Artigo Apresentaçao ICP-MS.pdf

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Talanta 76 (2008) 575579

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j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / t a l a n t a

Simultaneous determination of Cd, Cu, Mn, Ni, Pb and Zn in nail samples byinductively coupled plasma mass spectrometry (ICP-MS) aftertetramethylammonium hydroxide solubilization at room temperature:Comparison with ETAAS

Bruno L. Batista a, Jairo L. Rodrigues a, Juliana A. Nunes a, Luciano Tormen b,Adilson J. Curtius b, Fernando Barbosa Jr. a,

a Laboratorio de Toxicologia e Essencialidade de Metais, Depto. de Analises Clnicas , Toxico logicas e Bromatologicas, Faculdade

de Ciencias Farmaceuticas de Ribeirao Preto-USP, Avenida do Cafe s/n, Monte Alegre 14040-903, Ribeirao Preto-SP, Brazilb Universidade Federal de Santa Catarina, Depto. Qumica, Campus Trindade, 88040900 Florianopolis, SC, Brazil

a r t i c l e i n f o

Article history:

Received 14 February 2008

Received in revised form 24 March 2008

Accepted 26 March 2008

Available online 7 April 2008

Keywords:

ICP-MS

ETAAS

Nail samples

Trace elementsRapid determination

Tetramethylammonium hydroxide

a b s t r a c t

A simplemethodis describedforthe determination ofCd, Cu,Mn,Ni, PbandZn innails byusinginductivelycoupled plasma mass spectrometry (ICP-MS) or electrothermal atomic absorption spectrometry (ETAAS).

Prior to analysis,1020mg of nail samples were accurately weighed into (15 mL)conical tubes. Then,1 mLof 25% (w/v) tetramethylammonium hydroxide (TMAH) solution was added to the samples, incubated at

room temperature overnight and then further diluted to 10 mL with 1% (v/v) HNO3. After that, sampleswere directly analyzed. Rhodium was used as internal standard for ICP-MS analysis. Method detection

limits (3 s, n = 20) were 0.1, 3.0, 1.0, 4.5, 1.5, 5.0 ng g1 for Cd, Cu, Mn, Ni, Pb and Zn, respectively for ICP-MS, and 24, 26, 30, 143, 130 and 1000 ng g1, respectively for ETAAS. The key issue addressed here is the

elimination of the acid digestion prior to analysis. Moreover, with the use of the proposed method there

is a considerable improvement in the sample throughput comparing to the traditional methods usingmicrowave-assisted acid sample digestion prior to analysis. For validation purposes, six ordinary nailsamples were solubilized and then directly analyzed by ICP-MS and ETAAS, with no statistical differencebetween the two techniques at 95% level on applying the t-test.

2008 Elsevier B.V. All rights reserved.

1. Introduction

Nail is a biological specimen that has some advantages as abiomarker for trace elements exposure, especially because its col-lection is noninvasive and simple and because nail samples arevery stable after collection, not requiring special storage conditions

[1,2]. Trace elements in nails reflect long-term exposure becausethis compartment remains isolated from other metabolic activitiesin the body [2,3]. Because toenails are less affected by exogenousenvironmental contamination than fingernails, they have been pre-

ferred for exposure to toxic metals [3]. Toenails have a slowergrowth rate than fingernails (upto 50% slower, especially in winter)and thus may provide a longer integration of the exposure [24].

Atomic absorption spectrometry (AAS) [5,6] and inductively

coupled plasma emission spectrometry (ICP-OES) [7] are still thedominant analytical techniques used for trace element analysis

Corresponding author. Tel.: +55 16 36024701.

E-mail address: [email protected] (F. Barbosa Jr.).

in nail samples. However, the use of inductively coupled plasmamass spectrometry (ICP-MS) is becoming much more common in

clinical laboratory analysis [3,812]. Compared to electrothermalatomic absorption spectrometry(ETAAS) or ICP-OES, this techniquehas some distinct advantages, including simultaneous multiele-ment measurement capability coupled with very low detection

limits [7]. Moreover, it offers a wider linear dynamic range whichallows thedetermination of majorand trace elements at same sam-ple injection [810]. Additionally, compared to ICP-OES, ICP-MSprovides simpler spectral interpretation and isotopic information

[8].Sample preparation is a critical and laborious step for chemi-

cal elements determination in biological samples [1316]. In thecase of nails, usually, samples are digested in acid medium prior

to analysis. Wet digestion procedures in open [3] or closed ves-sels with microwave-assisted acid digestion [6,10] has been usedfor this purpose. However, both methods may be time-consumingfor laboratories operating in routine with large amount of sam-

ples to be analyzed and require the use of concentrated acids andcareful monitoring of digestion [11]. On the other hand, in clin-

0039-9140/$ see front matter 2008 Elsevier B.V. All rights reserved.

doi:10.1016/j.talanta.2008.03.046


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576 B.L. Batista et al. / Talanta 76 (2008) 575579

ical laboratories, more attention has been given to methods ofsample preparation with minimal handling and time consumption[17,18].

Nail is composed of keratin proteins. The sulfur in cysteinemolecules in adjacent keratin proteins link together in disulfidechemical bonds. These disulfide bonds are very strong and verydifficult to break apart. These disulfide chemical bonds linking the

keratins together are the key factor in the durability and resistanceof nail fiber. They are largely resistant to the action of acids butthe disulfide bonds can be broken apart by alkali solutions, makingthe nail weak. Tetramethylammonium hydroxide (TMAH), an alka-

line solution, has been previously used for sample pre-treatmentas an attractive alternative to microwave-assisted acid digestionof biological materials for the determination of trace elementsby different atomic spectrometry techniques [1926]. For exam-

ple, Pozebon et al. [24] used TMAH to dissolve biological samplesfor the determination of Tl, Pb, Ag, Hg and Cd by electrothermalvaporization inductively coupled plasma mass spectrometry (ETV-ICP-MS) and Moreton and Delves [25] used TMAH in one of their

diluents for the determination of total Hg in whole blood by ICP-MS.

The aim of this study was to develop a simple method for thedetermination of five elements in nail samples by ICP-MS, that can

also be applied for ETAAS analysis, using a sample solubilizationwith TMAH and then a dilute-and-shoot sample analysis.

2. Experimental

2.1. Reagents

All reagents used were of analytical-reagent grade (Sigma, St.Louis, MO, USA), except HNO3 (Sigma, St. Louis, MO, USA) whichwas previously purified in a quartz sub-boiling stills (Kurner)

before use. A clean laboratory and laminar-flow hood capa-ble of producing class 100 were used for preparing solutions.

High purity de-ionized water (resistivity 18.2 m cm) obtainedusing a Milli-Q water purification system (Millipore, Bedford,

MA, USA) was used throughout. All solutions were stored inhigh-density polyethylene bottles. Plastic bottles and glasswarematerials were cleaned by soaking in 10% (v/v) HNO3 for 24h, rins-ing five times with Milli-Q water and dried in a class 100 laminar

flow hood before use. All operations were performed in a cleanbench.

For the ETAAS method, stock solutions containing 1000 mg L1

of each element were obtained from PerkinElmer (PerkinElmer,

Norwalk, CT, USA). Analytical calibration standards were pre-pared daily over the range of 540; 0.55; 1.050; 1.010; 550;0.53.0g L1 for Pb, Cd,Cu, Mn, Ni and Zn respectivelyby suitableserial dilutions of each stock solution in 2.5% (w/v) TMAH + 1% (v/v)

HNO3. ForCd andPb determination, graphite tubes were previouslycoated with iridium according to the method proposed by Borgeset al. [27]. The modifier solution was prepared by the suitable dilu-tion of the 1000mgL1 Ir stock standard (PerkinElmer, Norwalk,

CT, USA).For the ICP-MS method, multielement stock solutions contain-

ing 1000mg L1 of each element were obtained from PerkinElmer(PerkinElmer, Norwalk, CT, USA). Analytical calibration standards

were prepared daily over the range of 020 g L1 for all elementsby suitable serial dilutions of multielement stock solution in 2.5%(w/v) TMAH+ 1% (v/v) HNO3. Rhodium was used as internal stan-dard at the concentration of 10g L1 Rh. The internal standard

was diluted from 1000mg L1 stock standard (PerkinElmer, Nor-walk, CT, USA). A rinse solution consisting of 0.005% (v/v) Triton

X-100

in 2% (v/v) nitric acid was prepared before each run.

Table 1

Optimalheatingprogramsfor thedeterminationof Cd,Cu,Mn, Ni,Pb andZn innailsafter TMAH solubilization (30L injection volume)

Temperature (C) Ramp (C s1) Hold (s) Gas flow rate (Lmin1)

90 5 15 250120 10 15 250

600a/700b/800c/1000d, e , f 20 15 250

20 1 3 250

1500a/1600b, c/2200d , e/2400f 0 5 01900b, c/2000a/2500d , e, f 1 5 250

a Zn.b Cd.c Pb.d Cu.e Ni.f Mn.

2.2. Instrumentation

2.2.1. Electrothermal atomic absorption spectrometric methodCadmium, copper, lead, manganese, nickel and zinc were

determined in nails by using a AAnalyst 100 atomic absorp-

tion spectrometer (PerkinElmer, Norwalk, CT, USA), equipped withan HGA 800 longitudinally heated graphite tube atomizer andan AS-72 autosampler (PerkinElmer). Deuterium-arc backgroundcorrection was employed to correct for non-specific absorption.All measurements were performed using integrated absorbance

(peak area). Hollow cathode lamps for Cd, Pb, Ni, Mn, Cu and Zn(PerkinElmer) were operated at 4, 10, 25,20, 15 and 15 mA,respec-tively, with a spectral bandwidth of 0.7, 0.7, 0.2, 0.2, 0.7 and 0.7 nm,respectively. The selected wavelengths were 228.8, 283.3, 232.0,

279.5, 324.8 and 213.9 nm for Cd, Pb, Ni, Mn, Cu, Zn, respectively.Argon 99.996% (White Martins, Sao Paulo, SP, Brazil) was usedas protective and purge gas. Pyrolytic graphite coated polycrys-talline electrographitetubes with totalpyrolytic graphite platforms

(PerkinElmer) were used throughout. Then, 30L of diluted sam-ples was directly deposited onto the Lvov platform. The usedheating programs used are given in Table 1.

2.2.2. Inductively coupled plasma mass spectrometry methodWe used a PE ELAN DRC II ICP-MS instrument (PerkinElmer

Life and Analytical Sciences) for the determination of elements in

nails. Typical daily instrumental parameters are given in Table 2.Although this instrument can be used in the DRC mode to removepolyatomic interferences, we operated it solely in standard mode,i.e., with the DRC valve vented, for the determination of metals.

Table 2

ICP-MS operating conditions

PerkinElmer Elan DRC II

Spray chamber Cyclonic

Nebulizer Meinhard

RF power (W) 1100

Ar nebulizer gas flow (L min1) 0.70.9 (optimized daily)

Measures

Scan mode Peak hopping

Resolution (amu) 0.7

Replicate time (s) 1

Dwell time (s) 50

Sweeps/reading 20Integration time (ms) 1000

Replicates 3

Isotopes 63Cu, 111Cd, 208Pb, 60Ni, 55Mn, 64Zn

Correction equations

Cadmium= 114Cd (0.027250 118Sn)

Lead=(204Pb [0.230074 202Hg])+ 206Pb + 207Pb + 208Pb


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Then, the proposed method can be used with simple quadrupoleICP-MS equipments. The ICP-MS was operated with Pt samplerand skimmer cones purchased from PerkinElmer. Argon 99.999%

(White Martins, Sao Paulo, SP, Brazil) was used. The instru-ment conditions and measurement parameters used are listed inTable 2.

2.3. Specimen collection and washing

Fingernails were clipped with a stainless steel cutting instru-ment providing samples ranging in mass from 10 to 20 mg. Then,they were washed in 1% Triton X-100 and acetone and dried in aclass 100 laminar-flow hood.

For comparison with literature data on background levels formetals in nails we also analyzed 35 human nail clippings collectedfrom healthy and non-exposed subjects. These specimens hadbeenobtained with informed consent from human subjects in accor-

dance with procedures approvedby our Institutional Review Board.

2.4. Specimen solubilization and dilution for GF AAS and ICP-MS

analysis

For both ETAAS and ICP-MS methods, cleaned nail clippings

(1020 mg) were accurately weighed into (15 mL) conical tubes.Then, 1 mL of 25% (w/v) TMAH solution was added to the samples,incubated at room temperature overnight, vortexed for 5min andthen the volume made upto 10 mL with 1% (v/v) HNO3. For ICP-MS

analysis, rhodium was added as internal standard to get 10 g L1

final concentration.

3. Results and discussion

Preliminary experiments were carried out to explore the fea-sibility of analyzing nail specimens (20 mg) incubated with 1 mL

of 25% (w/v) TMAH solution at room temperature for 4 h and thenthe volume made up to 10 mL with MilliQ water. For this experi-ment, calibrations in TMAH medium were used for both ETAAS andICP-MS analysis. However, there was a considerable instability of

signals from the standard solutions, probably due the formation ofmetal hydroxides in basic pH conditions. To avoid this, nail speci-mens were incubated with TMAH and then diluted in acid mediumto drop the pH. Nitric acid was selected for this experiment. Based

on optimization studies, betterconditions wereattained after incu-bation of nail samples (20 mg) with 1 mL of 25 (w/v) TMAH andthen the volume made up to 10 mL with 1% (v/v) HNO3. All sub-sequent work utilized this sample preparation protocol for both

techniques.

3.1. Solubilization time

The required time for clipping nail solubilization in TMAHmedium was checked. For this study, ordinary nail clipping sam-

ples collected from one volunteer was used. In each of five tubes, anail clipping sample (20 mg) was diluted with 1 mL of 25% (m/v)TMAH; thetubeswere incubated at room temperature for 1, 2, 4, 6,8 h and overnight and then mixed (vortex) for 5 min. Only 4 h incu-

bation time with TMAH at room temperature was good enough tosolubilize nail clippings. However, in case of routine analysis withlargeamount ofsamples,we recommendthat onemightleave sam-ples incubating overnight and in the next day dilute the samples

with HNO3 before the analysis. In this way, if ICP-MS is the tech-nique of choice, at least 500 samples can be prepared and analyzed

in 3 days.

Fig. 1. Pyrolysis and atomization curves for Cd (20 pg), Pb (0.4ng), Zn (20 pg), Mn

(100pg),Cu (100pg),Ni (400pg) andZn (20pg) in 2.5(v/v) TMAH+ 1.0% (v/v)HNO3medium.

3.2. Pyrolysis and atomization curves for the determination by

ETAAS

Pyrolysis and atomization curves were used to find the opti-mum heating program for lead, cadmium, copper, manganese andnickel in ETAAS analysis. Fig. 1, shows the pyrolysis and atomiza-tion curves for, Cd (20 pg), Cu (100 pg), Mn (100 pg), Ni (400pg)

Pb (0.4 ng) and Zn (20 pg) diluted first with TMAH and then with

HNO3 on the same manner of solubilized samples. For Pb and Cd,graphite furnaces were previously coated with Ir (permanent mod-ifier). Similar thermal behavior was also observed for the analytes

in solubilized matrix. Based on the curves, the heating programshown in Table 1 was used for the determination of the analytes innails by ETAAS.

3.3. Sample calibration

For both ICP-MS and ETAAS analysis, a simple method of cal-ibration by using external standards to simplify the analysis andimprove the sample throughput rate was applied. Standards werediluted in TMAH and HNO3 (see Section 2). For ICP-MS anal-

ysis, the use of internal standards is recommended in routine

analysis to compensate the possible drift during long-term runs.


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Table 3

Trace elements in nail samples by ETAAS and ICP-MS methods (n = 4, meanS.D.)

Samples Cu (ngg1) Ni (ng g1) Cd (ng g1) Pb (ng g1) Mn (ng g1) Zn (g g1)

ETAAS ICP-MS ETAAS ICP-MS ETAAS ICP-MS ETAAS ICP-MS ETAAS ICP-MS ETAAS ICP-MS

1 4799 10 6 4 939 130 580 76 670 83


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