975
Anal. Cham. 1988, 60, 975-978
is obtained through a combination of solution preconcentration
and optimization of sample introduction rate into the ICP nebulizer. Quantification was achieved by a combination of stable isotope dilution analysis and comparison to an external standard. This combination allowed us to determine a total of 37 elements simultaneously. The use of isotope dilution gives more reliable results since partial loss after equilibration would not alter the concentration as calculated using the isotope dilution equation (eq 1). The added spike also serves as an internal monitor of recovery since complete loss due to sorption or volatility can be detected by the absence of the spike isotope. The ICP-MS instrument was designed for high sample throughput of dilute solutions and was ideal for this application. Using the mass scan mode, we have essentially complete elemental coverage and it is possible to expand the number of elements determined by both isotope dilution and the use of external standards in future surveys. Registry No. Pb, 7439-92-1; Tl, 7440-28-0; Pt, 7440-06-4; W, 7440-33-7; Ce, 7440-45-1; Nd, 7440-00-8; Ba, 7440-39-3; Te, 13494-80-9; Sb, 7440-36-0; In, 7440-74-6; Cd, 7440-43-9; Pd,
7440-05-3; Mo, 7439-98-7; Zr, 7440-67-7; Sr, 7440-24-6; Rb, 7440-17-7; Ge, 7440-56-4; Ga, 7440-55-3; Zn, 7440-66-6; Cu, 7440-50-8; Ni, 7440-02-0; Fe, 7439-89-6; Cr, 7440-47-3; Ti, 744032-6; Mg, 7439-95-4; U, 7440-61-1; Sn, 7440-31-5; As, 7440-38-2; Co, 7440-48-4; Mn, 7439-96-5; V, 7440-62-2; Ca, 7440-70-2; Al, 7429-90-5; Na, 7440-23-5; B, 7440-42-8; Be, 7440-41-7; Li, 7439-93-2; H20, 7732-18-5; HC1, 7647-01-0; HN03, 7697-37-2; HC104, 760190-3; HF, 7664-39-3; H2S04, 7664-93-9.
LITERATURE CITED (1) Kuehner, E. C.; Alvarez, R.; Paulsen, P. J.; Murphy, T. J. Anal. Cham. 1972, 44, 2050-2056. (2) Moody, J. R.; Beary, E. S. Talanta 1982, 29, 1003-1010. (3) Federal Standard 209b, Government Services Administration, Boston, MA, 1973. (4) Longerlch, . P.; Strong, D. F.; Kantipuly, C. J. Can. J. Spectrosc. 1986, 31, 111-121.
Received for review September 14,1987. Accepted January 15, 1988. Certain commercial equipment, instruments, or materials are identified in this report to specify adequately the experimental procedure. Such identification does not imply recommendation or endorsement by the National Bureau of Standards, nor does it imply that the materials or equipment identified are necessarily the best for the purpose.
Quantitation of Ethyl Carbamate in Whiskey, Sherry, Port, and Wine by Gas Chromatography/Tandem Mass Spectrometry Using a Triple Quadrupole Mass Spectrometer William C. Brumley,*1 Benjamin J. Canas, Gracia A. Perfetti, Magdi M. Mossoba, James A. Sphon, and Paul E. Corneliussen Division of Contaminants Chemistry and Division of Food Chemistry and Technology, Food and Drug Administration, Washington, D.C. 20204
A procedure was developed for the quantitation of ethyl carbamate (EC) In whiskey, sherry, port, and wine using gas
chromatography/mass spectrometry/mass spectrometry (GC/MS/MS) on a triple quadrupole Instrument. An extraction/lsolatlon procedure yielded an ethyl acetate solution of EC, and an aliquot of this solution was Injected onto a bonded Carbowax capillary GC column for separation. The MS
technique used Isobutane chemical Ionization to produce an (M + H)+ Ion for EC and for the stable Isotope-labeled EC Internal standard. Quantitation was based on the daughter Ions of m/z 62 of EC and m/z 64 of labeled EC. Levels of EC between 3 and 330 ppb were found In a number of extracts and results were compared to GC/matrlx Isolation Fourier transform Infrared and GC/nltrogen/thermal energy analyzer quantitations as Independent determinative techniques. Recoveries of EC averaged >90% with a limit of quantitation of 1 ppb.
Ethyl carbamate (EC) or urethane is a carcinogen (1,2) that of continuing interest in carcinogenicity studies (3,4). Since EC can occur naturally in fermented foods and beverages (5, is
1 Present address: Environmental Protection Agency, P.O. Box 93478, Las Vegas, NV 89193.
6), its presence in alcoholic beverages is a public health problem. Recent reports from Health and Welfare Canada (7) indicated that levels found in some alcoholic beverages exceeded Canadian guidelines.
A rapid and reliable procedure for quantitating EC in various beverages is needed. A gas chromatographic (GC) determination of EC in wines was reported that used electron ionization (El) mass spectrometry (MS) for confirmation of identity (8). However, the method was relatively time-consuming and required substantial cleanup prior to GC/MS confirmation of EC in the extracts. Lofroth and Gejvall (9) reported a radioisotopic dilution technique. Walker et al. (10) and Ough (5) based their quantitations on a Coulsen electrolytic conductivity detector with confirmation by GC/MS. A recent report (11) described the use of two-dimensional capillary GC with a heart cut from the first column transferred to a second column and flame ionization detection. While this paper was undergoing review, two additional papers concerned with determination of EC by mass spectrometry were published (12, 13). Cairns et al. (12) used GC/MS under methane Cl conditions, with quantitations based on m/z 90 and 92 of an internal standard, which was, in fact, custom synthesized for the present authors. Cairns et al. (12) reported problems with accuracy, precision, and adequate sampling of the GC peak. The paper of Lau et al. (13) determined EC by high-resolution mass spectrometry on
This article not subject to U.S. Copyright. Published 1988 by the American Chemical Society
976
·
ANALYTICAL CHEMISTRY, VOL. 60, NO. 10, MAY 15, 1988
the basis of m/z 62 of EC. The isolation procedure was that adopted in this paper so that a direct comparison of MS procedures might be made. This paper presents a method for quantitating EC in alcoholic beverages using a direct capillary GC/MS/MS introduction of extracts and a stable isotope-labeled internal standard (ECL). The quantitations are accurate in spite of the presence of coextractives, and the precision compares favorably with the usual GC/MS approaches and with other determinative procedures using GC. Levels of EC are reported for a number of extracts and compared to findings obtained by using GC/matrix isolation (MI) Fourier transform infrared
9 HjNCOC¡H5 62
18
(FT-IR) and GC/nitrogen/thermal energy analyzer (N/TEA) results as independent quantitative approaches.
EXPERIMENTAL
¡
:
,
i
74
SECTION
Chemicals. All solvents were distilled in glass (Burdick & Jackson, Muskegon, MI). [^C^^Nj-EC (>99+ atom % each) was custom synthesized by MSD Isotopes, Montreal, Canada, for the Food and Drug Administration’s Division of Contaminants Chemistry, and today resides with that division. Chemical purity was checked by GC/MS. Isolation/Concentration. The procedure of Conacher et al. (14) was followed: 50 g of beverage was extracted with methylene chloride, the extract was dried with sodium sulfate and concentrated to 5 mL in ethyl acetate. At the 100 ppb level, a concentration of 1 ng of EC/µ would result. At the 1 ppb level, the sample was concentrated to 300 µ , resulting in a solution of 300 pg of EC/µ , of which 3 µ was injected. GC/MS/MS. A Finnigan MAT TSQ-46 instrument interfaced to an INCOS 2300 data system with TSQ software (Revision C)
The collision activated decomposition was monitored under experimental control of the data system. The instrument was sequentially set to monitor m/z 90,62, and 44 when Ql passed m/z 90 of EC, and m/z 92, 64, and 46 when Ql passed m/z 92 of ECL. Ion dwell times of 0.05 s per ion resulted in a total cycle time of about 0.38 s. After data acquisition, the original file was split into two files holding the decomposition of m/z 90 (EC) and m/z 92 (ECL), respectively. Quantitative results were based on the relative areas of m/z 62 (EC) and 64 (ECL) and the spiking level. The response factor for m/z 62 versus 64 was unity throughout the levels encountered in this work. Standard procedures suggest spiking samples at levels close to those expected for the measured quantity. The capillary column was 60-m SP-10 (bonded, Carbowax, Supelco, Bellefonte, PA), 0.25-µ film thickness, 0.25 mm i.d. Temperature was programmed at 40-150 °C at 12.5 °C/min and then to 250 °C at 20 °C/min; 180 °C injector; 220 °C interface/transfer line; 38 cm/s helium at 40 °C. Chemical ionization (Cl) isobutane pressure was 0.50 Torr: source temperature, 140 °C; 0.35 mA emission at 70 eV; 2 mTorr argon collision gas at -10 eV energy, pre-amp, multiplier, and conversion dynode at 10"8 A/V, -1400 V, and -5 kV, respectively. GC/MI/FT-IR. An MI/Cryolect/Sirius 100 Mattson FT-IR spectrometer was interfaced to a Model 5890 Hewlett-Packard gas chromatograph with a DBWAX-30W (J&W Scientific, Inc.) capillary column (30 m X 0.32 mm i.d., 0.25-µ film). The carrier gas was helium containing 1.5% argon. The effluent was split, with 20% going to a flame ionization detector, while the remaining 80% was sprayed onto a slowly rotating collector disk placed in a vacuum chamber and held at 11 K. MI FT-IR measurements were carried out after EC and ECL were deposited on the collector disk. Assignments of characteristic frequencies of EC and ECL observed under MI/FT-IR were based in part on published work (15-18). Quantitation was based on observed peak heights with base-line correction and the fortification level of ECL. Peak heights were derived from the intense and sharp bands at 1325 and 1298 cm"1 in EC and ECL that were assigned as the asymmetric C-O-C(O) stretching mode. Details of the GC/MI/FT-IR procedures will be published separately (19). GC/N/TEA. A Hewlett-Packard Model 5710A gas chromatograph interfaced to an N/TEA with 610R nitrogen converter was used. The column was 9 ft (2.7 m) X 4 mm i.d. glass packed with 10% EGA on 100/120 mesh Chromosorb-WHP: 200 °C injector; 100-130 °C at 1 °C/min oven; GC interface, 225 °C; pyrolyzer furnace, 825 °C; vacuum, 1.3-1.4 Torr; carrier gas, argon was used.
Figure 1. El mass spectra, 70 eV, source 140 °C of (A) EC and (B) ECL.
at 40 mL/min; cold trap, liquid nitrogen/pentane slush (-130 6C).
RESULTS AND DISCUSSION El Mass Spectra. The El mass spectrum of EC (Figure 1A) consists of the molecular ion at m/z 89 and principal fragment ions at m/z 74 (M CH3")+, 62 (M C2H3')+, 45 -
-
(C2H50)+, 44 (NH2CO)+, 31, 29, 27, and 18 (NH4)+. Suggestions, partly based on the mass spectrum of the ECL in
Figure IB, are made for structural assignments to certain of the fragment ions. The principal mass shifts observed between the spectra of EC and ECL are m/z 89 to 91, 62 to 64, 44 to 46, and 18 to 19. The loss of 27 u to produce m/z 62 in the spectrum of EC is verified as C2H3" and must involve, a double hydrogen rearrangement. The ion at m/z 44 must consist primarily of NH2CO+ in view of the mass shift to m/z 46 observed in the spectrum of ECL. The ion at m/z 45 has the same relative abundance in both spectra and presumably represents (C2H50)+. The ion at m/z 18 represents NH4+, as verified by the shift to m/z 19 (15N). These results are entirely consistent with the scheme proposed by Lau et al. (13) and serve to verify the chemical identity of ECL. Collisional activation (CA) mass spectra of m/z 89, 74, and 62 reveal the following relationships. The molecular ion at m/z 89 fragments primarily to m/z 62 and 45. The ion at m/z 74 fragments to m/z 44, presumably by loss of CH20, and m/z 62 also leads to m/z 44, presumably by loss of H20. MS/MS Quantitation. To confirm the presence of and to quantitate EC in extracts of various beverages, some enhancement of the ion indicating molecular weight was deemed appropriate. The Cl (CH4) mass spectrum of EC consists predominately of the (M + H)+ ion at m/z 90 and m/z 62.
ANALYTICAL CHEMISTRY, VOL. 60, NO. 10, MAY 15, 1988
·
977
Table I. EC (ppb) in Whiskey, Sherry, Port, and Wine beverage
N/TEA
MI/FT-IR
60.0 330 166
123 139 83 323 163
117 126 68 389 183
47.0 38.7 232 39.6
47 32 242 50
31.0 56.0 22.9 13.6
33 60
8.3 7.7 3.1 3.3 4.4 7.0
9 8 3 4 9
MS/MS
whiskey 1
2 3
4 5
116 108
sherry 1
2 3
4
port 1
2 3
4
2 3
4 5 6
5:40
1300
1350
5:50
1950
5:53
6:09
3000
SCAN
f:l?
TIME
22 16
wine 1
1306
·· j
7
Additional selectivity and specificity were necessary with the particular isolation procedure chosen, and two MS refinements were added. First, isobutane was used as the reagent gas to provide greater ionization selectivity for EC compared to matrix coextractives and to provide all of the ion current in the (M + H)+ ion. Second, the (M + H)+ ion of EC was subjected to CA with ions at m/z 90, 62, and 44 monitored. ECL was added as an internal standard before analytical workup and a parallel CA experiment was followed (m/z 92 yielding ions at 92, 64, and 46). Quantitation was based on the relative areas of m/z 62 (EC) and 64 (ECL) based on the known fortification of ECL. Specificity, accuracy, and precision, and applicability to a variety of matrices are of concern with this approach. Table I reports a number of quantitations by GC/MS/MS and provides comparisons of GC/MI/FT-IR and GC/N/TEA data. Both of these latter instrumental approaches have high specificity. Good agreement was reached among the different techniques. The accuracy of the GC/MS/MS quantitations was judged by both incurred EC residues and spiked beverages. The matrices consisted of whiskey, sherry, port, and wine, thereby providing evidence that the GC/MS/MS procedure is applicable to extracts from a variety of matrices. Generally, the GC/MS/MS value is the lowest in Table I, and this presumably results from its high specificity and lack of interferences. The decomposition of m/z 90 -*· 62 (92 -» 64) appears to be absolutely specific for EC (ECL) in the beverages using the isolation procedure. Figure 2 is a chromatogram of wine quantitated at 8 ppb EC. The GC/MS/MS approach very likely could be applied to extracts from other matrices without
modification. The precision of repeat analysis of the
same solution over the same day and over several days was determined. Whiskey, analyzed three times over several days, gave results of 38.5, 38.7, and 38.6 ppb EC. Four sherry solutions, analyzed two or three times per day, gave results of 47.0 and 47.6; 116 and 118; 108,109, and 107; and 60.0 and 63.3 ppb. The technique is highly reproducible and comparable to usual GC/MS quantitations (20). The precision of determinations for replicate injections was ±1%. Variability of the extraction/ isolation procedure was estimated from four whiskey samples that were analyzed in duplicate by GC/N/TEA. An average variation of 2.9% was obtained with an individual high of 6%
j
Figure 2. Ion chromatograms for (A) m/z 62 of EC and (B) m/z 64 of ECL in wine quantitated at 8 ppb.
Table II. Recovery of EC in Whiskey, Sherry, Port, and Wine Based on GC/N/TEA Determination beverage
spike, ppb
EC + spike, ppb
recovery,0 %
whiskey 1
2
4
100 200 500
212 315 823
95 87 99
77 154 154 77
121 167
428 132
97 85 121 104
68 105 59 49
94 112 92 86
56 62 57 51
96 104 108 90 84 100
sherry 1
2 3 4
port 1
2 3
4
38.5 38.5 38.5 38.5
wine 1
2 3
4 5
6 “
50 50 50 50 50 50
51 57
Calculated using data in Table I.
for EC levels from 80 to 343 ppb. Recoveries using the isolation procedure averaged 97% (Table II) and were verified in all four matrices by using GC/N/TEA determination. These data verify that the extraction quantitatively recovers EC from the beverage. Quantitations by MS/MS are automatically corrected for recoveries by beverage spiking with ECL before extraction. The only variability not corrected by the MS/MS procedure is that introduced by the spiking procedure. Reagent blanks
978
Anal. Chem. 1988, 60. 978-982
had
no measurable EC (
