UV-irradiation and MW-digestion pre-treatment of Port wine suitable for the determination of lead isotope ratios by inductively coupled plasma mass spectrometry C. Marisa R. Almeida and M. Teresa S. D. Vasconcelos* LAQUIPAI, Departamento de Quý�mica, Faculdade de CieÃncias, Universidade do Porto, Rua do Campo Alegre, 687, P4169-007 Porto, Portugal. E-mail: mtvascon@fc.up.pt Received 6th July 1999, Accepted 4th October 1999 An UV-irradiation pre-treatment for the determination of Pb isotope ratios (IRs) in Port wine by ICP-MS has been developed. Optimised conditions: mixtures of 20 ml Port wine and 120 ml 30% H2O2 (150 : 1 mixture) are UV-irradiated (mercury high pressure vapour lamp, 1000 W) in PTFE-capped silica tubes for 1.5 h; the sample is then Æltered and 10 times diluted with a 0.5% HNO3 solution.The procedure was compared with two other implemented methods: (1) UV-irradiation for 0.5 h of 10 ml mixture of 1 : 1 (v : v) Port wine and 30% H2O2, and dilution up to 50 ml with 0.5% HNO3 solution; and (2) high pressure MW-digestion of 1.5 ml aliquots of Port wine with 150 ml concentrated HNO3 and 1.5 ml 30% H2O2, and dilution up to 15 ml with de-ionised water.All these procedures were found to be suitable for determination of Pb IRs in Port wines. As the UVirradiation of 150 : 1 mixture of wine ±H2O2 has the advantage of requiring lower amounts of chemicals and providing larger volumes of solution for analysis of Port wine, it was selected for further studies. The method was tested for eight different samples of Port wine.A precision study showed that the standard deviation associated with the overall procedure (sample pre-treatment and subsequent determination) was mainly due to the ICP-MS determinations, the sample pre-treatment giving only a small contribution. Precisions (relative standard deviation) of about 0.3% for 207Pbz/206Pbz and for 208Pbz/206Pbz and about 0.8% for 204Pbz/206Pbz have been routinely obtained and shown to be sufÆcient to differentiate natural variations of the Pb isotope abundances in Port wine samples.Most of the lead (a toxic heavy metal) intake by man comes from food and beverages, while respiration through lungs and skin contributes only to a small extent. A small amount of the lead ingested can pass through the intestinal barrier and be transferred from the blood to the tissues where it is accumulated. The regular absorption of small amounts of lead may result in serious effects on human health, particularly in individuals at risk (people more susceptible to getting sick by reason of lead absorption). Therefore, efforts should be made in order to be able to control the levels of the metal intake.Lead contamination of wine is a problem dating back to ancient times. Along the oenological chain, contamination with lead can have multiple and diversiÆed origins: the soil of the vineyard, atmospheric precipitation, pesticides and fertilisers,1 materials used to produce, transport and store the wine, etc.The role of the different lead sources in the levels of the metal in the Ænal product is unknown but it is important to clarify this issue in order to be able to reduce the lead level in wine. Lead is composed of four stable isotopes, three of which are of radiogenic origin: the radioactive decay of 238U, 235U and 232Th generates, respectively, 206Pb, 207Pb and 208Pb. 204Pb is non-radiogenic.The respective abundances of these lead isotopes, originating from the genesis of the rocks and ore deposits, vary with geological ages and consequently with geographical locations.2 This property may be explored in order to identify the source of lead in a given sample. Until recently, Thermal Ionisation Mass Spectrometry (TIMS) has been considered the most suitable technique for determination of isotope ratios with enough precision to distinguish lead of different origins in a sample.ICP-MS is now also a suitable and convenient technique for that purpose3±9 being less time-consuming than TIMS.10 The application of ICP-MS to the determination of lead isotope ratios in wines is a very unexplored subject. As far as we know, there are only a few results published on table wines.11,12 A dilution with an acid solution is the only pre-treatment required for these type of wines.11,12 ICP-MS has never been applied before to fortiÆed wines in which the genuine Port wine, from Portugal, is included.Port wine is an aged fortiÆed wine, very rich in alcohol, particles in suspension and polymeric organic compounds, particularly sugars. All of these substances strongly interfere with ICP-MS measurements. Alcohol causes suppression of the signal (which drastically reduces the sensitivity) and signal instability.13 Although a signal depression does not necessarily prevent the isotopic ratio determinations, it may affect the measurement precision if the ion intensities of the different isotopes are low.The polymeric organic matter causes blockage of the injector tube and cones of the ICP, preventing the Øux of the ions of the analyte to the MS detector. Such blockage is the result of an incomplete pyrolysis of the sugars in the plasma and formation of residual carbon deposits (carbon build-up). Therefore, a simple dilution of the wine does not solve the problem of interference of the Port wine matrix in ICP-MS measurements, because the maximum dilution allowed on account of sensitivity (ten times) is not sufÆcient to reduce either the alcohol or the sugar to acceptable levels.Such problems could probably be reduced by the addition of a small stream of oxygen to the plasma region, but this procedure requires a speciÆc accessory not available in most of the ICP-MS apparatus. So, a more drastic sample pre-treatment of Port wine is required for ICP-MS measurements. The aim of this study was the development and optimisation of sample pre-treatment procedures suitable for the determination of lead isotope ratios in Port wine using ICP-MS, with precision sufÆcient to warrant the investigation of the provenance of the metal.Pre-treatment which requires less chemical oxidising, to J. Anal. At. Spectrom., 1999, 14, 1815±1821 1815 This journal is # The Royal Society of Chemistry 1999prevent contamination, and provides analytical signal intensity as high as possible in the wine samples, was investigated. Goossens et al.13 used classical acid digestion, in open vessels, for determination of total lead in a liqueur red wine by ICP-MS.As such methods are generally very time consuming and require the addition of high concentrations of reagents to the sample, procedures involving UV-irradiation that required less chemical addition were implemented. An UV-irradiation pre-treatment has been applied recently by Sanllorente et al.14 for determination of nickel in commercial table wines by differential-pulse adsorptive stripping voltammetry. For comparison purposes, a high pressure microwave digestion procedure was also implemented.The high pressure microwave digestion (HPMW-digestion) procedure has been used for determination of total metals concentrations in different types of samples using different analytical techniques, 15 including determination by ICP-MS of several trace metals in biological materials,16 chromium and nickel in human blood,17 rare earth elements in tea18 and arsenic, cadmium and lead in seafood products.19 In this paper the selected UV-irradiation pre-treatment is described in detail and the quality of the results it provides is discussed and compared with other implemented pretreatments requiring greater amounts of chemicals.Experimental Material and reagents Suprapure concentrated HNO3 (65% m/m, d~1.40 g ml21) and solution of 30% H2O2, p.a., from Merck, were used without further puriÆcation.A stock NIST SRM-981 Common Pb Isotopic Standard solution (1000 mg l21 of Pb) was prepared by dissolving a portion of the metal in 1% v/v HNO3. All the other reagents used were p.a. grade or equivalent. Standards solution were prepared daily from the stocks, in polyethylene tubes, by weight, with de-ionised water (resistivity w14 MV cm) or diluted HN, as necessary. All material was soaked in 20% v/v HNO3 for at least 24 h, rinsed several times with de-ionised water and dried in a Class 100 laminar Øow hood.The sample manipulation was carried out in a clean room with Class 100 Æltered air. Wine samples Eight samples of genuine Port wine from the Douro Region, North of Portugal, supplied (by the ``Instituto do Vinho do Porto'', Porto, Portugal) in small glass bottles, were used. They were of three different types: 2 Late Bottled Vintage (LBV) harvest of 1988 (LBV 88a and LBV 88b), 5 Dated wines (DP) harvests of 1935 (DP 35), 1952 (DP 52), 1969 (DP 69), 1974 (DP 74) and 1984 (DP 84), and one wine with an indication of age of 10 years (IA 10). LBV are wines from a single year of harvest (year of excellent quality) that are bottled between the fourth and sixth year after they were made; DP are also wines from a single year that are aged in wooden barrels for several years and can only be sold after they have attained 7 years of age; IA Port wines are similar in style to DP but, unlike the latter, are blended from wines of different years.All the wines studied were bottled in 1992. The UV-irradiation pre-treatment procedures implemented in this work were also applied to two other types of commercial available Portuguese fortiÆed wines: Madeira wine, from the island of Madeira, and Favaios wine, from the Douro Region. The wines were sampled by removing the cork and pouring the required volume into silica tubes of ca. 45 ml of capacity.UV-irradiation pre-treatment UV-irradiation of the samples was carried out with a 1000 W mercury high pressure vapour lamp (with 3 cm diameter and 6 cm length) from Osram, in polytetraØuoroethylene (PTFE) -capped silica tubes (with 2 cm diameter and 15 cm length) positioned concentrically around the lamp (distance between the lamp and the centre of the tubes was 5 cm). This system was inserted in a closed aluminium box, provided with appropriate ventilation, which protects workers against UV-radiation.The optimisation of the pre-treatment of the Port wine included the study of the inØuence on the analytical results of the following parameters: volume of sample (varied between 10 and 40 ml), time of UV-irradiation (between 0.5 and 2.0 h) and addition of different volumes of 30% H2O2 (wine :H2O2 proportions between 150 : 1 and 1 : 1 v/v). When the wine samples were hazed after UV-irradiation, they were Æltered through a 0.45 mm pore size Ælter of mixed cellulose esters, from Schleicher & Schuell, using a syringe with an appropriate Ælter holder. The treated samples were kept at 4 �C and analysed within 48 h, after dilution with 0.5% v/v HNO3 solution.For comparison purpose, dilution with de-ionised water or 1% HNO3 solution was also used. HPMW-digestion Aliquots of 1.5 ml of wine were digested with 150 ml concentrated HNO3 and 1.5 ml of 30% H2O2 in closed PTFE vessels in a high pressure microwave system MLS-1200 Mega, from Millestone, coupled to an exhaust EM-30 of the same brand.The digestion programme and procedure was based on that developed by Alimonti et al.17 for determination of Ni and Cr in blood by ICP-MS. After cooling to room temperature, the vessels were opened and the obtained solutions were quantitatively transferred to polyethylene tubes being diluted to 15 ml. The Ænal solutions were kept at 4 �C and analysed within 48 h. ICP-MS isotope ratios measurements The analytical measurements were carried out in a Perkin- Elmer SCIEX Elan 5000 ICP-MS (Perkin-Elmer, U» berlingen, Germany) apparatus equipped with a crossØow nebulizer, nickel cones and a peristaltic sample delivery pump.The operating conditions for ICP-MS measurements were optimised daily, by using a solution with 10 mg l21 of Mg, Rh and Pb and monitoring the isotopes 24Mg, 103Rh and 208Pb. Since doubly charge ions were not a concern and oxide ions were considered unlikely in the m/z 204-208 regions, the operating conditions that maximised the ion intensity for mass 208 were selected.Operating conditions used: RF power of 1200 W; sample uptake rate of 0.800 l min21; plasma Øow rate of 15.00 l min21, nebulizer Øow rate between 0.750 and 0.810 l min21, and auxiliary Øow rate of 0.800 l min21; the ions lens settings (in arbitrary units) were P~52, S2~24, B~70 and E1~15. For the optimisation of the data acquisition procedure, the inØuence of the instrument parameters: number of replicates (varied between 3 and 10, with one reading per replicate), dwell time (varied between 5 and 10 ms) and number of sweeps per reading (varied between 200 and 1500), on the three Pb isotope ratios (IRs) 204Pbz/206Pbz, 207Pbz/206Pbz and 208Pbz/206Pbz (determine individually or together) and on the respective relative standard deviation (RSD), obtained for a Pb isotopic standard solution (50 mg l21 Pb concentration) was studied.In the optimised procedure, in order to obtain the best precision (lowest RSD), only two Pb isotopes were measured each time, using 1500 sweeps per reading and a dwell time of 10 ms, and three replicates for each measurement were carried out.The Pb isotopes were measured using the peak hopping mode, at 1816 J. Anal. At. Spectrom., 1999, 14, 1815±1821normal resolution. The results of each measurement was the mean of the replicates with the respective standard deviation, after blank subtraction.Since 204Pb is the least abundant stable isotope of Pb, to obtain similar precision for the four isotopes this isotope signal was measured for twice (by choosing the ``time factor'' 2 in the parameter Æle of the ICP-MS software) as long as the other Pb isotopes' signals. Although wines usually have very low contents of Hg,12 the IR with the Pb isotope 204Pb may be altered if this correction is not performed. Therefore, a mathematical correction of 204Hg interference with 204Pb was systematically carried out (by the software of the equipment) for all the determinations: the net signal at m/z 202 (202Hgz) was multiplied by 0.229 (the 204Hg/202Hg natural abundance ratio) and then subtracted from the signal at m/z 204 (204Hgz plus 204Pbz).All the Pb IRs were corrected using the Pb isotopic standard20 (Pb concentration similar to that pre-estimated for the samples). In order to underscore a possible shift with time, every workday the standard was analysed Ærst and then after every two samples (for IR 204Pbz/206Pbz it was measured after every four samples, because it was more time consuming and for this IR the repeatability was higher than for the other two IRs).Due to this correction the standard deviation was calculated for each measurement according to propagation of errors (resulting from the determination of the Pb IR both in the sample and in the isotopic standard). The correction of the Pb IRs with Tl IR (constant natural IR 205Tl/203Tl~2.3871) was also carried out, as discussed elsewhere,21 but no signiÆcant differences were observed between the results obtained using both types of corrections.For signal stabilisation, a sample read delay of 1.5 min was chosen. Between solutions of samples or standard the sampling system was rinsed with 2% HNO3 for 1.5 min. Results and discussion Optimisation of a pre-treatment of Port wine for ICP-MS determinations Development of the UV-irradiation pre-treatment.Volumes of 10 ml of mixtures of Port wine and 30% H2O2, in v:v proportions of 150 : 1, 20 : 1, 5 : 1, 2 : 1 and 1 : 1, were UVirradiated, in parallel, for 0.5 h. The obtained solutions were diluted with a 0.5% HNO3 solution, in order to obtain a 10 times dilution of the wine, and the ion intensities of the different Pb isotopes were measured. That set of experiments was repeated for 1.0, 1.5 and 2.0 h of UV-irradiation. The inØuence of UV-irradiation time and wine: H2O2 proportions on the ion intensities of 207Pb and 206Pb isotopes are illustrated in Table 1.For the other Pb isotopes similar relative intensities and precisions were obtained. The different results were statistically compared through a test for comparison of several means, the least iÆcance difference (LSD) test.22 For 0.5 h of UV-irradiation the highest signal was obtained for the 1 : 1 mixture, indicating that the highest H2O2 concentration originated the most efÆcient elimination of matrix interference.For 1.0 h of irradiation no improvement of sensitivity was observed for the 1 : 1 mixture. In a few other solutions an improvement of the signals was observed but in all cases they were statistically lower than those obtained for 1 : 1 mixture. For 1.5 h of irradiation, statistically identical signals were obtained for 1 : 1, 20 : 1 and 150 : 1. For 5 : 1 and 2 : 1 mixtures lower signals were observed. Higher UV exposure time (2.0 h) improved neither the magnitude nor the precision of the signals, for any of the wine :H2O2 mixtures.Higher signals for 150 : 1 and 20 : 1 than for 5 : 1 and 2 : 1 mixtures were probably a result of evaporation of alcohol (an important interference in ICP-MS) during UV-irradiation which occurred only for the less diluted wine. In fact, after irradiation of 150 : 1 and 20 : 1 mixtures, their volumes were, respectively, 80% and 85% of the initial ones. The treated solutions where evaporation of the alcohol occurred became hazed and were Æltered before analysis.In terms of sensitivity, the 150 : 1 and 20 : 1 mixtures UVirradiated for 1.5 h and the 1 : 1 mixture UV-irradiated for 0.5 h provided statistically identical results (see Table 1). The 150 : 1 mixture has the advantage of enabling the treatment of a larger volume of wine per batch: up to 20 ml (higher volumes cannot be used because solution came out of the silica tube during the UV-irradiation due to the heating) provides about 160 ml of solution for analysis after a 10 times dilution of the treated solution (since about 4 ml evaporate during the UV-irradiation).For 1 : 1 wine :H2O2 mixture more than 10 ml (a quart of the vessel volume) cannot be used because a tumultuous release of gas occurs during UV-irradiation. Therefore, after a 10 times dilution of the wine, only 50 ml of Ænal solution is obtained. Larger solution volumes are useful when different analyses by ICP-MS have to be performed.The 150 : 1 mixture has the additional advantage of requiring much less volume of H2O2. One the other hand, the 1 : 1 mixture has the advantage of being less time consuming since it requires less time of UVTable 1 InØuence of UV-irradiation time and wine :H2O2 proportions on the ion intensities of 206Pb and 207Pb isotopes and on the respective precision, observed for a Port wine sample Wine :H2O2 Timea/h Haze after pre-treatment 206Pb ion intensityb61023/ion s21 207Pb ion intensityb61023/ion s21 150 : 1 0.5 No 8.4 (0.4) 7.34 (0.04) 1.0 Yesc 10.6 (0.1) 9.25 (0.03) 1.5 Yesc 13.08 (0.04) 11.39 (0.07) 20 : 1 0.5 No 11.1 (0.2) 9.74 (0.02) 1.0 No 11.5 (0.1) 10.04 (0.02) 1.5 Yesc 13.13 (0.04) 11.45 (0.02) 5 : 1 0.5 No 12.39 (0.06) 10.85 (0.07) 1.0 No 12.49 (0.02) 10.97 (0.04) 1.5 No 11.94 (0.03) 10.39 (0.04) 2 : 1 0.5 No 11.2 (0.2) 9.79 (0.02) 1.0 No 11.4 (0.1) 9.95 (0.04) 1.5 No 11.5 (0.1) 10.07 (0.02) 1 : 1 0.5 No 13.14 (0.05) 11.54 (0.07) 1.0 No 13.26 (0.02) 11.57 (0.01) 1.5 No 13.05 (0.03) 11.41 (0.07) aFor 2.0 h of UV-irradiation the results were statically identical to those of 1.5 h.bMean and standard deviation (n~3). cThe solutions were Æltered before the analysis. J. Anal. At. Spectrom., 1999, 14, 1815±1821 1817irradiation. The 20 : 1 mixture offers no advantages compared with the other two mixtures. For comparison purposes, UV-irradiation pre-treatments of 150 : 1 and 1 : 1 mixtures were applied, in parallel, to two Port wine samples.The results obtained for the different IRs were statistically identical (see Table 2). The same pre-treatments were also applied to other types of fortiÆed wines (one sample of Madeira wine and one sample of Favaios wine). The obtained results (not shown) suggested that both UV-irradiation procedures are also suitable for ICP-MS analysis of those fortiÆed wines. Since the 150 : 1 mixture requires lower addition of chemicals and can provide larger sample volume for analysis it was selected for subsequent studies.Optimisation of a HPMW-digestion. A more classical pretreatment procedure was also optimised and applied to Port wine. A sample volume of 1.5 ml was chosen, because it was a compromise between the maximum volume suitable for digestion in the closed vessels of the microwave system and the volume required for the ICP-MS determinations (after a 10 times dilution of the wine).The volumes of HNO3 and H2O2 used in the attack were varied between 150 ml and 1.5 ml and 150 ml and 3 ml, respectively. Volumes of 150 ml HNO3 and 1.5 ml H2O2 were selected because they provided clear solutions, and higher concentrations of this two reagents did not improved the analytical signals in ICP-MS. Comparison of MW-digestion and UV-irradiation procedures. The HP-MW-digestion and the UV-irradiation of 150:1 Port wine:H2O2 mixture pre-treatments were applied to Æve wines (DP 35, DP 74, LBV 88a, LBV 88b and IA 10), in parallel, for comparison.For each sample, two replicates were independently pre-treated by each procedure and the mean of the ICP-MS signals (three per replicate) and the respective RSD (n~6, calculated according to the propagation of errors) were obtained. The UV-irradiation procedure provided results with RSD between 0.045 and 0.330%, more precise than those obtained by microwave digestion whose RSD were between 0.100 and 1.82%.In Fig. 1 the results obtained for the various Pb IRs in samples pre-treated by both procedures are compared by linear regression. A linear least-squares adjustment of the global results for the different Pb IRs yield the equation: [Microwave]~( 1.003°0.003) [UV]2(0.0004°0.0037). When each IR was treated separately, the following equations were Table 2 Comparison, through the test of comparing two means,22 of the Pb IRs obtained with two UV-irradiation pre-treatments (see the text) (n~3) 207Pb/206Pba 208Pb/206Pba 204Pb/206Pba UV-irradiation (150 : 1) UV-irradiation (1 : 1) UV-irradiation (150 : 1) UV-irradiation (1 : 1) UV-irradiation (150 : 1) UV-irradiation (1 : 1) DP 52 0.857 (2) 0.859 (3) 2.101 (4) 2.103 (2) 0.0553 (3) 0.0550 (3) 0.858 (2) 0.857 (3) 2.102 (4) 2.100 (7) 0.0552 (1) 0.0552 (2) 0.858 (3) 0.858 (3) 2.100 (3) 2.098 (6) 0.0551 (3) 0.0551 (1) RSD: 0.106% RSD: 0.129% RSD: 0.063% RSD: 0.127% RSD: 0.105% RSD: 0.181% DP 69 0.866 (4) 0.865 (6) 2.100 (5) 2.109 (3) 0.0555 (2) 0.0554 (3) 0.866 (3) 0.864 (7) 2.099 (8) 2.099 (6) 0.0560 (4) 0.0555 (3) 0.866 (3) 0.866 (6) 2.094 (8) 2.103 (4) 0.0562 (2) 0.0554 (5) RSD: 0.027% RSD: 0.072% RSD: 0.165% RSD: 0.248% RSD: 0. 645% RSD: 0.104% aMean and standard deviation (calculated according to the propagation of errors, value affecting last digit) of each measurement. Fig. 1 Comparison, by linear regression, of the Pb IR obtained for Port wine samples pre-treated by 150 : 1 UV-irradiation and by microwave digestion (the °values are the 95% conÆdence limits). 1818 J.Anal. At. Spectrom., 1999, 14, 1815±1821obtained: [Microwave]~(0.98°0.44) [UV]2(0.02°0.37) for 207Pbz/206Pbz; [Microwave]~(1.0°0.8) [UV]2(0.03°1.62) for 208Pbz/206Pbz; and [Microwave]~(1.2°0.4) [UV]2(0.009°0.022) for 204Pbz/206Pbz. This statistical analysis showed no evidence of either relative or Æxed bias in the measured range. Therefore, both the proposed methods were considered to be acceptable for the determination of Pb IRs by ICP-MS.Analysis of blanks. The inØuence of the pre-treatment in the blank signals (ion intensities of the various Pb isotopes) is illustrated in Table 3. For comparison purposes, dilution of the pre-treated samples with de-ionised water or a 1% HNO3 solution were also carried out (see below) and the signals referring to those blanks (not submitted to the pre-treatment) and to a pre-treated wine sample are also shown. Although the ion intensities of Pb isotopes in the blank pre-treated by 150 : 1 UV-irradiation were signiÆcantly higher than those on the blank not UV-irradiated, they were signiÆcantly lower than those obtained for the other pre-treatments blanks, which included larger amounts of chemicals. A comparison of the signals for de-ionised water and the HNO3 solutions indicates that even the suprapure HNO3 contributes signiÆcantly to the blank signal. Nevertheless, Table 3 also shows that, in all cases, the blank signals were two to three orders of magnitude lower than the signals obtained for the Port wine samples.Precision of the Pb IR measurements The data acquisition procedure was optimised with the Pb isotopic standard NIST SRM-981, the Pb IRs of this Table 3 Comparison of the ion intensitiesa obtained for the different Pb isotopes in the blanks of the different pre-treatment procedures. Results for a Port wine solution were included for comparison 204Pb ion intensity/ion s21 206Pb ion intensity /ion s21 207Pb ion intensity /ion s21 208Pb ion intensity /ion s21 De-ionised H2O 3.1 (0.2) 7 (1) 8 (2) 10 (3) 0.5% HNO3 4.4 (0.7) 46 (1) 40 (1) 82 (3) 1% HNO3 6.2 (0.5) 56 (3) 46 (2) 111 (5) UV-irradiation (150 : 1) 17.7 (0.5) 143 (2) 134 (2) 259 (4) UV-irradiation (1 : 1) 24.1 (0.3) 385 (3) 339 (4) 818 (9) HPMW-digestion 30.5 (0.5) 537 (1) 465 (4) 1127 (3) DP 84 wine 7.67 (0.02)6102 141.6 (0.2)6102 123.8 (0.4)6102 301.5 (0.5)6102 aMean and standard deviation (n~3).Different replicates provided statistically identical results. Fig. 2 Variations of the IRs of the Pb isotopic standard NIST SRM- 981, observed over a period of 50 days (s~standard deviation of the mean). CertiÆed values for 207Pbz/206Pbz, 208Pbz/206Pbz and 204Pbz/206Pbz are 0.91464°0.00033, 2.1681°0.0008 and 0.059042°0.000037, respectively. Fig. 3 Pb IRs (mean and standard deviation, n~3), obtained for eight different samples of Port wine. The symbol (*) indicates a sample that displayed IRs signiÆcantly different from all the others, according to the least signiÆcance difference (LSD) test.22 The other symbols (#,+, %) indicate samples with IRs signiÆcantly different between themselves (for example, DP 35 and DP 84 displayed 207Pbz/206Pbz signiÆcantly different, indicated by %).J. Anal. At. Spectrom., 1999, 14, 1815±1821 1819standard (207Pbz/206Pbz, 208Pbz/206Pbz and 204Pbz/206Pbz, certiÆed values: 0.91464°0.00033, 2.1681°0.0008 and 0.059042°0.000037, respectively) being determined every working day.Considering a period of about two months, the means and respective RSDs of all the determinations (n~ 11±13) were calculated (see also Fig. 2): 207Pbz/206Pbz~ 0.920, RSD: 0.08%; 208Pbz/206Pbz~2.208, RSD: 0.17%; 204Pbz/206Pbz~0.0582, RSD: 0.38%. The precisions for long-term measurements were similar to those obtained for short-term measurements (except for 204Pb/206Pb ratio, to which short-term precision was lower) and even for single determinations of the respective Pb IR.The long-term precision obtained for the 207Pbz/206Pbz ratio was better than that obtained for the same Pb isotopic standard by Dean et al.11 and Campbell et al.,23 by using VG PlasmaQuad ICPMS apparatus, but was similar to that obtain by Halicz et al.10 using a Perkin-Elmer SCIEX Elan 6000 ICP-MS. For the 204Pbz/206Pbz ratio, a worse precision was obtained, compared to that obtained for the other two Pb IRs, probably due to poor counting statistic on the 204Pb isotope relative to the other Pb isotopes, since this is the least abundant isotope.Stroh et al.24 reported that the addition of acid to table wines increases the stability of the signals for Pb IRs determined by ICP-MS. In order to test the inØuence of acid in the signals, measurements were carried out, in parallel, in samples pretreated by the chosen procedure either not acidiÆed or acidiÆed with 0.5% or 1% HNO3.Three different Port wine samples and the Pb isotopic standard solution were analysed. It was observed that the acidiÆcation improved markedly the intensity of the ion intensities but not the precision of the Pb IRs (results not shown). However, no signiÆcant differences were observed, by the statistical test of comparison of two means,22 between the results obtained in solutions with 0.5% and 1% HNO3. From these results it was decided to acidify to 0.5% HNO3 all the subsequent solutions (samples, blanks and standards) before the ICP-MS measurements. In order to test the repeatability of the results obtained with the chosen pre-treatment procedure, three replicates of each wine sample were independently pre-treated and analysed.The respective mean and variance were calculated and compared with the mean and variance obtained for a single pre-treated sample which was analysed three times. All the experimental work was carried out in a single working day.Since the measurement of each Pb IR is affected by an error resulting from its own determination and that of the Pb isotopic standard, and since three analyses were performed for each wine, the variance associated with the respective mean was calculated according to propagation of errors. The obtained results, presented in Table 4, show that the variance obtained for the three independently pre-treated samples were of the same order of magnitude as that obtained for a single pretreatment. 204Pbz/206Pbz data referring to the wine samples DP 69 and LBV 88a are not presented due to insufÆcient sample volume but similar results are expected. These results indicated that the implemented pre-treatment did not contribute markedly to the variance of the overall method (pre-treatment plus determination). The decisive factor for the precision of the obtained Pb IRs being the ICP-MS determinations. Pb IRs in Port wines The three different Pb IRs (207Pbz/206Pbz, 208Pbz/206Pbz and 204Pbz/206Pbz) were determined in eight Port wine samples of different ages and types (Fig. 3). The RSDs associated with the mean values of the 207Pbz/206Pbz and 208Pbz/206Pbz ratios in the wine samples were between 0.15 and 0.50%, being lower than 0.3% in most of the cases. Similar precisions were obtained by Dean et al.11 and Table 4 Precision of the Pb isotope ratios observed in different Port wine samples 207Pb/206Pb Analysis of three aliquots of a single UV-irradiated solution Analysis of three independently UV-irradiated solutions s2 (UV-irradiation)a DP 52 Mean 0.858 0.858 s2 1.261025 1.8610-5 5.6610-6 DP 69 Mean 0.866 0.866 s2 2.461025 3.461025 1.061025 DP 74 Mean 0.858 0.860 s2 3.961025 3.661025 22.861026 DP 84 Mean 0.870 0.871 s2 3.761025 4.361025 6.361026 LBV 88a Mean 0.862 0.862 s2 1.261025 2.461025 1.261025 208Pb/206Pb DP 52 Mean 2.100 2.101 s2 7.861025 9.761025 1.961025 DP 69 Mean 2.099 2.097 s2 1.961024 1.661024 23.661025 DP 74 Mean 2.088 2.086 s2 1.861024 1.761024 21.261025 DP 84 Mean 2.092 2.093 s2 6.461025 1.261024 5.761025 LBV 88a Mean 2.084 2.080 s2 9.461025 1.361024 3.861025 204Pb/206Pbb DP 52 Mean 0.0552 0.0552 s2 1.161027 2.761027 1.361027 DP 74 Mean 0.0557 0.0556 s2 2.261027 2.561027 3.261028 DP 84 Mean 0.0557 0.0557 s2 2.461028 2.861027 2.661027 aDifference between the total variance (analysis of three independently UV-irradiated solution) and the determined variance (analysis of three aliquots of a single UV-irradiated solution).bNot measured in the wines DP 69 and LVB 88a, due to insufÆcient sample volume. 1820 J. Anal. At. Spectrom., 1999, 14, 1815±1821Augagneur et al.12 in table wines and were considered to be sufÆcient to differentiate natural variation of the Pb isotope abundances.12 For the 204Pbz/206Pbz ratio the RSDs were between 0.2 and 1%, seven of the eight samples displaying RSD values lower than 0.8%. Therefore, the precision of this IR was worse than that obtained for the remainders, presumably entirely due to poor counting statistic on the 204Pb isotope, relative to the other Pb isotopes, since this is the least abundant isotope. A similar result was observed by Goossens et al.25 in table wines and is typical of IRs involving an isotope of low abundance, such as 204Pb.26 With the purpose of testing if among the analysed Port wines signiÆcant differences in the values of the Pb IRs occurred, a test for comparison of several means, the LSD,22 was applied.As shown in Fig. 3, some signiÆcant differences were found (values of Pb IR indicated with the same symbol). For example, the LBV 88a wine displayed both 207Pbz/206Pbz and 208Pbz/206Pbz ratios signiÆcantly different from those of all the other wines and a 204Pbz/206Pbz ratio signiÆcantly different from that of the DP 74, DP 84 and LBV 88b wines. These results indicate that the precision of the Pb IRs obtained with the proposed method was sufÆcient to distinguish Pb isotopic composition in some of the Port wines analysed. SigniÆcant correlation (Pv0.05)22 between the age of DP wines and the 207Pbz/206Pbz ratio or the 204Pbz/206Pbz ratio were found, but at this stage no explanation can be provided for this observation.SigniÆcant correlation between the age of the wines and the 208Pbz/206Pbz ratio was not observed. The two LBV wines, although with the same age and designation, displayed signiÆcantly different Pb IRs.This indicates that the Pb present in the LBV 88a wine was from a source different of that of the LBV 88b. 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