Anal. Chem. 1986, 58, 2421-2425
2421
Supercritical Fluid Extraction and Direct Fluid Injection Mass Spectrometry for the Determination of Trichothecene Mycotoxins in Wheat Samples Henry T. Kalinoski, Harold R. Udseth, Bob W. Wright, and Richard D. Smith* Chemical Methods and Separation Group, Chemical Technology Department, Battelle, Pacific Northwest Laboratories, Richland, Washington 99352 The application of on-llne supercrltlcai fluid extractlon wlth chemlcal lonlzallon mass spectrometry and coHislon induced dissociation tandem mass spectrometry for the rapid Identlflcatlon of parts-permllllon levels of several trlchothecene mycotoxins Is demonstrated. Supercrltlcal carbon dioxlde Is shown to allow identlficatlon of mycotoxins wlth minimum sample handllng In complex natural matrices (e.g., wheat). Tandem mass spectrometry techniques are employed for unambiguous ldentlflcatlon of compounds of varying polarlty, and “false posltlves” from isobaric compounds are avoided. Capillary column supercritical fiuld chromatography-mass spectrometry of a supercrRlcal fluld extract of the same sample was also performed and detection limits In the partcper-blillon range appear feaslble.
The trichothecenes are a group of over 60 sesquiterpenoid mycotoxins produced by a diverse variety of imperfect fungi, including species in the genera Cepholosporium, Fusarium, Mycothecium, Stachybotris, Trichoderma, and Trichothecium. Structurally they are characterized by the 12,13-epoxytrichothec-9-ene ring system with subgroups classed on the basis of specific functionalities. As a group, the trichothecenes have been reported to show a wide range of biological activity ( 1 ) and have been involved in natural intoxications in humans and domestic animals following ingestion of moldy grains (1-3). The analytical chemistry of the trichothecenes has proven difficult, with purification usually accomplished by thin-layer or column chromatography (4-6). HPLC techniques are also used (7-9), although high levels of contamination (3-5, 10, 11) are a disadvantage. Gas chromatography-mass spectrometry (GC-MS), although effective, requires derivatization of the samples to ethers and esters (12-19) -prior to analysis. Further, the use of electron ionization (EI) for GC-MS often results in excessive fragmentation and limited molecular weight information for these compounds, while chemical ionization (CI) requires different derivatives for the variety of mycotoxins to obtain comparable sensitivity. The application of the thermospray HPLC interface with mass spectrometry (MS) (20)has been reported for the analysis of some Fusarium mycotoxins (3,and analysis of deoxynivalenol (DON) with negative ion CI-MS following thin-layer chromatography has been published (21). Capillary column supercritical fluid chromatography (SFC) combined with CI mass spectrometry has been demonstrated to be a fast, selective, and sensitive method for the separation and detection of trichothecene mycotoxins, including the higher molecular weight macrocyclic roridins and verrucarins (22-24). In this work we demonstrate the feasibility of direct extraction of underivatized, unpurified grain samples with a supercritical fluid such as COz and subsequent direct injection of the extract into a quadrupole mass spectrometer for analysis. The direct fluid injection interface (DFI) has been previously demonstrated as a monitor for supercritical fluid extractions (SFE) (25). The benefits of SFE-MS include a great reduction in sample handling and preparation with no
discrete extraction, purification, or derivatization, thus facilitating analysis and simplifying interpretation. This approach has the potential of providing either high-speed detection or a rapid screening analytical capability in conjunction with MS/MS methods. Another potential advantage of SFE is the selectivity provided by control of fluid density (and solvating power) through control of pressure. However, any natural substance presents complications due to concurrent extraction of wide classes of chemical compounds (with the selectivity of the extraction dependent upon fluid composition, temperature and pressure), resulting in a relatively large “background signal, which can ultimately determine detection limits. Extraction with supercritical carbon dioxide is compatible with a variety of CI reagents (23, 24, 26, 27), which allows a sensitive and selective means for ionizing the solute classes of interest. If the interfering effects of the sample matrix cannot be overcome by selective ionization, techniques based upon tandem mass spectrometry (28) can be used. Combined information from parent ions and the MS/MS fragmentation pattern can often greatly enhance selectivity and (often) sensitivity (28).
EXPERIMENTAL SECTION Instrumentation. The instrumentation used for SFE-MS, Figure 1, described in part previously (22, 23, 29), allowed for continuous “microscale”extraction from a small sample cell for direct mass spectrometric analysis. A Varian Model 8500 highpressure syringe pump supplied a pulse-free flow of fluid to the extraction cell. The stainless steel extraction cell had a volume of 680 pL and can contain as much as 0.5 g of dry sample. Samples were held by stainless steel frit filters, with a 0.5-pm filter on the mass spectrometer side to prevent passage of particles and possible plugging of the pressure (flow) restrictor. A 1-m length of deactivated, 50 pm i.d., fused silica capillary tubing was used for transfer to the restrictor. A short (35-50 mm) length of 8 pm fused silica capillary acted as a pressure restrictor to regulate fluid flow rates. Typical flow rates were 5-50 pL/min, with flow rates as high as 100 pL/min possible (25) (defined by the pumping speed of the vacuum system). The temperature of the extraction cell was maintained by a Hewlett-Packard gas chromatograph oven, and the DFI probe was air-heated and assisted by an auxiliary electrical heater. This arrangement allowed constant temperature (ic0.5 “C) to be maintained along the transfer line to the point of injection to avoid potential complications due to solute precipitation. The entire extract effluent from the DFI transfer line was injected into the CI region of an Extranuclear Laboratories (Pittsburgh, PA) “simultaneous” dual EI-CI source. An Extranuclear triple quadrupole mass spectrometer was used to obtain mass spectra under control of a Teknivent Corp. (St. Louis, MO) data system. Positive ion mass spectra were typically obtained using ammonia as the CI reagent. For collision-induced dissociation (CID) studies (28), the second quadrupole was operated in the “rf only” mode to act as a collision cell. Argon was employed as the target gas for CID, with a collision energy of 40 eV and 60-70% attenuation of the parent ion signal. The MS/MS approach allowed for reduction of the chemical ”noise” and improved detection limits in many instances. For supercritical fluid extractions, the pressure was ramped upward with the aim of obtaining selectivity based upon the
0003-2700/86/0358-2421$01.50/00 1986 American Chemical Society
2422
ANALYTICAL CHEMISTRY, VOL. 58, NO. 12, OCTOBER 1986
Analog and Ion Counting Electronics
Computer
-@
4
I
I
Programmer
-1-
Filter
High Pressure Syringe Pump
-
Mobile Phase Reservoir
Valve
Figure 1. Schematic of supercritical fluid extraction apparatus interfaced to the triple quadrupole mass spectrometer. variable solubility of higher molecular weight solutes with changes in density. Pressure ramp rates of 1, 2.5, and 5 bar/min, from 100 bar to a maximum of 300 bar, were generally used. Distilled and filtered COz was used as the supercritical fluid for the extraction at temperatures of either 61 "C, reduced temperature (TR) of 1.1,or 98 "C, TR = 1.2. The instrumentation used for SFC-MS was similar to that described for SFE-MS (22-27, 29). However, in place of the extraction cell a short, deactivated 4 m X 50 km, fused silica capillary coated with a nonextractable, cross-linked, 5% phenyl poly(methylphenylsi1oxane) (SE-54) stationary phase chromatographic column was used. Samples were injected with a Valco 0.ObwL (C14W) injector operated at ambient temperature with a flow splitter adjusted to a ratio of 1:lO. Positive ion ammonia CI mass spectra were obtained by use of an Extranuclear Laboratories single quadrupole mass spectrometer with the total chromatographic effluent axially introduced into the CI source (22). Chromatography was performed at 100 "C, and the fluid pressure was ramped at a rate of 10 bar/min starting from 100 bar. Samples. Wheat samples were doped with varying levels of three mycotoxins. One sample was spiked a year prior to analysis with 1 ppm deoxynivalenol (DON), determined by TLC analysis of the solution prior to preparation of the wheat sample. Another sample was prepared to contain 10 ppm each of diacetoxyscirpenol (DAS), T-2 toxin, and DON. This sample was analyzed approximately 6 months after preparation. Both samples were stored at -4 "C prior to analysis by SFE-MS. A third sample contained 27 ppm DON, 2.5 ppm T-2, and 1.4 ppm DAS and was analyzed, after drying (in air) to remove solvent, less than 72 h following preparation. Untreated wheat was used as a blank for extraction studies. Wheat extractions were also compared to mycotoxin extractions from samples deposited onto glass beads using the SFE-MS extraction cell in the same manner as the wheat samples. The 45-150 wm diameter beads were deactivated prior to use by refluxing with hexamethyldisilazane and trimethylchlorosilane for 16 h. Solutions of the mycotoxins were dried on the glass beads; the weight percent of the mycotoxin to the beads was 9 X for DAS, 1.9 X lo4 for DON, and 1.8 X for T-2. Positive ion mass spectra and positive ion CID spectra were obtained by using this mode of introduction.
100-
-
-
RESULTS AND DISCUSSION Figures 2-4 show the positive ion CID spectra of the three trichothecene mycotoxins, produced using arhmonia as the CI reagent and argon as the collision gas. Both DAS (Figure 2) and T-2 (Figure 3) show intense ammonium adduct ions (M + 18)' at m / t 384 (DAS) and m / z 484 (T-2). Daughters
20
Q1
-
384 3841
-
307
t
O--F?P+.y,.;
150
200
,I,, 260
-, mlz
a,
, ,-,-,-, 300
',
,-I;;.
350
, 4
Flgwe 2. Argon CID mass spectrum of the ammonium adduct ion (M 18)+-ofdlacetoxyscirpenoi (DAS), m l z 384.
+
g 100 I
-6
0 1 = 484
20-
n,c.
CHICH~I~
484
-250
300
350
400
460
500
mlz
Figure 3. Argon CID mass spectrum of the ammonium adduct ion (M
+ 18)'
of T-2 toxin, m / z 484.
from these ions a t m / z 307 [(M + NH, - OAc - HzO)+ for DAS] and m / z 305 [(M + NH4 - (C02CHzCHCH3)z)HOAc - H20)+for T-21 had sufficient intensity to be used for confirming the identity of the trichothecene mycotoxins from a complex matrix. These and other ions present in the CID mass spectra are also present in the more energetic isobutane CI and electron impact mass spectra of these compounds (I, 22, 24).
ANALYTICAL CHEMISTRY, VOL. 58, NO. 12, OCTOBER 1986
~
7-
200
"0
2423
250
300
0 -29, 100
250
300
350
400
450
400
450
miz :
0 150
L
-
r
j
.--
200
8100 250
298
300
Figure 4. Argon CID mass spectra of the ammonium adduct ion (M 18)', m l r 314 (A), and the protonated molecular ion (M + l)', m / r
+
80
297 (B), of deoxynivalenol (DON).
The more polar DON forms two major ions following ammonia chemical ionization (Figure 4), a protonated molecule (M + 1)' at m/z 297 and an ammonium adduct ion (M + 18)' a t m / z 314. Dissociation of these parent ions under the (collision energy limited) conditions utilized in this study produced few fragments. With m/z 314 as the parent (Figure 4A), CID led primarily to the protonated molecule at m / z 297 with a small contribution to the spectrum at m / z 249. CID of the m / z 297 ion produced a few additional fragment ions at m / z 279, 261, 231, 219, and 203. Extraction Studies. Carbon dioxide, the most widely utilized supercritical solvent, was used for extraction since sufficient solubility of the trichothecenes over a wide range of temperatures had previously been demonstrated (23, 24. 29). As a blank, approximately 300 mg of untreated wheat was extracted at 61 "C (1.1T,)while increasing pressure at a rate of 5 bar/min. The wheat matrix produced a large total ion signal and obviously an additional separation step would be required to identify a single component from this complex matrix (Figure 5A). The pressure ramp and flow rates used (1C-20 lL/min) did not appear to give a selective extraction, as some species detected early in this extraction were still detectable more than 2 h later. An "exhaustive" extraction of a selected component from this matrix with the present experimental arrangement would be difficult. This limitation results from a combination of factors which include (1)limitations upon mass transport to the fluid phase, (2) solubilities a t the selected conditions, and (3) experimental parameters (cell volume, flow rate, mixing, etc.). Correct selection of the experimental parameters and the physical state of the sample should allow a significant improvement in the selectivity of the extraction on a reasonable time scale. Mass transport limitations for specific matrices pose the most significant unknown. The positive ion mass spectrum of the unspiked wheat shows major "background" components from the matrix (Figure 5A). In the region where DON would appear ( m / z 297 and 314), strong interferences were present for the ions at mlz 298 and 312 and related signals. In the region where DAS would be detected (mlz 384) there were weak interferences, but at m / z 484 where T-2 would appear, the background was negligible. Figure 5B is a single scan obtained during extraction of wheat containing 10 ppm each DAS, DON, and T-2 toxin. In addition to the few very intense ions found in the blank
250
300
350 mir
Figure 5. Single scan NH, chemical ionization mass spectra acquired during on-line supercritical fluid extraction mass spectrometry of a blank (unspiked)wheat sample (A) and a wheat sample containing 10 ppm each of the mycotoxins DAS, DON, and T-2 toxin (B).
spectrum, signals are present at m / z 384 and mlz 484. The ion at m / z 484 can be assigned to the ammonium adduct ion, (M + 18)' of T-2 toxin due to the lack of any significant interference. An argon CID spectrum of the m / z 484 ion showed the expected fragmentation for T-2 yielding ions at m / z 305, 245, 365, 275, and 257. Unambiguous identification of the m / z 384 ions as the (M + 18)' ion of DAS was accomplished by obtaining the CID spectrum. The confirmatory fragment ion at mlz 307 was found was well as ions at m / z 366, 349, and 247. The complex background spectrum produced from the wheat matrix made DON much more difficult to detect. Compared with CID spectra of the standard, Table I, signals at m / z 314 and m / z 297 from the spiked sample showed poor agreement. Ions found in the spiked sample (mlz 280 in the m / z 297 spectrum or m / z 279 in the m / z 314 spectrum) were not observed in the standard and an ion in the standard (mlz 249) was not significant in the spiked sample. The mass spectrum of a wheat sample spiked at 1 ppm DON, contains a signal a t m / z 384, possible (M + 18)' for DAS. Where signals from DON would be expected, a number of ions were detected, so no direct determination could be made. The known fragments of DAS were not found in the CID spectrum of the m / z 384 ion, indicating the CID MS/MS approach is also useful in ruling out the misidentification of signals that are isobaric with the compound of interest. Monitoring a specific ionic fragmentation process from CID provided a sensitive, selective means for identifying DON in a complex matrix. The ion at mlz 249 in the CID spectrum of DON can be formed from either the mlz 314 or m / z 297 ions and appeared to be characteristic for DON. The m / z
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ANALYTICAL CHEMISTRY, VOL. 58, NO. 12, OCTOBER 1986
Table I. Relative Abundance Values of CID Daughter Ions from m / z 314 and m / z 297 Ions Obtained during COz Supercritical Fluid Extractions of Deoxynivalenol (DON) Standard and Wheat Sample Spiked at 10 ppm DON" relative abundance,*
relative abundance,b
%
%
m/z
DON std
314 297 279 278 265 261 249 247 245 231 227 219 203
100 277.8 3.9 N/D
N/D 3.3 10.3 N/D N/D 3.6 N/D 3.3 4.2
wheat sample 100 84.4 41.6 10.6 23.2 N/D N/D 22.3 5.5 N/D 3.9 N/D N/D
m/z
DON std
297 280 279 265 262 261 251 249 247 233 231 223 219 213 208 203
100 N/D 4.0 N/D N/D
3.0
N/D 7.5 N/D 1.2 5.7 N/D 1.9 1.1 N/D 3.5
wheat sample 100 127.9 N/D 6.0 5.5 N/D 4.0 N/D 30.2 N/D N/D 9.6 N/D N/D 6.3 N/D
" N / D signifies no signal greater than 1% of the base peak was detected. Normalized to uarent ion intensitv (=loo%).
249 ion was not present in the CID spectra of m / z 297 or m / z 314 of the 10 ppm spiked wheat sample discussed earlier but was found in the CID spectrum of the DON standard. With the first quadrupole set to select the ammonium adduct ion for DON and the third quadrupole offset by 65 daltons (allowing the m / z 314 to m / z 249 fragmentation to be monitored), DON was detected in a wheat sample that had been freshly spiked (less than 72 h before) with 27 ppm DON, 2.5 ppm T-2, and 1.4 ppm DAS. Preliminary full scan mass spectra of this sample contained ions at m/z 384 and m/z 484, confirming the presence of DAS and T-2 a t the low partsper-million levels. DON may strongly bind to the wheat matrix and, with time, may irreversibly bind or be chemically reacted or degraded so that it cannot be extracted as the DON molecule using a less polar solvent (e.g., COz). Although metabolites of DON may have been present, their identification and the ultimate fate of DON in wheat were not the aim of the present study. S u p e r c r i t i c a l F l u i d Chromatography-Mass Spectrometry. A few experiments were conducted to evaluate the practicality of capillary SFC-MS with actual extract samples containing trace levels of the trichothecenes. The ion chromatograms shown in Figure 6 were produced from an aliquot of an off-line COz supercritical fluid extract of wheat spiked at 10 ppm DAS, DON, and T-2 toxin. Chromatography was performed on a relatively short column (4 m) with a nonselective (SE-54) stationary phase. Positive ion ammonia CI was used with the m / z 150-490 mass range scanned at 2.5 s/scan to acquire mass spectra. Although chromatographic conditions were not optimized for the separation of the mycotoxins, two of the compounds were easily detected using COz SFC-MS. The selected ion chromatograms of m / z 484 and m/z 384 indicated well-defined chromatographic peaks for the DAS and T-2 toxin spikes. Analysis of the mass spectra obtained during the elution of these components confirmed the identity of these toxins. Although extraction efficiency from the wheat was not well characterized and the sample was split prior to injection into the column, rough detection limits for DAS and T-2 toxin may be estimated. Assuming 100% extraction efficiency and a 101 split following injection (at a concentration of each toxin of 20 ng/pL), the amount of each material injected on-column, and therefore into the mass spectrometer ion source (22),was
'"1
TIC
lW
m/z 297
h
r56
I/
0
6
15
10
h I\ 6
10
16
10
15
m / i 484 x 100
l"] 60
I
o r-5 Tim..
minute.
Flgure 6. Total ion chromatogram (TIC) and single ion chromatograms produced for an aliquot of a supercritical fluid extract of wheat containing 10 ppm each DAS, DON, and T-2 toxins using CO, SFC with NH, CI-MS.
120 pg from a 10-ppm spike on wheat. In previous work -0.1-pg detection limits for DAS and T-2 have been demonstrated using single ion monitoring (24),suggesting detection limits of 10 ppb may be achievable. This value compares well with other mass spectral techniques ( 4 , 7, 21) (1-150 ppb) which require more extensive sample preparation. From the chromatograms we concluded that the method can detect DAS and T-2 toxins at concentrations a t least 1 to 2 orders of magnitude lower than that used in this study. (The apparent lower sensitivity for the higher molecular weight T-2 toxin, based on comparison of meas of chromatographic signals, very likely reflects the decreased transmission efficiency of the mass spectrometer a t higher m / z values.) Further refinement of extraction, preconcentration, injection techniques, and improvement in column technology, currently under development in this laboratory, are anticipated to lead to significantly lower detection limits for these compounds. The ion chromatograms in Figure 6 also indicate that DON was not detected. Since capillary SFC of DON has been demonstrated previously (23),it is most likely that DON was not extracted from the wheat using supercritical COz. Although the solubility of DON in COz is approximately an order of magnitude smaller than for T-2, the wheat matrix may result in an impractically small partition coefficient. A large, well-defined signal was observed at m / z 314, (M 18)' for DON, but a corresponding (M + 1)' ion ( m / z 297) was not detected. Analysis of the mass spectra producing this signal also indicated that it was not related to DON. The large number of interfering compounds at m / z 297 and mlz 314 also made identification of DON difficult.
+
CONCLUSION The successful application of on-line and off-line SFE followed by CI-MS for identifying trichothecene mycotoxins was demonstrated. With minimum sample handling and preparation, the trichothecenes diacetoxyscirpenol (DAS) and T-2 toxin were readily identified in a wheat matrix. The added dimension of selectivity offered by collision induced dissociation tandem mass spectrometry allowed rapid detection
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, SFE 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.
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(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
