Determination of Polychlorinated Biphenyls in Milk Samples by

Anal. Chem. 2001, 73, 5858-5865

Determination of Polychlorinated Biphenyls in Milk Samples by Saponification-Solid-Phase Microextraction Marıá Llompart,* Manuel Pazos, Pedro Landıń, and Rafael Cela

Departamento de Quı´mica Analı´tica, Nutricio´ n y Bromatologıá, Facultad de Quı´mica, Universidad de Santiago de Compostela, E-15706, Santiago de Compostela, Spain

A saponification-HSSPME procedure has been developed for the extraction of PCBs from milk samples. Saponification of the samples improves the PCB extraction efficiency and allows attaining lower background. A mixed-level fractional design has been used to optimize the sample preparation process. Five variables have been considered: extraction time, agitation, kind of microextraction fiber, concentration, and volume of NaOH aqueous solution. Also the kinetic of the process has been studied with the two fibers (100-µm PDMS and 65-µm PDMS-DVB) included in this study. Analyses were performed on a gas chromatograph equipped with an electron capture detector and a gas chromatograph coupled to a mass selective detector working in MS-MS mode. The proposed method is simple and rapid, and yields high sensitivity, with detection limits below 1 ng/mL, good linearity, and reproducibility. The method has been applied to liquid milk samples with different fat content covering the whole commercial range, and it has been validated with powdered milk certified reference material. Polychlorinated biphenyls (PCBs) are a group of pollutants widely distributed in the environment due to their generous use in the past, their lipophilic character, and their chemical stability.1,2 Thus, PCBs have a long environmental half-life and tend to accumulate in the food chains; the highest concentrations were usually found in human beings and higher animals at the top of the food chain.3,4 Food, and especially fatty food, has been widely recognized as the main source of intake of toxic chemicals such as the PCBs.5 * Corresponding author: (phone) 34-981-563100, ext 14374; (fax) 34-981595012; (e-mail) [email protected]. (1) Erickson, M. D. Analytical Chemistry of PCBs; Lewis Publications: New York, 1997. (2) Ahlborg, H. G.; Becking, G. C.; Birnbaum, C. S.; Brouwer, A.; Drecks, H. J. G. M.; Feeley, M.; Golor, G.; Hanberg, A.; Larsen, G. C.; Lieh, A. K. D.; Safe, S. H.; Schlatter, C.; Waern, F.; Younes M.; Yrjanheikki, E. Chemosphere 1994, 28, 1049-1067. (3) Skaare, J. U.; Tuveng J. M.; Sande, H. A. Arch. Environ. Contam. Toxicol. 1988, 17, 55-63. (4) Galetin-Smith, R.; Pavkov S.; Roncevic, N. Bull. Environ. Contam. Toxicol. 1990, 45, 811-818. (5) Gallo, M. A., Scheuplein, R. J., Van der Heijden, K. A., Eds. Biological Basis for Risk Assessment of Dioxins and Related Biological Basis for Risk Assessment of Dioxins and Related Compounds; Banbury Report 35; Cold Spring Harbor Laboratory Press: Plainview, NY, 1991.

5858 Analytical Chemistry, Vol. 73, No. 24, December 15, 2001

Dairy products, and milk in particular, have received special interest due to their extensive and elevated consumption by the population.6 Several countries have established levels (recommended maximum limits, RMLs) for PCBs in dietary products such as fish (∼2000 ng/g), meats (ranging from 200 to 2000 ng/ g), and eggs (100-300 ng/g). For milk and dairy products, RMLs range from 200 (in Canada) to 1500 ng/g (Thailand). Germany has established RMLs for some congeners (PCBs 28, 52, 101, 180) in 8 ng/g of fat and 10 ng/g of food, for the food with more and less than 10% fat, respectively.7 The general analytical procedure for the determination of PCBs in full-fat milk includes four main steps: extraction from the matrix, preconcentration and cleanup steps, gas chromatographic separation, and detection. Conventional methods of extraction are liquid-liquid extraction and Soxhlet extraction.1 Also, solid-phase extraction (SPE) has been employed.8 The most common cleanup methods are as follows: shaking of the extract with concentrated sulfuric acid,9-12 Florisil,9-13 alumina,14,15 and silica gel8,13 and size exclusion chromatography (SEC).15,16 Also, saponification of the milky matrix has been often employed to separate the PCBs from the fat and to degrade some coexisting organochlorinated pesticides prior of the analysis by gas chromatography.1,17-18 Thus, the analytical control of PCBs in milk products is time-consuming and expensive. Furthermore, large volumes of organic solvents are used and significant amounts of residues are generated. (6) Mes J.; Newsome, W. H. Food Addit. Contam. 1989, 6, 365-375. (7) Angulo R. Residuos organoclorados persistentes en leche humana; Universidad de Cordoba, Servicios de publicaciones, 1998. (8) Pico Y.; Redondo M. J.; Font G.; Man ˜es J. J. Chromatogr., A 1995, 693, 339-346. (9) Galceran, M. T.; Santos, F. J.; Barcelo´, D.; Sańchez, J. J. Chromatogr., A 1993, 665, 275-284. (10) Ramos, L.; Eljarrat, E.; Hernańdez, L. M.; Rivera, J.; Gonza´lez, M. J. Anal. Chim. Acta 1999, 402, 241-252. (11) Ramos, L.; Torre, M.; Laborda F.; Marina, M. L. J. Chromatogr., A 1998, 823, 365-372. (12) Ramos, L.; Torre, M.; Marina, M. L. J. Chromatogr., A 1998, 815, 272277. (13) Krokos, F.; Creaser, C. S.; Wright C.; Startin, J. R. Fresenius J. Anal. Chem. 1997, 357, 732-742. (14) McLachlan, M. S.; Kinkel, M.; Reissinger, M.; Hippelein M.; Kamp, H. Environ. Pollut. 1994, 85, 337-343. (15) Tuinstra, L. G.; Traag, W. A.; van Rhijn, J. A.; vd Spreng, P. F. Chemosphere 1994, 29, 1858-1875. (16) Rimkus, G. G.; Rummler M.; Nausch, I. J. Chromatogr., A 1996, 737, 9-14. (17) Smedes, F.; Boer, J. de. Trends Anal. Chem. 1997, 16, 503-517. (18) Xiong, G.; He, X.; Zhang Z. Anal. Chim. Acta 2000, 413, 49-56. 10.1021/ac0106546 CCC: $20.00

© 2001 American Chemical Society Published on Web 11/14/2001

Arthur and Pawliszyn19 introduced solid-phase microextraction (SPME) in 1990 as a solvent-free sampling technique that reduces the steps of extraction, cleanup, and concentration to a unique step. SPME utilizes a small segment of fused-silica fiber coated with a polymeric phase to extract the analytes from the sample and to introduce them into a chromatographic system. Initially, SPME was used to analyze pollutants in water20,21 via direct extraction. Subsequently, SPME was applied to more complex matrixes, such as solid samples or biological fluids. With these types of samples, direct SPME is not recommended; nevertheless, the headspace mode (HSSPME) is a effective alternative to extracting volatile and semivolatile compounds from complex matrixes. Although recently HSSPME has been applied to an enormous variety of matrixes,22,24 to date, the number of publications that deal with application of SPME technology to milk samples is relatively low25,26 and most of them are related to the determination of flavors.27,28 One of them refers to a multianalyte method for the monitoring of polar (e.g., phenols) and less polar (chlorinated organic compounds including PCBs) analytes from breast milk.26 In that paper, the analytical conditions have been obviously developed to allow the multianalyte extractions and analysis. Although this is an interesting goal in screening studies, for routine control of PCBs in milk products, it should be better to have extraction and determination optimized conditions without sacrifice of detection limits or selectivity. Moreover, any proposal must be validated if certified reference materials of a matching matrix are available. In this paper, a simple and rapid saponification-HSSPME procedure has been developed for the extraction of PCBs from different milk samples. Saponification of the fats helps the transference of the PCBs from the sample to the microextraction fiber. Moreover, saponification acts as a cleanup step and then improving selectivity and reliability in peak identification. The different parameters affecting the analytical process performance have been optimized using a mixed-level factorial design. Analyses were performed on a gas chromatograph equipped with an electron capture detector (ECD) and a gas chromatograph coupled to a mass-selective detector working in mass spectrometry-mass spectrometry (MS-MS) mode, to achieve better limits of detection and selectivity. The proposed method yields high sensitivity, good linearity, precision, and accuracy. EXPERIMENTAL SECTION Reagents and Materials. The PCB congeners, 2,4,4’-trichlorobiphenyl (PCB-28), 2,2’,5,5’-tetrachlorobiphenyl (PCB-52), 2,2’,4,5,5’-pentachlorobiphenyl (PCB-101), 2,3,3’,4,4’-pentachlorobiphenyl (PCB-105), 2,3’,4,4’,5-pentachlorobiphenyl (PCB-118), (19) Arthur, C. L.; Pawliszyn, J. J. Anal. Chem. 1990, 62, 2145-2148. (20) Potter, D. W.; Pawliszyn, J. J. Environ. Sci. Technol. 1994, 28, 298-305. (21) Boyd-Boland, A. A.; Pawliszyn, J. J. Chromatogr. Sci. 1994, 704, 163-172. (22) Zhang, Z.; Pawliszyn, J. J. Anal. Chem. 1993, 65, 1843-1852. (23) Llompart, M.; Li, K.; Fingas, M. J. Microcolumn Sep. 1999, 11, 397-402. (24) Llompart, M.; Blanco, B.; Cela, R. J. Microcolumn Sep. 2000, 11, 23-32. (25) DeBruin, L. S.; Josephy P. D.; Pawliszyn, J. J. Anal. Chem. 1998, 70, 19861992. (26) Ro ¨hring, L.; Meisch, H.-U. Fresenius J. Anal. Chem. 2000, 366, 106-111. (27) Marsili, R. T. J. Agric. Food Chem. 1999, 47, 648-654. (28) Marsili R. T. J. Agric. Food Chem. 2000, 48, 3470-3475.

2,2′,3,4,4′,5′-hexachlorobiphenyl (PCB-138), 2,2′,3,4,4’,5’-hexachlorobiphenyl (PCB-153), 2,3,3’,4,4’,5’-hexachlorobiphenyl (PCB-156), and 2,2’,3,4,4’,5,5’-heptachlorobiphenyl (PCB-180) (PCB numbering according to IUPAC) were supplied by Ultra Scientific (North Kingstown, RI). Isooctane, acetone, and sodium hydroxide were obtained from Merck (Mollet del Valle´s, Barcelona, Spain). All the solvents and reagents were analytical grade. The full-fat milk (3.61% fat), half-fat milk (1.55% fat), and skimmed milk (0.34% fat) were purchased from local supermarkets. Spiked milk samples were prepared by addition of a small volume of acetone solutions containing the target analytes in the concentration range of 1-100 ng/mL. The spiked samples were homogenized in an ultrasonic bath for 5 min; later, the samples were kept at 4 °C for 24 h, to allow analyte-matrix interactions. Afterward, they were frozen at -20 °C until 1 h before the analysis. The certified reference material (CRM 450), used for the validation of the method, is real contaminated powdered milk with a certified content in PCB-52, PCB-101, PCB-118, PCB-156, and PCB-180. This material contains approximately 3.9% water and 25% fat, respectively. It is used after reconstituting and was supplied by the EC Community Bureau of Reference (BCR). HSSPME Extraction Procedure. Manual SPME holders were used with a 100-µm poly(dimethylsiloxane) (PDMS) and 65µm poly(dimethylsiloxane)-divinylbenzene (PDMS-DVB) fiber assembly (Supelco, Bellefonte, PA). The fibers were conditioned as recommended by the manufacturer. The samples were placed in headspace vials. When saponification was performed, a few milliliters of NaOH solution was added to the sample. The vial was sealed with a headspace aluminum cap furnished with a Teflon-faced septum, immersed in a water bath maintained at 100 °C, and let equilibrate for 6 min before HSSPME. Afterward, the fiber was exposed to the headspace over the sample for 5-240 min, depending on the experiment. The sample was magnetically agitated during sampling. Once the exposition period was finished, the fiber was immediately inserted into the GC injector and the chromatographic analysis was carried out. Desorption time was set at 5 min. Chromatographic Conditions. GC-ECD analysis were performed in a HP 5890 series II GC equipped with an electron capture detector and a split/splitless injector, operated by a HP Chemstation software. PCBs were separated on a 25 m length × 0.32 mm i.d., HP-1 column coated with a 0.17-µm film. The GC oven temperature program was as follows: 90 °C hold 2 min, rate 20 °C/min to 170 °C, hold for 7.5 min, rate 3 °C/min, to final temperature 280 °C, and hold for 5 min. N2 was employed as carrier and makeup gas, with a column flow of 1.2 mL/min at 90 °C. Split flow was set at 50 mL/min. Injector and ECD temperatures were 260 and 280 °C, respectively. Injector valve time was set at 2 min. The GC/MS-MS analyses were performed on a Varian 3800 gas chromatograph (Varian Chromatography Systems, Walnut Creek, CA) equipped with a 1079 split/splitless injector and a ion trap spectrometer (Varian Saturn 2000, Varian Chromatography Systems) with a waveboard for MS-MS analysis. The system was operated by Saturn GC/MS WorkStation v5.4 software. The MSMS detection method was adapted from reference.29 PCBs were Analytical Chemistry, Vol. 73, No. 24, December 15, 2001

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Figure 1. HSSPME-GC-ECD chromatograms of spiked milk samples: (A) skim milk; (B) full-fat milk; (C) full-fat milk after saponification. Peak identification: (1) PCB-28, (2) PCB-52, (3) PCB-101, (4) PCB-118, (5) PCB-105, (6) PCB-153, (7) PCB-138, (8) PCB-156, and (9) PCB-180.

separated on a 25 m length × 0.32 mm i.d., CPSil-8 column coated with a 0.25-µm film. The GC oven temperature program was as follows: 90 °C hold 2 min, rate 30 °C/min to 170 °C, hold for 10 min, rate 3 °C/min to 250 °C, rate 20 °C/min to a final temperature of 280 °C, and hold for 5 min. Helium was employed as carrier gas, with a constant column flow of 1.0 mL/min. Injector was programmed to return to the split mode after 2 min from the beginning of a run. Split flow was set at 50 mL/min. Injector temperature was held constant at 270 °C. Trap temperatures, manifold temperatures and transfer line temperatures were 250, 50, and 280 °C, respectively. (29) Brochu, Ch.; Moore, S.; Hamelin, G. Varian Application Note 64, March 2000.

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Analytical Chemistry, Vol. 73, No. 24, December 15, 2001

Table 1. Factor Levels in the Mixed-Level Fractional Design and Optimum Values levels key

factor

low

high

optimum

A B C D E

vol of NaOH (mL) concn of NaOH (%) time (min) fiber stirring

0.5 20 30 PDMS-DVB no

3.5 35 60 PDMS yes

2-3.5 20 60 PDMS-DVB yes

RESULTS AND DISCUSSION Preliminary Experiments. Influence of Fat Contents. Initial SPME experiments were performed using spiked skimmed and

Table 2 expt

vol of NaOH (mL)

1 2 3 4 5 6 7 8 9 10 11 12

0.5 3.5 3.5 0.5 3.5 0.5 0.5 3.5 2.0 2.0 2.0 2.0

a. Design Matrix in the Mixed-Level Fractional Design concn of NaOH (%) time (min) 20.0 20.0 35.0 20.0 35.0 35.0 35.0 20.0 35.0 20.0 20.0 35.0

60 30 30 30 60 60 30 60 30 30 60 60

fiber

stir

PDMS PDMS-DVB PDMS-DVB PDMS-DVB PDMS PDMS PDMS-DVB PDMS PDMS PDMS PDMS-DVB PDMS-DVB

no no yes no yes yes yes no no yes yes no

b. Response Values in Optimization Experiments response (area counts) run

PCB-28

PCB-52

PCB-101

PCB-118

PCB-105

PCB-153

PCB-138

PCB-156

PCB-180

1 2 3 4 5 6 7 8 9 10 11 12

980 950 1 424 410 2 709 990 421 821 1 085 460 1 096 020 2 260 501 852 233 776 758 1 038 570 4 402 370 2 599 081

237 115 240 803 511 484 63 534 328 503 304 545 418 615 250 296 153 235 312 406 851 813 444 112

196 294 143 535 340 514 40 815 475 522 340 608 246 640 294 436 124 689 299 428 708 507 316 287

131 798 177 086 301 969 28 143 544 098 310 320 191 526 266 487 88 742 259 751 625 116 238 178

164 224 287 349 470 628 40 552 864 604 461 059 290 138 384 803 104 794 368 233 1 012 671 338 537

191 600 186 291 325 493 31 105 708 150 453 419 227 531 397 218 131 503 372 527 747 394 268 463

156 256 188 678 331 261 32 812 689 307 428 688 213 593 374 207 107 122 351 075 775 739 275 042

111049 228168 254685 12208 677673 414970 191453 345934 75757 314973 738361 255127

77 478 127 450 174 313 18 378 417 062 297 861 119 448 260 409 54 580 226 709 478 827 170 360

half-fat milk samples. The concentration of added compound was between 10.8 and 43.2 ng/mL depending on the PCB congener. Two milliliters of sample was poured in a 22-mL glass vial that was sealed with a Teflon septum and an aluminum cap. The vial was introduced in a water bath at 100 °C and a 100-µm PDMS coated fiber was exposed to the headspace over the sample for 30 min while stirring with a magnetic stirring bar. Figure 1A shows the ECD chromatogram obtained for the skim milk. When the same experiments were performed on full-fat milk, the results were considerably lower, as can be seen in the chromatogram shown in Figure 1B. Also, in this kind of milk sample, the background appeared higher, and it increased after each SPME injection. This indicates that this simple procedure might be adequate for the analysis of PCBs in milk samples having low fat content; however, it is not adequate when the percentage of fat increases. This is quite logical because PCBs are more strongly retained in the sample matrix as the fat content increases, and SPME, even at 100 °C, is not an efficient technique. Taking into account these results, our objective was to develop a SPME procedure that improved the release of PCBs from the sample to the fiber coating irrespective of the fat content of the samples. Saponification of fats to their corresponding glycerols and carboxylates facilitates the release of PCBs from fatty matrixes and also can selectively degrade many other interfering substances without affecting the PCBs.1 Sets of preliminary HSSPME experiments were run after 2 mL of 20% NaOH was added to the samples. Figure 1C shows the chromatogram obtained by HSSPME for the full-fat milk with the addition of NaOH solution. When comparing chromatograms B and C in Figure 1, we can see the increase in response, as well as the lower background obtained, after saponification.

Optimization of the Saponification-HSSPME Process: Factorial Design. A factorial design was performed with the purpose of optimizing the saponification-HSSPME process. For this study, a spiked full-fat milk sample was used. Due to the fact that many factors can be considered as potentially affecting the analytical process, a progressive strategy was used. Five experimental factors were included in the factorial design approach. These factors were selected in view of the previous experiments and our knowledge regarding SPME, while other factors have been left for later study after approaching the optimum for the factor in the design. The experimental parameters included in the factorial design were the following: concentration and volume of NaOH, extraction time, stirring, and kind of fiber used. Two fibers were tested: a 100-µm PDMS fiber and a 65-µm PDMS-DVB fiber. In principle, both fibers should be adequate for the extraction of PCBs. We chose a mixed-level fraction 3 × 24-2 type IV resolution design, which involved 12 runs.30 In this design, main effects are clear of two-factor interactions, but these are partially confounded with other two-factor interaction effects. Thus, with a reasonable number of experiments, the statistical significance of all the main factors can be clearly established. All parameters were studied at two levels, with the exception of the volume of NaOH, which was studied at three levels. Table 1 lists the upper and lower levels assigned to each factor. Table 2a shows the design matrix and Table 2b the responses obtained for each analyte in each experiment. An analysis of the results given in Table 2b produces the standardized Pareto charts shown in Figure 2, where the factors and interactions that exhibited nonsignificant effects have already (30) Statgraphics Plus Manual, Experimental Design. Manugistics, 1997.


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Figure 2. Pareto chart for main and interaction effects. The vertical line indicates the statistical significance bound for the effects.

been removed. Pareto charts are graphs that show the amount of influence that each experimental factor has on the response arranged in order of decreasing influence. In a standardized Pareto chart, each effect is divided by its standard error. The chart also includes a vertical line for testing significance. An effect that goes beyond the critical line is significant. Figure 3 shows the main effect plots for some of the compounds (PCB-28 and PCB-180). In these plots, obtained by drawing a line between the low and the high levels of main factors, we can see the magnitude of the effect of each factor on the microextraction process, as well as the level of the factor that produces the highest response. 5862


Three patterns were identified regarding the target PCBs. Figure 2 has been divided into three graphs to show these patterns. As can be seen in Figures 2 and 3, the agitation of the sample was the most important factor for almost all the analytes. In all cases, this factor has a positive effect and it appears to be of increasing importance as the degree of chlorination of the PCBs increases. Also the extraction time was a significant factor for all the compounds, as might be expected. On the other hand, the type of fiber was only important for the lighter PCBs, mainly for PCB-28 and PCB-52. For these compounds, the PDMS-DVB fiber

Figure 3. Graphics showing the influence of main effects on the extraction of PCB-28 and PCB-180. The size and the slope of the lines indicate the magnitude and sign of the variation of the extraction efficiency with the factor level, respectively.

is more efficient than the PDMS fiber. The effect of the factor fiber appeared negative (see Figure 2) because PDMS-DVB was selected as its low level (see Table 1). For the highly chlorinated PCBs, the two fibers tested seem to have similar performance and this factor lacks statistical significance. The volume of NaOH was also a significant factor for most of the compounds, but especially for the high molecular weight

PCBs. This factor was the only one studied at three levels, and as we can see in Figure 3, the main effect plot curvature of this factor shows optimum experimental settings that vary (2-3.5 mL) depending on the PCBs to be extracted. The concentration of NaOH was only statistically significant for PCB-105 and PCB-180 This factor showed a negative effect, which means that the extraction efficiency decreases when the concentration of NaOH was at the high level. A possible explanation for this effect can be attributed to an increased density and viscosity of the milk-NaOH phase when NaOH concentration increases. This retards the kinetics of the HSSPME process, and consequently, the extraction efficiency decreases. The last column of Table 1 summarizes the optimum conditions for the HSSPME of PCBs from milk. These optimum values were similar for all the compounds with the exception of the volume of NaOH. In the group of congeners PCB-28, PCB-52, and PCB-101, this factor is statistically significant only for PCB-101. On the contrary, for the remaining PCBs studied, it is important to use a volume of NaOH of 3 mL. Thus, for the next studies, the volume of NaOH added to the sample was set at 3 mL. Extraction Time Profile. To investigate the PCB sorption behavior in both fibers, the extraction time profiles for the PCB congeners were established by plotting the detector response versus the extraction time. Different extraction times (15, 30, 60, 120, and 240 min) were used for HSSPME at 100 °C with the PDMS fiber and with the PDMS-DVB fiber. The concentration and volume of NaOH were 20% and 3 mL, respectively, and the system was magnetically stirred. The sample volume was 2 mL. Representative extraction time profiles for PCB-28, PCB-101, PCB138, and PCB-180 are shown in Figure 4. The HSSPME process is faster when the PDMS fiber is used instead of the PDMS-DVB one. With the first fiber, equilibrium is reached within the interval of time studied for most of the compounds. Only PCB-138, PCB-156, and PCB-180 have not reached equilibrium after 4 h. The PDMS-HSSPME process is especially slow for PCB-180, the most chlorinated of the conge-

Figure 4. Extraction time profile obtained with PDMS and PDMS-DVB fibers.


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Table 3. Picograms of PCBs Absorbed by the PDMS Fiber from Skim, Half-Fat, and Full-Fat Milk Samples amount absorbed (pg) PCB 28 52 101 118 105 153 138 156 180

skim milk 413.2 102.9 109.3 121.3 262.5 138.6 169.1 130.7 72.5

half-fat milk 143.7 42.3 40.6 39.0 79.8 42.6 52.4 37.6 20.8

Table 4. Comparison of the Amount of PCBs Absorbed by the PDMS Fiber with and without Saponification of Samples as a Function of the Fat Content ratio (with/without saponification)

fat milk 56.2 19.3 17.9 14.3 26.6 17.3 20.5 12.8 7.7

ners, and its kinetic curve is almost a straight line in the interval of time studied. For the trichloro and tetrachloro congeners (PCB28 and PCB-52), the response obtained is maximum for a sampling time of 30 min, decreasing slightly when the time increases. For the PDMS-DVB fiber, the microextraction process is still slower. Only PCB-28 and PCB-52 seem to have reached equilibrium before 4 h. For the other compounds, the system is still far from equilibrium after this period of time. Despite the slower kinetics, the response obtained for PCB-28, PCB-52, and PCB101 was higher than the one obtained with PDMS fiber, whatever the sampling time was. For the other congeners, response was similar with the two fibers when sampling time was not longer than 60 min. For longer sampling times, the response achieved with PDMS-DVB was higher. For PCB-180, response was similar with the two fibers in the interval of time studied. These results indicate that the capacity of the PDMS-DVB fiber is superior to the PDMS fiber. Because the purpose of this study was to develop a simple and fast method for the analysis of PCBs in milk, sampling time was set in 60 min, although greater sensitivity can be achieved with longer sampling times. For sampling times of 60 min, the response achieved with the PDMS-DVB fiber was higher for the least chlorinated PCBs. But an inspection of the chromatograms obtained using an ECD detector shows that the PDMS fiber is more selective, producing less background. On the other hand, when a more selective detector was used, (e.g., MS-MS detector), the level of the background was similar with the two fibers. Both fibers are adequate for the extraction of PCBs from milk, and the choice of one of them is conditioned by the GC detector. Effect of Saponification in Milk Samples with Different Fat Content. The saponification-HSSPME method has been applied to three kinds of milk with different fat content, all spiked with PCBs at the 10 ng/mL level. Table 3 reports the amounts of PCBs extracted from each sample. As can be seen, the method is more efficient the lower the fat content. For the skim milk, the amount of each congener extracted is between 5 and 10 times higher than for the full-fat milk. On the other hand, Table 4 compares the ratios of the above results to direct (without saponification) extraction. In Table 4, is evident that, for all kinds of samples tested, the saponification stage enables a more efficient SPME process, especially for the half-fat and full-fat milk samples. For these samples, the increase in response was between 4 and 9 times, depending on the PCB congener. 5864 Analytical Chemistry, Vol. 73, No. 24, December 15, 2001

PCB

skim milk

half-fat milk

fat milk

28 52 101 118 105 153 138

3.2 3.0 3.4 4.0 4.1 3.8 3.8

4.7 4.4 4.9 6.8 8.2 5.7 6.5

4.2 4.2 4.7 6.3 8.1 5.8 6.7

Figure 5. Effects of sample volume on the response for HSSPME.

Effect of Sample Volume. One parameter that has not been included in the experimental design although it could increase the total amount of PCBs extracted by the fiber is the sample volume. To check the effect of sample volume on the amount of PCBs extracted by the saponification-HSPME method, a set of experiments was carried out with 2, 6, and 40 mL of milk. The volume of NaOH added in each case was proportional to the sample volume (3, 9, and 60 mL, respectively). For the experiments with a low volume of sample (2 and 6 mL), the size of the sampling vials was 22 mL. For the experiments with 40 mL of sample, the size of the vials was 120 mL. Mean response values are shown in Figure 5. As can be seen, the extraction of a greater volume of milk does not improve the sensitivity of the process. Even for a sample size of 40 mL, responses obtained were lower than the ones obtained for the other volumes tested. Possible explanations for this behavior could be the slower SPME kinetics when sample volume increases, besides the lower efficiency of stirring, which also retards the process. Performance Evaluation and Validation of the Proposed Method. To evaluate the linearity of the HSSPME method, calibration studies were performed with PDMS and PDMS-DVB fibers, using spiked milk samples. Concentration ranges tested are given in Table 5. The correlation coefficients, also given in Table 5, demonstrated a directly proportional relationship between the extracted amount of PCBs and its initial concentration in the sample over a 50-fold concentration range. The precision of the experimental procedure was also evaluated. A series of five HSSPME consecutive analyses gave a relative standard deviation (RSD) between 5.6 and 10% and between 5.0 and 13% with the PDMS and PDMS-DVB coatings, respectively

Table 5. Linearity, Precision, and Sensitivity of the HSSPME Procedure

PCB-28 PCB-52 PCB-101 PCB-118 PCB-105 PCB-153 PCB-138 PCB-156 PCB-180 a

correlation coefficient

detection limit (S/N ) 3, ng/mL)

repeatability (% RSD)

concn range (ng/mL)

PDMS

PDM-DVB

PDMS

PDM-DVB

PDMSa

PDM-DVBb

2.30-115.2 0.58-28.9 0.75-37.6 0.99-49.5 1.80-89.8 1.53-76.3 1.89-94.6 2.20-110.0 1.90-95.2

0.997 0.998 0.997 0.997 0.994 0.994 0.997 0.996 0.996

0.999 0.998 0.998 0.999 0.999 0.999 0.999 0.997 0.997

10.4 6.5 5.7 6.9 8.0 11.0 8.6 5.6 5.9

13.1 5.0 9.4 7.4 7.2 5.4 5.2 6.5 8.0

0.021 0.016 0.015 0.020 0.017 0.027 0.026 0.029 0.040

0.014 0.019 0.041 0.11 0.11 0.30 0.14 0.47 0.71

GC-ECD. b GCMS-MS.

Table 6. Procedure Validation. Analysis of CRM 450 Certified Milk PDMS (GC-ECD) certified values (ng/g) PCB-28 PCB-52 PCB-118 PCB-153 PCB-138 PCB-156 PCB-180 a

1.16 ( 0.17 3.3 ( 0.4 19.0 ( 0.7 14.9a 1.62 ( 0.20 11.0 ( 0.7

PDMS-DVB (GC/MS-MS)

HSSPME values (ng/g)

recoveries (%)

HSSPME values (ng/g)

recoveries (%)

0.86 ( 0.06 3.19 ( 0.24 18.6 ( 1.1

75 97 98

66 106 103

1.64 ( 0.11 9.29 ( 0.26

0.79 ( 0.11 0.78 ( 0.07 3.50 ( 0.34 19.6 ( 1.5 15. 9 ( 1.0

102 88

12.8 ( 1.0

116

Not certified, value taken from ref 8.

(see Table 5). Detection limits are under 1 ng/mL for all the target PCBs with both fibers. A real contaminated milk sample with certified content in PCBs was analyzed. This material is full-fat powdered milk (CRM 450 purchased from BCR), which content in PCBs is given in Table 6. For the quantification of this sample, a standard addition protocol was performed by adding different amounts of the certified PCBs to the sample. HSSPME was performed using PDMS fiber. Analyses were carried out in the GC-ECD. Another standard addition protocol was performed to validate the HSSPME PDMS-DVB method using GC/MS-MS detection. In this study, besides the certified PCB congeners, PCB-28 and PCB-138, which can be detected in the material although not certified, were also

added to the sample to perform their quantification. The results obtained (see Table 6) were in good agreement with the certified values. ACKNOWLEDGMENT Financial support from the Xunta de Galicia (Consellerıá de Medio Ambiente), project PGIDT99MA23701 is gratefully acknowledged.

Received for review June 12, 2001. Accepted September 17, 2001. AC0106546


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Determination of Polychlorinated Biphenyls in Milk Samples by

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