Anal. Chem. 1998, 70, 1986-1992
Solid-Phase Microextraction of Monocyclic Aromatic Amines from Biological Fluids Lillian S. DeBruin,† P. David Josephy,† and Janusz B. Pawliszyn*,‡
GuelphsWaterloo Centre for Graduate Work in Chemistry (GWC), Department of Chemistry & Biochemistry, University of Guelph, Guelph, Ontario, Canada N1G 2W1, and Department of Chemistry, University of Waterloo, Waterloo, Ontario, Canada N2L 3G1
A solid-phase microextraction (SPME) protocol was developed for the quantitative analysis of monocyclic aromatic amines from biological fluids. The headspace SPME sampling technique was optimized for extraction and concentration of five target analytes (aniline, otoluidine, 2-chloroaniline, 2,6-dimethylaniline, 2,4,6trimethylaniline) from urine, blood, and milk. The temperature, pH, and ionic strength of the matrix sample were modified to allow maximum adsorption of the analytes onto the SPME fiber. This method is rapid yet sensitive and can be completed in 15 min on a 5-mL sample. SPME/GC/MS analysis yielded good reproducibility (RSD >11%) for each analyte from urine, blood, and milk. Method detection limits for the various biological fluids were determined and ranged from 0.40 ppb for 2,4,6trimethylaniline in urine to 7.7 ppb for aniline in blood. This SPME sampling protocol can be applied to the biomonitoring of monocyclic aromatic amines from occupational, environmental, and medical exposure. Many aromatic amines are of toxicological concern to humans. The chemical class of aromatic amines can be subdivided into monocyclic aromatic amines, polycyclic amines, and heterocyclic amines. Several polycyclic amines, such as benzidine, 4-aminobiphenyl, and naphthylamine, have been classified by the International Agency for Research on Cancer (IARC) as known human carcinogens. Epidemiological studies have suggested that monocyclic aromatic amines, such as o-toluidine (2-methylaniline) and 4-chloro-o-toluidine, may also be human carcinogens.1,2 Comprehensive animal bioassays have determined that many monocyclic aromatic amines and heterocyclic amines are rodent carcinogens.3,4 Monocyclic aromatic amines, or aniline derivatives, are intermediates in the production of rubber, plastics, polyurethane foams,
dyes, pesticides, and pharmaceuticals. Their extensive use in industrial processes warrants closer biological monitoring for these compounds in workers as well as in the general population. Occupational exposure to aromatic amines has been measured by the biological monitoring of the workers’ blood and urine.1,5 These compounds are also found as pollutants in the environment. Aniline and its methylated and ethylated derivatives are indoor pollutants arising from environmental tobacco smoke (“secondhand” smoke).6 Furthermore, aniline and toluidines have been detected in the urine of smokers and, at a lower level, in nonsmokers.7 Another route of exposure to these aniline derivatives is through the biotransformation of certain pesticides and pharmaceuticals. 2,6-Dimethylaniline has been detected in the urine of farmers applying a commercial formulation of the systemic fungicide metalaxyl [N-(2,6-dimethylphenyl)-N-(methyoxyacetyl)D,L-alanine methyl ester]8 and 2,6-dimethylaniline-hemoglobin adducts were found in the blood of patients treated with lidocaine [2-(dimethylamino)-N-(2,6-dimethylphenyl)acetamide] for cardiac arrhythmia and for local anesthesia.9 The use of prilocaine as a local anesthetic in oral surgery is now limited because o-toluidine, a metabolite of prilocaine, has induced methemoglobinemia in a number of patients.10 Burn patients treated with the antiseptic chlorhexidine [1,1′-hexamethylenebis[5-(4-chlorophenyl)biguanide]] had detectable levels of 4-chloroaniline in their urine.11 Occupational, environmental, and medical exposures to monocyclic aromatic amines have been observed through the biomonitoring of urine and blood. Methods are available for the isolation of aromatic amines from urine and blood, but these techniques are lengthy procedures involving solvent and/or solid-phase extractions. Breast milk is an excellent matrix for noninvasive biomonitoring,12 but human milk has not been used as a matrix from which to biomonitor aniline derivatives or any other aromatic
* To whom correspondence should be addressed: (e-mail) janusz@watsci. uwaterloo.ca; (fax) (519)746-0435. † University of Guelph. ‡ University of Waterloo. (1) Ward, E. M.; Sabbioni, G.; DeBord, D. G.; Teass, A. W.; Brown, K. K.; Talaska, G. G.; Roberts, D. R.; Ruder, A. M.; Streicher, R. P. J. Natl. Cancer Inst. 1996, 88, 1046-1052. (2) Popp, W.; Schmieding, W.; Speck, M.; Vahrenholz, C.; Norpoth, K. Br. J. Ind. Med. 1992, 49, 529-531. (3) Gold, L. S.; Slone, T. H.; Manley, N. B.; Bernstein, L. Environ. Health Perspect. 1991, 93, 233-246. (4) Ohgaki, H.; Takayama, S.; Sugimura, T. Mutat. Res. 1991, 259, 399-410.
(5) Teass, A. W.; DeBord, D. G.; Brown, K. K.; Cheever, K. L.; Stettler, L. E.; Savage, R. E.; Weigel, W. W.; Dankovic, D.; Ward, E. Int. Arch. Occup. Environ. Health 1993, 65, S115-S118. (6) Luceri, F.; Pieraccini, G.; Moneti, G.; Dolara, P. Toxicol. Ind. Health 1993, 9, 405-413. (7) El-Bayoumy, K.; Donahue, M.; Hecht. S. S.; Hoffman, D. Cancer Res. 1986, 46, 6064-6067. (8) Headley, J. V.; Maxwell, D. B.; Swyngedouw, C.; Purdy, J. R. J. AOAC Int. 1996, 79, 117-123. (9) Bryant, M. S.; Simmons, H. F.; Harrell, R. E.; Hinson, J. A. Carcinogenesis 1994, 15, 2287-2290. (10) Knobeloch, L.; Goldring, J.; LeMay, W.; Anderson, H. Morb. Mortal. Wkly. Rep. 1993, 43, 655-657. (11) Brougham, L. R.; Cheng, H.; Pittman, K. A. J. Chromatogr. 1986, 383, 365373.
1986 Analytical Chemistry, Vol. 70, No. 9, May 1, 1998
S0003-2700(97)00941-4 CCC: $15.00
© 1998 American Chemical Society Published on Web 04/08/1998
amines. Analysis of these compounds in human milk will test the hypothesis that aromatic amines are causally associated with breast cancer.13 Only a few milk biomonitoring studies (which were limited to volatile organic compounds) have been carried out on the occupational exposure of mother and nursing child.14,15 Environmental contamination of human milk by monocyclic aromatic amines may occur, but for many decades, the focus has been on highly lipophilic chlorinated pesticides and polychlorinated biphenyls.16 We have developed a rapid yet simple procedure for the sampling, analysis, and quantification of monocyclic aromatic amines from human milk, urine, and blood. This procedure utilizes the recently developed solvent-free sampling technique, solid-phase microextraction (SPME), which extracts and concentrates in one step and provides a simple route for analyte introduction into a chromatographic instrument.17 The commercially available SPME device is composed of a syringelike holder with a septum-piercing needle. Within the needle is a fine gauge stainless steel tube to which is attached a 1-cm fused-silica rod, coated with a specific polymeric coating. A detailed guide to the theory and practice of SPME has been published.18 Initially, SPME was used to extract environmental pollutants from water19,20 via direct extraction, but as applications expanded, more complex matrixes were studied, for which direct SPME could not be used. With biological matrixes, adsorption of biomacromolecules decreased the affinity for the analytes of interest. The use of headspace SPME sampling provided a simple yet elegant alternative to extracting volatile and semivolatile compounds from complex matrixes.21 Analytes with a sufficient vapor pressure partition from the sample matrix into the headspace and from the headspace to the fiber coating. For more complex matrixes, multiphase systems exist and the total amount of analyte (N) can be described by eq 1, where C0 is the initial
N ) C0VMT ) CfVf + ChVh +
∑C
MiVMi
(1)
analyte concentration in the total matrix volume, VMT. Each term in the sum is the product of the analyte concentration and the volume in the fiber, headspace, and each phase of the matrix. Cf, Ch, and CMi, are the analyte concentrations on the fiber, in the headspace, and in the ith phase of the matrix, respectively; Vf, Vh, and VMi are the volumes of the fiber, headspace, and ith phase of the matrix, respectively. At equilibrium, or at any other point in time, the amount the analyte on the fiber can be used to determine the concentration in the initial sample solution. With semivolatile compounds, the time to reach equilibrium for the (12) Sim, M. R.; McNeil, J. J. Am. J. Epidemiol. 1992, 136, 1-11. (13) Josephy, P. D. Mutagenesis 1996, 11, 3-7. (14) Byczkowski, J. Z.; Gearhart, J. M.; Fisher, J. W. Nutrition 1984, 10, 4348. (15) Pellizzari, E. D.; Hartwell, T. D.; Harris, B. S. H., III; Waddell, R. D.; Whitaker, D. A.; Erickson, M. D. Bull. Environ. Contam. Toxicol. 1982, 28, 322-328. (16) Jensen, A. A.; Slorach, S. A. Chemical contaminants in human milk; CRC Press: Boca Raton, FL, 1991; Chapter 5. (17) Arthur, C. L.; Pawliszyn, J. Anal. Chem. 1990, 62, 2145-2148. (18) Pawliszyn, J. Solid-phase microextractionstheory and practice; Wiley-VCH: New York, 1997. (19) Potter, D. W.; Pawliszyn, J. Environ. Sci. Technol. 1994, 28, 298-305. (20) Boyd-Boland, A. A.; Pawliszyn, J. B. J. Chromatogr. Sci 1995, 704, 163172. (21) Zhang, Z.; Pawliszyn, J. Anal. Chem. 1993, 65, 1843-1852.
multiphase system can be decreased by increasing the solution ionic strength and temperature. Several classes of chemical compounds have been extracted from complex matrixes with headspace SPME, including basic drugs from blood22 and urine,23 polycyclic aromatic hydrocarbons from soil samples,21 and phenols from sewage sludge.24 To date, aromatic amines have not been investigated by direct or headspace SPME. This work shows that SPME, with analysis using GC/MS, can be used for the sampling and quantification of monocyclic aromatic amines in urine, breast milk, and blood. The method is rapid and simple to employ and requires little sample preparation, yet yields high sensitivity and excellent reproducibility. EXPERIMENTAL SECTION Chemicals and Materials. Aniline, o-toluidine, and 2,6dimethylaniline were purchased from Aldrich (Sigma-Aldrich Canada, Oakville, ON), 2-chloroaniline was from Acros (Fisher, Nepean, ON) and 2,4,6-trimethylaniline was from Pfaltz & Bauer (Waterbury, CT). Methanol and dichloromethane were obtained from Caledon (Georgetown, ON). The SPME fibers (PDMS, polyacrylate, PDMS/divinylbenzene (DVB), carbowax/DVB, carboxen), and holder assemblies are available from Supelco (SigmaAldrich Canada). All fibers were conditioned as recommended by the manufacturer. Both urine and breast milk samples were obtained from nonsmoking healthy volunteers with no known exposure to aromatic amines. The urine samples were analyzed immediately. The milk samples were frozen and then stored at -70 °C. As required, the samples were thawed in a 20 °C water bath and thoroughly mixed before use. Equine blood (Large Animal Clinic, Ontario Veterinary College, Guelph, ON) was used fresh for all the blood studies. Instrumentation. A Varian 3400 GC with a flame ionization detector (FID) was used for all experiments to determine the optimized SPME conditions. The carrier gas was helium (40 cm/s measured at 45 °C). The detector flow rates were set to 300 mL/ min for air, 20 mL/min for helium (makeup gas), and 30 mL/ min for hydrogen. For the SPME injections, the injector was maintained at 230 °C and the detector at 250 °C. The column was held at 45 °C for 3.0 min, ramped at 10 °C/min to 130 °C and again ramped at 20 °C/min to 250 °C and held for 2.0 min. For the liquid injections, both the injector and detector were at 250 °C. The column temperature program was as follows: 40 °C for 5.0 min, 10 °C/min to 130 °C, 20 °C/min to 250 °C, and held for 2.0 min. The column for the GC/FID was a (5%) diphenyl (95%) dimethylsiloxane copolymer (30 m × 0.25 mm i.d., 0.25-µm film) column (PTE-5, Supelco). The method detection limits and precision measurements were completed on a HP 5890 Series II GC coupled to a HP5971 mass selective detector. Standard autotunes with perfluorotributylamine (PFTBA) were carried out on a daily basis. The carrier gas was helium velocity (18 cm/s measured at 45 °C). The injector was maintained at 220 °C. The column [(5%) diphenyl (95%) dimethylsiloxane copolymer, 30 m × 0.25 mm i.d., 0.25-µm film (DB5MS, J&W)] was held at 45 °C for 1.0 min, then ramped to 130 (22) Nagasawa, N.; Yashiki, M.; Iwasaki, Y.; Hara, K.; Kohima, T. Forensic Sci. Int. 1996, 78, 95-102. (23) Lord, H. L.; Pawliszyn, J. Anal. Chem. 1997, 69, 3899-3906. (24) Buchholz, K.; Pawliszyn, J. Anal. Chem. 1994, 66, 160-167.
Analytical Chemistry, Vol. 70, No. 9, May 1, 1998
1987
°C at 15 °C/min and then 25 °C/min to 250 °C, and held for 2 min. For both instruments, silanized narrow-bore injector liners (0.75-mm i.d.) for the SPME injections or silanized wide-bore injector liners for liquid injections were installed. Preparation of Standards and Samples. Standards containing the monocyclic aromatic amines, aniline, o-toluidine, 2,6dimethylaniline, 2,4,6-trimethylaniline, and 2-chloroaniline, were prepared in MeOH, ranging from 50 to 1000 µg/mL, for the SPME analyses and in CH2Cl2, ranging from 1 to 1000 µg/mL, for the FID calibration curve. For the SPME analyses, the spiked matrix samples were prepared by adding e0.1% of the appropriate standard in MeOH to the aqueous matrix. For example, a urine sample containing 100 ng/mL of each analyte was prepared by adding 100 µL of 100 µg/mL aniline, o-toluidine, 2-chloroaniline, 2,6-dimethylaniline, and 2,4,6-trimethylaniline standard to 100.00 mL of urine. The water- and urine-spiked samples were immediately analyzed by the SPME sampling protocol listed below. The spiked milk samples were centrifuged (1500g, 10 min, 4 °C) and then the supernatant (i.e., the skim milk fraction, fat removed) was analyzed. Similarly, the spiked blood samples were centrifuged and the plasma fraction was analyzed. SPME Sampling Protocol. SPME conditions were optimized for the five target compounds (aniline, o-toluidine, 2-chloroaniline, 2,6-dimethylaniline, 2,4,6-trimethylaniline). A magnetic stir bar (13 mm × 3.2 mm), salt, and a strong base solution (1 mL) (see below) were added to 17 mm × 60 mm glass vials with open-top phenolic cap and Teflon septum (Supelco, Sigma-Aldrich) before the spiked matrix samples (5 mL) were added. The sample preparation was stirred until homogeneous, at room temperature, and then preincubated for 3.0 min at the required temperature, before the fiber was inserted into the headspace above the sample. The sample vial was maintained at a constant temperature and stirring rate during the SPME extraction period. This was achieved by clamping the vial inside a water bath which was set upon a digital hot plate/stirrer with a temperature probe (Datapac Series 720, Barnstead/Thermolyne Corp., Dubuque, IA). After the required extraction period, the fiber was immediately introduced into the GC injector port. FID Calibration. The FID response as a function of amount injected was determined with standards containing the five monocyclic aromatic amine analytes in CH2Cl2. A five-point calibration curve was generated for each analyte, ranging from 1.8 to 1800 ng of analyte. From these calibration curves, the amount of analyte extracted onto the SPME fiber was calculated. Detection Limits, Precision, and Quantification by GC/ MS. Method detection limits and precision measurements were performed for the five analytes in water, urine, milk, and blood. The method detection limit (MDL) for each analyte was determined by analyzing spiked samples (eight replicates) and multiplying the standard deviation of the mean by the appropriate Student’s t value for a 99% confidence level with the appropriate degrees of freedom. For eight replicates and seven degrees of freedom, the Student’s t value is 2.998. The limit of detection (LOD) was established by analyzing eight replicate blank water samples and multiplying by three times the noise. Precision values expressed as the percentage of the relative standard deviation (RSD) were calculated by analyzing spiked samples at 1988 Analytical Chemistry, Vol. 70, No. 9, May 1, 1998
Table 1. Physicochemical Parameters of the Five Target Analytes analyte
pKba
log Kowa
aniline o-toluidine 2-chloroaniline 2,6-dimethylaniline 2,4,6-trimethylaniline
9.37 9.56 11.35 10.11 9.62
0.90 1.27 1.90 1.87 2.88b
a From refs 26 and 27. b Estimated for structural group contributions as outlined in ref 28.
concentrations 5-10 times the method detection limit. Quantification was by external calibration curve for water, and for the more complex and variable matrixes (urine, blood, milk), the method of standard addition was employed. RESULTS AND DISCUSSION Optimization of the SPME Conditions for Monocyclic Aromatic Amines. The objective was to develop and optimize SPME sampling conditions for the extraction and quantification of monocyclic aromatic amines from the biological fluids, urine, milk, and blood. Since the biological fluids are mainly aqueous, initial optimization for the five target analytes was developed with water as the matrix. Furthermore, headspace SPME was utilized because the complex nature of biological fluids and the modification of the matrix with strong base would result in damage to the fiber coating, if direct sampling were employed. To promote the partitioning of the analytes into the headspace, the pH, ionic strength, and temperature of the matrix were modified. (a) Sample Size and Fiber Selection. A sample volume of 5 mL and vial size of 17 mm × 60 mm were selected. The 5-mL sample size allows low detection limits to be achieved and relatively small volumes of the biological fluids to be collected. In the chosen vial, the headspace volume is much smaller than the total matrix volume and equilibrium between the headspace and the fiber coating is reached quickly. All of the commercially available SPME fibers (PDMS, polyacrylate, PDMS/DVB, carbowax/DVB, carboxen) were tested for efficiency of headspace extraction of monocyclic aromatic amines from water in preliminary studies. Based on the criteria of amount extracted onto the fiber and desorption from the fiber, the PDMS/DVB fiber performed best and was employed in further optimization studies. (b) pH and Ionic Strength. Monocyclic aromatic amines, which are weak bases (see Table 1), must be predominantly in the un-ionized state for extraction. Subsequently, all sampling solutions were adjusted to a pH greater than 13 by the addition of strong bases. Suitable bases were K3PO4, K2CO3, and KOH. For aqueous sampling solutions with a final concentration of 1.0 M base, the amount of analytes extracted onto the fiber was similar in each base. KOH was used for further protocol development since its solubility in aqueous solutions exceeds that of carbonate and phosphate. The amount extracted onto the fiber increased with increasing KOH concentration. A final concentration of 2.0 M KOH was employed for further extractions. The addition of a salt to an aqueous matrix increases the ionic strength of the solution. For many organic analytes, aqueous solubility decreases with increasing ionic strength, and thus, the
Figure 1. Temperature profile for headspace SPME of the five monocyclic aromatic amine analytes. Water samples containing 1000 ppb of each analyte were modified to 4.0 M NaCl and 2.0 M KOH. At various temperatures, 15-min headspace sampling with a PMDS/DVB fiber was completed for aniline (b), o-toluidine (9), 2-chloroaniline (2), 2,6-dimethylaniline (1), and 2,4,6-trimethylaniline ([).
Figure 2. Equilibration time profiles at 45 °C. Water samples containing 2000 ppb aniline (b), 1000 ppb o-toluidine (9), 500 ppb 2-chloroaniline (2), 500 ppb 2,6-dimethylaniline (1), and 200 ppb 2,4,6-trimethylaniline ([) were modified to 4.0 M NaCl and 2.0 M KOH. Headspace samplings with a PMDS/DVB fiber were completed for various time periods.
partitioning of the analytes from the aqueous solution to the headspace is improved. The ionic strength was increased by the addition of NaCl. (The divalent chloride salts were unsuitable because their corresponding hydroxides have limited solubility in aqueous solutions.) The effect of NaCl concentration was investigated over the range of 0-4.0 M. The amount of analyte extracted onto the fiber increased with salt concentration. A final concentration of 4.0 M NaCl was used for all further experiments. (c) Temperature. By increasing the temperature of the sample solution, the vapor pressure of the analyte is increased, allowing the partitioning of the analyte between the sample and the headspace to reach equilibrium more quickly. However, the distribution constant for the analyte between the headspace and fiber coating is also temperature-dependent. At excessively high temperatures, the affinity of the analyte for the fiber coating diminishes. An optimum temperature exists for the partitioning of the analyte among the sample matrix, headspace, and fiber coating, with the maximum loading onto the fiber. With the water matrix modified to final concentrations of 4.0 M NaCl and 2.0 M KOH, the effect of sample temperature on the headspace extraction was examined. The temperature of the sample was increased from 35 to 65 °C, in 10 °C increments. The optimum temperature for aniline, o-toluidine, 2-chloroaniline, and 2,6-dimethylaniline was 45 °C and for 2,4,6-trimethylaniline it was 65 °C (see Figure 1). (d) Extraction Time Profiles. Extraction time profiles from a water matrix, i.e., the amount of analyte loaded onto the fiber (measured as FID response) as a function of exposure time, were measured to establish equilibration times for each of the five analytes, at the optimum sampling temperature of 45 °C. The time to reach equilibrium was 10 min for aniline, 15 min for o-toluidine, 30 min for 2-chloroaniline and 2,6-dimethylaniline, and greater than 90 min for 2,4,6-trimethylaniline (see Figure 2). For a water sample, the equilibration time of an analyte is dependent on its distribution constants: the headspace/aqueous sample
distribution constant, Khs, which is directly related to its Henry’s law constant, and the fiber coating/headspace distribution constant, Kfh.21 Since the Henry’s law constants are similar for the aniline derivatives, the larger the Kfh, the longer the equilibration time. For quantitative analysis, it is not necessary for the analytes to have reached equilibrium, only for sufficient loading onto the fiber and exact reproducible extraction time. Therefore, a 15min extraction time was adopted, even though several analytes had not reached equilibrium at this time point, but the analytical sensitivity was sufficient. Also, this sampling time was similar to the chromatography run time, thus allowing for maximum sample through-put. In summary, for optimum SPME sampling of monocyclic aromatic amines from water, the matrix was modified to 4.0 M NaCl and 2.0 M KOH. The 5-mL sample was maintained at 45 °C and stirred at a rate of 1500 rpm, while the headspace was sampled with a PDMS/DVB fiber for 15 min. Analysis of Biological Fluids. The optimum headspace SPME sampling conditions for water were applied to the urine, blood, and breast milk matrixes. Treatment of whole milk and whole blood with strong base, salt, and heat as required for the SPME sampling resulted in the saponification of the milk fat and in the coagulation of the red blood cells, respectively. Furthermore, a low amount of analyte was extracted onto the fiber and the chromatographic resolution was poor, most likely a result of the extraction solution containing the base-hydrolyzed lipid components. Therefore, it was preferable to remove the lipids from the milk and blood samples by centrifugation prior to analysis. Since the monocyclic aromatic amines are not highly lipophilic, a significant proportion of the analytes remains in the aqueous skim milk and plasma fractions. In the biological fluids, metabolites such as the N-acetylated derivatives may also be found, but with the optimized sampling Analytical Chemistry, Vol. 70, No. 9, May 1, 1998
1989
protocol, the sample is treated with strong base. Therefore, any acetamide metabolites are hydrolyzed to the parent compound and the total aromatic amine concentration is quantified. It should be possible for the aromatic amines and their metabolites to be extracted separately from the same sample by adjusting the pH of the biological fluids. Initially, the matrix would be adjusted to a pH 5 and the acetamide metabolites could be extracted with the PDMS/DVB fiber and then the matrix would be treated with KOH to increase the pH. Now, the aromatic amines would be extracted. Amount of Analytes Adsorbed onto the PDMS/DVB Fiber. The amount of analyte extracted onto the fiber was determined from the FID response for the SPME injection and calculated from the FID calibration curve generated with the liquid injections. In preliminary studies with water as the matrix, equal amounts of the five analytes were spiked into the matrix, yet unequal amounts were adsorbed onto the fiber from the headspace extraction. The amount of analyte adsorbed onto the fiber was
2,4,6-trimethylaniline . 2-chloroaniline ∼ 2,6-dimethylaniline > o-toluidine > aniline
Table 2. Amount of Analyte on PDMS/DVB Fiber for Each of the Five Target Analytes, from the Various Matrixes, under the Optimized SPME Sampling Conditionsa amt on fiber (ng) analyte
amt spiked (ng)
water
urine
milk
blood
aniline o-toluidine 2-chloroaniline 2,6-dimethylaniline 2,4,6-trimethylaniline
10000 5000 2500 2500 1000
214 246 318 313 216
147 167 229 232 182
163 154 67 65 18
80 55 31 37 9
a Each spiked matrix sample (5 mL) contained 2000 ppb aniline, 1000 ppb o-toluidine, 500 ppb 2-chloroaniline, 500 ppb 2,6-dimethylaniline, and 200 ppb 2,4,6-trimethylaniline The skim milk and plasma fractions were analyzed (see text). The sample (5 mL) was modified to 4.0 M NaCl and 2.0 M KOH in a total of 6 mL. The sample was maintained at 45 °C and stirred at 1500 rpm while the headspace was sampled with a PDMS/DVB fiber for 15 min.
Table 3. Method Detection Limit (MDL) of the Monocyclic Aromatic Amines Analytes in the Various Matrixesa MDL (ppb)
which is consistent with the observation that, with increasing alkylation, there is an increased amount of analyte extracted onto the fiber at equilibrium.25 2,4,6-Trimethylaniline has the greatest affinity for the PDMS/DVB fiber. The affinity of 2,6-dimethylaniline and 2-chloroaniline was approximately half of 2,4,6-trimethylaniline’s affinity and o-toluidine and aniline approximately one-fifth. This affinity is expressed in Kfh. In a pure water sample, the amount of analyte on the fiber can be readily calculated from Khs and Kfh. For complex matrixes, the relationship is not as simple; partitioning of the analyte into the lipid and protein components must also be considered, and thus, it is difficult to determine Khs and Kfh. For simplicity, only the amount of each analyte adsorbed onto the fiber from each matrix was calculated (see Table 2). The concentrations of the analytes in the water matrix were adjusted so that the amounts adsorbed onto the fiber were similar. These analyte concentrations were also used for the biological fluids to allow comparison to the pure aqueous matrix. The amounts of target analytes extracted from urine, milk, and blood were less than those from water. As the biological matrix became more complex, less of the analytes were extracted onto the fiber. The urine matrix, which is the least complex, was most similar to the water extraction profile, followed by milk and then blood from which the least amount of analytes could be extracted from. For the more complex matrixes of milk and blood, the amount of an analyte in the matrix can be defined by eq 1. The various phases of the matrix (Mi) include the aqueous fraction (skim milk, plasma), the protein components (such as caseins in milk and serum albumen and hemoglobin in blood), and the lipid fraction (milk fat and red blood cell membranes). The amount of an (25) Martos, P.; Saraullo, A.; Pawliszyn, J. Anal. Chem. 1997, 69, 402-408. (26) Sabbioni, G. Chem.-Biol. Interact. 1992, 81, 91-117. (27) Weast, R. C., Ed. Handbook of Chemistry and Physics, 64th ed.; CRC Press: Boca Raton, FL, 1984. (28) Schwarzenbach, R. P.; Gschwend, P. M.; Imboden, D. M. Environmental Organic Chemistry; John Wiley & Sons: New York, 1993; Chapter 6.
1990 Analytical Chemistry, Vol. 70, No. 9, May 1, 1998
analyte
waterb
urinec
milkc
bloodc
aniline o-toluidine 2-chloroaniline 2,6-dimethylaniline 2,4,6-trimethylaniline
3.17 1.55 0.88 0.70 0.18
3.39 1.88 1.05 0.81 0.40
5.33 3.47 2.01 1.67 6.60
7.71 6.25 4.72 4.09 4.58
a The water and urine matrix samples were spiked with 20 ppb aniline, 10 ppb o-toluidine, 5 ppb 2-chloroaniline, 5 ppb 2,6-dimethylaniline, and 2 ppb 2,4,6-trimethylaniline. The milk and blood matrixes were spiked with 20 ppb aniline, 20 ppb o-toluidine, 20 ppb 2-chloroaniline, 20 ppb 2,6-dimethylaniline, and 20 ppb 2,4,6-trimethylaniline. The skim milk and plasma fractions were analyzed (see text). The sample (5 mL) was modified to 4.0 M NaCl and 2.0 M KOH in a total of 6 mL. The sample was maintained at 45 °C and stirred at 1500 rpm while the headspace was sampled with a PDMS/DVB fiber for 15 min. b External calibration curve. c Standard addition.
analyte partitioning into the lipids, or its lipophilicity, is directly related to its octanol-water partition coefficient (Kow). The log Kow of the analytes increases with increasing alkyl or halogen substitution (see Table 1). The more lipophilic compounds, 2,4,6trimethylaniline, 2,6-dimethylaniline, and 2-chloroaniline, partition to a greater extent than the other two analytes into the lipid components of the milk and blood matrixes and, subsequently, are removed to a greater extent by the centrifugation step. Yet, since the partition coefficients Kfh for these compounds are greater than for o-toluidine and aniline, significant amounts of 2,4,6trimethylaniline, 2,6-dimethylaniline, and 2-chloroaniline are adsorbed onto the fiber. Method Detection Limits and Method Validation by GC/ MS. All measurements to determine detection limits and precision for the five analytes were performed with the optimized headspace SPME protocol. Listed in Table 3 are the MDL values for each analyte in the various matrixes. The MDL values for water and urine are very similar; in both matrixes, the MDL for the monocyclic aromatic amines range from the sub-ppb to lowppb levels with the analyte sensitivity reflecting its partition coefficient. The limit of detection of each analyte by the SPME/
a
b
c
Figure 3. SPME/GC/MSD chromatograms from urine (panel a), blood (panel b), and milk (panel c) samples. The SPME conditions and the concentration of analytes spiked in the matrixes were as listed in Table 4. In each panel, the upper chromatogram is the total ion chromatogram (TIC) and the lower is the extracted ion chromatogram (ions 93, 106, 121, 127, and 135) corresponding to the base ion of each of the five analytes. Labeled peaks: A, aniline; B, o-toluidine; C, 2-chloroaniline; D, 2,6-dimethylaniline; and E, 2,4,6-trimethylaniline. See text for chromatographic conditions.
GC/MS procedure was determined with water as the matrix. The LOD was 0.317 ppb for aniline, 0.013 ppb for o-toluidine, 0.002 ppb for 2-chloroaniline, 0.008 ppb for 2,6-dimethylaniline, and 0.006 ppb for 2,4,6-trimethylaniline. For the more complex matrixes of milk and blood, which require centrifugation, the MDL values are slightly higher than those obtained with water and urine, and the values range from 1.67 to 7.71 ppb for the various analytes.
The method validation was completed with spiked matrix samples, since reference standards in the biological fluids were not available. Comparison to other methods was not made since there is no established method for monocyclic aromatic amines in various biological matrixes. Available methods are lengthy procedures and are limited to aniline and o-toluidine in urine.5,7 For the SPME protocol, precision values for all the matrixes were obtained at concentrations approximately 10 times higher than Analytical Chemistry, Vol. 70, No. 9, May 1, 1998
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Table 4. Method Validation Based on Analyte-Spiked Matrix Samplesa waterb analyte aniline o-toluidine 2-chloroaniline 2,6-dimethylaniline 2,4,6-trimethylaniline
urinec
milkc
bloodc
mean RSD mean RSD mean RSD mean RSD (ppb) (%) (ppb) (%) (ppb) (%) (ppb) (%) 53.0 27.3 13.4 13.0 5.1
6.5 6.0 4.7 5.3 4.4
49.3 8.5 26.7 5.8 13.1 2.7 11.8 3.3 6.19 10.6
46.6 44.7 45.5 51.6 42.1
8.9 6.3 7.2 8.6 9.3
37.4 52.5 47.9 45.7 47.2
9.7 6.7 7.1 5.5 6.0
a The water and urine matrixes were spiked with 50 ppb aniline, 25 ppb o-toluidine, 12.5 ppb 2-chloroaniline, 12.5 ppb 2,6-dimethylaniline, and 5 ppb 2,4,6-trimethylaniline. The milk and blood matrixes were spiked with 50 ppb aniline, 50 ppb o-toluidine, 50 ppb 2-chloroaniline, 50 ppb 2,6-dimethylaniline, and 50 ppb 2,4,6-trimethylaniline. The skim milk and plasma were analyzed (see text). The sample (5 mL) was modified to 4.0 M NaCl and 2.0 M KOH in a total of 6 mL. The sample was maintained at 45 °C and stirred at 1500 rpm while the headspace was sampled with a PDMS/DVB fiber for 15 min. b External calibration curve. c Standard addition.
the MDL and the results are shown in Table 4. The SPME/GC/ MS chromatograms for the various biological matrixes are shown in Figure 3. Precision values ranged from 2.7 to 10.9% RSD for the various analytes in the different matrixes. The relative standard deviations were slightly higher with the more complex matrixes of milk and blood than with the urine matrix. In a previous study, a butyl alcohol extraction of NaOH-modified urine yielded 12-14% RSD for 77 ppb aniline- and 102 ppb o-toluidinespiked urine.5 Headspace SPME sampling of spiked urine yielded
1992 Analytical Chemistry, Vol. 70, No. 9, May 1, 1998
8.5% RSD for 50 ppb aniline and 5.8% RSD for 25 ppb o-toluidine. In this work, quantification of analyte concentrations was completed by method the of standard addition, but the more rapid quantification method of isotope dilution would ideally be employed, should the stable isotopes of all the analytes become readily available. CONCLUSIONS This SPME protocol provides a new sampling method for the quantitative analysis of monocyclic aromatic amines in biological fluids and is rapid and sensitive. This solvent-free technique yields excellent reproducibility and low detection limits with a 15-min sampling time and a 5-mL sample size. It can be applied as a biomonitoring tool for urine, blood, and breast milk, in the investigation of occupational, environmental, and medical exposures to these analytes. Furthermore, the protocol can be modified to include the extraction of aromatic amine metabolites. ACKNOWLEDGMENT The authors thank Dr. Perry Martos for his helpful discussions. This work was supported by the Canadian Breast Cancer Foundation, Natural Sciences and Engineering Research Council of Canada, Varian, and Supelco.
Received for review August 26, 1997. Accepted January 28, 1998. AC9709413
