42

4

Research articleReceived: 30 July 2010 Revised: 24 September 2010 Accepted: 27 September 2010 Published online in Wiley Online Library: 3 October 2011

(wileyonlinelibrary.com) DOI 10.1002/xrs.1361

Determination of cobalt marker in cow ruminalfluid by EDXRF and SRTXRFEduardo de Almeida,a∗ Paulo Rogerio Massoni,a

Amauri Antonio Menegario,b Laudi Cunha Leite,c

Dante Pazzanese Duarte Lannac and Virgílio Franco do Nascimento Filhoa

This article describes a comparison of conventional energy-dispersive X-ray fluorescence (EDXRF) and synchrotron radiationtotal-reflection X-ray fluorescence (SRTXRF) for Co determination in ruminal fluid from Holstein cow. This element is used asmarker for animal nutrition studies. For EDXRF, 200 µl of the sample were dried on 6.35 µm Mylar film at 60 ◦C. The excitationwas carried out using an X-ray tube with Mo target and Zr filter operated at 30 kV/20 mA. For SRTXRF, 10 µl of the sample werepipetted on a Lucite carrier and dried at 60 ◦C. In both the techniques, Ga was used as internal standard and the acquisition timewas 200 s. The trueness of both techniques was evaluated through the standard addition method, the recoveries obtained bySRTXRF and EDXRF were 76 and 99%, and the limits of detection, 13 and 240 µg l−1, respectively. Copyright c© 2011 John Wiley& Sons, Ltd.

Introduction

Evaluation of the nutritive value of feed is essential for animalnutrition. A possible manner to obtain this knowledge involvesthe total feces collection of the animal, which is difficult andlaborious. As an alternative, the use of the marker method issuitable and it can afford important diet informations, such asquantity of food ingested, digestibility, the digested passage ratethrough the gastrointestinal tract and so forth.[1 – 2]

An ideal marker should fulfill certain requirements. It shouldneither be absorbed in the gastro intestinal tract nor affect (or beaffect by) the microbial population, and the analytical methods forits determination should be sensitive, specific and not interfereother analytical analysis.[3] Besides markers such as Cr and rareearths (Yb, Dy, Er, Tb), cobalt is a common and important markerfor animal nutritional studies, it is usually used as Co-EDTA.[1,4 – 12]

In addition, it should be mentioned that for ruminants, inwhich cow is included, an important digestive step takes placein their rumen compartment by anaerobic microorganisms.Cobalt (marker) evaluation in liquid-phase digesta of samplesfrom ruminant stomach affords important animal nutritioninformation such as fluid passage rate[6,11] and feed partialdigestibility assessment. This nutritional knowledge allows a betterruminal fermentation understanding and also a feed cow useimprovement. In this manner, the development of analyticalmethod for Co determination in ruminal fluid is notable. Theaim of this work is to compare two X-ray fluorescence energy-dispersive techniques (conventional EDXRF and SRTXRF) for Codetermination in ruminal fluid from cow.

Materials and Methods

Collection and sample processing

The Holstein cow ruminal fluid samples were filtered in onelayer of cheesecloth, centrifuged at 10 000 g for 30 min and thesupernatants stored at −20 ◦C for posterior analysis.

Energy-dispersive X-ray fluorescence (EDXRF)

Working standard solution and sample preparations. The Coworking standard solutions (0–25 mg l−1) were prepared from a1000 mg l−1 Co standard solution (Aldrich Inc.) proper dilution. Forquantitative analysis, the Ga internal standard was added to stan-dards at 25.0 mg l−1 (from 1025 mg l−1 Ga solution, Aldrich Inc.).This latter element was added to avoid geometrical error in the dry-ing sample step as well as uncertainties in the sample pipetting andat smaller extent variations of the X-ray excitation beam. Two hun-dred microliters of the standard solution was pipetted onto 6.35-µm thickness Mylar film (Spex Industries Inc.), using an XRF samplecup with 31.6 mm outer diameter (no. 1530, Chemplex IndustriesInc.), and dried overnight in a laboratory oven at 60 ◦C. The sameprocedure was used for sample analyses. Deionized water was usedthroughout this work. All samples were analyzed in duplicate.

Excitation/Detection. The excitation was carried out using anX-ray Mo tube Philips PW1316/92 model, with Zr filter, coupled toa high voltage generator Philips PW1830 operated at 30 kV/20 mA.The acquisition time was 200 s. It was used as a conventional X-rayspectrometer with a Si(Li) semiconductor detector and the X-ray

∗ Correspondence to: Eduardo de Almeida, Laboratorio de InstrumentacaoNuclear (LIN), Centro de Energia Nuclear na Agricultura (CENA), Universidadede Sao Paulo (USP), Avenida Centenario, no. 303, CEP 13400-970, Piracicaba,SP, Brazil. E-mail: edualm@usp.br

a Laboratorio de Instrumentacao Nuclear (LIN), Centro de Energia Nuclear naAgricultura (CENA), Universidade de Sao Paulo (USP), Avenida Centenario, no.303, CEP 13400-970, Piracicaba, SP, Brazil

b Centro de Estudos Ambientais (CEA), Universidade Estadual Paulista ‘‘Julio deMesquita Filho’’ (UNESP), Av. 24-A, no. 1515, CEP 13506-900, Rio Claro, SP, Brazil

c Laboratorio de Nutricao e Crescimento Animal (LNCA), Departamentode Zootecnia, Escola Superior de Agricultura ‘‘Luiz de Queiroz’’ (ESALQ),Universidade de Sao Paulo (USP), Av. Padua Dias, no. 11, CEP 13418-970,Piracicaba, SP, Brazil

X-Ray Spectrom. 2011, 40, 424–426 Copyright c© 2011 John Wiley & Sons, Ltd.

42

5

Determination of cobalt (employed as marker) in cow ruminal fluid

spectra were deconvoluted by AXIL software.[13] To minimize thescattered radiation produced by experimental arrangement, a5.0-mm diameter Al collimator was used in front of the detector.

Synchrotron radiation total-reflection X-ray fluorescence (SRTXRF)

Working standard solution and sample preparations. The Coworking standard solutions preparation for SRTXRF was the sameas EDXRF one, except for internal standard concentration, in whichGa was used at 5.12 mg l−1. Ten microliters of the standard solutionwas pipetted onto Lucite (Perspex) carrier and dried overnight ina laboratory oven at 60 ◦C. The same procedure was used forsample analyses. Deionized water was used throughout this work.All samples were analyzed in duplicate.

Excitation/Detection. A polychromatic X-ray beam from 4 to22 keV with 5 mm width and 0.1 mm height was utilized undertotal-reflection condition at the D09B-XRF beamline in the BrazilianSynchrotron Light Laboratory, Campinas, Sao Paulo, Brazil. Theacquisition time, detection system and spectra deconvolutionwere the same as EDXRF ones.

Quantification

In both techniques, the working standard solutions and thesamples could be considered as thin films, in which theabsorption and the enhancement effects are neglected. ThenCo concentration could be calculated as follows[14]:

ICo = SCo CCo (1)

Considering the internal standard, the same equation could bewritten for Ga:

IGa = SGa CGa (2)

where ICo is the characteristic Kα X-ray intensity (cps), SCo is thesensitivity (cps µg−1 ml) and CCo is the concentration (µg ml−1) forCo, and IGa is the characteristic Kα X-ray intensity (cps), SGa is thesensitivity (cps µg−1 ml) and CGa is the concentration (µg ml−1)for Ga.

From Eq. (1) and (2),

ICo CGa

IGa= SCo

SGaCCo (3)

in which

RCo = ICoCGa

IGaand S′

Co = SCo

SGa(4)

resulting

RCo = S′Co CCo (5)

where RCo is the relative intensity (µg ml−1) and S′Co is the relative

sensitivity (unitless) for Co element.The EDXRF and SRTXRF cobalt relative sensitivities (S′

Co) weredetermined experimentally by the slope of their respectiveanalytical curves, in which RCo was plotted against the CCo fromthe working standard solutions set.

0.1

1

10

0 2 64 8 10 12 14 16

Co

un

t ra

te (

cps)

Energy (keV)

K

CaAr

Cl

Mn

Fe

Co3.81

Ga25.6

Zn

Concentration, mg l-1

Br

Rb

Figure 1. EDXRF spectrum of a sample of ruminal fluid from cow containing3.81 mg l−1 of Co and 25.6 mg l−1 of Ga (internal standard).

Limit of detection

In both techniques, the Co limit of detection (LDCo) is given by:

LDCo = 3.

√ICo(BG)

t

CGa

IGaS′Co

(6)

where LDCo is the limit of detection (µg ml−1), ICo(BG) is thebackground intensity (cps) under the Kα X-ray peak for cobaltand t is the acquisition time (s).[14]

Trueness

The trueness of the analytical methods was evaluated by recoverytest. Ten micrograms of Co (10 µl of 1000 mg l−1 standard solution,Aldrich Inc.) was spiked in 1 ml of the sample for EDXRF. For SRTXRF,it was spiked 5 µg of Co (5 µl of 1000 mg l−1 standard solution,Aldrich Inc.) in the same sample volume.

Results

Fig. 1 presents an EDXRF spectrum of ruminal fluid from cowcontaining 3.81 mg l−1 of Co. Although Co-Kα is interfered byFe-Kβ peak, the AXIL software is able to separate them based onKα/Kβ ratio knowledge of each element. So, the EDXRF and SRTXRFtechniques are feasible for determination of Co in cow ruminalfluid as far as spectral interference is concerned. It is worthwhileto consider that neither digestion nor sample dilution was carriedout (except a small dilution due to Ga internal standard addition).

This spectrum also shows the presence of other elements inthe sample, such as Cl, K, Ca, Mn, Fe, Zn, Br and Rb. In case ofinterest, all these elements might be determined simultaneously,except for Mn, Br and Rb. For the latter ones, some modificationsin the method are demanded in order to increase their analyticalsensitivities, for instance: longer time acquisition, higher currentand/or voltage in the excitation condition and so forth. It is alsoworth mentioning that Zn–Kα is is slightly overlapped by Ga peakescape, but this interference is considered in the AXIL software forZn quantification.

It should be noted in this spectrum that the background(BG) rises above 10 keV significantly. In addition, the argoncharacteristic X-ray (2.96 keV) peak showed in the spectrum isdue to its presence in air atmosphere considering that EDXRF

X-Ray Spectrom. 2011, 40, 424–426 Copyright c© 2011 John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/xrs

42

6

E. de Almeida et al.

0.1

1

10

100

1000

0 2 4 6 8 10 12 14 16 18 20 22

Co

un

t ra

te (

cps)

Energy (keV)

P

Cl

KCa

Mn

Co3.71

Fe

Cu

Zn

Ga5.12

BrRb Sr

Concentration, mg l-1

Figure 2. SRTXRF spectrum of sample of ruminal fluid from cow containing3.71 mg l−1 of Co and 5.12 mg l−1 of Ga (internal standard).

Table 1. Co concentrations (mg l−1) and their respective standarddeviations (1σ ) in three cow ruminal fluid samples measured by EDXRFand SRTXRF techniques

Techniques Sample 01 Sample 02 Sample 03

EDXRF 3.81 ± 0.07 7.48 ± 0.11 5.8 ± 0.3

SRTXRF 3.71 ± 0.19 5.9 ± 0.4 3.90 ± 0.13

analysis was not carried out under controlled atmosphere orvacuum.

However, Fig. 2 presents a SRTXRF spectrum of ruminal fluidfrom cow containing 3.71 mg l−1 of Co (this spectrum and theEDXRF one, Fig. 1, are from the same sample). Several elementscan be observed in the sample, such as P, Cl, K, Ca, Mn, Fe,Cu, Zn, Br, Rb and Sr. In case of interest, all these elementsmight be determined simultaneously. Therefore, SRTXRF is ableto determine more elements than EDXRF, in which phosphorusshould be highlighted due to its importance in animal nutrition.The SRTXRF spectrum also shows a quite constant BG from 2to 14 keV, and above this energy the BG decreases as opposedto EDXRF (Fig. 1). This is explained due to the polychromaticSRTXRF (from 4 to 22 keV) and monochromatic EDXRF (17.45 keV)excitations.

Table 1 shows the Co concentrations (mg l−1) and their respec-tive standard deviations (1σ ) in three cow ruminal fluid samples byboth techniques. The paired Student’s t-test was utilized to assessthe results from SRTXRF and EDXRF. There were no significant dif-ferences (at 95% level) between the results from both techniques.

Concerning trueness evaluation, the recoveries for EDXRF andSRTXRF were 99 ± 9 and 76 ± 10%, respectively. There is nosignificant difference between recovery values at 95% level.

In literature, the direct and slurry analyses in biological samplesby TXRF are well discussed.[15 – 18] Karjou (2007), for example,compares TXRF results of the digested and suspended IAEA-A-13reference material sample, in which the quantitative results forK, Ca, Ni, Cu, Zn, Se, Rb and Pb were similar. However, Fe andBr concentrations were different considering these two samplepreparations due to iron adsorption on the walls of the Teflonbomb and bromine volatility which are avoided by direct analysis.

The LDCo were 13 and 240 µg l−1 by SRTXRF and EDXRF,respectively. This result was expected because synchrotron X-rayexcitation beam is several orders of magnitude brighter than thatone from an X-ray tube and, in addition, SRTXRF excitation beam

is collimated and linearly polarized.[19] It must be highlighted thatthe cobalt relative intensity is quite the same in both techniques;however, the Co BG to Ga intensity ratio is much smaller in theSRTRF, and consequently, a lower LD is obtained.

With reference to other analytical techniques for Co markerdetermination in ruminal fluid, atomic absorption spectroscopy(AAS) is commonly used.[6 – 12] However, these articles focus on thenutritional features of cobalt marker use mainly and AAS figuresof merit are not discussed.

Conclusion

In both techniques, the sample preparation is very straightforward,neither digestion nor other laborious and expensive steps wererequired. The trueness of these techniques was assessed throughthe standard addition method and the recoveries obtained bySRTXRF and EDXRF were 76 and 99% for cobalt element. TheSRTXRF allows an evaluation of a larger pool of elements inHolstein cow ruminal fluid due to its higher sensitivity and it has alower limit of detection for Co (13 µg l−1) than EDXRF (240 µg l−1),but this last technique has the advantage of being much cheaperand more easily accessible.

Acknowledgement

Research (partially) supported by LNLS - Brazilian SynchrotronLight Laboratory (D09-XRF-6648 Project).

References

[1] G. S. Lopes, A. R. A. Nogueira, P. V. Oliveira, J. A. Nobrega, Anal. Sci.1999, 15, 165.

[2] J. van Keulen, B. Young, J. Anim. Sci. 1977, 44, 282.[3] F. Vicente, A. Sarraseca, A. Vega, J. A. Guada, J. Sci. Food Agric. 2004,

84, 2035.[4] J. A. More, J. Anim. Sci. 1992, 70, 3528.[5] A. Sairanen, H. Khalili, J. I. Nousiainen, S. Ahvenjarvi, P. Huhtanen,

J. Dairy Sci. 2005, 88, 1443.[6] F. J. Mulligan, P. J. Caffrey, M. Rath, J. J. Callan, P. O. Brophy,

F. P. O’Mara, Livest. Prod. Sci. 2002, 77, 311.[7] A. P. Moloney, R. K. Wilson, B. C. Moloney, Anim. Feed Sci. Technol.

1994, 46, 215.[8] F. Loya-Olguin, L. Avendano-Reyes, A. M. Encinias, D. A. Walker,

N. A. Elam, S. A. Soto-Navarro, J. Anim. Sci. 2008, 86, 2749.[9] M. Dias, E. Detmann, M. I. Leao, S. M. Souza, S. C. Valadares Filho,

A. M. Vasconcelos, L. N. Renno, R. F. D. Valadares, R. Bras. Zootec.2007, 36, 689.

[10] S. A. Soto-Navarro, C. R. Krehbiel, G. C. Duff, M. L. Galyean,M. S. Brown, R. L. Steiner, J. Animal Sci. 2000, 78, 2215.

[11] E. L. Fredrickson, R. E. Estell, K. M. Havstad, W. L. Shupe,L. W. Murray, Livest. Prod. Sci. 1995, 42, 45.

[12] F. T. McCollum, M. L. Galyean, J. Anim. Sci. 1985, 60, 570.[13] P. van Espen, H. Nullens, F. Adam, Nucl. Instrum. Method. 1977, 142,

243.[14] V. F. Nascimento Filho, V. H. Poblete, P. S. Parreira, E. Matsumoto,

S. M. Simabuco, E. P. Espinoza, A. A. Navarro, Biol. Trace Elem. Res.1999, 71/72, 423.

[15] J. Karjou, Spectrochim. Acta Part B 2007, 62, 177.[16] L. M. Marco-Parra, E. A. Hernandez-Caraballo, Spectrochim. Acta Part

B. 2004, 59, 1077.[17] Ch. Zarkadas, A. G. Karydas, T. Paradellis, Spectrochim. Acta Part B.

2001, 56, 2219.[18] K. Barkacs, A. Varga, K. Gal-Solymons, G. Zaray, J. Anal. At. Spectrom.

1999, 14, 577.[19] P. Kregsamer, C. Streli, P. Wobrauschek, in Handbook of X-ray

Spectrometry: Methods and Techniques, (Eds: R. E. van Grieken,A. A. Markowicz), Marcel Dekker Inc.: New York, 2002, pp. 559.

wileyonlinelibrary.com/journal/xrs Copyright c© 2011 John Wiley & Sons, Ltd. X-Ray Spectrom. 2011, 40, 424–426