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INFORMATION
SHEET
Number 10/00 January
2001
Food Standards Agency UK -
2- MERCAPTOBENZOTHIAZOLE AND BENZOTHIAZOLE FROM RUBBER
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Summary
Background
Methodology
Results and Interpretation
References
Further Information
Spreadsheet Tables
- A survey of 236 samples of food and drink
found no trace of the chemicals 2-mercaptobenzothiazole and
benzothiazole which are known to be present in some rubber
components used in food processing equipment.
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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.
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Analysis
The methodology described below was based on a published method to
identify components in benzothiazole derivatives.2
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:
- Canned beers: MBT 0.012 mg/kg, BT 0.005
mg/kg
- Vending machine water: MBT 0.024 mg/kg,
BT 0.010 mg/kg
- Baby food: MBT 0.026 mg/kg, BT 0.017
mg/kg
- Long-life fruit juice: MBT 0.027 mg/kg,
BT 0.027 mg/kg
- Tinned fruit juice: MBT 0.043 mg/kg, BT
0.039 mg/kg
- Canned soft drink: MBT 0.042 mg/kg, BT
0.014 mg/kg
- Tinned fruit syrup: MBT 0.009 mg/kg, BT
0.005 mg/kg
- Yoghurt: MBT 0.034 mg/kg, BT 0.012 mg/kg
- Infant and evaporated milk: MBT 0.022
mg/kg, BT 0.029 mg/kg
- Bottled drinks and draught beer: MBT
0.019 mg/kg, BT 0.010 mg/kg
- Milk: MBT 0.036 mg/kg, BT 0.010 mg/kg
- Vending machine coffee: MBT 0.008 mg/kg,
BT 0.007 mg/kg
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Analytical quality assurance
For MBT or BT to be considered present in a sample extract, three
criteria had to be met:
- The ratios of retention time for MBT (at
m/z 168) or BT (m/z 136) compared with that for the elution
marker HBT (m/z 152) had to be within 2.5 per cent for
standards, 'spikes' and samples.
- The confirmatory ion at m/z 109 must also
be present at the correct retention time for MBT or BT.
- The signal:noise ratio of any putative
LC-MS peak must be greater than 3:1.
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.
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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.
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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.
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1. 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 Nobel House, 17 Smith Square, London, SW1P 3JR
(Tel. No. +44 (0) 20 7238 6575).
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 Davd Watson
Food Standards Agency
Chemical Safety and Toxicology Division
PO Box 31037
Room 212, Ergon House
17 Smith Square
London SW1P 3WG
Tel: +44 (0) 20 7238 6250
Fax: +44 (0) 20 7238 6124
E-mail: david.watson@foodstandards.gsi.gov.uk
A copy of the final report of this survey has been placed in the
library in Nobel House, 17 Smith Square, London, SW1P 3JR, UK;
Tel. No. +44 (0) 20 7238 6575. 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.
Table 1: Details of
Samples Obtained
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the Excel 5.0 version of Table 1
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Table 1 (if you have any other spreadsheet package)
These pages were last updated on 29
December 2000.
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