Continuous Ultrasound-Assisted Extraction Coupled to Flow Injection

A dynamic system for the continuous removal of ammonia from cigarettes with ultrasound assistance and iterative change of the flow direction of the ex...
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Anal. Chem. 2006, 78, 2297-2301

Continuous Ultrasound-Assisted Extraction Coupled to Flow Injection-Pervaporation, Derivatization, and Spectrophotometric Detection for the Determination of Ammonia in Cigarettes A. Caballo-Lo´pez and M. D. Luque de Castro*

Department of Analytical Chemistry, Annex C-3, Campus of Rabanales, University of Co´ rdoba, E-14071, Co´ rdoba, Spain

A dynamic system for the continuous removal of ammonia from cigarettes with ultrasound assistance and iterative change of the flow direction of the extractant through the sample cell has been developed. A 0.1-g sample of cigarette was subjected to 7 min of ultrasound-assisted extraction (application and duration of pulse 0.7 s, output amplitude 85% of the converter nominal amplitude), and 1 M NaOH solution was used both as extractant and as carrier in the dynamic system. The ultrasound-assisted extractor was coupled to a pervaporation unit through a flow injection interface in order to develop a fully automated method. In arriving at the pervaporator, the ammonia is transferred from the donor-carrier stream to an acceptor stream, where the classical Berthelot reaction takes placesthus favoring pervaporation. The blue complex formed is spectrophotometrically monitored at 655 nm. The method was applied to the determination of ammonia in a selection of 10 European cigarette brands and Kentucky Reference 2R4F cigarettes. Tobacco use contributes at present to ∼4 million deaths each year.1 The World Bank has estimated that continued tobacco consumption threatens to claim 7 million deaths per year in developing countries. Among more than 20 different alkaloids found in tobacco, nicotine is the most abundant and accounts for widespread human use of tobacco products throughout the world. The European Union has a list of over 600 additives that manufacturers may use in elaboration of their cigarettes.2 When smoked, cigarettes release over 4000 chemicals, a number of which are carcinogenic.3 Presently, ammonia is a common ingredient, usually found in the form of ammonium hydroxide or diammonium phosphate,4,5 the increased levels of which in tobacco lead to increased levels of * To whom correspondence should be addressed. Phone and Fax: 34957218615. E-mail: [email protected]. (1) Corrao, M. A.; Guindon, G. E.; Sharma, N.; Shokoohi, D. F. Tobacco Control Country Profiles; The American Cancer Society: Atlanta, GA, 2000. (2) Department of Health. London. March 2000R. (3) FN AQ2222. Mutagenic activity of flavour compounds; BN 400916808400916815; BAT. December 12, 1986. (4) Brand by brand ingredients. Philip Morris USA, http://www.philipmorrisusa.com/company_&_products/what_is_in_my_brand.asp. (5) Pelander, A.; Ojanpera, I.; Laks, S.; Rasanen, I.; Vuori, E. Anal. Chem. 2003, 75 (21), 5710-5718. 10.1021/ac051115u CCC: $33.50 Published on Web 02/21/2006

© 2006 American Chemical Society

ammonia in smoke.6-9 When ammonia is added, the nicotine converts from the acid into the base form, which vaporizes more easily from the smoke particles into the gas phase, is deposited in the mouth,10-14 and travels to the stomach (ingestion) where it is surely converted to protonated nicotine due to the high acidity in the stomach.15-19 In contrast, the nicotine in the particles is delivered to the lungs. Recent research suggests a delay of 1-2 min from the lung deposition to the brain.20,21 There are no data that demonstrate stronger physiological effects on human smokers of a given amount of nicotine in the presence of ammonia than in its absence. The review by Dixon et al.22 concludes from all the data available that ammonia in tobacco and ammonia in smoke do not increase nicotine bioavail(6) Armitage, A. K.; Dixon, M.; Frost, B. E.; Mariner, D. C.; Sinclair, N. M. Chem. Res. Toxicol. 2004, 17, 537-544. (7) Ellis, C. L.; Cox, R. H.; Callicutt, C. H.; Laffoon, S. W.; Podraza, K. F.; Seeman, J. I.; Kinser, R. D.; Farthing, D. E.; Hsu, F. H. In CORESTA Meeting; Innsbruck, 1999; p ST 2. (8) Frost, B. E.; Mariner, D. C.; Sinclair, N. M. In CORESTA; Brighton, England, 1998; pp 211-218. (9) Saint-Jalm, Y.; Duval, G.; Conte, T.; Bonnichon, I. In CORESTA; Portugal, 2000; Vol. 2000, p ST9. (10) Bergstrom, M.; Nordberg, A.; Lunell, E.; Antoni, G.; Langstrom, B. Clin. Pharmacol. Ther. 1995, 57, 309-317. (11) Lunell, E.; Bergstrom, M.; Antoni, G.; Langstrom, B.; Nordbert, A. Clin. Pharmacol. Ther. 1996, 593-594. (12) Lunell, E.; Molander, L.; Ekberg, K.; Wahren, J. Eur. J. Clin. Pharmacol. 2000, 55, 737-741. (13) Molander, L.; Lunell, E.; Andersson, S. B.; Kuylenstierna, F. Clin. Pharm. Ther. 1996, 59, 394-400. (14) Schuh, K. J.; Schuh, L. M.; Henningfield, J. E.; Stitzer, M. L. Psychopharmacology 1997, 130, 352-361. (15) Benowitz, N. L., Ed. Nicotine Safety and Toxicity; Oxford University Press: New York, 1998. (16) Benowitz, N. L. The Nature of Nicotine Addiction. In Smoking. Risk, Perception, & Policy; Slovic, P., Ed.; Sage Publications: Thousand Oaks, CA, 2001; pp 159-186. (17) Benowitz, N. L. Nicotine Pharmacology and Addiction, In Nicotine Safety and Toxicity; Benowitz, N. L., Ed.; Oxford University Press: New York, 1998; pp 3-16. (18) Henningfield, J. E.; Fant, R. V. Nicotine Delivery Systems: Implications for Abuse Potential, Medications Development, and Public Health. In Nicotine and Public Health; Ference, R., Slade, J., Room, R., Pope, M., Eds.; American Public Health Association: Washington, DC, 2000; pp 229-247. (19) Stratton, K., Shetty, P., Wallace, R., Bondurant, S., Eds. Clearing the Smoke. Assessing the Science Base for Tobacco Harm Reduction; Institute of Medicine, National Academy Press: Washington, DC, 2001. (20) Rose, J. E.; Behm, F. M.; Westman, E. C.; Coleman, R. E. Drug Alcohol Depend. 1999, 56, 99-107. (21) Brewer, B. G.; Roberts, A. M.; Rowell, P. P. Drug Alcohol Depend. 2004, 75, 193-198.

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ability to the bloodstream and brain of smokers, an opinion that is not held by Pankow23 though without human smoking data. Recently, research by Armitage et al. on smokers demonstrated that ∼99% of the nicotine in mainstream smoke inhaled by human smokers is retained by him/her, regardless of the level of ammonia in the smoke, for reasonable puff volumes;24 and also the recent research by Seeman et al. based on chemical and computational simulation corroborated the Armitage results.25 Many reports have focused on the analysis of nicotine in various matrixes, including biological fluids and tissues, by employing high-performance liquid chromatography,26 gas chromatography,27,28 or gas chromatography/mass spectrometry;29 however, only a limited number of methods for measuring ammonia levels in tobacco have been reported.30,31 The concomitant presence of lower levels of nicotine and higher levels of ammonia, along with numerous other chemical compounds in light cigarettes, impart a significant challenge for developing a simple and fast quantitative method. Analytical pervaporation seems to offer possibilities to implement on-line flow injection measurements of ammonia in the presence of nicotine and other chemical compounds. This separation technique can be defined as a combination of continuous evaporation and gas diffusion through a membrane in a single step.32 Analytical chemistry is one of the chemical areas where ultrasound radiation has been little used. This auxiliary energy could be a powerful aid for the acceleration of various steps of the analytical process. Although analytical chemists have shown little interest in the use of ultrasound, its potential usually surpasses that of other, conventional auxiliary energies. Thus, ultrasound is of great help in the pretreatment of solid samples as it facilitates and accelerates operations such as the extraction of organic and inorganic compounds, slurry dispersion, homogeneization, nebulization, crystallization, levitation, washing, derivatization, etc.33,34 Also, ultrasound-based detection is going to look for a place in the analytical arena.35 (22) Dixon, M.; Lambing, K.; Seeman, J. I. Beitr. Tabakforsch. Int. 2000, 19, 103-113. (23) Pankow, J. F. Chem. Res. Toxicol. 2001, 14, 1465-1481. (24) Armitage, A. K.; Dixon, M.; Frost, B. E.; Mariner, D. C.; Sinclair, N. M. Chem. Res. Toxicol. 2004, 17, 537-544. (25) Seeman, J. I.; Lipowicz, P. J.; Piade´, J. J.; Poget, L.; Sanders, E. B.; Snyder, J. P.; Trowbridge, C. G. Chem. Res. Toxicol. 2004, 17, 1020-1037. (26) Meger, M.; Meger-Kossien, I.; Schuler-Metz, A.; Janket, D.; Scherer, G. J. Chromatogr., B: Anal. Technol. Biomed. Life Sci. 2002, 778 (1-2), 251261. (27) Cai, J. B.; Liu, B. Z.; Lin, P.; Su, Q. D. J. Chromatogr., A 2003, 1017 (1-2), 187-193. (28) Yang, S. S.; Smetena, I.; Huang, C. B. Anal. Bioanal. Chem. 2002, 373 (8), 839-843. (29) Torano, J. S.; van-Kan, H. J. M. Analyst 2003, 128 (7), 838-843. (30) Morie, G. P. Tobacco Sci. 1972, 16, 167-179. (31) Collins, P. F.; Lawrence, W. W.; Williams, J. F. Beitr. Tabakforsch. Int. 1972, 6, 167-172. (32) Luque de Castro, M. D.; Papaefstathiou, I. Encyclopedia of Environmental Analysis and Remediation; John Wiley & Sons: Chichester, 1998; p 3462. (33) Luque de Castro, M. D.; Luque-Garcı´a, J. L. Acceleration and Automation of Solid Sample Treatment; Elsevier: Amsterdam, 2002. (34) Luque de Castro, M. D.; Priego-Capote, F. Analytical Applications of Ultrasound; Elsevier: Amsterdam, 2006. (35) Priego-Capote, F.; Luque de Castro, M. D. Trends Anal. Chem. 2004, 23, 644-653.

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There are just few papers where ultrasound probes have been used in continuous extraction systems.36 Four approaches can be used in ultrasound-assisted extraction when the extractant flows continuously through the sample, namely: (i) Open, singledirection continuous systems, where fresh extractant flows continuously through the sample, so the mass transfer is displaced to the solubilization of the analyte into the liquid phase. This dynamic mode has two mains disadvantages: extract dilution and increased compactness of the solid during extraction. (ii) Open, continuous systems with iterative change of the flow direction, where a preset volume of extractant is circulated forward and back through the sample by an appropriate program of the peristaltic pump. In this way, both dilution of the extract and undesirable increase of sample compactness in the extraction chamber and hence pressure increase in the dynamic system, are avoided. (iii) Closed, single-direction systems, where a preset volume of extractant is recirculated through the sample. In this way, dilution of the extract is avoided. (iv) Closed systems with iterative change of the flow direction, with programming of the impulsion system for changing the flow direction at preset intervals, thus avoiding both dilution of the extract and increased compactness of the sample. The aim of this work was to provide a new method for the determination of ammonia in cigarettes by combining different steps (namely, ultrasound-assisted, leaching, pervaporation, derivatization, and detection, through a flow injection interface) for full automation of the analytical process. EXPERIMENTAL SECTION Reagents and Samples. An ammonia stock standard solution of 1 g L-1 was prepared from ammonium chloride. The selection of donor and acceptor stream composition was based on a previous report37 on the modified Berthelot method. The reagents involved were as follows: 1 and 0.3 mol L-1 sodium hydroxide solutions for carrier and Berthelot reaction, respectively (Merck, Darmstadt, Germany), 2.5 g L-1 sodium dichloroisocyanurate (Sigma-Aldrich, Deisenhufen, Germany), 1 g L-1 sodium nitroprusside (SigmaAldrich), and 120 g L-1 sodium salicylate (Sigma-Aldrich). Kentucky Reference 2R4F cigarettes were obtained from the Kentucky Tobacco Research & Development Center, University of Kentucky, Lexington, KY.38 A selection of 10 European cigarette brands were obtained from a local tobacco shop. All cigarettes were conditioned at 60% relative humidity and 22 °C for 24 h prior to use. All working solutions were prepared daily using distilled water of high purity obtained from a Millipore (Bedford, MA) Milli-Q plus system and all safety precautions (gloves, mask, fume hood, etc.) were adopted. Instruments and Apparatus. Ultrasonic irradiation was applied by a Branson 450 sonifier (20 kHz, 450 W) equipped with a cylindrical titanium alloy probe (19.10-mm diameter), which was immersed in a water transmitting bath in which the extraction cell was placed. (36) Priego-Capote, F.; Luque de Castro, M. D. Trends Anal. Chem. 2004, 23, 829-838. (37) Muraki, H.; Higuchi, K.; Sasaki, M.; Korenaga, T.; Toˆei, K. Anal. Chim. Acta 1992, 261, 345-349. (38) Kentucky Reference 2R4F, Kentucky Tobacco Research & Development Center, University of Kentucky, Lexington, KY.

Figure 1. View and dimensions of the pervaporator chambers.

The flow injection-pervaporation manifold was constructed by a Gilson Minipuls-3 low-pressure peristaltic pump (Gilson, Worthington, OH), a 5054 Rheodyne low-pressure injection valve (Rheodyne, Cotati, CA), three-way standard connectors, PTFE tubing of 0.5- and 1.0-mm i.d. (Scharlau, Barcelona, Spain), and a Selecta (Barcelona, Spain) Ultraterm 6000383 water bath. The reaction coil RC1, consisted of a coiled Teflon tube of 0.5-mm i.d. and 60 mm in length. The reaction coil RC2 was made of Teflon tube of 1.0-mm i.d. and 200 mm in length. For all other connections 0.5-mm-i.d. tubing was used. Two Rheodyne low-pressure injection valvessone adapted to work as selection valvesa channel of the low-pressure peristaltic pump, and Teflon tubing of 0.8-mm i.d. were used to build the leaching system. An extraction chamber consisting of a stainless steel cylinder (64 mm × 7.4 mm i.d.) closed with screws and cotton filters at either end, which permitted the circulation of the leaching solvent through it, was also used. The pervaporation unit (Figure 1) consists of a lower, donor chamber 5.0 mm deep and hexagonal in shape for minimizing dead volumes and an upper, acceptor chamber 0.3 mm deep and spiral shape and a single layer of glass beads (3.6-mm diameter) used to partially fill the donor chamber. The two chambers were fitted with inlet and outlet orifices, and a metal membrane support was located between both. Firm contact between parts was achieved by screwing the pervaporation unit between aluminum supports with four screws. PTFE membranes (47-mm diameter and 1.5-mm thickness from Trace, Braunschweig, Germany) were also used. Method. The overall arrangement for ammonia ultrasoundassisted leaching, pervaporation, derivatization, and photometric detection is illustrated in Figure 2. The pervaporation unit employed in this work was built inhouse and presents a novelty in the acceptor chamber, with respect to the pervaporation units used so far. A spiral shape was machined in this block for dragging of analyte with minimum dispersion.39 An amount of 0.1 g of cigarette (paper included) was placed into the extraction cell, which was assembled in the system and immersed in the ultrasound transmitting liquid. Then, the loop of the injection valve IV2 (with a total volume of 4 mL) was filled with the leaching carrier (1 mol L-1 NaOH solution), after which valve IV2 was switched and the leaching carrier, impelled by the peristaltic pump, was circulated through the solid sample. By switching the selection valve SV, the dynamic system including the sample chamber and peristaltic pump became a closed system with the extractant impelled by the peristaltic pump at 0.6 mL (39) Cerda´, V. Personal communication.

Figure 2. Experimental setup for development of the extraction of ammonia from cigarettes. EC, extraction cell; IV, injection valve; SV, selection valve; W, waste; RC, reaction coil; UP, ultrasound probe; PU, pervaporation unit; R1, salicylate/nitroprusside reagent; R2, dichloroisocyanurate/sodium hydroxide reagent; MC, mixing coil; PP, peristaltic pump; C, carrier.

min-1 and the sample was irradiated for 7 min under ultrasonic irradiation (application and duration of pulse 0.7 s, output amplitude 85% of the converter nominal amplitude, with the probe placed 1 mm from the top surface of the extraction cell). During extraction, the direction of the leaching carrier was changed every complete turn, minimizing dilution of the extract as lower extractant volume is thus required and avoiding increased compactness of the sample in the extraction cell that could cause overpressure in the system. After extraction, valve SV was switched, and the ammonia extract was circulated through the mixing coil for homogenization and then filled the loop of the injection valve IV1 (with an injection volume of 1.5 mL). Then, IV1 was opened, and the ammonia extract reached the donor chamber of the pervaporation unit, evaporated into the headspace of the chamber, and diffused across the PTFE membrane into the acceptor solution. There, an R1 stream containing sodium salicylate and sodium nitroprusside developed the first step of the Berthelot reaction and carried the analyte to a three-way standard connector, where it merged with the R2 stream containing sodium dichloroisocyanurate in NaOH medium for development of the second step of the Berthelot reaction. The absorbance was monitored at 655 nm. The temperature of the donor and acceptor streams was controlled by immersing the reservoirs of the donor and reagent solutions R1 and R2, the pervaporation unit, and reaction coils RC1 and RC2 into a water bath at 37 °C. RESULTS AND DISCUSSION Optimization of the Method. First, the flow injectionpervaporation step was optimized and characterized by using an ammonium chloride solution; then, the optimum values found were adopted for monitoring the optimization of the extraction step. The ranges over which the variables were studied and their optimum values are given in Table 1. Optimization of the Pervaporation and Flow Injection Variables. Despite the fact that the method was optimized in previous research,40,41 the flow injection and chemical variables (40) Wang, L.; Cardwell, T. J.; Cattrall, R. W.; Luque de Castro, M. D.; Kolev, S. D. Anal. Chim. Acta 2000, 416, 177-184.

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Table 1. Ranges over Which the Variables Were Studied and Their Optimum Values variables pervaporation injection volume (µL) temperature (°C) spacer (mm) derivatization salicylate (R1), (g L-1) nitroprusside (R1), (g L-1) dichloroisocyanurate (R2), (g L-1) extraction flow rate (mL min-1) duration of the ultrasound pulse per second radiation amplitude (%) sonication time (min)

tested range 200-2000 25-50 2, 4, 6

optimum value 1500 37 6

80-150 0.4-1.2 1.5-3.5

120 1.0 2.5

0.6-1.4 50-90

0.6 70

50-90 1-10

85 7

were reoptimized here. Different sample volumes ranging from 0.2 to 2 mL were studied using an ammonium standard solution of 50 µg mL-1. As expected, small sample volumes increased the sample throughput, but to the detriment of the sensitivity. A sample volume of 1.5 mL was selected as a compromise between the conflicting requirements for high sensitivity and high sample throughput. The flow rate of the FI pump was set at 0.6 mL min-1 and the reactor lengths at 60 (RC1) and 200 cm (RC2), as in the previous research.41 Because of the volatility of the ammonia, increased temperatures only slightly favored the pervaporation step, which resulted in a slight vapor pressure difference that increased the permeation of the substance through the membrane. High temperatures are of little benefit for the sensitivity and development time of the nitroprusside-catalyzed reaction40 and, mainly, because potential interference caused by other compounds present in cigarettes increases with temperature. A working temperature of 37 °C was used as recommended elsewhere for the Berthelot reaction.40-42 The influence of the volume of the donor chamber on the sensitivity was studied by placing in it spacers of 2-, 4-, and 6-mm thickness. The presence of spacers lowered the sensitivity, since they increased the volume of the headspace but increased both lifetime of the PTFE membranes and reproducibility of the analysis, so that a 6-mm spacer was used. When a metal membrane support was used, the membrane lifetime was considerably prolonged; however, the sensitivity slightly decreased because the membrane support covers part of the membrane, thus reducing its active surface area. Optimization of the Ultrasound-Assisted Leaching Step. A multivariate optimization design was applied to study leaching step. The variables optimized were the ultrasound radiation amplitude, duration of the ultrasound pulse per second, extractant flow rate, and irradiation time. A solution of 1 mol L-1 NaOH was used as donor stream, as leacher, and as converter of ammonium ion into ammonia. The probe was placed 1 mm from the top surface of the extraction cell. In all cases, the probe position was the same. Brand 1 was the light cigarette used for optimization. (41) Wang, L.; Cardwell, T. J.; Cattrall, R. W.; Luque de Castro, M. D.; Kolev, S. D. Talanta 2003, 60, 1269-1275. (42) Searle, P. L. Analyst 1984, 109, 549-568.

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Statistical software43 was used to analyze the data from the experimental values. A half-fraction 24-1 type IV resolution design allowing 3 degrees of freedom involved eight randomized runs plus three centered points was selected for a screening study of the behavior of the main factors affecting the extraction process. The upper and lower values given to each factor were selected from the available data and experience gathered in the preliminary experiments. The conclusions of this first screening study were that the extractant flow rate and the duration of the ultrasound pulse per second were factors not statistically influential at the 95% confidence level in the ranges under study. However, the results showed better efficiency with the lower value tested for the extractant flow rate (0.6 mL min-1) and with the medium value tested for the duration of the ultrasound pulse per second (0.7 s). Thus, these values were selected for subsequent experiments. The other variables (namely, radiation amplitude and irradiation time) were factors statistically influential at the 95% confidence level for the removal of the target analyte. Higher values for both variables were tested using a central composite design based on a 22 full factorial design and the juxtaposition of a face-centered star design. This study was performed in order to obtain the response surface of ammonia considering the ultrasound radiation amplitude and the irradiation time of ultrasonic exposure. This design allowed 5 degrees of freedom and involved eight randomized runs plus three centered points. Analyzing the design for radiation amplitude and irradiation time, which were the factors that showed a maximum of the response surface, a second-order polynomial equation was obtained. The optimal values were obtained by equalizing to zero the first derivative of the polynomial. Optimum values of 85% and 7 min, for the ultrasound radiation amplitude and irradiation time, respectively, were obtained and used for subsequent experiments. VALIDATION OF THE METHOD Calibration of the Proposed Method. The detection and quantification limits, established as 3sb + xb/slope and 10sb + xb/ slope (where sb is the standard deviation and xb is the mean of 11 blank measurements) are 0.05 and 0.09 µg mL-1, respectively. The linear concentration range of the calibration curve covers from 0.1 to 10 µg mL-1, with a correlation coefficient, r2, of 0.999. Evaluation of the Precision of the Method. To evaluate the precision of the proposed method, within-laboratory reproducibility and repeatability were estimated in a single experimental setup with duplicates.44 The experiments were carried out using brand 1. In all experiments, the optimal values obtained for the variables were used. Two measurements of the analyte per day were carried out on 7 days. The repeatability and within-laboratory reproducibility, both expressed as relative standard deviation, were 2.5 and 4.2%, respectively. Interferences. Potential ammonia from amino acids degradation during the extraction step would cause not significant interference both because of the high ammonia-to-amino acid ratio (43) Statgraphics Plus for Windows v 2.1, Rockville, MD, 1992. (44) Massart, D. L.; Vanderginste, B. G. M.; Buydens, L. M. C.; De Jong, S.; Lewi, P. J.; Semeyers-Verbeke, J. Handbook of Chemometrics and Qualimetrics, Part A; Elsevier: Amsterdam, 1997.

Table 2. Results of Analyses brands

proposed method (µg/cigarette)a

B1 B2 B3 B4 B5 B6

14.0 ( 0.34 12.0 ( 0.29 5.3 ( 0.14 17.5 ( 0.41 12.9 ( 0.30 11.3 ( 0.19

brands

proposed method (µg/cigarette)a

B7 B8 B9 B10 2R4Fb

15.5 ( 0.39 9.8 ( 0.24 10.8 ( 0.27 6.6 ( 0.17 12.3 ( 0.33

a Mean ( standard deviation (n ) 3). b The value for 2R4F Kentucky reference cigarettes was of 11.02 ( 0.06 µg/cig.38

in tobacco and the mild working conditions. Other tobacco constituents are the so-called tobacco-specific nitrosamines (NNN, NNK, NAT, NAB), the boiling points of which are above 150 °C. This value, the high molecular weight of these compounds, and their concentration in tobacco (average value 1.5 ppm) make their interference improbable. In addition, the samples do not come into contact with the membrane in the pervaporation unit, thus avoiding membrane deterioration by the alkaline medium. Yield of the Extraction. The optimized method was applied to Kentucky Reference 2R4F cigarettes and to 10 different cigarette brands. The results are summarized in Table 2. CONCLUSIONS The ultrasound-assisted extraction, pervaporation continuous derivatization, and photometric detection assembly provide a selective method by which ammonia from cigarettes can be determined. The method combines typical advantages of flow

injection pervaporation and ultrasound, such as versatility, reproducibility, rapidity, and automation. The main aspects of the proposed method are as follows: (1) the combination of pervaporation flow injection with the Berthelot reaction is a reliable method for the determination of ammonia in cigarette containing aromatic amines; (2) automated, fast extraction of the target analyte; (3) the flow injection manifold enables both transport of the extract to the pervaporation chamber and continuous derivatization of the pervaporated species. The closed circuit enables us to minimize the extractant volume and thus analyte dilution. ACKNOWLEDGMENT The authors are grateful to the Spain’s Comisio´n Interministerial de Ciencia y Tecnologı´a (CICyT) for financial support (project BQU-2002-1333). NOTE ADDED AFTER ASAP PUBLICATION The following change was made after this paper was posted on the Web on February 21, 2006. In the section titled Optimization of the Ultrasound-Assisted Leaching Step and in Table 1, the description of the variable was changed from “percentage of application and duration of pulse of ultrasound exposure” to “duration of the ultrasound pulse per second.” The corrected version of the paper was again posted on the Web on March 2, 2006. Received for review June 23, 2005. Accepted January 31, 2006. AC051115U

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