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Determination of Methyl Carbamate and Ethyl Carbamate In Alcoholic Beverages and Other Fermented Foods
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Health Protection Branch Laboratories
Bureau of Chemical Safety
Ottawa
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(PDF Version - 339 K)Definition: This method is applicable to the quantitative determination of methyl carbamate and ethyl carbamate in alcoholic beverages, yogurt, soya sauce, buttermilk, breads and toast in accordance with Section 4 of the Food and Drugs Act.
Scope: This method has been evaluated using both an extraction tube and an alumina-Celite column followed by GLC with thermal energy analyzer (N - mode) detection and/or GC with high resolution MS. Recovery of spikes at the 10 to 50 ppb level ranged from 70 to 114% (Table 1).
THE ANALYST SHOULD BECOME FAMILIAR WITH THIS METHOD USING STANDARDS AND SPIKED SAMPLES BEFORE UNDERTAKING THE ANALYSIS OF UNKNOWNS.
Principle: Samples are extracted with dichloromethane using an extraction tube or an alumina-Celite column. The extract is concentrated with a Kuderna-Danish concentrator using a macro and then a micro Snyder column. The concentrate is analyzed by GLC-TEA (N - mode) or by GC-MS for the quantity of methyl carbamate and ethyl carbamate present. The concentrate for toast samples must be analyzed by GC-MS because of numerous N-containing compounds which interfere with the GLC-TEA analysis.
Caution: Ethyl carbamate and methyl carbamate are carcinogenic to laboratory animals and should be handled with caution. For details of other chemical hazards refer to the cautionary notes described in the Appendix, Laboratory Safety, of the current edition of Official Methods of Analysis, Association of Official Analytical Chemists, Arlington, VA, or other recognized texts respecting laboratory safety.
Apparatus:
1. Varian gas chromatograph (Model 3400), or equivalent, attached to a TEA (Model 502) with nitrogen converter (Model 610 Thermedics Inc., Woburn, MA) and equipped with: (a) two 30 m DB-Wax (1 mm film thickness) fused silica capillary columns (i.d., 0.53 mm) connected in series, or a 60 m column of the same type;
Operating conditions:
The GLC oven temperature was programmed as follows:
ii) then heated to 150°C at 3°C/min followed by further heating to 200°C at 25°C/min,
iii) hold at 200°C for 20 min.
TEA vacuum chamber pressure was 0.5 - 0.6 torr;
Injector, at 200°C;
Helium carrier gas, flow rate 8 mL/min;
GLC-nitrogen converter interface at 275°C;
Pyrolyzer furnace at 800°C.
2. GC-MS 2 Systems:
Both mass spectrometers operating in the electron impact mode;_lpfc163.html Both gas chromatographs equipped with a 30 m (i.d., 0.22mm) DB-Wax (0.25 mm film thickness) fused silica capillary column (J & W Scientific);_lpfc163.html
Operating conditions:
The GLC oven temperature was programmed as follows:
ii) then heated to 150°C at 5°C/min followed by further heating to 250°C at 50°C/min,
Other conditions:
ii) GC-MS transfer line 200°C,
iii) carrier gas He at 20 psi,
iv) ion source temperature 200°C,
v) electron energy ca 55 eV
3. Water bath capable of holding temperature at 55 to 60°C;
4. Kuderna-Danish (K-D) evaporative concentrator, 250 mL capacity, with 24/40 column connection and 19/22 lower joint, complete with springs (Kontes Cat. No. 570000);
5. K-D concentrator tube 4 mL capacity, with 19/22 joint, and 0.1 mL subdivisions from 0 to 2.0 (Kontes Cat. No. 570050); Verify accuracy of graduations;
6. Pennyhead stoppers (19/22 joint) for concentrator tubes (Kontes Cat. No. 850500);
7. Snyder column - 3 sections, 225 mm, with 24/40 joints (Kontes Cat. No. 503000);
8. Micro Snyder column, 3 chambers, with 19/22 joint (Kontes Cat. No. 569000-0319);
9. Micro syringes, 10 µL;
10. Chem Elut Extraction tubes, 20mL;
11. Chromatography column, 29.5 mm x 400 mm;
12. Tamping rod and funnel (Figure 1).
Reagents:
1. Glass-distilled, reagent grade solvents;
Dichloromethane (DCM), n-pentane, n-hexane, ethanol and ethyl acetate;
2. Sodium chloride (NaCl) reagent grade;
3. Sodium sulfate, anhydrous, reagent grade;
4. Celite 545 (not acid washed), prepared (Note 3);
5. Neutral alumina for column chromatography (ICN Biomedicals), deactivated (Note 4);
6. Distilled water (H2O);
7. Standards:
ethyl carbamate and methyl carbamate (Aldrich),
B. Dilute stock solutions 10.0 µmg/mL. Transfer 1 mL of stock solution to 100 mL volumetric flask and dilute to volume with ethyl acetate and mix well;
C. Working standards and spiking solutions: Prepare the following working standards and spiking solutions with appropriate serial dilutions of dilute stock solution with ethyl acetate: 0.05, 0.10, 0.50 and 1.0 µmg/mL.
Procedure: Sample Preparation and Extraction
A. Alcoholic Beverages
1. Transfer 10 mL aliquot of wines to Chem Elut tube and let equilibrate for 5 min (Note 5);
2. Wash contents of tube with 50 mL DCM:n-pentane solution (20:80), and discard washing;
3. Elute with a total of 100 mL DCM, adding 15 to 20 mL portions at a time;
4. Collect eluate directly in the K-D concentrator, as described in step E.3;
5. Add 0.5 mL ethanol or ethyl acetate as a keeper;
6. Proceed to concentrate the extract beginning step E.4 to E.14;
1. Place small glass wool plug in bottom of 29.5 mm x 400 mm chromatography column;
2. Add to column 10 g deactivated neutral alumina followed by 40 g anhydrous sodium sulfate;
3. Pipette 10 mL aliquot of sample (Note 5) into 250 mL beaker;
4. Add 15 g prepared Celite 545 and mix with spatula until uniform;
5. Place tamping rod and powder funnel in column with end of tamping rod extending through funnel opening;
6. Load, then tamp Celite-beverage mixture a little at a time until transfer is complete;
7. Wash column with 50 mL solution of DCM:n-pentane (20:80) (v/v) by pouring onto column through funnel before removing tamping rod;
8. Adjust stopcock for flow rate of 1-2 drops/sec and discard washing;
9. Add 100 mL DCM to beaker, swirl with spatula, and pour on to column through funnel;
10. Collect the 100 mL DCM eluate directly in the K-D concentrator, as described in step E.3;
11. Add 0.5 mL ethanol or ethyl acetate as a keeper;
12. Proceed to concentrate the extract beginning step E.4 to E.14;
B. Soy Sauce
C. Breads and Toast
2. Homogenize 10 g of sample with 15 mL of NaCl -saturated H2O;
3. Follow directions for celite-alumina column method (steps A.ii) 1 to A.ii) 10 inclusive) except in step A.ii) 3 use entire homogenate from step C.2 above, and in step A.ii) 7 wash column with 100 mL DCM:n-pentane solution (20:80) (v/v);
4. Add 0.5 mL ethanol, (do not use ethyl acetate) as a keeper (Note 6);
5. Proceed to concentrate the extract beginning step E.4 to E.14;
D. Yogurt and Buttermilk
2. Follow directions for celite-alumina column method (steps A.ii) 1 to A.ii) 10 inclusive) except in step A.ii) 3 use the entire homogenate from step D.1 above, and in step A.ii) 7 wash column with 100 mL DCM:n-pentane solution (20:80) (v/v);
3. Add 0.5 mL ethanol, (do not use ethyl acetate), as a keeper (Note 6);
4. Proceed to concentrate the extract beginning step E.4 to E.14;
E. Concentration of Eluate (Extract)
2. While connecting bottom tube, wet joint with DCM and attach springs;
3. Collect eluate (extract) directly in K-D concentrator assembly referred to above;
4. Add one tiny "Boileezer" (1-2 mm) to contents of flask;
5. Attach a 3-section Snyder column and concentrate extract to ca 4 mL by heating the flask in a water bath at 55 to 60°C (Note 7);
6. Raise flask above the water and allow condensed DCM in Snyder column to drain back into flask;
7. Add ca 1 mL of DCM in top of Snyder column and let drain into flask;
8. Disconnect the 4 mL concentrator tube from flask;
9. Add another tiny piece of "Boileezer" to contents and attach micro Snyder column and springs;
10. Concentrate extract to ca 0.8 mL by heating the concentrator tube in a water bath at 55 to 60°C (Note 8);
11. Rinse micro Snyder column with a few drops of DCM, and allow rinse to drain into tube;
12. Disconnect column;
13. Make extract volume up to 1.0 or 1.1 mL with DCM, but not more than 1.1 mL;
14. Stopper the tube, mix contents using vortex mixer and store at 4°C in the dark until analysis;
F. Phase Transfer and Liquid-Liquid Partitioning
For Bread, Toast, Yogurt and Buttermilk extracts only (Note 9)
2. Heat concentrator tube and contents in the water bath at 55 to 60°C until most of the DCM is driven off;
3. Remove the micro Snyder column and heat concentrator tube and contents for another 2 to 5 min in the water bath until the last traces of DCM disappear;
4. Transfer aqueous extract quantitatively; using a Pasteur pipette, into a 20 mL separatory funnel;
5. Rinse concentrator tube with two 2 mL portions of H2O, two 2 mL portions of n-hexane and transfer rinsings to the 20 mL separatory funnel;
6. Gently shake contents of separatory funnel for ca 2 min, then allow layers to separate;
7. Carefully draw off lower aqueous layer into a 15 mL centrifuge tube and make up to ca 10 mL with H2O;
8. Saturate the 10 mL solution with NaCl by adding ca 4 g NaCl;
9. Pour mixture onto a 20 mL Chem Elut extraction tube and let equilibrate for 5 min;
10. Elute with 5 x 20 mL portions of DCM and collect the eluate directly in the K-D concentrator, as described in step E.3; use portions of the DCM to rinse the 15 mL centrifuge tube and pour the rinse onto the Chem Elut extraction tube;
11. Add 0.5 mL of ethanol or ethyl acetate to the combined eluates;
12. Proceed to concentrate the extract beginning step E.4 to E.14;
G. Reagent Blanks
2. Use 10 mL 10% (v/v) alcohol in H2O as the reagent blank for alcoholic beverages in place of the sample aliquot;
3. Prepare blanks for the other sample matrices using the quantities of reagents specified but omitting the sample;
4. Carry blanks with the corresponding samples through the entire procedure ending at step E.14;
H. Establishing a Standard Curve
2. Using this attenuation, analyze 2 µmL aliquots, in duplicate, of each standard having concentration of 0.05, 0.10, 0.5 and 1.0 mg/mL (Note 10);
3. Accurately measure peak heights (± 0.1 cm) and calculate the average peak heights of 2 injections at each concentration (Note 11);
4. Plot standard curve peak height vs pg injected;
5. To prepare a standard curve for GC-MS, use a similar procedure as described above except inject 1 µmL aliquots.
I. Analysis of Samples
2. Measure and determine average peak height corresponding to the 2 µmL injection;
3. Compare this peak height with a recently prepared standard curve and determine which of the standard solutions, when injected under the same attenuation setting, produces the closest peak height;
4. Choose the standard solution, inject 2 µL aliquots, in duplicate, and determine average peak height;
5. For GC-MS analyses, inject 1 µL aliquots at all times.
J. Calculations
where
H1 is the average peak height in cm of sample
H2 is the average peak height in cm of the corresponding analyte standard
P is pg of analyte standard which produced H2
V1 is 2µL, except for GC-MS where 1µL is used
V2 is 1 mL
G is grams of sample taken.
Note 1. This GC-MS system is used for the analysis of alcoholic beverages, some bread and toast, and soy sauce extracts.
Note 2. This GC-MS system is used for the analysis of other toast, as well as yogurt, and buttermilk extracts.
Note 3. Heat Celite 545 for 16 h at 600°C, let cool to room temperature in a desiccator.
Note 4. Heat neutral alumina for 16 h at 400°C, and then cool to room temperature in a desiccator. Deactivate by mixing with 10% w/w H2O, store in a stoppered flask and let equilibrate overnight.
Note 5. Although a 10 mL aliquot of wines is taken, other samples must be prepared as follows before adding to extraction tube:
Dilute 5 mL of sherries to 10 mL with H2O, add 2 g of NaCl and mix well
Dilute 2 mL of beverages containing > 30% alcohol to 10 mL with H2O, add 2 g NaCl and mix well.
Note 6. The more polar ethanol must be used for more ready dissolution into the H2O phase as required for further purification by phase transfer and partitioning.
Note 7. Initially, maintain outside water level close to that of the DCM inside the flask and continue heating until the concentrated extract is ca 4 mL. If excessive boiling occurs during concentration, control it either by raising flask slightly or, by lowering the temperature of the water bath.
Note 8. Lift out or immerse tube in water to control boiling rate but do not lift tube completely for this will stop the action of the "Boileezer". Avoid overheating and excessive accumulation of DCM in the column chambers. Stop concentrating when the DCM level reaches 0.8 mL. Do not concentrate to less than 0.8 mL.
Carry out the final concentration step slowly taking at least 30 min. Finally, raise tube with bottom still touching the water, let liquid drain, and note volume to confirm that it is 0.8 mL. If volume is < 0.8 mL continue concentrating as before.
Note 9. The liquid-liquid partitioning procedure is required only for bread, toast, yogurt and buttermilk extracts to remove any lipids or fats which may be present.
Note 10. Before injection, draw out syringe plunger slightly and note exact volume of sample or standard to be injected. There must be an air gap between the solution to be injected and the rinsing solvent already inside the needle. During injection, be sure that no sample or standard is lost through back pressure. After injection, hold needle in septum for ca 5 s before withdrawing.
Note 11. If exactly 2 µL is not injected, make appropriate corrections to convert all peak heights equivalent to 2 µL injections.
Note 12. The attenuation could vary from 8 to 128 depending on concentration of analyte.
Reference:
1. Sen, N.P., Seaman, S.W., Boyle, M. and Weber, D. Methyl Carbamate and Ethyl Carbamate in Alcoholic Beverages and other Fermented Foods, Food Chemistry (accepted for publication)
2. Sen, N.P., Seaman, S.W. and Weber, D. A Method for the Determination of Methyl Carbamate and Ethyl Carbamate in Wines, Food Additives and Contaminants 9, 149, (1992)
3. Canas, B.J., Havert, D.C. and Joe, F.L. Jr. Rapid Gas Chromatographic Method for Determining Ethyl Carbamate in Alcoholic Beverages with Thermal Energy Analyzer Detection, J. Assoc. Offic. Anal. Chemists 71, 509 (1988)
Table 1
Recoveries of MC and EC from Alcoholic Beverages, Soy Sauce, Bread and Toasts, and Yogurt
| Sample | Spiking level(ppb) | Method used for analysisa | % recoveriesb | |
|---|---|---|---|---|
| MC | EC | |||
| Alcoholic beverages | ||||
| Wine | 10 | A | 74 | 114 |
| Wine | 20 | A | 75 | 103 |
| Wine | 20 | A | 94 | 101 |
| Wine | 20 | A | 71 | 98 |
| Gin | 50 | B | 85 | 96 |
| Vodka | 50 | B | 94 | 108 |
| Soy Sauce | ||||
| Mushroom soy sauce | 10 | B | 60 | 59 |
| Soy sauce | 20 | B | 98 | 93 |
| Soy sauce | 20 | B | 120 | 108 |
| Bread and toast | ||||
| White bread | 16 | C | 67 | 86 |
| White bread toast | 16 | C | 86 | 90 |
| Scone bread | 10 | C | 88 | 115 |
| Whole wheat bread toast | 16 | C | 76 | 71 |
| White bread toast | 10 | C | 96 | 114 |
| Italian style bread, dark toast | 36 | C | 69 | 96 |
| Yogurt | ||||
| Peach bottom natural yogurt | 16 | C | 71 | 80 |
| Blueberry bottom yogurt | 16 | C | 83 | 124 |
a Methods used: A, Chem Elut extraction followed by TEA (N) detection; B, Celite + neutral alumina (10% water content) clean-up followed by GC-MS; C, same as "B" with additional liquid-liquid partitioning to remove fats and lipids.
b Levels of MC and EC present in the unspiked samples were subtracted before calculating recoveries.
