Document Control # SPR-008-V1.0 Determination of 2-Imidazolidinethione in Fruits and Vegetables by GC/MSD 1 Scope and Field of Application This method is applicable to the analysis of 2-Imidazolidinethione (Ethylene Thiourea, ETU) in fruits and vegetables at the reporting limit of 0.01 g/g in the sample. 2 References 2.1 The Determination of Ethylenethiourea in Fruits & Vegetables, LSD No. P-RE-060-97(1)- ETU, Compliance, Lab Services Subdivision, Laboratory Services and Regional Operations Division, PMRA, Health Canada, Ottawa, March 31, 1997. 2.2 The Determination of 2-Imidazolidinethione in Fruit and Vegetables by GC/AED, Method CSP-007-V1.0, Calgary Laboratory, Canadian Food Inspection Agency, Calgary, Alberta, February, 28 1998. 3 Health and Safety WARNING The toxicity or carcinogenicity of each reagent and standard used in this method has not been precisely defined. Each chemical must be treated as a potential health hazard; therefore, exposure to these chemicals must be reduced to a minimum through the use of protective clothing, fume hoods, proper disposal of waste, etc. Appropriate safety procedures are documented in the current CFIA Laboratory Safety Manual. Pertinent information in the Material Safety Data Sheet (MSDS) on each chemical in regards to its potential hazard and control measures required must be consulted and evaluated prior to its use. 4 Principles and Theory The sample is extracted by methanol. The 2-Imidazolidinethione extracted is derivatized by the alkylation of the thiocarbonyl group using benzyl chloride to form Benzylthio-2-imidazoline. The matrix is then made acidic and washed with dichloromethane. The matrix is then made basic and the analyte is extracted using dichloromethane, concentrated and then derivatized further using Canadian Food Inspection Agency, Calgary Laboratory Page 1 of 7
Trifluroacetic Anhydride. The quantitation is performed by capillary gas-liquid chromatography (GC) with a mass selective detector (MSD). 5 Standards Nanogen Code Identity, Purity Source MAU 2-Imidazolidinethione, 99% Pestanal LEM* Leptophos, > 98% Pestanal * Internal standard All solutions prepared from analytical standards are kept in amber flasks and maintained at 4 C when not in use. 5.1 Working standard For a calibration curve, four standards are prepared at concentrations of 0.25, 0.50, 0.75 and 1.00 g/ml in the final extract. For routine analysis only the 0.50 g/m L standard is required. 5.2 Matrix working standard solution, 0.500 g/ml. Matrix working standard is prepared by transferring 0.500 ml of the 1.0 g/ml 2- imidazolidinethione (ETU) intermediate standard solution into the matrix extract at step 10.1.10 then carrying it through the remaining steps of the method. The final extract volume is brought to 1.0 ml with toluene and placed in a GC autosampler vial. The resulting concentration of the standard is 0.500 g/ml. 5.3 Internal standard Prepare a solution of 34.6 g/ml of Leptophos in toluene. 6 Reagents 6.1 Methanol, distilled in glass grade, Caledon or equivalent 6.2 Deionized water, Milli-Q by Millipore or equivalent 6.3 Hydrochloric acid, reagent grade, Caledon or equivalent 6.4 Sodium hydroxide, solution (50% w/w), Caledon or equivalent 6.5 Trifluroacetic anhydride, Fisher or equivalent 6.6 Sodium sulphate, anhydrous, Caledon or equivalent 6.7 Benzyl chloride, 99%+, Sigma or equivalent Page 2 of 7
6.8 Toluene, distilled in glass grade, Caledon or equivalent 6.9 Dichloromethane, distilled in glass grade, Caledon or equivalent. 6.10 Solutions and Materials 6.10.1 Trifluroacetic Anhydride (TFAA), 10% Prepare a 10% v/v solution by pipetting 5.00 ml of TFAA into 45.0 ml of toluene and mixing well. 7 Apparatus 7.1 Glassware and Labware 7.1.1 Glass Mason jars - 500 ml 7.1.2 Volumetric flasks: amber - 25.0 ml, 50.0 ml 7.1.3 3-Ball Snyder Condensers, 24/40 joint 7.1.4 Flat bottom boiling flasks, 24/40 glass joint - 500 ml 7.1.5 Graduated glass centrifuge tubes - 5 ml 7.1.6 Disposable syringes - 5 ml 7.1.7 Graduated cylinders - 5 ml, 50 ml, 100 ml 7.1.8 Hamilton digital syringes - 500 L, 100 L or equivalent 7.1.9 Millex-HV disposable filters - 0.45 m or equivalent 7.1.10 Class A pipets - 1 ml, 2 ml 7.1.11 Hewlett Packard GC vials and caps - 1.5 ml or equivalent 7.1.12 Powder funnels 7.1.13 Buchner funnels 7.1.14 Filter paper: #2 7.1.15 Boiling chips (anti-bumping granules) - BDH or equivalent 7.1.16 Separatory funnels - 250 ml 7.1.17 Graduated centrifuge tubes - 5 ml 7.1.18 Disposable pasteur pipets 7.1.19 Variable volume dispensers - 50 ml, 100 ml 7.1.20 Glass Wool - DMCS treated 7.2 Auxiliary Equipment 7.2.1 Electronic analytical balance, 4 decimal places, Mettler AE 200 or equivalent. 7.2.2 Electronic top loading balance, 3 decimal places, Mettler PM 400 or equivalent. 7.2.3 Vortex test tube mixer - Thermolyne or equivalent 7.2.4 Homogenizer, Dupont Model 17105 Omni-mixer Homogenizer or equivalent 7.2.5 Nitrogen evaporator, N-EVAP 112, (water bath at 40 C), Organomation Limited or equivalent 7.2.6 Rotary Evaporator (water bath at 40 C) - Brinkmann or equivalent 7.2.7 Heating mantles (hotplates) - Precision or equivalent Canadian Food Inspection Agency, Calgary Laboratory Page 3 of 7
7.2.8 Refrigeration cooler unit - Neslab RTE - 111 or equivalent 7.3 Analytical Instrumentation 7.3.1 Gas Chromatography/Mass Selective Detector Selective Detector (GC/MSD): Hewlett-Packard gas chrom atography 5890 Series II, with W indows Chemstation, 7673A autosampler, 5972 Mass Selective Detector or equivalent Analytical column: 25 metre HP Ultra 1, J&W, 0.20 mm I.D., 0.33 m film thickness or equivalent Retention gap: J&W Deactivated fused silica, 0.25 mm id. - 2m or equivalent Supelco GlassSeal connector - #2-0479 or equivalent Inlet liner - Cyclosplitter, Chromatographic Specialties - #RK20707 or equivalent 8 Sample Preparation The Calgary Laboratory is not responsible for initial sampling procedures. Representative samples supplied by client divisions are collected according to established guidelines. 8.1 Prepare the edible portions of the sample. 8.2 Blend the entire sample (1 kg) in food processor for two minutes. 8.3 Transfer each homogenate to a glass bottle (large Mason jar) and freeze until required for analysis. 8.4 Thaw frozen homogenate and mix well before taking any sub-sample. 9 Procedure 9.1 Extraction 9.1.1 A typical set of samples consists of the following: 1) 10 regular samples 2) One blank sample 3) A 2-Imidazolidinethione Spike: Add 0.5 ml of the 2-Imidazolidinethione spiking standard 1.00 g/ml to a blank extract at step 10.1.4. This corresponds to a 0.50 g spike for 2- Imidazolidinethione in the final extract. 9.1.2 Let the sample reach room temperature. 9.1.3 Mix well before weighting 9.1.4 In a 500 ml Mason jar, weigh at least 20.0g of sample. 9.1.5 Add 100 ml of methanol. 9.1.6 Homogenize for 1 minute. 9.1.7 Filter the extract using a Buchner funnel with a #2 filter paper. 9.1.8 Rinse the Mason jar twice with 20 ml of methanol and filter. 9.1.9 Quantitatively transfer the filtrate to a 500 ml boiling flask and rotovap @ 40 C until the sample has a volume of approximately 5 to 10 ml. (This is the point where the rate of evaporation of the solvent has decreased significantly). 9.1.10 The first derivatization step is carried out on every sample, standard and blank by adding 20 ml of Milli-Q water, 20 ml of methanol, a few boiling chips and 10 drops of benzyl chloride. Place a 3-ball Snyder condenser on the 500 ml boiling flask and place on a heating mantle or hotplate. Allow the samples to remain on Page 4 of 7
the hot plate for 1 hour. (The total time of 1 hour on the hot plate includes the period of time for the solution to come to a boil and a period of at least 40 minutes of vigorous reflexing.) 9.1.11 Remove the boiling flasks from the heating mantle or hot-plate and allow to cool for 10 min. Remove the Snyder condenser and add approximately 2.0 ml of conc. HCl and swirl to mix. 9.1.12 Place each sample in a 250 ml separatory funnel and rinse the boiling flask with 40 ml of Milli-Q water and then 40 ml of dichloromethane. Add the rinses to the separatory funnel. 9.1.13 W hile degassing the funnel in an inverted position, vigorously swirl the funnel. Allow the two layers to separate and discard the dichloromethane (lower layer). Repeat this step with another 40 ml of dichloromethane and again discard. (NOTE: A third 40 ml wash with dichloromethane should be used for matrices such as peppers and tomato paste with strong colours that are extractable.) 9.1.14 To each separatory funnel, add 5 ml of a 50% solution of sodium hydroxide and mix by swirling the flask. (WARNING: It should be noted that, while no significant reactions were observed with the addition of this strong base to the strongly acidic samples, caution should be exercised while carrying out this procedure.) 9.1.15 Add 100 ml of dichloromethane and vigorously shake the samples for 30 seconds. Pass the dichloromethane layer (lower layer ) through a powder funnel that is approximately half filled with anhydrous, granular sodium sulfate into a dry 500 ml boiling flask. 9.1.16 Repeat step 10.1.15 using 80 ml of dichloromethane. Rinse the sodium sulfate with approximately 30 ml of dichloromethane. 9.1.17 Rotovap the combined extract and rinses to a volume of approximately 2-3 ml, and then quantitatively transfer to a 15 ml centrifuge tube using toluene as a rinse. N-Evap the samples to a volume of approximately 50 L. 9.1.18 Derivatize the sam ples by adding 1.0 ml of the 10% TFAA solution in toluene to all sample extracts, standards and blanks, then cap and vortex and let stand at room temperature overnight. 9.1.19 At the end of the derivatization, mix each sample well on vortex mixer, quantitatively transfer and filter through a 0.45 Gelman filter using toluene into a dry 5 ml centrifuge tube. Rinse the syringe & filter with 2 mls of toluene into the centrifuge tube. Concentrate using the N-Evap to a volume of approximately 0.05 ml. Bring the sample to a volume of approximately 0.8 ml with toluene and add 80 L of internal standard (34.6 g/ml of Leptophos in toluene). Bring to a final volume of 1.0 ml with toluene and vortex. 9.1.20 Transfer the sample into an autosampler vial and cap tightly. The sample is now ready for GC analysis. 9.2 Determination Analyse the sample by GC/MSD by monitoring mass ions 288, 219, and 191 amu. Ion 288 is the quantifying ion due to the fact that it appears to be much more selective than the other ions Canadian Food Inspection Agency, Calgary Laboratory Page 5 of 7
9.2.1 Heated Zones Injection Mode Injector Temp. Transfer Line Temp. Splitless 250 C 280 C 9.2.2 Injection volume 1 L with purge on @ 0.50 min. 9.2.3 GC Temperature Program Initial Temp ( C) Initial Hold (min) Ramp ( C/min) Final Temp ( C) Final Hold (min) 70 1 10.0 25.0 240290 6 10 Critical Control Points 10.1 After the initial derivatization, it is strongly recommended that the cooled extracts be processed further through the subsequent steps to be: acidified, washed and extracted with dichloromethane. Lower recoveries may be observed, if the samples are allowed to sit at this step for any length of time in terms of hours. 10.2 In step 10.1.17, the rotovaping of the dichloromethane must not be allowed to go to dryness as the will cause low recoveries. 11 Calculations and Result Interpretation 11.1 Calculation An internal standard method is used. The concentration of 2-Imidazolidinethione is determined according to the following equation. [spl] ppm =area spl x area istd-std x [std]ng/ L x vol spl (ml) x 10 3 L/mL x 10-3 g/ng area std area istd-spl wgt spl (g) % recovery = ppm found x 100 % ppm spiked spl: std: istd-std: istd-spl: sample standard response of the internal standard in the 2-Imidazolidinethione standard response of the internal standard in the sam ple 11.2 Analyte Identity Confirmation Positive determinations are confirmed by GC/MSD by monitoring mass ions 288, 219, and 191 amu. Ion 288 is the quantifying ion used due to the fact that it appears to be much more selective than the other ions. 11.3 Calibration standards Page 6 of 7
A calibration curve using the 4 matrix standards described in Section 6.1 is run monthly to ensure operation in the linear range of the instrument. Routine analysis only requires a single point calibration at the m atrix standard #2 level. 12 Performance Standards 12.1 Analytical range is 0.010 g/g to 0.050 g/g. Samples with results higher than 0.050 g/g need to be retested at a lower weight. 12.2 Linearity Correlation coefficient of a standard curve should be 0.99 (technically 0.985) or better. Otherwise, samples must be re-analyzed. 12.3 Spike Recovery Recoveries should be 70% (technically 69.5%) or better. If the % recovery of the spiked sample is lower than 70%, analysis should be repeated. 12.4 Limit of Detection (LOD) is estimated to be 0.0019 g/g and Limit of Quantitation (LOQ) is estimated to 0.0057 g/g. 12.5 Reporting Limit is 0.01 g/g. Results below the Reporting Limit are to be reported as not detected. 12.6 Results are to be reported to the limit of up to three significant figures and up to the second decimal (e.g., 123 g/g, 12.3 g/g, 1.23 g/g, 0.12 g/g or 0.01 g/g) if found to contain 0.01 g/g or more. 13 Method Revision History Version Date Description Author P-RE-060-97(1)-ETU 31 March 1997 Determination of Ethylenethiourea in Fruits & Vegetables PMRA CSP-007-V1.0 28 February 1998 ETU by capillary gas-liquid chromatography with atomic emission detection (AED) Dave Wotherspoon SPR-008-V1.0 16 June 2004 ETU by capillary gas-liquid chromatography with mass selective detector (MSD) Doug Baker Canadian Food Inspection Agency, Calgary Laboratory Page 7 of 7