a n a l y t i c a c h i m i c a a c t a 6 2 4 ( 2 0 0 8 ) 5967

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Method validation for determination of arsenic, cadmium, chromium and lead in milk by means of dynamic reaction cell inductively coupled plasma mass spectrometry
S. DIlio , F. Petrucci, M. DAmato, M. Di Gregorio, O. Senofonte, N. Violante
Department of Environment and Primary Prevention, Istituto Superiore di Sanit, Viale Regina Elena 299, Rome, Italy

a r t i c l e
Article history:

i n f o

a b s t r a c t
With Regulation No. 1881/2006 the European Union xed a maximum level for lead in milk. Consequently, there is the need to determine very low concentration of elements that may be present in milk in trace and ultratrace levels. Quadrupole inductively coupled plasma mass spectrometry (Q-ICP-MS) combined with dynamic reaction cell (DRC) has been widely employed in order to reach very low concentration, requested for this product. Furthermore, the DRC technology can help in removing polyatomic and argon-based interferences. In the present study, a method for the determination of arsenic, cadmium, chromium and lead in bovine milk was validated according to the EU common standards by means of DRCICP-MS. The main parameters evaluated in the validation were: recovery, repeatability and within-laboratory reproducibility, detection and quantication limits, linearity range and measurement uncertainty. Additionally, stability studies of the analyte in solution and ruggedness studies were carried out. The results obtained for limit of detection (LoD) and limit of quantication (LoQ) in g kg1 were respectively: As, 3.1 and 9.5; Cd, 0.08 and 0.24; Cr, 0.229 and 0.693; Pb, 0.5 and 1.5. While for the recovery: As, 91%; Cd 96%; Cr 99%; Pb, 95%. As for the repeatability: As, 7%; Cd, 3%; Cr, 6%; Pb, 4%. 2008 Elsevier B.V. All rights reserved.

Received 13 May 2008 Received in revised form 11 June 2008 Accepted 12 June 2008 Published on line 24 June 2008 Keywords: Method validation Milk Trace elements Inductively coupled plasma mass spectrometry

1.

Introduction

The relevance of milk in the human diet is widely known because of its nutritional and immunological characteristics. Generally, two types of milk are mostly used for human consumption: milk of animal origin (cow, sheep, goat, etc.) and milk of human origin, being the latter a primary natural source for the nutrition of infants. Potentially toxic elements, deriving from environmental or accidental pollution, may be transferred in milk as a consequence of their ingestion through foodstuff and feed.

Therefore, together with essential macroelements, a certain number of toxic elements at trace and ultratrace levels could be found. With Commission Regulation No. 1881/2006 [1], the European Union established a maximum level in milk only for lead; although, in some EU countries, national action levels have been set for arsenic and cadmium, as well. The multi-elemental determination of various elements at very low concentrations still poses a challenge in terms of accuracy and precision for most analytical techniques. Nowadays, inductively coupled plasma mass spectrometry (ICP-MS) may be considered the most suited and fast technique for

Corresponding author. Tel.: +39 0649902349; fax: +39 0649902721. E-mail address: sonia.dilio@iss.it (S. DIlio). 0003-2670/$ see front matter 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.aca.2008.06.024

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ultratrace elemental analysis; mainly, this is due to its elevated sensitivity, high samples throughput and wide linearity range of concentration [2,3]. However, this technique suffers from unwanted spectral and non-spectral interferences, which might change with the elemental composition of the samples analysed [35]. Most of them cannot be solved by quadrupole ICP-MS because of its low resolution. Other powerful spectrometric techniques, such as sector eld ICP-MS and electrothermal vaporization ICP-MS, were employed to quantify essential and toxic elements in milk [610], in order to overcome the interferences. Furthermore, ICP-MS coupled with size exclusion chromatography was used in the speciation of chemical elements in breast and human milk [11,12], in order to study the inuence of age and area of residence on the presence of metals in milk. The assessment of the transfer factors, from food into human milk, was studied by means of ICP-MS for elements such us, Ag, Ce, Co, Cr, Ga, La, Mo, Nb, Ru, Sb, Th, Ti and U [13]. The recent use of the dynamic reaction cell technology combined with ICP-MS, has allowed the removal of the interferences with a minimum loss of sensitivity (DRCICP-MS). This technology may be considered an interesting alternative to the above-mentioned spectrometric techniques, because it offers various possibilities for the elements determination in different matrices. The removal of the interferences by dynamic reaction cell is mainly based on the reaction between ion molecules and suitable reaction gases [1418]. The validation of the analytical methods has become a basic prerequisite for those laboratories that work in the ofcial food control. By applying methods validated according to common procedures and performance criteria, the quality and comparability of the analytical results can be ensured [19]. There are several papers dealing with method validation in milk for categories of substances, like pharmaceuticals and pesticides, which via food chain can be found in milk, this is also true for aatoxins, due to the scarce hygienic treatment of milk [2022]. As concerns trace elements, there is a lack of validated methods in milk. The aim of this study is to validate a method for the determination of arsenic, cadmium, chromium and lead in bovine milk according to Commission Decision 2002/657/EC [19], UNI CEI EN ISO/IEC 17025/2005 [23], Commission Regulation (EC) No. 333/2007 [24] and by taking into account Commission Regulation (EC) No. 1881/2006 [1].

Table 1 Digestion procedure Step

1 2 3 Ramp Hold Cooling

Time (min)
10 20 20

Temperature ( C) Power (W)

200 200 1000 1000 0

2.
2.1.

Experimental
Material and method

Milk samples of 1.5 mL were transferred into previously decontaminated TFM vessels and digested by means of a Microwave oven (Ethos plus, Milestone, FKV, Sorisole, Bergamo, Italy) equipped with a probe for the internal temperature monitoring control of the samples (reference vessel). A number of ve different portions of 1 mL, sampled from the same tube, were weighed on analytical balance (Mod. BP 210 D, Sartorius Stedim Italy, Antella, Florence, Italy), in order to calculate the mean density of milk. This value was taken into account to calculate the nal concentration of the analytes in milk (ng g1 ). Numerous attempts were carried out to digest the milk by changing its amount from 1 to 2 mL and temperature programme. The right volume of milk was chosen as a compromise between reaching a sufcient sensitivity during the measure and obtaining a complete dissolution with a colourless solution. In fact, the presence of fat in milk has required a strong digestion programme, reported in Table 1, with a reagents mixture of 5 mL of Suprapur concentrated nitric acid 65% (v/v) (Merck, Darmstadt, Germany) and 1 mL of Suprapur hydrogen peroxide 40% (v/v) (Merck, Darmstadt, Germany). After cooling, the digested solutions were quantitatively transferred in Falcon tubes of 50 mL by adding high purity deionized water with a specic resistance 18 M cm (Easy Pure, PBI International, Milan, Italy) up to a weight of 20 g. The densities of the digested solutions were also measured by weighing 1 mL of those solutions. The digested samples were stored in a refrigerator at +4 C, except for the solutions to be destined to the stability study. The standard addition calibration method was used to quantify the elements of interest. Four standard additions were made and Rh was used as the internal standard at the concentration of 1 g L1 in calibrants and samples, in order to compensate any random uctuations of the signals. A sample dilution factor of 2 was applied. Single element 1000 mg L1 stock standard solutions of As, Cd, Cr, Se, Pb, Rh and Zn in 2% (v/v) HNO3 were utilised in the study (Spex Industries Inc., Edison, NJ, USA). For the study of the interferences, a standard solution of chlorine at about 1000 mg L1 was prepared by starting from Suprapur NaCl (Merck, Darmstadt, Germany).

2.2.
The method was validated on fresh pasteurised whole bovine milk (liquid form), whose applicability was also tested on seven different types of liquid milk, and one breast milk. Additionally, for cadmium and lead, the method was applied to seven milk powder infant formula. One litre of milk was purchased from the market. It was subsequently sub-sampled in aliquots of about 50 mL in Falcon tubes (polypropylene) and stored in a refrigerator at 20 C until the moment of the analysis.

Instrumentation

All measurements were performed by means of dynamic reaction cell inductively coupled plasma mass spectrometry DRCICP-MS (Elan DCR II, Perkin Elmer SCIEX, Norwalk, CT, USA). Arsenic, Cd, and Pb were quantied in standard mode, while Cr was determined by DRC with ammonia as the appropriate reaction gas (with a purity of 99.999%). Instrumental setting and data acquisition parameters for DRCICP-MS are reported in Table 2.

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Table 2 Instrumental parameters for DRCICP-MS

Spectrometer: Elan DRC II (PerkinElmer SCIEX, Norwalk, CT, USA) Sample uptake rate: 1.01.2 ml min1 Sample introduction: Meinhard nebulizer with cyclonic spry chamber RF power: 13501450 W Gas ow rates (L min1 ): plasma, 15; auxiliary, 1.0; nebulizer, 0.85 Dwell time (ms): 50100 Number of replicate: 35 Interface: Pt cones Extraction lens voltage: optimized for maximum I (56 Fe) Acceptance limit for optimization solution: Mg > 6000 cps per 1 g L1 , In > 30,000 cps per 1 g L1 , U > 20,000 cps per 1 g L1 , 220 < 2 cps, Ba2+ /Ba < 3%, CeO/Ce < 3% Cell Gas A: reaction gas NH3 for 52 Cr, internal standard 103 Rh, gas ow 0.60, RPq 0.65 Scanning mode: peak hopping Analytical masses (amu): standard mode 75 As, 114 Cd, 206 Pb, 207 Pb, 208 Pb, 103 Rh (internal standard), 82 Se, 118 Sn, 40 Ar37 Cl

Table 3 Levels of addition for the analytes ( g kg1 ) As

Level 1 Level 2 Level 3 20 30 40

Cd
10 15 20

Cr
10 15 20

Pb
10 20 30

2.3.

Method validation

Several parameters have been taken into account and evaluated for the in-house validation of method in milk, namely: selectivity/specicity, trueness by recovery at three level of concentration, repeatability and within-laboratory reproducibility at three level of concentration, instrumental/method detection limits (LoDs) and quantication limits (LoQs), range of linearity, standard measurement uncertainty. Furthermore, stability studies of the solution in four different storage conditions and ruggedness studies of the method were carried out. In this study, the denitions and procedure for validation parameters have been applied according to the EU standards for foodstuff [19,24]. During the validation, an internal quality control (IQC) procedure was adopted by checking the blanks level and the drift of the instrument readings. The evaluation of the blanks level was performed before starting the measurements on the matrix and the drift by measuring a single standard, as a reference, after the fourth calibrant and every 10 samples. A maximum drift of 10% was considered acceptable to the aim of the validation study.

BCR-150 and BCR-151 (high levels), NIST-1549 (non-fat milk powder). Since for As, Cd and Cr no maximum level in milk has been established so far at EU level, the concentrations, to be considered eligible for the additions in the whole validation, were based on the sensitivity of the analytical technique; especially in the cases of arsenic and chromium. The former, for its high ionization potential, and the latter, for the effect of signal lowering due to the reaction cell gas. The three levels of additions were selected on the basis of the criterion indicated in CD 657/2002 [19] as 1, 1.5 and 2 times these eligible concentrations. For lead, instead, CR No. 1881/2006 [1] sets 0.020 mg kg1 wet weight as the maximum level in raw milk, heat-treated milk, milk for the manufacture of milk-based products and for infant formulae and follow-on formulae; thus, an addition of 0.5, 1.0 and 1.5 the maximum level was chosen. Consequently, six independent aliquots of milk were spiked with the right amount of standard solution of As, Cd, Cr and Pb in three different levels of concentration ( g kg1 ) according to the scheme of Table 3, so as to obtain 18 spiked samples and 9 unspiked samples in order to quantify the natural level. All samples were digested according to the pre-established MW programme and then analysed. The recovery was calculated as follow: recovery% = 100 C spiked concentration

where C is the element concentration found. An acceptance limit between 90 and 110% was selected in compliance with the CD No. 657/2002 [19].

2.3.3.

Repeatability and within-laboratory reproducibility

2.3.1.

Selectivity/specicity

Both terms describe the extent to which a method uniquely reacts to a selected element. Then, selectivity studies must be performed in order to investigate the effect of potential interferent ions. The relevant discussion is reported in Section 3.1.

2.3.2.

Recovery study

The trueness of the method was assessed by using the recovery, due to the lack of Certied Reference Materials in liquid form for trace elements. On the contrary, a number of CRMs is offered on trace elements in milk powder, for example, the BCR-063R (representative of natural elemental levels in milk),

The repeatability and within-laboratory reproducibility were calculated as percentage variation coefcient (CV%) by analysing three independent sets of samples (six spiked and three unspiked) in three different days. As for the repeatability, no changes were applied to the analytical conditions; being repeatability the precision under repeatability conditions, as stated in CR No. 333/2007 [24] and CD No. 657/2002 [19]. In fact, repeatability conditions refer to those mean conditions where independent test results are obtained with the same method on the same sample in the same laboratory by the same operator using the same equipment in a short interval of time. The same set of samples at three levels of concentration used for the recovery was also employed to calculate the rst day of analysis for the repeatability. As concerns the within-laboratory reproducibility, being this the precision obtained in the same laboratory under stipulated (predetermined) conditions over long time intervals, some changes were applied and the measurements were carried out over a period of 6 weeks (3 days of analysis with an interval of 15 days). In particular, the rst day no changes were

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applied, the operator and the instrument were changed in the second day and, nally, a different kind of liquid milk was digested and analysed in the third day.

recovery of 10 samples was evaluated for the storage conditions.

2.3.7. 2.3.4. limits Instrumental/method detection and quantication

Ruggedness study (minor changes)

In order to calculate the instrumental/method limits of detection and quantication (LoDs and LoQs), the standard 3 and 10 approach were employed. A number of 20 pool reagents blanks/digested milk (diluted 1:2) and 20 pool reagents blanks/digested milk (diluted 1:2) spiked with 0.5 g L1 of all elements were prepared and analysed. The reagent blanks were used for the calculation of instrumental LoDs and LoQs, while digested milk was used for the calculation of method LoDs and LoQs. The nal values were multiplied by the dilution factor. Instead, for method LoDs and LoQs, nal dilution and weight were taken into account to calculate the nal values. The following formula was used: LoD = 3 sd Cspike Ispike I LoQ = 10 sd Cspike Ispike I

where C is the concentration expressed in g L1 , sd is the standard deviation and I is the signal intensity of the element. Requirements for LoD and LoQ are set in Table 5 of CR No. 333/2007 [24] for those elements for which a maximum level has been set. For example, as regards LoQ, it says: For inorganic tin less than 10 mg kg1 . For other elements less than one fth of the maximum level in Regulation (EC) No. 1881/2006 [1], except if the maximum level for lead is less than 100 g kg1 . For the latter, less than two fth of the maximum level. Therefore, only for lead, a requirement must be fullled and to this regard, LoQ must be lower than 0.008 mg kg1 .

The ruggedness study is strongly recommended when a method is being subjected to the validation process. It has to be carried out by selecting and varying any factor that may inuence the result of the measurement. The consequences of these minor reasonable variations have to be observed and evaluated by using the approach of Youden [19]. The approach consists in introducing several variations at once instead of studying one single variation at a time. In this work, the experiment design for ruggedness studies reported in Table 11 of the CD No. 657/2002 [19] was strictly followed. Therefore, the following seven different parameters were chosen and tested: (a) analytical instrumentation (two identical models of DRCICP-MS); (b) operator; (c) variable volume electronic micropipette (Eppendorf, Biohit); (d) dwell time; (e) replicates number; (f) peristaltic pump rate; (g) RF power. These studies were performed by spiking the milk digested solutions with 1 g L1 of As and Pb, and 0.5 g L1 of Cd and Cr. The solutions were mixed into a pool and the analyses were performed on six aliquots. The Youden approach applied revealed whether a minor variation of a parameter, or more than one, affected the method. According to this test, a method is robust when the SDi (standard deviation of the differences Di ) is signicantly smaller than the standard deviation of the method carried out under within-laboratory reproducibility conditions.

2.3.8.

Measurement uncertainty

2.3.5.

Range of linearity and calibration curve

Although ICP-MS is well known for beneting from a wide linear concentration range, this parameter was evaluated by checking the linear regression coefcient (r2 ) of a calibration curve constructed with 10 standard additions. A pool of digested milk was used for this test. The linearity of the calibration curve was considered acceptable when r2 > 0.999. In the whole validation, the calibration curve for the measurements was always prepared with at least ve points (blank not included), as recommended by CD No. 657/2002 [19].

2.3.6.

Stability study

A laboratory has to demonstrate the quality of the results produced and its tness for purpose by giving a measure of the condence that can be placed on the result. All possible sources of uncertainty have to be carefully identied. When the major contributions are detected, a good estimate of the measurement uncertainty can be made by concentrating effort on the largest and most signicant contributions. Afterwards, the uncertainty components are quantied and the combined uncertainty is calculated [25]. In this study, the following contributions to combined measurement uncertainty were selected: the preparation of the standard solutions (uf ), the standard uncertainty associated to the recovery (urec ) and the within-laboratory reproducibility of the measurements (um ). To the aim of the validation, each component was calculated as follows: rsd urec = n where rsd is the ratio between the standard deviation of the mean recovery and the mean recovery value and n is the number of spiked samples; the uncertainty uf associated with the preparation of the standard solutions was considered by the sum of the contributes: pipettes volume, scale and stock solutions: uf = C uv V
2

An insufcient stability of the analyte in the sample during storage may give rise to signicant deviations in the outcome of the result of the analysis [19]. The analyte stability can be characterised by analysing the sample after different storage conditions. To this end, a pool of digested milk solutions were spiked with 20 g L1 of As and Pb, and 10 g L1 of Cd and Cr. A volume of 2.5 mL of this pool was transferred in 160 Falcon tubes of 15 mL divided in four groups of 40 aliquots each, that were stored in four different conditions: in the dark, at 20 C, +4 C, +20 C and in the light at +20 C. A series of 10 samples for every storage condition was analysed each week for 4 weeks. For all elements, the mean

ub M

ucal / 3 Cal

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63

where uv is the standard uncertainty of the pipettes volume, ucal is the standard uncertainty of the stock standard solutions and ub is the standard uncertainty of the scale. Then, the combined uncertainty (uC ) was calculated as the square sum of the three contributes: uC = C u2 + u2 + u2 m rec f

In each run of analyses, the signal intensity of three different masses was measured as to control the efciency of the mathematical correction; the rst mass As(I) was taken as it was without correction, the second was corrected only for ArCl and the third one for ArCl and Se. The following correction equations were used: (a) (b)
75 As(II) = 75 As 3.127 40 Ar37 Cl 75 As(III) = 75 As 3.127 [40 Ar37 Cl (0.874 82 Se)]

While, the expanded measurement uncertainty (U) was: U = uC (x)k where k is the coverage factor of 2, which considers a normal distribution of measurements with a 95% condence level.

3.

Results and discussion

Inductively coupled plasma mass spectrometry is well known to be a very sensitive analytical technique in trace elements analyses, but the isotopes signals may suffer from the overlapping of potential interferences. The inuence of these interferences has to be investigated in order to assure the reliability of the analytical data produced. A full discussion on the interferences study is provided in this section. Furthermore, the fullment to the EU requirements for each parameter validated in this study is discussed hereafter together with some applications of the method to different kinds of milk.

3.1.

Interferences study

Since the mass spectrometer used in the present study is equipped with a dynamic reaction cell (DRC), chromium was determined by using this technology with ammonia as reaction gas, while, arsenic, cadmium and lead were quantied in standard mode. The DRC option should be used only when alternative techniques, able to remove the interferences, are not available; furthermore, its use leads to a loss of sensitivity that should be taken into account.

The two correction equations (a) and (b) were carefully evaluated by performing appropriate experiments. To this end, the inuence of chlorine concentration on the signal of arsenic was investigated by adding 50, 100, 150 and 200 mg L1 of standard solution of chlorine to the digested milk. It was found that the correction factor applied for arsenic, according to the equation (a), was able to tolerate a high content of chlorine in milk (up to 5 g L1 ) without affecting the signal. Afterwards, the inuence of selenium concentration on the signal was evaluated by adding 5, 10, 15 and 20 g L1 of standard solution of selenium to the digested milk. A signal increase on mass 75 As (III) was observed; nevertheless, the apparent concentration of arsenic, due to selenium spiked in milk, turned out to be lower than the LoQ of the method (LoQ: 9.5 g kg1 ). Specicity studies were additionally performed on the correction factor (b), applied for the overlapping of ZnO on 82 Se, by adding an increasing concentration of standard solution of Zn to the digested milk at the following concentrations: 200, 400, 600 and 800 g L1 . No increase was observed due to the addition of Zn on the signal of 75 As (III) and 82 Se. The evaluation of these interference studies led to the quantication of arsenic by selecting the rst correction factor for chlorine.

3.1.2.

Cadmium

3.1.1.

Arsenic

Quantication of As with quadrupole ICP-MS is affected by the occurrence of the typical molecular ion interference 40 Ar35 Cl. This can be solved by means of two different approaches: (a) use of reaction cell technology with oxygen as one of the most effective reaction gas; (b) standard mode analysis with application of corrective factors for 40 Ar37 Cl and 82 Se, calculated by taking into account the isotope abundances ratio 35 Cl/37 Cl and 77 Se/82 Se. The mathematical equations are capable to correct the signal of an analyte when the level of interfering element produces an interference signal 20% of total signals. In this study, the mathematical correction was preferred because the level of the interfering element was below the abovementioned percentage, and the use of another gas, besides that already foreseen for Cr (ammonia), would protract the analysis duration. In order to investigate the inuence of the overlapping ions on As signal, the additions of Cl, Se and Zn were chosen on the basis of the content determined in the diluted (1:1) digested milk by a preliminary semi-quantitative screening.

The content of oxides, which potentially interfered on the signal of Cd, was daily kept under control below 3% by calculating the oxides percentage on CeO/Ce masses. To this end, a standard solution containing 1 g L1 of Mg, In, U, Ce, Rh, Pb, Na, Fe, Ca, K and 10 g L1 of Ba, in 0.5% HNO3 was daily measured, and the optimization parameters were adjusted (i.e., nebulizer gas ow, lens voltage, RF power) in order to select the best instrument performance, according to the information provided by PerkinElmer (In > 30,000 cps, Mg > 5000 cps, mass 220 (background) < 30 cps, CeO/Ce < 0.03 and Ba2+ /Ba+ < 0.03). Moreover, the isobaric interference of 114 Sn was solved by using the following mathematical correction: 0.027250 118 Sn.

3.1.3.

Chromium

Strong argon-based interferences cannot be removed by simply using a mathematical correction equation. Then, the reaction cell technology became the only possibility to remove the interferences by means of different chemical reactions occurring among analytes and interfering ions. The appropriate reaction gas reacts with the species before they enter into the analyser. In this way, the determination of analytes at ultratrace level is allowed [1618]. Ammonia resulted to be particularly suited in the quantication of 52 Cr in order to solve the main interference 40 Ar12 C; in fact, the interfering ion is

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Table 4 LoD, LoQ and linearity range Parameters (units)

Instrumental LoD and LoQ ( g L1 ) LoD and LoQ of the method ( g kg1 ) Range of linearity ( g L1 )

As
0.12; 0.37 3.1; 9.5 0.253.0

Cd
0.004; 0.012 0.08; 0.24 0.1251.5

Cr
0.025; 0.076 0.229; 0.693 0.1251.5

Pb
0.01; 0.03 0.5; 1.5 0.253.0

Table 5 Method performance Parameters (%) Level 1

Recovery Repeatability Within-laboratory reproducibility Expanded uncertainty 91 7 6 18.3

As Level 2
97 5 5

Cd Level 3
92 7 6

Cr Level 3
92 4 5

Pb Level 3
96 6 9

Level 1
96 3 3 15.8

Level 2
95 2 4

Level 1
99 6 12 29.8

Level 2
91 3 8

Level 1
98 8 10 18.9

Level 2 Level 3
95 4 6 90 7 10

eliminated by charge transfer reaction [26]: I+ + R I + R+ in this case : ArC+ + NH3 ArC + NH+ 3

The optimization of the NH3 ow rate and the quadrupole rejection parameters q (RPq) was carried out using two digested milk sample diluted 1:2 with 1 g L1 of Rh as the internal standard (unspiked sample and spiked sample of 5 g L1 of Cr). The best ow rate resulted to be 0.60 mL min1 with RPq = 0.65.

3.1.4.

Lead

Since no major interferences occurred on lead isotopes, the standard mode analysis was carried out. Moreover, since in the natural state the abundance of lead isotopes is not uniform, as commonly adopted by the analysts, the analytical signal of lead was corrected for the sum of the most abundant isotopes, namely, 206 Pb, 207 Pb and 208 Pb.

Fig. 1 Contribution of each factor to the overall combined uncertainty for As, Cd, Cr and Pb.

3.2.

Validation parameters

The overall performance of the method proposed is summarised in Tables 4 and 5. For lead, CR no. 333/2007 [24] has xed a maximum value for LoQ of 8 g kg1 ; the present method achieved a value of 1.5 g kg1 , well below this limit. Other analytical techniques could hardly reach this very low quantication limit. The quantication limit for arsenic is high, while for the remaining elements the limits are quite satisfactory.

The results for the recovery tests were particularly successful being these in the acceptance range 90110%; especially considering the thermal microwave digestion programme used for milk samples. For all elements analysed, the CV% for the withinlaboratory reproducibility did not exceed the limit of 20%. The expanded standard measurement uncertainty, expressed as percentage and with a coverage factor of 2, was estimated according to the adopted procedure as follows: As, 18.3%; Cd, 15.8%; Cr, 29.8%; Pb, 18.9%. The percent contribution of each factor to the overall combined uncertainty was plotted in Fig. 1. As already described in the text, the following contributions were identied: uf the

Fig. 2 Recovery obtained during the time studied (4 weeks) for As.

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65

Fig. 3 Recovery obtained during the time studied (4 weeks) for Cd.

Fig. 4 Recovery obtained during the time studied (4 weeks) for Cr.

Fig. 5 Recovery obtained during the time studied (4 weeks) for Pb.

preparation of the standard solutions, urec the standard uncertainty associated to the recovery and um the within-laboratory reproducibility of the measurements. It can be observed that, for As, Cd and Pb, the lowest contribution for all elements is given from urec , while, the highest appears to be um and uf , conversely, in case of Cr, the highest contribution is due to um . The results of the stability studies are displayed in Figs. 2 through 5. The recoveries obtained during the study (4 weeks) are in good agreement with the recovery of the method

for all elements. In the case of Cr, a slight decrease in the recovery in the second week was observed for all storage conditions; nevertheless, this could be ascribed to signal instrumental uctuations, because for Cr no trend was observed. Table 6 reports data of the Youden test for the ruggedness studies. It can be inferred that the minor changes applied did not affect the method. According to the tness-for-purpose approach [24], the suitability of the methods for ofcial control can be assessed

Table 6 Results of the Youden test Element

As Cd Cr Pb

SDi
0.055 0.008 0.045 0.082

Within-laboratory reproducibility sd
1.1 0.3 1.3 1.2

Test result SDi < sd

Table 7 Measurement uncertainty check by the Fitness-for-purpose approach Element

As Cd Cr Pb

uM (mg kg1 )
4.3E03 2.0E03 2.0E03 4.0E03

uc (mg kg1 )
1.8E03 7.9E04 1.5E03 1.9E03

uM > uc

Passed

Passed

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Table 8 Results for milk samples analysed Milk sample As

Fresh pasteurised whole milk Fresh pasteurised milk partially skimmed Long-life whole milk Long-life partially skimmed milk Long-life partially skimmed milk low lactose Breast milk Long-life whole milk with Fe and vitamins added Milk powder infant formula 1 Milk powder infant formula 2 Milk powder infant formula 3 Milk powder infant formula 4 Milk powder infant formula 5 Milk powder infant formula 6 Milk powder infant formula 7 n.d.: not determined.

Concentration in g kg1 (n = 3) Cd
<LoQ <LoQ <LoQ <LoQ <LoQ <LoQ <LoQ 1.0 0.3 <LoQ 2.0 0.6 2.7 0.2 3.7 0.3 <LoQ 2.5 0.1

Cr

Pb

n.d. n.d. n.d. n.d. n.d. n.d. n.d.

n.d. n.d. n.d. n.d. n.d. n.d. n.d.

3.1 0.5 7.9 0.3 4.9 0.3 9.8 0.3 11.5 0.5 19.2 0.5 4.9 0.2 6.9 0.2

by checking if they are able to produce results with standard measurement uncertainties (uc ) less than the maximum standard measurement uncertainty (uM ) calculated using the formula reported in point C.3.3.2 [24]: LoD 2
2

Acknowledgement
The authors thank Daniela Pino for editorial assistance on the manuscript.

uM =

+ (C)

references

where uM is the maximum standard measurement uncertainty; LoD is the limit of detection of the method; C is the concentration of interest; is a numeric factor to be used depending on the value of C. In this case, Table 7 illustrates that the present method well meets the performance criteria set out by the regulation.

3.3.

Application of the method to milk samples

The four elements were quantied in six different kinds of milk in liquid form and one sample of breast milk sampled from a donor. At the same time, seven different milk powders, infant formula, were analysed only for the content of cadmium and lead, being these elements tested for the recoveries (94% for Cd, and 100% for Pb). In fact, for chromium and arsenic, no supplementary interference studies were carried out on milk powder form, and the recovery tests were applied only to those elements that are not strongly interfered. The results for three independent samples for each kind of milk are listed in Table 8.

4.

Conclusion

The validated method presented in this paper shows to meet the performance criteria and the requirements set in the regulations of the European Union for method validation to be used in ofcial food control. Furthermore, the present method offers satisfactory detection limits due to the powerful spectrometric analytical technique employed and provides a precise and accurate method with high sample throughput for determination of As, Cd, Cr and Pb in milk.

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