Supported Liquid Membrane Enrichment Combined With Atomic Absorption Spectrometry for the Determination of Lead in Urine Nii-Kotey Djanea, Ingvar A. Bergdahlb, Kuria Ndung’ua, Andrejs Sch�utzb, Gillis Johanssona and Lennart Mathiasson*a a Department of Analytical Chemistry, University of Lund, P.O. Box 124, S-221 00 Lund, Sweden b Department of Occupational and Environmental Medicine, University Hospital, S-221 85 Lund, Sweden Supported liquid membrane (SLM) methodology was used for sample clean-up and enrichment of lead in urine prior to determination by AAS.Lead ions at pH 3 were extracted across a membrane solution containing 40% m/m di-2-ethylhexylphosphoric acid dissolved in kerosene and back-extracted into an acceptor solution of 1 mol l21 nitric acid. The mechanism of mass transfer is a proton gradient across the membrane. The concentration range investigated was between 5 and 80 ng ml21 and the extraction time was varied between 0.5 and 4 h, leading to enrichment factors of up to 200.The extraction efficiency was about 95%. The detection limit, expressed as 3s of five replicate determinations, using a 45 min enrichment of a urine sample low in lead, was 0.1 and 6.0 mg l21 for ETAAS, and FAAS respectively. The results obtained by the developed method agreed with those obtained by direct ICP-MS determinations for reference urine samples and samples from occupationally lead-exposed workers. The linear correlation coefficient was 0.97, the slope of the regression line was 1.06 and the intercept was 20.37 mg l21.The 95% confidence intervals for the slope and the intercept were 0.95 to 1.18 and 23.9 to 3.1, respectively. The results at the 95% confidence level for reference urine material were of 91 ± 1.5 and 92 ± 2.0 mg l21 for ICP-MS and SLM–AAS, respectively, which agreed well with the recommended value of 90 mg l21 (range 83–97 mg l21). Keywords: Lead; urine; supported liquid membranes; atomic absorption spectrometry; inductively coupled plasma mass spectrometry; carry-over effects Different enrichment techniques have been described for the determination of trace levels of heavy metals in urine, e.g., preconcentration by electrodeposition in combination with electrothermal atomic absorption spectrometry (ETTAS)1–3 and stripping techniques.4–6 Sample preparation methods based on liquid–liquid extraction7–10 are now gradually being replaced by solid-phase extractions. In the latter case, the facilitated online connection to the final analysis equipment increases the possibilities for automated analysis.11,12 Another modern approach to sample clean-up and enrichment is to use supported liquid membrane (SLM) methodology.This technique has been shown to give efficient clean-up of organic analytes in urine and plasma samples.13 In a recent paper,14 we reported the use of di- 2-ethylhexylphosphoric acid (DEHPA) as the extractant in an SLM-based procedure for the determination of some heavy metal ions including lead in both reagent water and natural water samples.The use of a proton gradient as the main transport mechanism eliminates the use of buffers that are often contaminated with the analytes of interest. In this study, we demonstrated the possibility of extending the DEHPA extraction system to the determination of lead in a biological matrix, urine. For comparison, direct determination of lead in dilute urine samples was carried out using inductively coupled plasma mass spectrometry (ICP-MS).Experimental Equipment Atomic absorption spectrometry The ETAAS system was a Varian AA-1475, fitted with a GTA- 95 graphite tube atomiser and a programmable sample dispenser (Varian, Victoria, Australia). Deuterium lamp background correction was used. The furnace programme is given in Table 1. The flame AAS (FAAS) system was a Varian AA-6. Parameter settings, such as lamp current and wavelength, were those recommended by the manufacturer. Inductively coupled plasma The possibility of using ICP-MS for the direct determination of metals in environmental and biological samples has been demonstrated in a number of studies.15–18 For lead in blood samples, ICP-MS has proved to be very useful.19,20 The ICPMS system used was a Plasma Quad PQ2 Plus (Fison Elemental, Winsford, Cheshire, UK).SLM equipment The SLM device used was similar to one described previously, 21 except that a modification was made to facilitate membrane replacements.The device consists of two identical circular PTFE blocks (diameter 120 mm, thickness 15 mm) with grooves cut in an Archimedes spiral (depth 0.25 mm, width 1.5 mm, length 250 cm), giving channels with a total volume of 1 ml. A porous PTFE membrane with a polyethylene backing (pore size 0.2 mm, porosity 0.70, total thickness 175 mm, of which 115 mm is the polyethylene backing; Type FG, Millipore, Table 1 Graphite furnace programme: wavelength 217.0 nm, lamp current 4.0 mA, slit 1.0 nm, slit height normal, background correction on.Pyrolytic graphite-coated graphite tubes were used Argon gas Tempera- Ramp Hold flow rate/ Step ture/°C time/s time/s l min21 Drying 90 5.0 10.0 3.0 Drying 120 5.0 30.0 3.0 Ashing 400 5.0 20.0 3.0 Ashing 400 0.0 2.0 0.1 Atomization 2000 1.0 2.0 0.1 Cleaning 2200 1.0 1.0 3.0 Analyst, October 1997, Vol. 122 (1073–1077) 1073Bedford, MA, USA) was impregnated by soaking it for at least 15 min in kerosene containing 40% m/m of DEHPA. The membrane was then placed between the two PTFE blocks and clamped with a stainless-steel screw in the middle.The screw was covered with a polyetherether ketone (PEEK) tube to prevent contact between the steel surface and the solutions in the membrane channels. Chemicals All solutions were prepared from either Suprapur or analyticalreagent grade chemicals and high-purity water was obtained from a MilliQ-RO4 unit (Millipore).Polyethene bottles were used throughout and were cleaned with 4 m HNO3 for at least 2 d and rinsed with high-purity water before use. Nitric acid (Suprapur) was obtained from Merck (Darmstadt, Germany) DEHPA (95%) from Sigma Chemicals (St. Louis, MO, USA), kerosene from Kebo Lab (Malm�o, Sweden) and lead nitrate stock standard solution (1000 mg ml21) from BDH (Poole, Dorset, UK). Freeze-dried urine (batch 403125, Seronorm, Nycomed, Oslo, Norway) was used as a reference material.The recommended value of lead was 90 (range 83–97) mg l21. A synthetic urine sample was prepared by dissolving 2.08 g of creatinine (Sigma), 1.25 g of urea (BDH), 14.1 g of NaCl, 2.8 g of KCl, 0.794 g of CaCl2·2H2O, 0.88 g of MgSO4·7H2O and 1.9 ml of ammonia solution (all from Merck) in high-purity water and diluting to 1000 ml.22 Sampling and Sample Pre-treatment Urine samples were obtained from lead-exposed workers at a secondary lead smeltery.Each individual was provided with a 2 l polyethylene bottle and was instructed to collect urine at home, but not in the smeltery. All urine samples used in the preliminary experiments were obtained from occupationally non-exposed subjects. The urine samples were preserved by adjusting the pH to 3.0 with concentrated nitric acid to prevent the precipitation of calcium phosphate and the consequent loss of heavy metals due to coprecipitation or adsorption on the precipitate.Usually, urine has a pH value in the range 5.0–6.5, and 2.5–3.0 ml of concentrated nitric acid were needed for each litre of urine to adjust the pH to 3.0. The freeze-dried reference urine samples had a pH below 3.0 after reconstitution with highpurity water. Prior to SLM–AAS analysis the pH was adjusted to 3.0 with ammonia solution. The final concentration of analyte in these samples solution was 20 mg l21. These samples were processed in the same manner as the other samples for 45 min and at a flow-rate of 0.9 ml min21.For the ICP-MS determinations, the reconstituted freeze-dried urine sample was analysed after a 10-fold dilution. Enrichment and AAS Determinations Fig. 1 shows the set-up for preconcentration of lead. It is similar to that described previously.14 Briefly, the sample is pumped at a flow rate of 1 ml min21 with the peristaltic pump A. After thrichment, the acceptor solution is pumped with the peristaltic pump B to a vial placed in a fraction collector (D).The pneumatic valve A directs the sample stream to the donor channel in the SLM unit, and valve B directs fresh solution to the acceptor channel and keeps a closed loop on the stagnant acceptor during the enrichment step. Cleaning of the tube between valve C and the fraction collector D is done with peristaltic pump C for off-line analysis using ETAAS or FAAS. Normally, 2 ml of acceptor solution are used to transfer the enriched sample to a vial.To reduce possible carry-over effects due to slow kinetics at the membrane–acceptor interface, the acceptor channel was usually washed with 10 ml of the stripping solution at a flow rate of 1.0 ml min21 after each preconcentration procedure. pH changes in the acceptor during the extraction process were monitored as described previously.14 Experiments concerning analyte recovery were carried out on natural urine samples spiked with known concentrations of lead.The extent of carry-over effects was investigated by collecting and analysing 2 ml fractions of the acceptor solution, collected 0, 5 and 10 min after the preconcentration. Each collection procedure took about 160 s. ICP-MS Determinations The urine samples were acidified by adding 2% v/v concentrated nitric acid (Aristar, BDH), and left overnight. Lead was determined according to a method used previously for whole blood.19 Briefly, the samples were analysed after a 10-fold dilution with a solution containing EDTA, Triton X-100 and ammonia., The samples were introduced into the ICP-MS equipment in a flow injection-like manner (segmented flow mode; 14 s uptake, 24 s acquisition).Bismuth and thallium were used as internal standards. Validation The validation of the proposed method was carried out by comparing the results of ICP-MS and SLM–AAS determinations. Results and Discussion Influence of Enrichment Time on Acceptor pH During the use of DEHPA as carrier in the extraction of lead from natural waters, a donor pH of 3 and an acceptor pH of 0 have been found to be suitable for the mass transfer of analytes to the acceptor side.14 Accordingly, these conditions were chosen for the investigation of the influence of the enrichment and the pH of the acceptor solution.Fig. 2 shows the influence of enrichment time on acceptor pH. It is seen that the pH on the acceptor side rises steadily and reaches a value of about 1.6 after approximately 4 h.Extraction Efficiency Table 2 gives the extraction efficiencies from synthetic urine and diluted spiked natural urine. The extraction efficiencies for lead in spiked natural urine and synthetic urine are 93 and 95%, respectively, which are in general slightly higher than those obtained in diluted urine. Using a two-tailed Student’s t-test, the Fig. 1 Experimental set-up for metal enrichment. 1074 Analyst, October 1997, Vol. 122differences between diluted and undiluted urine samples was not significant at the 95% confidence level except for the samples diluted to 30 and 50%.However, the difference is not significant at the 99% confidence level. Hence there is no reason to dilute the urine samples as this will lead to higher detection limits. Further, it may be concluded that physiologically normal variations between the urine samples will not influence the extraction efficiency. For reliable quantification it is important to work under conditions where the enrichment factor is constant.The influence of the enrichment time on the extraction efficiency at the normal sample flow rate of 1 ml min21 is shown in Table 3. The extraction efficiency (Table 3) is independent of the enrichment time up to at least 2 h. The value at 4 h is lower but the difference between this value and the other values in Table 3 is not significant at the 95% confidence level using a twotailed Student’s t-test. We have found previously that the pH gradient across the membrane should be about 2 pH units in order to keep the mass transfer rate constant.14 After 4 h, the difference is about 1.4 pH units (Fig. 2). However, the driving force is the difference in H+ concentration across the membrane. Since the pH scale is logarithmic, the difference in pH may be relatively smaller at high H+ concentrations, without giving significant changes in extraction efficiency, which is clearly demonstrated here. The change in extraction efficiency with time depending on the increase in pH on the acceptor side does not seriously affect the long-term stability.Experiments have been run continuously at a flow rate of 1 ml min21 on the same membrane for periods of up to 48 h with replacement of the acceptor solution every 2.5 h without any significant changes in extraction efficiency. In this respect, the behaviour is similar to what has been observed previously for lead in reagent water and natural waters, where the long-term stability was confirmed for periods of at least 30 and 200 h for natural and reagent waters, respectively.14 The detection limit is inversely related to the enrichment time as long as the extraction efficiency is independent of time. A 4 h run at 1 ml min21 (with negligible decrease in extraction efficiency) results in a processed sample volume of 240 ml.With an acceptor volume of approximately 1.0 ml and an extraction efficiency for lead of about 90%, an enrichment factor of more than 200 is obtained.Carry-over Effects in the Membrane System In some SLM systems, the mass transfer kinetics at the membrane–acceptor interface can be slow, which has been shown previously, e.g., in the enrichment of surfactants using ion-pair formation.23 It is therefore necessary in the investigation of new SLM applications always to consider the extent of carry-over effects, which may occur as a result of limited mass transfer kinetics in the actual system.Table 4 shows the extraction efficiencies of lead at different elution times after the completion of an enrichment. The first 2 ml fraction was collected immediately after the enrichment, the second after a waiting time of 5 min, and the third after 10 min. From Table 4, it may be concluded that a 10 min wash of the acceptor channel is sufficient to remove about 98% of the enriched lead. This makes memory effects negligible, provided that the concentration difference between samples is reasonable.Quantification Effects of initial donor concentration on lead recovery The concentration dependence was tested by spiking urine samples with lead in the concentration range 5–80 mg l21 and performing a 30 min SLM enrichment at a flow rate of 1.0 ml min21 followed by analysis by ETAAS for spiked Fig. 2 Change in acceptor pH with time during SLM processing of a urine sample at pH 3.0 with a flow rate of 1 ml min21. Acceptor solution, 1.0 m nitric acid; membrane liquid, 40% m/m DEHPA in kerosene.Table 2 Extraction efficiency of lead in spiked synthetic urine and spiked natural urine of various dilutions in high-purity water (n = 5). Standard deviations are given in parentheses. Lead concentration, 50 mg l21; sample pH, 3.0; acceptor solution, 1.0 m HNO3; enrichment time, 60 min; flow rate approximately 1.0 ml min21 Urine in Extraction Urine in Extraction water (%) efficiency (%) water (%) efficiency (%) 5 91.6 (3.1) 60 90.5 (2.4) 10 88.5 (5.5) 70 88.9 (3.2) 20 89.0 (3.7) 80 94.4 (8.5) 30 88.5 (2.5) 90 93.5 (2.9) 40 90.0 (3.8) 100 93.2 (2.9) 50 89.2 (2.1) Synthetic urine 95.3 (3.0) Table 3 Extraction efficiency of lead in spiked natural urine at different enrichment times. Standard deviations are given in parentheses.Lead concentration 50 mg l21; sample, pH 3.0; acceptor solution, 1.0 m HNO3; sample flow rate, approximately 1.0 ml min21 Extraction efficiency (%) Time/h (SD) n 0.5 94.3 (1.3) 6 1.0 94.7 (1.4) 6 2.0 95.3 (1.2) 6 4.0 93.5 (4.0) 4 Table 4 Carry-over effects using SLM methodology for enriching lead in a spiked urine sample investigated using two different porous membrane filters.Two enrichments on each filter. Average values are given. Standard deviations are given in parentheses. Lead concentration, 5.5 mg l21; membrane liquid, 40% m/m DEHPA in kerosene; sample pH, 3.0; acceptor solution, 1.0 m HNO3; flow rate 0.90–1.0 ml min21; enrichment time, 100 min.The eluted volume from the acceptor after each waiting time was 2 ml Waiting time/ Extraction efficiency min (%) 0 93.1 (0.5) 93.0 (0.8) 5 5.3 (1.1) 4.3 (1.5) 10 0.4 (2.0) 0.5 (1.0) Analyst, October 1997, Vol. 122 1075concentrations below 25 mg l21 and FAAS for higher concentrations. The results are given in Table 5. Applying a Student’s t-test for the differences in Table 5 between the recoveries obtained at different concentrations and the average recovery value of 93.8% revealed that there were no significant differences at the 95% confidence level for any of the concentrations in the range considered. Detection limit The detection limit was determined as three times the standard deviation of five replicate determinations of lead in a urine sample from an occupationally unexposed individual.In two different experiments with enrichment times of 45 min, with a flow rate of 1 ml min21 and ETAAS determination, the detection limit was 0.1 mg l21.This value is well below the concentrations found in unexposed individuals. The corresponding value for FAAS is 6.0 mg l21. This is sufficient for measuring samples from workers with moderate lead exposure. The concentration of lead in occupationally exposed workers is normally above 10 mg l21. Validation In the validation using the reference urine samples, SLM–AAS gave a lead value of 92.0 ± 2.0 mg l21 (five replicates) at the 95% confidence level assuming a Student’s t distribution.This was in accordance with the recommended value of 90 (range 83–97) mg l21 (five replicates). The corresponding result for ICP-MS was 91.0 ± 1.5 mg l21 (five replicates) at the 95% confidence level. The results obtained with SLM–AAS and ICP-MS agreed well with each other and with the certified values. The plot of the results in this comparison (samples from lead-exposed workers) is illustrated in Fig. 3. Linear correlation24 based on 22 samples gave a slope of 1.06, an intercept of 20.37 mg l21 and a correlation coefficient of 0.97.The 95% confidence intervals for the slope and intercept were 0.95 to 1.18 and 23.9 to 3.1, respectively. Since these intervals include the ideal values of 1 and 0 for the slope and the intercept, respectively, there is no evidence of a systematic difference between the two sets of results. The relative deviations between the ICP-MS and the SLM– AAS values are larger for low concentrations.At low concentrations, lead was determined by ETAAS in the enriched samples. The larger deviations within this concentration range (below 20 mg l21) are probably due to technical problems related to the background correction in ETAAS. In some experiments using ETAAS, RSD values of !20% were encountered. Since FAAS generally gives good precision (RSD Å 4% in the considered concentration interval here), the final analysis could be based entirely on FAAS by prolonging the enrichment time for low-concentration samples.The longer times needed do not necessarily decrease the sample throughput. With a multi-channel system in an off-line configuration, many samples can be simultaneously enriched by the SLM technique with low labour input. The relatively low cost of FAAS equipment, low operational cost, robustness and good stability of the FAAS system together with short analysis times makes SLM–FAAS a powerful alternative. The results in Tables 2, 4 and 5 indicate that neither the matrix composition nor the initial analyte concentration influences the extraction efficiency.However, these experiments were performed on the same sample bulk. In the comparison experiments, samples were collected from different individuals. The overall agreement between the results obtained with SLM– FAAS and ICP-MS in this comparison indicates that individual variations in the urine composition do not influence the results in any significant way.Furthermore, the present sample cleanup process using SLM does not seem to affect the results in any significant way. Using SLM with a cation-exchange extractant, negatively charged lead complexes, for example, could have been excluded from the membrane transport process. Even species which under the given conditions in the donor solution can form neutral complexes with lead might not contribute to the mass transfer of lead across the membrane, thereby decreasing the extraction efficiency to values below 100%.The most important compounds to be considered are probably chloride, phosphate, sulfate, citrate and oxalate ions, which are normally present in undiluted urine at concentrations of about 6000, 1200, 180 and 10–50 mg l21, respectively. Also, high concentrations of urea and creatinine could have some effects. However, these changes must be small. In the validation experiment the creatinine concentration varied between 5.2 and 20.9 mm.According to the literature,25 the normal variation of creatinine in urine is 0.13–0.22 mm kg21. Hence for a person with a mass of 70 kg, the normal level of creatinine should vary from 9.1 to 15.4 mm. Obviously the variation of creatinine in the samples investigated exceeded this range without leading to a significant influence on the extraction efficiency of lead. Concerning urea, the dilution experiments in Table 3 show that dilution has a negligible effect on the recovery of lead.The range was further investigated by comparing the recovery of lead in synthetic urine samples with normal urea concentrations (20 mm) with samples with about three times higher (62 mm) concentrations. It was found that the difference between the average values (based on three determinations at 20 mm and six determinations at 62 mm) were less than 1%. Hence the method developed seems to be well suited for clinical measurements. Table 5 Recovery of lead in natural urine spiked with different lead concentrations.The background concentration of lead, 0.9 mg l21, obtained as an average for six unspiked natural urine samples, was subtracted before calculating the extraction efficiency. The number of experiments at each concentration was six. Standard deviations are given in parentheses. Lead concentration, 5–80 mg l21; sample pH, 3.0; acceptor solution, 1.0 m HNO3; enrichment time, 30 min; flow rate, 1.0 ml min21 Lead concentration/ Recovery mg l21 (%) 5 92.4 (3.5) 10 94.8 (5.9) 20 94.0 (5.8) 50 95.3 (1.7) 80 92.7 (6.5) Fig. 3 Validation of the SLM–AAS methodology for lead determination by comparison with results obtained with ICP-MS. Sample pH, 3.0; acceptor solution, 1.0 m nitric acid; membrane liquid, 40% m/m DEHPA in kerosene. 1076 Analyst, October 1997, Vol. 122Conclusion We have shown that SLM combined with AAS can be used for the determination of lead in urine samples. By extending the enrichment time to 2 h, a detection limit of 2 mg l21 is obtainable using FAAS, thus covering the whole range of interest at occupational exposure.The methodology could be extended to other metals such as Cd, Cu and Cr, which will also give high extraction efficiencies in the chosen SLM system. For ICP-MS determinations, the flexibility of the SLM technique may be potentially useful when sample clean-up procedures are necessary, to avoid spectral interferences from certain elements.This work was made possible by financial support from the Swedish Environmental Protection Agency and the Medical Faculty at Lund University. The authors are grateful to Docent J. Å. J�onsson for fruitful suggestions. They also thank workers of Boliden Bergs�oe AB, Landskrona, for providing the urine samples. Mr. A. Ekholm and Mr. F. Malcus are acknowledged for skilful technical assistance. References 1 Zhang, G., Li, J., Fu, D., Hao, D., and Xiang, P., Talanta, 1993, 40, 409. 2 Boxing, X., Ming, X. T., Neng, S. M., and Ahi, F. Y., Talanta, 1985, 32, 1016. 3 Wolff, E. W., Landy, M. P., and Pell, D. A., Anal. Chem., 1981, 53, 1566. 4 Connor, M., Dempsey, E., Smyth, M. R., and Richardson, D. H. S., Electroanalysis, 1991, 3, 331. 5 Agraz, R., Seilla, M. T., Pinilla, J. M., and Hernandez, L., Electroanalysis, 1991 393. 6 Agraz, R., Sevilla, M. T., and Hernandez, L., Anal. Chim. Acta, 1993, 273, 205. 7 Bruland, K. W., Franks, R. P., Knauer, G. A., and Martin, J.H., Anal. Chim. Acta, 1979, 105, 233. 8 Pedersen, B., Willems, M., and Jørgensen, S., Analyst, 1980, 105, 119. 9 Smith, C. L., Motooka, J. M., and Willson, W. R., Anal. Lett., 1984, 17, 1715. 10 Ping, L., Matsumoto, K., and Fuwa, K., Anal. Chim. Acta, 1983, 147, 205. 11 Sperling, M., Yin, X., and Weiz, B., J. Anal. At. Spectrom., 1991, 6, 615. 12 Malamas, F., Bengtsson, M., and Johansson, G., Anal. Chim. Acta, 1984, 160, 1. 13 Lindegråd, B., J�onsson, J.-Å., and Mathiasson, L., J.Chromatogr., 1992, 573, 191. 14 Djane, N.-K., Ndung’u, K., Malcus, F., Johansson, G., and Mathiasson, L., Fresenius’ J. Anal. Chem., 1997, 358, 822. 15 Lu, P.-L., Huang, K.-S., and Jiang, S.-J., Anal. Chim. Acta, 1993, 284, 181. 16 Houk, R. S., Fassel, V. A., Svec, H. J., Gray, A. L., and Taylor, C. E., Anal. Chem., 1980, 52, 2283. 17 Douglas, D. J., and Houk, R. S., Prog. Anal. At. Spectrom., 1985, 8, 1. 18 Vanhoe, H., J. Trace Elem. Electrolytes Health Dis., 1993, 7, 131. 19 Bergdahl, I. A., Schutz, A., Geraldsson, L., Jensen, A., and Skerfving, S., Scand. J. Work Environ. Health, in the press. 20 Sch�utz, A., Bergdahl, I. A., Ekholm, A., and Skerfving, S., J. Occup. Med., 1996, 53, 736. 22 Papantoni, M., Djane, N.-K., Ndung’u, K., J�onsson, J.-Å., and Mathiasson, L., Analyst, 1995, 120, 1471. 22 Krushevska, A., Barnes, R. M., and Amarasiriwaradena, C., Analyst, 1993, 118, 1175. 23 Miliotis, T., Knutsson, M., J�onsson, J. Å., and Mathiasson, L., Int.J. Environ. Anal. Chem., 1996, 64, 35. 24 Miller, J. C., and Miller, J. N., Statistics for Analytical Chemistry, Ellis Horwood, Chichester, 3rd edn., 1994. 25 The Merck Manual of Diagnosis and Therapy, ed. Berkow, R., and Fletcher, J. A., Merck, Rahway, NJ, 16th edn., 1992. Paper 7/02340E Received April 7, 1997 Accepted June 23, 1997 Analyst, October 1997, Vol. 122 1077 Supported Liquid Membrane Enrichment Combined With Atomic Absorption Spectrometry for the Determination of Lead in Urine Nii-Kotey Djanea, Ingvar A.Bergdahlb, Kuria Ndung’ua, Andrejs Sch�utzb, Gillis Johanssona and Lennart Mathiasson*a a Department of Analytical Chemistry, University of Lund, P.O. Box 124, S-221 00 Lund, Sweden b Department of Occupational and Environmental Medicine, University Hospital, S-221 85 Lund, Sweden Supported liquid membrane (SLM) methodology was used for sample clean-up and enrichment of lead in urine prior to determination by AAS.Lead ions at pH 3 were extracted across a membrane solution containing 40% m/m di-2-ethylhexylphosphoric acid dissolved in kerosene and back-extracted into an acceptor solution of 1 mol l21 nitric acid. The mechanism of mass transfer is a proton gradient across the membrane. The concentration range investigated was between 5 and 80 ng ml21 and the extraction time was varied between 0.5 and 4 h, leading to enrichment factors of up to 200. The extraction efficiency was about 95%.The detection limit, expressed as 3s of five replicate determinations, using a 45 min enrichment of a urine sample low in lead, was 0.1 and 6.0 mg l21 for ETAAS, and FAAS respectively. The results obtained by the developed method agreed with those obtained by direct ICP-MS determinations for reference urine samples and samples from occupationally lead-exposed workers. The linear correlation coefficient was 0.97, the slope of the regression line was 1.06 and the intercept was 20.37 mg l21.The 95% confidence intervals for the slope and the intercept were 0.95 to 1.18 and 23.9 to 3.1, respectively. The results at the 95% confidence level for reference urine material were of 91 ± 1.5 and 92 ± 2.0 mg l21 for ICP-MS and SLM–AAS, respectively, which agreed well with the recommended value of 90 mg l21 (range 83–97 mg l21). Keywords: Lead; urine; supported liquid membranes; atomic absorption spectrometry; inductively coupled plasma mass spectrometry; carry-over effects Different enrichment techniques have been described for the determination of trace levels of heavy metals in urine, e.g., preconcentration by electrodeposition in combination with electrothermal atomic absorption spectrometry (ETTAS)1–3 and stripping techniques.4–6 Sample preparation methods based on liquid–liquid extraction7–10 are now gradually being replaced by solid-phase extractions.In the latter case, the facilitated online connection to the final analysis equipment increases the possibilities for automated analysis.11,12 Another modern approach to sample clean-up and enrichment is to use supported liquid membrane (SLM) methodology.This technique has been shown to give efficient clean-up of organic analytes in urine and plasma samples.13 In a recent paper,14 we reported the use of di- 2-ethylhexylphosphoric acid (DEHPA) as the extractant in an SLM-based procedure for the determination of some heavy metal ions including lead in both reagent water and natural water samples. The use of a proton gradient as the main transport mechanism eliminates the use of buffers that are often contaminated with the analytes of interest.In this study, we demonstrated the possibility of extending the DEHPA extraction system to the determination of lead in a biological matrix, urine. For comparison, direct determination of lead in dilute urine samples was carried out using inductively coupled plasma mass spectrometry (ICP-MS).Experimental Equipment Atomic absorption spectrometry The ETAAS system was a Varian AA-1475, fitted with a GTA- 95 graphite tube atomiser and a programmable sample dispenser (Varian, Victoria, Australia). Deuterium lamp background correction was used. The furnace programme is given in Table 1. The flame AAS (FAAS) system was a Varian AA-6. Parameter settings, such as lamp current and wavelength, were those recommended by the manufacturer. Inductively coupled plasma The possibility of using ICP-MS for the direct determination of metals in environmental and biological samples has been demonstrated in a number of studies.15–18 For lead in blood samples, ICP-MS has proved to be very useful.19,20 The ICPMS system used was a Plasma Quad PQ2 Plus (Fison Elemental, Winsford, Cheshire, UK).SLM equipment The SLM device used was similar to one described previously, 21 except that a modification was made to facilitate membrane replacements. The device consists of two identical circular PTFE blocks (diameter 120 mm, thickness 15 mm) with grooves cut in an Archimedes spiral (depth 0.25 mm, width 1.5 mm, length 250 cm), giving channels with a total volume of 1 ml.A porous PTFE membrane with a polyethylene backing (pore size 0.2 mm, porosity 0.70, total thickness 175 mm, of which 115 mm is the polyethylene backing; Type FG, Millipore, Table 1 Graphite furnace programme: wavelength 217.0 nm, lamp current 4.0 mA, slit 1.0 nm, slit height normal, background correction on.Pyrolytic graphite-coated graphite tubes were used Argon gas Tempera- Ramp Hold flow rate/ Step ture/°C time/s time/s l min21 Drying 90 5.0 10.0 3.0 Drying 120 5.0 30.0 3.0 Ashing 400 5.0 20.0 3.0 Ashing 400 0.0 2.0 0.1 Atomization 2000 1.0 2.0 0.1 Cleaning 2200 1.0 1.0 3.0 Analyst, October 1997, Vol. 122 (1073–1077) 1073Bedford, MA, USA) was impregnated by soaking it for at least 15 min in kerosene containing 40% m/m of DEHPA.The membrane was then placed between the two PTFE blocks and clamped with a stainless-steel screw in the middle. The screw was covered with a polyetherether ketone (PEEK) tube to prevent contact between the steel surface and the solutions in the membrane channels. Chemicals All solutions were prepared from either Suprapur or analyticalreagent grade chemicals and high-purity water was obtained from a MilliQ-RO4 unit (Millipore). Polyethene bottles were used throughout and were cleaned with 4 m HNO3 for at least 2 d and rinsed with high-purity water before use.Nitric acid (Suprapur) was obtained from Merck (Darmstadt, Germany) DEHPA (95%) from Sigma Chemicals (St. Louis, MO, USA), kerosene from Kebo Lab (Malm�nd lead nitrate stock standard solution (1000 mg ml21) from BDH (Poole, Dorset, UK). Freeze-dried urine (batch 403125, Seronorm, Nycomed, Oslo, Norway) was used as a reference material. The recommended value of lead was 90 (range 83–97) mg l21.A synthetic urine sample was prepared by dissolving 2.08 g of creatinine (Sigma), 1.25 g of urea (BDH), 14.1 g of NaCl, 2.8 g of KCl, 0.794 g of CaCl2·2H2O, 0.88 g of MgSO4·7H2O and 1.9 ml of ammonia solution (all from Merck) in high-purity water and diluting to 1000 ml.22 Sampling and Sample Pre-treatment Urine samples were obtained from lead-exposed workers at a secondary lead smeltery. Each individual was provided with a 2 l polyethylene bottle and was instructed to collect urine at home, but not in the smeltery.All urine samples used in the preliminary experiments were obtained from occupationally non-exposed subjects. The urine samples were preserved by adjusting the pH to 3.0 with concentrated nitric acid to prevent the precipitation of calcium phosphate and the consequent loss of heavy metals due to coprecipitation or adsorption on the precipitate. Usually, urine has a pH value in the range 5.0–6.5, and 2.5–3.0 ml of concentrated nitric acid were needed for each litre of urine to adjust the pH to 3.0.The freeze-dried reference urine samples had a pH below 3.0 after reconstitution with highpurity water. Prior to SLM–AAS analysis the pH was adjusted to 3.0 with ammonia solution. The final concentration of analyte in these samples solution was 20 mg l21. These samples were processed in the same manner as the other samples for 45 min and at a flow-rate of 0.9 ml min21. For the ICP-MS determinations, the reconstituted freeze-dried urine sample was analysed after a 10-fold dilution.Enrichment and AAS Determinations Fig. 1 shows the set-up for preconcentration of lead. It is similar to that described previously.14 Briefly, the sample is pumped at a flow rate of 1 ml min21 with the peristaltic pump A. After the enrichment, the acceptor solution is pumped with the peristaltic pump B to a vial placed in a fraction collector (D). The pneumatic valve A directs the sample stream to the donor channel in the SLM unit, and valve B directs fresh solution to the acceptor channel and keeps a closed loop on the stagnant acceptor during the enrichment step.Cleaning of the tube between valve C and the fraction collector D is done with peristaltic pump C for off-line analysis using ETAAS or FAAS. Normally, 2 ml of acceptor solution are used to transfer the enriched sample to a vial. To reduce possible carry-over effects due to slow kinetics at the membrane–acceptor interface, the acceptor channel was usually washed with 10 ml of the stripping solution at a flow rate of 1.0 ml min21 after each preconcentration procedure.pH changes in the acceptor during the extraction process were monitored as described previously.14 Experiments concerning analyte recovery were carried out on natural urine samples spiked with known concentrations of lead. The extent of carry-over effects was investigated by collecting and analysing 2 ml fractions of the acceptor solution, collected 0, 5 and 10 min after the preconcentration.Each collection procedure took about 160 s. ICP-MS Determinations The urine samples were acidified by adding 2% v/v concentrated nitric acid (Aristar, BDH), and left overnight. Lead was determined according to a method used previously for whole blood.19 Briefly, the samples were analysed after a 10-fold dilution with a solution containing EDTA, Triton X-100 and ammonia., The samples were introduced into the ICP-MS equipment in a flow injection-like manner (segmented flow mode; 14 s uptake, 24 s acquisition).Bismuth and thallium were used as internal standards. Validation The validation of the proposed method was carried out by comparing the results of ICP-MS and SLM–AAS determinations. Results and Discussion Influence of Enrichment Time on Acceptor pH During the use of DEHPA as carrier in the extraction of lead from natural waters, a donor pH of 3 and an acceptor pH of 0 have been found to be suitable for the mass transfer of analytes to the acceptor side.14 Accordingly, these conditions were chosen for the investigation of the influence of the enrichment and the pH of the acceptor solution.Fig. 2 shows the influence of enrichment time on acceptor pH. It is seen that the pH on the acceptor side rises steadily and reaches a value of about 1.6 after approximately 4 h. Extraction Efficiency Table 2 gives the extraction efficiencies from synthetic urine and diluted spiked natural urine.The extraction efficiencies for lead in spiked natural urine and synthetic urine are 93 and 95%, respectively, which are in general slightly higher than those obtained in diluted urine. Using a two-tailed Student’s t-test, the Fig. 1 Experimental set-up for metal enrichment. 1074 Analyst, October 1997, Vol. 122differences between diluted and undiluted urine samples was not significant at the 95% confidence level except for the samples diluted to 30 and 50%.However, the difference is not significant at the 99% confidence level. Hence there is no reason to dilute the urine samples as this will lead to higher detection limits. Further, it may be concluded that physiologically normal variations between the urine samples will not influence the extraction efficiency. For reliable quantification it is important to work under conditions where the enrichment factor is constant. The influence of the enrichment time on the extraction efficiency at the normal sample flow rate of 1 ml min21 is shown in Table 3.The extraction efficiency (Table 3) is independent of the enrichment time up to at least 2 h. The value at 4 h is lower but the difference between this value and the other values in Table 3 is not significant at the 95% confidence level using a twotailed Student’s t-test. We have found previously that the pH gradient across the membrane should be about 2 pH units in order to keep the mass transfer rate constant.14 After 4 h, the difference is about 1.4 pH units (Fig. 2). However, the driving force is the difference in H+ concentration across the membrane. Since the pH scale is logarithmic, the difference in pH may be relatively smaller at high H+ concentrations, without giving significant changes in extraction efficiency, which is clearly demonstrated here. The change in extraction efficiency with time depending on the increase in pH on the acceptor side does not seriously affect the long-term stability.Experiments have been run continuously at a flow rate of 1 ml min21 on the same membrane for periods of up to 48 h with replacement of the acceptor solution every 2.5 h without any significant changes in extraction efficiency. In this respect, the behaviour is similar to what has been observed previously for lead in reagent water and natural waters, where the long-term stability was confirmed for periods of at least 30 and 200 h for natural and reagent waters, respectively.14 The detection limit is inversely related to the enrichment time as long as the extraction efficiency is independent of time.A 4 h run at 1 ml min21 (with negligible decrease in extraction efficiency) results in a processed sample volume of 240 ml. With an acceptor volume of approximately 1.0 ml and an extraction efficiency for lead of about 90%, an enrichment factor of more than 200 is obtained. Carry-over Effects in the Membrane System In some SLM systems, the mass transfer kinetics at the membrane–acceptor interface can be slow, which has been shown previously, e.g., in the enrichment of surfactants using ion-pair formation.23 It is therefore necessary in the investigation of new SLM applications always to consider the extent of carry-over effects, which may occur as a result of limited mass transfer kinetics in the actual system.Table 4 shows the extraction efficiencies of lead at different elution times after the completion of an enrichment. The first 2 ml fraction was collected immediately after the enrichment, the second after a waiting time of 5 min, and the third after 10 min.From Table 4, it may be concluded that a 10 min wash of the acceptor channel is sufficient to remove about 98% of the enriched lead. This makes memory effects negligible, provided that the concentration difference between samples is reasonable. Quantification Effects of initial donor concentration on lead recovery The concentration dependence was tested by spiking urine samples with lead in the concentration range 5–80 mg l21 and performing a 30 min SLM enrichment at a flow rate of 1.0 ml min21 followed by analysis by ETAAS for spiked Fig. 2 Change in acceptor pH with time during SLM processing of a urine sample at pH 3.0 with a flow rate of 1 ml min21. Acceptor solution, 1.0 m nitric acid; membrane liquid, 40% m/m DEHPA in kerosene. Table 2 Extraction efficiency of lead in spiked synthetic urine and spiked natural urine of various dilutions in high-purity water (n = 5).Standard deviations are given in parentheses. Lead concentration, 50 mg l21; sample pH, 3.0; acceptor solution, 1.0 m HNO3; enrichment time, 60 min; flow rate approximately 1.0 ml min21 Urine in Extraction Urine in Extraction water (%) efficiency (%) water (%) efficiency (%) 5 91.6 (3.1) 60 90.5 (2.4) 10 88.5 (5.5) 70 88.9 (3.2) 20 89.0 (3.7) 80 94.4 (8.5) 30 88.5 (2.5) 90 93.5 (2.9) 40 90.0 (3.8) 100 93.2 (2.9) 50 89.2 (2.1) Synthetic urine 95.3 (3.0) Table 3 Extraction efficiency of lead in spiked natural urine at different enrichment times.Standard deviations are given in parentheses. Lead concentration 50 mg l21; sample, pH 3.0; acceptor solution, 1.0 m HNO3; sample flow rate, approximately 1.0 ml min21 Extraction efficiency (%) Time/h (SD) n 0.5 94.3 (1.3) 6 1.0 94.7 (1.4) 6 2.0 95.3 (1.2) 6 4.0 93.5 (4.0) 4 Table 4 Carry-over effects using SLM methodology for enriching lead in a spiked urine sample investigated using two different porous membrane filters.Two enrichments on each filter. Average values are given. Standard deviations are given in parentheses. Lead concentration, 5.5 mg l21; membrane liquid, 40% m/m DEHPA in kerosene; sample pH, 3.0; acceptor solution, 1.0 m HNO3; flow rate 0.90–1.0 ml min21; enrichment time, 100 min. The eluted volume from the acceptor after each waiting time was 2 ml Waiting time/ Extraction efficiency min (%) 0 93.1 (0.5) 93.0 (0.8) 5 5.3 (1.1) 4.3 (1.5) 10 0.4 (2.0) 0.5 (1.0) Analyst, October 1997, Vol. 122 1075concentrations below 25 mg l21 and FAAS for higher concentrations. The results are given in Table 5.Applying a Student’s t-test for the differences in Table 5 between the recoveries obtained at different concentrations and the average recovery value of 93.8% revealed that there were no significant differences at the 95% confidence level for any of the concentrations in the range considered.Detection limit The detection limit was determined as three times the standard deviation of five replicate determinations of lead in a urine sample from an occupationally unexposed individual. In two different experiments with enrichment times of 45 min, with a flow rate of 1 ml min21 and ETAAS determination, the detection limit was 0.1 mg l21. This value is well below the concentrations found in unexposed individuals.The corresponding value for FAAS is 6.0 mg l21. This is sufficient for measuring samples from workers with moderate lead exposure. The concentration of lead in occupationally exposed workers is normally above 10 mg l21. Validation In the validation using the reference urine samples, SLM–AAS gave a lead value of 92.0 ± 2.0 mg l21 (five replicates) at the 95% confidence level assuming a Student’s t distribution. This was in accordance with the recommended value of 90 (range 83–97) mg l21 (five replicates).The corresponding result for ICP-MS was 91.0 ± 1.5 mg l21 (five replicates) at the 95% confidence level. The results obtained with SLM–AAS and ICP-MS agreed well with each other and with the certified values. The plot of the results in this comparison (samples from lead-exposed workers) is illustrated in Fig. 3. Linear correlation24 based on 22 samples gave a slope of 1.06, an intercept of 20.37 mg l21 and a correlation coefficient of 0.97.The 95% confidence intervals for the slope and intercept were 0.95 to 1.18 and 23.9 to 3.1, respectively. Since these intervals include the ideal values of 1 and 0 for the slope and the intercept, respectively, there is no evidence of a systematic difference between the two sets of results. The relative deviations between the ICP-MS and the SLM– AAS values are larger for low concentrations. At low concentrations, lead was determined by ETAAS in the enriched samples.The larger deviations within this concentration range (below 20 mg l21) are probably due to technical problems related to the background correction in ETAAS. In some experiments using ETAAS, RSD values of !20% were encountered. Since FAAS generally gives good precision (RSD Å 4% in the considered concentration interval here), the final analysis could be based entirely on FAAS by prolonging the enrichment time for low-concentration samples. The longer times needed do not necessarily decrease the sample throughput.With a multi-channel system in an off-line configuration, many samples can be simultaneously enriched by the SLM technique with low labour input. The relatively low cost of FAAS equipment, low operational cost, robustness and good stability of the FAAS system together with short analysis times makes SLM–FAAS a powerful alternative. The results in Tables 2, 4 and 5 indicate that neither the matrix composition nor the initial analyte concentration influences the extraction efficiency.However, these experiments were performed on the same sample bulk. In the comparison experiments, samples were collected from different individuals. The overall agreement between the results obtained with SLM– FAAS and ICP-MS in this comparison indicates that individual variations in the urine composition do not influence the results in any significant way. Furthermore, the present sample cleanup process using SLM does not seem to affect the results in any significant way. Using SLM with a cation-exchange extractant, negatively charged lead complexes, for example, could have been excluded from the membrane transport process. Even species which under the given conditions in the donor solution can form neutral complexes with lead might not contribute to the mass transfer of lead across the membrane, thereby decreasing the extraction efficiency to values below 100%.The most important compounds to be considered are probably chloride, phosphate, sulfate, citrate and oxalate ions, which are normally present in undiluted urine at concentrations of about 6000, 1200, 180 and 10–50 mg l21, respectively. Also, high concentrations of urea and creatinine could have some effects.However, these changes must be small. In the validation experiment the creatinine concentration varied between 5.2 and 20.9 mm. According to the literature,25 the normal variation of creatinine in urine is 0.13–0.22 mm kg21. Hence for a person with a mass of 70 kg, the normal level of creatinine should vary from 9.1 to 15.4 mm.Obviously the variation of creatinine in the samples investigated exceeded this range without leading to a significant influence on the extraction efficiency of lead. Concerning urea, the dilution experiments in Table 3 show that dilution has a negligible effect on the recovery of lead. The range was further investigated by comparing the recovery of lead in synthetic urine samples with normal urea concentrations (20 mm) with samples with about three times higher (62 mm) concentrations.It was found that the difference between the average values (based on three determinations at 20 mm and six determinations at 62 mm) were less than 1%. Hence the method developed seems to be well suited for clinical measurements. Table 5 Recovery of lead in natural urine spiked with different lead concentrations. The background concentration of lead, 0.9 mg l21, obtained as an average for six unspiked natural urine samples, was subtracted before calculating the extraction efficiency.The number of experiments at each concentration was six. Standard deviations are given in parentheses. Lead concentration, 5–80 mg l21; sample pH, 3.0; acceptor solution, 1.0 m HNO3; enrichment time, 30 min; flow rate, 1.0 ml min21 Lead concentration/ Recovery mg l21 (%) 5 92.4 (3.5) 10 94.8 (5.9) 20 94.0 (5.8) 50 95.3 (1.7) 80 92.7 (6.5) Fig. 3 Validation of the SLM–AAS methodology for lead determination by comparison with results obtained with ICP-MS. Sample pH, 3.0; acceptor solution, 1.0 m nitric acid; membrane liquid, 40% m/m DEHPA in kerosene. 1076 Analyst, October 1997, Vol. 122Conclusion We have shown that SLM combined with AAS can be used for the determination of lead in urine samples. By extending the enrichment time to 2 h, a detection limit of 2 mg l21 is obtainable using FAAS, thus covering the whole range of interest at occupational exposure.The methodology could be extended to other metals such as Cd, Cu and Cr, which will also give high extraction efficiencies in the chosen SLM system. For ICP-MS determinations, the flexibility of the SLM technique may be potentially useful when sample clean-up procedures are necessary, to avoid spectral interferences from certain elements. This work was made possible by financial support from the Swedish Environmental Protection Agency and the Medical Faculty at Lund University. The authors are grateful to Docent J. Å. J�onsson for fruitful suggestions. They also thank workers of Boliden Bergs�oe AB, Landskrona, for providing the urine samples. Mr. A. Ekholm and Mr. F. Malcus are acknowledged for skilful technical assistance. 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