Anal. Chem. 2004, 76, 6313-6320
Measurement of Selected Polybrominated Diphenyl Ethers, Polybrominated and Polychlorinated Biphenyls, and Organochlorine Pesticides in Human Serum and Milk Using Comprehensive Two-Dimensional Gas Chromatography Isotope Dilution Time-of-Flight Mass Spectrometry Jean-Franc¸ ois Focant,* Andreas Sjo 1 din, Wayman E. Turner, and Donald G. Patterson, Jr.
Organic Analytical Toxicology (OAT), Division of Laboratory Sciences (DLS), National Center for Environmental Health (NCEH), Centers for Disease Control and Prevention (CDC), 4770 Buford Highway, NE Mail Stop F-17, Atlanta, Georgia 30341
A new method using comprehensive two-dimensional gas chromatography and isotope dilution time-of-flight mass spectrometry (GC×GC-IDTOFMS) for the simultaneous measurement of selected polychlorinated biphenyls (PCBs), organochlorine pesticides (OCPs), and brominated flame retardants is presented. In contrast to the reference methods based on classical GC/MS, a single injection of the extract containing all compounds of interest results in accurate identification and quantification. Using GC×GC ensures the chromatographic separation of most compounds, and TOFMS allows mass spectral deconvolution of coeluting compounds as well as the use of 13C-labeled internal standards for quantification. Isotope ratio measurements of the most intense ions for both native and labels ensure the required specificity. The use of this new method with an automated sample preparation procedure developed at the Centers for Disease Control and Prevention (CDC) for the analysis of human serum and milk compared favorably to conventional isotope-dilution one-dimensional gas chromatography-high-resolution mass spectrometry (GC-IDHRMS) for the different human serum and milk pools tested. The instrumental detection limits ranged between 0.5 pg/µL and 10 pg/µL and the method detection limits ranged between 1 and 15 pg/µL (N ) 59 analytes). The reproducibility of the method was almost as good as with GC-IDHRMS, the relative standard deviations ranging between 1 and 11% for OCPs measured in human serum. OCP, PBDE, and PCB levels measured using the two methods were highly correlated, and the deviations between the two methods were below 20% for most analytes with concentrations above 1 ng/g milk lipids.
* Corresponding author. Current address: Mass Spectrometry Laboratory, University of Lie`ge, Alle´e de la Chimie, B6c, B-4000 Lie`ge, Belgium. Phone: +32 4 366 35 31. Fax: +32 4 366 43 87. E-mail:
[email protected]. 10.1021/ac048959i CCC: $27.50 Published on Web 10/06/2004
© 2004 American Chemical Society
Polychlorinated biphenyls (PCBs) and organochlorine pesticides (OCPs) are some of the persistent organic pollutants that have been the most studied in the past years. Like many other organizations involved in human monitoring, the Centers for Disease Control and Prevention (CDC) has been measuring most of these analytes in human serum and milk for 25 years using several different approaches.1-6 More recently, polybrominated diphenyl ethers (oxides) (PBDEs or PBDPOs), a class of brominated flame retardants (BFRs), have been described as emerging persistent toxic compounds.7,8 Although little is still known about their toxic effects in humans, recent reports indicate serious concerns regarding possible developmental neurotoxicity and endocrine disruption.9-11 They have been reported in environmental and human samples at varying increasing levels, indicating the pertinence of their monitoring. In Europe, the current levels of PBDEs in humans are usually more than 1 order of magnitude lower than the levels of PCBs.12 However, levels of PBDEs measured in the North (1) Burse, V. W.; Patterson, D. G., Jr.; Brock, J. W.; Needham, L. L. Toxicol. Ind. Health 1996, 12, 481-498. (2) Brock, J. W.; Burse, V. W.; Ashley, D. L.; Najam, A. R.; Green, V. E.; Korver, M. P.; Powell, M. K.; Hodge, C. C.; Needham, L. L. J. Anal. Toxicol. 1996, 20, 528-536. (3) DiPietro, E. S.; Lapeza, C. R., Jr.; Cash, T. P.; Turner, W. E.; Green, V. E.; Gill, J. B.; Patterson, D. G., Jr. Organohalogen Compd. 1997, 31, 26-31. (4) Barr, J. R.; Maggio, V. L.; Barr, D. B.; Turner, W. E.; Sjo¨din, A.; Sandau, C. D.; Pirkle, J. L.; Needham, L. L.; Patterson, D. G., Jr. J. Chromatogr., B 2003, 794, 137-148. (5) Sandau, C. D.; Sjo ¨din, A.; Waterman, A. L.; Roman, W.; Davis, M. D.; Patterson, D. G., Jr. Extraction of PCBs, persistent pesticides and their hydroxylated metabolites from plasma using semi-automated solid-phase extraction and cleanup technique. Unpublished work. (6) Sandau, C. D.; Sjo ¨din, A.; Davis, M. D.; Barr, J. R.; Maggio, V. L.; Waterman, A. L.; Preston, K. E.; Preau Jr, J. L.; Barr, D.; Needham, L. L.; Patterson, D. G., Jr. Anal. Chem. 2003, 75, 71-77. (7) de Wit, C. A. Chemosphere 2002, 46, 583-624. (8) Rahman, F.; Langford, K. H.; Scrimshaw, M. D.; Lester, J. N. Sci. Total Environ. 2001, 275, 1-17. (9) Eriksson, P.; Jakobsson, E.; Fredriksson, A. Environ. Health Perspect. 2001, 109, 903-908. (10) Legler, J.; Brouwer, A. Environ. Int. 2003, 29, 879-885. (11) Darnerud, P. O. Environ. Int. 2003, 29, 841-853.
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American population are at least 1 order of magnitude higher compared with European populations.13 Therefore, in the United States, PBDEs, PCBs, and OCPs are found at similar levels in humans. These compounds are lipophilic, are concentrated in the lipid compartments, and might easily be isolated from the sample matrix by using similar sample preparation steps.14,15 The CDC target group of compounds consists in 38 human marker PCBs,4 11 persistent OCPs,6 and 10 prominent BFRs,14 and the chromatographic separation is challenging. Fractionation in subclasses during the sample preparation step, multiple injections, or both are required to perform the measurements.16,17 A few reports have described the simultaneous measurement of a subfraction of this multianalyte group using gas chromatography (GC),4,14,18 but to the best of our knowledge, a comprehensive analytical method has never been reported that allows the simultaneous measurement of the entire set of 59 compounds. A potential tool for this particular task is comprehensive twodimensional gas chromatography (GC×GC).19-21 This technique involves the serial coupling of two GC columns using an interface, called a “modulator”, which is responsible for efficiently and continuously sampling narrow bands of all the species eluting from the first column (first dimension, 1D) and reinjecting them into the second column (second dimension, 2D). The separation achieved in 1D is maintained through the entire separation process (conservation rule) because of the high sampling rate (modulation frequency, PM) of the modulator. After a very fast (2-10 s) separation in 2D, high-speed chromatograms are recorded at the detector. They consist of slices that can be combined to describe the elution pattern by means of contour plots in the chromatographic separation space. Peak widths in 2D are in the range of 50-200 ms. In contrast with heart-cutting 2DGC,22 the whole sample undergoes separation in the 2D using the GC×GC technique. The resulting increased peak capacity is well suited to accommodate complex mixtures of compounds,23-27 and GC×GC has previously been used for the separation of complex mixtures of selected PCBs.28-33 (12) Darnerud, P. O.; Eriksen, G. S.; Jo´hannesson, T.; Larsen, P. B.; Viluhsela, M. Environ. Health Perspect. 2001, 109, 49-68. (13) Sjo ¨din, A.; Patterson, D. G., Jr.; Bergman, A° . Environ. Int. 2003, 29, 829839. (14) Sjo ¨din, A.; Jones, R. S.; Lapeza, C. R., Jr.; Focant, J.-F.; McGahee, E. E., III; Patterson, D. G., Jr. Anal. Chem. 2003, 76, 1921-1927. (15) Sjo ¨din, A.; McGahee, E. E., III; Focant, J.-F.; Jones, R. S.; Lapeza, C. R., Jr.; Zhang, Y.; Patterson, D. G., Jr. Anal. Chem. 2004, 76, 4508-4514. (16) Barr, J. R.; Green, V. E.; Lapeza, C. R., Jr.; Maggio, V. L.; Turner, W. E.; Woolfit, A. R.; Grainger, J.; Needham, L. L.; Patterson, D. G., Jr. Organohalogen Compd. 1997, 31, 276-281. (17) Pirard, C.; De Pauw, E.; Focant, J.-F. J. Chromatogr., A 2003, 998, 169181. (18) Covaci, A.; de Boer, J.; Ryan, J. J.; Voorspoels, S.; Schepens, P. Anal. Chem. 2002, 74, 790-798. (19) Phillips, J. B.; Beens, J. J. Chromatogr., A 1999, 856, 331-347. (20) Marriott, P.; Shellie, R. Trends Anal. Chem. 2002, 21, 573-583. (21) Dimandja, J.-M. D. Anal. Chem. 2004, 76, 167A-174A. (22) Simmons, M. C.; Snyder, L. R. Anal. Chem. 1958, 30, 32-35. (23) Koryta´r, P.; van Stee, L. L. P.; Leonards, P. E. G.; de Boer, J.; Brinkman, U. A. Th. J. Chromatogr., A 2003, 994, 179-189. (24) Marriott, P. J.; Haglund, P.; Ong, R. C. Y. Clin. Chim. Acta 2003, 328, 1-19. (25) Marriott, P. J.; Shellie, R.; Cornwell, C. J. Chromatogr., A 2001, 936, 1-22. (26) Blomberg, J.; Schoenmakers, P. J.; Brinkman, U. A. T h. J. Chromatogr., A 2002, 972, 137-173. (27) Adahchour, M.; van Stee, L. L. P.; Beens, J.; Vreuls, J. J.; Batenburg, M. A.; Brinkman, U. A. Th. J. Chromatogr., A, in press.
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Time-of-flight mass spectrometers (TOFMS) are the detectors of choice when high acquisition scan rates are required,34 and they became the mass analyzers of choice in high-speed GC/ MS.35-37 The high scanning rate of the TOFMS has been used to ensure accurate characterization of the 2D narrow peaks produced by GC×GC.38,39 Another advantage of TOFMS is that all ion fragments represent the same point on the chromatographic peak profile, ensuring that ion ratios remain the same across the peaks. This spectral continuity allows mass spectral deconvolution of overlapping peaks when the fragmentation patterns are different. Deconvoluted ion currents (DICs) can be used to solve chromatographic coelution problems in the MS domain. The coupling of GC×GC with TOFMS results in an analytical tool offering high separation power based on the combined use of chromatographic and mass spectral resolution. Additionally, chromatographic zone compression after the modulation process results in a signal enhancement, which improves TOFMS instrumental detection limits (IDLs). The TOFMS detection is sometimes described as a true third dimension of a GC×GC-TOFMS system.21 Isotope dilution (ID) based on 13C-labeled compounds is commonly used in conjunction with electron impact for the accurate measurement and quantification of PCBs and OCPs4,6 at low parts-per-billion levels. PBDEs can be either measured in the same way14,17 or by electron capture negative ionization MS,40 which does not allow ID because bromine isotopes m/z 79 and 81 are the only available ions to monitor. This paper describes the development and application of a GC×GC-IDTOFMS method for the simultaneous measurement of a multianalyte group of 59 compounds targeted in biomonitoring of human serum and milk. EXPERIMENTAL SECTION Standards, Chemicals, and Supplies. The compounds considered in the present study are highly toxic, and their measurement should only be attempted by properly trained personnel. All standards should be handled with great care. The use of a BSL-2 laboratory is required to work with human body fluids where the presence of infectious agents may be unknown. Related wastes have to be decontaminated before disposal by an approved decontamination method such as autoclaving. (28) Dimandja, J.-M. D.; Grainger, J.; Patterson, D. G., Jr. Organohalogen Compd. 1999, 40, 23-26. (29) Koryta´r, P.; Leonards, P. E. G.; de Boer, J.; Brinkman, U. A. Th. J. Chromatogr., A 2002, 958, 203-218. (30) Focant, J.-F.; Dimandja, J.-M.; Clouden, G. C.; Grainger, J.; Patterson, D. G., Jr. Pittcon 2002, New Orleans, March 17-22, 2002; Abstract 283. (31) Focant, J.-F.; Sjo ¨din, A.; Patterson, D. G., Jr. J. Chromatogr., A 2003, 1019, 143-156. (32) Harju, M.; Danielsson, C.; Haglund, P. J. Chromatogr., A 2003, 1040, 111126. (33) Focant, J.-F.; Sjo ¨din, A.; Patterson, D. G., Jr. J. Chromatogr., A 2003, 1040, 227-238. (34) Holland, J. F.; Enke, C. G.; Allison, J.; Stults, J. T.; Pinkston, J. D.; Newcome, B.; Watson, J. T. Anal. Chem. 1983, 55, 997A-1012A. (35) Leclercq, P. A.; Cramers, C. A. Mass Spectrom. Rev. 1998, 17, 37-49. (36) Leonard, C.; Sacks, R. Anal. Chem. 1999, 71, 5177-5184. (37) Focant, J.-F.; Cochran, J. W.; Dimandja, J.-M. D.; DePauw, E.; Sjo ¨din, A.; Turner, W. E.; Patterson, D. G., Jr. Analyst 2004, 129, 331-336. (38) Shellie, R.; Marriott, P.; Morrison, P. Anal. Chem. 2001, 73, 1336-1344. (39) van Deursen, M.; Beens, J.; Reijenga, J.; Lipman, P.; Cramers, C.; Blomberg, J. J. High Resolut. Chromatogr. 2000, 23, 507-510. (40) Sellstro ¨m, U.; Jansson, B.; Kierkegaard, A.; de Witt, C.; Odsjo¨, T.; Olsson, M. Chemosphere 1993, 26, 1703-1718.
All standard solutions were purchased from Cambridge Isotope Laboratories (Andover, MA). The EC-5022 PCB 10-points calibration standard solution contained a mixture of 38 native PCBs: IUPAC no. 18, 28, 44, 49, 52, 66, 74, 87, 99, 101, 105, 110, 118, 128, 138, 146, 149, 151, 153, 156, 157, 158, 167, 170, 172, 177, 178, 180, 183, 187, 189, 194, 195, 196, 201, 203, 206, and 209 spanning the concentration range 0.5-1000 pg/µL; as well as 21 13C -labeled PCBs: IUPAC no. 28, 32, 52, 70, 101, 105, 111, 118, 12 128, 138, 153, 156, 157, 167, 170, 178, 180, 189, 194, 206, and 209 at a concentration of 75 pg/µL in nonane. The EC-5087 13C12labeled PCB internal standard spiking solution contained a mixture of the same 21 13C12-labeled PCBs at a concentration of 7.5 pg/ µL in methanol (Table S-1). The EO-5159 BFR 10-points calibration standard solution contained a mixture of native compounds, spanning the concentration range 0.2-2000 pg/µL, and 13C12labeled analytes, at a concentration of 75 pg/µL. The solution contained the following PBDEs: IUPAC no. 17, 28, 47, 66, 85, 99, 100, 153, 154, 183, 203, and 209, as well as CB-153 and the polybrominated biphenyl BB-153. The EO-5158 13C12-labeled BFR internal standard spiking solution contained the following 13C12labeled PBDEs: UIPAC no. 28, 47, 99, 100, 153, 154, 183, 209, and 13C12-labeled BB-153 at a concentration of 7.5 pg/µL in methanol. The ES-5019 persistent OCP 8-points calibration standard solution contained the following mixture of native and 13C -labeled (n ) 6, 10, or 12)6 analytes: hexachlorobenzene n (HCB), β-hexachlorocyclohexane (β-HCCH), γ-hexachlorocyclohexane (γ-HCCH), heptachlor epoxide, oxychlordane, transnonachlor, 1,2,3,4,10,10-hexachloro-6,7-epoxy-1,4,4a,5,6,7,8,8aoctahydro-endo-1,4-exo-5,8-dimethanonaphthalene (dieldrin), 1,1,1trichloro-2-(2-chlorophenyl)-2-(4-chlorophenyl)ethane (o,p′-DDT), 1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane (p,p′-DDT), 1,1a,2,2,3,3a,4,5,5,5a,5b,6-dodecachlorooctahydro-1,3,4-metheno-1H-cyclobuta[cd]pentalene (mirex), and 1,1-dichloro-2,2-bis(4-chlorophenyl)ethylene (p,p′-DDE). Native compounds were in the concentration range of 5-1000 pg/µL and 13Cn-labeled compounds at a concentration of 100 pg/µL, except for 13C12-p,p′-DDE which was at a concentration of 250 pg/µL. The ES-5177 13Cn- labeled OCP internal standard spiking solution contained the same analytes as the calibration solution at a concentration of 10 pg/µL in methanol. The EO-5169 recovery spiking standard contained the following labeled compounds: 1,2,3,4-tetrachlorodibenzo-p-dioxin (13C6-1,2,3,4-TCDD, 2.5 pg/µL), 13C12-BDE-77 (7.5 pg/µL), and 13C -CB-208 (10 pg/µL). The solvent for the recovery standard 12 was n-hexane containing 10% and 2 vol % nonane and dodecane, respectively. We prepared the multianalyte calibration solution (59 native compounds) (Table S-1) by combining equal volumes of the EC-5022, EO-5159, and ES-5019 solutions and by reducing the final two-thirds volume by evaporation of the nonane. Reagents and solvents were the highest possible grade available or were intended for pesticide residue analysis and were only used after background levels of target analytes were measured to be nonsignificant regarding sample analysis. All details concerning consumables, as well as the glassware precleaning, are available elsewhere.14 Samples. Human serum samples were from a pool collected from 15 individuals in 2002 in three U.S. cities (Philadelphia, PA; Memphis, TN; Miami, FL) and obtained from the Memphis, Interstate blood bank. A mixture of water (3.5 mL) and calf serum
(0.5 mL) (Bibco BRL, Grand Island, NY) was used as a serum blank. Three human milk pools were analyzed. Pool A was obtained from the Mothers’ milk bank (Denver, CO) which was collected from two individuals in 2002; pools B and C both corresponded to 10 specimens collected in 2003 in California and in North Carolina, respectively. A 10-fold water-diluted bovine milk obtained in a local supermarket was used as method blank samples. Serum and milk were aliquoted in 10-mL portions upon arrival at CDC to minimize the number of freeze-thaw cycles and stored at -70 °C. Extraction and Cleanup. A semiautomated extraction and cleanup method has recently been developed for the measurement of the PCBs, OCPs, and BFRs in human serum and milk and is described in detail elsewhere.6,14,15 Briefly, serum pretreatment with formic acid and water, as well as spiking with internal standards, was performed using the Gilson 215 liquid handler (Gilson, Middleton, WI). Solid-phase extraction and cleanup were performed on a Zymark RapidTrace Automated SPE workstation (Zymark Corp., Hopkinton, MA). Custom-made Oasis HLB SPE cartridges (3 mL, 540 mg) (Waters Corp., Milford, MA) were used for extraction of 4-mL serum samples.6,14 The elution of the compounds of interest was accomplished using 15 mL of dichloromethane. Removal of coextracted biogenic materials was performed on a two-layered custom-made SPE cartridge (3 mL) packed with 100 mg of silica and 1000 mg of sulfuric acid silica. The elution of analytes was obtained by passing 11 mL of hexane through the cartridge. All extracts were evaporated using a Labconco RapidVap evaporation system (Labconco Corp., Kansas City, MO), transferred to GC vials containing the recovery standard, and subsequently evaporated to a volume of 10 µL prior to GC/MS analyses. The nonane and dodecane present in the recovery standard acted as a keeper for volatile components. QA/ QC criteria, reproducibility, and precision data, as well as details on the serum enzymatic lipid determinations, are available in a previous report.14 Human milk samples were pasteurized prior to analysis to eliminate the exposure of the laboratorians to viruses and bacteria. The extraction step was performed on 1 g of milk by matrix solid-phase dispersion using diatomaceous earth (3mL cartridges, 900 mg). The eluting volume was 12 mL of dichloromethane.15 The amount of extracted lipids was determined gravimetrically using an analytical balance. The same protocol as for serum was used for the remaining cleanup of human milk samples.14 Gas Chromatography Isotope Dilution High-Resolution Mass Spectrometry (GC-IDHRMS). GC-IDHRMS measurements were performed on a MAT95XP instrument (ThermoFinnigan MAT, Bremen, Germany) interfaced with a 6890N gas chromatograph (Agilent Technologies, Atlanta, GA). Measurements of PBDEs were carried out using a 15 m × 0.25 mm i.d. × 0.10 µm df DB-5HT capillary column (J&W Scientific, Folsom, CA). PCBs and OCPs were separated on a 30 m × 0.25 mm i.d. × 0.25 µm df DB-5MS capillary column (J&W Scientific), in two separate injections. For PBDEs, the oven was programmed from 140 (1 min) to 320 °C with a ramp rate of 10 °C/min. For PCBs, the oven was programmed from 100 (0.6 min) to 200 °C with a ramp rate of 25 °C/min for 5.0 min, then to 250 °C with a ramp rate of 4.5 °C/min, and then to 320 °C with a ramp rate of 50 °C/min for 5.0 min. For OCPs, the oven was programmed from Analytical Chemistry, Vol. 76, No. 21, November 1, 2004
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100 (1.0 min) to 220 °C with a ramp rate of 20 °C/min for 4.0 min and then to 300 °C with a ramp rate of 8.0 °C/min for 3.0 min. Splitless injections were carried out with an injector temperature of 275 °C. The source temperature was 280 °C in the electron impact mode using a filament bias of 40 eV. Details of the GCIDHRMS analyses are given elsewhere.14 Comprehensive Two-Dimensional Gas Chromatography Isotope Dilution Time-of-Flight Mass Spectrometry. The GC×GC-TOFMS instrument was the Pegasus 4D (Leco Corp., St Joseph, MI). This system was based on a nonmoving quadruple jet dual-stage modulator made of two cold nitrogen jets and two pulsed hot-air jets responsible for trapping and refocusing of compounds eluting from the 1D column. This modulator was mounted in an Agilent 6890 GC oven, and liquid nitrogen was used to create the cold jets. Details regarding the system have been reported elsewhere.21,31 The automated 1.2-µL injections were performed using a MultiPurposeSampler MPS2 (Gerstel Inc., Baltimore, MD). The GC inlet temperature was 280 °C for splitless injections. The purge time was 120 s at a flow of 20 mL/min. Carrier gas was helium, and a constant flow of 0.8 mL/min was used. The GC column set used was made from the combination of a 15 m × 0.25 mm i.d. DB-1 100% dimethylpolysiloxane (J&W Scientific) with a film thickness of 0.25 µm as 1D and a 1.2 m × 0.10 mm i.d. high-temperature HT-8 (8% Phenyl)-polycarboranesiloxane (SGE, Austin, TX) with a film thickness of 0.10 µm as 2D. Deactivated universal presstight connectors (Restek Corp., Bellefonte, PA) were used for connecting the capillary columns. During chromatographic separation, the primary GC oven was programmed as follows: 90 °C for 1 min, then to 150 °C at 10 °C/min, then to 250 °C at 3 °C/min, and finally to 300 °C at 5 °C/min. The 2D column was coiled into the secondary oven that was 40 °C higher than the primary oven and was operated in the isoramping mode. The temperature of the modulator had an offset of 60 °C compared with the temperature of the primary GC oven. Modulation was carried out on the very beginning of the 2D column. The modulator period P was 3 s with a hot-pulse M duration set at 700 ms and a cooling time between stages of 800 ms. The transfer line connecting the secondary column and the MS source was operated at a temperature of 250 °C. The source temperature was 250 °C with a filament bias voltage of -70 eV. The unit m/z resolution mass spectrometer was capable of acquiring 5000 transients/s, which results in 500 summed complete mass spectra/s for the mass range from 10 to 1000 m/z. The data acquisition rate was set at 60 spectra/s for a collected mass range from 100 to 750 m/z. The detector voltage was 1800 V. Data collection and processing were achieved using the 2.10 version of Leco ChromaTOF software provided with the instrument. Peak apex finding was performed automatically and manually corrected when required. The combination of slices corresponding to a compound was performed by comparing the mass spectra under preestablished match criteria. Spectral searching was performed using the National Institute of Standards and Technology (NIST) library available with the software, as well as through the custom-built 13C-labeled compound library.
Figure 1. Comprehensive two-dimensional gas chromatography results for a contour plot of total ion chromatogram of (A) the 100 pg/µL native compound multianalytes calibration solution (see Table S-3 for the significance of numbers) and (B) a real human serum sample. The shaded surface plot and the reconstructed onedimensional trace in (C) are based on specific extracted ion current (Table S-2) for the same human serum sample as in (B). The 2D scale was shifted by 1.5 s.
RESULTS AND DISCUSSION The chromatographic parameters for the separation of the 59 investigated compounds were selected to ensure a minimum of
coelutions in an acceptable time scale of 50 min (1.2 compound/ min, 1 h cycle time) (Figure 1A). The short 1D column length
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Table 1. Calculation Process for the Isotope Dilution Quantification of GC×GC-IDTOFMS 13C-label
compound BDE-99-1 BDE-99-2b BDE-99-3c BDE-99-4 sum ratio calcd by averaging the 4 ratios calcd for each sep peaks
1t
R
(s)
2493 2496 2499 2502
2t
R
(s)
1.72 1.68 1.71 1.65
12C-native
area
S/N
area
4755 20523 18341 3940 47559
22 102 78 21
786 4351 6335 2210 13682
S/N ratioa 4 20 26 10
0.17 0.21 0.35 0.56 0.29d 0.32e
a Ratio of 12C-native over 13C-label. b Base peak for 13C-label. c Base peak for natives. d Value based on the ratio of the value issued from the summation of all 12C-native areas over the value issued from the summation of all 13C-label areas. e Similar weight was given to small peaks with high integration variability than to more intense peaks.
Figure 2. Region of the chromatogram of a real human serum sample (A) where Mirex and 2,2′,3,3′,4,4′,5-heptachlorobiphenyl (CB170) elute. Extracted ion chromatograms are based on specific extracted ion current (Table S-2) for those two compounds (12C-native and 13C-labeled). Peaks: (1) Mirex, (2) CB-170, (3) phthlalate, (4) siloxane bleed, (5) tetracosane. The dashed Gaussian shapes are artificial and are only shown to help to locate the elution windows of the two compounds. (B) Expanded section showing one of the slices illustrating chromatographic and mass spectral resolution of the two compounds and their corresponding 13C-labels (left) as well as the corresponding contour plots (right).
separated the compounds depending on their boiling point while the small piece of 2D column acted more selectively toward the compound classes. The 2D column was also responsible for the isolation of the strong matrix-related interference background in an area of the chromatographic separation space where no compounds of interest were present (Figure 1B,C). Because of the mass spectral deconvolution capability of the TOFMS, coeluting and closely eluting compound identification was carried out by mean of DICs based on specific clusters of masses (Table S-2). The two most intense ions of the mass cluster were used to calculate and compare isotopic ratios against theoretical values (tolerance of 20%). In conjunction with two retention time (tR) checks (1tR and 2tR), this ensured the specificity of the unit-mass resolution TOFMS instrument. The summation of the mass cluster intensities resulted in an improvement of the IDLs while high specificity was maintained. Figure 2 illustrates the ID measurement of two close eluters, Mirex (1tR ) 2271 s and 2tR ) 1.53 s) and CB-170 (1tR ) 2274 s and 2tR ) 1.65 s), which
were separated from interfering compounds in 2D. For ID quantification, the five to six chromatographic slices produced by the modulation process for each compound (12C-native and 13Clabel) were integrated. Slices exhibiting identical mass spectral characteristics were combined and considered as representing the same compound. Area values of each combined slice were summed up, and the 12C/13C ratio was calculated. This approach had the advantage of taking all slices with a preset signal-to-noise ratio (S/N) value greater than 3 into account in the quantification procedure, which appeared to be important. Attempts to simplify the integration process by limiting the calculation to the most intense peak of each cluster (the base peak) were not successful because of the slight shift in retention time always observed between 12C-native and 13C-label compounds. This is illustrated in Table 1 for BDE-99. In addition, calculating individual ratios and extracting an average ratio value resulted in erroneous ratio values (0.32 versus 0.29) that should not be used because similar weight was given to small peaks with high integration variability than to more intense peaks. A value based on the ratio of the value issued from the summation of all native areas over the value issued from the summation of all 13C-label areas (0.29) is more accurate and was used for the quantification procedure. The efficiency of the modulation process was improved by using extended hot-pulse durations to ensure proper reinjection of the low-boiling compounds after cryogenic trapping during modulation (e.g., 13C12-BDE-153, m/z 656). Although a short modulation period (PM ) 3 s) was used, one wraparound took place inside the time window where the first compound (β-HCH) and the last compound (BDE-153) eluted. This had no consequences on the chromatographic resolution, and a seconddimension retention time (2tR) correction of 1.5 s was used during the study to simplify chromatogram displays. Calibrations were carried out using the homemade multianalyte 10-points calibration solution (N ) 5) (59 12C-native and 37 13Clabel compounds). Good correlation coefficients were obtained for all the compounds, including the ones that did not have a 13Clabel and for which a close-eluter 13C-label with a similar structure was used for quantification (Table S-3). The calibration range spanned 3 orders of magnitude (0.5-2000 pg/µL), and all unknown sample levels were included in this range. The IDLs were determined by injecting the smallest quantity of analytes Analytical Chemistry, Vol. 76, No. 21, November 1, 2004
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Figure 3. QA/QC plots for (A) 2,2′,4,4′-tetrabromodiphenyl ether (BDE-47), (B) 2,2′,4,4′,6-pentabromodiphenyl ether (BDE-100), and (C) 2,2′,4,4′,5,5′-hexachlorobiphenyl (CB-153) (pg/g of fresh weight) in nonfortified human serum QC samples. The dashed lines represent the confidence intervals calculated on the basis of (2 and (3 standard deviations (see text for details).
that gave a S/N greater than 3 for the base (most intense) peak of the cluster consistently over several injections (N ) 5) (Figure S-1). They ranged between 0.5 and 10 pg/µL (Table S-3). The influence of the current sample preparation method on the measurements was verified by spiking the calibration curve standard (N ) 3 per point in calibration curve) into extracted and cleaned up serum samples not spiked with any internal standards. The slope of the calibration curve was not significantly affected by any remaining matrix interferences originating from the samples (data not shown). Method detection limits (MDLs) were determined by spiking processed bovine serum samples with the calibration standards and by measuring the smallest quantity of analytes that gave a S/N greater than 3. They ranged between 1 and 15 pg/µL. Due to the broad range of collected masses (100-750 m/z), the high scanning rate (60 scans/s), the elevated number of analytes (N ) 59, plus 13C-labels), and the relatively short PM (3 s), such calibration file sizes were in the range of 10-15 Gb. This obviously significantly slowed access to the data for reviewing, backup, and transfer. An alternative was to carry out separate calibrations for each compound class and to perform independent data processing once the sample extracts have been injected. This option became necessary when processing samples that contain levels of different orders of magnitude. For example, if levels of polychlorinated dibenzo-p-dioxins and polychlorinated dibenzofurans would have to be measured, low-concentration standards have to be used to ensure adequate calibration in the working range.41 When analyzing European samples for PBDEs, it requires lower calibration standards because the EU levels are much lower than the U.S. levels. 6318 Analytical Chemistry, Vol. 76, No. 21, November 1, 2004
Figure 4. Comparison of comprehensive GC×GC-IDTOFMS and GC-IDHRMS results for a nonfortified human serum pool.
The new method was tested against the reference GCIDHRMS validated procedure, which consisted of three separate injections with three different GC temperature programs. A quality control human serum pool extensively used for GC-IDHRMS QA/QC purposes in routine analysis was used as a reference material for method development. Figure 3A-C shows the QA/ QC charts corresponding to BDE-47, BDE-100, and CB-153, respectively. The left side of the figure corresponds to GCIDHRMS values obtained during a 6-month period, and the right side plots are for GC×GC-IDTOFMS over a period of 1 month. Both measurement methods were applied on samples that were extracted and cleaned-up using the same procedure.14 The QA/ QC charts have two different mean values, one based on measurements by GC-IDHRMS and the other based on GC×GCIDTOFMS data. Two independent sets of control limits were calculated based on the respective standard deviation (SD) values (mean plus and minus 2SD and 3SD), and classical “Westgard rules” were applied to estimate the quality of the data.42 Statistical (41) Focant, J.-F.; Reiner, E. J.; MacPherson, K.; Kolic, T.; Sjo ¨din, A.; Patterson, D. G., Jr.; Reese, S. L.; Dorman, F. L.; Cochran, J. W. Talanta 2004, 63, 1231-1240. (42) Westgard, J. O.; Barry, P. L.; Hunt, M. R.; Groth, T. Clin. Chem. 1981, 27, 493-501.
Table 2. Comparison between GC×GC-IDTOFMS and the GC-IDHRMS Methods for the Measurement (ng/g of Lipids) of Selected PBDEs, OCPs, and PCBs in Natural Human Milk Sample Pools pool A GC-HRMS n)8
pool B
GC×GC-TOFMS n)4
analyte
mean
SEMa
mean
SEM
BDE-28 BDE-47 BDE-100 BDE-99 BB-153 BDE-154 BDE-85 BDE-153 ΣBDEs CB-28 CB-74 CB-99 CB-101 CB-105 CB-118 CB-138 CB-146 CB-153 CB-156 CB-170 CB-177 CB-180 CB-187 HCB β-HCH γ-HCH hept. epox.c oxychlor.d t-nona.e o,p′-DDT p,p′-DDT Mirex p,p′-DDE
6.6 230.4 46.1 71.6 0.7 5.9 7.9 18.5 386.9 2.9 5.5 4.3 0.4 1.8 6.9 11.7 1.3 11.6 1.3 intf 0.5 4.4 1.8 13.9 7.2 0.8 12.2 15.9 21.3 1.9 7.6 0.4 150.0
0.6 8.2 1.7 2.9 0.1 0.2 1.0 1.4 36.5 0.6 0.1 0.4 0.1 0.0 0.2 0.3 0.1 0.5 0.0 0.2 0.0 0.2 0.1 0.7 0.5 0.1 0.9 1.0 2.0 0.2 0.5 0.0 9.9
7.7 227.4 49.4 76.7 1.0 4.9 6.1 18.6 390.8 2.8 6.4 4.1 0.6 2.0 6.9 8.6 1.7 11.0 1.3 1.9 0.7 4.8 1.8 17.0 9.3 1.4 8.5 16.8 21.1 2.3 11.4 1.1 153.7
0.2 17.4 0.9 1.6 0.3 0.4 0.3 0.3 16.2 0.5 0.4 0.5 0.1 0.1 0.1 1.2 0.7 0.5 0.2 0.3 0.2 0.4 0.2 0.6 0.3 0.1 0.3 0.5 1.6 0.6 0.3 0.5 5.2
e
diffb (%) 15.3 1.3 7.3 7.1 52.7 17.3 22.9 0.7 1.0 1.3 16.5 4.1 44.0 8.9 0.3 26.1 33.3 4.9 0.3 50.1 9.7 3.7 22.3 29.1 84.1 30.3 5.6 1.0 17.6 49.1 147.3 2.4
GC-HRMS n)3
pool C
GC×GC-TOFMS n)6
mean
SEM
mean
SEM
12.7 284.5 45.6 74.0 1.2 3.7 6.9 21.4 448.8 2.1 6.7 4.6 0.3 2.1 10.6 33.0 4.6 35.4 3.5 7.0 1.7 14.6 5.5 13.2 33.5 0.6 3.3 10.1 12.4 0.9 6.7 0.4 272.9
0.9 14.0 2.2 2.6 0.0 0.2 0.2 0.9 20.9 0.6 1.0 0.7 0.1 0.3 1.3 4.0 0.5 4.2 0.4 0.5 0.2 1.9 0.8 1.7 4.9 0.1 0.4 1.4 1.6 0.1 0.9 0.0 34.0
11.7 308.3 53.9 81.7 1.6 4.7 7.4 25.8 493.5 1.6 5.8 3.8 0.4 1.9 8.4 20.1 6.3 34.5 3.0 6.3 1.7 13.8 4.3 16.0 42.0 ndg 1.3 10.8 11.8 1.4 8.8 0.4 286.6
1.0 24.4 3.7 6.4 0.5 1.1 0.8 5.4 39.0 1.1 1.2 0.6 0.1 0.1 0.9 2.1 1.1 2.6 0.2 0.1 0.2 0.8 1.1 2.1 3.8 ndg 0.1 1.1 0.6 0.2 1.4 0.2 7.0
diff (%) 8.3 8.4 18.3 10.4 27.7 27.3 7.1 20.4 9.9 27.0 13.2 17.7 23.6 8.9 21.0 39.3 36.7 2.6 13.3 10.2 2.4 5.2 22.4 21.2 25.4 62.0 7.0 4.9 54.1 30.5 8.0 5.0
GC-HRMS n)3
GC×GC-TOFMS n)3
mean
SEM
mean
SEM
diff (%)
2.9 64.0 11.4 19.3 6.7 1.1 1.6 9.2 109.5 2.3 6.2 5.8 0.5 1.8 8.3 20.6 2.5 22.6 2.7 4.9 0.8 10.9 4.0 11.1 9.0 0.8 10.0 19.2 31.1 0.8 5.9 1.3 133.8
0.1 2.8 0.6 0.7 0.4 0.0 0.0 0.4 4.7 0.6 0.1 0.4 0.1 0.0 0.2 0.3 0.1 0.5 0.0 0.2 0.0 0.2 0.1 0.3 0.3 0.0 0.5 1.0 0.9 0.0 0.0 0.0 1.3
4.0 65.7 11.4 19.7 9.0 0.9
0.5 0.4 0.7 0.6 0.7 0.2
41.0 2.6 0.3 1.9 33.6 18.3
9.8 111.5 2.6 6.4 5.5 0.9 1.9 7.8 14.7 3.0 19.7 2.6 4.7 1.1 10.6 4.1 13.1 12.5 1.2 9.4 19.9 33.3 1.5 10.4 1.4 139.8
0.9 2.1 0.5 0.4 0.5 0.1 0.1 0.1 1.2 0.7 0.5 0.2 0.3 0.2 0.4 0.2 1.3 0.4 0.3 0.4 1.0 1.6 0.6 1.0 0.8 8.8
6.1 1.8 10.4 3.0 4.5 95.5 4.7 6.6 28.7 15.9 12.9 5.6 5.3 26.9 2.1 1.3 17.8 38.7 46.8 5.3 3.6 7.2 95.0 75.5 6.6 4.5
a SEM, standard error of the mean. b Percentage difference between the mean results of the two methods. c Heptachlor epoxide. d Oxychlordane. trans-Nonachlor. f int, interference. g nd, not detected.
distributions of the control measurements were identical for both methods. The calculated mean values were similar for both methods with a coefficient of variance (CV) of 0.8 and 2.5% for BDE-47 and CB-153, respectively, and a CV of 10.8% for BDE-100 (concentration below 50 pg/g fresh weight). Higher SD values were found using GC×GC-IDTOFMS (13.1, 4.0, and 11.9 pg/g fresh weight versus 7.5, 1.4, and 6.8 pg/g of fresh weight for BDE47, BDE-100, and CB-153 by GC×GC-IDTOFMS and GCIDHRMS, respectively). This difference can at least be partly explained by the low-mass resolution of the TOFMS instrument as well as the higher variations during the integration process due to multiple peak measurement after GC×GC modulation. Nevertheless, SD values below 15% for the measurement of picogram-range levels are acceptable and can be used for implementation of confidence intervals for QA/QC monitoring purposes. Figure 4 shows the comparison of the new instrumental method against the reference data for OCPs, PBDEs, and selected PCBs. For OCPs, the relative standard deviations (RSDs) for GC×GC-IDTOFMS ranged between 1 and 11%, except for γ-HCCH and o,p′-DDT (RSDs were 15 and 46%, respectively), which had levels that were close to the IDLs (5 pg/µL). Heptachlor epoxide and dieldrin, two acid-labile compounds, were
not isolated during the sample preparation procedure (see Experimental Section), and no data are available to compare both methods. RSDs for the GC-IDHRMS method ranged from 1 to 7%. For PBDEs, RSDs ranged from 3.5 to 10% for detected congeners (BDE-47, BDE-100, BDE-99, BDE-153). These PBDE congeners are the most prominent because they are the major constituents of the technical pentaBDE technical mixtures commonly used in North America and are likely to be found in human specimens. PCB levels measured using the two methods were highly correlated. One major difference appeared for CB-138 and CB-158, which were coeluting congeners in the GC-IDHRMS method. These two congeners were separated by GC×GCIDTOFMS and had to be summed together for comparison. No reason was found that accounted for the difference between the two methods for these congeners. Results from congener measurements in several human milk pools are listed in Table 2. The results are expressed on a lipid weight basis, which resulted in slightly higher RSD values. The reproducibility for the measurements was similar for both methods. The percent deviations between the two methods were acceptable and below 20% for most analytes with concentrations above 1 ng/g of lipid. Discussions about the patterns and levels in the milk pool are available elsewhere.15 Analytical Chemistry, Vol. 76, No. 21, November 1, 2004
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CONCLUSION The GC×GC-IDTOFMS results demonstrate the efficiency of this multianalyte measurement approach for such important matrixes as serum and milk for human biomonitoring. This singleinjection method satisfactorily correlates with the well-established and accepted reference GC-IDHRMS methods, which requires at least three separate injections to report all the analytes considered in the present study. Potential interfering compounds are separated from analytes of interests in the chromatographic GC×GC space due to the increased peak capacity, ensuring sufficient specificity for the low-mass-resolution TOFMS instrument. On a practical point of view, although the required financial investment is significantly lower for the GC×GC-TOFMS system compared to the GC-HRMS system, a much higher manual input is still required in order to process and handle the data under good QA/QC guidelines. Future improvements of the processing software coupled to the robustness of the GC×GC and TOFMS hardware will contribute to make this technique a viable alternative in routine conditions. The comprehensive aspect of the TOFMS full-scan mass spectral acquisition additionally opens the possibility for identification of other (un)expected halogenated toxicants that are potentially present in human samples. This method is a well suited
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Analytical Chemistry, Vol. 76, No. 21, November 1, 2004
analytical tool in an area where additional toxicants (polybrominated dibenzo-p-dioxins and furans (PBDD/Fs), toxaphenes, paraffins, fluorinated compounds) are likely to be added soon. ACKNOWLEDGMENT Chester R. Lapeza, Richard S. Jones, Ernest E. McGahee, III, Yalin Zhang, and Susan Welch are deeply appreciated for their help in sample preparation and GC-IDHRMS measurements. J.-F.F. and R.S. Jones were financially supported by the Oak Ridge Institute for Science and Education (ORISE), a Department of Energy (DOE) facility managed by Oak Ridge Associated Universities (ORAU). SUPPORTING INFORMATION AVAILABLE Tables S-1, S2, and S-3 containing complete listings of ID standards, sets of specific ions used for DIC traces, analyte retention times, correlation coefficients, and instrumental detection limits. Figure S-1, the signal evolution when reaching detection limits. This material is available free of charge via the Internet at http://pubs.acs.org. Received for review July 16, 2004. Accepted August 24, 2004. AC048959I
