Speciation of Arsenic Animal Feed Additives by Microbore High-performance Liquid Chromatography with Inductively Coupled Plasma Mass Spectrometry Spiros A. Pergantis*†, Edward M. Heithmar and Thomas A. Hinners US Environmental Protection Agency, National Exposure Research Laboratory, Environmental Sciences Division, P.O. Box 93478, Las Vegas, NV 89193-3478, USA Phenylarsonic compounds have been used as poultry and swine feed additives for the purpose of growth promotion and disease prevention.Owing to the lack of suitable analytical methods, however, knowledge of their metabolism, environmental fate and impact remains incomplete. In order to compensate for this, analytical procedures were developed that allow the speciation of arsenic animal feed additives by using microbore high-performance liquid chromatography (mHPLC) coupled on-line with ICP-MS. More specifically, reversed-phase (RP) chromatographic methods were optimised to achieve the separation of various phenylarsonic acids from each other and from the more toxic inorganic arsenic compounds.This mode of chromatography, however, exhibits limitations, especially in the presence of naturally occurring organoarsenic compounds. The application of RP ion-pairing chromatography eliminates such shortcomings by minimising the co-elution of arsenic species. In general, the mHPLC–ICP-MS methods developed in this study provide high selectivity, extremely good sensitivity, low limits of detection (low-ppb or sub-pg amounts of As), require small sample volumes ( < 1 ml), minimise waste and operate most efficiently under low mobile-phase flow rates (15–40 ml min21), which are compatible for use with other types of mass spectrometers, e.g., electrospray.Reference materials containing naturally occurring arsenic compounds were spiked with phenylarsonic compounds and then analysed by using the procedures developed in this study. Keywords: Arsenic; speciation; inductively coupled plasma mass spectrometry; microbore high-performance liquid chromatography; reference materials; animal feed additives A number of phenylarsonic compounds have been shown to control cecal coccidiosis in poultry, and also act as growth promoters, providing improved feed conversion, better feathering and increased egg production and pigmentation. 4-Hydroxy- 3-nitrophenylarsonic acid (roxarsone), p-arsanilic acid (p-ASA), 4-nitrophenylarsonic acid (4-NPAA), p-ureidophenylarsonic acid (p-UPAA) and benzenearsonic acid have all been used for such purposes and, with the exception of p- UPAA, are still in use today. Variation in the substituents on the aromatic ring results in differences in the growth-promoting and disease-controlling effects of the compounds.Thus, roxarsone and p-ASA are approved as animal feed additives for both poultry and swine, whereas 4-NPAA and p-UPAA are approved only for controlling blackhead disease in turkeys.1–3 Even though studies pertaining to the metabolism,4 toxicity5,6 and excretion of these compounds have been conducted, their physiological role, environmental fate and impact are still not well understood.In order to improve our understanding of these processes, it is required that analytical methods be developed that will allow for the speciation of phenylarsonic compounds and their metabolites. So far, only a limited number of methods have been developed and used for the determination of this class of compounds. Most of these methods are for the targeted analysis of arsenic animal feed additive compounds, and are therefore not necessarily suitable for the detection of potential metabolites.For example, gas chromatography with flame ionization detection, which has been used for the determination of p-UPAA and p-ASA, is not suitable for the determination of roxarsone and is also interference prone.7 A spectrophotometric method for the determination of p-UPAA in the animal feed additive Carbasone has also been reported.8 This method involves a coupling reaction with N-1-naphthylethylenediamine.The colored product that forms is extracted with butanol and subsequently measured photometrically; again, interferences can cause difficulties with this determination. Thin-layer chromatography has been used for the separation and identification of roxarsone, p-ASA, 4-NPAA and p-UPAA; chromogenic reagents were used for detection.9 HPLC has also been used to separate some arylarsenicals, but not specifically those used as animal-feed additives.10–12 Also, an LC method has been developed for the detection and quantification of roxarsone in poultry feed.The drug is extracted by means of a phosphate buffer and determined using solid-phase extraction in combination with reversed-phase (RP) LC with UV detection.13 HPLC coupled on-line with ICP-MS has been used for the determination of roxarsone14,15 and other arsenic animal feed additive compounds.14 More recently, a number of MS methods have been tested as a means to characterize structurally arsenic animal feed additives.16–18 In this paper we report on the development of microbore HPLC (mHPLC)–ICP-MS methods, which allow the speciation of arsenic animal feed additives present in environmental and biological samples.Of primary interest was the development of methods that allow us to differentiate between arsenic animal feed additives and naturally occurring arsenic compounds, and also provide information concerning the presence of potential metabolites.Experimental Instrumentation A VG PlasmaQuad II STE ICP-MS system (VG Elemental, Winsford, Cheshire, UK) was used for elemental detection. The quadrupole mass analyser was operated in the single-ion monitoring mode (m/z 75) for the determination of arsenic. The microscale flow injection (mFI) and mHPLC set-ups have been described in detail in previous work.14 A 0.5 or 1 ml internal loop injector (Valco Instruments, Houston, TX, USA) was used for both mFI and mHPLC.A Model 100DM syringe pump (Isco, Lincoln, NE, USA) was used to deliver microflows between 15 and 80 ml min21. † Present address: Department of Chemistry, Birkbeck College, University of London, Gordon House, 29 Gordon Square, London, UK WC1H 0PP. Analyst, October 1997, Vol. 122 (1063–1068) 1063Chemicals and Reagents Inorganic arsenic standard solutions used for the mFI–ICP-MS experiments were prepared by diluting 1000 ppm standards (Inorganic Ventures, Lakewood, NJ, USA) with de-ionized water (18 MW) acidified to 0.05% v/v with doubly distilled nitric acid (Seastar, Sidney, BC, Canada).The mFI carrier was also 0.05% nitric acid. Although 1% nitric acid is more commonly used in ICP-MS, a lower acid concentration was used in this study to avoid possible corrosion of the stainlesssteel syringe pump. No problems with memory effects were caused by the use of 0.05% nitric acid carrier.To minimise contributions to the blanks from leached pump materials, the small-diameter steel tubing and valves on the pump were replaced with Teflon hardware. The following certified reference materials were analysed for their arsenic species by using mHPLC–ICP-MS: Trace Elements in Water [National Institute of Standards and Technology (NIST) Standard Reference Material (SRM) 1643a]; Trace Metals in Drinking Water (High-Purity Standards, Charleston, SC, USA); Toxic Metals in Urine (NIST SRM 2670n); and San Joaquin Soil (NIST SRM 2709).Soil extractions were performed by using 0.3 m H3PO4 and sonicating for 60 min. All solutions were filtered with 0.45 mm syringe filters (nylon Acrodisc, Gelman Sciences, Ann Arbor, MI, USA) and analysed without further dilution. The separation of arsenic compounds by using RP-mHPLC was accomplished using a mobile phase consisting of 0.1% trifluoroacetic acid (Sigma, St.Louis, MO, USA) and 5–10% v/v methanol in water. For the RP ion-pairing (IP) mHPLC separations, tetrabutylammonium hydroxide (Alfa Products, Danvers, MA, USA) was used as the ion-pairing reagent at concentrations ranging from 1 to 5 mm. Malonic acid (Sigma) was used to adjust the mobile phase pH. The analytical mHPLC column, used for both RP and RP-IP chromatography, was a 150 mm 3 1 mm id stainless-steel column packed with Spherisorb 3 mm C18 material (Isco).HPLC mobile phases were filtered and de-gassed using a 35 mm all-glass filter holder (Millipore, Bedford, MA, USA), fitted with a 0.45 mm hydrophilic nylon filter (Cuno, Meriden, CT, USA) for aqueous solvents and a 0.5 mm PTFE filter (Cole-Parmer, Chicago, IL, USA) for organic solvents. Chromatographic standards were prepared from 1000 mg l21 As aqueous solutions of the following arsenic compounds: parsanilic acid (p-ASA) (Eastman Organic Chemicals, Rochester, NY, USA), 3-nitro-4-hydroxyphenylarsonic acid (roxarsone) (ICN Biochemicals, Cleveland, OH, USA), 4-nitrophenylarsonic acid (4-NPAA) (Aldrich, Milwaukee, WI, USA), dimethylarsinic acid (DMA) (Sigma), 4-hydroxyphenylarsonic acid (4-OH) (Eastman Kodak), disodium methylarsonate (MMA) (Chemical Service, West Chester, PA, USA), sodium meta-arsenite (Aite) (Sigma), and sodium arsenate (Aate) (Sigma).Procedures Time resolved acquisition (TRA) software provided by VG was used to acquire data during mHPLC–ICP-MS experiments.Data files of response versus time were exported as ASCII files and analysed using commercial spreadsheet software. The mFI– and mHPLC–ICP-MS response was optimised for the detection of arsenic at m/z 75 using carrier flow rates of 80 and 40 ml min21, respectively. The optimisation was carried out by employing a 60 ml loop fitted on a ceramic injector (SVI- 6U7) (Analyticon Instruments, Springfield, NJ, USA). The large volume loop provided quasi-continuous sample introduction, which lasted long enough for signal optimisation to be achieved.For the HPLC optimisation a 60 ml loop was connected post-column; this provided a signal equivalent to that obtained in the continuous flow mode. All instrument parameters were varied iteratively to reach the apparent optimum response. Results and Discussion Separation of Arsenic Animal Feed Additives Using RP- mHPLC–ICP-MS RP-mHPLC methods were developed and used for the separation of four phenylarsonic acids and two inorganic arsenic compounds.A mobile phase consisting of 0.1% trifluoroacetic acid (TFA) in water, with a variable percentage (5–15% v/v) of methanol, was used for their separation. The mHPLC stationary phase consisted of silica based C18 material (3 mm particle diameter, 12% carbon loading). The chromatograms obtained are presented in Fig. 1. Present in the injected sample was 4-hydroxyphenylarsonic acid (4-OH). Even though this compound has not been used as an animal feed additive, it may prove to be a decomposition product or metabolite of roxarsone or may adequately serve as a chromatographic internal standard.As mentioned previously, the RP-mHPLC method developed allows for the separation of inorganic arsenic species from various of the phenylarsonic acids. This separation is of particular importance, primarily because of the relatively high toxicity of the inorganic arsenic species. Thus, the RP chromatographic method can be used to monitor arsenic animal feed additives for the presence of inorganic arsenic impurities, and also to detect possible degradation of the phenylarsonic acids to their respective inorganic forms.As shown in Fig. 1, the extensive retention of the NO2-containing arsenicals (roxarsone and 4-NPAA) by the LC stationary phase results in excessive Fig. 1 RP-mHPLC–ICP-MS traces for six arsenic compounds. The mobile phase consisted of water (0.1% TFA) and 5–15% methanol, applied at a flow rate of 40 ml min21. 1064 Analyst, October 1997, Vol. 122peak tailing. A larger amount of methanol in the mobile phase reduces peak tailing significantly, but also provides poorer overall resolution. In addition, the methanol content affects the sensitivity of the ICP-MS detector. Signal enhancement for arsenic in the presence of organic solvents has been observed and reported extensively by others.19,20 This occurrence has major implications, especially when using gradient-elution methods which make use of variable amounts of organic solvents in conjunction with HPLC–ICP-MS.It has been proposed that the presence of organic solvents in the plasma alters the energetics of the ICP. Even when the introduction of organic solvent into the plasma is discontinued, a relatively long period of time is required before the ICP-MS sensitivity returns to its pre-solvent level. To explore further the possible use of RP chromatography for the speciation of phenylarsonic acids in environmental samples, we investigated the effects of naturally occurring arsenicals on the RP chromatograms.The RP-mHPLC separation of various phenylarsonic acids (arsenic animal feed additives) from naturally occurring arsenic compounds is presented in Fig. 2. These chromatograms were obtained with no methanol present in the mobile phase. Less efficient separations occurred in the presence of even small amounts of methanol ( < 0.5%). The mobile phase flow rate also had a pronounced effect on separation efficiency. The best separation was obtained at the lowest-tested flow rate (15 ml min21). The main drawback, however, was the incomplete separation of DMA from p-ASA. In addition, excessive peak broadening occurred in the low flow rate regime ( < 30 ml min21).Flow rates > 55 ml min21 were not considered, mainly because of the resulting high back-pressure ( > 3000 psi), which could potentially damage the mHPLC column.It should also be mentioned that when the mobile phase does not contain any methanol, the phenylarsonic acids roxarsone and 4-NPAA do not elute from the HPLC column. Thus, a preliminary chromatographic run using a mobile phase containing 5–15% methanol must be conducted in order to investigate the presence of the two nitro-containing arsenicals (roxarsone and 4-NPAA). Separation of Arsenic Animal Feed Additives Using RP-IP- mHPLC–ICP-MS Because RP chromatography provides poor separation of the arsenic animal feed additives from some of the naturally occurring arsenic compounds (Aite, Aate, MMA and DMA), alternative chromatographic modes of separation were investigated and further optimised to provide efficient separations.To accomplish this, RP-IP-mHPLC was further investigated. To assist in reaching the optimum conditions required for such separations, a plot of the apparent charge (Qapp) of each of the arsenic compounds as a function of mobile phase pH was constructed (Fig. 3). This plot provides a qualitative estimate of the extent of interaction of the anion-pairing reagent with the arsenic-containing analyte under various pH conditions. Qapp for arsenate was calculated as follows: Qapp AsO HAsO H AsO AsO HAsO H AsO H AsO = - + + + + + - - - - - - 3 2 4 3 4 2 2 4 4 3 4 2 2 4 3 4 [ ] [ ] [ ] [ ] [ ] [ ] [ ] The Qapp values for the other arsenic compounds were derived similarly.The pKa values21 used for these calculations were as follows: pKa[Aite] = 9.2, pKa[Aate] = 2.2, 6.98, 11.5, pKa[DMA] = 1.3,22 6.3, pKa[MMA] = 3.41, 8.18, pKa[p- ASA] = 2, 4.02, 8.92. The chromatogram presented in Fig. 4, obtained under RPIP- mHPLC conditions, exhibits sufficient separation of p-ASA Fig. 2 RP-mHPLC–ICP-MS traces for p-ASA (D) and 4-OH (E) in the presence of naturally occurring arsenic compounds: arsenite and arsenate (A), methylarsonic acid (B) and dimethylarsinic acid (C).The mobile phase consisted of water (0.1% TFA). The flow rates varied between 15 and 55 ml min21. Fig. 3 Apparent charge of arsenic compounds as a function of solution pH. Arrows indicate normal operating range of pH for reversed-phase anionpairing chromatography of arsenic acids. Fig. 4 RP-IP-mHPLC–ICP-MS traces for five arsenic compounds. The mobile phase consisted of 1 mm tetrabutylammonium hydroxide and 0.5% methanol in de-ionised water (pH 5.28), applied at a flow rate of 40 ml min21.Analyst, October 1997, Vol. 122 1065from four other arsenic compounds which have been found to occur naturally in various environmental samples. The elution order of these compounds at pH 5.3 is in agreement with that predicted by the Qapp versus pH plot (Fig. 3). A compound with high Qapp is expected to interact to a greater extent with the tetrabutylammonium ion-pairing reagent. Stronger interaction results in longer retention times. However, it should be noted that factors other than pH significantly influence the chromatographic behavior of the arsenic compounds.The most important of these factors include the ion-pairing reagent concentration and the methanol content of the mobile phase. Separation of p-ASA from Arsenic Compounds Present in Reference Materials Initially the effects of large amounts of NaCl on the chromatographic separation of arsenic compounds were investigated. The resulting chromatograms from these experiments are presented in Fig. 5. It was observed that the presence of 0.1% NaCl in the sample had a significant effect on the chromatographic separations (retention times, peak shapes), particularly when a low ion-pairing concentration (1–2 mm TBAH) was used. In order to minimise this problem, which is especially pronounced when speciating arsenic in biological materials such as urine, 5 mm TBAH was used. Also, the mobile phase pH was adjusted to allow for the separation of chloride from the other arsenic compounds, thus eliminating potential interferences caused by ArCl+.Freeze-dried urine (NIST SRM 2670) containing ‘normal levels’ of arsenic (as indicated by NIST) was analysed for its arsenic content. The freeze-dried urine sample was reconstituted with de-ionized water and then spiked with p-ASA and 4-OH. The resulting chromatogram is shown in Fig. 6. Resolution between DMA and MMA was sacrificed by appropriately adjusting the mobile phase pH in order to achieve improved separation between the Cl2 and arsenate species present.The determination of arsenate is of considerable importance mainly because its toxicity is substantially higher than that of MMA or DMA. Good separation of 4-OH, used as an internal standard in this case, was also accomplished. It should be noted that the original (non-spiked) urine SRM did not contain any measurable amounts of p-ASA or 4-OH. It should also be noted that the RP anion-pairing chromatography used in this study is not capable of differentiating between arsenobetaine and arsenite, as these two compounds co-elute.Also, there exists the possibility that other non-retained arsenic species may co-elute in this front-end peak. Arsenobetaine is normally present in human urine following the consumption of seafood.23 Previous investigations using this particular reference material have shown that indeed the first peak is the result of arsenite and arsenobetaine.18,24 Solutions referred to as Trace Elements in Water (NIST SRM 1643a) and Trace Metals in Drinking Water (High-Purity Standards) were also spiked with p-ASA and 4-OH, and subsequently analysed for their content of arsenic species (Fig. 7).The spiked arsenicals were separated from arsenite and arsenate, which were originally present in the water reference materials. Furthermore, San Joaquin Soil (NIST SRM 2709) was extracted using 0.3 m H3PO4 and then spiked with p-ASA and 4-OH.The resulting chromatogram is presented in Fig. 8. The soil extract, prior to spiking, was found to contain only arsenate as the single arsenic compound present. The limits of detection for each arsenic compound, achieved under various modes of chromatography, are summarised in Table 1. Because of the extremely high sensitivity of the ICPMS detector, the chromatographic methods described are eminently suitable for the development of methodologies for the determination of arsenic animal feed additives in real samples.These methods are particularly suited for the analysis of samples containing limited amounts of analyte. mFI–ICP-MS of Arsenic Animal Feed Additives In previous work, a mFI–ICP-MS technique was developed and applied to the determination of total arsenic.14 Of interest in this study was to investigate further any sensitivity variations observed when analysing arsenic animal feed additives by using mFI–ICP-MS. A number of reports have claimed sensitivity variations for other organoarsenic compounds determined using continuous-flow ICP-MS, particularly for arsenicals present in marine organisms.20 So far, no sensitivity data have been reported regarding arsenic animal feed additives.Fig. 9 shows the peaks obtained for the four phenylarsenical compounds discussed in this paper. Each peak corresponds to an Fig. 5 Effect of salt and ion-pair reagent concentrations on the separation of four arsenic compounds.Fig. 6 RP-IP-mHPLC–ICP-MS trace for urine (NIST SRM 2670n) spiked with p-ASA and 4-OH. The mobile phase consisted of 0.5% v/v methanol and 5 mm TBAH at pH 5.8, applied at a flow rate of 40 ml min21. 1066 Analyst, October 1997, Vol. 122injection of 80 pg of As (1 ml injections of a phenylarsenical solution containing 80 ppb of As). The fact that no sensitivity variations were observed permits the use of mFI–ICP-MS for the quantification of arsenic animal feed additives regardless of which phenylarsonic species are present in the sample and without their prior conversion into a common species by employing some form of digestion.Conclusions We have demonstrated the development and successful application of a variety of chromatographic methods for the identification of arsenic animal feed additives. More specifically, RP- mHPLC was used for the separation of arsenic animal feed additives from inorganic arsenic species. This mode of chromatography, however, did not allow the complete separation of arsenic animal-feed additives from naturally occurring organoarsenic compounds.RP–IP-mHPLC was used successfully for this purpose. Furthermore, the use of mHPLC offers the advantage of operating at very low flow rates (15–40 ml min21), thus allowing for minimisation of waste and the use of small sample volumes, and also offers flow rate compatibility with other mass spectrometric detectors such as electrospray and continuous-flow fast atom bombardment mass spectrometry.The latter feature may prove to be a great benefit for the structural identification of metabolites or decomposition products for which synthetic standards are not yet available. The use of the ICP-MS detector offers additional benefits, such as high selectivity and extremely good sensitivity, for the speciation of trace levels of arsenic in samples of environmental and biological origin. The US Environmental Protection Agency (EPA), through its Office of Research and Development (ORD), funded the research described here.This paper has been subjected to the Agency’s peer review and has been approved as an EPA publication. The US Government has a non-exclusive, royaltyfree license in and to any copyright covering this paper. Mention of trade names or commercial products does not constitute Fig. 7 RP-IP-mHPLC–ICP-MS traces for A, water (NIST SRM 1643a) spiked with p-ASA and 4-OH and B, water (High-Purity Standards, Drinking Water) spiked with p-ASA and 4-OH.The mobile phase consisted of 0.5% v/v methanol and 5 mm TBAH at pH 5.8, applied at a flow rate of 40 ml min21. Fig. 8 RP-IP-mHPLC–ICP-MS trace for soil extract (NIST SRM 2709 San Joaquin Soil) spiked with p-ASA and 4-OH. The mobile phase consisted of 0.5% v/v methanol and 5 mm TBAH at pH 5.8, applied at a flow rate of 40 ml min21. Table 1 Limits of detection (defined as three times the standard deviation of the background) for arsenic compounds obtained under RP- and RP-IP-m- HPLC–ICP-MS conditions RP-IPRP- mHPLC– mHPLC– ICP-MS ICP-MS pg ml21 pg ml21 As pg As As pg As Arsenite (Aite) 0.10 0.10 0.6 0.6 Arsenate (Aate) 0.10 0.10 0.4 0.4 p-Arsanilic acid (p-ASA) 0.10 0.10 0.9 0.9 4-Hydroxyphenylarsonic acid (4-OH) 0.10 0.10 0.8 0.8 3-Nitro-4-hydroxyphenylarsonic acid (roxarsone) 0.12 0.12 n.d.* n.d. 4-Nitrophenylarsonic acid (4-NPAA) 0.26 0.26 n.d. n.d. * n.d.: not determined because the compounds do not elute from column under the specified conditions.Fig. 9 mFI–ICP-MS of arsenic animal feed additives. Each peak represents the injection of 1 ml of a solution of 80 pg ml21 As. Analyst, October 1997, Vol. 122 1067endorsement or recommendation for use. This work was performed while S.A.P. held a National Research Council/ CRD-LV Research Associateship. References 1 Gilbert, F. R., Wells, G. A. H., and Gunning, R. F., Vet. Rec., 1981, 109, 158. 2 Drugs Directorate, Health and Welfare Canada, Ottawa, Canada, 1981. 3 Compendium of Medicating Ingredients Brochures, Agriculture Canada, Ottawa, 5th edn., 1984. 4 Aschbacher, P. W., and Feil, V. J., J. Agric. Food Chem., 1991, 39, 146. 5 Edmonds, M. S., and Baker, D. H., J. Anim. Sci., 1986, 63, 553. 6 Rice, D. A., McMurray, C. H., McCracken, R. M., Bryson, D. G., and Maybin, R., Vet. Rec., 1980, 106, 312. 7 Weston, R. E., Wheals, B. B., and Kensett, M. J., Analyst, 1971, 96, 601. 8 Hoodless, R. A., and Tarrant, K. R., Analyst, 1973, 98, 502. 9 Morrison, J. L., J. Agric. Food Chem., 1968, 16, 704. 10 Maruo, M., Hirayama, N., Wada, H., and Kuwamoto, T., J. Chromatogr., 1989, 466, 379. 11 Hirayama, N., and Kuwamoto, T., J. Chromatogr., 1988, 457, 415. 12 Dodd, M., PhD Thesis, University of British Columbia, Vancouver, 1988. 13 Sapp, R. E., and Davidson, S., J. AOAC Int., 1993, 76, 956. 14 Pergantis, S. A., Heithmar, E. M., and Hinners, T. A., Anal. Chem., 1995, 67, 4530. 15 Dean, J. R., Ebdon, L., Foulkes, M. E., Crews, H. M., and Massey, R. C., J. Anal. At. Spectrom., 1994, 9, 615. 16 Pergantis, S. A., Cullen, W. R., Chow, D. T., and Eigendorf, G. K., J. Chromatogr. A, 1997, 764, 211. 17 Pergantis, S. A., Cullen, W. R., and Eigendorf, G. K., Biol. Mass Spectrom., 1994, 23, 749. 18 Pergantis, S. A., Winnik, W., and Betwoski, D., J. Anal. At. Spectrom., 1997, 12, 531. 19 Allain, P., Jaunault, L., Mauras, Y., Mermet, J.-M., and Delaporte, T., Anal.Chem., 1991, 63, 1497. 20 Larsen, E. H., and St�urup, S., J. Anal. At. Spectrom., 1994, 9, 1099. 21 Dean, J. A., in Lange’s Handbook of Chemistry, McGraw-Hill, New York, 13th edn., 1985. 22 Hansen, S. H., Larsen, E. H., Pritzl, G., and Cornett, C., J. Anal. At. Spectrom., 1992, 7, 629. 23 Le, X. C., Cullen, W. R., and Reimer, K. J., Clin. Chem., 1994, 40, 617. 24 Pergantis, S. A., Momplaisir, G.-M., Heithmar, E. M., and Hinners, T. A., in Proceedings of the 44th ASMS Conference on Mass Spectrometry and Allied Topics, Portland, Oregon, May 12–16, 1996, American Society for Mass Spectrometry, East Lansing, MI, USA, p. 21. Paper 7/02691I Received April 21, 1997 Accepted July 14, 1997 1068 Analyst, October 1997, Vol. 122 Speciation of Arsenic Animal Feed Additives by Microbore High-performance Liquid Chromatography with Inductively Coupled Plasma Mass Spectrometry Spiros A. Pergantis*†, Edward M. Heithmar and Thomas A. Hinners US Environmental Protection Agency, National Exposure Research Laboratory, Environmental Sciences Division, P.O.Box 93478, Las Vegas, NV 89193-3478, USA Phenylarsonic compounds have been used as poultry and swine feed additives for the purpose of growth promotion and disease prevention. Owing to the lack of suitable analytical methods, however, knowledge of their metabolism, environmental fate and impact remains incomplete. In order to compensate for this, analytical procedures were developed that allow the speciation of arsenic animal feed additives by using microbore high-performance liquid chromatography (mHPLC) coupled on-line with ICP-MS.More specifically, reversed-phase (RP) chromatographic methods were optimised to achieve the separation of various phenylarsonic acids from each other and from the more toxic inorganic arsenic compounds. This mode of chromatography, however, exhibits limitations, especially in the presence of naturally occurring organoarsenic compounds.The application of RP ion-pairing chromatography eliminates such shortcomings by minimising the co-elution of arsenic species. In general, the mHPLC–ICP-MS methods developed in this study provide high selectivity, extremely good sensitivity, low limits of detection (low-ppb or sub-pg amounts of As), require small sample volumes ( < 1 ml), minimise waste and operate most efficiently under low mobile-phase flow rates (15–40 ml min21), which are compatible for use with other types of mass spectrometers, e.g., electrospray.Reference materials containing naturally occurring arsenic compounds were spiked with phenylarsonic compounds and then analysed by using the procedures developed in this study. Keywords: Arsenic; speciation; inductively coupled plasma mass spectrometry; microbore high-performance liquid chromatography; reference materials; animal feed additives A number of phenylarsonic compounds have been shown to control cecal coccidiosis in poultry, and also act as growth promoters, providing improved feed conversion, better feathering and increased egg production and pigmentation. 4-Hydroxy- 3-nitrophenylarsonic acid (roxarsone), p-arsanilic acid (p-ASA), 4-nitrophenylarsonic acid (4-NPAA), p-ureidophenylarsonic acid (p-UPAA) and benzenearsonic acid have all been used for such purposes and, with the exception of p- UPAA, are still in use today. Variation in the substituents on the aromatic ring results in differences in the growth-promoting and disease-controlling effects of the compounds. Thus, roxarsone and p-ASA are approved as animal feed additives for both poultry and swine, whereas 4-NPAA and p-UPAA are approved only for controlling blackhead disease in turkeys.1–3 Even though studies pertaining to the metabolism,4 toxicity5,6 and excretion of these compounds have been conducted, their physiological role, environmental fate and impact are still not well understood. In order to improve our understanding of these processes, it is required that analytical methods be developed that will allow for the speciation of phenylarsonic compounds and their metabolites.So far, only a limited number of methods have been developed and used for the determination of this class of compounds. Most of these methods are for the targeted analysis of arsenic animal feed additive compounds, and are therefore not necessarily suitable for the detection of potential metabolites.For example, gas chromatography with flame ionization detection, which has been used for the determination of p-UPAA and p-ASA, is not suitable for the determination of roxarsone and is also interference prone.7 A spectrophotometric method for the determination of p-UPAA in the animal feed additive Carbasone has also been reported.8 This method involves a coupling reaction with N-1-naphthylethylenediamine. The colored product that forms is extracted with butanol and subsequently measured photometrically; again, interferences can cause difficulties with this determination.Thin-layer chromatography has been used for the separation and identification of roxarsone, p-ASA, 4-NPAA and p-UPAA; chromogenic reagents were used for detection.9 HPLC has also been used to separate some arylarsenicals, but not specifically those used as animal-feed additives.10–12 Also, an LC method has been developed for the detection and quantification of roxarsone in poultry feed.The drug is extracted by means of a phosphate buffer and determined using solid-phase extraction in combination with reversed-phase (RP) LC with UV detection.13 HPLC coupled on-line with ICP-MS has been used for the determination of roxarsone14,15 and other arsenic animal feed additive compounds.14 More recently, a number of MS methods have been tested as a means to characterize structurally arsenic animal feed additives.16–18 In this paper we report on the development of microbore HPLC (mHPLC)–ICP-MS methods, which allow the speciation of arsenic animal feed additives present in environmental and biological samples.Of primary interest was the development of methods that allow us to differentiate between arsenic animal feed additives and naturally occurring arsenic compounds, and also provide information concerning the presence of potential metabolites. Experimental Instrumentation A VG PlasmaQuad II STE ICP-MS system (VG Elemental, Winsford, Cheshire, UK) was used for elemental detection.The quadrupole mass analyser was operated in the single-ion monitoring mode (m/z 75) for the determination of arsenic. The microscale flow injection (mFI) and mHPLC set-ups have been described in detail in previo work.14 A 0.5 or 1 ml internal loop injector (Valco Instruments, Houston, TX, USA) was used for both mFI and mHPLC. A Model 100DM syringe pump (Isco, Lincoln, NE, USA) was used to deliver microflows between 15 and 80 ml min21.† Present address: Department of Chemistry, Birkbeck College, University of London, Gordon House, 29 Gordon Square, London, UK WC1H 0PP. Analyst, October 1997, Vol. 122 (1063–1068) 1063Chemicals and Reagents Inorganic arsenic standard solutions used for the mFI–ICP-MS experiments were prepared by diluting 1000 ppm standards (Inorganic Ventures, Lakewood, NJ, USA) with de-ionized water (18 MW) acidified to 0.05% v/v with doubly distilled nitric acid (Seastar, Sidney, BC, Canada).The mFI carrier was also 0.05% nitric acid. Although 1% nitric acid is more commonly used in ICP-MS, a lower acid concentration was used in this study to avoid possible corrosion of the stainlesssteel syringe pump. No problems with memory effects were caused by the use of 0.05% nitric acid carrier. To minimise contributions to the blanks from leached pump materials, the small-diameter steel tubing and valves on the pump were replaced with Teflon hardware.The following certified reference materials were analysed for their arsenic species by using mHPLC–ICP-MS: Trace Elements in Water [National Institute of Standards and Technology (NIST) Standard Reference Material (SRM) 1643a]; Trace Metals in Drinking Water (High-Purity Standards, Charleston, SC, USA); Toxic Metals in Urine (NIST SRM 2670n); and San Joaquin Soil (NIST SRM 2709). Soil extractions were performed by using 0.3 m H3PO4 and sonicating for 60 min.All solutions were filtered with 0.45 mm syringe filters (nylon Acrodisc, Gelman Sciences, Ann Arbor, MI, USA) and analysed without further dilution. The separation of arsenic compounds by using RP-mHPLC was accomplished using a mobile phase consisting of 0.1% trifluoroacetic acid (Sigma, St. Louis, MO, USA) and 5–10% v/v methanol in water. For the RP ion-pairing (IP) mHPLC separations, tetrabutylammonium hydroxide (Alfa Products, Danvers, MA, USA) was used as the ion-pairing reagent at concentrations ranging from 1 to 5 mm.Malonic acid (Sigma) was used to adjust the mobile phase pH. The analytical mHPLC column, used for both RP and RP-IP chromatography, was a 150 mm 3 1 mm id stainless-steel column packed with Spherisorb 3 mm C18 material (Isco). HPLC mobile phases were filtered and de-gassed using a 35 mm all-glass filter holder (Millipore, Bedford, MA, USA), fitted with a 0.45 mm hydrophilic nylon filter (Cuno, Meriden, CT, USA) for aqueous solvents and a 0.5 mm PTFE filter (Cole-Parmer, Chicago, IL, USA) for organic solvents.Chromatographic standards were prepared from 1000 mg l21 As aqueous solutions of the following arsenic compounds: parsanilic acid (p-ASA) (Eastman Organic Chemicals, Rochester, NY, USA), 3-nitro-4-hydroxyphenylarsonic acid (roxarsone) (ICN Biochemicals, Cleveland, OH, USA), 4-nitrophenylarsonic acid (4-NPAA) (Aldrich, Milwaukee, WI, USA), dimethylarsinic acid (DMA) (Sigma), 4-hydroxyphenylarsonic acid (4-OH) (Eastman Kodak), disodium methylarsonate (MMA) (Chemical Service, West Chester, PA, USA), sodium meta-arsenite (Aite) (Sigma), and sodium arsenate (Aate) (Sigma).Procedures Time resolved acquisition (TRA) software provided by VG was used to acquire data during mHPLC–ICP-MS experiments. Data files of response versus time were exported as ASCII files and analysed using commercial spreadsheet software. The mFI– and mHPLC–ICP-MS response was optimised for the detection of arsenic at m/z 75 using carrier flow rates of 80 and 40 ml min21, respectively.The optimisation was carried out by employing a 60 ml loop fitted on a ceramic injector (SVI- 6U7) (Analyticon Instruments, Springfield, NJ, USA). The large volume loop provided quasi-continuous sample introduction, which lasted long enough for signal optimisation to be achieved. For the HPLC optimisation a 60 ml loop was connected post-column; this provided a signal equivalent to that obtained in the continuous flow mode.All instrument parameters were varied iteratively to reach the apparent optimum response. Results and Discussion Separation of Arsenic Animal Feed Additives Using RP- mHPLC–ICP-MS RP-mHPLC methods were developed and used for the separation of four phenylarsonic acids and two inorganic arsenic compounds. A mobile phase consisting of 0.1% trifluoroacetic acid (TFA) in water, with a variable percentage (5–15% v/v) of methanol, was used for their separation.The mHPLC stationary phase consisted of silica based C18 material (3 mm particle diameter, 12% carbon loading). The chromatograms obtained are presented in Fig. 1. Present in the injected sample was 4-hydroxyphenylarsonic acid (4-OH). Even though this compound has not been used as an animal feed additive, it may prove to be a decomposition product or metabolite of roxarsone or may adequately serve as a chromatographic internal standard. As mentioned previously, the RP-mHPLC method developed allows for the separation of inorganic arsenic species from various of the phenylarsonic acids.This separation is of particular importance, primarily because of the relatively high toxicity of the inorganic arsenic species. Thus, the RP chromatographic method can be used to monitor arsenic animal feed additives for the presence of inorganic arsenic impurities, and also to detect possible degradation of the phenylarsonic acids to their respective inorganic forms. As shown in Fig. 1, the extensive retention of the NO2-containing arsenicals (roxarsone and 4-NPAA) by the LC stationary phase results in excessive Fig. 1 RP-mHPLC–ICP-MS traces for six arsenic compounds. The mobile phase consisted of water (0.1% TFA) and 5–15% methanol, applied at a flow rate of 40 ml min21. 1064 Analyst, October 1997, Vol. 122peak tailing. A larger amount of methanol in the mobile phase reduces peak tailing significantly, but also provides poorer overall resolution.In addition, the methanol content affects the sensitivity of the ICP-MS detector. Signal enhancement for arsenic in the presence of organic solvents has been observed and reported extensively by others.19,20 This occurrence has major implications, especially when using gradient-elution methods which make use of variable amounts of organic solvents in conjunction with HPLC–ICP-MS. It has been proposed that the presence of organic solvents in the plasma alters the energetics of the ICP.Even when the introduction of organic solvent into the plasma is discontinued, a relatively long period of time is required before the ICP-MS sensitivity returns to its pre-solvent level. To explore further the possible use of RP chromatography for the speciation of phenylarsonic acids in environmental samples, we investigated the effects of naturally occurring arsenicals on the RP chromatograms. The RP-mHPLC separation of various phenylarsonic acids (arsenic animal feed additives) from naturally occurring arsenic compounds is presented in Fig. 2. These chromatograms were obtained with no methanol present in the mobile phase. Less efficient separations occurred in the presence of even small amounts of methanol ( < 0.5%). The mobile phase flow rate also had a pronounced effect on separation efficiency. The best separation was obtained at the lowest-tested flow rate (15 ml min21). The main drawback, however, was the incomplete separation of DMA from p-ASA.In addition, excessive peak broadening occurred in the low flow rate regime ( < 30 ml min21). Flow rates > 55 ml min21 were not considered, mainly because of the resulting high back-pressure ( > 3000 psi), which could potentially damage the mHPLC column. It should also be mentioned that when the mobile phase does not contain any methanol, the phenylarsonic acids roxarsone and 4-NPAA do not elute from the HPLC column. Thus, a preliminary chromatographic run using a mobile phase containing 5–15% methanol must be conducted in order to investigate the presence of the two nitro-containing arsenicals (roxarsone and 4-NPAA).Separation of Arsenic Animal Feed Additives Using RP-IP- mHPLC–ICP-MS Because RP chromatography provides poor separation of the arsenic animal feed additives from some of the naturally occurring arsenic compounds (Aite, Aate, MMA and DMA), alternative chromatographic modes of separation were investigated and further optimised to provide efficient separations.To accomplish this, RP-IP-mHPLC was further investigated. To assist in reaching the optimum conditions required for such separations, a plot of the apparent charge (Qapp) of each of the arsenic compounds as a function of mobile phase pH was constructed (Fig. 3). This plot provides a qualitative estimate of the extent of interaction of the anion-pairing reagent with the arsenic-containing analyte under various pH conditions.Qapp for arsenate was calculated as follows: Qapp AsO HAsO H AsO AsO HAsO H AsO H AsO = - + + + + + - - - - - - 3 2 4 3 4 2 2 4 4 3 4 2 2 4 3 4 [ ] [ ] [ ] [ ] [ ] [ ] [ ] The Qapp values for the other arsenic compounds were derived similarly. The pKa values21 used for these calculations were as follows: pKa[Aite] = 9.2, pKa[Aate] = 2.2, 6.98, 11.5, pKa[DMA] = 1.3,22 6.3, pKa[MMA] = 3.41, 8.18, pKa[p- ASA] = 2, 4.02, 8.92. The chromatogram presented in Fig. 4, obtained under RPIP- mHPLC conditions, exhibits sufficient separation of p-ASA Fig. 2 RP-mHPLC–ICP-MS traces for p-ASA (D) and 4-OH (E) in the presence of naturally occurring arsenic compounds: arsenite and arsenate (A), methylarsonic acid (B) and dimethylarsinic acid (C). The mobile phase consisted of water (0.1% TFA). The flow rates varied between 15 and 55 ml min21. Fig. 3 Apparent charge of arsenic compounds as a function of solution pH. Arrows indicate normal operating range of pH for reversed-phase anionpairing chromatography of arsenic acids.Fig. 4 RP-IP-mHPLC–ICP-MS traces for five arsenic compounds. The mobile phase consisted of 1 mm tetrabutylammonium hydroxide and 0.5% methanol in de-ionised water (pH 5.28), applied at a flow rate of 40 ml min21. Analyst, October 1997, Vol. 122 1065from four other arsenic compounds which have been found to occur naturally in various environmental samples. The elution order of these compounds at pH 5.3 is in agreement with that predicted by the Qapp versus pH plot (Fig. 3). A compound with high Qapp is expected to interact to a greater extent with the tetrabutylammonium ion-pairing reagent. Stronger interaction results in longer retention times. However, it should be noted that factors other than pH significantly influence the chromatographic behavior of the arsenic compounds. The most important of these factors include the ion-pairing reagent concentration and the methanol content of the mobile phase.Separation of p-ASA from Arsenic Compounds Present in Reference Materials Initially the effects of large amounts of NaCl on the chromatographic separation of arsenic compounds were investigated. The resulting chromatograms from these experiments are presented in Fig. 5. It was observed that the presence of 0.1% NaCl in the sample had a significant effect on the chromatographic separations (retention times, peak shapes), particularly when a low ion-pairing concentration (1–2 mm TBAH) was used.In order to minimise this problem, which is especially pronounced when speciating arsenic in biological materials such as urine, 5 mm TBAH was used. Also, the mobile phase pH was adjusted to allow for the separation of chloride from the other arsenic compounds, thus eliminating potential interferences caused by ArCl+. Freeze-dried urine (NIST SRM 2670) containing ‘normal levels’ of arsenic (as indicated by NIST) was analysed for its arsenic content.The freeze-dried urine sample was reconstituted with de-ionized water and then spiked with p-ASA and 4-OH. The resulting chromatogram is shown in Fig. 6. Resolution between DMA and MMA was sacrificed by appropriately adjusting the mobile phase pH in order to achieve improved separation between the Cl2 and arsenate species present. The determination of arsenate is of considerable importance mainly because its toxicity is substantially higher than that of MMA or DMA.Good separation of 4-OH, used as an internal standard in this case, was also accomplished. It should be noted that the original (non-spiked) urine SRM did not contain any measurable amounts of p-ASA or 4-OH. It should also be noted that the RP anion-pairing chromatography used in this study is not capable of differentiating between arsenobetaine and arsenite, as these two compounds co-elute. Also, there exists the possibility that other non-retained arsenic species may co-elute in this front-end peak. Arsenobetaine is normally present in human urine following the consumption of seafood.23 Previous investigations using this particular reference material have shown that indeed the first peak is the result of arsenite and arsenobetaine.18,24 Solutions referred to as Trace Elements in Water (NIST SRM 1643a) and Trace Metals in Drinking Water (High-Purity Standards) were also spiked with p-ASA and 4-OH, and subsequently analysed for their content of arsenic species (Fig. 7). The spiked arsenicals were separated from arsenite and arsenate, which were originally present in the water reference materials. Furthermore, San Joaquin Soil (NIST SRM 2709) was extracted using 0.3 m H3PO4 and then spiked with p-ASA and 4-OH. The resulting chromatogram is presented in Fig. 8. The soil extract, prior to spiking, was found to contain only arsenate as the single arsenic compound present. The limits of detection for each arsenic compound, achieved under various modes of chromatography, are summarised in Table 1. Because of the extremely high sensitivity of the ICPMS detector, the chromatographic methods described are eminently suitable for the development of methodologies for the determination of arsenic animal feed additives in real samples. These methods are particularly suited for the analysis of samples containing limited amounts of analyte.mFI–ICP-MS of Arsenic Animal Feed Additives In previous work, a mFI–ICP-MS technique was developed and applied to the determination of total arsenic.14 Of interest in this study was to investigate further any sensitivity variations observed when analysing arsenic animal feed additives by using mFI–ICP-MS.A number of reports have claimed sensitivity variations for other organoarsenic compounds determined using continuous-flow ICP-MS, particularly for arsenicals present in marine organisms.20 So far, no sensitivity data have been reported regarding arsenic animal feed additives.Fig. 9 shows the peaks obtained for the four phenylarsenical compounds discussed in this paper. Each peak corresponds to an Fig. 5 Effect of salt and ion-pair reagent concentrations on the separation of four arsenic compounds. Fig. 6 RP-IP-mHPLC–ICP-MS trace for urine (NIST SRM 2670n) spiked with p-ASA and 4-OH. The mobile phase consisted of 0.5% v/v methanol and 5 mm TBAH at pH 5.8, applied at a flow rate of 40 ml min21. 1066 Analyst, October 1997, Vol. 122injection of 80 pg of As (1 ml injections of a phenylarsenical solution containing 80 ppb of As). The fact that no sensitivity variations were observed permits the use of mFI–ICP-MS for the quantification of arsenic animal feed additives regardless of which phenylarsonic species are present in the sample and without their prior conversion into a common species by employing some form of digestion. Conclusions We have demonstrated the development and successful application of a variety of chromatographic methods for the identification of arsenic animal feed additives.More specifically, RP- mHPLC was used for the separation of arsenic animal feed additives from inorganic arsenic species. This mode of chromatography, however, did not allow the complete separation of arsenic animal-feed additives from naturally occurring organoarsenic compounds. RP–IP-mHPLC was used successfully for this purpose. Furthermore, the use of mHPLC offers the advantage of operating at very low flow rates (15–40 ml min21), thus allowing for minimisation of waste and the use of small sample volumes, and also offers flow rate compatibility with other mass spectrometric detectors such as electrospray and continuous-flow fast atom bombardment mass spectrometry.The latter feature may prove to be a great benefit for the structural identification of metabolites or decomposition products for which synthetic standards are not yet available.The use of the ICP-MS detector offers additional benefits, such as high selectivity and extremely good sensitivity, for the speciation of trace levels of arsenic in samples of environmental and biological origin. The US Environmental Protection Agency (EPA), through its Office of Research and Development (ORD), funded the research described here. This paper has been subjected to the Agency’s peer review and has been approved as an EPA publication. The US Government has a non-exclusive, royaltyfree license in and to any copyright covering this paper.Mention of trade names or commercial products does not constitute Fig. 7 RP-IP-mHPLC–ICP-MS traces for A, water (NIST SRM 1643a) spiked with p-ASA and 4-OH and B, water (High-Purity Standards, Drinking Water) spiked with p-ASA and 4-OH. The mobile phase consisted of 0.5% v/v methanol and 5 mm TBAH at pH 5.8, applied at a flow rate of 40 ml min21. Fig. 8 RP-IP-mHPLC–ICP-MS trace for soil extract (NIST SRM 2709 San Joaquin Soil) spiked with p-ASA and 4-OH.The mobile phase consisted of 0.5% v/v methanol and 5 mm TBAH at pH 5.8, applied at a flow rate of 40 ml min21. Table 1 Limits of detection (defined as three times the standard deviation of the background) for arsenic compounds obtained under RP- and RP-IP-m- HPLC–ICP-MS conditions RP-IPRP- mHPLC– mHPLC– ICP-MS ICP-MS pg ml21 pg ml21 As pg As As pg As Arsenite (Aite) 0.10 0.10 0.6 0.6 Arsenate (Aate) 0.10 0.10 0.4 0.4 p-Arsanilic acid (p-ASA) 0.10 0.10 0.9 0.9 4-Hydroxyphenylarsonic acid (4-OH) 0.10 0.10 0.8 0.8 3-Nitro-4-hydroxyphenylarsonic acid (roxarsone) 0.12 0.12 n.d.* n.d. 4-Nitrophenylarsonic acid (4-NPAA) 0.26 0.26 n.d. n.d. * n.d.: not determined because the compounds do not elute from column under the specified conditions. Fig. 9 mFI–ICP-MS of arsenic animal feed additives. Each peak represents the injection of 1 ml of a solution of 80 pg ml21 As. Analyst, October 1997, Vol. 122 1067endorsement or recommendation for use. This work was performed while S.A.P. held a National Research Council/ CRD-LV Research Associateship. References 1 Gilbert, F. R., Wells, G. A. H., and Gunning, R. 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A., in Proceedings of the 44th ASMS Conference on Mass Spectrometry and Allied Topics, Portland, Oregon, May 12–16, 1996, American Society for Mass Spectrometry, East Lansing, MI, USA, p. 21. Paper 7/02691I Received April 21, 1997 Accepted July 14, 1997 1068 Analyst, October 1997, Vol. 1