Food Chemistry 218 (2017) 348–355

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Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Analytical Methods

Multi-element analysis of vegetal foodstuff by means of low power total

reflection X-ray fluorescence (TXRF) spectrometry
Rogerta Dalipi a, Eva Marguí b,⇑, Laura Borgese a, Laura E. Depero a
a
Department of Mechanical and Industrial Engineering, University of Brescia, 25123 Brescia, Italy
b
Department of Chemistry, University of Girona, Campus Montilivi, 17071 Girona, Spain

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

Article history: In this work, an analytical methodology in combination with a low power benchtop total reflection X-ray
Received 19 November 2015 fluorescence (TXRF) system has been developed for multi-elemental analysis of vegetal materials. An easy
Received in revised form 12 May 2016 and rapid sample preparation consisting in suspending 20 mg of sample in de-ionized water showed to
Accepted 5 September 2016
be the most suitable for this kind of samples. However, for comparison purposes, two digestion proce-
Available online 7 September 2016
dures were also applied.
A set of fifteen certified reference materials and three real vegetal samples were employed for the
Keywords:
quantitative determination of K, Ca, Mn, Fe, Cu, Zn, Br, Rb, Sr, Pb, As and Sn. All the parameters affecting
TXRF
Vegetal foodstuff
sample preparation and TXRF measurements conditions were carefully evaluated. Accurate and precise
Multi-element analysis results were obtained for mid-high Z elements (Mn-Sr) using internal standardization as quantification
Suspension approach, while for light Z elements (K and Ca), due to absorption effects, an improvement of analytical
results by external calibration was necessary.
Ó 2016 Elsevier Ltd. All rights reserved.

1. Introduction
Human health and life quality is strongly dependent on the
quality of food consumed daily. Food is the main source of essen-
tial, major and trace elements. De la Guardia proposed a classifica-
tion for elemental composition of food. This classification includes
major essential elements, Ca, Cl, C, H, Mg, N, O, P, K, Na, S; trace
essential elements, F, I, Fe, Si, Zn; ultra-trace essential elements,
Cr, Co, Cu, Mn, Mo, Ni, Se, V; non-essential trace elements, B, Ti,
Sb, As, Ba, Ce, Ge, Rb, Sr; and potentially toxic trace elements; Al,
As, Sb, Cd, Cr, Cu, Pb, Hg, Th, Sn, U (Miguel de la Guardia, 2015).
Vegetation is the primary recipient of elements from the envi-
ronment (soil, water, air) that are transferred via roots and foliage.
Vegetables are an essential part of diet and are consumed both
cooked and raw by humans. Some metals present in vegetables
are biochemically important from health point of view. Metals like
Co, Cr, Cu, Fe, Mn, Mo, Se and Zn help in regulating human meta-
bolism (Jolly, Islam, & Akbar, 2013). Therefore, essential elements
are required for normal plant growing, animal and human nutri-
tion, but high concentration intake can be toxic (Fahad et al.,
2015; Singh et al., 2011; Vincevica-Gaile & Klavins, 2013). More-
over, these elements are transferred to humans directly through
⇑ Corresponding author.
E-mail address: eva.margui@udg.edu (E. Marguí).
fruits and vegetables, or indirectly through milk and meat from
those animals that consume the plants. For this reason, the ele-
mental composition of vegetable foodstuff is important for safety
and nutritional purposes (Fraga, 2005).
Commonly, spectroscopic techniques such as inductively cou-
pled plasma-mass spectrometry (ICP-MS) (Alam, Snow, & Tanaka,
2003; Barbosa et al., 2015; Gałuszka, Krzciuk, & Migaszewski,
2015; Islam, Ahmed, & Habibullah-Al-Mamun, 2015; Nardi et al.,
2009; Peake et al., 2015), ICP atomic emission spectrometry
(AES) (Alam et al., 2003; Babalola & Akinwande, 2014;
Balabanova, Stafilov, & Bačeva, 2015) and flame atomic absorption
spectrometry (FAAS) (Chandio, Talpur, Khan, Afridi, & Khaskheli,
2013; Luis-González et al., 2015; Shrivas & Jaiswal, 2013) have
been used for elemental analysis of vegetation material. However,
the use of these techniques usually requires sample preparation
procedures for total destruction of the organic matrix by chemical
treatments. Sample digestion is a critical step in most of the analyt-
ical methods used for routine elemental determination in plant
samples. Dry ashing and wet digestion methods are the most
widely employed procedures for this purpose (Akinyele &
Shokunbi, 2015). Both methods are time-consuming and losses of
analytes by volatilization are common. Alternatively, microwave
ovens have been used in closed systems to provide faster and more
efficient sample digestion, to reduce the amount of reagents
employed as well as to avoid sample contamination and losses of
analytes by volatilization (Smith & Arsenault, 1996).

http://dx.doi.org/10.1016/j.foodchem.2016.09.022
0308-8146/Ó 2016 Elsevier Ltd. All rights reserved.
 R. Dalipi et al. / Food Chemistry 218 (2017) 348–355
Nowadays, there is an increasing demand of methods that
implicate less or no sample pretreatment for multi-elemental anal-
ysis of foodstuff samples, particularly on a routine basis, and even
for screening purposes.
Most X-ray fluorescence techniques are adequate for this pur-
pose since they present the possibility to perform direct and
multi-element analysis of solid samples, qualitative, semi-
quantitative and quantitative determination in a wide dynamic
range and are really cost effective (Marguí, Queralt, & Hidalgo,
2009). Among them, total reflection X-ray fluorescence (TXRF)
which is a variation of energy dispersive X-ray fluorescence spec-
trometry shows a higher sensitivity and limits of detection at
ppb level (lg g
1 and lg L
1) (West et al., 2012). The main reason
for this is that the glancing angle of the primary X-ray beam is very
small (0.1°), and the detector is positioned very close to the sam-
ple, which results in lowered background noise and improved
detection limits (Streli, 2006). To perform analysis under total-
reflection conditions, samples must be provided as thin films,
depositing 5–50 lL of sample on a reflective carrier. This procedure
excludes matrix effects, such as absorption and secondary excita-
tion, and thus, the quantification in TXRF analysis can be done
directly by internal standardization.
Moreover, TXRF has several advantages over other multi-
elemental spectrometric techniques (ICP-MS or ICP-AES) such as
low amount of sample to perform the analysis (few lL or ng), fast
and easier quantification by internal standardization and no matrix
effects. Besides, new low power benchtop TXRF systems are really
cost-effective since they do not require gas or cooling media.
TXRF has been largely employed for the analysis of foodstuff
including vegetal material, demonstrating thus the suitability of
this technique for this field of analysis (Borgese, Bilo, Dalipi,
Bontempi, & Depero, 2015). However, most of the papers published
so far deal with decomposition of samples prior to TXRF analysis
and employing large-scaled TXRF instrumentations with high-
power X-ray tubes (Antoine, Hoo Fung, Grant, Dennis, & Lalor,
2012; Grant, Dennis, Antoine, Hoo-Fung, & Lalor, 2013; Günther
& von Bohlen, 1990, 1991; Günther, von Bohlen, & Strompen,
1995; Marcó Parra, 2011; Vives, Moreira, Brienza, Zucchi, &
Nascimento Filho, 2006).
The aim of this research was to develop a fast and reliable ana-
lytical method based on the use of TXRF for multi-element analysis
of vegetal material. For this purpose, a set of different certified and
real vegetal samples was employed. Firstly, analytical conditions
for TXRF measurements of suspensions (internal standardization,
sample amount and deposition volume, dispersing agent and son-
ication effect) were evaluated and discussed. Effect of measuring
time on precision and limits of detection was also studied. An accu-
rate evaluation of the quantification procedure and method accu-
racy was performed by comparing the TXRF values with certified
ones. Furthermore, obtained results using the developed method
(suspension + TXRF), were also compared with those obtained
using more sophisticated sample treatments (digestion + TXRF).
2. Experimental
2.1. Materials and reagents
Stock solutions of 1000 mg L
1 (ROMIL PrimAg@ Mono-
component reference solutions) were used to prepare Ga, Rh and
Y internal standard solutions. Ultrapure de-ionized water for
preparation of sample suspensions, dilution of stock solutions
and microwave digested samples was obtained from a Milli-Q
purifier system (Millipore Corp., Bedford, Massachusetts). TritonTM
X-100 (laboratory grade, Sigma-Aldrich) was used for the prepara-
tion of sample suspensions. Nitric acid (69%, HIPERPUR, Panreac),
349
hydrogen peroxide solution (P30%, TraceSELECTÒ, Sigma-
Aldrich) and hydrochloric acid (37%, Panreac, AppliChem) were
used for the microwave and block heater digestion procedures.
Silicone solution in isopropanol (Serva GmbH & Co, Germany)
was used to coat all the quartz glass disc reflectors in order to
obtain a hydrophobic film so as to facilitate sample deposition.
2.2. Vegetal food samples
Different certified reference materials (CRMs) and real vegetal
samples were employed to study the suitability of the proposed
methodology for multi-elemental analysis. The plant tissues ana-
lyzed were: SRM 1515 (apple leaves), SRM 1547 (peach leaves),
SRM 1567A (wheat flour), SRM 1568A (rice flour), SRM 1570A (spi-
nach leaves), SRM 1572 (citrus leaves) purchased from National
Institute of Standards & Technology, NCS ZC 73011 (soy bean),
NCS ZC 73012 (cabbage), NCS ZC 73013 (spinach), NCS ZC 73031
(carrot), NCS ZC 73032 (celery), NCS ZC 73033 (scallion), NCS ZC
73036 (green tea) from China National Analysis Center for Iron
and Steel, INCT-TL-1 (black tea) from Institute of Nuclear Chem-
istry and Technology and NMIJ 7405-a (seaweed) from National
Institute of Advanced Industrial Science and Technology. According
to certificates the particle size was 75 lm for SRM 1515, SRM 1547
and SRM 1570A, <425 lm for SRM 1567A and SRM 1568A,
<250 lm for SRM 1572 and for all the NCS CRM samples less than
177 lm. No data was available for INCT-TL-1 and NMIJ 7405-a.
Three real vegetal samples RM 001 (mushrooms), RM 003
(strawberries) and RM 004 (peeled canned tomatoes) were pro-
vided by the Italian National Agency for New Technologies, Energy
and Sustainable Economic Development.
2.3. Apparatus and instrumentation
An Ethos Plus Milestone microwave with HPR-1000/10S high
pressure rotor (Sorisole, Bergamo, Italy) and a dry block heater
for tubes with digital electronic control of temperature and time
(Tembloc, Selecta) were employed for acid digestion of samples.
An ultrasonic bath (Selecta) was used for the sonication of sample
suspensions. A stereoscopic optical microscope from (NIKON SMZ-
1000) and a scanning electron microscope (SEM) from (Zeiss DSM
960 Germany) were used for morphological study of sample sus-
pensions deposited on quartz reflectors. TXRF analysis of all sam-
ples was performed using a benchtop TXRF system (S2 PICOFOX,
Bruker AXS Microanalysis GmbH, Berlin, Germany) equipped with
a 50 W X-ray tube with a tungsten (W) anode and multilayer
monochromator (35.0 keV). Using such configuration elements
from K to U can be identified. The characteristic radiation emitted
by the elements present in the sample is detected by a silicon drift
detector with an active area of 10 mm2 and a resolution of
146.72 eV (Mn Ka). The measurements were performed working
at 50 kV and 1000 lA. A measurement time of 2000 s was selected
as a compromise between precision and total analysis time. More-
over, the TXRF used in this work is equipped with a cassette chan-
ger for 25 samples and the measurements are performed in air
environment. For comparison purposes, an Agilent ICP-OES 5100
Synchronous Vertical Dual View (SVDV) spectrometer was used
for elemental determination of real vegetal samples after applying
a microwave digestion to the samples (see Section 2.4.2). The ele-
ment wavelengths (nm) were: K (769.897), Ca (393.366), Mn
(257.610), Fe (238.204), Cu (324.754), Zn (213.857), Rb (780.026)
and Sr (407.771). The plasma was operated with 12 L min
1
plasma gas and the plasma configuration elements were: radial
(K, Ca) and axial (other elements). The type of detector was silicon
based multichannel array CCD (charge coupled device). Other
parameters were: 1200 W RF power, concentric nebulizer type
and polychromatic wavelength selector.
350 R. Dalipi et al. / Food Chemistry 218 (2017) 348–355
2.4. Sample preparation and TXRF analysis
2.4.1. Preparation of suspensions and morphological study
A preliminary study was performed to select the most suitable
amount of sample and disperser agent to suspend the vegetal sam-
ple. According to the obtained results (see Section 3.1 for details),
finally, sample suspensions were prepared by weighing 20 mg of
sample and adding 1 mL of de-ionized water containing 10 lg of
Ga as internal standard. Duplicates were prepared for each sample
and 5 min sonication in ultrasonic bath was applied. After that,
10 lL of the internal standardized sample was deposited on a
quartz glass reflector and dried using an infrared lamp for the later
TXRF analysis.
A morphological study of the sample suspensions deposited
on quartz reflectors was performed by optical and scanning
electron microscopy (SEM). In the first case, a stereoscopic
microscope using transmitted light was used. For SEM measure-
ments, the samples were sputtered with a 22.5 nm gold layer at
1.5 kV using a current of 30 mA and a deposition time of 2 min.
Digital images were collected and processed by ESPIRIT 1.9
Bruker program.
2.4.2. Microwave digestion
A microwave acid digestion, based on the EPA method 3052,
was employed for the preparation of vegetal samples. About of
250 mg of sample was added in PTFE vessel with 9 mL of nitric acid
and 1 mL of hydrogen peroxide. The vessels were closed and
heated following a two-stage digestion program consisting of a
first step of 5 min to reach 180 °C and a second step of 10 min at
180 °C. After cooling, digested sample solutions were transferred
to a 30 mL flask and brought to volume with ultrapure de-
ionized water. From each sample digest, an aliquot of 1 mL was for-
tified with a suitable volume of a Ga solution (internal standard) to
have a final concentration of 10 mg L
1. The sample deposition vol-
ume and drying mode to perform TXRF analysis were the same as
for the suspended samples.
2.4.3. Digestion in tube block heater
Digestion of low amount of vegetal samples was also tested
using a dry block heater for polypropylene vials. The digestion pro-
tocol was performed following a recently published paper
(Fernandez-Ruiz, Redrejo, Friedrich, Ramos, & Fernandez, 2014).
For all vegetal samples, about 20 mg amount were weighed in ster-
ile polypropylene vials of 2 mL with cover. To each sample was
added 1 mL of aqua regia (HCl/HNO3, 3:1) and 10 lg of Ga as inter-
nal standard. Firstly, the samples were orbital agitated for 2 h and
then heated to 90 °C for up to 2 h until concentrated to a volume of
500 lL. For a complete digestion of vegetal samples, 20 lL of
hydrogen peroxide and 500 lL of nitric acid were added. Samples
were heated again at 90 °C until a concentrated volume of 100 lL
was reached. In this way the organic matrix was completely
destroyed and the sample digest result in more concentrated metal
content. The sample deposition volume and drying mode to per-
form TXRF analysis were the same as for the suspended and micro-
wave digested samples.
3. Results and discussion
3.1. Evaluation of analytical conditions for TXRF measurements of
suspensions
In order to evaluate the best analytical conditions to analyze
vegetal suspensions by TXRF, the certified reference material
SRM 1570A (spinach) was used.
3.1.1. Internal standardization
In the case of TXRF, quantification can be performed directly by
internal standardization (Expression 1) and thus, external calibra-
tion is usually not needed.


Nj C pi Spi
Ci ¼ ðExpression1Þ
Npi Si
where Ci: analyte concentration, Ni: analyte net peak area, Cpi: IS
concentration, Spi: instrumental sensitivity for the IS, Npi: IS net
peak area, Si: instrumental sensitivity for the analyte.
An ideal element to be used as internal standard should not be
present in the original sample, not interfere with the target ele-
ments and have an adequate XRF response. According to that,
and after checking the non-presence of Ga, Rh and Y, we tested
these elements as internal standard candidates. It is important to
remind that the TXRF system used in this study is equipped with
a W anode to generate X-rays and allows the determination of
Rh using its K-lines that is not possible when employing the most
commonly used Mo target. From the results of elemental quantifi-
cation obtained by using Ga, Rh and Y as IS in SRM 1570A sample
(data not shown) there were no statistically significant differences
and consequently Ga was chosen to be used as IS for further TXRF
analysis since it can be used in both Mo and W TXRF systems.
3.1.2. Sample amount
The amount of sample used for preparation of suspensions can
significantly influence TXRF analysis. To perform analysis under
total reflection conditions, samples must be provided as thin layers
on a reflective carrier. For this reason, the thickness of the depos-
ited sample, such as the amount of vegetal suspension is consid-
ered as a critical parameter regarding the matrix effects and can
really affect the elemental determination. In Fig. 1 are shown the
elemental recoveries (%) obtained by analyzing 10, 20 and 50 mg
of the SRM 1570A sample dispersed in 1 mL de-ionized water.
When using low amount (10 mg) of sample a higher dispersion
of the data is observed, especially for low content elements like
Mn and Cu. On the contrary, for larger amounts (20 or 50 mg) there
are not statistically significant differences on elemental recoveries
but for 50 mg the resulting sample on the reflector was too thick
and thus we have the risk of not being under total reflection con-
ditions anymore. In view of this situation, and considering the
repeatability of the obtained results, an amount of 20 mg of vegetal
sample was used in further experiments.
3.1.3. Dispersing agent and morphological study
The type of dispersing agent used to prepare the suspension is
also an important parameter to be tested. Some contributions have
shown that the homogeneity of the suspension can be improved if
using a surfactant solution to prepare the suspension (De La Calle,
1000
10 mg 20 mg 50 mg
100
Recovery (%)
10
1
K Ca Mn Fe Cu Zn Br Rb Sr
Fig. 1. Effect of sample amount (10, 20 and 50 mg) on elemental recoveries (%)
(analyzed sample: CRM SRM 1570A). Error bars represent the standard deviation of
duplicate samples.
 R. Dalipi et al. / Food Chemistry 218 (2017) 348–355
Cabaleiro, Romero, Lavilla, & Bendicho, 2013; Stosnach, 2005).
However, in a recent paper, we also demonstrated that for some
kind of solid samples results for suspension analysis can be
improved if using diluted nitric acid instead (Bahadir, Bulut,
Hidalgo, Soylak, & Marguí, 2015). In view of these previous find-
ings, in the present contribution, three types of dispersing agents
were tested to prepare the suspensions: de-ionized water, a dilu-
tion of 1% TritonTM X-100 in de-ionized water and a solution of
nitric acid 2 M. Moreover, in addition to compare the quantitative
results obtained using the different dispersant agents, we per-
formed a morphological study of the deposited drop on the reflec-
tor to study in more detail the homogeneity of the analyzed
sample. The morphological study was performed using a stereo-
scopic optical microscope in transmitted light and scanning elec-
tron microscopy (SEM) to check the homogeneity of the sample
spot. In Fig. 2 are shown the images obtained by both microscopes
for a 10 lL sample spot in quartz glass reflector for each type of
solution used to prepare the suspensions. The diameter of the sam-
ple spot on the sample carrier has to be within the beam size
(10 mm) for complete exposure of the drop to the X-ray beam.
For that, only a few microliters (5–20 lL) of suspension solution
should be deposited on the sample carrier. In our case the diameter
of the sample spot was smaller (3.685 mm) and better homogene-
ity was observed when de-ionized water was used for the prepara-
tion of suspension. Moreover, from the zoomed images obtained by
SEM it can be seen that in the case of nitric acid we have a non
homogeneous sample spot, where more particles are concentrated
in the borders and, as it is stated above, this is very critical in total
reflection conditions. Taking into account the obtained results, de-
ionized water was chosen to be used as a dispersing agent for the
preparation of all vegetal sample suspensions.
3.1.4. Sample deposition volume
After sample pretreatment and hydrophobization of quartz
glass sample carrier with silicone solution, sample deposition is
performed. In the case of suspensions, it is recommended that only
a few microliters (5–20 lL) of solution are deposited on sample
carrier obtaining a thin layer (<500 lm) in order to avoid matrix
effects and possible damage of TXRF detector (De La Calle et al.,
2013). The influence of sample deposition was studied at different
volumes: 5, 5 + 5 and 10 lL. For the 5 + 5 lL sample volume, two
Fig. 2. SEM and optical microscope images in transmitted light for a 10 lL SRM 1570A sample spot in quartz glass reflector prepared by using de-ionized water, nitric acid
(2 M) and TritonTM X-100 (1%) as disperser agents.
351
subsequent volumes of 5 lL were deposited on the same sample
carrier and the droplet was dried between successive depositions.
No statistically significant differences were observed in elemental
determination for the studied depositions volumes. Anyway, the
amount of the sample deposited on the reflector, affect also the
limits of detection (LODs) of the elements to be determined. The
LODs were calculated for each spectrum using the following
Expression 2:
pffiffiffiffiffiffiffiffiffiffi
3Ci Nbkg
LOD ¼ ðExpression2Þ
Ni
where Ci is the concentration of a given analyte; Nbkg is the back-
ground count rate and Ni is the net count rate of the analyte. This
equation is analogous to the 3r definition of the LOD.
Better LODs were obtained using higher volumes (5 + 5 or
10 lL) and for practical reasons we selected 10 lL as suitable depo-
sition volume for TXRF analysis.
3.1.5. Measurement time
The measuring time has a significant influence on precision,
which was evaluated in terms of relative standard deviations
(RSD), and on LODs. The RSD values (five replicates) and LODs for
elements determined in the SRM 1570A applying 200, 500, 2000
and 3000 s as measuring time were calculated (data not shown).
It was observed that depending on the concentration level, the
measuring time has high impact on RSD values. For high content
elements like K, Ca and Fe there was not any significant improve-
ment on precision if measurements were performed on 200 s com-
pared to longer measurement times. On the contrary, for low
content elements, especially Cu, a longer measuring time was fun-
damental to achieve an acceptable precision. Furthermore, LODs
decreased significantly when measurements were performed on
longer measurement time. Anyway, there was not a real difference
between the results obtained by 2000 s compared to 3000 s. As a
consequence 2000 s was fixed as suitable for TXRF measurements.
3.1.6. Sonication effect
During suspension preparation, sedimentation errors should be
minimized and for this purpose a stirring system is generally
required in order to obtain homogeneous and stable suspensions.
Usually, manual shaking, vortex or magnetic agitation and bath
352 R. Dalipi et al. / Food Chemistry 218 (2017) 348–355

sonication are employed (De La Calle et al., 2013). It has been analyze the suspensions without performing any sonication proce-
demonstrated that when ultrasound energy is applied, agglomera- dure and at different sonication times: 5, 10 and 15 min. No influ-
tion is avoided or reduced and fragmentation and erosion of parti- ence of sonication on elemental determination was observed but
cles may occur (Bendicho et al., 2012). Therefore, we decided to limits of detection were decreased (26%) if sample suspensions

Table 1
Concentration range of elements (mg kg
1) in CRMs and values of parameters for linear regression (LR) analysis between TXRF data and certified values.

Element Parameters for LR № of CRMs Concentration range (mg kg
1)

2
R A B
K 0.9953 223.654 0.749 11 1280–29,000
Ca 0.9917
103.587 0.932 11 118–22,800
Mn 0.9980 0.655 1.047 12 9.4–1570
Fe 0.9963
6.908 0.927 9 7.4–1010
Cu 0.9505 0.207 1.046 7 2.7–24
Zn 0.9922 0.455 1.043 10 11.2–82.3
Br 0.9796 0.147 1.173 10 1.8–20
Rb 0.9990 0.678 0.999 11 0.68–89
Sr 0.9986
1.926 1.105 9 9.9–213

Regression analysis: LR model, TXRF = A + B Certified (A: Intercept, B: Slope); 95% confidence level.

Table 2
Comparison of certified/indicative values with the obtained suspension-TXRF results using internal standardization and external calibration as quantitative approaches.

Element Sample Certified values (mg kg
1) Internal standardization (mg kg
1) External calibration (mg kg
1)
K NCS ZC 73012 (cabbage) 15500 ± 600 11890 ± 60 15600 ± 100
NCS ZC 73013 (spinach) 24900 ± 1100 18300 ± 100 24200 ± 100
* **
RM 001 (mushrooms) 40440 ± 40 33000 ± 2000 44000 ± 2000
*
RM 003 (strawberries) 14600 ± 100 10700 ± 200 13960 ± 250
* **
RM 004 (peeled canned tomatoes) 43800 ± 100 33100 ± 400 43900 ± 600
Ca NCS ZC 73012 (cabbage) 7000 ± 200 6600 ± 300 7200 ± 300
NCS ZC 73013 (spinach) 6600 ± 300 6000 ± 1000 7000 ± 1000
*
RM 001 (mushrooms) 740 ± 20 760 ± 70 900 ± 100
*
RM 003 (strawberries) 2400 ± 100 1875 ± 6 2123 ± 6
*
RM 004 (peeled canned tomatoes) 1520 ± 10 1100 ± 100 1300 ± 100
Mn NCS ZC 73012 (cabbage) 18.7 ± 0.8 18 ± 3 17 ± 3
NCS ZC 73013 (spinach) 41 ± 3 40 ± 10 40 ± 10
*
RM 001 (mushrooms) 4.73 ± 0.02 – –
*
RM 003 (strawberries) 23.7 ± 0.4 28 ± 1 26 ± 1
*
RM 004 (peeled canned tomatoes) 11.46 ± 0.02 15 ± 1 14 ± 1
Fe NCS ZC 73012 (cabbage) 98 ± 10 68 ± 2 81 ± 3
NCS ZC 73013 (spinach) 540 ± 20 600 ± 100 600 ± 100
*
RM 001 (mushrooms) 33 ± 3 40 ± 10 50 ± 4
*
RM 003 (strawberries) 17.5 ± 3.5 22 ± 3 31 ± 3
*
RM 004 (peeled canned tomatoes) 37 ± 1 42 ± 2 53 ± 2
Cu NCS ZC 73012 (cabbage) 2.7 ± 0.2 – –
NCS ZC 73013 (spinach) 8.9 ± 0.4 9±3 8.1 ± 2.5
*
RM 001 (mushrooms) 24.7 ± 1.4 21 ± 2 20 ± 2
*
RM 003 (strawberries) 3.8 ± 0.1 4±2 4±2
*
RM 004 (peeled canned tomatoes) 12.0 ± 0.3 14 ± 5 13 ± 5
Zn NCS ZC 73012 (cabbage) 26 ± 2 30.6 ± 0.5 28.9 ± 0.5
NCS ZC 73013 (spinach) 35.3 ± 1.5 43 ± 3 41 ± 3
*
RM 001 (mushrooms) 48.7 ± 0.3 58 ± 4 55 ± 4
*
RM 003 (strawberries) 8.3 ± 0.4 12 ± 1 12 ± 1
*
RM 004 (peeled canned tomatoes) 18.0 ± 0.2 19 ± 1 18 ± 1
Br NCS ZC 73012 (cabbage) 6.0 ± 1.3 7±3 6±2
NCS ZC 73013 (spinach) 10 ± 2 12.0 ± 2.5 10 ± 2
RM 001 (mushrooms) – 11 ± 1 9±1
**
RM 003 (strawberries) – 76 ± 2 64.5 ± 1.5
**
RM 004 (peeled canned tomatoes) – 33.3 ± 0.5 28.2 ± 0.4
Rb NCS ZC 73012 (cabbage) 19.6 ± 1.0 19 ± 2 18 ± 2
NCS ZC 73013 (spinach) 30 ± 2 29 ± 1 28 ± 1.0
* **
RM 001 (mushrooms) 102 ± 6 121 ± 5 117 ± 1
*
RM 003 (strawberries) 3.338 ± 0.001 3.2 ± 0.2 2.5 ± 0.2
*
RM 004 (peeled canned tomatoes) 12 ± 0.3 11.1 ± 0.1 10.5 ± 0.1
Sr NCS ZC 73012 (cabbage) 48 ± 3 50 ± 3 46 ± 2
NCS ZC 73013 (spinach) 87 ± 5 90 ± 10 80 ± 10
*
RM 001 (mushrooms) 3.05 ± 0.01 4±1 5±1
*
RM 003 (strawberries) 12.5 ± 0.1 14.7 ± 0.4 15.1 ± 0.3
*
RM 004 (peeled canned tomatoes) 6.60 ± 0.01 6.9 ± 0.3 7.9 ± 0.2
*
Only reference-determined by ICP-OES.
**
Out of linear range.
 R. Dalipi et al. / Food Chemistry 218 (2017) 348–355 353

were treated for at least 5 min. No more improvement on LODs correlation between the TXRF and certified/reference values can be
was observed for longer sonication time. For this reason, 5 min achieved using both quantification approaches.
sonication was established as a necessary procedure before per-
forming TXRF measurements. 3.3. Comparison of TXRF results obtained with different sample
preparation procedures
3.2. Suspension analysis of vegetal food samples by TXRF
Furthermore, we compared the results of TXRF analysis
In order to evaluate the proposed analytical methodology, a obtained by different sample preparation procedures: the devel-
linear regression (LR) analysis was performed between TXRF data oped method of suspension, and sample digestion. Two different
(using internal standardization as quantitative approach) and cer- digestion procedures were performed: microwave digestion and
tified values of 12 analyzed CRMs (SRM 1515, SRM 1547, SRM digestion in a tube block heater. This latter one allows digesting
1567A, SRM 1568A, SRM 1570A, SRM 1572, NCS ZC 73011, NCS very small amount of sample (10–70 mg) in polypropylene vials
ZC 73031, NCS ZC 73032, NCS ZC 73033, NCS ZC 73036, INCT- (see Section 2.4.3). The samples are concentrated to a final volume
TL-1). Two CRMs (NCS ZC 73012 and NCS ZC 73013), were of 100 lL resulting in higher concentrations. Six vegetal samples
excluded on purpose from the linear regression dataset. Indeed, were selected. In Fig. 3, as an example, the typical raw spectra
the corresponding certified values were used to check the reliabil- for NCS ZC 73013 sample obtained using the three different sample
ity of the external calibration procedure. In Table 1, parameters treatments studied in the present work: block heater (BH) diges-
for the linear regression as well as the concentration range tion, suspension and microwave (MW) digestion, are displayed.
studied for each element are displayed. For most elements, good As it was expected, the produced background and intensity of all
agreement (R2 > 0.99) between TXRF datasets and certified values elements is higher for digested sample in block heater. This is
was obtained and no statistical differences at 95% of confidence due to the more concentrated sample since the resulting final vol-
level were found. The lower correlation coefficient values for Cu ume was only 100 lL. The lowest background is observed on the
(R2 = 0.95) and Br (R2 = 0.98) can be related with their very low TXRF spectra of microwave digested sample due to the matrix
content (close to the limit of detection) in most of the studied decomposition by acids, the high dilution effect with de-ionized
CRMs samples. As it can be seen, the slope value for linear regres- water, and thus, more homogeneous films are formed on sample
sion of K was significantly lower than 1.0. This fact can be carriers. The Si K-lines from the quartz glass reflectors and Ka
explained taking into account absorption effects of light Z ele- and Kb peaks of Ga used as internal standard are observed. In addi-
ments when TXRF measurements are performed in air conditions tion, when a W target is used to produce X-rays, a continuum radi-
(Streli, 2006). ation is produced. Results of quantitative analysis are reported in
In order to compare the quality of the results obtained by inter- Fig. 4. For all the investigated samples, no statistically significant
nal standardization and external calibration (using linear regres- differences are observed. It’s worth noting that accurate determi-
sion parameters reported in Table 1), we calculated the nation of toxic elements like Pb (NCS ZC 73013), As (NMIJ 7405-
elemental concentration of five vegetal samples: 2 CRMs (NCS ZC a) and Sn (RM 004) was achieved with all the employed sample
73012 and NCS ZC 73013) and 3 real vegetal samples (RM 001, treatments. In particular, Sn is a high Z element that cannot be
RM 002 and RM 003) with both quantification modes. Results measured with the commonly TXRF systems equipped with Mo
obtained are presented in Table 2. tube.
As it can be seen, inaccurate results with low recoveries were The digestion in block heater was carried out in open vials due
obtained for K (R  75%) and Ca (R  89%) when using internal to the high pressure created, but a high loss of K and Ca was
standardization. However, the results of these elements can be sig- observed in all studied samples. Nevertheless, this procedure was
nificantly improved when using external calibration. This means successful for the determination of really low content elements
that for an accurate determination of light elements in such kind like Ni in the NMIJ 7405-a sample. According to the results, this
of samples, an external calibration correction is necessary. For pre-concentration procedure may be very useful in cases where
other elements, in the atomic weight range from Mn to Sr, a good the content of trace metals is very low and conventional methods
KK

BH digeson
Suspension
MW digeson
Cl K
K K + Ca K

Ga K (IS)

W bremsstrahlung
cps

Fe K

Sr K
Si K

Ca K

Ga K
SK

Rb K
Zn K
Fe K
Mn K

Sr K
Cu K

Br K

Rb K

2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36
Energy (keV)

Fig. 3. TXRF raw spectra for the analysis of NCS ZC 73013 regarding three different sample treatments: block heater (BH) digestion, suspension and microwave (MW)
digestion.
354 R. Dalipi et al. / Food Chemistry 218 (2017) 348–355

100000 Suspension 100000 Suspension

NCS ZC 73012 (cabbage) NCS ZC 73013 (spinach)
MW digeson MW digeson
10000 BH digeson 10000 BH digeson
Cerfied Cerfied
Concentraon (mg kg-1 )

Concentraon (mg kg-1 )

1000 1000

100 100

10 10

1 1
K Ca Mn Fe Cu Zn Br Rb Sr K Ca Mn Fe Cu Zn Br Rb Sr Pb

100000 Suspension 100000 Suspension

NMIJ 7405-a (seaweed) RM 001 (mushrooms)
MW digeson MW digeson
10000 BH digeson BH digeson
10000
Reference
Concentraon (mg kg-1 )

Concentraon (mg kg-1 )

Cerfied

1000 1000

100 100

10 10

1 1
K Ca Mn Fe Ni Zn As Rb Sr K Ca Fe Cu Zn Rb Sr

100000 Suspension 100000 Suspension

RM 003 (strawberries) RM 004 (peeled c. tomatoes)
MW digeson MW digeson
10000 BH digeson 10000 BH digeson
Concentraon (mg kg-1 )

Concentraon (mg kg-1 )

Reference Reference

1000 1000

100 100

10 10

1 1
K Ca Mn Fe Cu Zn Rb Sr K Ca Mn Fe Cu Zn Rb Sr Sn

Fig. 4. Comparison of elemental determination of three CRMs (NCS ZC 73012, NCS ZC 73013, NMIJ 7405-a) and three real vegetal samples (RM 001, RM 003, RM 004)
regarding to three different sample treatments: block heater (BH) digestion, suspension and microwave (MW) digestion. Error bars represent the standard deviation of
duplicate samples.

like microwave digestion cannot be employed, due to the higher compared with other procedures and selected as the best one. It
dilution factor. In fact, after calculating the detection limits using is easy to operate and the risk of contamination and analyte
the three sample treatment approaches, it was found that limits losses are reduced in comparison with conventional procedures,
of detection were lower when using the digestion in a tube block such as acid digestion. Good correlation was achieved between
heater. certified concentration values and those obtained by TXRF using
On the basis of our results, suspension of powdered solid sam- internal standardization as quantification method for elements
ples combined with TXRF analysis is a successful procedure for with Z in the range from Mn to Sr. However, due to the limited
multi-elemental analysis of vegetation foodstuffs. It is faster than sensitivity of W TXRF system and working conditions in
digestion and does not involve the use of dangerous reagents. Con- atmospheric air, inaccurate results were obtained for light-mid
sidering the accuracy levels that can be achieved suspension may Z elements, as K and Ca. In this case, an external calibration
be recommended as a standard procedure for this kind of samples. was necessary to improve the analytical quality of the results.
TXRF has several advantages over other spectroscopic techniques
4. Conclusions including the low sample amount required and low reagents and
consumables since no gas or cooling media are needed for
In this work, we have developed a rapid, simple and reliable operation. Considering the accuracy levels that were achieved
methodology for multi-elemental analysis of vegetal foodstuffs in the present study, sample suspension followed by TXRF
by means of a benchtop low-power W TXRF system. Sample analysis may be recommended as a standard procedure for
preparation by suspension in simply de-ionized water was multi-elemental analysis of vegetable foodstuffs.
 R. Dalipi et al. / Food Chemistry 218 (2017) 348–355
Acknowledgments
This work was supported by the Spanish National Research Pro-
gram (Project ref. CGL2013-48802-C3-2-R).
The authors acknowledge Claudia Zoani of the Italian National
Agency for New Technologies, Energy and Sustainable Economic
Development.
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