Anal. Chem. 1986, 58, 2425-2428
and identification without exhaustive extraction. Misidentifications of compounds displaying ions isobaric with the mycotoxins (“false positive”) was also avoided by using MS/MS methods. The three mycotoxins exhibited varying detection limits imposed by the sample matrix rather than sample size. DON appeared the most difficult to identify due to the more polar nature of the compound, the greater number of interferences present, and low extraction efficiency. If more complete separation and characterization are required, or if samples cannot be brought to the laboratory as they are collected for mass spectral analysis, S F E can, of course, be performed “off-line”. The collected extract can then be injected onto a capillary chromatographic column for analysis by SFC-MS. A further extension of the SFE-MS method would ideally aim at on-column deposition for SFC following extraction of a sample with a supercritical fluid. The development of such a procedure may lead to further significant improvements in detection limits for the mycotoxins. Extraction from a complex matrix to which the more polar DON is, apparently, tightly bound would also be enhanced by the use of modified fluid mixtures or more polar supercritical fluids. Improvements in injection techniques, allowing greater sample volume to be handled, and column technology, allowing higher sample loading, need to be addressed for more sensitive analysis of extracts.
ACKNOWLEDGMENT We thank Paul Bossle of the U.S. Army Chemical Research and Development Center, Aberdeen, MD, for many helpful comments and discussion of this work and supplies of samples used. Registry No. DON, 51481-10-8; DAS, 2270-40-8;T-2 toxin, 21259-20-1; COZ, 124-98-9. LITERATURE CITED Cole, R. J.; Cox. R. H. Handbook of Toxic Fungal Metabolites; Academic Press: New York, 1981. Mirocha, C. J.; Pathre, S. V.; Behrens, J. J . Assoc. O f f .Anal. Chem 1976, 5 9 , 221-223. Mirocha, C. J.; Pathre, S. V.; Christensen, C. M. Mycotoxic fungi, Mycotoxins, Mycotoxicose -An Encyclopedic Handbook ; Syllie, T. D.. Morehouse. L. D., Eds.; Marcel Dekker: New York, 1977: Vol. 1.
2425
(4) Chaytor, J. P.; Saxby, J. M. J . Chromatogr. 1982, 237, 107-113. (5) Ikediobi. C. U.; Hsu, I. C.;Bamburg, J. T.; Strong, F. M. Anal. Biochem. 1971, 4 3 , 327-340. (6) Trucksess, M. W.; Neshein. S. J.; Eppley, R. M. J . Assoc. Off. Anal. Chem. 1984, 6 7 . 40-43. (7) Voyksner, R. D.; Hagler. W. M.; Tycykowska, K.; Haney, C. A. HRC C C , J . High Resolut . Chromatogr . Chromatogr , Commun , 1985, 8 , 119-125. (8) Bennett, G. A.; Peterson, R. E.; Plattner, R. D.; Shotwell, 0. L. J . Am. OilChem. S e c . 1981, 5 8 , 1002A-1005A. (9) Chang, H. L.; DeVries. J. W.; Larson, P. A,: Patel. H. H. J . Assoc. O f f Anal-Chem. 1984, 6 7 , 52-54. Blakely, C. R.; Carmody. J. J.: Vestal, M. L. J . Am. Chem. SOC. 1980, 102, 5931-5933. Schmidt, R.; Dose, K. J . Anal. Toxicoi. 1984, 8 , 43. Schmidt. R.; Ziegenhagen, E.; Dose, K. J . Chromatogr. 1981, 212, 370. Brumley, W. C.; Andrzejewski, D.; Trucksess. E. W.; Preifuse, P. A,; Roach, J. A. G.; Eppley, R. M.; Thomas, F. S.;Thorpe, C. W.; Sphon, J. A. Biomed. Mass Spectrom. 1982, 9 . 451-458. Tatsuno. T.; Ohtsubo. K.: Saito, M. Pure Appl. Chem. 1973, 3 5 , 309-313. Mirocha, C. J.; Pathre, S. V.; Schauerhamer, B.; Christensen, C. Appl. Environ. Microbiol. 1976, 3 2 , 553-556. Pathre. S. V.; Mirocha, C. J. Appl. Environ. Microbiol. 1978, 3 5 . 992-994. Vesonder, R. F.; Ciegler. A.; Rogers, R. F.; Burgridge, K. A,; Bothost, R. J.; Jensen, A. H. Appl. Environ. Microbiol. 1978. 3 6 , 885-888. Kuroda, H.; Mori. T.; Nishioka, C.; Okasaki, H.; Takagi, M. J . Food Hyg. SOC.Jpn. 1979, 2 0 , 137-142. Scott, P. M.; Lau, P. Y.; Kahere, S. R. J . Assoc. Off. Anal. Chem. 1981, 6 4 , 1364-1371. Bennett, G. A.; Stubblefield, R. D.; Shannon, G. M.; Shotwell, 0.L. J . Assoc. O f f . Anal. Chem. 1983, 6 6 , 1478-1480. Bromiey, W. C.; Trucksess, M.; Alder, S. H.; Cohen, C. K.; White, K. D.: Sphon. J. A. J . Agric. Food. Chem. 1985, 3 3 , 326-330. Smith. R. D.; Udseth, H. R. Anal. Chem. 1983, 5 5 , 2266-2272. Smith, R. D.; Udseth, H. R. Biomed. Mass Spectrom. 1983, 10, 577-580. Smith, R. D.; Udseth, H. R.; Wright, B. W. J . Chromatogr. Sci. 1985. 2 3 , 192-199. Smith, R. D.;Felix, W. D.; Fjeldsted, J. C.; Lee, J. L. Anal. Chem. 1982, 5 4 , 1883-1885. Kalinoski, H. T.; Wright, B. W.; Smith, R. D. Biomed. Environ. Mass Spectrom. 1986, 13, 33-45. Smith, R. D.; Kalinoski, H. T.; Udseth, H. R.; Wright, B. W. Anal. Chem. 1984, 5 6 , 2476-2480. Yost, R. A.; Enke, C. G. J . Am. Chem. SOC.1978, 100, 2274-2275. Smith, R. D.; Udseth, H. R.; Wright, B. W. Supercritical Fluid Technoiogy; Penninger, J. M. L., Radosz. M., McHugh, M. A,, Krukonis, V. J., Eds.; Elsevier: Amsterdam, 1985; pp 191-223.
RECEIVED for review March 26, 1986. Accepted June 4, 1986. The authors gratefully acknowledge the support of the United States Army through Contract No. DAAKll-84-c-0007.
Determination of Acrylamide in Sugar by Thermospray Liquid Chromatography/Mass Spectrometry S. S. Cuti6* and G . J. Kallos The n o w Chemical Company, Analytical Laboratories, 574 Building, Midland, Michigan 48667
Results presented in this paper illustrate the versatility of the thermospray liquid chromatographlc/mass spectrometric technique (TSP LC/NIS) for the determination of trace levels of acrylamide In sugar. The acrylamide is derlvatlred with bromine, separated by multidhnensionalreversed-phase iiquld chromatography, and detected wlth mass spectrometry through a thermospray Interface. Thls technlque Is shown to be superior to the movhg-belt Interface in Hs ablilty to provide molecular mass lnformatlon from a thermally iablle low volatimy compound. The llmlt of detection has been determined to be on the order of 200 pptr (parts per trillion)
Recent interest has focused on development of analytical 0003-2700/86/0358-2425$01.50/0
methods for the determination of acrylamide in sugar samples. The interest has been generated because this compound was reported ( I , 2 ) to show some biological activity. Since acrylamide monomer is the raw material for making polyacrylamide, residual monomer may be present in polyacrylamide which is used in the sugar industry as a flocculant. Lime is added to sugar syrups and reacted, a t high temperatures, to destroy naturally occurring amides found in the sugar, forming soluble lime salts and ammonia. Water-soluble polymers, including polyacrylamides, are added to flocculate the lime ( 3 ) . Thus, the possible presence of acrylamide monomer in sugar products is of interest. An analytical technique for determining parts-per-trillion levels of this monomer in refined sugar has, therefore, been developed. Initial efforts to determine acrylamide in refined sugar by (C 1986 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 58, NO. 12, OCTOBER 19
liquid chromatography techniques developed in our laboratory were found t o be unacceptable due to interferences present in the sugar matrix. Sugar is known to contain numerous nitrogenous compounds, such as asparagine and glutamine at the high parts-per-million level, which could interfere with the analysis of acrylamide. As a result, a multidimensional liquid chromatographic/thermospray mass spectrometric procedure has been developed and validated for the determination of acrylamide in sugar samples. T h e acrylamide is derivatized with bromine, separated by multidimensional reversed-phase liquid chromatography, and detected with a mass spectrometer that is coupled t o a conventional thermospray interface. T h e derivatization with bromine was optimized by Hashimoto ( 4 ) who used gas chromatography with electron capture detection for the analysis; however, the method suffered from detector contamination and loss of sensitivity. Brown and Rhead (5) tried LC/UV to eliminate t h e problems associated with the electron capture detector, but the procedure still suffered from interferences. Limits of about 200 pptr (parts per trillon) for an on-column injection can be achieved without any evidence of thermal decomposition, using multidimensional reversed-phase liquid chromatography/mass spectrometry coupled with a thermospray interface detection.
EXPERIMENTAL SECTION Equipment. The LC setup consisted of a Varian ternary gradient liquid chromatograph, Model 5560 (Varian, 220 Humboldt, Sunnyvale, CA), with UV-200 programmable wavelength UV-vis detector, and a Waters M-6000 pump (Waters Associates, Milford, MA) that was used for the postcolumn addition of buffers. The samples were injected with a Rheodyne 7125 injector and separated on a 25 cm X 4.6 mm Partisil 10 ODs-2 column (Whatman Chemical Separation, Inc., Clifton, NJ). A similar column was used in the multidimensional separation with a Rheodyne 7001 pneumatic actuated second valve to switch the chromatographic flow. The Valco zero dead volume tee for postcolumn addition of reagent was located between the column and TSP interface. A 0.5-pm filter was also installed in-line before the TSP interface. The TSP interface (Vestec, Houston, TX) was installed on a Finnigan 4500 quadrupole mass spectrometer. The interface included a temperature controller and readout. The temperature zones monitored were the vaporizer, TI, vaporizer tip, T,, source block, T3,and aerosol, T4. This interface did not require any splitting of the LC effluent. The moving-belt interface (Finnigan MAT Co., San Jose, CA) was installed on a Finnigan 4500 quadrupole mass spectrometer. The electron impact and methane chemical ionization mass spectra were obtained on the same instrument through the solids direct probe. The data were collected and processed with an INCOS data system (Finnigan MAT Co., San Jose, CA). Instrument Operation. The mass calibration of the mass spectrometer was verified with poly(ethy1ene glycol) standard. The TSP interface was optimized for the best stability and intensity of the solvent-buffer ions a t mass range 15-150 on the oscilloscope. The optimal temperature for 40% methanol in water of 1 mL/min in multidimensional separation and postcolumn addition of 0.3 mL/min 0.25 M trifluoroacetic acid were TI 130 "C, T, 197 " C , T3243 "C, and T4229 "C. The amount of sample injected was 100 pL. The LC-MS conditions for the acrylamide linearity study were as follows: column, 4.6 mm X 250 mm Partisil 10 ODs-2; eluant, 65% methanol in water; flow, 1mL/min; injection, 100 p L ; reagent postcolumn eluant, 0.25 M trifluoroacetic acid, 0.3 mL/min; vaporizer TI 130 "C, vaporizer tip T2200 "C, block source T3252 "C, and aerosol T4 229 "C. The mass spectrometer was operated in the selected ion recording mode covering ions a t m / z 230, 232, and 234 for the dibromopropionamide derivative and m / z 72 and 90 for acrylamide. The standard of dibromopropionamide analyzed using the moving belt interface was injected into a 4.6 mm X 250 mm Partisil 10 ODs-2 column with flow of 1 mL/min, 40% methanol/water where 50% was split onto the belt and deposited via
a nebulizer assembly. The temperature for the vaporizer and cleanup heaters was set at 240 "C and 250 "C, respectively. The IR lamp was 80%. Methane was added as a makeup gas to obtain chemical ionization data. The electron impact and methane chemical ionization mass spectral data were obtained through the solids probe inlet with temperature being programmed from 30 "C to 300 "C at 30 "C/ min. Preparation of Samples. Sugar (50 g) and potassium bromide (50 g) were dissolved in Millipore filtered water (450 mL) in a 32-02 narrow mouth bottle fitted with a polyseal cap, and 2.5 mL of concentrated hydrobromic acid was added. Saturated bromine water (25 mL) was added to the mixture. The bottle was then placed in a refrigerator a t 0 "C overnight. The excess bromine was decomposed by adding 1M sodium thiosulfate dropwise until the yellow color disappeared. To the mixture, 75 g of sodium sulfate was added. It was then extracted with 3 70-mL portions of ethyl acetate, that has been purified through silica gel-alumina, in a flatbed shaker for 3 min each. The organic layer was separated in a separatory funnel and placed in a 250-mL beaker. To the ethyl acetate, 5 g of sodium sulfate was added and the ethyl acetate was decanted to another beaker. The ethyl acetate was evaporated to 10 mL on a heating plate. The final 10 mL of ethyl acetate was evaporated to dryness in a Reacti-vial under nitrogen. Water was added and the sample was ready for analysis. Dibromopropionamide is sensitive to temperature and pH changes. The evaporation of the ethyl acetate on the heating plate should be discontinued prior to dryness (10 mL) and the final evaporation should be performed under nitrogen. Additionally, the heating plate should be maintained at 65 "C and the pH of the samples should be maintained between 1 and 7 for maximum yield. Reagents. Bromine, potassium bromide, and anhydrous sodium sulfate, ACS grade, were received from the local Fisher Scientific supplier. The silica gel, chromatographic grade silicic acid Bio-Si1 A and basic alumina, chromatographic aluminum oxide AG-10, were available from Bio-Rad Laboratories, Richmond, CA. Hydrobromic acid and sodium thiosulfate pentahydrate crystals were supplied by J. T. Baker, Phillisburg, NJ. Ethyl acetate "Distilled in Glass Quality" was available through Burdick and Jackson, Muskegon, MI. The saturated bromine water was prepared by shaking bromine in Millipore filtered water and allowing it to stand in a refrigerator until use. The acrylamide monomer, electrophoresis grade, was available from Eastman Kodak Co., Rochester, NY. The dibromoproprionamide derivative was synthesized and purified a t Dow. Glassware. The glassware was cleaned with Pierce RBS-35 detergent and rinsed with deionized water and methanol and then placed in an oven a t 400 "C for 1 h. Reagent Purification. Silica Gel-Alumina. Silica gel (50 g) and basic alumina (20 g) were placed in a Pyrex tube with appropriate dimensions to fit in a temperature-controlled clamshell furnace. Methanol (50 mL) and dichloromethane (50 mL) were added to the top of the column. After the solvents eluted, the tube was placed in the furnace, the inlet glass joint was connected, and the nitrogen flow was started. When the solvents were totally removed, the furnace was heated to 200 "C and the temperature was maintained for 2 h. The furnace was in a fume hood and the entire operation was performed with the hood doors closed. Potassium Bromide. Potassium bromide (100 g) was placed in a quartz tube with appropriate dimensions to fit in a temperature-controlled clam-shell furnace. After being placed in the furnace, the inlet glass joint was connected and the nitrogen flow started. The furnace was heated to 400 "C and the temperature maintained for 1 h. Sodium Sulfate. Sodium sulfate (100 g) was placed in a quartz tube with appropriate dimensions to fit in a temperature-controlled clam-shell furnace. After the tube was placed in the furnace, the inlet glass joint was connected and the nitrogen flow started. The furnace was heated to 600 "C and the temperature maintained for 1 h. Nitrogen. Contaminants present in cylinder gases interfere with analysis a t the ultratrace level (6). Two columns in series were added to the nitrogen line to remove interferences. Column 1 was packed with molecular sieve to remove water vapor and residual hydrocarbons. Column 2 was a hydrocarbon trap
ANALYTICAL CHEMISTRY, VOL. 58, NO. 12, OCTOBER 1986
available from Anspec (Ann Arbor, MI). This column is recommended for the removal of a variety of organics. Hood for Ultratrace Analysis. The fume hood was dedicated to this analysis and it was decontaminated by washing down with water and methanol before use. Disposable Pasteur Pipets. The disposable Pasteur pipeta used in this analysis were thoroughly cleaned after unpacking. Two packages of pipets were placed in a 3-L glass beaker and rinsed with deionized water. After the water was removed, the beaker was filled with methanol and placed in an ultrasonic bath for 15 min. After the methanol was removed, the beaker was placed in an oven (for glass drying) at 100 "C overnight. The beaker was kept covered with aluminum foil while in use. Ethyl Acetate. The ethyl acetate was purified by passing it through a bed containing 50 g of silica gel and 20 g of basic alumina, which were previously purified as it is explained before. Characterizationof Dibromopropionamide Standard. The dibromopropionamidestandard was characterized by DSC, NMR, and LC/MS. Liquid Chromatographic Calibration. Dibromopropionamide decomposes in water; however, it is very stable in acetonitrile. The stock solution was prepared in acetonitrile and the working standards were prepared in water by appropriate dilution of the stock solution on a daily basis. The liquid chromatograph was calibrated every morning to determine the exact time to switch the valve to inject the dibromopropionamide into the second column. The retention time of dibromopropionamide was very reproducible over a period of 2 months.
RESULTS AND DISCUSSION Initial data obtained from standards of acrylamide by the thermospray liquid chromatography/mass spectrometry (TSP LC/MS) technique (7) had shown promise for good sensitivity and selectivity. Both intense protonated molecular ion a t m / z 72 and water adduct ion a t m/z 90 were monitored under MID conditions to obtain the calibration of acrylamide in water. The MS detector response was found to be linear with a dynamic range of at least 3 orders of magnitude. However, the direct procedure was found t o be unacceptable in the complex sugar matrix. An alternate route was to derivatize the acrylamide t o dibromopropionamide for easy extraction out of the sugar matrix and to analyze by T S P LC/MS. A linear calibration curve of this derivative was obtained with similar dynamic range as that of acrylamide. The calibration study was carried out in the entire parts-per-billion and lowparts-per-million range. The precision of the TSP LC/MS procedure for the dibromopropionamide derivative was found to be 13.2% for lo. The application of t h e TSP L C / M S appears to be more suitable than the moving belt for the solution of this problem, since the brominated derivative was found to be thermally unstable. In order to establish proper ionization conditions for the analysis of this compound by the moving belt LC/MS, electron and methane chemical ionization mass spectra were obtained through the solids direct probe. The electron impact mass spectrum (Figure l a ) of the dibromopropionamide exhibits a very weak molecular ion at m / z 229 and several fragment ions that would not be desirable for selective ion monitoring. The methane chemical ionization maw spectra obtained by the solids probe and LC/MS moving belt introduction as shown in Figure 2 exhibit a protonated molecular ion a t m / z 230 (2Br) and intense protonated acrylamide ion a t m / t 72 particularly more pronounced in the moving belt interface. There is strong evidence that significant decomposition occurs by these techniques. However, the thermospray ionization mass spectrum shown in Figure l b did not show any significant thermal decomposition but did show a very intense protonated molecular ion a t m / z 230 (2Br), when trifluoroacetic acid was used. In the determination of dibromopropionamide derivative in sugar, the trifluoroacetic acid reagent was introduced
2427
%1
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Flgwe 1. (a)Electron impact mass spectrum of dibromopropionamide. (b) Thermospray ionization mass spectrum of dibromopropionamide: eluent, 40:60 CH,OH:H,O, 1 mL/min; postcolumn buffer, 0.25 M trifluoroacetic acid in water 0.3 mL/min; T , 149 O C ; T, 251 O C ; T , 251 OC;
T P 301
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Figure 2. Methane chemical ionization mass spectra of dibromo-
propionamide: (a) direct probe inlet; (b) LClMS moving-belt inlet. through postcolumn addition, since it was found to provide much improved sensitivity. Then the protonated molecular ions a t mlz 230,232, and 234 were monitored for the analysis. Originally in this study, a single Partisil 10 ODS-2 (4.6 mm X 250 mm) column was coupled to the thermospray LC/MS system. As shown in Figure 3a, the sugar blank contains many interferences. Therefore, a multidimensional chromatographic LC approach was applied with the use of two Partisill0 ODs-2 columns via a switching valve to eliminate most of the interferences. A cut from the first column that corresponded to the dibromopropionamide retention time was placed into the second column. Most of the interferences present in the sugar blank were removed, as can be seen in Figure 3b. The
2428
ANALYTICAL CHEMISTRY, VOL. 58, NO. 12, OCTOBER 1986 492
ion time of dibromopropionamide I9
L l 8 1. 9 1287
1
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Figure 5. Thermospray LC/MS total ion chromatograms and mass spectra of dibromopropionamide: (a) sample sugar spiked with 100 ng of acrylamide and then derivatized and concentrated 500X; (b) standard dibromopropionamide, 96 ppb; (c) mass spectrum of spiked sample: (d) mass spectrum of dibromopropionamide standard.
Table I. Recovery Data of Acrylamide in Sugar Samples
sample
ID
a m t added, pptr
water blank I
1 500
1 1000
I 1500
I 2000
I 2500
Scan
Figure 3. LC/MS total ion chromatogram of sugar sample: (a) sugar blank single LC column; (b) sugar blank, multidimensional liquid chromatography. IC/
$ / - f 2
5
4
5
200 800 800 800 2000
6
2000
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% recovered, corrected for
pptr
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320 30
15
240
30
320 270
40
890 610
45 31
34
The valve and column arrangement are shown in Figure 4. A typical total ion chromatogram of a spiked sample and a standard of 96 ppb dibromopropionamide derivative is shown in Figure 5. In the recovery experiments, acrylamide in methanol was added to the sugar samples and shaken for 30 min until equilibration was obtained. The results of the recovery experiments are shown in Table I. The variability of the values reported for the recoveries may be a function of the derivatization procedure and stability of the dibromo derivative in water.
ACKNOWLEDGMENT We thank P. Smith and D. Armentrout for their assistance in determining the structure of dibromopropionamide. Figure 4. Block diagram of multidimensional switching setup: (A, E) pumps, (1, 2) LC columns, (d) detector, ( V l ) Rheodyne 7120 valve, (V2) Rheodyne 7001 pneumatic actuated valve, (W) waste; (a) load, (b)
inject, (c) load.
column switching technique improved the analysis by removing the interferences; however, the manual switching of the valve introduced pressure upsets into the mass spectrometer with eventual shutdown on every injection. The installation of a pneumatic actuated valve (8)improved the switching speed of the valve and significantly decreased the surge in pressure.
LITERATURE CITED (1) Dow report "Recent Acrylamide Monomer Toxicity Data and Its Importance To You The Monomer Customer"; November 1984. (2) Johnson, K. A.; Gorzlnskl, S. J.; Bodnar, K. M.; Campbell, R. A,; Wolf, C. H.; Froedman, M. A.; Mast, R . W., submitted for p'ublication in Toxicol Appl Pharmacol (3) McGlnnis, R. A. "Beet Sugar Technology"; published by Beet Sugar Development Foundation, Fori Colllns, CO, pp 62, 178-179, 249-256. (4) Hashimoto, A. Analysf (London) 1978, 707, 932-938. (5) Brown, L.; Rhead, M. Analyst (London) 1979, 704, 391-399 (6) Nestrick, T. J.; Lamparski, L. L. Anal. Chem. 1981, 5 3 , 122. (7) Blakley, C. R.; Vestal, M. L. Anal. Chem. 1983, 5 5 , 750-754. (8) Harvey, M. C.; Stearns, S. D. Anal. Chem. 1984, 5 6 , 837-839.
.
.
.
RECEIVED for review February 3,1986. Accepted June 6,1986.
