Analysis of Butylated Hydroxytoluene in Food with Headspace Trap-GC/MS

a p p l i c at i o n N o t e
Gas Chromatography/
Mass Spectrometry
Meng Yuan
PerkinElmer, Inc.
Shelton, CT 06484 USA
Analysis of Butylated
Hydroxytoluene in
Food with Headspace
Butylated hydroxytoluene
(BHT, 2,6-di-tert-butyl-4-methylphenol)
is a common food additive. BHT is found
in many types of food including butter,
meats, cereals, chewing gum, baked
goods, snack foods, dehydrated potatoes
and beverages. It is used to preserve food
odor, color and flavor. BHT is oxidized
preferentially in fats or oils, protecting
the foods from spoilage.
Concern exists that long-term human consumption of BHT may have potential
health risks. It has undergone the additive application and review process
required by the U.S. Food and Drug Administration (FDA); the committee concluded
that no evidence in the available information on BHT demonstrates a hazard to
the public when it is used at levels that are now current and in the manner now
practiced. However, uncertainties exist requiring that additional studies should
be conducted.1 The chemical properties which make BHT an excellent preservative
may also be implicated in health effects. The oxidative characteristics and
metabolites of BHT may contribute to carcinogenicity. Some people may have
difficulty metabolizing BHT, resulting in health and behavioral changes.
This application note will demonstrate a fast and easy analytical
technique to determine the amount of BHT in foods.
Headspace (HS) sample introduction is used because it provides
a means to analyze food without any sample preparation.
Headspace eliminates the need for solvents and other samplepreparation steps to reduce cost and complexity of extraction.
In this application note, an adsorbent trap is used to
concentrate the headspace sample and increase sensitivity,
allowing for low-level detection or small sample sizes.
The analysis is carried out with gas chromatography mass
spectrometry (GC/MS) – this will allow us to resolve the BHT
from other volatile compounds in the food matrices and
provide positive identification of the BHT with mass spectral
data. Calibration of the system and analysis of food samples
will be demonstrated.
The instrumental platform for this application is the
TurboMatrix™ HS Trap coupled to a Clarus® 680 GC/MS,
both platforms from PerkinElmer. The transfer line of the
HS was directly connected to the Elite™-17ms column with a
universal butt connector. The samples are heated in a sealed
vial at 80 ˚C for 30 minutes to drive the BHT from the food
into the headspace. Using automated headspace technology,
the gas is extracted from the vial, concentrated on an
adsorbent trap (PerkinElmer® Air Toxics), and injected into
the GC/MS system. Table 1 shows the detailed instrumental
setup parameters for the HS Trap-GC/MS system.
Table 1. Instrument Parameters.
Sample Introduction PerkinElmer
TurboMatrix HS-40 Trap
Needle Temperature 90 ˚C
Transfer Line Temperature 110 ˚C
Oven Temperature 80 ˚C
Trap Low Temperature 40 ˚C
Trap High Temperature 280 ˚C
Dry Purge (Helium) 5 min
Trap Hold Time 6 min
Desorb Time 0.5 min
Thermostatting Time Gas Chromatograph PerkinElmer Clarus 680 GC
Headspace Connector Universal Connector
Inlet Temperature
150 ˚C
Oven Program Initial Temp
50 ˚C
Hold Time 1 1 min
Ramp 1 25 ˚C/min to 280 ˚C
Hold Time 2 1.8 min
Vacuum Compensation On
Headspace Control On
30 min
Column Elite-17ms
30 m x 0.25 mm x 0.25 μm
Pressurization Time 1 min
Carrier Gas Helium
Decay Time 2 min
Mass Spectrometer PerkinElmer Clarus 600 MS
Column Pressure 17 psi
Mass Range 45-300 u
Vial Pressure 35 psi
Solvent Delay Time 0.1 min
Desorb Pressure 10 psi
Scan Time 0.20 sec
Universal Capillary Column Connector Part No. N9302149
InterScan Delay Time 0.02 sec
Transfer Line Fused Silica 2 m x 320 μm
Transfer Line Temperature 240 °C
Source Temperature 200 °C
Multiplier 500 V
Calibration-Standards Preparation
A 10 ng/μL standard stock solution was prepared by diluting
0.1 mL of a 1000 μg/mL BHT standard to 10 mL with
methanol. 1 ng/μL, 2 ng/μL and 5 ng/μL standard working
solutions were prepared by diluting 0.1 mL, 0.2 mL and
0.5 mL of a 10 ng/μL BHT standard to 1 mL with methanol.
20 ng/μL, 50 ng/μL and 100 ng/μL standard working solution
was prepared by diluting 0.02 mL, 0.05 mL and 0.1 mL of
a 1000 μg/mL BHT standard to 1 mL with methanol.
The working curve was prepared by injecting 1 μL of each
working standard solution into headspace vials. Working
calibration standards at 1, 2, 5, 10, 20, 50, and 100 ng
were prepared fresh each day.
One gram of each food sample purchased at local Shanghai
markets were placed into the headspace vials. All headspace
vials were sealed immediately and transferred to the
headspace-trap vial tray.
Figure 1. Example chromatogram of a 100 ng standard injection of BHT.
Results and Discussion
The instrument calibration included seven calibration levels
in the working curve; the response of this calibration curve
was linear (Table 2). Additionally, the method is precise
throughout the calibration range, as demonstrated by the
relative standard deviation of 3.2% at the calibration limit
(1 ng, n=5) and 1.9% at 10 ng (n=5).
Figure 1 is an extracted ion chromatogram, of m/z 205,
from the analysis of a 100 ng BHT standard. Figure 2
demonstrates the spectral data of BHT which matches
exactly the fragmentation of BHT in the NIST® spectral library.
Following the calibration of the system, five food samples
were analyzed: a cracker, powdered coffee creamer, instant
noodles, sausage, and tea leaves. The BHT concentrations
are quantified (Table 3). BHT concentration in the food
samples analyzed here was below the quantitation limit
of 1 ng/g. It can be seen in Figure 3 (Page 4) that the BHT
peak is easily identified in the sample analysis. Each sample
was analyzed in triplicate – the area reproducibility achieved
(Table 3) demonstrates that the method remains very precise,
even below the quantitation limit.
Figure 2. Background subtracted spectra from the analysis of a BHT standard.
Table 2. Calibration Table for BHT.
Time (min)
Ion 1
Ion 2
(n=5 at 1 ng)
(n=5 at 10 ng)
Table 3. %RSD of BHT in Food Samples.
BHT (ng/g) in 1 g of Sample
BHT (ng/g) in
1 g of Duplicate
BHT (ng/g) in
1 g of Triplicate
Mean (ng/g)
Coffee Creamer
Instant Noodles
Tea Leaves
Figure 3. Resultant chromatogram from the analysis of instant noodles for BHT.
BHT is a common food additive used to prevent spoilage.
Analysis of BHT is needed for both food quality and safety
reasons. Food is often a complicated sample matrix which is
time consuming to prepare and analyze. This method uses
headspace technology to virtually eliminate sample preparation
and reduce the cost and labor of the analysis. In addition to
eliminating sample preparation, the method is both sensitive
and precise as demonstrated by the analysis of standard
reference materials and a variety of food samples. The
throughput of the system is further improved by the Clarus
680 GC/MS with a fast-cooling GC oven, further improving
throughput and productivity. The MS data provides positive
confirmation of BHT in sample matrices.
1.Database of Select Committee on GRAS Substances
(SCOGS) Reviews-Butylated Hydroxytoluene (BHT),
available from
PerkinElmer, Inc.
940 Winter Street
Waltham, MA 02451 USA
P: (800) 762-4000 or
(+1) 203-925-4602
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