1. 2003 EUROPEAN WINTER CONFERENCE on PLASMA SPECTROCHEMISTRY,
Garmisch-Partenkirchen , January 2003
“New evidence obtained on Refractory Arsenicals in estuarine waters by HPLC-MW-HG-QFAAS, Ultrafiltration and LC-ESI-MSn”
Bettencourt, A M M de*, S G Capelo*, C M Carapeto*, M H Florêncio, M L Gomes , L F Vilas Boas and B R Larsen**,
* Environmental Biogeochemistry Group-IMAR, Colégio Luis A. Verney, Rua Romão Ramalho, 59, Évora,Portugal
 Departamento de Química e Bioquímica, Faculdade de Ciências, Universidade de Lisboa, Campo Grande, Edifício C8, 3º piso, Lisboa
Instituto de Tecnologia Química e Biológica (ITQB), Quinta do Marquês, 2870 Oeiras, Portugal
** Institute for Environment and Sustainability, Joint Research Centre, European Commission, I Ispra (VA), Italy
Introduction
The results of the optimization of the HPLC-ESI-MSn technique -for a number of organoarsenic compounds likely to occur in natural waters - showed that the peaks separated by HPLC on natural water (or its extracts) could not be analyzed directly by mass spectrometry without further purification (Bettencourt et al., 2000, Larsen et al., 2001).
A new approach to the identification of Arsenic refractory species in natural waters combining ultrafiltration with HPLC-MW-QF-AAS and HPLC-ESI-MSn, was therefore developed (Bettencourt et al., for publ). Arsenic compounds were identified by Tandem Mass Spectrometry. .
Experimental Conditions
A water sample processed as before (Bettencourt et al., 2000) was further purified through an ultrafiltration cascade with Pall Filtron Microsep Centrifugal Devices, equipped with Omega membranes of 10 and 1KD MWCO. These were conditioned with 2x OH2 Milli-Q water N OHNa Suprapur + 2xOH2 Milli-Q water and the extracts, centrifuged at 5000 g and 7500 g, during 40 and 80 minutes, respectively. The ultrafiltrates were then concentrated to a final volume of l .
Aliquots (20 µL) were loop injected into a Thermo Separation HPLC coupled to a Finnigan Mat LCQ ion-trap mass spectrometer.
Results
Figs. 1 a) and 2a) depict the CID MS2 spectra of AB and AC, obtained in the purified fractions F32 and F42, respectively, and Fig 1 b) and 2b) those obtained for the corresponding standards.
Trimethylarsine oxide (TMAO) (as well as Arsenate, MMAV and DMAV) was also identified in some of these fractions by CID MS2. Former HPLC-ICP/MS and new LC-ESI-MS2 evidence still suggest that Tetramethylarsonium ion (TMA) may be present in these extracts at trace level. An unidentified compound detected at m/z=193 and fragmented by CID MS2, could eventually be an arsenical moiety.
Fig.1
Fig.2
Table 1
\MS2species
HPLC-fraction AB AC TMAO m/z = Total RefAs
I dentified/total Re
fAs in the fract
F ng ng ng ng/11ng (32%)
F ng ng
ng /18.6 ng
(1.9%)
F ng Traces < ng
mg /12.5 ng
(1%)
F ng Traces Traces ng
<0.01 ng < 0.01 ng /20.3 ng
(0.07%)
Total n
/62.4 ng (6.4%)
RT in the LC-ESI-MSn  .
Table 1 shows the main arsenic compounds identified with the indication of the fractions in which they have been identified, the quantities measured and Retention times for the HPLC-ESI-MSn system. )
Discussion
It follows from Figs 1-2 that the CID MS2 fragmentation pattern of arsenical species present in the extracts (Figs. 1a) and 2 a)) are entirely similar to the CID MS2 fragmentation pattern of the corresponding standards of AB and AC (Figs.1 b and 2 b)) which implies identical chemical structures.
 Therefore AB and AC, might be present in the water extracts brought to analysis and are likely constituents of the refractory fraction under scrutiny or of their break-down products.
 These findings confirm what was formerly obtained for Arsenocholine, Arsenobetaine and TMAO using Mass-Fragmentometry, Pyrolysis GC-MS, ESI-MS HPLC-ICP/MS and HG-QFAAS (Christakopoulos, 1988, Bettencourt, 1988, Bettencourt et al., 1994, Duarte et al., 1995, Florêncio et al., 1997b).
Abnormal retention times in the HPLC chromatograms obtained might be explained by matrix effects in the HPLC-MW-QF-AAS system. Nevertheless the presence of AC and AB in a number of different separated peaks suggests that part of that AC may come from heavier precursors, and that AC degraded, during the processing of the samples, to AB, that in turn degraded to TMAO.
Acknowledgments
The authors acknowledge the financial support of Fundação para a Ciência e Tecnologia (FCT) under the contracts FESTA (PRAXIS 2/2.1/MAR/1715/95) and FESTA II (Sapiens, PCTI/1999/MGS/34457). They are also indebted to the Institute of Environment, JRC, EU, Ispra, Italy, for the use of laboratory facilities.
References
Bettencourt, A M de, C Carapeto, M H Florêncio, M L R Gomes, L F Vilas Boas e B Larsen (2002), “Identification of Arseocholine in Estuarine Waters by Tandem-Mass Spectrometry”, (Poster) Annual Meeting, IMAR, Museu do Mar-Rei D. Carlos, Cascais, de Novembro de 2002
Bettencourt, A M M de, S G Capelo, C M Carapeto, M H Florêncio, M L Gomes, L F Vilas Boas and B R Larsen (2003), On the nature of Refractory Arsenicals in estuarine surface waters. New evidence obtained by HPLC-MW-HG-QFAAS, Ultrafiltration and LC-ESI-MSn (to be submitted to ABC or JAAS)