Spectrochimica Acta Part B 57 (2002) 20952102

Determination of total arsenic by batch hydride generation atomic absorption spectrometry in injectable drugs containing high levels of Sb(V) as N-methylglucamine antimonate
Erico Marlon de Moraes Floresa,*, Fabiana E. Barcelos da Silvaa, Eliane Pereira dos Santosa, Favero Reisdorfer Paulaa, Juliano Smanioto Barina, Renato Zanellaa, Valderi Luiz Dresslera, Celso Figueiredo Bittencourtb
b a Departamento de Qumica, Universidade Federal de Santa Maria, 97105-900 Santa Maria, RS, Brazil Departamento de Farmacia Industrial, Universidade Federal de Santa Maria, 97105-900 Santa Maria, RS, Brazil

Received 17 May 2002; accepted 13 August 2002

Abstract A procedure for the determination of arsenic by batch hydride generation atomic absorption spectrometry (HG AAS) in commercial samples of injectable drugs, containing high concentrations of Sb(V), is described. The procedure is based on the complexing effect for Sb of citric, oxalic and acetic acids as reaction media. Aqua regia was used for sample digestion prior to As determination by HG AAS. The following experimental conditions for the determination of total As, as As(V), were evaluated: the acid medium and its concentration, sodium tetrahydroborate concentration, purge time, and influence of the different oxidation states of As. The effect of the delay time after mixing of sample and acid solution was also studied. Optimized conditions were: 10% (myv) citric acid, 1.5% (my v) sodium tetrahydroborate solution and 30 s for purge time. A delay time of 1 h was required after the digested sample had been mixed with citric acid, before As determination could be carried out. No interference on As(III) and As(V) signals was observed in the presence of up to 1 mg Sb(V). The tolerance limits for Ni(II), Cu(II) and Pb(II) were 1 mg, 100 mg and 100 mg, respectively. Recovery tests for As(III) and As(V) resulted in values between 97 and 101%. Characteristic mass and detection limit (3s), using the recommended conditions, were 0.52 and 0.8 ng, respectively, for total As. 2002 Elsevier Science B.V. All rights reserved.

Keywords: Arsenic determination; Hydride generation atomic absorption spectrometry; Sb(V) interference; Injectable drugs; Leishmaniasis

This paper was presented at the 7th Rio Symposium on Atomic Spectrometry, held in Florianopolis, Brazil, April 2002 and is published in the Special Issue of Spectrochimica Acta Part B, dedicated to that conference. *Corresponding author. Tel.: q55-16-261-5611; fax: q55-55-220-8054. E-mail address: flores@quimica.ufsm.br (E.M. de Moraes Flores). 0584-8547/02/$ - see front matter 2002 Elsevier Science B.V. All rights reserved. PII: S 0 5 8 4 - 8 5 4 7 0 2 . 0 0 1 7 8 - 7

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1. Introduction Leishmaniasis is an inflammatory disease, occurring in tropical regions, which affects 12 million people worldwide, and 1.52 million new cases of leishmaniasis are estimated to occur annually w1x. Leishmaniasis is endemic in some regions and it is generally treated with drugs based on high concentrations of pentavalent antimony. Formulations based on N-methylglucamine antimonate (meglumine antimonate) are the drugs of choice, and over 2.5 million of ampoules are commercialized per year in Brazil, where approximately 30 000 people are reported annually to suffer from this disease w2x. Drugs based on meglumine antimonate are sold in ampoules of 5 ml containing a nominal concentration of 85 000 mg ly1 of Sb(V). Determination of inorganic impurities is of critical importance in the quality control of drugs and medicines because several trace elements have toxic effects, and some of them could decrease the stability of active ingredients even at ultra-trace concentrations w3x. In this aspect, the determination of low levels of arsenic is important w4x, and its presence in pharmaceutical products can be a risk factor. Moreover, the presence of arsenic in parenteral pharmaceutical dosage forms, such as meglumine antimonate, is critical because in this form its bioavailability in systemic blood circulation is strongly increased in comparison to other forms of application. Trace element contamination of drugs and their intermediates may be introduced by several ways, such as through raw materials, reagents, solvents and equipment used for the synthesis. Many of these impurities can also be introduced after the synthesis, and could be indicators of non-adequate handling and storage or be used as fingerprints for the source of drug raw material w3x. All pharmacopoeias include a test for heavy metals, which is commonly carried out by sulfide precipitation in a weakly acidic medium. The test should also be suitable for the determination of arsenic w5,6x, but it could be give consistent results only with dangerously high As concentrations. In recent years, the analytical control of injectable drugs has become more important in view of improvements

in the production of pharmaceutical raw materials and recent, more restrictive demands regarding levels for toxic impurities w7x. Hydride generation atomic absorption spectrometry (HG AAS) has been used thoroughly for the quantitative determination of arsenic in a variety of samples. However, work related to the use of this technique for As determination in drugs is scarce. One of the limitations of HG AAS for arsenic determination in antimonial drugs is due to the strong interference from the very high Sb(V) concentration (;85 000 mg ly1 Sb). The interference of Sb on As is a well known problem in HG AAS w810x. Dittrich and Mandry w11x found that the signal suppression was 10% for an As:Sb ratio of 1:1000, using a quartz tube atomizer. Narasaki and Ikeda w12x reported that a 500-fold excess of antimony over arsenic could be tolerated without the use of masking agents. Petrick and Krivan w13x found that 1000 mg of Sb completely suppressed the absorbance signal of 50 ng of As(V). In view of its complexing effect, citric acid, as well as other hydroxy-acids, have been used for speciation studies for Sb(III) and Sb(V) determination by HG AAS w1417x. Moreover, citric acid is able to complex Sb species w17x and, in view of this, its use can be an alternative to minimize Sb interference on the determination of As by HG AAS. Based on the above, a procedure for determination of total arsenic in drugs used for leishmaniasis treatment containing very high levels of Sb(V) as meglumine antimonate is proposed. The effect of the reaction medium and other operating conditions for As determination were investigated. The extent of interference caused by transition elements was also studied. In this work the total As content was investigated to meet the specification of current pharmacopoeias. The optimized procedure was applied to the determination of As in commercial lots of meglumine antimonate. 2. Experimental 2.1. Instrumentation A Perkin-Elmer Model 3030 atomic absorption spectrometer with deuterium background correction, equipped with an arsenic hollow cathode

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Table 1 Figures of merit and analytical parameters for the proposed procedure for As determination in meglumine antimonate by HG AAS Wavelength (nm) Volume of 10% myv citric acid (ml) NaBH4 concentration (% m yv) Purge time (s) Integration time (s) Characteristic mass (ng) Limit of detection (absolute, 50 ml), 3s (ng) Limit of detection in sample, 3s (mg ly1) Typical correlation coefficient Linear range (ng) Contact time between 10%, myv citric acid and test solution (h) 193.7 (bandpass 0.7 nm) 10 1.5 30 20 0.51 0.80 0.42 0.9988 up to 125 1

lamp, operated at 18 mA, was used throughout. The equipment was coupled to a Perkin-Elmer MHS-10 batch hydride generation accessory. Argon (99.996%, White Martins, Brazil) was used as purge gas. The quartz cell was periodically cleaned by immersion in a 1.4 mol ly1 HNO3q 0.3 mol ly1 HF solution for 30 s. Integrated absorbance was used instead of peak height for measurement. The analytical parameters are summarized in Table 1. Results for commercial samples of meglumine antimonate were compared with those obtained by using inductively coupled plasma optical emission spectrometry with conventional nebulization (Perkin-Elmer Optima 2000 DV, using the conditions recommended by the manufacturer and the 188.979-nm wavelength). 2.2. Reagents All chemicals used were of analytical reagent grade obtained from Merck (Darmstadt, Germany). Concentrated nitric acid (65% m y v) and hydrochloric acid (37% m y v) were doubly distilled in a subboilling still (Berghof Model BSP 929, Germany). Milli-Q water (18.2 MV cm) was used to prepare all solutions. All glass apparatus were soaked in 2% (m y v) HNO3 and 4% (m y v) HCl for 48 h before using, and thoroughly washed with water before use. Stock standard solutions, 1000 mg ly1 of As(III) and As(V), were prepared from NaAsO2 and Na2HAsO47H2O salts. Working solutions of As(III) and As(V) were prepared daily by dilution with 6 mol ly1 HCl. Stock standard solutions, of 1000 mg ly1 Sb(V) were prepared by dissolving KSb(OH)6 in 6 mol ly1 HCl.

Working solutions of Sb(V) were prepared daily just before use by an appropriate dilution with 6 mol ly1 HCl. Sodium tetrahydroborate solutions were prepared in water and stabilized with 1% m y v NaOH. For interference studies, diluted solutions of Pb(II), Ni(II), Cu(II), and Bi(II) were prepared from 1000 mg ly1 stock solutions (Merck Titrisol Cat. No. 109969, 109989, 109987 and 109898, respectively). 2.3. Samples and sample treatment In this work four lots of commercial samples (A and B) were analyzed, which are commercialized in ampoules of 5 ml, containing approximately 85 000 mg ly1 of Sb(V) as meglumine antimonate in aqueous solution. Sample aliquots of 1 ml were digested with 8 ml of a 3q1 mixture of conc. HCl and conc. HNO3 in 50-ml glass tubes in a digestion block at 150 8C for 1 h. After cooling, sample digests were diluted with 6 mol ly1 HCl. 2.4. Procedure In order to optimize the conditions for the proposed procedure some media for hydride generation were studied for the determination of total arsenic as As(V). Solutions with 50 ng As(V), 50 ng As(V)q200 mg Sb(V), and commercial sample solutions with a nominal concentration of 85 000 mg ly1 Sb(V), spiked with 50 ng As(III), were investigated. Of the commercial samples, a volume of digested solution correspondent to a mass of 200 mg was placed into the reaction vessel. The

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arsenic spike, as As(III), in this solution was made before digestion. The influence of acetic, oxalic and citric acids (concentrations varying from 1 to 20%, m y v) on the As signal in the presence of Sb was investigated as medium for hydride generation. The influence of the contact time between digested sample solution and acid solution was also studied. The effect of the different oxidation states of Sb on the signal of As(III) and As(V) was investigated maintaining the amount of arsenic constant and increasing the quantity of antimony in the reaction vessel. 3. Results and discussion 3.1. Digestion of commercial samples of meglumine antimonate The determination of As in samples with an Sb(V) concentration of approximately 85 000 mg ly1 by HG AAS is not an easy task. In earlier experiments with solutions containing the equivalent of 200 mg Sb(V), the signal for As, as As(V), was almost completely suppressed. The presence of meglumine, a complexing agent, in commercial samples could bring additional drawbacks due to foam formation. A direct determination of As in these samples by HG AAS, hence, is not possible, and a dilution of the solutions could impair the limit of detection. An alternative could be to digest commercial samples with a suitable medium, avoiding the precipitation of Sb oxides y hydroxides and to complex Sb species to prevent generation of stibine. In addition, if a wet oxidation step is applied, foam formation is minimized and arsenic in the final solution is present in a defined oxidation state as As(V). Based on these aspects, a digestion procedure was evaluated to decompose the meglumine in commercial samples. The conditions studied were: kind and amount of acid, temperature, time of digestion, and the recommended procedure was to digest 1 ml of commercial sample with a 3q1 mixture of concentrated hydrochloric and nitric acids in 50-ml glass tubes heated at 150 8C for 1 h. After cooling, sample digests were diluted to 20 ml with 6 mol ly1 HCl, and aliquots of 50 ml were used for subsequent studies, but higher vol-

umes up to 200 ml could be used without changes. Spikes with As(III) or As(V) solutions were made for the final digested solutions (final concentration of 1 mg mly1). The volume of 50 ml of digested commercial solution corresponds to 200 mg Sb(V) according to the nominal value for the drugs. Recovery tests with the addition of arsenic solution as As(III) were performed to evaluate the proposed digestion procedure and the results were between 98.9 and 100.3%. 3.2. Influence of acid medium on the As signal Hydroxyacids have been used as reaction medium in the selective determination of antimony species by HG AAS previously w14,16x. Related to this, the effect of 10% m y v (concentration arbitrarily chosen for initial tests) solutions of acetic, oxalic and citric acids on the integrated absorbance for As was investigated. The contact time between solutions was varied from 1 to 24 h. Tests were made with 50 ng As(V), 50 ng As(V)q200 mg Sb(V) solutions and digested commercial sample solutions spiked with 50 ng of arsenic as As(III). Acid solutions (10 ml) were added using an automatic volumetric syringe and the total added volume of NaBH4 solution was 2.5 ml. Acetic acid has been suggested as medium for the selective determination of Sb(III), preventing stibine generation from Sb(V) w17x, and hence was investigated in this study to minimize possible interferences on arsenic determination. However, in acetic acid medium results were strongly influenced by the presence of 200 mg Sb(V) for analytical and As-spiked digested commercial sample solutions. In the latter, the arsenic signal was suppressed by more than 90%, independent of the contact time between 0 and 24 h. In the presence of oxalic acid the overall sensitivity was approximately 10% better, and the As signal was not influenced by the presence of Sb for analytical As(V)qSb(V) solutions, independent of the contact time studied. However, in the As-spiked digested commercial solution, the As signal was decreased, for all contact times studied, between 25% (immediately), and 5% (contact time of 1 h).

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Fig. 1. Influence of citric acid concentration on As determination by HG AAS (1.5% myv NaBH4 , 1 h delay time, 10 s purge time). Vertical bars are relative standard deviation (ns5).

The use of citric acid has been reported in the literature as medium for studies concerning speciation analysis w1416,18,19x. In this work, preliminary tests using citric acid were more promising than with the other acids. In the presence of 10% m y v citric acid no difference was observed between signals for As(V) in matrix-free solution and in the presence of 200 mg Sb(V) for all time intervals investigated. However, for the As-spiked digested commercial sample solution, the signal was 38% lower when the determination was made just after citric acid addition. No difference was observed for the three solutions for contact time of 1, 6 and 24 h, and the integrated absorbance signals were approximately 0.44 s. The relative standard deviation with all three contact times was always less than 5%. This might be related to the strong complexing effect of citric acid on Sb(V) reported in Refs. w16,18x, as confirmed by other authors w19x. A contact time of 1 h before the measurement was kept for all further investigations. First, the effect of citric acid concentration was investigated, using solutions with 1, 5, 10 and 20% m y v citric acid. As can be seen in Fig. 1 citric acid concentrations of 1 and 5% m y v are

not sufficient to reduce the interference of Sb(V) on As from As(V)q200 mg Sb(V) and As-spiked digested commercial solution. Moreover, the results with 1 and 5% m y v citric acid were erratic and not very reproducible. For citric acid concentrations of 10 and 20% m y v, all integrated absorbance signals were practically the same and a standard deviation approximately 3% was observed for all solutions. Thus, a concentration of 10% m y v citric acid was chosen for subsequent studies. These results could be explained by a reaction of citric acid with Sb(V), maybe by bonding through terminal carboxylic acid groups w18x that could complex inorganic Sb(V), minimizing the generation of stibine. The influence of sodium tetrahydroborate concentration was investigated for As determination by HG AAS using 10% m y v citric acid with a contact time of 1 h. A dependence of the As signal on the reductant concentration between 0.5 and 2% m y v was observed. For all three investigated solutions: 50 ng As(V), 50 ng As(V)q200 mg Sb(V) and 50 ng As-spiked digested commercial solution, signals increased approximately 50% in going from 0.5 to 1.5% m y v sodium tetrahydroborate, and decreased 31% with a concentration of

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Fig. 2. Interference of (a) Pb and (b) Bi, Cu and Ni on As signals by the proposed procedure (10% myv citric acid solution, 10 s purge time, 1 h delay time). Axis correspondent to interferent mass is in logarithmic scale. Vertical bars represent the relative standard deviation (ns4).

2% m y v NaBH4. No background signals were recorded, and peaks were completely integrated within 15 s (a total integration time of 20 s was used in this work). As the signals were very similar for the three analyzed solutions, it appears that there is no difference in the overall process (generation, transport and atomization) for the three solutions under these conditions. No difference in the As signal was observed either for purge times from 15 to 45 s for all solutions. The pH value of the solutions after the addition of citric acid was 1.6. At this pH stibine could be generated from Sb(V) if the generation step were only a pH controlled process. However, it has not been observed in previous work w20x that the generation of SbH3 could be dominated by the complexation of Sb(V) with citric acid, and the effect of pH would be limited to a minor role. Similar results have also been observed previously w16,21x. The nominal oxidation state of antimony in meglumine antimonate is Sb(V). Moreover, under the oxidizing conditions under which the digestion step was performed, probably any Sb(III) species has been converted to Sb(V). The influence of an increasing mass of Sb(V) on both, As(III) and

As(V) signals was investigated. A difference of approximately 55% between the overall sensitivity for As(III) and As(V) was observed, and the integrated absorbance signals for 50 ng of the two species were 0.75 and 0.43 s, respectively. However, practically no effect (-9%) was observed up to 1 mg Sb(V) added in reaction vessel for both species. This fact is one more indicative for the strong complexation of Sb(V) with citric acid in the liquid phase. These results were surprising, and to the knowledge of these authors there is no report in literature concerning the determination of As in the presence of such high levels of Sb. 3.3. Influence of interferents Some commercial lots of meglumine antimonate were previously analyzed and results have shown the presence of some contaminants such as Bi, Cu, Ni and Pb. Thus, an interference study was carried out to make sure that these elements have no influence on the determination of As(V) by the proposed batch HG AAS procedure. It can be seen in Fig. 2 that up to 1000 mg of Ni have no influence on the signals for As(V). For Cu and Bi a small decrease was observed with masses higher

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than 10 and 100 mg, respectively. Lead caused a more pronounced decrease in the As(V) signal at masses higher than 100 mg Pb. The conditions for the proposed procedure, especially for Ni, a wellknown interferent on As determination by HG AAS, were considered suitable. The tolerance limits w22x for Sb(V), Ni(II), Cu(II) and Pb(II) were 1 mg, 1 mg, 100 and 100 mg, respectively. In the proposed procedure arsenic was determined as As(V), and no prereduction step was attempted in view of the fact that reduction of As(V) to As(III) is generally more difficult than that of Sb(V) to Sb(III), and the generated Sb(III) could interfere more strongly with the As determination. 3.4. Figures of merit The developed procedure was suitable for arsenic determination by HG AAS in the presence of high concentrations of pentavalent Sb in samples of meglumine antimonate. Table 1 presents the recommended conditions for the proposed procedure. The linear range extended up to 125 ng As(V), and a relative standard deviation of 2.8% was obtained. Recovery tests in commercial samples were made by adding As(III) analytical solutions to samples (corresponding to 5 and 25 ng As in the final aliquots) before the digestion step. Arsenic was determined by the proposed procedure and recoveries were between 97 and 101% (ns 5). With the proposed procedure the determination of As in a high concentration of Sb(V), up to 1 mg Sb(V), was possible without decrease of analytical signals. This value is high when compared to others procedures described in the literature. Four lots of commercial meglumine antimonate solutions were analyzed using the recommended conditions. Results for As concentration were between less than 0.48 and 1.27"0.08 mg ly1 for manufacturer A; the lots from manufacturer B were contaminated with As between 9.6"0.7 and 78.9"2.7 mg ly1. These results were very similar to those obtained by inductively coupled plasma optical emission spectrometry after sample dilution with water, with an agreement between 98 and 102.5%. As the meglumine antimonate is administrated in an injectable form, the presence of As is a high risk factor for people under treatment. In

view of this, arsenic control must be made routinely to ascertain the quality of drugs used for leishmaniasis treatment. Acknowledgments Authors thank to FAPERGS, CNPq, Farmaco Brasileira, ANVISA (to Dr Gonzalo Vecina peia Neto) for supporting this study and to Dr Joao Alfredo Medeiros (UFRJ) for comments and suggestions. References
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