Food Analytical Methods

https://doi.org/10.1007/s12161-018-1272-4

Application of MALDI-TOFMS Combined with Partial Least Square

Regression for the Determination of Mercury and Copper in Canned
Tuna, Using Dithizone as the Complexing Agent and Ag(I)
as Internal Standard
Manuel Mendez Garcia 1 & Kazimierz Wrobel 1 & Alejandra Sarahi Ramirez Segovia 1 & Eunice Yanez Barrientos 1 &
Alma Rosa Corrales Escobosa 1 & Oracio Serrano 1 & Francisco Javier Acevedo Aguilar 1 & Katarzyna Wrobel 1

Received: 8 February 2018 / Accepted: 23 April 2018

# Springer Science+Business Media, LLC, part of Springer Nature 2018

Abstract
In this work, a procedure for the determination of mercury and copper in canned tuna samples is presented, based on the
extraction of their respective dithizone complexes and quantification by conventional MALDI-TOFMS with the aid of partial
least square regression (PLS2). Methanolic dithizone solution (1 ml, 0.01% m/v) containing Ag(I) as internal standard was added
to the calibration solutions and to the acid-digested samples (5 ml, pH 3); copper, mercury, and silver complexes were extracted to
cyclohexane (1.5 ml), and after evaporation, the residues were re-constituted in acetone (100 μl). The samples were prepared by
dried droplet crystallization with α-cyano-4-hydroxycinnamic acid. The experimental and MALDI-TOFMS instrument operat-
ing conditions were set up under criteria of as high as possible detectability and repeatability. When univariate calibration was
carried out using the most intense ion of Cu or Hg complex normalized by internal standard, determination seemed feasible yet
precision of the results was poor. Taking baseline-corrected spectra in m/z range 560–725, PLS2 model was constructed for the
prediction of two elements with the method quantification limits 63 μg kg−1 for Cu and 75 μg kg−1 for Hg, respectively. For
different brands of canned tuna, the results obtained by MALDI-TOFMS combined with PLS2 and by ICP-MS were in good
agreement. The proposed procedure is an attractive alternative for the determination of trace metals in foods because of the
procedural simplicity, micro-scale dimension, high speed of data acquisition/processing, and the cost of instrument operation
lower as compared to any atomic spectrometry technique.

Keywords MALDI-TOFMS . Mercury . Copper . Fish . Dithizone . Internal standard . Partial least square regression

Introduction detection power, wide dynamic range, multielemental and iso-

Determination of trace metals/metalloids in food is highly
demanded, and there is a range of atomic spectrometry tech-
niques available for this analytical task (Wrobel et al. 2018).
Among them, inductively coupled plasma-mass spectrometry
(ICP-MS) is considered a gold standard owing to its high
Electronic supplementary material The online version of this article
(https://doi.org/10.1007/s12161-018-1272-4) contains supplementary
material, which is available to authorized users.
* Katarzyna Wrobel
katarzyn@ugto.mx
1 multitarget capability for polar and non-polar species in a wide
Department of Chemistry, University of Guanajuato, L de Retana
No. 5 36000 Guanajuato Mexico
topic capabilities; however, spectral interferences and high
cost of instrument operation have often been mentioned as
inconvenient aspects (Agatemor and Beauchemin 2011;
Engelhard 2011; Lum and Leung 2016; Wrobel et al. 2018).
As an alternative, high-resolution mass spectrometry (HRMS)
with a soft ionization source can been used in quantitative
approach with additional advantage of confirmation/
identification of chemical species in a variety of matrices
(Keith-Roach 2010; Maki 2012; Schaumloffel and Tholey
2011; Tsednee et al. 2016).
The main features of matrix assisted laser desorption/
ionization high-resolution mass spectrometry (MALDI-
HRMS) encompass exceptional speed of data acquisition,
range of m/z values, micro-scale procedural simplicity, lack of

memory effects, and high sensitivity. This technique has
proved its potential in the analysis of metal-containing large
molecules such as metalloproteins, natural metal adducts with
biomolecules, and metal-tags intentionally attached for better
selectivity and detectability of certain molecules (Abdelhamid
and Wu 2014a; Arruda and Azevedo 2009; Rigueira et al.
2016; Schwarz et al. 2014; Yang et al. 2017). Furthermore,
MALDI-HRMS has also been used to study reaction mecha-
nisms and for structural elucidation of metal complexes (Furia
et al. 2014; Santos et al. 2015; Wei et al. 2015; Wyatt 2011).
On the other hand, applications for the quantification of small
metal-containing molecules (m/z below 800) have been scarce
(Ivanova and Spiteller 2014; Minakata et al. 2014) mainly
because of troublesome background noise caused by matrix-
related ions and uncertain homogeneity of sample co-
crystallization with matrix (Dreisewerd 2014; Maki 2012).
Over the past years, research has been focused on the en-
hancement of analytical performance of laser desorption/
ionization (LDI) for small molecules. Different mixtures of
commercial matrices and new chemical compounds have been
tested (Dreisewerd 2014; Hou et al. 2014; Petkovic et al.
2009; van Kampen et al. 2011); in parallel, several surface-
assisted LDI techniques were developed and some of them
used for metals determination (Abdelhamid and Wu 2014b;
Bergman et al. 2014; Grechnikov et al. 2014; Hou et al. 2016;
Obena et al. 2011; Valdés et al. 2016). These techniques are
free from volatile matrices and provide good tolerance to salts
or other contaminants; however, the efficiency of analyte
desorption/ionization strongly depends on the chemical nature
and the size of nanomaterial applied and has often been re-
ported as poorer with respect to the conventional matrix
assisted LDI (Arakawa and Kawasaki 2010; Picca et al.
2017; Silina et al. 2015; Tang et al. 2009). Despite recent
advances, standard MALDI-HRMS still seems a convenient
and easy to perform technique for routine analysis of small
molecules in the real-world samples (Wang and Giese 2017).
In this work, different brands of canned tuna commer-
cialized in Mexico were analyzed as food items. Mercury
was selected as a typical toxic metal accumulating in
aquatic organisms and entering the food chain (Kim et al.
2016). Essential role of the second element, copper, is well
documented but its homeostasis may be disrupted due to
insufficient or excessive dietary intake leading to the in-
crease of oxidative stress and enhanced risk of several re-
lated pathologies (Bhattacharjee et al. 2017; Jomova and
Valko 2011). The determination of two elements had been
undertaken by conventional MALDI-time of flight mass
spectrometry (TOFMS). The strategy applied to counter-
balance typical limitations of this technique in quantitative
analysis of small molecules and to achieve analytical per-
formance comparable to the actual ICP-MS standard in-
cluded the following aspects: (i) extraction of analytes as
their dithizone complexes and elimination of dithizone
Food Anal. Methods
excess, (ii) application of silver (Ag(I)) as internal stan-
dard, (iii) selection of suitable chemical matrix, (iv) setting
the sample and matrix amounts and tuning MALDI-
TOFMS instrument operating parameters to achieve as
high as possible signal-to-noise ratio, and (v) application
of partial least square method (PLS2) for multivariate pre-
diction of Hg and Cu. Of note, dithizone has been selected
due to its tunable selectivity; at low pH, it forms complexes
only with copper, mercury, silver, palladium, and gold;
these complexes are uncharged in acidic conditions hence
easily extracted to organic solvents (Bousnall and
McBryde 1960; Burger 1973; Cheng and Dong 2005;
Irving and Iwantscheff 1980; Paradkar and Williams
1994; Yang et al. 2010). Once the final conditions were
established, the results obtained in the analysis of several
tuna samples were in good agreement with ICP-MS data.
The proposed MALDI-TOFMS procedure with PLS2
quantification seems an attractive alternative for the deter-
mination of trace metals in foods because of micro-scale
dimension, simplicity and a high speed of data acquisition/
processing, lack of memory effects related with the intro-
duction system, and lower cost of the instrument operation
as compared to any atomic spectrometry technique.
Materials and Methods
Reagents and Samples
All chemicals were of analytical reagent grade except for
dithizone (Dz). Deionized water (18.2 MΩ cm, Labconco),
HPLC-grade methanol, acetone, acetonitrile, cyclohexane,
and chloroform (Sigma) were used throughout.
Stock standard solutions of copper, mercury, and silver
(1000 mg l−1 each) were from Sigma and inductively coupled
plasma mass spectrometry (ICP-MS) internal standard mix
from Agilent Technologies. The following Sigma reagents
were used: α-cyano-4-hydroxycinnamic acid (HCCA), nitric
acid, hydrochloric acid, ammonium hydroxide, sodium hy-
droxide, hydrogen peroxide, ammonium formate, and ethyl-
enediaminetetraacetic acid disodium salt (EDTA). Practical
grade Dz was also from Sigma (D-5130).
Certified reference material DORM-2 (dogfish muscle, cer-
tified value for total mercury: 4.64 ± 0.26 μg g−1) was from
National Research Council, Canada; NIST1572 citrus leaves
with reference values for Cu 16.5 ± 1.0 μg g−1 and for Hg 80
± 20 μg g−1 were also used.
Six canned tuna products (A-F) were purchased in a local
market in Guanajuato city, Mexico, and were freeze-dried pri-
or to analysis. Freeze-dried red snapper tissue was used for the
evaluation of the quantification limits of MALDI-TOFMS;
ICP-MS analysis of this sample showed Cu concentration
183 ± 5 μg kg−1, and Hg was not detected.
Food Anal. Methods
Cleaning Procedures
Dithizone reagent is prone to oxidation and it usually contains
traces of metal ions (Irving and Iwantscheff 1980). In this
work, freshly prepared purified solution of dithizone 0.01%
m/v in methanol was always used. In brief, 250 mg Dz was
extracted with 50 ml of chloroform (vortex, 5 min); the organ-
ic phase was taken, and 25 ml of ammonium hydroxide (1%
m/v) was added. Back-extracted Dz was precipitated by
dropwise addition of hydrochloric acid 6 mol l−1 and the pellet
was recovered (Landry and Fonseca Redondo 1954). The pro-
cedure was repeated three times, and the stock solution was
prepared by dissolving 50 mg of the dried powder in 50 ml of
methanol (0.1% m/v).
Glass tubes for microwave digestion (10 ml) and BlueCap
polypropylene falcon tubes (15 ml) were cleaned by soaking
in 10% v/v nitric acid and rinsing with deionized water.
Residual trace metal impurities were complexed with
dithizone (0.01% m/v in methanol) and extracted to chloro-
form. Finally, the tubes were rinsed three times with deionized
water.
Sample Digestion and ICP-MS Determination of Hg
and Cu
An aliquot (200 mg) of the freeze-dried material was placed in
a glass tube; 250 μl of internal standard solution (2 mg l−1
each of In, Y, Bi, Rh; 5 mg l−1 of Sc; and 10 mg l−1 of Li), 1 ml
of concentrated nitric acid, and 750 μl of deionized water were
added. Microwave-assisted digestion was carried out using
Discover SP-D system (CEM) with the following program:
temperature 150 °C, 3 min ramp, 6 min hold, pressure
300 psi, Bhigh^ agitation. Afterwards, the samples were cen-
trifuged (13,000×g, 10 min), 200 μl portions were diluted 25-
fold with deionized water and introduced to the ICP-MS sys-
tem. An inductively coupled plasma mass spectrometer
(Model 7500ce; Agilent Technologies) with a Meinhard neb-
ulizer and Peltier-cooled spray chamber (2 °C) was used with
the previously reported instrumental operating conditions
(Gomez Ojeda et al. 2015; Maldonado Santoyo et al. 2011).
The isotopes 63Cu, 65Cu, 200Hg, 202Hg, 107Ag, and 109Ag were
monitored and standardized to 89Y and 209Bi signals, respec-
tively. Calibration was performed using mixed standard solu-
tions containing 0, 0.5, 1.0, 2.0, 5.0, 10, 25, and 50 μg l−1 of
Hg and Cu and the internal standards, Y and Bi (10 μg l−1
each). The instrumental detection limits were 51 ng l−1 for Cu
and 150 ng l−1 for Hg; the method detection limits evaluated
using 20 times diluted digests of red snapper tissue were 11
and 32 ng g−1, respectively (ICH 2012). For accuracy
checking, Citrus Leaves NIST 1572 reference material was
used; copper and mercury concentrations found in triplicate
analysis of this material, 16.3 ± 0.5 and 76 ± 2.4 μg g−1, were
in good agreement with the certified values 16.5 ± 1.0 and 80
± 20 μg g−1, respectively. For total mercury in DORM-2 ref-
erence material, 4.62 ± 0.09 μg g−1 was found in triplicate
analysis, also in consistency with the certified value
(4.64 ± 0.26 μg g−1).
Analysis of Dithizone Complexes by ESI-QTOF-MS
and MS/MS
Standard solutions containing individual metal ions (500 μl,
5 mg l−1 Cu, Hg or Ag) were prepared adjusting pH 3 with
ammonium formate 1 mol l−1, 100 μl of dithizone (0.01% m/v
in methanol) was added to each tube, and extraction was car-
ried out with 500 μl of cyclohexane. The organic phase was
recovered and dithizone excess eliminated by vortex with
200 μl of ammonium hydroxide 5% v/v. The organic phase
was evaporated (SpeedVac, 30 °C) and was re-constituted in
500 μl of acetonitrile. The obtained solutions were further
diluted with acentonitrile/0.1% HCOOH to obtain final metal
concentration 100 μg l−1 and were introduced by direct infu-
sion (3 μl min−1) to the electrospray ionization source of a
quadrupole-time of flight mass spectrometer (maXis impact
ESI-QTOF-MS equipped with Data Analysis 4.1, Bruker
Daltonics). ESI was operated in a positive mode with ion
spray voltage 4500 V, nitrogen dry gas 4 l min−1, drying tem-
perature 180 °C, and nebulizing gas pressure 0.4 bar. External
mass calibration was accomplished based on sodium formate
clusters.
Proposed MALDI-TOFMS Procedure
For microwave-assisted digestion, 200 mg of the freeze-dried
material was placed in a glass tube together with 1 ml of water
and 1 ml of concentrated nitric acid; the digestion program
was applied as described above. Next, the tubes were trans-
ferred to a heating block, two 500 μl portions of hydrogen
peroxide were successively added, and the samples were kept
at 90 °C for 1 h to complete mineralization. Finally, the sam-
ples were passed to 15-ml falcon tubes, pH 3 was adjusted by
addition of ammonium formate 1 mol l−1 and sodium hydrox-
ide 10 mol l −1 , and the volume was brought to 5 ml.
Calibration solutions were prepared keeping the same volume
of 5 ml and pH 3 (nitric acid, ammonium formate, sodium
hydroxide). For univariate calibration, the mixed standard so-
lutions contained copper and mercury, each at 0, 10, 20, 40,
and 60 μg l−1. For partial least square method (PLS2), the
training set of 15 solutions was prepared containing the con-
centrations 0, 10, 20, 40, and 60 μg l−1 of each analyte and
according to the half factorial design. For complexation, 1 ml
of dithizone 0.01% m/v in methanol was added; it contained
internal standard (Ag(I)) to ensure concentration 25 μg l−1 of
silver in all analyzed solutions. Extraction was carried out
with 1.5 ml cyclohexane; after vortex (1 min), organic phase
was recovered, and the excess of dithizone was eliminated by

back extraction to 500 μl of ammonium hydroxide (pH > 11).
Finally, organic solvent was evaporated (30 °C, SpeedVac)
and the residue was reconstituted in 100 μl of acetone.
A model AutoFlex speed MALDI-TOF-MS from Bruker
Daltonics with flexControl 3.4 and flexAnalysis 3.4 software
was used. This instrument is equipped with a smartbeam™-II
Nd:YAG laser (335, 532, 1064 nm). For dried droplet crystal-
lization, 0.6 μl of calibration or sample extract was deposited
on a ground steel target followed by 1 μl of HCCA matrix
(2 mg ml−1 in acetone/water 98:2) and dried at room temper-
ature. MS spectra were acquired in the m/z range 560–725 in a
positive reflector mode, using 20% laser intensity and fre-
quency of 1000 Hz. For each spot, a sum spectrum was accu-
mulated from ten replicates of 50 laser shots applied with
small spiral measuring raster. For internal mass calibration,
monoisotopic m/z value 615.044 corresponding to Ag(I)-Dz
was used.
Statistical Methods
The recorded MALDI-TOFMS spectra were baseline-
corrected, saved in Excel 2010, and directly imported to The
Unscrambler 7.0 (CAMO). This software was used to perform
partial least square regression (PLS2).
For univariate calibration, linear regression functions were
obtained using Microsoft Excel 2010.
All analyses were carried out in three independent repli-
cates, concentrations are provided on a dry mass basis of fish
tissue, and mean values with respective standard deviations
were calculated using Microsoft Excel 2010. The results ob-
tained by MALDI-TOFMS and by ICP-MS were statistically
compared using unpaired t test (Statistica for Windows,
Statsoft Inc.) at significance level p < 0.05.
Results and Discussion
Selection of the Internal Standard, Metal
Complexation, and Extraction Conditions
Dithizone forms uncharged 2:1 complexes with copper at
pH 1–8 (Cheng and Dong 2005) and with mercury at pH 1–
4 (Yang et al. 2010). As mentioned in the BIntroduction^ sec-
tion, other metal ions reacting with dithizone in acid medium
(pH < 5) with formation of uncharged complexes include sil-
ver (Ag(I)), palladium (Pd(II)), and gold (Au(I)) (Bousnall and
McBryde 1960; Burger 1973; Cheng and Dong 2005; Irving
and Iwantscheff 1980; Paradkar and Williams 1994; Yang
et al. 2010); among them, Ag(I) was selected as the internal
standard in this work. Noteworthy, Ag, Pd, and Au were not
detected by ICP-MS in any of the acid digested samples.
Dithizone complexes of three metals (Cu(II), Hg(II), Ag(I))
were extracted to organic phase; this procedural step helps to
Food Anal. Methods
eliminate potentially interfering polar or charged species and
allows for preconcentration; it is also important that complex-
ation moves analyte signals in mass spectrum toward higher
m/z values. The experimental conditions for complexation and
extraction were adopted from previous studies (Irving and
Iwantscheff 1980) and were also tested by UV/Vis spectro-
photometry. For three dithizone complexes extracted to cyclo-
hexane, relatively high signal intensity (absorbance measured
at maximum) was obtained around pH 3 (absorption spectra
and the effect of pH are shown in Fig. 1S, electronic supple-
mentary material (ESM)).
For the analysis of acid digested food samples, pH 3 was
adjusted by addition of ammonium formate and sodium hy-
droxide, yielding the final volume of 5 ml. Noteworthy, more
efficient sample digestion was needed for MALDI procedure
as compared to ICP-MS determination, because internal stan-
dard (Ag(I)) presents high affinity to organic species interfer-
ing with dithizone complex formation. Indeed, when nitric
acid digest was additionally heated with hydrogen peroxide,
Ag(I)-Dz signals in mass spectra presented good repeatability
and intensity enabling for setting Ag(I) concentration of
25 μg l−1 as referred to 5 ml of the calibration and sample
solutions.
Among various solvents suitable for the extraction of
metal-dithizone complexes, cyclohexane was selected be-
cause of poor ligand solubility in this solvent (0.14 g l−1)
(Irving and Iwantscheff 1980). The solvent volume of 1 ml
was used to facilitate its further evaporation in a reasonable
time; any signal fluctuation due to changes in extraction effi-
ciency should be compensated using IS. Of note, even low
concentration of dithizone in organic phase caused back-
ground noise in low m/z range of mass spectra; therefore, its
elimination by back-extraction to ammonium hydroxide was
necessary.
Mass Spectra of Cu, Hg, and Ag Complexes
with Dithizone
For ESI-QTOF-MS, individual complexes were obtained (de-
tailed description provided in the BMaterials and Methods^
section). Formation of singly charged ions was confirmed by
exact mass measurement; the mass errors were as follows:
0.2 ppm for Cu (C 2 6 H 2 0 CuN 8 S 2 ), 2.7 ppm for Hg
(C26H21HgN8S2), and 1.6 ppm for Ag (C26H20AgN8S2). In
Fig. 1, experimental isotopic patterns are compared against
in silico calculated spectra and the respective molecular struc-
tures are presented (MS/MS spectra with fragments assigna-
tion are shown in Fig. 2S, ESM). Our results indicate that
single-charged ionic species in a gas-phase present ligand-
to-metal stoichiometry 2:1 and that no fragmentation occurred
in ESI source, in agreement with the previous study (Ross
et al. 1998). During electrospray ionization, charge rearrange-
ment within the molecules is not unusual and, in this work,
Food Anal. Methods

Int.x10 4 Experimental: ESI(+) -QTOF-MS +MS

(a) C26H20CuN8S2 1+
571.0542
N N
4
N S N 1+
N S
Cu
+(I)
N
3 573.0529
N N 1+
2 572.0570
1+
574.0557 1+
1 1+
575.0515 576.0521
0
Int.x10 4. Experimental: MALDI -TOF-MS +MS Theoretical C26H20CuN 8S2, 571.0543
1.50 571.06 2500 1+
571.0543
1.25 2000
1.00 573.06
1500 1+
573.0527
0.75
572.07 1000 1+
0.50 572.0569 1+
574.06
575.07 500 574.0549 1+
0.25 576.07 575.0518
1+
576.0522
0
571 572 573 574 575 576 577 m/z 571 572 573 574 575 576 m/z

Int.x10 4 Experimental: ESI(+) -QTOF-MS 1+ +MS

(b) C26H21HgN8S2 711.1051
5 1+
N N 709.1027
N
S N 4 1+
Hg
+(II)
N 1+ 710.1047
NH S 708.1022
N N 3 1+
1+ 712.1079 1+
707.1007 713.1090
2 1+
714.1117
1
Int. Experimental: MALDI -TOF-MS +MS 0 C26H21HgN 8S2, 711.1032
711.12 Theoretical 1+
709.12 711.1031
4000 1+
708.12 2000
709.1012
3000 710.11 1+
1500 1+ 710.1027
708.1010
2000 1+
712.10 1+ 1+
707.10 713.12 1000 712.1049 713.1045
707.0993
1000 714.36 1+
500 714.1069
0
0
706 708 710 712 714 m/z 706 708 710 712 714 m/z

(c) C26H20AgN8S2 Int.x10 5. Experimental: ESI(+) -QTOF-MS

1+ +MS
1+ 617.0306
4 615.0308

N N
3
N S N
+(I)
Ag
N S N
2 1+ 1+
N N
616.0333 618.0331
1 1+
619.0321

0
Int.x10 4 Experimental: MALDI -TOF-MS +MS 2500 Theoretical 1+
C26H20AgN 8S2, 615.0298
1+ 617.0294
1.0 615.01 617.01 615.0298
2000
0.8
1500
0.6
1000 1+ 1+
0.4 616.02 618.02 616.0324 618.0318
1+
0.2 619.00 500 619.0287 1+
620.0293
0.0 0
614 615 616 617 618 619 620 m/z 614 615 616 617 618 619 620 m/z

Fig. 1 Experimental isotopic patterns obtained for dithizone complexes by ESI-QTOF-MS and by MALDI-TOF-MS; in silico calculated spectra and the
proposed structures of ions are also presented for a copper, b mercury, and c silver

reduction of Cu(II) to Cu(I) was observed as reported before ESI-QTOFMS results suggested that the structure of metal
not only for dithizone complex (Ross et al. 1998) but also for complexes would also be preserved in MALDI process.
Cu(II) bound to amino acids (Xu et al. 1996), peptides Indeed, for each of the three metals, the most intense metal-
(Lavanant et al. 1998), 1,2,4-triazoles (Dytrtova et al. 2011), containing signals in MALDI-TOFMS spectra were consis-
or other organic ligands (Gianelli et al. 2001; Turecek 2007). tent with m/z values and with isotopic patterns obtained using

electrospray ionization. Experimental isotopic patterns are
shown in Fig. 1; for clarity of presentation, spectra for indi-
vidual standard solutions (20 μg l−1 Cu(II), 20 μg l−1 Hg(II),
25 μg l−1 Ag(I)) were acquired using finally selected condi-
tions of the proposed MALDI-TOFMS procedure (BMaterials
and Methods^ section).
In the view of not fully understood redox processes oc-
curring during soft ionization, it is particularly interesting
that this same gas-phase Cu(I)-Dz ion was formed in ESI
source and in MALDI. For Cu(II) complexes analyzed by
ESI, inner electron transfer from ligand or electron transfer
from solvent molecule has been considered (Gianelli et al.
2001; Turecek 2007); owing to the solvent-free conditions
in MALDI, our results point to the inner electron transfer
from ligand to metal. On the other hand, we have not found
any data on the reduction of Hg(II) or Ag(I) during soft
ionization of their organic complexes and in this work,
such reduction was not observed.
Even though two ionization techniques could be used for
the determination of Cu and Hg in form of their dithizone
complexes, MALDI was selected because of its high through-
put and speed, better ionization efficiency, no memory effects,
higher tolerance to sample matrix, and lower cost of analysis
(van Kampen et al. 2011). Green chemistry aspect of MALDI,
in terms of substantially reduced use of organic solvents,
should also be mentioned (Gałuszka et al. 2013).
Setup of MALDI-TOFMS Conditions
Dithizone and its complexes with three metal ions present
absorption bands in the vicinity of the smartbeam™-II
Nd:YAG laser excitation wavelength 532 nm (Fig. 1S); there-
fore, possibility of the matrix-free desorption/ionization was
first explored. Since no metal containing signals were ob-
served in mass spectra for metal ions at their concentration
of 1 mg l−1, 2,5-dihydroxybenzoic acid (2,5-DHB), α-
cyano-4-hydroxycinnamic acid (HCCA), and universal matrix
(1:1 2,5-DHB and HCCA mix) were tested as typical matrices
recommended for small molecules in a positive ionization
mode (Cohen and Gusev 2002). In the application of HCCA
and 2,5-DHB for peptide mass fingerprinting, the reported
optimal excitation wavelengths were 340–355 and 320–
335 nm, respectively (Soltwisch et al. 2012), matching the
smartbeam™-II Nd:YAG laser wavelength 355 nm; these
two matrices and their mix have also been successfully ap-
plied in the analysis of metal-containing compounds (Cohen
and Gusev 2002; Wyatt 2011). Initially, the matrices were
prepared according to the Bruker Guide to MALDI Sample
Preparation (Bruker, #702557) and were mixed 1:1 with ace-
tone solution of metal complexes for the dried droplet co-
crystallization. Using standard sample preparation, the best
peak intensity, single-charged ion formation and no fragmen-
tation of metal complexes were observed for HCCA; hence,
Food Anal. Methods
(a) 4
x10 571.05
1.5 Cu(I)-Dz
Ag(I)-Dz
Intensity, cps
617.01
1.0
Hg(II)-Dz
711.11
0.5
(b)
0
1.5
Intensity, cps
1.0
0.5
0
580 600 620 640 660 680 700 m/z
Fig. 2 MALDI-TOF-MS spectra obtained using finally selected
experimental and instrument operating conditions for a standard
solution containing Cu(II), Hg(II) (20 μg L −1 each), and Ag(I)
(25 μg l−1) and b blank solution without IS
this matrix was selected for further method development.
Noteworthy, our choice was consistent with the recent recom-
mendations for quantitative analysis of small molecules
(Wang and Giese 2017).
The next task was to ensure homogenous co-crystallization
and the highest possible signal-to-noise ratio. Keeping HCCA
concentration at 10 mg ml−1 and concentration of metal ions in
the standard solution at 100 μg l−1, different sample prepara-
tion techniques on a ground steel target and different solvents
were tested. Based on these experiments, it was decided to
deposit successively sample and matrix solutions and to use
acetone as a volatile solvent in which both the matrix and
metal complexes are soluble (Irving and Iwantscheff 1980;
Padliya and Wood 2004). It was also noted that without acid-
ification of HCCA solution with trifluoroacetic or formic acid,
signal intensities for metal complexes were relatively higher.
Likewise reported elsewhere, addition of nitrocellulose
(Donegan et al. 2005) improved homogeneity but also caused
signal suppression; therefore, co-crystallization additives were
not further considered. Fine-tuning of sample-to-matrix ratio
was carried out (Padliya and Wood 2004); after evaporation of
cyclohexane, metal complexes (100 μg l−1 of each metal)
were re-constituted in 20, 40, 100, 200, or 400 μl of acetone
and 0.6 μl of the obtained solution was mixed with 1 μl of
HCCA 10 mg ml−1 without dilution or diluted 2, 3, 5, 10, and
20 times with acetone. Mass spectra were obtained using sin-
gle cycle of 50 laser shots, and the detection limits (DLs) were
estimated for each preparation; the lowest DLs were 0.4 μg l−1
for Cu, 0.3 μg l −1 for Hg, and 0.8 μg l −1 for Ag and
corresponded to the sample re-constituted in 100 μl (5-fold
dilution with respect to 20 μl, which was the minimum ace-
tone volume required for re-constitution after solvent evapo-
ration) and 5-fold diluted matrix. The results of these
Food Anal. Methods

Fig. 3 Repeatability of MALDI- (a) (b)

6
TOF-MS spectra obtained for

Intensity x 103, cps

Intensity x 103, cps
standard solutions prepared in 5 8
three different days (10 μg l−1 Cu, Ag(I)-Dz
Cu(I)-Dz
20 μg l−1 Hg, 25 μg l−1 Ag): a 4 6
Cu(I)-Dz, b Ag(I)-Dz; c Hg(II)- 3
Dz. d Mean values of signal 4
intensities measured in four 2
replicates for each metal complex 2
1
with respective standard
deviations (m/z value indicated on 0 0
each bar) 570 572 574 576 m/z 614 616 618 620 m/z

(c) 10 (d)
4

Intensity x 103, cps

Intensity x 103, cps

8
3
Hg(II)-Dz
6

m/z 617
m/z 615
2
4

m/z 573
m/z 571
1

m/z 711
m/z 709
2

0 0
706 708 710 712 714 m/z Cu(I)-Dz Ag(I)-Dz Hg(II)-Dz

experiments are presented in ESM; for better clarity of presen-
tation, Fig. 3S shows spectra with photos of the respective
spots obtained for 5-fold diluted matrix (final concentration
2 mg ml−1) while varying dilution of the sample. Mass spectra
and photos shown in Fig. 4S were acquired for 5-fold diluted
sample (evaporated extract plus 100 μl acetone) and different
matrix dilutions. Detection limits were estimated based on
three signal-to-noise ratios and are shown in Fig. 5S; of note,
these values cannot be considered true detection limits be-
cause they were obtained using single laser shot series for
different dilutions of relatively high concentration of metal
ions (100 μg l−1).
Finally, laser fluence, the number of shots, and the mode of
their application were adjusted under criterion of as high as
possible signal-to-noise ratio and good repeatability (the se-
lected conditions are listed in the BMaterials and Methods^
Table 1 Analytical parameters evaluated for univariate linear
regression calibration using the most intense signals of Cu and Hg, with
and without normalization by IS (m/z 617)
Parameter Cu (m/z 571) Hg (m/z 711)
Without IS With IS Without IS
section). As often reported before, low laser fluency with rel-
atively few shots per series were beneficial to keep low spec-
tral noise and to get sound repeatability whereas summation of
signals, obtained in successive series, provided enhanced
sensitivity.
Mass spectra obtained for blank and for standard mix
(20 μg l−1 of Cu and Hg, 25 μg l−1 Ag) using the final
conditions are presented in Fig. 2; it can be observed that
ions of protonated Cu, Hg and IS molecules appear in m/z
regions free from high background signals. In Fig. 3a–c,
spectra of metal complexes in the standard solutions
(10 μg l−1 Cu, 20 μg l−1 Hg, 25 μg l−1 Ag) prepared inde-
pendently in four different days are shown whereas in Fig.
3d, mean values for the most intense signals acquired in
these measurements together with respective standard devi-
ations are given. Relative standard deviations were as fol-
lows: 3.7% for Cu (m/z 571.06), 6.2% for Hg (m/z 711.12),
and 6.5% for Ag (m/z 617.01). Taking Cu and Hg signals
normalized by IS, relative standard deviations decreased
(3.1% for Cu and 2.2% for Hg), indicating that application
of Ag(I)-Dz as internal standard provided improved preci-
sion of the entire procedure.
With IS

R2 0.9854 0.9958 0.9856 0.9955 Univariate Calibration

DL (μg l−1) 0.96 0.71 1.4 1.1
QL (μg l−1) 3.2 2.4 4.7 3.6 Each calibration solution was repeatedly deposited on three
CV, % (20 μg l−1) 7.4 3.8 8.4 5.7 spots of the MALDI target. Mass spectra were then acquired
Method DL (μg kg−1) 24 18 35 28 from each spot in three successive replicates, and all individ-
Method QL (μg kg−1) 80 59 117 91 ual intensities of the most abundant signal were used to obtain
linear regression function for each metal. In Table 1, analytical
 Food Anal. Methods

2.5 (a) 8 (b) (c)

709.10 711.10 8
571.05 615.04 617.04
2.0
573.05 6

Intensity x 104, cps

6
1.5
Cu(I)-Dz Hg(II)-Dz Ag(I)-Dz
4 4
1.0

0.5 2 2

0 0 0
570 572 574 576 706 708 710 712 714 614 616 618 620
m/z m/z m/z

Fig. 4 Metal-dithizone signals obtained by MALDI-TOF-MS procedure for aqueous standard solution containing 20 μg l−1 of Cu(II) and Hg(II)
for fresh strip fin (blue); this same sample with standard addition (red). All three solutions contained IS (25 μg l−1 of Ag as Ag(I)-Dz). a
(20 μg l−1 of Cu(II) and Hg(II) in the acid digested sample) (black) and Cu(I)-Dz. b Hg(II)-Dz. c Ag(I)-Dz (color figure online)

parameters are presented that were evaluated using analyte
signal directly or after normalization by IS (m/z 617.01). As
can be observed, the calibration detection limits were around
one part per billion, making the procedure attractive for trace
element determination. Noteworthy, application of Ag
dithizone complex as IS resulted in the improved linearity of
calibration function, slightly better detection and quantifica-
tion limits and better repeatability (Table 1). Method detection
and quantification limits were evaluated based on calibration
performed in the presence of the sample matrix (10-fold
diluted acid digest of red snapper sample) (ICH 2012). For
accuracy checking, standard addition method was applied.
Specifically, aliquots of Cu and Hg standard solutions
(100 ng of each metal) were added to 200 mg of freeze dried
sample of the canned tuna A, yielding 20 μg l−1 of each metal
in the solution to which dithizone with IS was added or
500 μg kg−1 in the freeze-dried sample. In Fig. 4, spectra of
metal complexes obtained in the sample without and with
standard addition are presented; spectra of standard solution
containing 20 μg l−1 Cu, Hg, and 25 μg l−1 of Ag are also
included. Since natural abundance of 202Hg is 29.74% as com-
pared to 69.15% for 63Cu, taking these two isotopes for uni-
variate quantification, about two times higher signal intensi-
ties were obtained for copper complex at m/z 571 with respect
to mercury complex at m/z 711. Taking IS-normalized signals,
the percentage recoveries 101 and 91% obtained for Cu and
Hg, respectively, were indicative of acceptable accuracy. The
quantitative results for the method of standard addition are
presented in Table 2 together with Cu and Hg concentrations
determined in this same sample by ICP-MS; a good consis-
tency between two methods is clearly observed; however, pre-
cision of MALDI-TOFMS results obtained by univariate cal-
ibration was substantially poorer. Finally, the concentration
found for Hg in DORM-2 was 4.89 ± 0.47 μg g−1, in agree-
ment with the certified value 4.64 ± 0.26 μg g−1 (t test,
p < 0.05).

Table 2 Five replicates of Cu and Hg determination in canned tuna sample BA^ by univariate MALDI-TOFMS procedure and by ICP-MS. For
MALDI-TOFMS, results obtained in this sample after standard addition and percentage recoveries of the two elements are also presented

Element Element concentration (μg kg−1) Recovery (%) ICP-MS result (μg kg−1)

Sample Sample + 100 nga

Copper 188 693 101 242.3

236 801 113 244.6
263 768 101 251.9
290 680 78 248.4
201 761 112 255.1
Mean ± SD 236 ± 38 741 ± 46 101 ± 13 248.5 ± 4.7
Mercury 720 1117 79.4 667.2
605 1180 115 679.1
655 1076 84.1 673.5
694 1052 71.5 669.3
668 1128 104 665.8
Mean ± SD 668 ± 39 1121 ± 55 91 ± 16% 671.0 ± 4.8
a
Five hundred micrograms per kilogram added to the freeze-dried sample
Food Anal. Methods

Table 3 Analytical parameters evaluated for PLS2 model constructed for Cu and Hg determination, based on MALDI-TOFMS spectra

Analysis stage Parameter Cu Hg

Calibration Slope 0.9896 0.9879

R2 0.9948 0.9939
RMSEC (%) 1.261 1.555
Cross validation slope 0.9899 0.9897
R2 0.9936 0.9923
RMSEP (%) 1.667 2.088
Prediction SD for 5 blank sample predictions (μg l−1) 0.240 0.297
Method QL (μg kg−1) 63 75
Sample A predicted 259 ± 23 643 ± 21
Sample A + 500 μg kg−1 predicted 779 ± 25 1123 ± 35
Recovery (%) 104 96
DORM-2 (4.64 ± 0.26 μg Hg g−1) 2.41 ± 0.18 4.38 ± 0.31

Even though sound analytical performance was obtained,
univariate quantification presents important drawbacks since
it is based on the signal intensities measured for a single ion,
ignoring analytical information existing in the entire spectrum
and potentially correlated with analyte concentration.
Furthermore, manually integrated analyte signals need to be
normalized by IS and two calibration functions must be com-
puted independently for Hg and Cu. To circumvent these lim-
itations, multivariate calibration was performed by means of
partial least square method (PLS2) which uses baseline-
corrected entire spectra in a suitable m/z range.
Multivariate Calibration (Partial Least Square Method)
PLS2 algorithm utilizes mass spectra of the calibration solu-
tions containing varying concentrations and proportions of the
analytes to generate latent variables (components) that allow
for collecting the spectral data of high relevance in predicting
concentrations of two analytes while reducing data
dimensionality (Mehmood et al. 2012). This particular multi-
variate method was selected, due to its capability to handle
multicollinearities inevitable when continuous data, such as
mass spectra, are generated (Alves et al. 2014; Nicolaou and
Xu 2011). In the construction of PLS2 model (Unscrambler
7.0), X matrix consisted of the mass spectra acquired for cal-
ibration set of solutions whereas Y matrix contained concen-
trations of Cu and Hg in each of these solutions. It was ob-
served that elimination of signals appearing outside the isoto-
pic patterns of the compounds of interest had no important
effect on the regression coefficients; hence, the entire spectra
(m/z 560–725) were used for the construction of PLS2 model.
In the next step, the number of components was varied; each
model was cross-validated by leaving one sample at a time;
according to the results, five components were selected for
each analyte (Unscrambler screenshots showing PLS2 model
are presented in ESM: Fig. 6S for Cu and Fig. 7S for Hg,
respectively). In Table 3, analytical parameters obtained for
this model are resumed; good linearity (R2 > 0.99) and similar

Table 4 Results of Hg and Cu determination in different brands of MS (3). Mean values with respective standard deviations are presented,
canned tuna commercialized in Mexico, obtained by MALDI-TOFMS based on three independent replicates
with IS and univariate calibration (1), by PLS2 method (2), and by ICP-

Brand Cu, mean ± SD (μg kg−1) Hg, mean ± SD (μg kg−1)

1 2 3 1 2 3

A 236 ± 38 259 ± 23 249 ±5 668 ± 39 643 ± 21* 671 ± 5

B 364 ± 38 358 ± 20 355 ± 9 385 ±47 383 ± 21 391 ± 11
C 386 ± 19 390 ± 14 378 ± 11 651 ± 56 668 ± 14 673 ± 9
D 730 ± 22 722 ± 11 716 ± 13 636 ± 58 671 ± 23 657 ± 12
E 778 ± 45 746 ± 16 754 ± 13 198 ± 18 197 ± 10 184 ± 10
F 654 ± 31 659 ± 14 680 ± 12 134 ± 11* 103 ± 5 106 ± 7
DORM-2a 2.48 ± 0.42 2.41 ± 0.18 2.45 ± 0.11 4.89 ± 0.47 4.55 ± 0.32 4.62 ± 0.09
a
Concentration given in micrograms per gram
*Statistically significant difference with respect to ICP-MS result (t test, p < 0.05)

slopes were obtained for calibration as compared to cross-
validation; however, root mean square errors of prediction
(RMSEP 1.667 for Cu and 2.088 for Hg) were higher than
errors of calibration (RMSEC 1.261 for Cu and 1.555 for Hg).
Method quantification limits were calculated as ten standard
deviations obtained for five prediction replicates performed on
blank solution (10-fold diluted acid digest of red snapper
tissue) (Zhang and Harrington 2015); the values 63 μg kg−1
for Cu and 75 μg kg−1 for Hg were similar as those attained for
univariate calibration (59 μg kg−1 for Cu and 91 μg kg−1 for
Hg). Likewise described in the BMaterials and Methods^ sec-
tion, sample A was used for recovery experiments, yielding
acceptable values of 104% for Cu and 96.0% for Hg.
Accuracy was also demonstrated by Hg prediction in the cer-
tified reference material DORM-2, as indicated in Table 3
(prediction plots shown in Figs. 6S and 7S, ESM). Finally, it
was observed that elimination of IS signals from mass spectra
(m/z range 610–620) clearly deteriorated PLS2 results (see
Figs. 8S and 9S in ESM) which points out that the algorithm
used sample-to-sample fluctuation of IS signals during con-
struction of regression functions for the two analytes.
Analysis of Canned Tuna Commercialized in Mexico
Six different brands were analyzed by MALDI-TOFMS pro-
cedure using univariate calibration and PLS2 method. In
Table 4, the results are presented together with those obtained
by ICP-MS. For each method, mean value with respective
standard deviation is presented based on three independent
replicates. Good consistency of means is clearly observed
with no statistical differences found between the proposed
procedures and the ICP-MS (t test, p < 0.05), except for Hg
results in sample A (PLS2) and sample F (univariate calibra-
tion). It should be stressed however that the best precision
corresponded to ICP-MS though the application of PLS2 en-
abled for enhanced precision of MALDI-TOFMS with respect
to univariate calibration.
Few previous studies informed on Hg and/or Cu concen-
trations in canned tuna fish marketed in different countries, as
briefly reviewed by Mol (Mol 2011). Our ICP-MS results
obtained for Cu (249–754 μg kg−1) were within the ranges
reported in Turkey (0.08–11.9 mg kg−1) (Mol 2011; Tuzen
and Soylak 2007) and in Kingdom of Saudi Arabia (0.13–
1.87 mg kg−1) (Ashraf et al. 2006) yet tended to exceed the
concentrations reported in the USA (0.01–0.51 mg kg−1)
(Ikem and Egeibor 2005); of note, seafood is considered a
good source of this essential element and some authors cite
concentration of 30 mg kg−1 as internationally approved in
fishery products (Mol 2011). As to the highly toxic mercury,
the US Food and Drug Administration has a regulatory action
level of methylmercury in fish at 1 mg kg−1 wet mass (FDA
2001); the ICP-MS results obtained for total Hg in this study
were in the range 106–673 μg kg−1 (as referred to the freeze-
Food Anal. Methods
dried mass), indicating that some products were at least close
to the above action level. On the other hand, the concentra-
tions of Hg found in this work were consistent with those
reported in Turkey (from not detected to 1.14 μg kg−1) (Mol
2011), Libya (200–660 μg kg−1) (Voegborlo et al. 1999), and
in the USA (53–740 μg kg−1) (Ikem and Egeibor 2005) but
slightly higher than in Iran (43–253 μg kg−1) (Khansari et al.
2005). Important differences in Hg concentrations were de-
tected among analyzed brands of canned tuna; there was more
than six times lower concentration found in brand E as com-
pared to brand B which in turn corresponded to the highest Hg
concentration. Our results confirm that heavy metal determi-
nation in canned fish is necessary in food quality control, and
the feasibility of MALDI-TOFMS for such purpose has been
demonstrated.
Conclusions
Many applications of laser desorption/ionization techniques
for the analysis of small molecules by high-resolution mass
spectrometry have been proposed in the last decade; however,
to the best of our knowledge, this is the first study using
conventional MALDI-TOFMS for the quantification of
metals in form of their complexes with organic ligand and it
is also the first attempt for using partial least square regression
(PLS2) for this purpose. For two heavy metals of interest,
dithizone was used due to its selectivity toward Hg(II) and
Cu(II) at slightly acidic conditions (pH 3) yielding formation
of uncharged complexes; among other metal ions reacting
with dithizone in similar conditions (Ag(I), Pd(II), Au(III)),
silver was used as internal standard. Formation of a gas-phase
singly charged metal complex ions with stoichiometry 2:1 was
confirmed by ESI-QTOFMS and MALDI-TOFMS analysis.
Typical problems related with the inhomogeneity of sample
co-crystallization with matrix, high background in relatively
low m/z region and poor repeatability, were addressed. In the
first place, polar and ionic species present in the sample were
eliminated by extraction of metal complexes to cyclohexane
whereas application of Ag(I) as internal standard allowed for
enhanced analytical performance especially in terms of im-
proved precision. Several variables such as relative amount
of sample and HCCA matrix on the MALDI target and instru-
ment operating conditions were set in course of systematic
experiments. Quantification was carried out by univariate cal-
ibration taking the ion of the highest abundance for each an-
alyte and for IS, whereas the entire spectra were used to con-
struct PLS2 model. In the latter case, traditional normalization
by IS was not necessary; apparently, PLS2 algorithm was
capable to modulate signal fluctuation based on IS signals
present in all mass spectra. In the analysis of six brands of
canned tuna fish, good agreement was obtained between
MALDI-TOFMS procedure with PLS2 quantification and
Food Anal. Methods
ICP-MS data. The proposed procedure can be recommended
as a high-throughput, simple, and affordable alternative for
ICP-MS which, currently, is a gold standard in the field of
trace element analysis. It should be stressed that instrument
operating cost for MALDI-TOFMS is lower as compared to
any atomic spectrometry technique.
Funding This study was funded by CONACYT, Mexico (grants 123732
and 253879) and by the University of Guanajuato (grants 800/2016 and
721/2016).
Compliance with Ethical Standards
Conflict of Interest Manuel Mendez Garcia declares that he has no
conflict of interest. Kazimierz Wrobel declares that he has no conflict of
interest. Alejandra Sarahi Ramirez Segovia declares that she has no con-
flict of interest. Eunice Yanez Barrientos declares that she has no conflict
of interest. Alma Rosa Corrales Escobosa declares that she has no conflict
of interest. Oracio Serrano declares that he has no conflict of interest.
Francisco Javier Acevedo Aguilar declares that he has no conflict of
interest. Katarzyna Wrobel declares that she has no conflict of interest.
Ethical Approval This article does not contain any studies with human
participants or animals performed by any author.
Informed Consent Not applicable.
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