Anal. Chem. 1987, 59, 2055-2059
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Residue Levels of Ethyl Carbamate in Wines and Spirits by Gas Chromatography and Mass SpectrometryIMass Spectrometry Thomas Cairns,* Emil G . Siegmund, Milton A. Luke, and Gregory M. Doose
Department of Health and Human Services, Food and Drug Administration, Office of Regulatory Affairs, Los Angeles District Laboratory, 1521 West Pic0 Boulevard, Los Angeles, California 90015
Analytical difficulties encountered In the detection of ethyl carbamate at low levels In a wide varlety of wlnes and spirits have been overcome by adoptlng a single multlresldue methodology followed by a Florlsli column cleanup. Primary detectlon has been accompllshed by gas chromatography using the Hall electrolytic conductivity detector in the nltrogen mode. Recovery studies conducted at concentration levels below 1 ppm have indicated that results are quantitative. Wlth the availability of a stable lsotoplcally labeled ethyl carbamate reference standard, both conflrmatlon and quantltatlon were carried out by chemlcai lonltatlon mass spectrometry uslng argon coiiislon activated dlssoclatlon technlques.
Approval for the continued use of diethyl pyrocarbonate (DEPC) in beverages as an antimicrobial food additive was rescinded in 1972 ( I ) . Two years later, the incidence of trace levels of ethyl carbamate (also known as urethane) in wines and other fermented beverages was tentatively attributed to the use of DEPC in reaction with low levels of ammonia (2). Subsequently, data were also presented to support the theory that ethyl carbamate could be naturally produced in fermenting food and beverages via ethanolysis of carbamoyl phosphate associated with the urea cycle that occurs in all normal cell growth (3-5). Since DEPC had been disapproved for use in 1972, the later data strongly suggested that trace levels of ethyl carbamate were a byproduct resulting from the fermentation process. More than a decade had passed since this controversy had surfaced, and the collection of current data on trace levels of ethyl carbamate was desirable. The analytical methods, however, used in these earlier studies relied heavily on complicated extraction methods, sample cleanup, and sample derivatization (2-4, 6) and usually employed a flame ionization detector. More recently, the use of methylation followed by gas chromatography with nitrogen-phosphorus thermionic detection and capillary gas chromatography (7-9) have been employed for the analysis of ethyl carbamate in alcoholic beverages. A recent FDA surveillance program (1986) to evaluate the trace levels of ethyl carbamate, in various wines and spirits prompted the development of a number of innovative analytical protocols to allow gathering of reliable data. The Luke et al. extraction procedure ( l o ) ,a rapid multiresidue methodology currently in use in this laboratory for analyzing domestic and imported food and feed commodities, was investigated as a potential simplified extraction method for ethyl carbamate. Gas chromatography with the Hall electrolytic conductivity detector in the nitrogen mode (HECD-N) was used for detection and quantification in conjunction with mass spectrometry for confirmation and check quantification. With the availability of a commercial stable isotope of ethyl carbamate labeled with both lSN and 13Cat the amino group and the carbonyl, respectively, the effect of potential interferences was explored. Quantification of ethyl carbamate by gas
chromatography, mass spectrometry, and mass spectrometry/mass spectrometry (MS/MS) was directly compared. In this paper we report the joint application of these revised analytical protocols to the incidences of ethyl carbamate in over 80 wines and spirits. EXPERIMENTAL SECTION Reagents. All solvents were pesticide grade (Burdick & Jackson Lab., Inc., Muskegon, MI) except for ethyl ether (Mallinckrodt, Paris, KY, nanograde with 2% absolute ethanol added). Procedural blanks were performed with solvents prior to use to ensure the absence of interferences. The stable isotope of ethyl carbamate (13C1,15N1)was obtained from Merck, Sharp and Dome. Complete details on other reagents employed have been previously published (10). Gas Chromatography. All chromatographic data were acquired on a Tracor Model 560 gas chromatograph equipped with the Hall Model 700A electrolytic conductivity detector (HECD) in the nitrogen mode (10). Operating conditions were as follows: 120 cm X 2 mm i.d. glass column packed with 10% Carbowax 20M on 80/100 mesh Supelcoport; carrier gas, 90 mL of ultrapure H2/min; column inlet temperature, 180 "C; column temperature, 130 "C (isothermal); detector temperature, 250 "C; furnace, 950 "C; solvent flow, 0.35 mL/min 50% 1-propanol/HPLC pure water; reaction gas, ultrapure H2 at 60 mL/min. Mass Spectrometry. Mass spectra were recorded with a Finnigan Model 45A triple-stage quadrupole mass spectrometer equipped with a chemical ionization source and Incos data system. Operating conditions for residue samples and synthetic reference standards were as follows: 15 m X 0.53 mm i.d. 0.5-pm Stabilwax (chemically bonded CWBOM),Restek Corp.; helium flow rate, 2 mL/min, programmed at 15 "C/min from 60 " C to 150 "C; 1pL of solution injected in the splitless mode. Typical instrument parameters used for Q1 were as follows: extractor, -24 V; lens, -108 V; electron energy, 70 V; quad entrance, -5 V; source pressure, 300 mtorr; electron multiplier set at 1050 V, source 170 "C. Methane gas was added at the ion source to about 0.5 torr and adjusted to maximize the intensity of [CH5+].. Collision activated dissociation studies were carried out by using argon as the collision gas (1mtorr) with the collision energy set at -10 V. Dwell time for multiple reaction monitoring (MRM) in the MS/MS mode was 100 ms. Sample Preparation. A 30-g sample (size of sample can be adjusted, in necessary, as long as solvent/sample ratios are maintained) was shaken with 60 mL of pesticide grade acetone. Eighty milliliters of this mixture was transferred to a 1-L separatory funnel containing 7 g of NaCl, 100 mL of dichloromethane, and 100 mL of petroleum ether. For liqueurs, 10 mL of distilled water was also added at this stage to reduce the density of the layers because of the higher sugar content of liqueurs. After the sample was shaken vigorously for 45 s, the layers were allowed to settle before draining off the lower aqueous layer to a second 1-L separatory funnel for reextraction by an additional 100-ML portion of dichloromethane. The organic layers were combined and dried by passage through sodium sulfate into a KudernaDanish evaporator with a Mills tube attached. The aqueous layer from the second extraction was also reextracted in a similar manner and finally combined with the other two organic layers after drying by passage through sodium sulfate. The sodium sulfate used for sequential drying of the three organic portions was rinsed with 50 mL of dichloromethane and collected in the Kuderna-Danish evaporator. Boiling chips were added, a Synder
This article not subject to U S . Copyright. Published 1987 by the American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 59, NO. 17, SEPTEMBER 1, 1987
Table I. Summary of Residue Findings of Ethyl Carbamate in Various Wines and Spirits samples examined
Carbernet sauvignon bergundy chenin blanc fume blanc chardonnay white wine Chablis pinot noir red wine zinfandel rose sherry port other wines burbon liquers
9
1
-
3 5 3 1 12 7 10 1 1
2 3
totals
89
lo00 ppb
totals 3 3 3 5
5 8 4 6 9
4
1
12 5 8
1 1
24
15
9
12
-
51
a The count given in a column covers a concentration range that starts at a value greater than that of the previous column heading (in ppm) and extends to the current column heading. Ethyl carbonate was not detected in all samples analyzed and hence the total number of samamles with residues may be smaller.
column was attached and the extracts were concentrated on a steam bath by placing just the tip of the Mills tube into the bath. The volume was not allowed to go below 6 mL at any time. This extract was then transferred to a 1WmL graduated cylinder and petroleum ether added to make up to the 100 mL mark. The 100 mL of diluted extract was added to a prewashed (100 mL petroleum ether) properly activated Florisil column (IO)and the column subsequently eluted with 200 mL of 15% ethyl ether/ petroleum ether. The eluate was discarded. Subsequent elutions (200 mL) with 50% ethyl ether/petroleum ether and ethyl ether were combined in a Kuderna-Danish evaporator with a Mills tube attached. Extract volume (400 mL) was then reduced on a steam bath to 6 mL as described above. Finally, 30 mL of acetone was added and the volume reduced once again to no less than 6 mL (to ensure the solubility of ethyl carbamate). Stable Isotope Spikes. The isotopically labeled ethyl carbamate was initially prepared as a stock solution at the concentration level of 1mg/mL in acetone. To spike the 6-mL wine and liqueur extracts prepared for primary analysis by GC, a 1:lO dilution of the stock solution was prepared. Samples were spiked by adding various microliter volumes of this stock solution, predetermined to be equal to the amount of ethyl carbamate found in the extract by GC. This approach was employed to process only positive samples detected by GC. Addition to the whole wine might have precluded an accurate determination by GC due t o two main reasons-coelution by the spiked labeled ethyl carbamate and nonlinearity of the detector over the unknown increased concentration range caused by the addition.
RESULTS AND DISCUSSION Gas Chromatography. Eighty-nine different samples of domestic and imported wines and spirits collected at random were extracted and examined by use of the Hall electrolytic conductivity detector (HECD) in the nitrogen mode. Application of an element-selective detector had the distinct advantage of nonsensitivity to those non-nitrogen-containing flavor components that can be coextracted along with the ethyl carbamate. In the case of a white wine the sample background recorded (Figure 1)for a blank possessed a number of unresolved nitrogenous compounds that had the potential of providing interferences below a detection level of 10 ppb. In spite of the short retention time for ethyl carbamate under the experimental conditions selected, the sensitivity of the method was determined to be about 20 ppb. Attempts to improve the level of sensitivity by further concentration of the sample extract resulted in loss of incurred ethyl carbamate. With the wide variety of sample matrices selected for study it was not surprising to observe that some wines had a greater
Ethyl Carbamate
I
I
B
I
i YL,
Spiked White Wine
1i Figure 1. Gas chromatograms of (A) white wine extract containing less than 10 ppb ethyl carbamate (5.5 mg sample injected on column) and (6) the same white wine extract fortified with 83 ppb ethyl car-
bamate. number of nitrogen-containing compounds. For example, examination of a cocktail sherry containing about 300 ppb of ethyl carbamate (Figure 2 ) revealed the presence of several additional compounds eluting after ethyl carbamate. Quantification of ethyl carbamate was carried out by external standardization using peak heights. Care was exercised to match peak heights to within 10% due to the nonlinearity of the detector a t the concentration levels studied. Several duplicate and triplicate determinations were performed and found to be within 10% utilizing this technique. The residue results obtained for the eighty-nine samples examined are illustrated in Table I. More than two-thirds of the samples examined contained ethyl carbamate. Fortified recovery studies using a few selected matrix types (Table 11) indicated that the methodology selected was ac-
ANALYTICAL CHEMISTRY, VOL. 59, NO. 17, SEPTEMBER 1, 1987
2057
Y .
!
1 . 3 .
4 D
.
90
I
A
y
fj
64
Ethyl Carbamate
mn+ 92
s
,
R
Ethyl
'j
arbamate
Collision Activated Dissociation (Argon)
E
s
fi
B
14
Parent Ion
4
92
I
d Y
Reference Standard
il'
\
Figure 3. Mass spectral data obtained under methane chemical ionization conditions for (A) reference standard of ethyl carbamate; (B) stable labeled isotope (I3C and "N) of ethyl carbamate [M 21; and (C) collision activated dissociation of MH' ( m / z 92) of stable isotope
+
of ethyl carbamate. Figure 2. Gas chromatograms of (A) cocktail sherry extract containing 100 ppb of ethyl carbamate (6.1 mg sample injected on column) and (B) 630 pg of ethyl carbamate reference standard.
Table 11. Fortified Recovery Studies of Ethyl Carbamate in Various Wines and Spirits by Gas Chromatography
generic commodity whisky sherry liquer
wine zinfandel
ethyl carbamate levels, ppb incurred fortified recovery," % 81 48 268 385 42 40
NDb
101 61 249 398 61 61 83
Expressed relative to fortified level. as samde blank. (I
76 84 82 108 108 108 107
* ND, not detected; used
ceptable. The lower recovery results of the whisky samples could be due to the additional concentration steps with petroleum ether and acetone employed to remove the dichloromethane and eliminate the Florisil cleanup (10) during the development of the method. While the whisky samples showed no interferences, wines and liqueurs did exhibit potential interferences. Fortified recovery studies were found to be significantly improved when the cleanup was added due to both a reduction in the time and the number of concentration steps required. Mass Spectrometry. It is desirable and often necessary to confirm the presence of a particular residue by mass spectrometry to support less selective analytical procedures. For example, in the case of the residues of ethyl carbamate in wines and spirits at the low trace levels detected by gas chromatography, it was vitally important to confirm the structural identity of the eluting peak employed in the quantitative analysis. However, the dramatic transition in elution profiles from using an element-sensitive gas chromatographic detector to mass spectrometric detection can cause
some problems. Perhaps the most notable is the impaired ability to detect the compound of interest among so many other possible procedural contaminants and sample background components. Two experimental techniques have assisted in overcoming these problems in a routine fashion: first, the use of chemical ionization (CI) to favor production of protonated molecular ions with minimal fragmentation, and second, the ability to monitor only these ions of interest and attempt to parallel the GC element-sensitive detector. In the case of ethyl carbamate, only two significant ions were observed under methane CI conditions (Figure 3A), the protonated molecular ion at m / z 90 and a fragment ion at m / z 62 (loss of 28 amu). The presence of only two ions at the correct retention time cannot always be considered as providing the ideal criteria for unambiguous proof of identity. However, with the ability to select the protonated molecular ion for collision-activated dissociation (CAD) experiments, daughter ions can be produced that are structurally related to the parent ion, thereby providing a more sophisticated level of confirmation. In the case of ethyl carbamate the CAD daughter spectrum yielded several ions for structural confirmation, m / z 29,44, and 62, including a residual parent ion contribution at m / z 90 (Figures 3C and 4). All extracts determined by GC to contain ethyl carbamate were confirmed in this manner. Samples covering a range of product and a range of concentration of ethyl carbamate were then chosen for the quantification study. With the availability of a stable labeled isotope of ethyl carbamate [M + 21 for addition to sample extracts, the possibility of performing a concurrent quantitative analysis during confirmation was possible (Figure 3B). Spikes were calculated to be as equal as possible to the level previously detected by gas chromatography. The following two approaches to quantification were explored. First, the conventional technique of the ion ratio measurements of the two protonated molecular ions (Le., m / z 90 to 92) was adopted by using simple methane CI mass spectrometry (Table 111). The correlation of results obtained by
ANALYTICAL CHEMISTRY, VOL. 59, NO. 17, SEPTEMBER 1, 1987
2058 r
> 376 ppb
Table 111. Comparison of Quantitative Results Obtained by Gas Chromatography and Mass Spectrometry for Ethyl Carbamate in Wines and Spirits
m/z 29
-.u,
levels of ethyl carbamate, ppb isotope spike GC-HECD level GC/MS MS/MS"
commodity
i ". ,
,
,
,
,
,
,
,
,
m/z , 62 ,
,
/ m/z 90 I
---
) ,
Scan Number 5 5 0 Tlme (mln r e d 4 02
600
TIC 650 446
4 24
700 5 08
BOO
750 5 30
5 52
Flgwe 4. Typical selected ion detection chromatograms obtained for ions expected for ethyl carbamate ( m / z90, 62, 44,and 29) from an extract of golden cream sherry (376 ppb) under argon collision activated dissociation conditions using mlz 90 as the parent ion.
this method agreed with those from conventional packed column gas chromatography using the HECD-N. Use of the megabore column employed in GC/MS rather than the packed column employed for gas chromatography avoided potential interferences from such coextractables. This correlation of quantitative results by two different methods has provided the necessary validation of the single multiresidue approach with an element-sensitive GC detector. Second, the approach to quantitation using daughter ions derived via CAD was explored for the first time. This approach to quantification had the advantage that observed interferences experienced in monitoring the m / z 90 and 92 ions in simple CI mass spectrometry would be removed by monitoring the daughter ion ratio, m / z 62 to m / z 64 by MS/MS (Table 111). Comparison of quantitative data obtained via simple methane ionization and CAD daughter ion ratio measurements in several selected samples covering the range of concentrations encountered revealed that the correlation was not as accurate as anticipated. Several reasons for the lack of accuracy in the CAD results may account for the observed experimental data. Since collision-activated dissociation studies are based on the cross sectional concentration of the parent ion relative to the col-
I , , , , ,
Reference Standards
Reference Standards 200 ppb
white wine zinfandel cider pinot noir cream sherry cream sherry cream sherry cream sherry cream sherry dry sherry dry sherry sherry golden sherry port marsala
230 70 56 70 68 118 120 135 450 53 240 90 385 166 321
230 70 58 70 70 119 123 136 448 53 238 90 385 164 321
217 51 54 64 68 110 123 131 478 66 220 95 376 182 313
111
117 516 70 224 109 175
'Only a selected seven samples covering the concentration range 70-516 ppb were selected for quantification using daughter ions derived from the protonated molecular ions.
lision gas, there is the possibility that at any given point in time (the dwell time for detection of the resulting daughter ion) collisions might result in a lesser or greater abundance of daughter ions than during a previous time frame. Repetitive experiments with the same extract indicated that a relative standard deviation in excess of 15% could be experienced by using area measurements. With the triple quadrupole instrument, experiments were conducted such that only the protonated molecular ion for ethyl carbamate (mlz 90) was permitted to pass into the second quadrupole (Q2) for collision with argon gas. The third quadrupole ($3) was then operated in a multiple ion detection mode for mlz 90 and the major daughter ion at m/z 62. The time frame for a single experiment was 0.4 s. A similar experiment was conducted by using the protonated molecular ion (mlz 92) for the stable labeled isotope and detecting m / z 92 and 64. Therefore, the total time spent in monitoring the four ions of interest in two consecutive experiments was 0.8 s. Since the narrow elution profile of the ethyl carbamate on the megabore column selected for separation was about 10 s, Dr Sherr Extract
Cream Sherry Extract
1-
516 ppb
m/z 62 , ,
m/z 64 ,
,
,
,
m/z 9 0
,
,
,,,,, ,
,
,b
rn/z 92
TIC
Scan N u m b e r
650 Scan N u m b e r
B
C
120
J
A
I
Scan Number
D
Figure 5. Multiple reaction monitoring chromatograms for ethyl carbamate and the stable-labeled isotope illustrating the raw data elution profiles before data accumulation into individual mass chromatograms.
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Anal. Chem. 1987, 59, 2059-2063
only four to five measurements of this type can be made. Figure 5 illustrates the raw data obtained during typical CAD experimental examination of various samples and extracts. It can be clearly observed that, in all cases, only five to six measurements for quantification purposes are obtained for each ion under the experimental protocol. In the case of the total ion chromatogram (TIC) detected through Q3, the number of data points is doubled since each point represents only half of the total experiment. In the case of reference standards (Figure 5A,B), the isotopically labeled ethyl carbamate was spiked at a slightly higher concentration than the unlabeled compound and the TIC reflected that increased level via higher alternate scan intensities that represented the total intensity observed for m / z 92 and 64. For the incurred residues (Figure 5C,D) the stable isotope was spiked at the same concentration level detected by gas chromatography with HECD-N and hence the TIC reflected an equality between the intensity of combined m / z 90 + 62 and combined m / z 92 64. Closer examination of the raw data illustrated in Figure 5A strongly suggests that the daughter ion ratio ( m / z 62:mlz 64) can be effectively used to a concentration level as low as 50 ppb of ethyl carbamate. However, the individual ratio measurements derived from each single experiment conducted within the 0.8-s-scan time frame have indicated a deviation from the expected results. It could be argued that what happens to m / z 90 should also happen to m / z 92 from the stable isotope. It should be remembered, however, that under CAD the measurement of the intensity of m / z 62 takes place about 0.5 s prior to measurement of the corresponding m / z 64. Since CAD experiments are concentration dependent, the possibility exists of a flaw in reliable quantification via isotope dilution techniques at low concentrations. While the observed
+
raw data (Figure 5) clearly indicated an elution profile under CAD, there are data points within those profiles that are of less intensity than those predicted for a smooth Gaussian shape. These discrepancies are evidence to support the notion that optimization of CAD experimental conditions (collision gas pressure and collision energy) plays an important role in ensuring precision and accuracy in quantitative results at low trace levels. In the present study, however, the elution profiles obtained by use of the megabore column were necessary to ensure sensitivity of detection to below the 30 ppb level. The exploration of daughter ions for quantitative analysis has revealed several interesting experimental factors that need to be closely monitored to ensure acceptable results. This abiality to rely on daughter ions (derived from protonated molecular ions) for quantitative purposes is an advancement in trace analysis via elimination of interfering ions observed in single-stage instruments. Registry No. Ethyl carbamate, 51-79-6. LITERATURE CITED (1) Fed. Regist. 1972, 37(149), 15426. (2) Walker, G.; Winterlin, W.; Fouda, H.; Seiber, J. J. Agric. FoodChem. 1974, 22, 944. (3) Ough, C. S.J . Agric. FoodChem. 1976, 24, 323. (4) Ough, C. S. J. Agric. FoodChem. 1976, 2 4 , 328. (5) Ough, C. S. Chem. Eng. News 1987, 6 5 , 19. (6) Joe, F. L.; Kline, D. A.; Miletta, E. L.; Roach, J. A,; Roseboro, E. L.; Fario, T. J. Assoc. Off. Anal. Chem. 1977, 60, 509. (7) Bailey, R.; North, D.; Myatt, D. J. Chromatogr. 1986, 369, 193. (8) Dennis, M. J.; Howarth, Massey, R. C.; Parker, I. J. Chromatogr. 1986, 369, 199. (9) Bertrand, A,; Triquet-Pissard, R. Conn. Vigne V;n. 1986, 2 0 , 131. (10) Luke, M. A.; Froberg, J. E.; Doose, G. M.; Masumoto, H. T. J. Assoc. Off. Anal. Chem. 1981, 64, 1107.
RECEIVED for review October 7,1986. Accepted April 2,1987.
Spin-Deposited Submicrometer Films of Organic Molecules for Secondary Ion Mass Spectrometry Studies G . Siive,* P. Hfikansson, and B. U. R. Sundqvist Department of Radiation Sciences, Uppsala University, Box 535, S-751 21 Uppsala, Sweden U. Jiinsson' and G . Olofsson
Laboratory of Applied Physics, National Defence Research Institute, Department 4, S-901 82 U m e i , Sweden M. Malmquist' Division of Radiation Biology, National Defence Research Institute, Department 4, S-901 82 U m e i , Sweden Spin deposition is presented as a new sample preparation technique for producing submicrometer films of labile moiecules for mechanism studies of plasma desorption mass spectrometry (PDMS). Films of valine, Giy-Ala-Leu, and porcine insulin 1-400 nm thick were spindeposlted on silicon substrates with hydrophilic surfaces. PDYS, ellipsometry, and scanning electron microscopy showed that for film thicknesses greater than about 30 nm the films had a constant refractive index and were of fiat topography with a loose organic molecule air solvent structure that decomposes upon exposure to high humidity. The new technique has made it possible to study the film thickness dependence of secondary ion yields. Present address: Pharmacia Bio-sensor AB, S-75182 Uppsala, Sweden.
The preparation of submicrometer solid films of involatile and thermally labile organic molecules is an important step in secondary ion mass spectrometry (SIMS) (1) and the high-energy primary ion version of SIMS, e.g. plasma desorption mass spectrometry (PDMS) (2,3).One question of fundamental interest in the study of these techniques is how the organic molecule desorption yields are correlated with sample film thickness. Samples of biomolecules in PDMS studies are usually prepared for analysis by using the electrospray technique (4).This method, however, gives clusterlike deposits of micrometer dimensions of the molecules on the surface, thereby making film thickness determinations unreliable. We present here a spin-deposition technique whereby it is possible to obtain homogeneous films of organic molecules in the submicrometer thickness range. Spin deposition is a well-known technique for processing of semiconductors where
0003-2700/87/0359-2059$01.50/00 1987 American Chemical Society
