Food Anal.
Methods
https://doi.org/10.1007/s12161-017-1086-9
Direct Determination of Ca, K and Mg in Cassava Flour Samples
by Laser-Induced Breakdown Spectroscopy (LIBS)
Vinicius Câmara Costa 1 & Diego Victor de Babos 1 &
Francisco Wendel Batista de Aquino 1 & Alex Virgílio 1 & Fábio Alan Carqueija Amorim 2 &
Edenir Rodrigues Pereira-Filho 1
Received: 31 August 2017 / Accepted: 27 October 2017
# Springer Science+Business Media, LLC 2017
Abstract This study aims to develop a method for direct de-
termination of Ca, K, and Mg in cassava flour samples by
laser-induced breakdown spectroscopy (LIBS). LIBS instru-
mental parameters were evaluated using factorial design with
a central point. The variables evaluated were delay time (0.0;
1.0 and 1.8 μs) and fluence (81; 2037 and 4074 Jcm−2): both
were tested in three different values. The best results obtained
were with a delay time of 1.8 μs and a fluence of 4074 Jcm−2.
The proposed calibration model obtained using LIBS data
presented a good correlation with reference values obtained
by inductively coupled plasma atomic emission spectrometry
(ICP OES). The predictive capabilities, based on the standard
error of validation (SEV), were 117, 530 and 77 mg kg−1 for
Ca, K, and Mg, respectively. The proposed LIBS method was
applied to the analysis of 90 cassava flour samples, and the
results showed concentrations of Ca, K, and Mg of 352–1845,
1285–3187, and 320–1242 mg kg−1, respectively.
Keywords LIBS . Cassava flour . Direct determination .
Univariate calibration . Normalization mode
Introduction
Cassava flour represents one of the main nutrient sources in
the diet of populations in developing countries, especially
* Edenir Rodrigues Pereira-Filho
erpf@ufscar.br
1
Group of Applied Instrumental Analysis, Department of Chemistry,
Federal University of São Carlos, São Carlos, São Paulo 13565-905,
Brazil
2
Department of Exact and Technological Sciences, State University of
Santa Cruz, State, Ilhéus, Bahia 45662-900, Brazil
those with low incomes (FAO 2015). It is a rich source of
fiber, carbohydrates, vitamins, and minerals. The main essen-
tial elements available in cassava flour are Ca, K, and Mg (Da
Silva et al. 2017).
Several analytical techniques, such as flame atomic absorp-
tion spectrometry (FAAS) (Amorim et al. 2016; Amorim et al.
2017), inductively coupled plasma optical emission spectrom-
etry (ICP OES) (Linhares and Nóbrega 2017), and inductively
coupled plasma-mass spectrometry (ICP-MS) (Pinheiro et al.
2017), have been applied to determine the elemental concen-
tration in food samples (Korn et al. 2008). In general, these
techniques require a sample pre-treatment to convert the solid
material to a homogeneous aqueous solution through the pro-
cedures of acid digestion (Ferreira et al. 2013; Korn et al.
2008). In addition, the sample is diluted promoting a loss in
sensitivity and, depending on the analytical technique used for
measurement, the analyte becomes undetectable. Systematic
errors can also be introduced due to contamination or loss by
volatilization, which affect the accuracy of the results (Bizzi
et al. 2017).
In contrast to intensive sample preparation procedures in-
volving acid digestion, the direct analysis of solids with min-
imal manipulation of the sample is a promising alternative
(Jantzi et al. 2016). In this sense, laser-induced breakdown
spectroscopy (LIBS) is an attractive technique that has been
used in direct analysis of several types of food samples. More
specifically, LIBS was used for the determination of such
nutrients as Ca, K, and Mg in powdered milk and solid dietary
supplements (Augusto et al. 2017), P, K, Ca, Mg, S, Fe, Cu,
Mn, and Zn in wheat flour (Peruchi et al. 2014), and the
determination of Ca in breakfast cereals (Ferreira et al.
2010), among others (Andrade et al. 2017; Bilge et al. 2015;
Hedwig et al. 2016). The success of LIBS for those studies
include some advantages, such as direct analysis with minimal
or no sample preparation, the use of small quantities of sample

(less than 1 g), multi-element capability, high analytical fre-
quency and lower consumption of reagents, and generation of
waste.
However, the direct analysis of solid samples by LIBS
presents several difficulties, such as: (i) reproduction of the
data related to the process of ablation, (ii) formation of the
plasma, (iii) microheterogeneity, (iv) matrix effects, and (v)
the absence of solid standards for calibration when masses in
the range of nanogram or microgram are used (Cremers and
Radziemski 2013; Musazzi and Perini 2014). A widely used
strategy approach to quantitative analysis in LIBS relies on the
use of calibration curves prepared with certified reference ma-
terials (CRMs) (Fantoni et al. 2008) and this procedure has
been used for plant analysis purposes (Santos et al. 2012). In
practical terms, however, the lack of similarity of the physical
and chemical properties of CRM such as density and porosity,
which is often not similar of the samples set, may impair the
results (Santos et al. 2012).
To overcome these limitations, several authors have used
spectra obtained from LIBS, associating the application of
chemometric tools, normalization or standardization strategies
(Castro and Pereira-Filho 2016) and the selection of variables
(El Haddad et al. 2014; Guezenoc et al. 2017) for the purposes
of identification/classification (Costa et al. 2017; Neiva et al.
2016) and calibration using univariate and multivariate strat-
egies (Andrade and Pereira-Filho 2016; Andrade et al. 2017;
Augusto et al. 2017). In this sense, the goal of this study is to
develop a simple and fast method for the direct analysis of
cassava flour samples using LIBS for the determination Ca,
Mg, and K contents. Univariate calibration strategies were
tested and combined to build linear regression models.
Experimental
Samples
Ninety samples of handmade cassava flour of the white and
yellow types were purchased directly from flour houses in
different cities of southern Bahia State, Brazil. Posteriorly,
the samples were ground in a ball mill, model 8000 FL
SHIMIDT (Spex Sample Prep, USA) to reduce particle size.
Later, the samples were placed in polyethylene bottles that had
been decontaminated previously and were stored in
desiccators.
Sample Preparation
To establish the univariate calibration models, the concentra-
tions of Ca, K, and Mg obtained by ICP OES after acid diges-
tion were used as reference values (n = 3). For the sample
digestions, a 50.0-mL digester block with PFA-closed vessels
(perfluoroalkoxy, Savillex, MN, USA) was used. The
Food Anal. Methods
digestion procedure (n = 3) was performed by accurately
weighing 200 mg of cassava flour into PFA vessels followed
by the addition of 2.0 mL of 65% v/v HNO3. The vessel was
kept overnight (ca. 16 h) at room temperature. Then, 2.0 mL
of 30% v/v H2O2 were added, and the solution was heated at
100 °C for 180 min. To check the performance of the proce-
dure, NIST CRM1568b (rice flour) was also digested. In ad-
dition, recovery experiments were employed for the evalua-
tion of the sample preparation procedure. In the recovery tests,
known concentration of analytes were added to the samples
before the digestion procedure.
Instrumentation
An initial solid sample inspection was made using an ARL
Perform’X from Thermo (Thermo Fisher Scientific, Madison,
WI, USA) wavelength-dispersive X-ray fluorescence (WD-
XRF) to perform a fast chemical evaluation. The instrument
analytical conditions were 30 kV and 80 mA for voltage and
current, respectively. For all elements evaluated in this study,
only the electronic transition, Kα, was considered. During the
initial chemical inspection using WDXRF, powdered cassava
samples were directly placed in the sample holders and, using
a He atmosphere, Ca, K, and Mg signals were observed
confirming its presence. We initially sought to obtain calibra-
tion models for these elements, but we could not get a good
agreement between the reference values by ICP OES and the
values predicted by WD-XRF.
LIBS spectra were obtained using a J200 LIBS system
(Applied Spectra, Fremont, CA, USA) controlled by the
Axiom 2.5 software (Applied Spectra). This instrument con-
sists of a 1064 nm Nd: YAG laser and a 6-channel charge-
coupled device (CCD) spectrometer recording spectral infor-
mation from 186 to 1042 nm. Channel 1 goes from 186 to
309 nm, channel 2 from 309 to 460 nm, channel 3 from 460 to
588 nm, channel 4 from 588 to 692 nm, channel 5 from 692 to
884 nm, and channel 6 from 884 to 1042 nm. The spectral
resolution is < 0.1 nm for UV to VIS and < 0.12 nm for VIS to
NIR. The Axiom 2.5 software from the same manufacturer
controlled the operational parameters of the equipment.
These parameters were the laser pulse energy ranging from 0
to 100 mJ, the gate delay (the time interval between the inci-
dence of the laser pulse and the start of signal recording by the
spectrometer) ranging from 0 to 2 μs, and the spot size ranging
from 50 to 250 μm. The Axiom 2.5 software also manages the
movement of the sample, assisted by an automated XYZ stage
and a 1280 × 1024 complementary metal-oxide semiconduc-
tor (CMOS) color camera imaging system. At 1.05 ms, the
software establishes the gate width, which is the time the
spectrometer registers the emission signals.
To establish the regression models, concentrations of Ca,
K, and Mg, obtained with ICP OES (iCAP 6000, Thermo
Scientific, Waltham, MA, USA), were used as reference
Food Anal. Methods
values (n = 3), after acid digestion of the samples. This instru-
ment allows sequential emission signal collection using both
axial and radial viewings. Argon (99.996%, White Martins-
Praxair, Sertãozinho, SP, Brazil) was used as the plasma gas
for ICP OES. The instrumental parameters are shown in
Table 1.
Reagents and Solutions
Ultrapure water (18.2 MΩ cm resistivity) produced by a Milli-
Q® Plus Total Water System (Millipore Corp., Bedford, MA,
USA) was used to prepare all the solutions. Nitric acid
(HNO3) was previously purified using a sub-boiling distilla-
tion system Distillacid™ BSB-939-IR (Berghof, Eningen,
Germany), and hydrogen peroxide (H2O2) 30% w/w (Synth,
Diadema, SP, Brazil) was used for the digestion of samples.
All glassware and polypropylene flasks were washed with
soap, soaked in 10% v/v HNO3 for 24 h, and rinsed with
deionized water prior to use. The multi-element standard so-
lutions were prepared daily by appropriate dilution of
1000 mg L−1 Ca, K, and Mg stock solutions (Qhemis,
Jundiaí, SP, Brazil) and used for construction of the calibration
curves.
Optimization of the Instrumental Conditions of LIBS
The cassava flour samples were weighed (ca. 500 mg) on an
analytical balance (model AY 220, Shimadzu, Kyoto, Japan)
to facilitate LIBS analysis. The samples were pressed at
10 t in. −1 to form pellets that are 12 mm in diameter.
Pelletized samples numbered 7, 22, 56, 75, 81, and 85 were
prepared in duplicate for preliminary studies using LIBS (op-
timization of the system). The instrumental parameters of the
LIBS were evaluated through the use of factorial design with a
central point. The variables evaluated were the delay time
(0.0; 1.0 and 1.8 μs) and the fluence (81; 2037 and
4074 Jcm−2). For each pellet, approximately 600 spectra were
obtained at different regions of the samples. These spectra
were obtained in 15 lines, and in each one, approximately 40
laser pulses were obtained. The additional laser settings used
were a scan length of 8 mm, a laser repetition rate of 5.0 Hz,
and a speed of 1.0 mm s−1.
As a response to factorial design, the highest signal-to-
noise and signal-to-background ratios (SNR and SBR, respec-
tively) were evaluated for all lines of Ca, K, and Mg moni-
tored. To allow for simultaneous optimization of the responses
obtained, a mathematical approach based on the desirability
functions applied in optimizing multi-response experiments
that was proposed by Derringer and Suich was used
(Derringer and Suich 1980). The approach first converts each
response (yi) into an individual desirability function (di),
which varies within the range of 0 ≤ di ≤ 1. If the response is
desired, the function value is di = 1, and if the response is
outside the acceptable region, di = 0. The individual desirabil-
ity (di) is calculated according to Eq. 1:
8
> 0 if y < L
<
y−L s
d¼ if L ≤ y≤ t ð1Þ
>
: T −L
1 if y > T
where L is the lowest acceptable value for the response, T is
the target value, and s is the weight (when equal to 1 = linear
desirability function). In this study, L values were the lowest
of the SBR in a set of experiments for each element and T
values were the highest of the SBR for each element.
Later, the overall desirability (OD) is calculated according
to Eq. 2:
pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
OD ¼ m d 1 d 2 …d m ð2Þ
where m is the number of response variables.
Analytical Parameters for the Proposed Method
Using LIBS
Reference concentrations obtained after sample digestion and
ICP OES determinations were used for construction of linear
models. LIBS calibration models were obtained from the re-
gression of emission signals and reference concentrations. The
predictive capability of the LIBS calibration models were
based on the lower standard error of calibration (SEC) and
standard of error validation (SEV) for all emission lines and
analytes tested (Augusto et al. 2017).
The limit of detection (LOD) and limit of quantification
(LOQ) for LIBS were calculated according to Eqs.3 and 4,
respectively.
3  SDBackground noise
LOD ¼ ð3Þ
b
10  SDBackground noise
LOQ ¼ ð4Þ
b
where SD is the standard deviation of the background noise,
measured in the surroundings of the selected emission line,
and b is the slope of the calibration curve. The LOD and LOQ
were calculated for each sample and the samples with the
lowest, average, and highest concentrations of Ca, K, and
Mg were selected to obtain LOD and LOQ ranges for the
LIBS method.
Data Collection and Evaluation
For evaluation and processing of the dataset, Microsoft Excel
was used for calculation of the univariate calibration models
for LIBS. Aurora software (applied spectra) and information
 Food Anal. Methods

Table 1 Operational parameters

for ICP OES determinations Instrument parameter Operational conditions

Radio frequency applied power (kW) 1.15

Integration time for low emission line (s) 15
Integration time for high emission line (s) 5
Sample introduction flow rate (mL min−1) 2.1
Pump stabilization time (s) 5
Argon auxiliary flow rate (L min−1) 0.5
Argon plasma flow rate (L min−1) 12
Argon nebulizer flow rate (L min−1) 0.7
Replicates 3
Elements and wavelengths (nm); Caa (II 317.9), bK (I 766.4
and I 769.8) and cMg (II 279.5, II
280.2 and I 285.2)

view modes: axial and radiala , only axialb , only radialc

from the National Institute of Standards and Technology was 1568, was employed for evaluation of the accuracy of the
used for the identification of the LIBS emission lines. analytical procedure. After comparison between the certified
MATLAB software version 2009a (MathWorks, Natick, and the determined (n = 3) concentrations, the obtained recov-
MA) and two custom-programmed routines were used for ery values were 106% (for Ca), 99% (for K), and 94% (for
the preliminary inspection of data (Castro and Pereira-Filho Mg). Statistical evaluation, using the Student t test, showed
2016): (1) LIBS_treat was applied to detect eventual outliers that there is no significant difference between both obtained
in the spectra. In this case, for each sample (rows in the data and the certified values at 95% confidence level, confirming
matrix), the standard deviation, area, and maximum and the accuracy of this method. In addition, the accuracy was also
Euclidean norm were calculated. If an outlier was detected evaluated through addition and recovery experiments.
(e.g., standard deviation equal to 0), this spectrum was re- Samples were spiked with standard solutions of Ca, K, and
moved by the analyst, and then 12 normalization modes were Mg before acid digestion for evaluating the accuracy of meth-
calculated. (2) libs_par was applied after normalization using od and the obtained recoveries were in the range of 90 and
the previous routine. The routine libs_par enables the calcula- 111% for all elements.
tion of signal-to-background ratio (SBR) for both signal area
and the height for a specified emission line. To use this rou-
tine, it is necessary to establish the emission line intervals that Optimization of LIBS Parameters
contain the background and the signal. Additional details
about normalization modes are available in the publication A factorial design (experiments 1–9) with a central point was
of Castro and Pereira-Filho (2016). used to optimize the LIBS instrument parameters (delay time
This process was required because LIBS spectra are sensi- and fluence). For the experiments performed in the variables
tive to several potential problems, including variations in the
Table 2 Matrix of factorial design with the variables evaluated for
sample surface, the stability of the laser, and the interaction LIBS and results obtained from overall desirability (OD)
between the laser and the sample (Tognoni and Cristoforetti
2016). The efficiency of these normalizations was assessed Experiment Delay time Fluence OD
comparing the SEC for the calibration models. Another im-
Coded Real/ (μs) Coded Real (Jcm−2)
portant parameter considered was the emission line selection.
Several emission lines were tested for each analyte; the best 1 −1 0 − 0.02 2037 0.10
line was that presented the lowest SEC value. 2 −1 0 −1 81 0.08
3 0.111 1 − 0.02 2037 0
4 0.111 1 −1 81 0
Results and Discussion 5 1 1.8 − 0.02 2037 0
6 1 1.8 −1 81 0
ICP OES Determinations 7 −1 0 1 4074 0.10
8 0.111 1 1 4074 0.37
After acid digestion of the samples, determinations of Ca, K, 9 1 1.8 1 4074 0.51
and Mg were carried by ICP OES. CRM of rice flour, NIST
Food Anal. Methods

highest SBR, area, and height). Thus, the individual desirabil-

ity was combined into a single response (the overall desirabil-
ity, OD, see Eq. 2). The experimental matrix for factorial de-
sign with the responses as functions of OD is presented in
Table 2. It is possible to observe that the highest OD value is
found for experiment 9, and thus, the optimized conditions for
delay time 1.8 μs and 4074 Jcm−2 (80 mJ of laser pulse energy
and 50 μm of spot size). Figure 1 shows the contour plot with
the most appropriate condition marked with a star.

Emission Line Selection and Calibration Models

The calibration models obtained for Ca, K, and Mg using

Fig. 1 Contour graph for variables fluency and delay time LIBS were obtained from 90 samples (60 for calibration and
30 for validation).To obtain calibration models, 12 normaliza-
tion modes were employed for the signal after obtaining ap-
evaluation, the individual desirability (see Eq. 1) was calcu- proximately 600 spectra for each sample: signal average
lated as a function of SBR, signal area, and height for the lines (Norm_1), signal normalized by individual norm and then
of Ca II 393.36, K I 766.49, and Mg II 279.55. In this case, averaged (Norm_2), normalized area (Norm_3) and maxi-
each response was coded from 0 (undesired response; the mum (Norm_4) and then averaged, signal sum (Norm_5),
lowest SBR, area, and height) to 1 (desired response; the signal sum after normalization by individual norm

800
(a) Calcium Area 16,000
(b) Potassium Area
Height Height
700 15,000

600 14,000

13,000
SEC (mg kg-1)

500
SEC (mg kg-1)

12,000
400

6,000
300

4,000
200
2,000
100
0
_1

_2

_3

0
_4

_5

_6

_7

_8

_9

2
_1

_2

_3

_4

_5

_7

1
_6

_8

_9

2
0

_1

_1

_1
_1

_1
_1

m
m

m
m

m
or

or

or

or

or

or

or

or

or
m
m

m
or

or

or

or

or

or

or

or

or

or

or

or
N

N
or
or

or
N

N
N

Normalization mode

350
(c) Magnesium Area
Height
300

250
SEC (mg kg-1)

200

150

100

50
_2

_6
_1

_3

_4

_5

_7

_8

_9

2
_1

_1

_1
m
m

m
m
or

or

or

or

or

or

or

or

or

or

or

or
N

Normalization mode
Fig. 2 Standard error of calibration (SEC) using the normalization modes proposed as functions of area and height for (a) Ca; (b) K, and (c) Mg
 Food Anal. Methods

(Norm_6), area (Norm_7) and maximum (Norm_8) and sig- SEC, SEV, LOD, LOQ, and relative standard deviation (%
nal average (Norm_9 and Norm_11) and sum (Norm_10 and RSD). In addition to the equation for the univariate linear
Norm_12) after normalization by C signals (I 193.09 and I model, we included the normalization mode selected, emis-
247.85 nm). sion line (nm), and signal type. Figure 4 presents the reference
The SEC was the criteria to select the best emission lines versus predicted concentrations for the three analytes, and
and the best normalization to obtain the calibration models. good concordance was observed between both the values ob-
The monitored lines for Ca: II 314.88, II 317.93, II 393.36, II tained by ICP OES and the concentrations predicted by LIBS
316.84, II 422.67, I 527.05, I 643.90, I 643.25, I 649.37; for K: for Ca (Fig. 4a), K (Fig. 4b), and Mg (Fig. 4c).
I 766.49, I 769.89; and for Mg: II 279.55, II 280.27, I 285.21 The proposed method was applied to the determination
were chosen using the Aurora software (Applied Spectra). It is of Ca, K, and Mg in ninety samples of cassava flour.
necessary to evaluate several emission lines because the spec- Table 4 presents the results obtained for all samples. The
tra generated via LIBS can have different spectral interfer- concentration ranges found were as follows: 352–
ences for each analyte (Augusto et al. 2017). 1845 mg kg−1 for Ca, 1112–6187 mg kg−1 for K, and
The emission lines that presented the lowest SEC were as 361–1242 mg kg−1 for Mg. According to the recommend-
follows: Ca I 527.05, K I 766.49 and Mg I 285.21 nm. In ed daily intake (RDI) for adults established by the
Fig. 2, it is possible to check that the best results (lowest B r a z i l i a n N a t i on a l H e a l t h S u r v e i l l a n c e A g e n c y
SEC) for Ca (Fig. 2a) were obtained with a signal normalized (ANVISA), intakes for Ca, K, and Mg may be considered
by maximum and then averaged (Norm_4) using signal adequate at 1000; 4700, and 260 mg day−1, respectively
height; for K (Fig. 2b) with the sum (Norm_5) using signal (ANVISA, Brazil 2005). Considering a 100 g portion of
area; and for Mg (Fig. 2c) using the average (Norm_1) using the sample and the concentration ranges found, it was
signal area. The peak profiles for the emission lines for Ca, K, possible to calculate the percentage range of recommend-
and Mg in functions for normalization models are shown in ed daily intake (%RDI) for the samples analyzed in this
Fig. 3 (a–c, respectively). study. This range varied from 3.5–18% for Ca, 2.3–13%
The analytical parameters obtained for the models of uni- for K, and 13–47% for Mg, thus evidencing the high
variate calibration are shown in Table 3. These include the nutritional potential of cassava flour.

0.060 10,000
(a) Ca I 527.05 (b) K I 766.49
(normalized by average maximum)

0.045 7,500
(normalized by sum)
Signal intensity

Signal intensity

0.030 5,000

2,500
0.015

0
0.000
526.8 526.9 527.0 527.1 527.2 527.3 765.75 766.00 766.25 766.50 766.75 767.00
Wavelength (nm) Wavelength (nm)

2,300
(c) Mg I 285.21

1,725
(normalized by average)
Signal intensity

1,150

575

285.0 285.1 285.2 285.3 285.4 285.5

Wavelength (nm)

Fig. 3 Emission peak profiles selected for (a) Ca, (b) K, and (c) Mg to calculate the univariate regression models
Food Anal. Methods

Table 3 Calibrations for

univariate model parameters Parameters Analyte
calculated for Ca, K, and Mg
Ca K Mg

Normalization mode Average maximum Sum Average

selected
Emission line (nm) 527.05 766.49 285.21
Signal type Height Area Area
Linear model (y = ax + b, R) [Ca] = 2.29.10−5x + [K] = 1671× + 4.92.106 [Mg] = 4.62× + 127
0.01 R = 0.9496 R = 0.9496 R = 0.9491
SEC (mg kg−1) 104 464 66
SEV (mg kg−1) 117 530 77
LOD (mg kg−1) 45–80 105–200 25–60
LOQ (mg kg−1) 150–266 349–666 83–200
RSD (%) 3–13 1–15 0.9–11
Analyte concentration range 352–1845 1112–6187 361–1242
(mg kg−1)

RSD relative standard deviation, SEC standard error of calibration, SEV standard error of validation, LOD limit of
detection, LOQ limit of quantification

2,100 6,500

(a) (b)
Ca predicted concentration (LIBS, mg kg )

K predicted concentration (LIBS, mg kg )

-1

-1

1,850
5,500

1,600
4,500
1,350

1,100 3,500

850 Calibration Calibration

Validation 2,500 Validation
Calibration Calibration
600
Validation Validation
1,500
350
[Ca] Calibration = (-1.2 ± 31) + (1.00 ± 0.03)x [K] = Calibration = (0.97 ± 156) + (0.99 ± 0.04)x
[Ca] Validation = (12 ± 56) + (0.94 ± 0.06)x [K] = Validation = (-43 ± 233) + (0.95 ± 0.06)x
100 500
100 350 600 850 1,100 1,350 1,600 1,850 2,100 500 1,500 2,500 3,500 4,500 5,500 6,500
-1 -1
Ca reference concentration (ICP OES, mg kg ) K reference concentration (ICP OES, mg kg )

1,450
(c)
Mg predicted concentration (LIBS, mg kg )
-1

1,250

1,050

850

650
Calibration
Validation
450 Calibration
Validation

250
[Mg] = Calibration = (1.13 ± 28) + (1.00 ± 0.03)x
[Mg] = Validation = (74 ± 36) + (0.93 ± 0.05)x
50
50 250 450 650 850 1,050 1,250 1,450
-1
Mg reference concentration (ICP OES, mg kg )
Fig. 4 Reference versus predicted concentrations for (a) Ca, (b) K, and (c) Mg. Calibration and validation samples are represented by circles and
squares, respectively
 Food Anal. Methods

Table 4 Concentrations (mg kg−1) determined (average ± standard deviation, n = 3) and recovery for Ca, K and Mg in cassava flour sample by the
reference (ICP OES) and proposed procedures (LIBS)

Sample Ca K Mg

Reference Determined Trueness Reference Determined Trueness Reference Determined Trueness

concentration concentration (%) concentration concentration (%) concentration concentration (%)
(ICP OES) (LIBS) (ICP OES) (LIBS) (ICP OES) (LIBS)

Cassava flour, calibration database

1 1004 ± 85 879 ± 111 88 3290 ± 145 3814 ± 179 116 897 ± 28 835 ± 17 93
2 604 ± 42 430 ± 39 71 4142 ± 205 4897 ± 309 118 732 ± 30 755 ± 24 103
3 567 ± 50 482 ± 45 85 2756 ± 85 2663 ± 22 97 803 ± 65 871 ± 71 108
4 1236 ± 85 1325 ± 124 107 3163 ± 142 2683 ± 171 85 1220 ± 85 1242 ± 52 102
5 895 ± 75 878 ± 24 98 3126 ± 111 2497 ± 82 80 540 ± 32 543 ± 55 100
6 868 ± 65 749 ± 109 86 3940 ± 358 4194 ± 406 106 800 ± 47 879 ± 20 110
7 1791 ± 98 1644 ± 87 92 2234 ± 98 2316 ± 118 104 598 ± 25 680 ± 38 114
8 765 ± 48 856 ± 23 112 2541 ± 196 2283 ± 119 90 700 ± 52 760 ± 40 109
9 739 ± 42 780 ± 60 105 3594 ± 274 3295 ± 383 92 1050 ± 60 1096 ± 28 104
10 1786 ± 70 1845 ± 86 103 3945 ± 359 3947 ± 460 100 965 ± 64 1082 ± 83 112
11 472 ± 38 385 ± 41 81 3373 ± 358 3580 ± 428 106 565 ± 29 498 ± 21 88
12 711 ± 48 842 ± 45 118 3927 ± 254 4446 ± 421 113 632 ± 18 673 ± 24 106
13 654 ± 51 578 ± 42 88 2823 ± 134 2360 ± 57 84 754 ± 53 784 ± 71 104
14 681 ± 48 739 ± 44 108 6050 ± 423 6187 ± 725 102 701 ± 46 785 ± 51 112
15 515 ± 55 602 ± 73 117 3102 ± 401 3734 ± 573 120 803 ± 40 836 ± 37 104
16 685 ± 38 781 ± 50 114 3650 ± 219 3798 ± 558 104 505 ± 32 509 ± 37 101
17 915 ± 101 864 ± 83 94 3202 ± 401 3310 ± 474 103 320 ± 20 361 ± 26 113
18 665 ± 44 597 ± 54 90 5289 ± 325 5476 ± 565 104 795 ± 47 742 ± 61 93
19 623 ± 60 554 ± 69 89 5034 ± 414 5416 ± 565 108 748 ± 53 736 ± 60 98
20 650 ± 58 752 ± 56 116 4076 ± 369 3843 ± 581 94 407 ± 26 404 ± 39 99
21 616 ± 42 695 ± 46 113 2398 ± 302 2118 ± 284 88 825 ± 61 762 ± 44 92
22 605 ± 55 630 ± 40 104 2587 ± 169 2626 ± 182 102 802 ± 58 710 ± 50 89
23 486 ± 42 418 ± 43 86 2312 ± 285 2519 ± 329 109 763 ± 35 714 ± 24 93
24 520 ± 51 381 ± 44 73 3476 ± 152 3912 ± 89 113 750 ± 36 684 ± 38 91
25 416 ± 33 480 ± 48 115 3050 ± 102 3350 ± 177 110 712 ± 40 700 ± 61 98
26 798 ± 75 885 ± 113 111 2182 ± 146 1888 ± 250 87 854 ± 38 901 ± 37 105
27 687 ± 65 829 ± 89 121 3915 ± 186 3240 ± 246 83 889 ± 42 961 ± 38 108
28 1653 ± 78 1653 ± 87 100 3391 ± 207 3076 ± 389 91 880 ± 42 900 ± 35 102
29 415 ± 36 415 ± 60 100 4667 ± 258 4750 ± 314 102 773 ± 18 783 ± 16 101
30 470 ± 43 352 ± 44 76 4321 ± 394 4480 ± 565 104 785 ± 32 791 ± 43 101
31 860 ± 80 798 ± 90 93 2421 ± 124 2290 ± 246 95 922 ± 49 951 ± 59 103
32 744 ± 70 853 ± 85 115 2387 ± 136 2771 ± 34 116 938 ± 45 958 ± 20 102
33 1100 ± 85 1088 ± 52 99 4300 ± 258 4596 ± 117 107 900 ± 25 964 ± 9 107
34 1075 ± 89 1164 ± 132 108 5403 ± 305 5007 ± 413 93 946 ± 34 955 ± 74 101
35 892 ± 63 891 ± 64 100 4785 ± 304 5000 ± 379 104 1098 ± 75 1061 ± 14 97
36 575 ± 62 660 ± 70 115 2345 ± 185 1981 ± 262 84 1042 ± 50 1000 ± 36 96
37 480 ± 44 412 ± 50 86 2889 ± 174 2330 ± 91 81 888 ± 38 909 ± 50 102
38 498 ± 35 441 ± 27 89 2158 ± 203 1960 ± 337 91 769 ± 32 797 ± 62 104
39 735 ± 49 854 ± 55 116 2577 ± 98 2665 ± 135 103 1100 ± 64 1113 ± 42 101
40 1162 ± 101 1250 ± 114 108 5900 ± 302 5482 ± 359 93 603 ± 27 631 ± 52 105
41 585 ± 25 631 ± 31 108 3058 ± 110 3327 ± 329 109 848 ± 40 887 ± 46 104
42 563 ± 34 520 ± 75 92 3452 ± 247 3454 ± 408 100 526 ± 22 468 ± 46 89
43 786 ± 30 886 ± 56 113 5928 ± 185 5325 ± 99 90 1116 ± 63 1045 ± 52 94
44 1082 ± 75 1189 ± 109 110 2645 ± 102 2588 ± 224 98 614 ± 49 557 ± 8 91
Food Anal. Methods

Table 4 (continued)

Sample Ca K Mg

Reference Determined Trueness Reference Determined Trueness Reference Determined Trueness

concentration concentration (%) concentration concentration (%) concentration concentration (%)
(ICP OES) (LIBS) (ICP OES) (LIBS) (ICP OES) (LIBS)

45 861 ± 50 915 ± 52 106 4529 ± 312 4783 ± 443 106 1001 ± 50 1041 ± 51 95
46 525 ± 43 511 ± 71 97 3158 ± 296 3095 ± 430 98 605 ± 29 551 ± 46 91
47 724 ± 36 775 ± 65 107 3128 ± 252 3098 ± 317 99 541 ± 31 471 ± 36 87
48 695 ± 44 698 ± 47 100 3658 ± 184 3810 ± 322 104 753 ± 37 840 ± 10 111
49 998 ± 48 1032 ± 38 103 2485 ± 132 2237 ± 247 90 659 ± 30 584 ± 26 89
50 1553 ± 71 1422 ± 80 92 4325 ± 214 4783 ± 403 111 1154 ± 60 1041 ± 51 90
51 832 ± 40 806 ± 36 97 2059 ± 114 3008 ± 381 151 730 ± 32 757 ± 71 104
52 845 ± 32 853 ± 60 101 3056 ± 136 3108 ± 382 102 896 ± 52 918 ± 17 102
53 1005 ± 59 1119 ± 42 111 2654 ± 113 2554 ± 378 96 670 ± 37 711 ± 15 106
54 759 ± 78 750 ± 84 99 2801 ± 136 2697 ± 22 96 702 ± 25 752 ± 76 107
55 559 ± 36 498 ± 27 89 3098 ± 168 2997 ± 395 97 612 ± 25 578 ± 3 94
56 1125 ± 84 1013 ± 51 90 2837 ± 101 2645 ± 194 97 1105 ± 68 1051 ± 94 95
57 452 ± 22 384 ± 39 89 3408 ± 185 3500 ± 564 103 542 ± 27 498 ± 21 92
58 993 ± 63 897 ± 54 90 1285 ± 96 1102 ± 108 86 400 ± 28 393 ± 33 98
59 1098 ± 85 1182 ± 65 108 1489 ± 85 1415 ± 69 95 554 ± 26 447 ± 15 80
60 899 ± 29 763 ± 58 85 4702 ± 158 4019 ± 191 85 748 ± 38 758 ± 57 101
Cassava flour, validation database
61 525 ± 44 454 ± 59 86 2976 ± 142 2989 ± 151 100 996 ± 45 1061 ± 17 106
62 419 ± 24 357 ± 51 84 2459 ± 185 2733 ± 252 111 1082 ± 50 1106 ± 34 102
63 445 ± 31 512 ± 60 115 3200 ± 225 3175 ± 358 99 825 ± 35 895 ± 52 108
64 699 ± 49 643 ± 62 92 2058 ± 152 1767 ± 186 86 575 ± 39 581 ± 54 101
65 752 ± 61 824 ± 56 110 3398 ± 274 3453 ± 321 102 425 ± 30 434 ± 35 102
66 835 ± 52 905 ± 63 108 2758 ± 186 2976 ± 297 108 510 ± 34 608 ± 54 119
67 625 ± 42 691 ± 18 111 1459 ± 99 1119 ± 116 77 902 ± 39 878 ± 25 97
68 1008 ± 85 920 ± 14 91 4845 ± 314 4366 ± 351 90 476 ± 25 567 ± 36 119
69 649 ± 65 727 ± 29 112 5001 ± 480 4840 ± 530 97 688 ± 42 758 ± 57 110
70 1248 ± 86 1053 ± 41 84 4398 ± 336 3550 ± 329 81 670 ± 32 785 ± 43 117
71 725 ± 32 599 ± 30 83 4258 ± 300 3512 ± 299 83 809 ± 36 923 ± 66 114
72 803 ± 32 883 ± 47 110 3325 ± 254 2401 ± 242 72 476 ± 28 531 ± 53 107
73 1323 ± 44 1214 ± 33 92 4500 ± 333 4711 ± 352 105 515 ± 32 551 ± 19 118
74 575 ± 48 466 ± 52 81 4125 ± 298 4140 ± 534 100 505 ± 39 596 ± 51 118
75 867 ± 51 889 ± 43 102 4345 ± 310 4400 ± 475 101 589 ± 35 590 ± 61 100
76 650 ± 36 442 ± 22 68 1685 ± 89 1484 ± 188 88 631 ± 40 632 ± 69 100
77 545 ± 41 490 ± 45 90 4501 ± 302 4434 ± 347 98 421 ± 32 392 ± 30 93
78 998 ± 42 1037 ± 51 108 2614 ± 152 2022 ± 201 77 854 ± 40 867 ± 15 101
79 515 ± 44 476 ± 37 92 4987 ± 236 5255 ± 177 105 400 ± 42 506 ± 14 126
80 498 ± 25 395 ± 36 79 2598 ± 69 2840 ± 46 109 604 ± 55 701 ± 65 116
81 1221 ± 74 1157 ± 61 95 3035 ± 196 2337 ± 276 77 444 ± 37 467 ± 43 105
82 485 ± 24 370 ± 48 76 1625 ± 136 1515 ± 177 93 1206 ± 60 1097 ± 49 91
83 648 ± 51 712 ± 60 110 2569 ± 221 2573 ± 280 100 602 ± 31 520 ± 43 86
84 1600 ± 60 1642 ± 72 103 4628 ± 369 3765 ± 297 81 845 ± 42 910 ± 38 108
85 1250 ± 63 1077 ± 63 85 2457 ± 201 2700 ± 300 110 450 ± 35 405 ± 38 90
86 900 ± 58 1137 ± 74 126 3552 ± 184 3407 ± 301 96 845 ± 38 777 ± 3 92
87 952 ± 46 845 ± 60 89 3252 ± 200 3108 ± 385 96 900 ± 53 919 ± 17 102
88 1015 ± 98 941 ± 80 93 2798 ± 163 2373 ± 285 85 907 ± 48 957 ± 52 105

Table 4 (continued)
Sample Ca K Mg
Reference Determined Trueness Reference
concentration concentration (%) concentration
(ICP OES) (LIBS) (ICP OES)
89 1200 ± 77 1079 ± 111 90 4225 ± 345
90 605 ± 40 742 ± 41 123 3998 ± 258
Conclusions
The proposed method is fast and suitable for implementation
in the direct determination of Ca, K, and Mg in cassava flour.
The normalization process of the raw data plays an important
role in the quality of the results. The proposed models present-
ed high prediction rates for the concentrations of Ca, K, and
Mg with high correlation coefficients and low SEC and LOD
for univariate models. The sample throughput is approximate-
ly 30 samples per hour, and the proposed method is environ-
mentally friendly as fewer reagents, such as acids and sol-
vents, are used, thus reducing the waste generation.
Acknowledgments This study was supported by the São Paulo
Research Foundation (FAPESP, 2016/01513-0) and the Conselho
Nacional de Desenvolvimento Científico e Tecnológico (CNPq,
401074/2014-5 and 305637/2015-0). The authors are grateful to
Analítica and Thermo Scientific for their instrument loans (ICP OES
and WDXRF).
Compliance with Ethical Standards This is an original research arti-
cle that has neither been published previously nor considered presently
for publication elsewhere.
All authors named in the manuscript are entitled to the authorship and
have approved the final version of the submitted manuscript.
Conflict of Interest Vinicius Câmara Costa declares that he has no
conflict of interest. Diego Victor de Babos declares that he has no conflict
of interest. Francisco Wendel Batista de Aquino declares that he has no
conflict of interest. Alex Virgilio declares that he has no conflict of inter-
est. Fábio Alan Carqueija Amorim declares that he has no conflict of
interest. Edenir Rodrigues Pereira-Filho declares that he has no conflict
of interest.
Ethical Approval This article does not contain any studies involving
human participants or animals performed by any of the authors.
Informed Consent Informed consent was obtained from all individual
participants included in the study.
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