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Anal Bioanal Chem (2003) 377 : 316–321 DOI 10.1007/s00216-003-2051-6

ORIGINAL PAPER

ORIGINAL PAPER

R. M. García-Rey · R. Quiles-Zafra · M. D. Luque de Castro

New methods for acceleration of meat sample preparation prior to determination of the metal content by atomic absorption spectrometry

Received: 26 March 2003 / Revised: 29 April 2003 / Accepted: 9 May 2003 / Published online: 19 July 2003 © Springer-Verlag 2003

Abstract Focused microwave-assisted digestion and ul- trasound leaching have been applied for the extraction of Pb, Cd, Cr, Cu, Fe, Zn, Ca, and Mg from raw meat. Semi- membranous muscle (SM) of raw pig ham was used for optimizing both the digestion and extraction steps by mul- tivariate approaches. The detection and quantification lim- its were 0.5 and 0.9 µg kg 1 for Pb, 0.06 and 0.1 µg kg 1 for Cd, 0.2 and 1.2 µg kg 1 for Cr, 0.4 and 3 µg kg 1 for Cu, 0.04 and 0.1 mg kg 1 for Fe, 0.012 and 0.017 mg kg 1 for Zn, 0.3 and 0.4 mg kg 1 for Ca, and 0.01 and 0.03 mg kg 1 for Mg. The precision, expressed as relative standard de- viation (RSD), ranged between 2.5 and 9.6% for focused microwave-assisted digestion and between 3.5 and 10.6% for ultrasound leaching. The methods were then compared with a reference method and applied to a certified refer- ence material (bovine muscle 184, from the BCR). The t-test, applied to the results obtained from focused micro- wave-assisted digestion, revealed that they are in agree- ment (p>0.01) with the certified and estimated values in the case of Pb, Cd, Cr, Cu, Fe, Ca, Mg, and Zn but not in that of Fe. In the case of ultrasound leaching, only the ex- traction of Pb, Cu, and Ca was quantitative. The method based on microwave digestion provides more accurate and precise results than ultrasound leaching. These new procedures have many advantages with regards to con- ventional methods, namely, reduction of the extraction time, simplification of the process, avoidance of chemical emissions to the atmosphere, and no losses of metals by volatilization.

Keywords

Focused microwave-assisted digestion · Ultrasound

Meat analysis · Pork · Sample preparation ·

R. M. García-Rey · M. D. L. de Castro ()

Department of Analytical Chemistry, Marie Curie Building, Campus de Rabanales, University of Córdoba, 14071 Córdoba, Spain e-mail: QA1LUCAM@uco.es

R. Quiles-Zafra

Department of Environmental Protection and Waste Management,

Junta de Comunidades de Castilla La Mancha, Toledo, Spain

leaching · Metal determination · Atomic absorption spectrometry · Multivariate optimisation

Introduction

The determination of trace elements in food is of great rel- evance because the minerals are essential in vital pro- cesses in humans [1]. In fact, knowledge of the relation- ship between the mineral content of the diet and some dis- eases like hypertension, osteoporosis, etc., has increased interest in the mineral content of food [2, 3, 4, 5, 6]. Meat and meat products constitute an important part of the hu- man diet [7] and their metal content influences the quality of the final product and thus acceptance by consumers. An aspect to be taken into account by manufacturers of meat products (particularly dry cured-ham producers) is the relationship between the content of some metals (namely, Ca, Mg, Zn, Fe, etc.) and the appearance of quality de- fects such as pasty textures or defective colour. Further- more, one of the most important consequences of envi- ronmental pollution on humans is the intake of toxic ele- ments through the diet. Metals are essential in small amounts and toxic at high levels (Fe, Cu, Zn, etc.) and the intake of food containing metals such as Pb and Cd has proved cumulative character [8, 9, 10]. Special pro- grammes have been carried out in many countries with the purpose of avoiding the distribution of foodstuffs that could be a risk to human health if consumed [11]. Most methods proposed to determine the metal content in meat products use atomic absorption spectrometry. De- struction or removal of the organic matter is mandatory prior to determination of trace elements. The decomposi- tion or mineralization of the sample can be carried out by different methods, namely: calcination [12], wet digestion with acid mixtures [13] in either open vessels [14] or closed systems [15], etc. These methods involve some shortcom- ings, such as long manipulation time [12, 16, 17] large con- sumption of chemicals, losses of elements by volatilization and fume emissions. The use of a domestic microwave oven with containers of either stainless steel or PTFE

(polytetrafluoroethylene) equipped with a sealing pres- sure-resistant system [18, 19] provides short lixiviation times and reduces emission of gases, but involves serious safety problems [15, 20]. With a view of overcoming these shortcomings, procedures based on either focused micro- wave-assisted digestion or ultrasound-assisted leaching have been proposed [21]. The use of focused-microwave digestors and ultrasound probes endows with key features the methods for solid sample preparation. Firstly, reduc- tion of the time required for the digestion or the extraction step; secondly, simplification of the process; and, thirdly, avoidance of chemicals emissions to the atmosphere [22, 23]. This last aspect fulfils the present policy for reducing environmental contamination. Furthermore, ultrasound leaching diminishes matrix effects because the analyte is removed from non-extractable and potentially interfering matrix components [24]. The aim of this research was to check the potential of two alternative energies, namely focused microwaves [25] and ultrasound [26, 27] for pig meat treatment prior to monitoring the metal content.

Experimental

Apparatus and instruments

A Soxwave-100 focused microwave digester (Prolabo, Fontenay- sous-Bois, France) with a maximum irradiation power of 300 W, controlled by a TX 32 device (Prolabo) was used. Ultrasonic irradiation was applied by means of a Branson 450 sonifier (20 kHz, 400 W) equipped with a cylindrical titanium al- loy probe (12.7 mm diameter), which was immersed in a water bath in which the extraction cell was placed. An extraction cham- ber consisting of a stainless steel cylinder (10 cm in length and 3 mm i.d.), closed with screws at either end, was used. A Hobersal model HD-150 (maximum temperature 1200 °C) furnace (Forns Hobersal, Barcelona, Spain) was used to carry out the reference method. An atomic absorption spectrometer with deuterium background correction and an SPS-5 autosampler from Varian (Spectraa 110, USA) was used to determine the metal content. The analyses of Pb, Cd, Cr and Cu were carried out by graphite furnace atomic ab- sorption spectrophotometry (GFAAS). Pyrolytic platforms were used for Pb and Cd measurements, which were unnecessary in the case of Cr and Cu. Zn, Fe, Ca and Mg were determined by flame atomic absorption spectrophotometry (FAAS) with a 10 cm burner head and standard air–acetylene flame. Varian hollow cathode lamps were used as radiation sources in all cases.

Reagents and standards

317

High purity de-ionized water (resistivity 18.2 mOhm cm) obtained from a Milli-Q water purification system (Millipore, Bedford, MA, USA) was used throughout. All glassware and containers used were cleaned in 10% HNO 3 for a minimum of 24 h and rinsed with ultrapure water before use. Analytical reagent grade 65% HNO 3 (Panreac, Barcelona, Spain) was used. Stock solutions (1 g L 1 each metal) were prepared using 99.5% granular Pb (Aldrich, Mil- waukee, WI, USA), Cd powder (Merck, Darmstadt, Germany), K 2 Cr 2 O 7 for analysis (Panreac), CuSO 4 for analysis (Panreac), granular Zn (Aldrich), granular Fe (Aldrich), CaCO 3 for analysis (Panreac) and granular Mg (Aldrich). The mixture of 1.8 mL 0.2% NH 4 (H 2 PO 4 )+200 µL 1% Mg(NO 3 ) 2 was used as a chemical modifier for Pb and Cd determinations by GFAAS. Sr(NO 3 ) 2 for analysis at a final concentration of 2 g L 1 was used as releasing agent in Ca and Mg determinations. Argon, (Carburos Metálicos, Barcelona, Spain) was used as a protective gas in GFAAS, and air and acetylene (Carburos Metáli- cos) as oxidizing and fuel gas, respectively, in FAAS.

Samples

Semimembranosus muscle (SM) – raw pig ham – was used for de- velopment of the methods. Three samples of SM from different pigs were then used for comparison with the reference method. Bovine muscle, a CRM (BCR-184), was used for validating the methods.

Sample preparation

Focused microwave-assisted digestion

SM sample (1 g) was introduced into the extraction vessel together with 10 mL HNO 3 (50% v/v). The vessel was connected to a distil- lation column and then irradiated with a microwave power of 300 W for 3 min. The suspension thus obtained was filtered through Albet no. 135 paper and diluted to 25 mL in a volumetric flask with de- ionized water. Potential losses by filtering were checked by the standard addition method and no losses were detected.

Ultrasound leaching

SM sample (1 g) was introduced into the extraction chamber to- gether with 10 mL HNO 3 (15% v/v) and then immersed into a wa- ter bath at room temperature. The solid sample was subject to ul- trasound irradiation for 10 min (duty cycle 0.1 s, output amplitude 70% of the converter nominal amplitude, with the probe placed 1 mm from the top surface of the extraction cell). The extract was filtered through Albet no. 135 paper and diluted to 25 mL in a vol- umetric flask with deionized water.

Table 1

Values of the instrumental parameters for the determination of Pb, Cd, Cr, and Cu by GFAAS

Parameter

Pb

Cd

Cr

Cu

λ (nm) A (mA) SW (nm) Background correction T dry stage (°C) Time (s) T pyrolysis stage (°C) Time (s) T atomisation stage (°C) Time (s)

217

228.8

357.9

327.4

5

4

7

4

1

0.5

0.2

0.5

Yes

Yes

No

No

95

110

140

95

110

135

85

95

120

85

95

120

5

80

5

5

50

10

5

30

10

5

40

10

700

650

1000

800

27.5

30

17

8

2150

1800

2600

2300

3

3.5

3.2

3

318

Table 2

Values of the instrumental parameters for the determina-

Table 3 Tested ranges in (a) the first and (b) the second factorial design and optimal values for the variables related to the extraction step assisted by focused microwaves

tion of Zn, Fe, Mg, and Ca by FAAS

 

Parameter

Zn

Fe

Mg

Ca

λ (nm) A (mA) SW (nm) Background correction

213.9

248.3

285.2

422.7

5

5

4

10

0.1

0.1

0.1

0.1

Yes

Yes

Yes

No

Reference method

Variable

Tested range

Optimum

 

value

 

(a) First

(b) Second

factorial

factorial

design

design

HNO 3 concn (%) Digestion time (min) H 2 O 2 (%)

20–40

30–50

50

5–15

3–9

3

0–10

0

0

SM sample (2 g) was ashed in a furnace at 450 °C for 12 h, using the AOAC method [28]. The ash was dissolved in 2.5 mL concen- trated nitric acid and diluted to 50 mL with deionized water.

Determination procedure

After extraction Pb, Cd, Cr, and Cu were determined by GFAAS (instrumental conditions in Table 1). A final stage at higher tem- perature was applied in all cases in order to remove all extract residues from the tube, thus avoiding possible memory effects be- tween both samples and replicates. Zn, Fe, Mg, and Ca were deter- mined by FAAS (instrumental conditions in Table 2). All measure- ments were the average of at least three replicates.

Results and discussion

After checking the determination procedure for the target analytes (linear range of the calibration curve, precision, etc.) the research was focused on establishing a method for removal of the metals from meat which was faster, cleaner, and required less consumption of reagents than those presently used. Two types of energy were used to achieve a drastic reduction of both the time and analyte losses common in long sample manipulation.

screening study of the behaviour of the main factors af- fecting the extraction process. The upper and lower values given to each factor were selected from the available data and experience gathered in the preliminary experiments. The ranges over which the variables were studied and the optimum values found are listed in Table 3a. The conclusions were that both the HNO 3 concentra-

tion and digestion times were significant and positive but their interaction was antagonist. The addition of H 2 O 2 was not significant.

In view of these results, a full two level factorial de-

sign was performed for HNO 3 concentration and digestion time only. This second design involved a total of 2 2= 4 ex- periments plus three centred points. Table 3b summarizes the values used for the design. The study showed a positive and significant effect for

HNO 3 concentration and a positive but non-significant ef- fect for digestion time. Thus, the highest HNO 3 concen- tration without fume production (50%) and the shortest digestion time providing full digestion (3 min) were the optimum values.

Optimisation of focused microwave-assisted digestion

A preliminary study involving different concentrations and volumes of HNO 3 , and different sample amounts and di- gestion times was performed. Nitric acid was selected as extractant because it is the most frequently used acid for mineralization by a microwave oven [20]. This acid be- haves ideally under microwave energy irradiation, has a relatively low boiling point, can be obtained in high pu- rity, and lacks of the hazards and inconveniences of other acids such as perchloric acid. The results showed that concentrations of HNO 3 higher than 55% must not be used because fume production. The extractant volume is not significant, so the minimum value tested (10 mL) was used. One gram of sample gave the best results, requiring a digestion time of 3 min. Then, a multivariate approach was designed for opti- misation of variables such as HNO 3 concentration, diges- tion time, and H 2 O 2 . The addition of H 2 O 2 was assayed because of its positive effect on the digestion of meat products [29, 30]. A full factorial design 2 3 involving eight randomized runs plus three centred points was built for a

Optimisation of ultrasound leaching

A preliminary study of the HNO 3 concentration and ex- traction time was performed. HNO 3 concentrations higher than 20% and extraction times longer than 20 min did not improve the extraction yield. One-gram portions of sam- ple were used in all instances with the view of subsequent comparison of this extraction method with digestion based on microwave assistance. The variables which affect ultrasound-assisted extrac- tion are probe position, ultrasound radiation amplitude, percentage of duty cycle of ultrasound exposure, sonica- tion time, acid concentration, and extractant volume. The probe position was defined as the distance between the tip

horn of the ultrasound probe and the top surface of the ex- traction cell.

A half fractionated factorial design 2 61 type V resolu-

tion allowing 3 d.f. and involving 32 randomized runs plus three centred points was built for a screening study of the behaviour of the main factors affecting the extraction process. The ranges for HNO 3 concentration and extrac- tion time were selected from the preliminary experiments. The values used for the rest of the variables covered the

Table 4 Tested ranges in (a) the first and (b) the second factorial design and optimal values for the variables related to the extraction step using ultrasound

Variable

Tested range

Optimum

value

(a) First

(b) Second

factorial factorial

 

design

design

Probe position (mm) Radiation amplitude(%) Duty cycle (%) Sonication time (min) HNO 3 concentration (%) Extractant volume (mL)

1–20

1

1

10–70

70

70

30–90

10–30

10

1–10

10–20

10

5–15

15

15

2–7

5–10

10

whole range provided by the ultrasound system (Table 4a). The conclusions of this screening study were that the ultrasound radiation amplitude and the acid concentration had positive effects but they were not statistically influen- tial factors. Thus, the highest values tested for these vari- ables (namely, 70 and 15%, respectively) were selected for subsequent experiments. The probe distance had a nega- tive but non-significant effect; so the minimum value tested (1 mm) was selected for subsequent experiments. The ex- traction time, the volume of extractant, and the duty cycle were significant factors in the ranges under study. The ex- traction time and the volume of extractant had a positive influence; meanwhile the effect of the duty cycle was neg- ative. Therefore, a new design was performed in order to evaluate the effect of using higher values of both extrac- tion time and extractant volume and lower values of the duty cycle. A full two-levels factorial design involving a total of eleven experiments (2 3 =8 experiments plus three centred points) was selected as a second experimental design. The ranges within which the variables were studied are shown in Table 4b. The analysis of the design showed that ex- traction time and extractant volume were significant and positive and their interaction was significant but antago- nist. The duty cycle had a negative but non-significant ef- fect, and its interaction with the extraction time was neg-

319

ative but non-significant; the interaction with the volume extractant was negative and significant. In view of these results, the highest extractant volume (10 mL) and the shortest both sonication time (10 min) and duty cycle (10%) were selected and used for further experiments.

Features of the methods

The detection and quantification limits were calculated by use of the equations LD=µ bl ±3s bl and LC=µ bl ±10s bl , re- spectively, where µ bl is the mean and s bl the standard de- viation of the blank responses obtained from the analyses of 10 blanks. The values obtained for the detection and quantification limits were 0.5 and 0.9 µg kg 1 for Pb, 0.06 and 0.1 µg kg 1 for Cd, 0.2 and 1.2 µg kg 1 for Cr, 0.4 and 3 µg kg 1 for Cu, 0.04 and 0.1 mg kg 1 for Fe, 0.012 and 0.017 mg kg 1 for Zn, 0.3 and 0.4 mg kg 1 for Ca, and 0.01 and 0.03 mg kg 1 for Mg. The precision, expressed as relative standard deviation (RSD) of five independent analyses of the same raw meat sample (SM), was studied for the two proposed methods and the reference method (Table 5). For the digested sam- ple obtained with microwave-assistance the precision for the metals determined by FAAS ranged between 3.4 and 5.7% and that of the metals determined by GFAAS ranged between 2.5 and 9.6%. For the extracts obtained with ultra- sounds-assistance, the precision for the metals determined by FAAS ranged between 3.5 and 8.6% and that of the met- als determined by GFAAS ranged between 5.4 and 10.6%.

Va lidation of the methods

To check the validity of the proposed methods for the de- termination of Pb, Cd, Cr, Cu, Fe, Zn, Ca, and Mg in raw pig meat, they were compared with a reference method. The results obtained by applying them to three samples are shown in Table 6. Each result is the mean of three repli- cates. As can be seen, the focused microwave-assisted di- gestion provides more accurate and precise results than ultrasound leaching for the eight metals.

Table 5 Repeatability as rela- tive standard deviation (%RSD) of the reference (RM), micro- wave (MWM) and ultrasound (USM) methods for each metal

a Five independent digestions/ extractions of the same raw pork meat sample (SM)

Metal

Method

Mean (n=5 a )

%RSD

Metal

Method

Mean (n=5 a )

%RSD

Zn

RM

20.5

1.7

Pb

RM

0.14

9.5

MWM

24.4

4.3

MWM

0.14

9.6

USM

15.5

7.6

USM

0.21

5.4

Fe

RM

25.9

5.1

Cd

RM

0.039

8.8

MWM

26

3.4

MWM

0.037

5.6

USM

16.7

8.6

USM

0.042

10.6

Mg

RM

206.

1.9

Cr

RM

0.37

6.6

MWM

227.9

4.6

MWM

0.36

6.3

USM

126.8

6.3

USM

0.57

10.4

Ca

RM

99.2

2.4

Cu

RM

2.15

6.4

MWM

106.9

5.7

MWM

2.17

2.5

USM

81.5

3.5

USM

2.24

8.7

320

Table 6

Metal contents of three samples of raw pork meat (SM) using the reference (RM), microwave (MWM) and ultrasound (USM)

methods (the mean and standard deviation are from three independent digestions/extractions of each sample)

Sample

1

2

3

Metal

RM

MWM

USM

RM

MWM

USM

RM

MWM

USM

Pb

0.13±0.01 0.140±0.001 0.17±0.01 0.18±0.02 0.15±0.01 0.13±0.009 0.094±0.01 0.093±0.002 0.085±0.004

Cd

0.015±0.002 0.015±0.002 0.022±0.003 0.016±0.001 0.015±0.001 0.019±0.002 0.0096±0.0007 0.010±0.002 0.013±0.002

Cr

0.49±0.03 0.55±0.06 9.1±1.9

 

0.52±0.01

0.54±0.04

2.3±0.2

0.56±0.02

0.52±0.01

1.7±0.2

Cu

1.9±0.1

2±0.1

1.8±0.4

2.2±0.7

2.3±0.1

1.9±0.5

2.5±0.6

2.54±0.01

2.1±0.3

Zn

22.9±3

25.9±1

19.4±2.7

22.8±1.4

24.9±2

24.7±2.4

21.9±1.7

24.9±2.6

26.3±3.6

Fe

24.2±0.4 27.5±0.4 16.6±1.2 27.5±0.4 26.8±0.8 12.5±0.5 31.8±0.5

30.7±1.3

18.9±1.4

Ca

170±2

171±2

166±11

188±4

187±4

170±1

160±1

162±5

150±6

Mg

223±1

213±0.3

219±3

215±0.8

212±1

189±5

225±2

224±3

199±10

Table 7

Validation of the methods (values obtained as the average of three independent digestions/extractions)

 

CRM 184

Certified value

Estimated

Found value

 

Efficiency

Found value

 

Efficiency

 

value

(MW)

(%)

(US)

(%)

Pb (mg kg 1 ) Cd (mg kg 1 ) Cr (mg kg 1 )

0.239±0.011

0.253±0.036

a

106

0.251±0.017

a

105

0.013±0.002

0.015±0.001

a

112

0.016±0.001

a

121

0.076

0.076±0.003

a

99

Cu (mg kg 1 ) Fe (mg kg 1 )

2.36±0.06

2.55±0.3 a

108

2.22±0.09 a

 

94

79±2

92±2.6 a

116

64±2 a

80

Zn

(mg kg 1 ) (mg kg 1 )

166±3

150±4

a

91

101±3

a

61

Ca

150

156±9

a

104

153±4

a

102

Mg (mg kg 1 )

1020

1008±16 a

 

99

652±10 a

 

64

a Standard deviation obtained for n=3

The optimized conditions for both methods were ap- plied to 1 g of a CRM (bovine muscle, 184 from the BCR). The extraction efficiencies for the eight metals are listed in Table 7. The t-test was applied to the results obtained with focused microwave-assisted digestion and it revealed that they are in agreement (p>0.01) with the certified and estimated values in all cases, except for Fe. The results obtained with ultrasound leaching are in agreement (p> 0.05) with the certified and estimated values in the case of Pb, Cd, Cu, and Ca, but not for Fe, Zn, and Mg, for which the extraction efficiency ranged between 61 and 80%. Contamination of the extract by metals from the sample chamber was checked by blank extractions. The chamber causes contamination only from Cr.

Conclusions

Focused microwave-assistance allows fast, easy, and clean digestion of the raw pig ham. The results obtained with the proposed method and with dry digestion were similar, thus allowing the substitution of tedious and/or error-prone handling methods for extracting metals from meat. There are no differences between the results ob- tained with the proposed method and the reference method for fresh meat samples for all the metals under study, non- significant differences between the content found and the certified value of the lyophilized CRM 184 exist for all the metals except Fe. The extraction efficiency ranges from 91 to 116% for the eight metals. These reasons make

focused microwave-assisted digestion of raw meat an ex- cellent method for sample preparation prior to atomic ab- sorption monitoring of these metals. The slightly different behaviour of the CRM as com- pared with the raw meat samples could be due to the dif- ferent texture and humidity of the natural samples and the lyophilized CRM. Ultrasound-assisted leaching provides acceptable re- sults for the extraction of Pb, Cd, Cu, and Ca – extraction efficiencies of 105, 94, and 102%, respectively – but not in the case of Cr, Cd, Fe, Zn, and Mg. This method re- duces the extraction time, avoids chemicals emission and losses of metals and diminishes matrix interference, so it is useful for extraction of Pb, Cu and Ca from raw meat.

Acknowledgements Financial support from Spanish Comisión Interministerial de Ciencia y Tecnología (CICyT) is gratefully ac- knowledged (Proyect BQU2002–01333). The R&D and Quality Department of Campofrío Alimentación S.A., especially Juan Ángel García, Julio Tapiador, Roberto Rodríguez, and Jesús Ro- dríguez, are thanked for supplying the samples and CRM.

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