Determination of Trace Amounts of Vanadyl Porphyrin in Marine Mussel Tissues by High-performance Liquid Chromatography With Both Ultraviolet/Visible and Inductively Coupled Plasma Atomic Emission Spectrometric Detection Paola Rivaro* and Roberto Frache Sezione di Chimica Analitica e Ambientale, Dipartimento di Chimica e Chimica Industriale, Universit`a di Genova, via Dodecaneso, 31-16146 Genoa, Italy An HPLC method with UV/VIS and ICP-AES detection is described for the determination of vanadyl porphyrins extracted from biological samples. A detection limit of 50 ng of vanadium was obtained.The method was used to determine these compounds following their extraction from tissues of mussels treated in laboratory experiments and collected during a ‘Mussel Watch Programme’. This allowed some conclusions about vanadium speciation in marine organisms to be made. In the tissues of mussels, collected at several sites of the monitored area, which showed high vanadium concentrations, it was possible to establish the presence of this metal in the form of organometallic compounds.Keywords: Vanadyl porphyrins; speciation; liquid chromatography; inductively coupled plasma atomic emission spectrometry; hyphenated technique; biological samples; mussel watch programme Vanadium enters the marine environment through natural processes, atmospheric fallout and human activity and it is believed that in sea-water it exists primarily as orthovanadate ions.1 Oil spills can also indroduce this element into the marine environment.In petroleum, vanadium is present in the form of organometallic compounds called vanadyl porphyrins,2,3 a group of macrocyclic aromatics consisting of a porphyrin ring, to which a metal ion is bound. Etioporphyrins and deoxophylloerythroporphyrins, with a total carbon number ranging from 29 to 39, are the most abundant series found. In the past, porphyrins have been used as biomarkers in the study of the origin and formation of petroleum.4 Recently, in environmental studies, attention has been focused on their possible use in ‘fingerprinting’ polluting oil residues (tar balls), because they are much more resistant to biodegradation and weathering than the hydrocarbon fraction.3 Despite the low concentrations in sea-water, ranging from 0.3 to 3.2 mg l21, vanadium is accumulated to relatively high levels in certain marine organisms, such as ascidians and certain molluscs.5 At low concentrations it is an essential trace element in animals and plants, but it also has some toxic or inhibitory effects, as it is able to inhibit the enzymes involved in the cationic transport across the cell membranes.6 Although information exists on vanadium levels in certain marine organisms, little is known about its speciation; there is a need, therefore, for more detailed knowledge of the chemical form of this element, in order to be able to assess this metal toxin in aquatic animals fully.Marine mussels have been widely used as biomonitors of heavy metal pollution in coastal areas. From December 1994 to October 1995 a ‘Mussel Watch Programme–Regione Liguria’ was carried out to assess the quality of water of the Ligurian Sea and to identify the possible sources of metal pollution. Metal concentrations were measured in mussels (Mytilus galloprovincialis, Lam) collected in different months at eight sampling sites of dissimilar water qualities as reported in Fig. 1. Vanadium was one of the metals taken into consideration; as shown under Results and Discussion (Fig. 4) its concentrations in mussels sampled in the Genoa oil port area and close to the town of Cogoleto were statistically significantly different from the values found at all the other sampling sites. Therefore, an investigation of vanadium speciation was conducted to identify the forms in which vanadium was present in these samples, i.e., whether as inorganic or organometallic (vanadyl porphyrin) species.Several methods have been proposed to separate different classes of petro porphyrins in oil, using either high-performance liquid chromatography (HPLC)7,8 or gas chromatography (GC)9,10 to separate the different classes of compound, coupled with different detection techniques such as atomic absorption spectrometry (AAS),11 inductively coupled plasma atomic emission spectrometry (ICP-AES)8,10 and inductively coupled plasma mass spectrometry (ICP-MS).12,13 We could not find any references to the extraction and determination of vanadyl porphyrins in matrices other than oil.In fact, all the studies published on vanadium distribution in sea-water1 or in marine organisms5,6,14 refer only to the total amount of vanadium. This work describes a method for the extraction of vanadyl porphyrins from a biological matrix (mussel tissues), and the use of ICP-AES coupled to ultraviolet/visible (UV/VIS) spectrometry as detection techniques to determine the extracted vanadium-containing compounds. The results of the application of the method to the samples collected in the above-mentioned environmental monitoring programme are also presented.Fig. 1 Sampling sites of ‘Mussel Watch Programme—Regione Liguria’. Sampling site locations: 1, Lavagna; 2, Paraggi; 3, Bogliasco; 4, Genova Darsena; 5, Genova Oil Port; 6, Pr`a; 7, Vesima; and 8, Cogoleto. Analyst, October 1997, Vol. 122 (1069–1072) 1069Experimental Reagents The methanol used was of HPLC-grade; all the other chemicals were of analytical-reagent grade and were obtained from Sigma (St.Louis, MO, USA). Vanadyl porphyrin standards (V-etio-; V-octaethyl-; and V phthalo cyanylporphyrins) were provided by ‘Stazione Sperimentale per i Combustibili’, (S. Donato Milanese, Italy). Working solutions were prepared by diluting the standards with toluene and were stable for several days. The vanadium solutions were prepared by diluting a 1000 ppm stock solution (SpectrosoL-grade, BDH, Poole, Dorset, UK).De-ionized water from a Milli-Q system (Millipore, Watford, Hertfordshire, UK) was used throughout. Apparatus HPLC The HPLC instrument used was a Varian LC system 5000 equipped with a 200 ml Rheodyne (Cotati, CA, USA) injector. Vanadyl porphyrin separations were performed on a 5 mm LiChrospher RP-8 column (250 3 4.6 mm id) (Bischoff Chromatography, Leonberg, Germany), operated at room temperature.The flow rate was 0.8 ml min21 and no gradient elution devices were used. The mobile phase was methanol– water (9 + 1). The UV/VIS detector was set at the maximum absorbance wavelength (406 nm for V-etio- and V-octaethylporphyrins or 700 nm for V phthalocyanylporphyrins). ICP-AES A Jobin–Yvon 24 spectrometer (Jobin–Yvon, Longjumeau, Paris, France) was used both to determine the total vanadium in mussel tissues and as a detector for HPLC in vanadium speciation studies.In the latter case, the ICP-AES parameters were: power, 700 W; principal argon flow rate, 16 l min21; coolant, 0.2 l min21; nebulizer, 0.7 l min21. At the HPLC–ICPAES interface, a Cetac USN 5000 ultrasonic nebulizer with the thermostat set at 140 °C and the cryostat at 28 °C was used. The working wavelength for vanadium was V(II) 292.402 nm. Sample Preparation Field experiments Mussels were collected in different months (December 1994, March 1995, June 1995, July 1995, October 1995) at eight sampling sites of the ‘Mussel Watch Programme’ as reported in Fig. 1. The tissues of 20 animals, removed from the shells, were homogenized using a Turrax high-speed homogenizer and were stored at 220 °C until analysis. Laboratory experiments This part of the work was necessary to verify whether mussels were able to concentrate vanadium in their tissues as organometallic compounds, whether any degradation processes occurred and to test the procedure for extracting vanadyl porphyrins from biological samples.Mussels, of 4–6 cm in length, obtained from a marine farm located near La Spezia, were exposed to an environment containing 100 mg l 21 V as an octaethylporphyrin solution, in a static sea-water system for 6 d. The mussels were first left in poly(propylene) tanks in seasonally adjusted, artificial seawater (15 °C) prepared according to the method of La Roche et al.15 The porphyrin solution was added daily, after changing the water.Some mussels were not exposed to the treatment and they were considered as control samples. Tissues were removed from shells, homogenized and stored at 220 °C . Analytical Procedure Total vanadium A 2 g amount of homogenized tissue (wet mass) was boiled in 10 ml of 1 m nitric acid for 2 h at 70 °C in a reflux system. The acid solution was filtered through a Schleicher–Schull blueband filter (i.e., 2 mm pore size) and diluted to the final volume (20 ml) with de-ionized water.The accuracy of the procedure was checked using the standard additions method. Extraction of vanadyl porphyrins A 2 g amount of homogenized tissue (wet mass) was added to 2 g of solid Na2SO4 and left at 60 °C overnight. The extraction from the tissue was accomplished with a Soxhlet apparatus, using toluene (200 ml for each sample). Eight extraction cycles were necessary to obtain reproducible results. The toluene was collected and evaporated to dryness by using a vacuum rotary evaporator.The samples were re-dissolved in 1 ml of methanol and 200 ml aliquots were injected onto the HPLC column. The accuracy of the method was tested by analyses of sub-samples of equal mass spiked with vanadyl porphyrin standard solution. The spiking experiments were carried out by adding to the homogenized tissue a few drops of V-etio- and V-octaethylporphyrin solutions, in order to obtain additions of 2 and 4 mg l21 as V. The usual extraction procedure was then carried out.A recovery of about 80% was determined. Results and Discussion In Fig. 2 the chromatograms of a standard mixture of V-etioand V-octaethylporphyrins are shown. As can be seen, the retention times of the vanadium peaks on the ICP-AES chromatogram are slighly shifted with respect to the UV peaks, because of the connecting tube between the output of the UV/VIS detector and the plasma torch (a dead volume of about 1 ml). The UV/VIS detector was fixed at 406 nm, because the considered porphyrins had their maximum absorbance at this wavelength.The use of an ultrasonic nebulizer allowed greater flexibility in the setting-up of a chromatographic method than the classical Meinhard nebulizer. The ultrasonic nebulizer leads to a greater lowering of the organic modifier concentration reaching the plasma torch, with a positive effect on the detection limits, owing to the low noise. Fig. 2 Chromatograms of a standard solution mixture of V-etio- (peak A) and V-octaethyl (peak B) porphyrins. 1: UV/VIS detection at 406 nm; 2: ICP-AES signal of V(II) at 292.402 nm. 1070 Analyst, October 1997, Vol. 122It should be noted that when the chromatographic method was first set up, the separations were performed using acetonitrile–water (45 + 55) as eluent. However, when the HPLC–UV/VIS detector system was coupled with the ICP-AES instrument, this organic modifier caused instability of the plasma torch, reducing its excitation properties and consequently the analytical response, while also creating a strong background signal.This drawback was overcome by using methanol, albeit in a higher percentage in order to obtain the same retention times. Under these conditions, a detection limit of 50 ng of vanadium (3s) was obtained. The detection limit was determined by injecting 200 ml aliquots of standard solutions of different concentrations onto the HPLC column. The calibration range was linear up to 1000 ng of vanadium and the relative standard deviations (n = 5) ranged from 7 to 3%.In order to verify the applicability of the extraction and determination methods to real samples, vanadyl porphyrins in tissues of mussels exposed in laboratory experiments to an octaethylporphyrin solution were determined. In Table 1, the total vanadium concentrations found in the analysed tissues (i.e., shell, total tissues, gills, digestive glands) at the end of the incubation time are reported.As can be seen, a higher vanadium concentration was found in the digestive glands. Because data on concentration factors of organometallic vanadium compounds in marine organisms were not available in the literature, these results were compared with the results of experiments in which the animals were exposed to vanadium in an inorganic form. The concentration factor for the digestive glands was higher than the values reported in the literature, whereas for the other tissues no significant differences were found.5 In Fig. 3 the chromatograms of a digestive gland extract of mussels exposed for 6 d are shown. In terms of retention time, the peaks correspond to the octaethylporphyrin present in tissues. Examination of the chromatograms revealed that all the vanadium in the samples was present as an organometallic compound, as in the ICP-AES signal, no vanadium peaks, other than this, were found. Moreover, it was established that, after 6 d of exposure, no transformation or degradation processes had occurred.The vanadium concentration calculated on the basis of peak area is in agreement with the total amount of vanadium found, taking into account the recovery value of the extraction. However, the procedure was considered suitable for the determination of organometallic vanadium species in marine organisms and was used to identify the forms in which vanadium was present in mussel samples collected during the ‘Mussel Watch Programme—Regione Liguria’.In Fig. 4 the result of the determination of total vanadium in tissues of mussels sampled in June 1995 is shown. As can be seen, the tissues of the animals collected in the Genoa Oil Port area and close to the town of Cogoleto had vanadium contents of 0.51 and 0.84 mg g 21, respectively, which were significantly higher than the concentrations found in all the other samples. Differences were tested by means of a Student’s t-test and found to be significant at P < 0.05.To these samples and to a control sample (i.e., mussels obtained from the marine farm) the determination and extraction procedures were applied for speciation purposes. The results are shown in Fig. 5. The chromatograms of both samples show that vanadium is present as vanadyl porphyrin; in terms of retention time, the peaks correspond to this compound. This result was expected for the sample collected in the oil port. This sample shows a peak which appears to consist of two different, but very closely eluting peaks; this would indicate either the presence of two different types of vanadyl porphyrin or a degradation product.On the other hand, the sample collected near the town of Cogoleto showed unequivocally the presence of vanadium as an organometallic compound in sea-water. This fact could be explained by taking into account that in this Table 1 Concentration of total vanadium (ng g21 wet mass) in tissues of mussel not exposed (control) and in mussel after 6 d exposure to water containing V-octaethylporphyrin (100 mg l21 as V).Data represent the mean ± standard deviation of three replicates ng g21 V Concentration Tissue Control Exposed factor Shell 50 ± 2 1370 ± 200 13.7 Soft parts (whole) 20 ± 2 700 ± 80 7 Gills 10 ± 1 520 ± 40 5.2 Digestive glands 60 ± 4 3790 ± 180 37.9 Fig. 3 Chromatograms of a mussel digestive gland after exposure to Voctaethylporphyrin for 6 d. 1: UV/VIS detection at 406 nm; 2: ICP-AES signal of V(II) at 292.402 nm.Fig. 4 Concentration levels of total vanadium (mg g 21 wet mass) found in tissues of mussels collected during the ‘Mussel Watch Programme— Regione Liguria’ (June 1995 sampling). Fig. 5 Chromatograms of mussel tissues sampled in the Genova Oil Port (A) and near Cogoleto (B) compared with a control sample (C). 1: UV/VIS detection at 406 nm; 2: ICP-AES signal of V(II) at 292.402 nm. Analyst, October 1997, Vol. 122 1071location the oil tanker Haven sank in 1992 and that oil losses are probably still occurring.Conclusion The procedures used to extract and determine organometallic vanadium compounds in biological samples were suitable for application to environmental studies. The use of a coupled detection technique, UV/VIS–ICP-AES, was helpful in giving information on vanadium speciation in tissues of marine mussels collected during a ‘Mussel Watch Programme’, allowing a more detailed knowledge of the chemical form of this element to be obtained.The presence of vanadyl porphyrins, which are characteristic compounds of petroleum, suggested that oil spills were probably responsible for the high concentration of vanadium found at some sites of the monitored area. This work was financially supported by Regione Liguria. We are also indebted to Dr. Paolo Tittarelli of the Stazione Sperimentale per i Combustibili for providing us with the vanadyl porphyrin standards. References 1 Jeandel, C., Caisso, M., and Minster, J.F., Mar. Chem., 1987, 21, 51. 2 de Waal, W. A. J., Heemstra, S., Kraak, J. C., and Jonker, R., J., Chromatographia, 1990, 30, 38. 3 Rankin, J. C., Czernuszewich, R. S., Org. Geochem., 1993, 20, 521. 4 Quirke, J. M. E., Eglinton, G., and Maxwell, J. R., J. Am. Chem. Soc., 1979, 101, 7693. 5 Miramand, P., Guary, J. C., and Fowler, S. W., Mar. Biol., 1980, 65, 281. 6 Unsal, M., Mar. Pollut. Bull., 1982, 13, 139. 7 Sundararaman, P., Anal.Chem., 1985, 57, 2204. 8 Xu, H., and Lesage, S., J. Chromatogr., 1992, 607, 139. 9 Kaur, S., Gill, J. P., Evershed, R. P., Eglinton, G., and Maxwell, J. R., J. Chromatogr., 1989, 473, 135. 10 Zeng, Y., Seeley, J. A., Dowling, T. M., and Uden, P. C., J. High Res. Chrom., 1992, 15, 669. 11 Fish, R. H., and Komlenic, J. J., Anal. Chem., 1984, 56, 584. 12 Pretorius, W. G., Foulkes, M., Ebdon, L., and Rowland, S. J., J. High Resolut. Chromatogr., 1993, 16, 157. 13 Ebdon, L., Evans, E.H., Pretorius W. G., and Rowland, S. J., J. Anal. At. Spectrom., 1994, 9, 939. 14 Miramand, P., and Bentley, D., Mar. Biol., 1992, 114, 404. 15 La Roche, G., Eisler, R., and Tarzwell, C. M., J. Water Pollut. Control Fed., 1970, 42, 1982. Paper 7/02568H Received April 15, 1997 Accepted June 23, 1997 1072 Analyst, October 1997, Vol. 122 Determination of Trace Amounts of Vanadyl Porphyrin in Marine Mussel Tissues by High-performance Liquid Chromatography With Both Ultraviolet/Visible and Inductively Coupled Plasma Atomic Emission Spectrometric Detection Paola Rivaro* and Roberto Frache Sezione di Chimica Analitica e Ambientale, Dipartimento di Chimica e Chimica Industriale, Universit`a di Genova, via Dodecaneso, 31-16146 Genoa, Italy An HPLC method with UV/VIS and ICP-AES detection is described for the determination of vanadyl porphyrins extracted from biological samples. A detection limit of 50 ng of vanadium was obtained.The method was used to determine these compounds following their extraction from tissues of mussels treated in laboratory experiments and collected during a ‘Mussel Watch Programme’. This allowed some conclusions about vanadium speciation in marine organisms to be made.In the tissues of mussels, collected at several sites of the monitored area, which showed high vanadium concentrations, it was possible to establish the presence of this metal in the form of organometallic compounds.Keywords: Vanadyl porphyrins; speciation; liquid chromatography; inductively coupled plasma atomic emission spectrometry; hyphenated technique; biological samples; mussel watch programme Vanadium enters the marine environment through natural processes, atmospheric fallout and human activity and it is believed that in sea-water it exists primarily as orthovanadate ions.1 Oil spills can also indroduce this element into the marine environment. In petroleum, vanadium is present in the form of organometallic compounds called vanadyl porphyrins,2,3 a group of macrocyclic aromatics consisting of a porphyrin ring, to which a metal ion is bound.Etioporphyrins and deoxophylloerythroporphyrins, with a total carbon number ranging from 29 to 39, are the most abundant series found. In the past, porphyrins have been used as biomarkers in the study of the origin and formation of petroleum.4 Recently, in environmental studies, attention has been focused on their possible use in ‘fingerprinting’ polluting oil residues (tar balls), because they are much more resistant to biodegradation and weathering than the hydrocarbon fraction.3 Despite the low concentrations in sea-water, ranging from 0.3 to 3.2 mg l21, vanadium is accumulated to relatively high levels in certain marine organisms, such as ascidians and certain molluscs.5 At low concentrations it is an essential trace element in animals and plants, but it also has some toxic or inhibitory effects, as it is able to inhibit the enzymes involved in the cationic transport across the cell membranes.6 Although information exists on vanadium levels in certain marine organisms, little is known about its speciation; there is a need, therefore, for more detailed knowledge of the chemical form of this element, in order to be able to assess this metal toxin in aquatic animals fully.Marine mussels have been widely used as biomonitors of heavy metal pollution in coastal areas.From December 1994 to October 1995 a ‘Mussel Watch Programme–Regione Liguria’ was carried out to assess the quality of water of the Ligurian Sea and to identify the possible sources of metal pollution. Metal concentrations were measured in mussels (Mytilus galloprovincialis, Lam) collected in different months at eight sampling sites of dissimilar water qualities as reported in Fig. 1. Vanadium was one of the metals taken into consideration; as shown under Results and Discussion (Fig. 4) its concentrations in mussels sampled in the Genoa oil port area and close to the town of Cogoleto were statistically significantly different from the values found at all the other sampling sites.Therefore, an investigation of vanadium speciation was conducted to identify the forms in which vanadium was present in these samples, i.e., whether as inorganic or organometallic (vanadyl porphyrin) species. Several methods have been proposed to separate different classes of petro porphyrins in oil, using either high-performance liquid chromatography (HPLC)7,8 or gas chromatography (GC)9,10 to separate the different classes of compound, coupled with different detection techniques such as atomic absorption spectrometry (AAS),11 inductively coupled plasma atomic emission spectrometry (ICP-AES)8,10 and inductively coupled plasma mass spectrometry (ICP-MS).12,13 We could not find any references to the extraction and determination of vanadyl porphyrins in matrices other than oil.In fact, all the studies published on vanadium distribution in sea-water1 or in marine organisms5,6,14 refer only to the total amount of vanadium. This work describes a method for the extraction of vanadyl porphyrins from a biological matrix (mussel tissues), and the use of ICP-AES coupled to ultraviolet/visible (UV/VIS) spectrometry as detection techniques to determine the extracted vanadium-containing compounds. The results of the application of the method to the samples collected in the above-mentioned environmental monitoring programme are also presented.Fig. 1 Sampling sites of ‘Mussel Watch Programme—Regione Liguria’. Sampling site locations: 1, Lavagna; 2, Paraggi; 3, Bogliasco; 4, Genova Darsena; 5, Genova Oil Port; 6, Pr`a; 7, Vesima; and 8, Cogoleto. Analyst, October 1997, Vol. 122 (1069–1072) 1069Experimental Reagents The methanol used was of HPLC-grade; all the other chemicals were of analytical-reagent grade and were obtained from Sigma (St.Louis, MO, USA). Vanadyl porphyrin standards (V-etio-; V-octaethyl-; and V phthalo cyanylporphyrins) were provided by ‘Stazione Sperimentale per i Combustibili’, (S. Donato Milanese, Italy). Working solutions were prepared by diluting the standards with toluene and were stable for several days. The vanadium solutions were prepared by diluting a 1000 ppm stock solution (SpectrosoL-grade, BDH, Poole, Dorset, UK). De-ionized water from a Milli-Q system (Millipore, Watford, Hertfordshire, UK) was used throughout.Apparatus HPLC The HPLC instrument used was a Varian LC system 5000 equipped with a 200 ml Rheodyne (Cotati, CA, USA) injector. Vanadyl porphyrin separations were performed on a 5 mm LiChrospher RP-8 column (250 3 4.6 mm id) (Bischoff Chromatography, Leonberg, Germany), operated at room temperature. The flow rate was 0.8 ml min21 and no gradient elution devices were used. The mobile phase was methanol– water (9 + 1).The UV/VIS detector was set at the maximum absorbance wavelength (406 nm for V-etio- and V-octaethylporphyrins or 700 nm for V phthalocyanylporphyrins). ICP-AES A Jobin–Yvon 24 spectrometer (Jobin–Yvon, Longjumeau, Paris, France) was used both to determine the total vanadium in mussel tissues and as a detector for HPLC in vanadium speciation studies. In the latter case, the ICP-AES parameters were: power, 700 W; principal argon flow rate, 16 l min21; coolant, 0.2 l min21; nebulizer, 0.7 l min21.At the HPLC–ICPAES interface, a Cetac USN 5000 ultrasonic nebulizer with the thermostat set at 140 °C and the cryostat at 28 °C was used. The working wavelength for vanadium was V(II) 292.402 nm. Sample Preparation Field experiments Mussels were collected in different months (December 1994, March 1995, June 1995, July 1995, October 1995) at eight sampling sites of the ‘Mussel Watch Programme’ as reported in Fig. 1. The tissues of 20 animals, removed from the shells, were homogenized using a Turrax high-speed homogenizer and were stored at 220 °C until analysis.Laboratory experiments This part of the work was necessary to verify whether mussels were able to concentrate vanadium in their tissues as organometallic compounds, whether any degradation processes occurred and to test the procedure for extracting vanadyl porphyrins from biological samples. Mussels, of 4–6 cm in length, obtained from a marine farm located near La Spezia, were exposed to an environment containing 100 mg l 21 V as an octaethylporphyrin solution, in a static sea-water system for 6 d.The mussels were first left in poly(propylene) tanks in seasonally adjusted, artificial seawater (15 °C) prepared according to the method of La Roche et al.15 The porphyrin solution was added daily, after changing the water. Some mussels were not exposed to the treatment and they were considered as control samples.Tissues were removed from shells, homogenized and stored at 220 °C . Analytical Procedure Total vanadium A 2 g amount of homogenized tissue (wet mass) was boiled in 10 ml of 1 m nitric acid for 2 h at 70 °C in a reflux system. The acid solution was filtered through a Schleicher–Schull blueband filter (i.e., 2 mm pore size) and diluted to the final volume (20 ml) with de-ionized water. The accuracy of the procedure was checked using the standard additions method. Extraction of vanadyl porphyrins A 2 g amount of homogenized tissue (wet mass) was added to 2 g of solid Na2SO4 and left at 60 °C overnight.The extraction from the tissue was accomplished with a Soxhlet apparatus, using toluene (200 ml for each sample). Eight extraction cycles were necessary to obtain reproducible results. The toluene was collected and evaporated to dryness by using a vacuum rotary evaporator. The samples were re-dissolved in 1 ml of methanol and 200 ml aliquots were injected onto the HPLC column. The accuracy of the method was tested by analyses of sub-samples of equal mass spiked with vanadyl porphyrin standard solution.The spiking experiments were carried out by adding to the homogenized tissue a few drops of V-etio- and V-octaethylporphyrin solutions, in order to obtain additions of 2 and 4 mg l21 as V. The usual extraction procedure was then carried out. A recovery of about 80% was determined. Results and Discussion In Fig. 2 the chromatograms of a standard mixture of V-etioand V-octaethylporphyrins are shown.As can be seen, the retention times of the vanadium peaks on the ICP-AES chromatogram are slighly shifted with respect to the UV peaks, because of the connecting tube between the output of the UV/VIS detector and the plasma torch (a dead volume of about 1 ml). The UV/VIS detector was fixed at 406 nm, because the considered porphyrins had their maximum absorbance at this wavelength. The use of an ultrasonic nebulizer allowed greater flexibility in the setting-up of a chromatographic method than the classical Meinhard nebulizer.The ultrasonic nebulizer leads to a greater lowering of the organic modifier concentration reaching the plasma torch, with a positive effect on the detection limits, owing to the low noise. Fig. 2 Chromatograms of a standard solution mixture of V-etio- (peak A) and V-octaethyl (peak B) porphyrins. 1: UV/VIS detection at 406 nm; 2: ICP-AES signal of V(II) at 292.402 nm. 1070 Analyst, October 1997, Vol. 122It should be noted that when the chromatographic method was first set up, the separations were performed using acetonitrile–water (45 + 55) as eluent. However, when the HPLC–UV/VIS detector system was coupled with the ICP-AES instrument, this organic modifier caused instability of the plasma torch, reducing its excitation properties and consequently the analytical response, while also creating a strong background signal.This drawback was overcome by using methanol, albeit in a higher percentage in order to obtain the same retention times. Under these conditions, a detection limit of 50 ng of vanadium (3s) was obtained. The detection limit was determined by injecting 200 ml aliquots of standard solutions of different concentrations onto the HPLC column. The calibration range was linear up to 1000 ng of vanadium and the relative standard deviations (n = 5) ranged from 7 to 3%. In order to verify the applicability of the extraction and determination methods to real samples, vanadyl porphyrins in tissues of mussels exposed in laboratory experiments to an octaethylporphyrin solution were determined.In Table 1, the total vanadium concentrations found in the analysed tissues (i.e., shell, total tissues, gills, digestive glands) at the end of the incubation time are reported. As can be seen, a higher vanadium concentration was found in the digestive glands. Because data on concentration factors of organometallic vanadium compounds in marine organisms were not available in the literature, these results were compared with the results of experiments in which the animals were exposed to vanadium in an inorganic form.The concentration factor for the digestive glands was higher than the values reported in the literature, whereas for the other tissues no significant differences were found.5 In Fig. 3 the chromatograms of a digestive gland extract of mussels exposed for 6 d are shown.In terms of retention time, the peaks correspond to the octaethylporphyrin present in tissues. Examination of the chromatograms revealed that all the vanadium in the samples was present as an organometallic compound, as in the ICP-AES signal, no vanadium peaks, other than this, were found. Moreover, it was established that, after 6 d of exposure, no transformation or degradation processes had occurred. The vanadium concentration calculated on the basis of peak area is in agreement with the total amount of vanadium found, taking into account the recovery value of the extraction.However, the procedure was considered suitable for the determination of organometallic vanadium species in marine organisms and was used to identify the forms in which vanadium was present in mussel samples collected during the ‘Mussel Watch Programme—Regione Liguria’. In Fig. 4 the result of the determination of total vanadium in tissues of mussels sampled in June 1995 is shown.As can be seen, the tissues of the animals collected in the Genoa Oil Port area and close to the town of Cogoleto had vanadium contents of 0.51 and 0.84 mg g 21, respectively, which were significantly higher than the concentrations found in all the other samples. Differences were tested by means of a Student’s t-test and found to be significant at P < 0.05. To these samples and to a control sample (i.e., mussels obtained from the marine farm) the determination and extraction procedures were applied for speciation purposes.The results are shown in Fig. 5. The chromatograms of both samples show that vanadium is present as vanadyl porphyrin; in terms of retention time, the peaks correspond to this compound. This result was expected for the sample collected in the oil port. This sample shows a peak which appears to consist of two different, but very closely eluting peaks; this would indicate either the presence of two different types of vanadyl porphyrin or a degradation product.On the other hand, the sample collected near the town of Cogoleto showed unequivocally the presence of vanadium as an organometallic compound in sea-water. This fact could be explained by taking into account that in this Table 1 Concentration of total vanadium (ng g21 wet mass) in tissues of mussel not exposed (control) and in mussel after 6 d exposure to water containing V-octaethylporphyrin (100 mg l21 as V).Data represent the mean ± standard deviation of three replicates ng g21 V Concentration Tissue Control Exposed factor Shell 50 ± 2 1370 ± 200 13.7 Soft parts (whole) 20 ± 2 700 ± 80 7 Gills 10 ± 1 520 ± 40 5.2 Digestive glands 60 ± 4 3790 ± 180 37.9 Fig. 3 Chromatograms of a mussel digestive gland after exposure to Voctaethylporphyrin for 6 d. 1: UV/VIS detection at 406 nm; 2: ICP-AES signal of V(II) at 292.402 nm. Fig. 4 Concentration levels of total vanadium (mg g 21 wet mass) found in tissues of mussels collected during the ‘Mussel Watch Programme— Regione Liguria’ (June 1995 sampling).Fig. 5 Chromatograms of mussel tissues sampled in the Genova Oil Port (A) and near Cogoleto (B) compared with a control sample (C). 1: UV/VIS detection at 406 nm; 2: ICP-AES signal of V(II) at 292.402 nm. Analyst, October 1997, Vol. 122 1071location the oil tanker Haven sank in 1992 and that oil losses are probably still occurring. Conclusion The procedures used to extract and determine organometallic vanadium compounds in biological samples were suitable for application to environmental studies. The use of a coupled detection technique, UV/VIS–ICP-AES, was helpful in giving information on vanadium speciation in tissues of marine mussels collected during a ‘Mussel Watch Programme’, allowing a more detailed knowledge of the chemical form of this element to be obtained.The presence of vanadyl porphyrins, which are characteristic compounds of petroleum, suggested that oil spills were probably responsible for the high concentration of vanadium found at some sites of the monitored area. This work was financially supported by Regione Liguria. We are also indebted to Dr. Paolo Tittarelli of the Stazione Sperimentale per i Combustibili for providing us with the vanadyl porphyrin standards. References 1 Jeandel, C., Caisso, M., and Minster, J. F., Mar. Chem., 1987, 21, 51. 2 de Waal, W. A. J., Heemstra, S., Kraak, J. C., and Jonker, R., J., Chromatographia, 1990, 30, 38. 3 Rankin, J. C., Czernuszewich, R. S., Org. Geochem., 1993, 20, 521. 4 Quirke, J. M. 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Paper 7/02568H Received April 15, 1997 Accepted June 23, 1997 1072 Analyst, October 1997, Vol. 122