments calculated from the mass-dependent isotopic fractionation
law [3]. The isotope composition of a given molecule was calculated
using the stepwise matrix multiplication algorithm described by
Kubinyi [4]. Then, the spreadsheet approach described by Kragten
[5] was employed to calculate the uncertainties of the molecular
isotope distributions. We selected Kragten instead of Monte-Carlo
simulations because of the smaller number of isotope distribution
calculations required.
The recent publication of “Comments on the uncertainties in
isotope patterns of molecules” by Meija and Mester [6], in which
alternative solutions for the uncertainties in the isotope patterns
of simple carbon-only clusters are presented, prompted us to write
this response letter. In their paper, Meija and Mester [6] indicate
some shortfalls of the first-order error propagation model when
applied to binomial distributions of carbon atoms and present ques-
∗ Corresponding author. Tel.: +34 985 10 34 84; fax: +34 985 10 31 25.
E-mail address: jiga@uniovi.es (J.I. Garcia Alonso).
tions about the validity of our first-order approach based on the
Kragten procedure. We would like here to take advantage of the
calculations provided by Meija and Mester and apply our procedure
to the same clusters to either validate our approach or understand
its limitations and pitfalls.
When determining the mass isotopomer distribution of a gen-
eral biomolecule of formula Cm Hn Op Nq Sr we need to calculate every
term of the polynomial expansion of:
(A12C + A13C )m × (A1H + A2H )n × (A16O + A17O + A18O )p
×(A14N + A15N )q × (A32S + A33S + A34S + A36S )r
where Axy indicates isotope abundances at nominal mass x and
element symbol y. Then, terms of equal nominal mass need to be
added together to calculate the isotope distribution. The matrix
algorithm described by Kubinyi [4] for complex molecules is an
elegant solution to the mass isotopomer distribution calculations,
which can be easily implemented in Visual Basic for Excel [1].
However, direct solutions for first-order error propagation mod-
els cannot be computed for these complex molecules. For small
organic molecules, however, their isotope distribution (or mass iso-
topomer distribution) is dictated mainly by the isotope distribution
of the carbon cluster and an alternative model was developed by
Meija and Mester [6] to predict the uncertainties of their isotope
distributions.
For simple carbon-only clusters, of formula Cm , the isotope
abundance for any combination of (m − i) atoms of 12 C and (i) atoms
of 13 C in the cluster is given by the binomial distribution where i

0003-2670/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.aca.2011.03.004
178 J.I. Garcia Alonso, P. Rodríguez-González / Analytica Chimica Acta 694 (2011) 177–180

can change from 0 to m: 0,16

u+
m! 0,14 u average
Ai = Ai Am−i (1)
i!(m − i)! 13C 12C
0,12

Relative uncertainty
where Ai is the isotope abundance of the mass isotopomer con- u-
0,1 M+2
taining i atoms of 13 C. According to Meija and Mester [6], the
differentiation of the binomial distribution equation for carbon- 0,08
only clusters using first order Taylor expansion leads to the
0,06
following equation for the relative uncertainty (uA /A) in the abun- M+1
dance of the mass isotopomer containing i 13 C atoms: 0,04
  u
uAi  i  A 0,02 M
= m −  × A 12C (2)
Ai A13C 12C
0
0 50 100 150 200 250 300
For small organic molecules, such as phenol, most of the uncer-
Number of carbon atoms
tainty in the isotope composition of the different mass isotopomers
is due only to the natural variability of carbon [1,6]. Meija and Fig. 1. Comparison of Kragten approximation (points) with the results of Eq. (2)
Mester [6] recognise that, for small organic molecules up to about (black lines) for the relative uncertainties of Cm carbon clusters (m = 10–300) for M
C80 , first-order error propagation is adequate (see Fig. 1b in [6]) (i = 0) (䊉), M+1 (i = 1) (䊉) and M+2 (i = 2) () mass isotopomers with i being the
number of 13 C atoms in the cluster.
when carbon is the only source of uncertainty.

2. Failures of the first-order error propagation model
The calculation model shown in Eqs. (1) and (2) using first-order
error propagation fails for a cluster containing 93 carbon atoms
(C93 ) [6]. The uncertainty of the isotopomer containing 1 atom
of 13 C (at M+1 mass) is predicted to be almost 0 when applying
Eq. (2). Meija and Mester indicated that, for certain number of m
carbon atoms in the molecule and for certain number of i 13 C iso-
topes in the molecule, the uncertainties calculated using Eq. (2)
are much smaller than those calculated using Monte-Carlo simula-
tions. In these cases, Eq. (2) leads to negligible uncertainties, which
are called “black holes” in Ref. [6]. For those special combinations
a second-order error propagation equation or even a third-order
equation need to be used if correct results are to be obtained [6].
The fact that the first-order equation for error propagation (Eq. (2))
fails for these particular combinations of m and i made Meija and
Mester pose doubts on the uncertainty results obtained by us using
the Kragten approximation and first-order error propagation par-
ticularly for large biomolecules such as peptide NIST 8327 (C68 ) and
bovine insulin (C254 ).
In order to compare our procedure [1] with that of Meija and
Mester [6], we have calculated here the relative uncertainties for
the isotope abundances of different Cm clusters (from m = 10 to
m = 300) containing different numbers of 13 C atoms in the clus-
ter (from i = 0 to i = 2) using both Eq. (2) from Meija and Mester [6]
and our approximation using Kragten. Then, the results were com-
pared with those given in [6] using second- and third-order error
propagation calculations. Please note that the Kragten procedure
determines the first derivative of Eq. (1) by adding and/or subtract-
ing the standard uncertainties of the isotope abundances of carbon
As can be observed, for small values of m (up to ca. m = 50) the
uncertainty values calculated adding and subtracting the standard
uncertainties (u+ and u− respectively) are very similar and in agree-
ment with the results of Eq. (2). For large values of m (ca. m > 100
for i = 0 and i = 1) there is an increasing divergence between the u+
and u− values. However, in all cases the u average values are in
agreement with Eq. (2) for any combination of m and i studied. In
our previous paper [1] we have already indicated that, when the
u+ and u− values differed, the u average values could be a good
estimation of the propagated uncertainties. The results shown in
Fig. 1 indicate that results in agreement with the expected values
can be obtained when the u average is calculated for this simple
carbon-only model.
In our previous paper [1] we observed that the relative uncer-
tainties for the M+1 isotopomer were, in many cases, lower than
the uncertainties for the M isotopomer. The results shown in Fig. 1
here and those given in Ref. [6] confirm these findings. Meija and
Mester [6] indicated that this behaviour would occur for Cm car-
bon clusters when m > 47 and this is in agreement with the Kragten
results shown in Fig. 1.
The minima in the relative uncertainties found at masses 93 for
M+1 and 186 for M+2 correspond to the so-called “black holes” and
when the first-order error propagation law (Eq. (2)) fails to pro-
vide correct results [6]. Figs. 2 and 3 show an expanded version
of Fig. 1 focusing on those regions where the first-order equation
fails (mass 93 for M+1 and 186 for M+2). As can be observed from
0,01
0,009

using Eq. (3): 0,008

Relative uncertainty

0,007
∂Ai Ai (A12C ± uA12C ) − Ai (A12C )
≈ (3) 0,006
∂A12C uA12C
0,005
Basically, Eq. (1) is applied both for the nominal A12C abundance
0,004
and for that when the uncertainty uA12C is added or subtracted. As
commented in our previous work [1], the original paper by Kragten 0,003

[5] indicated that a “linearity test can be performed both by adding 0,002
and subtracting the standard uncertainties”. Please note that when 0,001
subtracting the standard uncertainties the denominator in Eq. (3)
0
should also be negative. Fig. 1 shows the results obtained using 80 85 90 95 100 105 110
Kragten approximation (both adding and subtracting the uncer- Number of carbon atoms
tainties) in comparison with the numerical results obtained using
Fig. 2. Comparison of the Kragten approximation using u+ and u− (white points)
Eq. (2). For comparison purposes, we have used here the standard
and u average (black points) with the first-order solution (thick line, Eq. (2)) for
uncertainties employed in Ref. √ [6] for carbon assuming a uniform the relative uncertainties of Cm carbon clusters (m = 80–110) for the M+1 mass
distribution (uA12C = 0.0008/ 3). isotopomers.
 J.I. Garcia Alonso, P. Rodríguez-González / Analytica Chimica Acta 694 (2011) 177–180
0,02
0,018
0,016
Relative uncertainty
0,014
0,012
0,01
0,008
0,006
0,004
0,002
0
150 160 170 180 190 200 210
Number of carbon atoms
Fig. 3. Comparison of the Kragten approximation using u+ and u− (white points)
and u average (black points) with the first-order solution (thick line, Eq. (2)) for
the relative uncertainties of Cm carbon clusters (m = 150–220) for the M+2 mass
isotopomers.
the thick lines in Figs. 2 and 3, Eq. (2) provides very low uncer-
tainty values for clusters in the vicinity of C93 and C186 for M+1 and
M+2 respectively as indicated by Meija and Mester [6]. We have
also plotted here the u+, u− and the u average values calculated
using the Kragten procedure. As can be observed, the uncertainty
values provided by Kragten as u average do not fall into the “black
hole” region of the first-order error function. The relative uncer-
tainty for C93 at the M+1 isotopomer was computed to be 0.091%,
a value which is very close to that calculated by Meija and Mester
[6] for this mass isotopomer using both second- and third-order
error propagation and Monte-Carlo simulations (0.084%). For the
M+2 isotopomer in the C186 carbon cluster our relative uncertainty
was 0.18%. No comparative values were given in [6] for this clus-
ter. Thus, it can be concluded that Kragten uncertainty propagation
for carbon-only clusters provide uncertainty values (u average) in
agreement with the calculated values even in those regions when
the first-order error propagation law fails.
3. Extension of the carbon-only model to large
biomolecules
The basic assumption in the comments from Meija and Mester
[6] is that the natural variability of carbon is the only source of
uncertainty in organic molecules. From our calculations [1] using
bovine insulin (C254 H377 N65 O75 S6 ) as test molecule the carbon
natural variability in mammals only accounts for ca. 50% of the
mass isotopomer distribution uncertainty for this molecule (range
31–67% depending on M+i). Under those circumstances, the sim-
ple analytical solution of the uncertainty propagation model will
not provide satisfactory results as already recognised by these
authors [6]. Monte-Carlo simulations could be an alternative but
will require much longer computation time than the Kragten
approximation as the number of isotope distributions calculated
need to be much higher with Monte-Carlo.
4. Applications in isotope dilution mass spectrometry
The main reason behind our calculations of the uncertain-
ties in the isotope composition of molecules [1] is their possible
use in error propagation calculations for isotope dilution analy-
sis. In our laboratory we are trying to develop routine isotope
dilution procedures for organic molecules, which do not require
the establishment of a methodological calibration graph. For this
purpose, we need to select adequate isotopic labelling of the
molecules to avoid isotopic effects either during sample prepara-
179
tion or chromatographic separations. Both minimal 13 C labelling
[7] and heteroatom labelling [8] are being studied in combination
with isotope pattern deconvolution. Using this alternative proce-
dure, the uncertainties in the isotope composition of the natural
and isotopically labelled molecule need to be taken into account
[7]. The fact that for large organic molecules the relative uncertain-
ties for the M+1 or M+2 isotopomers are lower than that for the M
isotopomer (see Fig. 1) was indicated in our paper [1] as a point to
take into account when selecting the masses for isotope dilution
calculations.
As properly detected by Meija and Mester [6] the example given
in our paper, the relative quantitation of a peptide by formaldehyde
220 reductive dimethylation, was not the right choice. The isotope com-
position of the labelled and unlabelled peptide will be almost the
same making the selection of the measurement masses irrelevant
for uncertainty propagation. Originally, we intended to present a
metrological example in which a peptide of natural isotope com-
position will be determined using a synthetic peptide, labelled
with several 13 C atoms, for isotope dilution analysis [9]. Under
those circumstances the isotope composition of the natural abun-
dance and labelled peptide could be slightly different. Then, the
selection of the measurement masses will be relevant if the iso-
tope composition of the peptides was required for isotope dilution
analysis.
5. Correlation of isotope abundances
In our previous paper [1] we have employed the mass-
dependent fractionation law [3] to calculate the correlation factors,
r, between the isotope abundances from some selected tri- and
tetra-isotopic elements (O, Mg, Si, S, Fe, Cr) in order to include
these correlations in the error propagation model. This was done
because previous models published for the calculation of corre-
lation factors [10] failed to provide meaningful results for tri- a
tetra-isotopic elements. In our calculations [1] we did not intend to
provide a general universal solution for the correlation problem as
mentioned in Meija and Mester [6]. The explanations given in our
paper for the correlations found for O, Mg, Si, S, Fe and Cr were par-
ticular to these elements and not to be taken as a general rule. The
exceptions indicated by Meija and Mester [6] to our explanations
(tellurium, platinum and tungsten) are not really exceptions for tri-
or tetra-isotopic elements as all those elements contain more than
4 isotopes.
6. Conclusions
The publication of the “Comments to the uncertainties in isotope
patterns of molecules” by Meija and Mester [6] was an opportunity
to check and validate the results obtained in our previous publi-
cation [1]. In their comments, Meija and Mester concluded with
this sentence: “For this reason, when evaluating the uncertainties in
isotope patterns of molecules using the first-order error propagation
model (Eq. (2)) in conjunction with the Kragten’s method, we are gen-
erating approximate solutions for an approximate model”. We have
demonstrated here that Kragten procedure can be applied safely
for the calculation of the uncertainties in the mass isotopomer dis-
tribution of molecules. It is true that it is an approximate model but
one model able to produce accurate solutions. The final uncertain-
ties calculated using the u average values were in agreement with
the model for carbon-only clusters developed by Meija and Mester.
Additionally, Kragten procedure provided good results even when
the first-order error propagation law failed for C93 an C186 clusters
(“black holes”).
Obviously, the model given by Meija and Mester [6] cannot be
applied when the isotope composition of carbon is not the only
180 J.I. Garcia Alonso, P. Rodríguez-González / Analytica Chimica Acta 694 (2011) 177–180
source of uncertainty. The contribution of carbon to the total uncer-
tainty of a given isotopomer can be easily calculated using Kragten
procedure [1]. We have observed that for small proteins of natural
origin, when the isotope composition of carbon is that of mammals
(1.09 ± 0.02% 13 C abundance), carbon is no longer the main source
of uncertainty and the model for carbon-only clusters should not
be applied.
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Isotopes, The Mineral Society of America, Washington, 2004.
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