Anal. Chem.
m a , 6 0 , 945-948
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CORRESPONDENCE Application of Gas Chromatography/Matrix Isolation/Fourier Transform Infrared Spectrometry to the Determination of Ethyl Carbamate in Alcoholic Beverages and Foods Sir: Confirmation of analyte identity a t trace levels has been the eminent domain of mass spectrometry (MS). While infrared (IR)spectrometry offers (at least) equally unequivocal and complementary confirmatory information, its limits of sensitivity in the past precluded most such applications. The recent commercial availability of gas chromatography/matrix isolation/Fourier transform infrared (GC/MI/FT-IR) spectrometer systems (I) has made possible the IR spectral determination of subnanogram quantities of analytes (2). This increase in sensitivity results from the capability for depositing samples on microscopic surface areas and signal averaging of spectra for extended periods of time, as well as the extremely sharp band shapes due to the absence of rotational spectral contributions. It is noted that a sensitivity of 2.4 ng for nitrobenzene has been achieved by capillary GC/FT-IR microspectrometry at subambient temperature (3). We have used GC/MI/FT-IR to confirm and quantitate ethyl carbamate (EC) in alcoholic beverages and foods at part-per-billion levels using isotopicdy labeled EC (ECL) as an internal standard. EC (urethane) is a natural product of many fermentation processes and may be present in alcoholic beverages and foods. Since EC is an animal carcinogen (4, 5) methods of production must be controlled to minimize the levels in foods and alcoholic beverages. A number of methods for extracting and quantitating EC using different techniques have been reported (6-10).Monitoring at the levels of interest has in many cases been difficult because of the presence of materials that interfere in the determinative technique. Since some difficulties with interferences were encountered in preliminary MS, the application of GC/MI/FT-IR methods was also investigated. EXPERIMENTAL SECTION EC (urethane, 99+%) was obtained from Aldrich Chemical Co., Inc., Milwaukee, WI. [13C1,15N]-EC(99+ atom % each) was custom synthesized by MSD Isotopes, Montreal, Canada. All solvents were purchased from American Burdick & Jackson, Muskegon, MI. The extraction/cleanup procedure of Conacher et al. (10) was used for whiskey, bread, and yogurt, while the method of Canas et al. (11)was used for beer and soy sauce. Blue cheese was first vacuum distilled and then extracted by the Conacher et al. (10) method. A Mattson (Madison, WI) Sirius 100 FT-IR spectrometer equipped with an MI Cryolect operating at 12 K that was interfaced to a Model 5890 Hewlett-Packard (Sunnydale, CA) gas chromatograph was used. For the GC separation a DBWAX-BOW (J&W Scientific, Inc., Folsum, CA) capillary column (30 m,0.32 mm i.d., 0.25-pm film) was held at 50 "C for 5 min after injection and then ramped at 5 OC/min to a final temperature of 150 O C . The injection mode was splitless. The injector and flame ionization detector (FID) temperatures were 200 and 250 "C, respectively. Spectra were recorded at 4 cm-' resolution and the scan time for 300 scans was 2 min, 43 8. A Finnigan MAT TSQ-46 spectrometer interfaced to an INCOS 2300 data system with TSQ software (revision C) was used for the MS determinations. The experiment monitored the collision activated decomposition of m / z 90 and 92 with multiple ion detection at m / z 90, 62, and 44 for EC and m / z 92,64, and 46
for ECL. Quantitation was based on the ratio of the areas of m/z 62 and 64 and the spiked level of ECL. Detaih of the GC/MS/MS experiment will be published separately (12).
RESULTS AND DISCUSSION FT-IR measurements were carried out after EC had been deposited on the collector disk. Sharp MI/FT-IR bands were observed for EC (Figures 1and 2A). Spectra of EC observed at subhanogram levels still allowed positive identification using the five strongest bands: the two strong bands a t 1763 and 1326 cm-' and the three medium intensity bands at 1583,1373, and 1093 cm-' (Figure 1). ECL was used as an internal standard (Figure 2B). The FT-IRspectra for all of the extracts were identical with those obtained for authentic standards of EC, confirming the identity of EC in the products studied (Figures 2C and 3B). The retention time far EC was 20.08 min (Figure 3A) when a DBWAX-BOW column was used. Initial trials with different conditions and capillary columns (Analabs GB-20-m, 0.32-mm id., 0.25-pm film, 15-m, and 25m) gave shorter retention times for EC. However, for most products, other interfering compounds in the extracts coeluted with EC in the initial trials. The characteristic frequencies of EC and ECL observed by MI/FT-IR as well as those found in solution (carbon disulfide, CSz) (Figure 3C) and the solid state (KBr) (Figure 3D) are given in Table I. The characteristic vibration modes are associated with the -OCONHzgrouping and are sensitive to the physical state and environment of the molecule. The N-H and the carbonyl stretching frequencies for EC listed in Table I show that a shift to higher frequencies is obtained as the phase changes from solid (KBr) to either liquid (CSz)or MI, consistent with the loss of intermolecular hydrogen bonding. The relative shifta of the N-H stretching vibrations are greater than those of the C=O stretch frequencies. The two weak bands around 3500 cm-' are assigned to the asymmetric and symmetric stretching vibrations expected for an NHz group attached to a carbonyl (13). The observed C = O stretch band for EC falls in the same region as ester carbonyl bands, which are higher than those expected for the structurally related 4 0 - N H - amide group (14). This is because the C-0 bond is less polar in carbamates than in amides due t~ the additional delocalization over the ester oxygen atom. Although only one C=O band was found for EC (Table I), two closely spaced bands at 1709 and 1719 cm-' have been observed for the MI spectrum of ECL (Figure 2B). Absorptions at 1786 and 1766 cm-' have also been reported for methyl carbamate (MC) in the vapor phase (15), which indicates that MC and EC may exist as two conformers. However, for MC some additional contribution to the C=O stretch band from the OCN bend has been suggested (16). In a study of 32 carbamates, five N-methyl- or N-phenylcarbamates exhibited two bands in the 1700-1740 cm-' range in KBr solid phases (17). An MI/FT-IR investigation of carbamates with different substituents may clarify the origin of the bands in the carbonyl region.
This article not subject to U.S. Copyright. Published 1988 by the American Chemical Soclety
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ANALYTICAL CHEMISTRY, VOL. 60, NO. 9, MAY 1, 1988
Table I. Assignment of Observed Frequencies (cm-I) for E t h y l Carbamate
matrix isolation
carbon disulfide
KBr
EC
ECL
EC
ECL
EC
ECL
N-H stretch asym sYm CH, asym stretch C=O stretch
3579 (w)4 3461 (w) 3002 (w) 1763 (s)
3547 (w) 3432 (w) 2979 (w) 1740 (9)
3537 (w) 3427 (w) 2979 (w) 1694 (9)
3425 (m) 3334 (w) 2987 (w) 1692 (9)
3415 (m) 3310 (w) 2991 (w) 1643 (9)
NH2 in-plane def
1583 (m)
3569 (w) 3456 (w) 3003 (w) 1719 (m) 1709 (m) 1574 (m)
1616 (m)
1607 (m)
1489 (vw) 1378 (m)
1488 (vw) 1374 (m)
1372 (m)
1368 (m)
1487 (w) 1382 (m)
1486 (w) 1377 (m)
1326 (5) 1093 (m)
1298 (9) 1089 (m)
1324 (s) 1069 (m)
1297 (s) 1065 (m)
1343 (m) 1080 (m)
1330 (m) 1074 (m)
CH3 bend =Ym SYm C-0-CO stretch asym sYm
Key: w, weak; m, medium; s, strong. ETHYL CaRBAMA7E
I O 9npl
A
c L - - -
A
c 035
0
I
H,N 2 033
-
c
-
0.0015
5
a
a
0
CH,
CH,
ETHYL CARBAMATE
0
A b
C
025
3 023
1
0 0 ?3C ?3C
i,
_--1326 CY-!
0 001C
I
c
0 0005
0 0000 L--
7
dOCC
3500
-
7
3000
af:t
:
I
2500
2000
I5C0
2 :
1
H,N H,N
0
C C
0 0
CH, CH,
I
_ , ' -,/--,
CH, CH,
-,A
/ _ I ' C I I A N D N15 LABELED ETHYL CARBAMATE
!293
EM-1
1000
WaYe"""Der E T H Y L CAiiBAMATE
10 6ngl
:,
3:p
i
- 3"E
I
CY-1
A
c
0 0008 0
6
I
LIQUOR SAMPLE N4C HAngl
0 0006
-/
~
C
0 0004
1298 CM-1 C-13 6 N-15 L A 8 E L E 3 E T H Y L CARBAMATE :NTERNAL STANOARO
1 I
0 0002
1
0 0000
1328 C H - 1 hAT:VE
-0 0002
4000
3500
3000
2500
2000
1500
1000
WaVeiUmbei
Flgure 1. Subnanogram FT-IR spectra of EC standard acquired by coadding 300 scans (4 cm-'): (A) 0.9 ng; (6)0.6 ng.
The NH2 group also gave rise to the in-plane scissors deformation mode near 1600 cm-l. Absorptions between 1300 and lo00 cm-l are the result of the asymmetric and symmetric C-0-CO stretch vibrations of the ester function. The C-N stretching mode in carbamates is expected at a higher frequency than a normal C-N stretch due to the partial double bond character of C-N in carbamates (17). The procedure for quantitative determination of EC in argon matrices was to apply a base-line correction and then observe the peak heights for the sharp and intense bands at 1326 and 1298 cm-' for EC and ECL, respectively (Figure 2). The observed band intensities in milliabsorbance units were plotted against the quantity of extract injected into the gas
I 4C3L
I
3503
30C0
2530
20C0
15CC
!003
*avenunter
Flgure 2. MI/FT-IR spectra of 300 scans of the test sample interferograms observed for (A) EC standard, (6)ECL standard, and (C) whiskey that contained 1.4 ng of EC. ECL, which coeiutes with EC, was used as internal standard. The sharp bands at 1326 and 1298 cm-' for EC and ECL, respectively, were used for quantitation. The amount of EC relative to that of ECL is different for different test samples.
chromatograph, which is postcolumn split between the Cryolect for IR detection and the FID. Calibrations plots have been generated for injected amounts of EC and ECL in the range 0.5-8.0 ng. The variability data in terms of standard deviation and percent relative standard deviation are given in Table 11. The standard deviation generally increases with the level, which indicates that precision of repeat analysis is
ANALYTICAL CHEMISTRY, VOL. 60, NO. 9, MAY 1, 1988
947
Table 11. Variability of FT-IR Quantitation of EC and ECL
compound
amt injected, ng
replicate value absorbance, mA
SD
% RSD
EC
0.5 1.0 2.0 6.0 8.0
1.1 2.2 4.3 12.4 18.0
1.1 2.1 4.2 12.0 16.0
1.0 2.4 3.7 13.9 17.2
1.1 1.8 3.5
0.06 0.3 0.4 1.0 1.0
5.5 11.8 9.8 7.9 5.9
ECL
0.5 1.0 2.0 6.0 8.0
1.0 1.8 4.2 12.2 16.3
1.0 2.0 4.0 12.4 15.9
1.1 1.9 3.7 11.5 16.2
1.0
0.05 0.1 0.3 0.5 0.2
4.9 5.3 6.3 3.9 1.3
W H I S K E Y E X T R A C T S A M P L E SC
A
Table 111. Recovery Data of EC in Whiskey Based on FT-IR Determination
I
recovery,
EC corr loo%,
PPb
ECL found, ppb
70
ppb
112 110 64.0 346 168
96.0 173 96.0 446 184
96.0 86.5 96.0 89.2 92.0
117 126 68.0 389 183
ECL spike,
found,
EC
sample
ppb
2c 3c 4c 5c 6C
100 200 100 500 200
~~
Table IV. EC Levels (ppb) in Whiskey
sample
GC/MI/FT-IR
GC/MS/MS
ratio IR/MS
2c
117 126 68.0 389 183
116 108 60.0 330 166
1.00 1.17 1.13 1.18 1.10
0 015
3c 4c 5c 6C
0 010
0 005
c
0 . 1 4 1
ETHYL CARBAMATUCSZ
0
0.10
0.06
0.04
D A
l1 66
-
1 4000
ETHYL CARBAMATUKBr
3500
3000
2500
2000
1500
1000
Ya"e""mQeP
Flgure 3. (A) Truncated FID chromatogram (retention time 20.08min) and (B) FT-IR spectrum of whiskey extract that contained 7.5 ng of EC. FT-IR EC spectra observed in (C)CS, and (D) KBr phases are shown for comparison.
probably level dependent. The linear regression equations were y = -0.14 (f0.42) + 2.15 (f0.10)x for EC and y = -0.06 (f0.19) + 2.02 (f0.04)x for ECL. The lower and upper confidence intervals for the intercept were -0.56 and +0.28 for EC and -0.25 and +0.12 for ECL, which encompassed zero a t the 95% two-tail confidence level; therefore there is no indication of systematic bias. Regression correlation coefficients of r = 0.996 and 0.999 for EC and ECL, respectively, were obtained. The absolute minimum and maximum percent deviations of the predicted from the observed absorbance values were 1.18 and 18.67 for EC and 0.56 and 8.79 for ECL, respectively. Spike levels and recovery data of EC in five whiskey samples are given in Table 111. The EC levels obtained by GC/ MI/FT-IR are in agreement with those found by GC/MS/MS (Table IV). Linear regression analysis was carried out on EC quantitation of these five samples by GC/MI/FT-IR axis) relative to GC/MS/MS ( x axis). The linear regression equation of the fitted line was y = -10.83 (f26.36) +1.20 (f0.15)~and the 95% confidence bounds for t h e y intercept were -37.19 and +15.52 which encompassed zero, while those for the slope were +1.06 and f1.35. Since the slope intervals did not encompass the theoretical slope of 1.00, there was a small positive amount of concentration-dependent bias in the GC/MI/FT-IR quantitation, besides random errors. This agreement between IR and MS data supports the quantitation based on GC/MI/FT-IR. Preliminary results from the products analyzed are consistent with the presence of only negligible levels (not detected or < l o ppb) of EC in foods containing a fermentation product or resulting from fermentation processes, including cheese, bread, yogurt, and soy sauce. The operation of this system, particularly for quantitative measurements, is neither simple nor routine. As more refined
Anal. Chem. 1988, 60, 948-950
948
protocols for optical alignment and focusing are developed, significant improvements in ease of operation of the system are expected. The ultimate functional sensitivity of this technique has yet to be reached. This work demonstrates the applicability of GC/MI/FT-IR spectrometry in trace residue determination. Other applications, including isomer identification, where IR spectra have considerable advantages over mass spectra, are in progress. ACKNOWLEDGMENT The authors thank B. J. Canas for preparing EC extracts and R. H. Albert for statistical analysis. Registry No. EC, 51-79-6.
Walker, G.; Winterlin, W.; Fouda, H.; Seiber, J. J. Agrlc. F w d Chem. 1974, 22, 944-947. Ough, C. S . J. Agric. FoodChem. 1978, 2 4 , 323-328. Joe, F. L.; Kllne, D. A.; Miletta, E. M.; Roach, J. A. G.; Roseboro, E. L.; Fazio, T. J. Assoc. OW. Anal. Chem. 1977, 6 0 , 509-516. Conacher, H. B. S.; Page, B. D.; Lau, B. P.-Y.; Lawrence, J. F.: Bailey, R.: Colwav. P.: Hanchav. ,. J.-P.:. Mori.. B. J. Assoc. Off. Anal. Chem. 1087, 70,. 749-751. Canas, B. J.; Havery, D. C.; Joe, F. L. J. Assoc. Off. Anal. Chem., in press. Brumley, W. C.; Canas, 8. J.; Perfetti, G. A,; Mossoba, M. M.; Sphon, J. A.; Corneliussen, P. A. Anal. Chem., in press. Bellamy, L. J.; Williams, R. L. Spectrochim. Acta 1957, 9 , 341-345. Pinchas, S.;Ben Ishai, D. J. Chem. Soc. 1960, 676-679. Cutmore, E. A.; Hallam, H. E. Spectrochim. Acta, ParfA 1969, 2 5 A , 1767-1784. Carter, J. C.; Devia, J. E. Spectrochim. Acta, Part A 1973, 2 9 A , 623-632. Chen, J.-Y. T.; Benson, W. R. J. Assoc. Off. Anal. Chem. 1986, 4 9 , 412-452.
LITERATURE CITED Bourne, S.;Reedy, G. T.; Coffey, P. J.; Mattson, D. A m . Lab. (Fairfield, Conn.) 1984, 16, 90-101. Reedy, G. T.; Ettlnger. D. G.; Schenider, J. F. Anal. Chem. 1985, 5 7 , 1602-1609. Fuoco, R.; Shafer, K. H.; Griffiths, P. R. Anal. Chem 1986, 5 8 , 3250-3254. Nettleship, A.; Henshaw, P. S.; Meyer, H. L. J. Natl. Cancer Inst. (U.S.) 1943, 4 , 309-319. Schlatter, J. 1986 IUPAC EURO FOOD TOX I1 Metting, Zurich, Switzerland, 1986; pp 249-254. Lofroth, G.; Gejvall, T. Science 1971, 774, 1248-1250.
M. M. Mossoba* J. T. Chen W. C. Brumley S. W. Page Division of Contaminants Chemistry Food and Drug Administration Washington, D.C. 20204
RECEIVED for review July 17, 1987. Accepted January 7,1988.
Two-Dimensional Liquid Chromatographic Method for Resolution of Prostaglandin Enantiomers Sir: Several recent reviews have documented the growth and importance of chiral high-performance liquid chromatography (HPLC) separations (1-6). The vast majority of communications have described approaches which resolve racemates with one asymmetric carbon. The present report describes an HPLC system which yields selectivity for the active enantiomer of the PGEl prostaglandin, methyl (lla,13E)-(f)-11,16-dihydroxy-16-methyl-9-oxoprost-l3-en1-oate (misoprostol), which has two asymmetric carbons (carbons 11 and 16). Samples consist of four diastereomeric forms (two pairs of enantiomers):
Misoprostol is the antiulcer agent in the prescription drug Cytotec. The pharmacologically active form is the llR,16S enantiomer. Selectivity for the active enantiomer has been achieved with a two-dimensional HPLC method that uses column switching to combine a reversed-phase diastereomer separation and a chiral separation. The method has been developed to provide a basis for determining the active/inactive enantiomer ratio in control and degraded misoprostol samples.
EXPERIMENTAL SECTION Appartus. The system employed for resolution of the misoprostol enantiomers coupled two liquid chromatographs. System A consisted of a Hewlett-Packard 1080 liquid chromatograph equipped with a Waters 490 variable-wavelength detector. System B consisted of a Beckman 114M pump and a Kratos 757 detector. The effluent from system A was directed through a two-position, six-port valve (Valco Instruments Co. Inc., Houston, TX)equipped with a 250-pL injection loop. The peak detector of the Waters 490 detector was programmed on 40-min after injection of the misoprostol sample into system A. The detector output was also programmed to autozero at 40-min. As the first misoprostol diastereomeric pair eluted from system A, the peak detector actuated the two-position valve and injected the contents of the loop into system B. A peak detect threshold of 0.01 absorbance unit was used. The valve was programmed to the original position 80 min after the system A fraction was switched into system B. Peak area measurements were made with an in-house chromatographic data management system. Reagents. Misoprostol samples were provided by the Chemical Development Department of Searle Research and Development. All solvents and chemicals used in the study were reagent grade. Procedures. Chromatography Conditions. Conditions for system A were as follows: mobile phase, methanol/water, 3/2 (v/v); flow, 0.7 mL/min; column, Supelcosil ODS,3 pm (Supelco, Bellefonte, PA); column dimensions, 150 X 4.6 mm (i.d.); column temperature, 28 O C ; detection wavelength, 205 nm; injection volume 20 pL. Chromatography conditions for system B were as follows: mobile phase, 2-propanol/0.02 M pH 5.7 phosphate buffer, 1/24 (v/v); flow, 0.4 mL/min; column, LKB EnantioPac (LKB Instruments, Inc., Gaithersburg, MD); column dimensions 100 X 4.6 mm (i.d.); detection, 205 nm. Sample Preparation. Misoprostol chemical samples were 1.0 mg/mL in system A mobile phase. Control samples were stored at -20 OC. Samples were degraded by storage at 30 O C for 33 weeks. Degraded samples were degraded to an 80% level relative to the control samples.
0003-2700/88/0360-0948$01.50/00 1988 American Chemical Society
