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Analytical, Nutritional and Clinical Methods

Chromium determination in foods by quadrupole inductivelycoupled plasmamass spectrometry with ultrasonic nebulization

Francesco Cubadda*, Silvana Giovannangeli, Francesca Iosi,Andrea Raggi, Paolo Stacchini

Istituto Superiore di Sanita` , Laboratorio Alimenti, Viale Regina Elena 299, 00161 Rome, Italy

Received 20 August 2002; received in revised form 10 December 2002; accepted 10 December 2002

Abstract

The analytical issues connected with chromium determination in foodstuffs by quadrupole inductively coupled plasmamass

spectrometry (Q-ICP-MS) were addressed, including signal stability, spectral interferences and the use of mathematical correction

equations. The analytical performance was compared to that of electrothermal atomisationatomic absorption spectrometry (ETA-

AAS), selected as reference method. Five food certified reference materials (CRMs), including two meat-based CRMs not previously

characterized for their Cr content, were included in the study. The use of ultrasonic nebulization (UN) and the adoption of 53Cr as

analytical mass allowed precise and accurate results to be obtained by Q-ICP-MS, with lower detection limits than ETA-AAS.

# 2003 Elsevier Science Ltd. All rights reserved.

Keywords: Chromium; Food analysis; Electrothermal atomisationatomic absorption spectrometry; Inductively coupled plasmamass spectrometry;

Ultrasonic nebulization; Certified reference materials

1. Introduction

Chromium is an essential element for humans having

a role in maintaining normal glucose tolerance in the

organism (Expert consultation WHO/FAO/IAEA,

1996). It potentiates the action of insulin and thus acts

on carbohydrate, lipid and protein metabolism. Chro-

mium dietary intake has been estimated in the range

2856 mg/day in many countries, but some surveys found

remarkably higher levels (Anke, Muller, Trupschuch,

Seifert, Jaritz, Holzinger, & Anke 2000; Lukaski, 2000;

Tripathi, Raghunath, Vinod Kumar, & Krishna-

moorthy, 1998; Ysart et al., 2000). These intakes arelikely to meet nutritional requirements for healthy indi-

viduals, however chromium supplements have been

introduced on the market.

On the other hand, Cr is widely recognized as a

potential food contaminant. Stainless steel may contain

chromium at relatively high percentages. The metal or

its compounds are also used in electroplating and in

surface treatment of food cans. Therefore Cr migration

from cookware and cans has been postulated, even

though only small quantities have been generally

observed in foodstuff as a result of leaching (Berg,

Petersen, Pedersen, Petersen, & Madsen, 2000; Flint &

Packirisamy, 1997; Jorhem & Slorach, 1987; Smart &

Sherlock, 1985).

Organic chromium compounds such as Cr picolinate

have been reported to improve carcass characteristics

and growth performance of breeding animals, especially

in stressed individuals, and are proposed as supplements

in swine production (Lindemann, 1999). However, theuse of such compounds in zootechny is not author-

ized. Of the inorganic forms of chromium, Cr (VI) is

considerably more toxic than Cr (III)which is the

prevailing form in foodsand is classified as carcino-

genic to humans (IARC, 1990). Hexavalent chromium

is recognized as a hazardous water pollutant in envir-

onments degraded by industrial activities releasing

chromate compounds.

In the light of both the nutritional and the toxi-

cological importance of Cr, the determination of its

levels in foodstuffs is a matter of interest worldwide. In

0308-8146/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved.

doi:10.1016/S0308-8146(03)00002-5

Food Chemistry 81 (2003) 463468

www.elsevier.com/locate/foodchem

* Corresponding author. Tel.: +39-06-49902740; fax: +39-06-

49387101.

E-mail address: [email protected] (F. Cubadda).
http://www.elsevier.com/locate/foodchem/a4.3dmailto:[email protected]:[email protected]://www.elsevier.com/locate/foodchem/a4.3d

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the past, considerable difficulties have been experienced

in obtaining reliable analytical data for this metal,

whatever the technique used for detection (Reilly, 1991;

Veillon & Patterson, 1999). These obstacles have been

overcome due to the developments in the field of

analytical techniques for trace metal detection and

analytical quality assurance; still Cr analysis at lowlevels is considered a challenge to the skill of the ana-

lyst. In the food control field, this situation is reflected

by the paucity of reference materials with certified

values for Cr, which in turn limits the possibility of

adequately verify the accuracy of Cr analyses carried

out by laboratories.

In the last decade, inductively coupled plasmamass

spectrometry has emerged as a powerful tool for the

analysis of trace elements in all biological matrices

including food, allowing rapid multielemental analyses

to be performed with very low detection limits. This

technique has the sensitivity required for Cr detection in

foodstuffs, but suffers from heavy spectral interferencesaffecting the major Cr isotopes utilized for the quantifi-

cation of the element. Highly biased results were

obtained in certification campaigns of food and

environmental reference materials where ICP-MS was

used without accounting for these interferences (Larsen,

Pedersen, & McLaren, 1997; Quevauviller, van Raa-

phorst, & Muntau, 1996).

A number of analytical approaches have been pro-

posed to deal with this drawback, including higher

resolution powers (sector-type ICP-MS instruments),

alternative sample preparation methods (i.e. suited dis-

solution, separation procedures), use of mixed gases,and, in very recent times, the exploitation of new

modified instruments equipped with collisional or

dynamic reaction cells (Lam, McLaren, & Methven,

1995; Neubauer & Vo llkopf, 1999; Vanhaecke &

Moens, 1999).

In the present work, the results of a study carried out

with the aim to elucidate the different analytical factors

affecting chromium determination in foodstuffs by ICP-

MS are presented. The final goal of our investigation

was to find out if there were robust and feasible analy-

tical solutions which allowed routine chromium deter-

mination for food control purposes without complex

changes in instrumentation and spectrometer operatingconditions or lengthy procedures for sample treatment

prior to analytical measurement, i.e. while taking

advantage of the high sample throughput enabled from

microwave (MW) closed vessel digestion followed by

ICP-MS detection.

Five food certified reference materials, spanning three

orders of magnitude of Cr concentrations, were utilized

in the study. Electrothermal atomisationatomic

absorption spectrometry was selected as a bench mark

in order to evaluate the overall performance of the

analytical method developed.

2. Experimental

2.1. Samples and reagents

The five reference and certified reference materials

were: the RM 8436 (Durum wheat flour) and the SRM

1577b (Bovine liver)provided by the US NationalInstitute of Standards and Technology (NIST)the

BCR CRM 278R (Mussel tissue) and 184 (Bovine mus-

cle)provided by the European Institute for Reference

Materials and Measurements (IRMM)and DORM-2

(Dogfish muscle), obtained from the Canadian Institute

for National Measurement StandardsNational

Research Council (INMS-NRC).

Calibration standards were prepared from a 1000 mg

Cr l1 stock solution (BDH, Poole, England) by dilu-

tion with high purity deionized water (Milli-Q, Milli-

pore, Molsheim, France). The standard for ETA-AAS

and ICP-MS measurements were 0.5% v/v and 3% in

ultrapure concentrated HNO3 (Carlo Erba Reagenti,Milan, Italy), respectively. The reagents used in sample

digestion were ultrapure concentrated HNO3 and H2O2(Merck, Darmstadt, Germany). In the study of spectral

interferences, ultrapure Na2CO3 and NaCl (Merck,

Darmstadt, Germany), were used as carbon and chlor-

ine sources, respectively.

2.2. Sample preparation

Sample treatment, including the digestion procedure

by a Milestone MLS-1200 Mega MW oven (FKV, Ber-

gamo, Italy), was described elsewhere (Cubadda, Raggi,Testoni, & Zanasi, 2002). Sample weight was 0.200.55

g depending on the material. Each CRM was digested in

triplicate and made up to 2550 ml in polypropylene

disposable tubes with high purity deionized water.

2.3. Instrumentation and analytical measurements

For ETA-AAS analyses, a SIMAA 6000 spectrometer

(Perkin-Elmer, Norwalk, CT, USA) with inverse long-

itudinal Zeeman-effect background correction and a

transversely heated furnace was used. The instrument

was equipped with a AS 72 autosampler (Perkin-Elmer,

Norwalk, CT, USA). A furnace program with ashingand atomization temperatures of 1400 and 2400 C,

respectively, was used. Calibration was performed with

the method of standard addition. Other instrumental

details and operating conditions are summarized in

Table 1.

For ICP-MS measurements, a quadrupole Sciex Elan

6000 ICP-MS (Perkin-Elmer, Norwalk, CT, USA),

equipped with a ASX-500 autosampler model 510 and a

ADX-500 autodilutor (both from CETAC Tech-

nologies, Omaha, NE, USA), was used. Two sample

introduction systems were employed in this study: a

464 F. Cubadda et al. / Food Chemistry 81 (2003) 463468

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pneumatic nebulizer of the cross-flow type with a Scott

type spray chamber and an ultrasonic nebulizerU-5000AT+ (CETAC Technologies, Omaha, NE,

USA). Calibration was performed with both external

standards and the method of standard addition. Diluted

solutions were prepared with the same nitric acid con-

centration of calibration standards (3% v/v). Rhodium

was selected as internal standard for correction of

matrix effects and instrumental drift. Before operating

the instrument, a warm up time of 3 h since plasma

ignition was adopted throughout. Other details on the

instrumentation and the operating conditions are sum-

marized in Table 1.

Both in ETA-AAS and ICP-MS measurements,

digestion blanks were analysed together with samples

belonging to the same analytical batch. Standards were

run regularly after 68 sample measurements. Meanelement concentrations together with standard devia-

tions were calculated after blank subtraction. In ICP-

MS analyses, each measurement was done after 2 min of

rinsing with a HNO3 solution (5% v/v) to overcome

memory effects from preceding samples and an addi-

tional 1 min of sample pumping to allow stabilization of

the instrument response. In each analytical run, the iso-

topes 13C and 37Cl were selected for monitoring C and

Cl signal (masses 12 and 35 were avoided in order to

prevent exceedingly intense signals).

3. Results and discussion

The chromium concentrations of the five CRMs

measured by ETA-AAS are shown in Table 2. The cer-

tified or best estimated values are shown in the same

table. For CRM 184 only a range of indicative values is

available, while for SRM 1577b no information is given

from the supplier about Cr levels.

For the CRMs with a certified (best estimated) value,

good agreement was observed with the ETS-AAS

results. The found values were all inside the confidence

interval of the certified values and close enough to the

means. As regards reproducibility, the coefficients ofvariation were on average equal to 3.8% (7.0% maxi-

mum value in RM 8436).

The results of the ICP-MS determinations with con-

ventional nebulization and without correction for spec-

tral interferences are shown in Table 3. Chromium has

four stable isotopes, but only the two more abun-

dant52Cr and 53Cr (natural abundance 83.8% and

9.5%, respectively)were selected for analyses. The

Table 1

Instrumental operating conditions for ETA-AAS and ICP-MS

ETA-AAS

Instrument Perkin-Elmer SIMAA 6000

Wavelength 357.9 nm

Slit width 0.7 nm

Sample injection volume 10 ml

Absorbance measurement mode Peak areaGraphite tubes Pyrolytic with Lvov platform

Matrix modifier Mg(NO3)2

ICP-MS

Instrument Perkin-Elmer Sciex Elan 6000

Plasma

RF generator Frequency: 40 MHz, power output

1000 W

Ar flow rate (L min1) Pla sma: 16, Au xil iary: 0 .9;

Nebulizer: 0.9

Solution uptake rate 1 ml min1

Interface

Sampler cone Nickel, i.d.: 1.1 mm

Skimmer cone Nickel, i.d.: 0.9 mm

Interface: 4 torr,Quadrupole: 2105 torra

Scanning conditions Dwell time 100 ms, sweeps/reading 20,

readings/replicate 3, number of

replicates 3

Scanning mode Peak hopping

Internal standard 103Rh

Analytical masses 52Cr, 53Cr

Masses for interference correction 13C, 37Cl

Table 2

Results of chromium determination in food certified reference materials (mg g1 dry wt.)

Certified reference material Certified valuesa

Found values with ETA-AASd

Found values with UN-Q-ICP-MSe

Mean c.i.b Mean S.D. Mean S.D.

RM 8436 (Durum wheat flour) 0.023 0.009 0.025 0.002 0.026 0.002

CRM184 (Bovine muscle) [0.0760.153]c 0.098 0.004 0.095 0.005

SRM 1577b (Bovine liver) 0.30 0.01 0.31 0.01

CRM 278R (Mussel tissue) 0.78 0.06 0.79 0.02 0.80 0.02

DORM-2 (Dogfish muscle) 34.7 5.5 31.9 0.5 33.0 0.5

a Best estimated value for RM 8436.b Uncertainty as half-width of the 95% confidence interval of the mean.c Indicative range.d Detection limit: 0.17 mg l1 (estimated on the basis of the 3s criterion from the standard deviation (s) of 10 digestion blank determinations carried out

during the same analytical run).e Isotope 53. Detection limit: 0.08 mg l1 (calculated as above).

F. Cubadda et al. / Food Chemistry 81 (2003) 463468 465

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other two isotopes, 50Cr and 54Cr, were ruled out after a

preliminary study, which showed the difficulty in

obtaining a stable signal and reliable analyte quantifi-

cation in real samples. These isotopes suffer from theisobaric interferences of50Ti, 50V and 54Fe, respectively.

Generally, these overlaps can be overcome by elemental

correction equations based on relative natural abun-

dances (e.g. 50Cr=50M[0.739726 (47Ti)+0.002506

(51V)). However, correction by elemental equations was

ineffective because of the low abundance of the two

isotopes and the severity of isobaric and polyatomic

interferences at m/z 50 and 54.

While free of isobaric interferences from other ele-

ments, 52Cr and 53Cr are susceptible to potential inter-

ferences by a number of polyatomic species (52Cr:36

Ar16

O,38

Ar14

N,36

Ar15

NH,40

Ar12

C,35

Cl17

O,35Cl16OH, 37Cl15N, 34S18O, 36S16O; 53Cr: 36Ar17O,36Ar16OH, 38Ar15N, 38Ar14NH, 40Ar13C, 37Cl16O,35Cl17OH, 35Cl18O, 36S17O). The contribution of poly-

atomic ions generated by reagent solutions or the

plasma (i.e. ArO, ArOH, ArN, ArNH) is appreciable

only at low Cr concentrations and, in principle, can be

corrected for by blank subtraction. On the other hand,

the sulphur-containing species are unable to sig-

nificantly interfere with Cr determination at the S levels

found in the food matrices analysed in this study.

Different is the case of carbon argides arising from the

residual carbon content of food after destruction of the

organic matrix by MW digestion. The interference fromArC is severe (especially that of 40Ar12 on mass 52) and

hampers accurate chromium determination in all the

selected CRMs with the only exception on DORM-2,

where the very high levels of the analyte make it almost

negligible (Table 3). The apparent analyte concen-

trations originated by a carbon solution of 100 mg l1 in

the experimental conditions adopted in this study and

with the standard pneumatic nebulization as sample

introduction system are shown in Table 4. If one con-

sider that with MW digestion residual carbon contents

of sample solutions can be of several tenths of mg l1, it

is clear how the real Cr levels of the original matrix can

be completely obscured by the apparent concentration

arising from 40Ar12.

The interference of 40Ar13C on 53Crmeasured as

apparent analyte concentrationsis about eight times

lower. However, this isotope is 10 times more suscep-

tible than 52Cr to the influence of chlorine-containing

polyatomic ions (Table 4), and the analyst should be

aware of this when analysing, for instance, seafood.Also the use of chlorine-containing acids in the diges-

tion procedure contributes to the chlorine content of

digestates and thus should be avoided.

The simplest way to attempt a solution to these inter-

ference problems is the use of correction equations,

which subtract to the intensities of the analytes the sig-

nal resulting from interfering polyatomic ions (Ashley,

1992; Violante, Petrucci, Delle Femmine, & Caroli

1998). As an example, the correction equation for the

determination of 53Cr was: 53Cr=53M(37ClCF),

where the correction factor CF was equal to the ratio of

the signal at mass 53 (37

Cl16

O) and 37 (Cl) produced bya pure Cl solution. CFs were calculated using both net

(i.e. blank subtracted) and total intensities as deter-

mined by the analysis of carbon- and chlorine-contain-

ing solutions before each analytical run. Afterwards, a

system of correction equations was entered into the

instrument software.

The results obtained (four analytical runs on different

days) were enough accurate but were sufficiently precise

only for DORM-2 and CRM 278R, the materials with

the highest Cr levels. Coefficients of variation of 10

19% were obtained for the other CRMs. In particular,

in the case of RM 8436 (Cr certified value: 0.0230.009

mg kg1), inter-run CVs as high as 36% were obtained(three non-consecutive measurements in each run),

while the intra-run CV was equal to 19%.

Several reasons account for this poor precision. When

correction equations are used, the invariability of the

ratio of the signal intensities included in the CF is

assumed. However, this ratio is determined through a

separate series of measurements before the analytical

run and variations can occur especially if, subsequently,

long analyses are performed. A way to minimize and

control this phenomenon is to start the analysis soon after

the determination of the CF and to check periodically the

Table 3

Biased results for chromium determination in food certified reference

materials (mg g1 dry wt.)a

Certified reference material Found values

52Cr 53Cr

Mean S.D. Mean S.D.

RM 8436 (Durum wheat flour) 0.35 0.02 0.076 0.006

CRM184 (Bovine muscle) 0.58 0.05 0.17 0.02

SRM 1577b (Bovine liver) 0.70 0.05 0.33 0.03

CRM 278R (Mussel tissue) 1.54 0.08 1.44 0.09

DORM-2 (Dogfish muscle) 34.6 0.4 34.4 0.5

a Analysis performed with Q-ICP-MS, pneumatic nebulization, no

correction for spectral interferences.

Table 4

Apparent analyte concentrations in mg l1 originated at mass 52 and

53 by carbon and chlorine solutions. Experimental conditions: stan-

dard instrumental settings, pneumatic nebulization

Isotope Major

interferences

C 100 mg l1 Cl 100 mg l1

Mean S.D. Mean S.D.

52Cr 40Ar12, 35Cl16OH 16.5 2.0 0.2 0.153Cr 40Ar13C, 37Cl16O 2.0 0.5 2.3 0.4


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efficacy of the correction by analysing solutions con-

taining the interfering element(s). If the signal ratio is

constant and the correction works well, the apparent

analyte concentration for the corrected masses should

be zero. Another weakness of the use of correction

equations is that the intensity of the interference is

assumed to be the same in the digestates and in theaqueous solutions used for CF calculation, which in

some cases may be not entirely true. For all these rea-

sons mathematical equations generally give reliable

results when only a minor part of the apparent concen-

tration is due to interference, as happens for DORM-2

and CRM 278R and as previously demonstrated in the

case of the Ca-containing polyatomic species interfering

with Fe, Co, Ni (Cubadda et al., 2002).

Another difficulty of Cr determination when low

analyte levels (

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4. Conclusion

Chromium analysis of foodstuffs not rich in Cl and

with a simple organic matrix can be carried out after

MW digestion with Q-ICP-MS resorting to both 52Cr

and 53Cr, if the analyte level in the digestates is >100 mg

l

1

, and to

53

Cr if the analyte is in the range 10100 mgl1. At lower Cr concentrations a correction equation

must be used in order to compensate for the ArC inter-

ference. In this latter case, the best results are obtained

with 52Cr and the correction is effective above 23 mg

l1, whereas it leads to imprecise results for lower ana-

lyte levels. Accurate and precise Cr determination down

to 0.1 mg l1 and below can be performed resorting to

ultrasonic nebulization and selecting the 53Cr isotope as

analytical mass, with an additional correction for ClO if

significant amounts of Cl are present in the sample.

Following these findings, the chromium levels of five

food CRMs, including two meat-based CRMs not pre-

viously characterized for their Cr content, could beascertained by Q-ICP-MS. The selected CRMs covered

a wide range of analyte concentrations and were repre-

sentative of different staple food (meat, seafood, cereal

grains). The results obtained by UN-Q-ICP-MS were in

good agreement with those obtained by ETA-AAS and

with the available certified (or best estimated) values for

the selected CRMs.

Acknowledgements

This work, which was carried out as a part of theproject Alimenti di origine animale: valutazione dei

residui di sostanze chimiche impiegate in zootecnia,

was supported by a grant from the Ministero della

Salute (Ministry of Health) of Italy.

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