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Food Survey Information Sheet
This survey was carried out to test whether 2-mercaptobenzothiazole (MBT) and benzothiazole (BT) migrate from rubber during the manufacture and storage of food or drink. Research has shown that this might happen. 1 Both MBT and BT migrated from rubber into laboratory solvents, but it was not clear whether they migrate into food or drink. 1
The main types of rubber components where residues of MBT and BT may be found include natural rubber or nitrile rubber seals and gaskets used in food processing equipment. MBT is formed from two accelerators used in vulcanising rubber: 2-mercaptobenzothiazyl disulphide (MBTS) and N-cyclohexyl-2-benzothiazole sulphenamide (CBS). 1 BT is formed by splitting a disulphide bridge in MBTS. 1
Chemical migration from rubber into food is controlled in Great Britain under the general provisions of The Materials and Articles in Contact with Food Regulations 1987 and of the Food Safety Act 1990. Similar controls apply in Northern Ireland.
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Sampling
Two hundred and thirty-six retail samples of aqueous food and drink were purchased between January and August 2000 (Table 1). Each sample was assigned a unique sample log-in number. Duplicates were obtained except for six samples of bottled milk. Samples were stored at ambient temperature (approximately 20°C; stored away from sunlight), in a refrigerator (less than 10°C) or in a freezer (less than -20°C) as appropriate. Samples were mixed thoroughly by shaking and/or stirring (as appropriate) before removing sub-samples for analysis.
Analysis
The methodology described below was based on a published method to identify components in benzothiazole derivatives.
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Analysis was for MBT and BT. In developing the methodology described below it was confirmed that both MBTS and CBS are unstable and break down rapidly during food analysis to give MBT and BT as the main products. If BT or MBT were to be detected in any sample, then that sample would be re-tested for residues of CBS or MBTS.
Two extraction regimes were employed for the food samples, one for milk, yoghurt and infant formulae and one for other foodstuffs. For milk, yoghurt and infant formulae, hydroxybenzothiazole (HBT) (0.12 ml, 0.005 mg/ml solution) was added, as an elution marker, to an extraction vial (capacity more than 12 ml), and evaporated to dryness. Sample (2 ml or 2 g as appropriate) was added to the vial followed by a few drops (approx. 0.5 ml) of trifluoroacetic acid which resulted in a protein precipitate forming on the bottom of the vial. Acetonitrile (9.5 ml) was added to give a total volume of 12 ml. The samples were shaken for 15 minutes on a roller bed and centrifuged for 5 minutes at 3,000 rpm. One ml of the top, solvent layer, was filtered through a 0.45 micron filter ready for analysis.
For samples of other foodstuffs 0.1 ml HBT (0.005 mg/ml solution) was added, as an elution marker, to an extraction vial (capacity more than 12 ml), and evaporated to dryness. Two ml or 2 g of sample was added to the vial followed by glacial acetic acid (1 ml) and acetonitrile (7 ml). The samples were sonicated for 5 minutes and then centrifuged for 5 minutes (3,000 rpm). An aliquot (1 ml) was filtered through a 0.45 micron filter ready for analysis.
HPLC was performed using a Columbus (ODS) analytical column (250 x 2 mm) and guard column (30 x 2 mm) utilising a gradient mobile phase (mobile phase A was 1 per cent glacial acetic acid in water, mobile phase B was 1 per cent glacial acetic acid in acetonitrile) at a flow rate of 0.3 ml/min. Mass spectral data were recorded in the positive ionisation mode. The ions monitored were at m/z 168 (MBT), 152 (HBT), and 136 (BT). The ion at m/z 109 was also included as a confirmation ion for all 3 compounds should analyte be found in any of the samples.
Quantification was based on external calibration. Once linearity had been established over a range of 25-500 ng/ml (correlation coefficients more than 0.98), single point bracketing calibration (100 ng/ml solvent-based standard) was used for quantification. All sample types were 'spiked' with BT and MBT at 0.5 mg/kg (0.2 ml of a 0.005 mg/ml solution in acetone) added to sample (2 g or 2 ml) prior to extraction, carried out as described above, to determine the analytical recovery. Thus, the recovery figures will include both the physical recovery of the analyte plus any matrix suppression or enhancement on the ion signal within the source of the mass spectrometer. A standard solution for comparison with peak areas in sample extracts was prepared from a stock 1:1 mix of 0.1 mg/ml MBT and BT solutions in acetone.
The instrumental limit of detection (LoD) was calculated from the 3:1 signal:noise ratio obtained for batch blank samples. The noise of the LC-MS instrument and therefore the LoD depended on the sample type. When the instrumental response was corrected for the average recovery for each analytical batch, the LoDs for MBT and BT in the samples were as follows:
Analytical quality assurance
For MBT or BT to be considered present in a sample extract, three criteria had to be met:
Each batch of samples was of one food type. A typical batch size was 20 samples. Each analytical batch included at least two method blanks along with four 'spiked' samples to measure the recovery for that food type. Due to the instability of MBT and BT on long-term storage, an in-house reference material was not produced. To compensate for this at least four 'spikes' were included with each analytical batch.
To check on the accuracy of the methodology, samples of several different food types were 'spiked' by an independent laboratory and supplied 'blind' to the surveying laboratory for analysis. Samples (2 g) were 'spiked' using undisclosed volumes of a stock solution of MBT and BT at 0.005 mg/ml. These samples were then extracted using the method described above for other foodstuffs, and analysed by LC-MS. The results for nine check samples analysed 'blind' were reported and then the 'spiking' levels disclosed for an assessment of the results. For MBT 'spiked' at seven levels in the range 0.1 to 0.2 mg/kg, the laboratory found 81 to 106 per cent of the expected concentration with a mean of 91 per cent. For BT 'spiked' at seven levels in the range 0.1 to 0.2 mg/kg, the laboratory found 80 to 103 per cent of the expected concentration with a mean of 90 per cent.
An assessment of the batch recovery of MBT and BT during the survey was carried out by quantifying 'spiked' samples against solvent standards. For 61 'spiked' extracts the mean recovery was 82 per cent for MBT and 87 per cent for BT.
The within-batch precision of the analytical method for BT and MBT analysis was assessed from the batch recovery for 'spiked' samples prepared in-house. For the samples (n=61) 'spiked' with MBT at 0.5 mg/kg the precision was in the range 3 to 34 per cent with an average for all analytical batches of 18 per cent. For the samples (n=61) 'spiked' with BT at 0.5 mg/kg the precision was in the range 1 to 24 per cent with an average for all analytical batches of 7 per cent.
Reporting
Brand names are reported as this survey was carried out in accordance with guidelines for reporting survey results published in the
Food Safety Information Bulletin
in September 1997. The absence of a particular brand from Table 1 means only that the brand was not included in the survey.
MBT and BT were not detected in any of the samples. This provides strong evidence that these substances do not migrate into food or drink. MBT and BT would have been expected to be found in retail food and drink samples in this survey, if they had migrated from rubber during food production or storage. Contamination of food or drink with MBT and BT might occur via the environment, but this appears unlikely from the results of this survey.
Back to top1 . J. Sidwell and R.B. Simpson. Migration studies - food contact elastomeric materials, The evaluation of rubbers and elastomers for food contact use (FS 2219) (1997). Final project report available from the library in Aviation House, 125 Kingsway, London WC2B 6NH (Tel. No. +44 (0) 20 7276 8187)
2 . W. M. A. Niessen, C. C. McCarney, P.E.G. Moult, U.R. Tjaden, and J. Van der Greef, Liquid chromatography-mass spectrometry for the identification of minor components in benzothiazole derivatives. J. Chromatography , 1993, 647 , 107-119
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Dr David Watson
Food Standards Agency
Chemical Safety and Toxicology Division
Room 516, Aviation House
125 Kingsway
London WC2B
6NH
Tel: +44 (0) 20 7276 8537
Fax: +44 (0) 20 7276 8514
Email: david.watson@foodstandards.gsi.gov.uk
A copy of the full report of this survey has been placed in the library in Aviation House, 125 Kingsway, London, WC2B 6NH (Tel. No. +44 (0) 20 7276 8187). If you wish to consult a copy please contact the library for an appointment giving at least 24 hours notice or alternatively copies can be obtained from the library; a charge will be made to cover photocopying and postage.
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