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Indirect determination of sodium diclofenac, sodium dipyrone and calciumgluconate in injection drugs using digital image-based (webcam) flameemission spectrometric method

Wellington da Silva Lyra, F�atima Aparecida Castriani Sanches, Francisco Antonio da Silva Cunha,Paulo Henrique Goncalves Dias Diniz, Sherlan Guimaraes Lemos, Edvan Cirino da Silvaand Mario Cesar Ugulino de Araujo*

Received 31st March 2011, Accepted 7th June 2011

DOI: 10.1039/c1ay05197k

This paper proposes a digital image-based flame emission spectrometric (DIB-FES) method for indirect

determination of sodium diclofenac, sodium dipyrone and calcium gluconate in injectable forms. The

proposed DIB-FES method uses digital images obtained from a webcam, based on the RGB

(Red-Green-Blue) system. It offers a simple and inexpensive way to quantify these organic substances

using the radiation emitted by the alkaline and earth-alkaline metals present in their formulae.

Analytical curves were constructed on the basis of the relationship between RGB values and calibration

solution concentrations. The results showed no statistical difference between the proposed and

reference methods when applying the paired t-test at a 95% confidence level. The proposed DIB-FES

method also performed well in terms of the figures of merit LOD, LOQ, linear range, precision, and the

accuracy as revealed by recovery tests.

Introduction

In recent years stricter regulations have led to increasing

demands by the pharmaceutical industry for fast and simple

analytic assays.1 The industry is always seeking such methods to

guarantee drug content, and identify adulterated medicines

which represent health risks.2 The need is for selective, fast, low

cost analytical methods of quality control for pharmaceutical

formulations.3

In the latest editions of the American, Brazilian and British

Pharmacopoeia4–6 articles describing titrimetric and chromato-

graphic methods for drug determination predominate. However,

these methods are laborious, time-consuming, and generate large

quantities of residues while using equally large quantities of

reagents and (toxic) organic solvents. On the other hand, spec-

troanalytical methods have been developed for pharmaceutical

and biomedical analyses which have certain advantages such as

low operational/maintenance costs and simple analytic

procedure.

In the context of spectroanalytical methods, the trend is

toward atomic absorption (AAS) and inductively coupled

plasma (ICP) for indirect determination of drugs, because of

their high sensitivity and speed extending the reach of these

techniques.7

Departamento de Qu�ımica, CCEN, Universidade Federal da Para�ıba, P. O.Box 5093, 58051-970 Joao Pessoa, Brazil. E-mail: laqa@quimica.ufpb.br;Fax: +55 83 3216 7438; Tel: +55 83 3216 7438

This journal is ª The Royal Society of Chemistry 2011

According to Yebra,7 the indirect determination of drugs is

carried out by prior reaction between the analyte and a metal

cation acting as a precipitant. The concentration estimate may be

carried out as follows: (i) the precipitant is extracted in organic

solvent and the analyte concentration is estimated by means of

measurements performed in the organic phase, (ii) an excess of

the metal cation is added to the sample and concentration of the

analyte is determined in the supernatant, (iii) redox reaction

followed by a precipitation and solvent extraction. Despite good

results normally provided by AAS and ICP indirect determina-

tions, these methods as well as the titrimetric and chromato-

graphic methods employ reagents and organic solvents. Despite

a variety of methodologies for determination of these drugs,

none of them uses counter-ions. Our study highlights the use of

the counter-ion and only sodium diclofenac was indirectly

determined by AAS8,9 using the approach described by Yebra.7

Recently, an innovative, simple and low-cost method which is

based on digital images obtained through analyses by flame

emission spectrometry was proposed.10 Digital images of the

radiation emitted by the analyte in an air–butane flame were

captured using a digital camera (webcam) whose detection is

based on the RGB (Red-Green-Blue) colour system. In order to

build linear analytical curves, a mathematical model was

proposed on the basis of the RGB-based values (analytical

response) and defined as the norm for position vectors in RGB

three-dimensional space.

The goal of the present paper is to propose a digital image-

based flame emission spectrometric (DIB-FES) method for

Anal. Methods, 2011, 3, 1975–1980 | 1975

Fig. 1 Routine of the software for image treatment.

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indirect determination of sodium diclofenac, sodium dipyrone

and calcium gluconate in their injectable forms. The DIB-FES

method offers a simple and inexpensive way to determine organic

substances based on the radiation emitted by the alkaline and

earth-alkaline metals present in their formulae. For this reason,

the salts stoichiometry of the analyzed drugs must also be taken

into account. The following stoichiometric ratios between

metallic cation and organic anion: sodium diclofenac (1 : 1),

sodium dipyrone (1 : 1) and calcium gluconate (1 : 2) were used.

The choice of analytical determinations above was motivated

by the importance that these drugs share. Sodium diclofenac is

a relatively safe and effective non-steroidal drug with

pronounced anti-rheumatic, anti-inflammatory, analgesic and

antipyretic properties.11 On the other hand, sodium dipyrone is

an active constituent of dozens of pharmaceutical preparations

and widely used as an antipyretic and analgesic drug.12 Finally,

calcium gluconate is employed in the treatment of hypocalcemia

for conditions requiring prompt increase in blood plasma

calcium (exchange transfusions).13 It must also be emphasized

that in injection solutions there is only the analyte and dilution

water. Even in high concentration the present analytes do not

cause great changes in the physico-chemical properties of the

water and no matrix effect will be observed in analytical deter-

minations of this study. Furthermore the absence of other metals

in the sample is a guarantee that no spectral interference will be

observed in analytical determinations of this study.

Material and methods

Reagents, solutions, and samples

All chemicals were of analytical grade and deionized water

(Milli-Q Millipore system) was employed throughout the work.

Stock solutions (500 mg L�1) of both sodium diclofenac and

sodium dipyrone 99% (Sigma, St Louis, MO, USA), and 2000 mg

L�1 of calcium gluconate 98% (Aldrich, Milwaukee, WI, USA)

were prepared by dissolution of suitable amounts from the

respective salts in 1% v/v HNO3 solution (Sigma, St Louis, MO,

USA). The calibration solutions with five levels of concentration

of sodium diclofenac (24.9–124.5 mg L�1) (1.8–9.0 mg L�1 in

terms of sodium), sodium dipyrone (27.5–137.5 mg L�1) (1.8–

9.0 mg L�1 in terms of sodium) and calcium gluconate (282.0–

1410.0 mg L�1) (172.8–864.2 mg L�1 in terms of CaOH) were also

prepared by suitable dilutions from their respective stock solu-

tions with 1% v/v HNO3 solution.

The concentration ranges of the calibration solutions

mentioned above were defined taking into account the linear

response range for calcium and sodium, which has already been

studied and reported elsewhere.10 Five brands of injection drugs

with a nominal content of 25 mg mL�1 of sodium diclofenac,

500 mg mL�1 of sodium dipyrone and 10% (m/v) of calcium

gluconate were purchased from local drugstores. Before analysis,

all samples were suitably diluted with 1% v/v HNO3 solution in

order to read the analytical signals in the linear response range of

the method.

Apparatus

Details of the system assembled for digital image acquisition are

described by Lyra and coworkers.10 In order to capture the

1976 | Anal. Methods, 2011, 3, 1975–1980

digital images, a model Instant Creative webcam was employed,

which was connected to a universal serial bus (USB) inlet of

a Pentium III 650 MHz microcomputer (PC). This webcam was

configured to capture 24-bit digital images (16.7 million colours)

at a rate of 34 images s�1 and 352 pixels � 288 pixels of spatial

resolution. The images were captured by means of the software

written in Delphi (version 3.0). A flame emission photometer

(model NK-2004 Digimed) was also coupled to the system. The

optimum conditions found for maximum sensitivity with an air–

butane flame were: butane pressure ¼ 3.5 psi, air pressure ¼20 psi, air flow rate ¼ 9 L min�1 and sample aspiration flow

rate ¼ 4 mL min�1. The images were captured at 2.5 cm over the

burner of the photometer (oxidant region of the flame).

Treatment of digital images, RGB-based value, DIB-FES

analytical curves and figures of merit

The treatment of the captured digital images was made by means

of second software also written in Delphi (version 3.0). The

routine with the working stages of this software is illustrated in

Fig. 1 and is similar to that used elswhere.10,14,15 Initially the user

selects, with the computer mouse, the most homogeneous region

in the image (in the middle) which will define the coordinates of

the selected region and will also be used in all other images. Then

the software scans all the pixels column by column to extract the

RGB component for each pixel and calculate a mean integer

value of each RGB component. These mean values are used in

the RGB-based value calculation (analytical response) as

described below.

The RGB-based values were calculated by means of a mathe-

matical model developed from the concept of vector norm v,10

‘‘kk’’, calculated as:

kvk ¼ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiR

2

s�b þ G2

s�b þ B2

s�b

q(1)

where �Rs–b, �Gs–b and �Bs–b result from the difference between the�Rs, �Gs and �Bs average values obtained from digital images of

the calibration solutions and samples and �Rb, �Gb and �Bb from the

blank. As a result, a linear relationship was observed between the

analyte concentration (C) in the calibration solution (or sample)

and RGB-based value, for which the following equation is valid:

This journal is ª The Royal Society of Chemistry 2011

Table 1 Reference methods for the chosen analytes in Pharmacopoeias

Analyte American4 Brazilian5 British6

Sodiumdiclofenac

Potentiometrictitrationin non-aqueousmedium

UV-Visspectrophotometryin methanolmedium

Liquidchromatography

Sodiumdipyrone

Not described Redox titration Not described

Calciumgluconate

Not described Complexometrictitration

Complexometrictitration

Fig. 2 Digital images captured from the flame emission of sodium

diclofenac (a), sodium dipyrone (b) and calcium gluconate (c) in the

calibration solutions used to build DIB-FES linear analytical curves.

Fig. 3 Vectors of each calibration solution (v1 to v5) and the values of

where �Rs–b and �Gs–b for the digital images of sodium diclofenac (a),

sodium dipyrone (b) and calcium gluconate (c).

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kvk ¼ kC (2)

As demonstrated in previous work,10 eqn (2) provides the basis

for building DIB-FES analytical curves, establishing a linear

relationship between kvk (RBG-based value adopted as analy-

tical response) and analyte concentration in calibration solu-

tions. Furthermore, the vectors associated with the digital images

from each analyte should be positioned on the same support line

in the RGB three-dimensional space.

The figures of merit were estimated according to the metho-

dology developed and reported by Lyra and coworkers.10

Reference methods

Table 1 shows the recommended reference methods for the

chosen drugs. As can be seen only in Brazilian Pharmacopoeia

the methods for the three drugs are described, so these methods

had to be followed. In the reference method for the determina-

tion of sodium diclofenac an aliquot of the sample is diluted in

methanol and absorbance measurements at 258 nm were carried

out against methanol as blank. In the reference method for the

determination of sodium dipyrone an aliquot of the sample in

acetic acid medium is titrated against iodine standard solution

using starch as indicator and the temperature must be kept below

15 �C. In the reference method for the determination calcium

gluconate an aliquot of the sample is alkalinized by addition of

NaOH solution and then titrated against EDTA standard solu-

tion using calcon as indicator. This procedure is the same as

described in the Brazilian and British Pharmacopoeias.

Recovery tests

The accuracy and the presence of some kind of interference were

evaluated by means of recovery tests.16 In this study the spike

strategy was used. An aliquot of the sample and an aliquot of the

stock standard solution of the analyte are mixed and diluted up

to a final volume in a volumetric flask in order to read the

analytical signals in the linear response range of the method

(spiked sample). In another volumetric flask the same aliquot of

the sample is diluted to the same final volume (original sample).

The recovery or recovery factor is calculated according to the

following equation:

RA ¼ ½xAðOþ SÞ � xAðOÞ�xAðSÞ (3)

where xA(O + S) is the spiked concentration of the analyte, xA(O)

is the original concentration of the analyte and xA(S) is the spike

concentration of the analyte.

This journal is ª The Royal Society of Chemistry 2011

The spike concentrations for sodium diclofenac, sodium

dipyrone and calcium gluconate were 10.1, 11.3 and 334.7 mg L�1

respectively.

Results and discussion

Digital images and emission phenomenon

Fig. 2 shows digital images of the radiation emitted by sodium

diclofenac (a), sodium dipyrone (b) and calcium gluconate (c)

from five calibration solutions with different concentrations. The

first image of each sequence is of background radiation emitted

while the blank solution (1% v/v HNO3 solution) is sampled and

introduced into the air–butane flame.

Digital images in lines (a) and (b) of Fig. 2 present the yellow

secondary colour, which results from a balanced mixture of the

primary colours red + green.17 This radiation corresponds to the

resonance lines (D duplet) at 589.6 and 589.0 nm in the sodium

spectrum, resulting from the electronic transition from the 3p

excited states (2P1/2 and 2P3/2 spectroscopic terms) to the 3s

ground state (2S1/2 spectroscopic term).18 It is worth noting that

the images in sequences of Fig. 2(a) and (b) have the same

intensity, because the calibration solutions have the same

concentrations in terms of sodium ions.

Anal. Methods, 2011, 3, 1975–1980 | 1977

Fig. 4 DIB-FES analytical curves and residue distributions from the models developed for each analyzed drug: (a) sodium diclofenac, (b) sodium

dipyrone and (c) calcium gluconate.

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Fig. 2(c) presents digital images with an orange tertiary colour,

which results from the mixture of red + green with a larger

contribution of red. This orange radiation is associated with the

emission band from the CaOH species commonly generated in an

air–butane flame.19 So the measurements were made in terms of

CaOH because the temperature of the flame is not suitable to

generate atomic calcium.

In all cases presented in Fig. 2, the intensity of the colour rises

as the analyte concentration in the calibration solutions

increases. Consequently, an increasing concentration of the

emitting species leads to a proportional increase in the amount of

photons reaching the webcam’s phototransducer.10

Table 2 ANOVA for the fit to a linear model (y ¼ a + bX) of DIB-FESanalytical curves (Fig. 3)

Analyte Sourcea SS MS

Sodium diclofenac Regression (1) 7.33 � 104 7.33 � 104

Residual (13) 9.57 0.74Lack of fit (3) 1.94 0.65Pure error (10) 7.63 0.76

Sodium dipyrone Regression (1) 7.47 � 104 7.47 � 104

Residual (13) 10.05 0.77Lack of fit (3) 3.66 1.22Pure error (10) 6.38 0.64

Calcium gluconate Regression (1) 4.64 � 104 4.64 � 104

Relationships between kvk and �Rs–b, �Gs–b and �Bs–b values

Fig. 3(a) and (b) show that the vectors v1–v5, which are associated

with digital images from five calibration solutions of sodium

diclofenac (a) and sodium dipyrone (b) with equal concentra-

tions of sodium ions. As can be seen, all five vectors are placed on

the same straight line whose direction coincides with the bisectrix

line between the �Rs–b and �Gs–b axis. This finding can be explained

by considering the fact �Rs–b and �Gs–b values present equivalent

contributions for the norm of the vectors v1–v5.

Fig. 3(c) shows that �Rs and �Gs values from the calcium

gluconate calibration solutions also contribute to the vector

norm, but in an unequal manner. In this case, the straight line

onto which the vectors v1–v5 are localized is closer to the �Rs–b axis

due to the larger contribution of this component.

Residual (13) 6.32 0.49Lack of fit (3) 1.37 0.46Pure error (10) 4.95 0.50

a Values between parentheses show the number of degrees of freedom.

DIB-FES analytical curves and figures of merit

Fig. 4 shows the DIB-FES analytical curves for the indirect

quantification of sodium diclofenac, sodium dipyrone and

1978 | Anal. Methods, 2011, 3, 1975–1980

calcium gluconate in injectable forms and their corresponding

residual plots. A linear relationship between the analytical

response (RGB-based value) and the concentrations of the cali-

bration solutions is observed for the three analytes. Before

building residual plots, a variance analysis (ANOVA) was

implemented in order to validate the linear calibration models.

For this purpose, the F-test for lack of fit was applied according

to recommendations found elsewhere.20 Moreover, the experi-

mental design should also anticipate genuine repeat measure-

ments in at least one of the concentration levels. In this study, the

analytical curves were constructed on the basis of three repeated

measurements in five levels. The values of pure errors and lack of

fit were calculated by using the mean squares (MS) presented in

Table 2.

This journal is ª The Royal Society of Chemistry 2011

Table 3 Confidence intervals for the model parameters and figures of merit for the proposed method

Confidence intervals for model parameters (y ¼ a + bX)Values of figures of merit/mg L�1

Analyte a � t13s(a) b � t13s(b) LOD LOQ

Sodium diclofenac 0.01 � 7.01 1.98 � 0.08 0.6 2.1Sodium dipyrone �0.04 � 1.99 1.81 � 0.02 0.9 3.0Calcium gluconate �0.02 � 1.59 1.4 � 10�1 � 1.7 � 10�3 2.5 8.4

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Regarding the residual plots in Fig. 4, they seem homoge-

neous, they did not vary as the concentration increased and they

were almost symmetrical spread around the central value for

most of the repeated measurements. These characteristics are

also in agreement to residual plots observed in a previous study.10

Such finding may be ascribed to the trivariate nature of the

monitored signal and data treatment procedure.

It can be seen that in all cases, the values of MSlack of fit/

MSpure error are smaller than the point of F-distribution at a 95%

confidence level with equivalent degrees of freedom. This finding

indicates that there is no evidence of lack of fit for linear models.

This inference is corroborated by an analysis of the residual

plots (Fig. 4), in which random distribution of the residuals

around zero for all the analytes was observed, indicating a good

fit. It is worth noting that ANOVA in Table 1 also shows that the

results of linear regressions are significant. Values of MSregression/

MSresidual are much larger than the value of the point of F-

distribution at a 95% confidence level with corresponding degrees

of freedom. The confidence intervals of the calibration model

parameters (y ¼ a + bX) for the three analytes are shown in

Table 3 at a 95% confidence level.

After the ANOVA test and analysis of the residual plots, the

figures of merit were estimated according to IUPAC recom-

mendation21 (eqn (4) and (5)) using the mathematical approach

Table 4 Results of the indirect determinations of sodium diclofenac, sodium d

Samples Proposed method

Sodium diclofenac (mg mL�1)(1) 25.1 � 0.2(2) 25.0 � 0.2(3) 24.9 � 0.1(4) 25.0 � 0.1(5) 24.9 � 0.2Overall RSD 0.2Sodium dipyrone (mg mL�1)(1) 496.9 � 1.4(2) 501.6 � 3.6(3) 496.9 � 4.9(4) 496.1 � 2.4(5) 500.8 � 2.4Overall RSD 3.2Calcium gluconate % (m/v)(1) 9.90 � 0.02(2) 9.80 � 0.02(3) 9.98 � 0.02(4) 9.99 � 0.02(5) 9.94 � 0.02Overall RSD 0.02

This journal is ª The Royal Society of Chemistry 2011

developed by Lyra and co-workers (eqn (6)).10 For this reason, 20

digital images from the blank of each analyte were acquired and

used to estimate the blank standard deviation (sb)

LOD ¼ 3sb

b(4)

LOQ ¼ 10sb

b(5)

sb ¼ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiðRbsRÞ2þðGbsGÞ2þðBbsBÞ2

R2

b þ G2

b þ B2

b

vuut (6)

Table 3 shows that the proposed method presents low values for

limit of detection (LOD) and limit of quantification (LOQ). The

DIB-FES method also presents good performance with respect

to linear response ranges. The proposed method presents wide

linear ranges, especially for calcium gluconate.

Analytical determinations by using the proposed method

Table 4 shows that proposed DIB-FES and reference methods

yielded similar results in the determination of the three analytes.

In fact, no statistical difference was verified between the results

ipyrone and calcium gluconate by using proposed and reference methods

Reference method Recovery (%)

25.0 � 0.4 98 � 224.8 � 0.5 102 � 225.0 � 0.5 100 � 224.9 � 0.4 98 � 224.9 � 0.3 104 � 20.4 —

493.3 � 8.2 97 � 3498.6 � 16.4 99 � 2498.6 � 8.2 99 � 3498.1 � 14.2 102 � 2502.0 � 8.2 103 � 311.6 —

9.86 � 0.04 98 � 39.81 � 0.04 100 � 29.91 � 0.02 101 � 19.96 � 0.05 103 � 29.97 � 0.02 98 � 20.04 —

Anal. Methods, 2011, 3, 1975–1980 | 1979

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by applying the paired t-test at a 95% confidence level. However,

the proposed method was more precise as revealed by the smaller

values of overall RSD (n¼ 3). Such precision must be ascribed to

the multivariate nature of the monitored signal in RGB image-

based analytical determinations.10 Regarding the accuracy and

the presence of interferences, the proposed method was also

evaluated by employing recovery tests and results are also shown

in Table 4.

The DIB-FES method is suitable for the chosen samples

considering that most of the recovery percentages were between

97 and 104%.

Conclusions

This study demonstrates the feasibility of using a DIB-FES

method for analysis of pharmaceutical formulations. The

proposed method offers simple and inexpensive indirect deter-

mination of injection drug concentrations, by combined use of

digital images and flame emission photometry. The good results

in accordance with previous studies10 reinforce the idea of

replacement of interference filters by webcams in the detection

system in flame photometry.

In terms of the estimated figures of merit, the DIB-FES

method presented satisfactory performance in every determina-

tion. Special attention must be given to the high accuracy of the

results, which was revealed by the recovery tests, and that the

samples used to assess the accuracy of the proposed method

present neither spectral interference nor matrix effect.

Since the proposed methodology employs an inexpensive

webcam for detection and dispenses with the use of a wavelength

selector, it can reduce costs considerably, and simplify the

instrumentation. In spite of the advantages, the absence of

a wavelength selector makes the response of the webcam more

susceptible to spectral interference and/or matrix effects, espe-

cially in determinations involving complex matrices. In order to

overcome these drawbacks, multivariate calibration methods

and the Generalized Standard Addition Method (GSAM) could

be implemented.22,23

The proposed method may be applied in the determination of

any organic substance since it is in a salt form whose metal cation

is capable of emitting radiation in the visible region and is indi-

cated for drug quality control in industry line production, but

not for adulteration screening analysis because of poor

discrimination power caused by counter-ions.

1980 | Anal. Methods, 2011, 3, 1975–1980

Acknowledgements

The authors thank the Brazilian agency CNPq for scholarships.

The authors also thank David Harding, a Pharmacy student here

in Para�ıba, Brazil, native of California for reviewing the English

in this article.

References

1 Z. Song and N. Zhang, Talanta, 2003, 60, 161.2 P. L. Weinert, L. Pezza and H. R. Pezza, J. Braz. Chem. Soc., 2007,18, 846.

3 F. A. A. Matias, M. M. D. C. Vila and M. Tubino, J. Braz. Chem.Soc., 2004, 15, 327.

4 The United States Pharmacopoeia—National Formulary, USP-NFXXIII.

5 Farmacop�eia Brasileira, Atheneu Editora, Sao Paulo, 4th edn, 1988,pp. 144, 145 and 916.

6 European Pharmacopoeia, Council of Europe, Strasbourg, 3rd edn,1997.

7 M. C. Yebra, TrAC, Trends Anal. Chem., 2000, 19, 629.8 G. Alpdo�gan and S. Sungur, Anal. Lett., 1999, 14, 2799.9 M. M. Issa, R. M. Nejem, M. Al-kholy, N. S. El-Abadia, R. S. Hellesand A. A. Saleh, J. Serb. Chem. Soc., 2008, 73, 569.

10 W. S. Lyra, V. B. Santos, A. G. G. Dion�ızio, V. L. Martins,L. F. Almeida, E. N. Gaiao, P. H. G. D. Diniz, E. C. Silva andM. C. U. Ara�ujo, Talanta, 2009, 77, 1584.

11 R. S. Rocha, A. A. G. F. Beati, J. G. Oliveira and M. R. V. Lanza,Quim. Nova, 2009, 32, 354.

12 L. Pezza, M. Tubino, C. B. Melios and H. R. Pezza, Anal. Sci., 2000,16, 313.

13 C. E. Ariyan and J. A. Sosa, Crit. Care Med., 2004, 32,S146.

14 E. N. Gaiao, V. L.Martins,W. S. Lyra, L. F. Almeida, E. C. Silva andM. C. U. Ara�ujo, Anal. Chim. Acta, 2006, 570, 283.

15 A. R. Torres, W. S. Lyra, S. I. E. Andrade, R. A. N. Andrade,E. C. Silva, M. C. U. Ara�ujo and E. N. Gaiao, Talanta, 2011, 84,601.

16 D. T. Burns, K. Danzer and A. Townshend, Pure Appl. Chem., 2002,74, 2201.

17 R. S. Berns, Principles of Colour Technology, Wiley, New York, 3rdedn, 2000.

18 B. Weltz and M. Sperling, Atomic Absorption Spectrometry, Wiley-VCH, Weinheim, 1999.

19 D. A. Skoog and J. J. Leary, Principles of Instrumental Analysis,Saunders College Publishing, New York, 4th edn, 1992.

20 N. R. Draper and H. Smith, Applied Regression Analysis, Wiley, NewYork, 3rd edn, 1998.

21 IUPAC, Pure Appl. Chem., 1976, 45, 99.22 B. E. H. Saxberg and B. R. Kowalski, Anal. Chem., 1979, 51,

1031.23 E. C. Silva, V. L. Martins, A. F. Ara�ujo and M. C. U. Ara�ujo, Anal.

Sci., 1999, 15, 1235.

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