Journal of Analytical Toxicology, Vol. 31, July/August 2007
Rapid and UnambiguousIdentification of Melamine in
Contaminated Pet Food Basedon Mass Spectrometry
with Four Degreesof Confirmation
Teresa M. Vail, Patrick R. Jones, and O. David Sparkman*
University of the Pacific, Stockton, California
Abstract I
A method for analyzing pet food without sample processingis
described for rapid identification of melamine based on mass
spectrometry (MS) usingsoft ionization by direct analysisin real
time (DART) to provide accurate measurement of massand
isotope-peak intensities,in-source collisionally activated
dissociation (CAD) fragmentation, and determination of active
hydrogens. Usually, MS analysesbased on other than electron
ionization (El) spectra can be suspectbecauseof the limited
amount of information provided by a single massspectral peak (or
very few peaks). In such cases,additional degrees of confirmation
are desirable to increase confidence in the experimental results.
Chromatographic retention time can provide a degree of
confidence; however, this requires time and, in some cases,
detailed sample processing.Currently, the United States Food and
Drug Administration usesa gas chromatography-El-MS technique
for the determination of melamine in pet food that involvessample
extraction and derivatization prior to a lengthy chromatographic
separation. In the method described here, identification is also
confirmed through a determination of the number of active
hydrogen atoms in the analyte molecule achieved by
hydrogen/deuterium (H/D) exchange by treatment with deuterium
oxide (D20) at the initial stage of analysis.Cross-correlationof
these four experimental data provides an unambiguous
identification of melamine in contaminated pet food without the
need for any sample preparation or chromatography.Limits of
detection and the validity of the H/D exchange method as a
confirmatory technique are also presented.
Introduction
Melamine, chemically known as 2,4,6-triamino-l,3,5-triazine, is commonly used as a monomer in the production of
plastic resin used in the fabrication of dishes, furniture, and
* Author to whom correspondenceshould be addressed:ChemistryDepartment,
University of the Pacific, 360~ PacificAve., Stockton,CA 95211.
E-mail: [email protected].
304
flooring (1). Melaminehas recently been detected in substances
used in pet food and actual samples of the pet food, instigating
a recall of over 60 million cans and packages in the U.S.,
Canada, and Mexicomanufactured by MenuFoods (Streetsville,
ON, Canada). The melamine contamination has been found to
be associatedwith wheat gluten imported from a Chinese company, Xuzhou Anying Biologic TechnologyDevelopment (Pei
County, Jiangsu, China). Apparently,some Asian countries use
melamine as a fertilizer because of its rich nitrogen content.
The tainted wheat gluten was imported by a Canadian company, ChemNutra, Inc., that supplies wheat gluten to Menu
Foods. Wheat gluten is a common pet foodcomponent, used as
a binder and source of protein. The contaminated foods have
reportedly caused the death of more than 14 animals to date
(2).
In view of the melamine's apparent toxicityin canines and felines, there is renewed concern regarding possible human
health ramifications if melamine were to enter the human
food supply. According to the CarcinogenicPotency Project at
UC Berkeley, melamine is not toxic to rodents, a commonly
used model to relate to human effects (3). Further, in March
1989, melamine was removed from the list of toxic chemicals
under Section 313 of Title III of SARA(Superfund Amendment Reauthorization Act) "because there is insufficient evidence to establish that the substance may cause, or can reasonably be anticipated to cause, adverse effects to human
health or the environment" as stated by Melamine Chemicals,
Inc. (4).
United States Food and Drug Administration (U.S.FDA) laboratories have used analytical tools such as infrared spectroscopy, liquid (LC)and gas chromatography(GC), mass spectrometry (MS), and GC-MS to identify melamine as a
contaminant in the wheat gluten used in the pet food (5).
Each of these analyses requires extensive sample preparation
because the pet food is a complicated matrix composed of
many compounds. In order to confirm the presence of
melamine, even when other methods are initially used to detect
its presence, U.S. FDA laboratories used methanol extraction
and trimethylsilyl derivatization, which requires over 4 h of
Reproduction (photocopying) of editorial content of this journal is prohibited without publisher's permission.
Journal of Analytical Toxicology, Vol. 31, July/August 2007
sample preparation (6) before the GC-MS analysis can be carried out, requiring another hour, including a required blank
analysis. Therefore, a time-efficient method of confrming the
presence of food contaminants is most advantageous. The
method reported here is a rapid analysis technique using the
direct analysis in real time (DART) ion source, which can
ionize samples in the solid, liquid, or gaseous phase. The U.S.
FDA actually used DART (in one case interfaced to a Thermo
Fisher LTQ-FTMS and in another to a triple-quadrupole instrument) in the analysis of melamine, but required confirmation by the cited GC-MS method, which was the rate-limiting step in producing results (7).
There are many ionization processes taking place in the
DART interface; however, the ions encountered most often
are protonated analyte molecules. Metastable atoms of He produced by a corona discharge interact with water molecules in
air forming protonated water clusters, which participate in
ion molecule reactions with the analyte to produce the protonated analyte molecules (8). Ionization of samples using the
DART is possible even in the presence of complicated matrices with little to no sample preparation (9). The DART,when
interfaced to a JEOL AccuTOI~-MS, allows for accurate mass
measurements to the nearest 0.0001 Da, which translates into
an elemental composition for compounds that have a nominal
mass of < 200 Da. The AccuTOFalso providesaccurate isotopepeak intensities, which can be used to further confirm the elemental composition of the analyte.
After rings-plus-double-bond calculations and formula
searches using utilities in the National Institute of Standards
and Technology (NIST) Mass Spectral Search Program and
the NIST05 Mass Spectral Database, possible compounds can
be proposed based on these elemental compositions (10). Traditionally, a compound's structural identity can be verified
using in-source collisionally activated dissociation (CAD), a
third degree of analyte confirmation. For the fourth degree of
analyte confirmation, the presence of active hydrogens in the
structure that will exchange with deuterium upon contact
with deuterium oxide (D20) is exploited. In 1980, Hunt and
Sethi (11) showed that hydrogen/deuterium (H/D) exchange is
an appropriate structural analysis technique for small
molecules. To demonstrate the validity of the H/D exchange
method in the DARTinterface, experiments were carried out
in situ using commercially available amino acids and the constitutional isomers dipropylene glycol (DPG) and diethylene
glycol monoethyl ether (DEGE) as test compounds.
Using the optimized technique, melamine was detected and
identified in contaminated ALPO| Prime Cuts In Gravy
through cross-correlation of results obtained in a matter of
minutes by the four experimental procedures described here.
Experimental
Reagents and materials
The amino acids, melamine, DPG, deuterium oxide, and
DEGE were purchased from Sigma-Aldrich (St. Louis, MO).
The HR/GC methanol and HPLC water were purchased from
VWR (Brisbane, CA). On March 30, 2007, the voluntary recall
of contaminated pet food was expanded to include ALPOPrime
Cuts In Gravy having four-digit code dates of 7037 through
7053 followed by a plant code 1159 with a "Best Before Feb.
2009" date (12). One contaminated can, London Grill
70441159, and two uncontaminated cans, Stew with Beef &
Vegetables 70121159 and Gourmet Dinner 63041159 (all purchased from the same commercial source, at the same time,
prior to the recall), were tested. The pet food was composed of
solid chunks measuring approximately 2 cmx 1.5 cm x 0.75
cm suspended in viscous gravy.
MS and sample preparation
All mass spectral analyses were performed on an AccuTOF
LC-MS (JEOL, Peabody,[VIA)equipped with a DARTion source
(Ion Sense, Peabody, ]VIA).The DARTsource was operated with
a helium flow of 4.26 L/rain heated to 200~ The DARTdischarge needle was set to 5629 V in the positive ionization
mode. The interface Orifice-1 was operated at 20 V. In order to
produce in-source CAD fragmentation, Orifice-1 was also operated at 40 V and 60 V. The operating resolving power (R) of
the AccuTOF is approximately 6000 (FWHM). The mass spectral acquisition range for these experiments was m/z 50 to
1000. Spectra were acquired at rate of 2.13 spectra/s with a
delay time of 3 ms and a data sampling interval of 0.5 ns per
point without the use of data compression. Spectra of polyethylene glycol (PEG) were acquired for each data file to provide an
internal accurate mass calibration.
All standards and gravy of pet food were analyzed using disposable glass melting-point capillaries purchased from VWR
Figure I. Side view of interface between DART ion source and JEOLAccuTOF mass spectrometer (A). Pari Plus nebulizer is in the foreground of
the picture. Tube from nebulizer is being held in the interface by the
stainless steel sample holder provided by Ion Sense. Opposite side of
interface (B). Stainless steel cone on left side of picture is Orifice-1. The
insulating ceramic cap from which He flows is on the right side of the
picture.
305
Journal of Analytical Toxicology, Vol. 31, July/August 2007
Table I. Solutions of Melamine used to Determine LOD of Exact Mass Measurement, In-Source CAD Fragmentation, and
H/D Exchange Confirmation*
Concentration
of Melamine
(ng/pL)
Measured
ExactMass
• Error(ppm)
100
127.0728
-+3.1
CentroidedIn-SourceCAD
MassSpectra
Orifice-1 Operatedat 60 V
Profile MassSpectraof Melaminein
Presenceof D20 (Numberof H/D
ExchangesMarkedwithAsterisks)
J
*
[M+HI ~ I
i
*7 exchanges
.
i
*
mtz
75
127.0726
+4.7
i
r-"~[M+ HI.
FI
*S exchanges
L"
al/r
50
~
it". ~ + Hr
127.0725
+ 5.5
.
.
-
[
1
i
I
i
.
": lU * H]'
25
~ [ M + Hi*
127,0722
+ 7.9
FI
!
!
I
*3 exchanges
i,
m~
20
i
" ~ [M * HI+
FI
127,0723
_+7.1
m~
i
*2 exchanges
,
m/z
Ir IM + H]*
10
127.0721
+ 8.7
FI
i
~ + HI"
/~
m/z
1
127.0719
_+10.2
I
*2 exchanges
m/z
i
~r'~ IM * HI"
'JIM + H1i
*2 exchanges
I
i
9
1
4
i
~Vz
m/:
* All peaks representing protonated molecules are labeled with (M + H] § Mass spectral peaks labeled with FI at nominal m/z 85 are thought to represent fragment ions consistent
with the loss of cyanamide from the protonated melamine molecule, [M + H - CH2N2]§
306
Journal of Analytical Toxicology, Vol. 31, July/August 2007
surface of the glass melting-point capillary (a volume estimated to be 1 pL). To analyze the chunks of pet food, stainless
steel tweezers were used to extract small portions that were
then held in the DART ion source.
In order to induce H/D exchange, the interface was saturated
with D20 using a Pari Plus nebulizer. Approximately 2 mL of
deuterium oxide was poured into the nebulizer's cartridge. Ni-
(Brisbane, CA). The amino acids were dissolved in methanol to
produce solutions that had a concentration of 10 mg/mL. All
other compounds were analyzed in pure form (liquid or solid).
In order to analyze the samples, the sealed end of the meltingpoint capillaries were dipped into the liquids and then placed
in the helium gas stream of the DARTion source. The amount
of sample analyzed was the amount that adhered to the outer
Table II. Four Amino Acids (AA) used as Test Compounds*
Amino
Acid (AA)
Proline
Protonated AA Structure
(Active Hydrogen Atoms Circled)
Profile Mass Spectra of AA with H/D Exchange
2
I"
[M + H]*
3
~.ts hL
, ~
" ~ '
Alanine
4
[M + H] *
i
k~
. . .~
A.
....
'~s
....
9
"
~s
"
'~
Asparagine
5
/
6
I
'
9 1~ ~'
"
"
' i~ A . . . .
111
Lysine
5
1
[M +LHI§ !
I.k
"
" ib
"
1
1~
i,o
" ~
"
"
I~"
"
Ik
* Active hydrogenatoms,which will exchangewith deuterium,are circledon the structures.The peak representingthe protonatedmoleculein eachspectrumis labeledwith
[M + H]§ The massspectralpeakspresentbecauseof H/D exchangeare labeledwith the numberof hydrogenatomsexchangedwith deuterium.
307
Journal of Analytical Toxicology, Vol, 31, July/August 2007
trogen gas was used to nebulize the liquid with regulator set at
500 kPa with a flow rate of 150 L/rain. The exit tube of the nebulizer was held in the interface using a sample holder provided
by Ion Sense (Figure 1). Saturation of the interface is accomplished in less than a minute. Recovery to a normal vapor
state (no D20 present) also takes less than 1 min. The condition
of the interface with respect to saturation with D20 or no D20
is determined by observing the real-time total-ion-current
monitor.
To determine the limit of detection (LOD) of melamine in air
with Orifice-1 operating at 20 V and 60 V and in the presence
of D20, melamine was dissolved in a mixture of water and
methanol (3:1, v/v) at multiple concentrations (Table I). At
concentrations higher than listed, the signal was saturated altering the exact mass measurement and isotope-peak intensity
ratios of the signal. Three samples of each solution were analyzed consecutively in air and in the presence of D20.
The analytical process involves the acquisition of two sets of
spectra. Each spectral set consists of placing a drop of a PEG
(PEG 600/1000) solution suspended from the sealed end of a
glass melting-point capillary (as explained) in the interface to
provide a mass-to-charge ratio calibration scale over the range
of 50 to 1000 requiring about 30 s. The sample is then introduced into the interface requiring another 30 s of acquisition
time. The interface is flooded with the D20 vapor (< i min) followed by acquisition of spectra for another 30 s. This is a total
analysis time of approximately 3 min. A separate acquisition for
in-source CAD with its own mass-to-charge ratio calibration
scale is required. This acquisition requires another 1-1.5 rain.
F,affme~ Io.
IM 9ii1.
A
Data analysis is accomplished in another 3-4 rain. The total
time from opening the can of pet food to completing the analysis is < 10 min.
Results and Discussion
In high-resolution MS, accurate mass measurement and
isotope-peak intensities can provide sufficient information to
determine the elemental composition of a compound. From
the elemental composition, the number of rings plus doublebonds can be calculated to begin to get an idea of the structure
of the compound. In order to verify possible structures of ions
representing intact molecules formed by 'soft ionization' techniques, fragmentation spectra are often produced by in-source
CAD or MS-MS. Because in-source CAD fragmentation on the
AccuTOF is induced by increasing the Orifice-1 voltage, any
and all ions are fragmented. Such fragmentation spectra may
be inconclusive for identification; thus, additional structural information may be required such as that provided by H/D exchange.
Amino acids
Amino acids were analyzed in open air (saturated with H20)
and with the interface saturated with D20. Each active hydrogen, n, as well as the proton associated with the protonated molecule is predicted to exchange with deuterium to
form n + 1 ions. When the amino acids were analyzed, clusters
of mass spectral peaks representing the protonated molecule
and successively larger numbers of exchanged hydrogen atoms
were detected as predicted. The results are shown in Table II.
These results validate the reliability of in situ H/D exchange in
the DART interface.
uTon~3
l
m/:
,~
lu 9 .l'
oi
ii
i
I~L
iI ~
9
,~
Figure 2. Mass spectra acquired using in-source CAD with Orifice-I operated at 40 V of an unknown compound (A). The base peak represents
a fragment ion at m/z 59.0471. The protonated molecule peak labeled
[M + HI + is still present at m/z 135.0976 with a 80% relative intensity.
A mass spectral peak at m/z 117.0853 with a 45% relative intensity is
thought to represent an ion formed by the loss of water from the protonated molecule. This spectrum is presented in the centroided mode. Mass
spectrum of the protonated molecule region presented in the profile
mode of the unknown compound analyzed in the presence of D20 (B).
The protonated molecule peak is labeled [M + H] +. The mass spectral
peaks due to H/D exchange are labeled with the number of hydrogen
atoms exchanged with deuterium.
308
Unknown identification
In an ana]ysis unrelated to the pet food, an unknown compound captured on filter paper was analyzed using the
DART/AccuTOF instrument. The unknown's protonated
molecule peak was observed at m/z 135.0976. Using accurate
mass measurement, the elemental composition, within + 5
mmu, was determined to be C6H1503 . This elemental composition was confirmed through the use of isotope-peak intensity
ratios. A Formula Search on C6H1403 using the NIST05 Mass
Spectral Database produced 13 candidate compounds. Compounds that had no synonyms or commercial names and were
not common to multiple databases were eliminated from consideration. The in-source CAD fragmentation spectrum of the
unknown showed a mass spectral peak at m/z 117.0853 when
Orifice-1 was set to 40 V and 60 V; this mass spectral peak was
consistent with a loss of a molecule of water from the protonated molecule, [M + H - H20] § indicating the unknown compound must contain at least one hydroxyl group (Figure 2A).
Two constitutional isomers that are commercially common
and logically related to the possible origin of the unknown
compound were good candidates for the unknown's identity;
one contained a single hydroxyl group (DEGE, a common solvent used to break down adhesives), and the other contained
Journal of Analytical Toxicology, Vol. 31, July/August 2007
two hydroxyl groups (DPG, a common component of plasticizers, flavors, and fragrances).
Unfortunately, the in-source CAD spectrum did not provide
a way to differentiate between the two candidate compounds by
either the nominal mass or the accurate mass of the fragment
ions. At the time of the initial analyses, authentic compounds
of the two analytes were not available to determine if the spectrum produced by in-source CADwas the same for both compounds. The dilemma was resolved using in-source H/D exchange on the DART.The unknown analyte's mass spectrum
(Figure 2B) obtained through H/D exchange exhibited a cluster
of peaks representing the protonated molecule and three other
peaks representing an n + i exchange where n was 2. This
meant that the analyte had to have two active hydrogen atoms,
that is, the analyte had to be DPG.
Authentic samples of both DPG and DEGE were obtained.
The in-source CAD fragmentation spectra of these authentic
samples produced mass spectral peaks representing the protonated molecule and the loss of water from the protonated
molecule, as expected (Figures 3A and B, respectively). The
spectra also showed unique fragment ions for DPG (nominal
m/z 59) and DEGE (nominal m/z 73 and 89). The in-source
CAD fragmentation spectra of the two authentic compounds
were different enough to differentiate between these constitutional isomers. The in-source CAD mass spectrum of the unknown matched the in-source CAD mass spectrum of DPG obtained at the same Orifice-1 voltage.
Any ambiguity associated with the possibility of other anaA
Fl~nalu
lytes being present was eliminated through the use of H/D exchange. The mass spectrum obtained through H/D exchange
for both compounds is shown in Figure 4. The DEGE spectrum
obtained with H/D exchange shows a peak representing the
protonated molecule at m/z 135.1024 as well as two mass spectral peaks representing two consecutive H/D exchanges. The
DPG mass spectrum obtained using H/D exchange exhibits
the protonated molecule peak at rn/z 135.1024 as well as three
mass spectral peaks representing three consecutive H/D exchanges. Each compound's spectrum obtained with H/D exchange was compared to the H/D exchange spectrum of the unknown compound captured on filter paper. Both the unknown
and the DPG spectra exhibit a cluster of peaks representing the
protonated molecule and three consecutive H/D exchanges.
Because of the similarity of peak clusters between the spectra
obtained using the H/D exchange for the DPG and the unknown on the filters, the unknown was confirmed by cross-correlation of results from four different sets of results to be
dipropylene glycol.
Pet food analysis
The DARTAccuTOF mass spectral analysis of the contaminated pet food (liquid and solid) resulted in spectra that exhibited a base peak at rn/z 127.0715; such a mass spectral peak
was absent in the spectra obtained from liquid and solid
aliquots taken from the two cans of the uncontaminated pet
food (Figure 5). The calculated exact mass of the [M + H]§ of
melamine is 127.0732 Da (as determined using the "Isotope
Ratio Calculator" in the MS Tools software available from
ChemSW, Fairfield, CA),whereas the measured accurate mass
of the [M + HI§ of the melamine standard was 127.0713 Da
(Figure 6A), yielding a difference of 1.9 mmu. It should be
IM 9 H + ~ O ] "
+i
Ill 9 Iq.
Ilil + H r
f1~,1024 " " , b
"~%"n JI@;'|
~§
13@*l@1@
r+lgmN~
"l
L
7
,
Fi.,-,.or
1l~,.Ewz~z
Figure 3. Mass spectra obtained using in-source CAD with Orifice-1 operated at 40 V of DPG (dipropylene glycol) (A) and DEGE (diethylene
glycol monoethyl ether) (B). The base peak in the DPG spectrum at m/z
59.0547 representsa fragment ion. The protonated molecule peak is present at m/z 135.0995 with 35% relative intensity. The mass spectral
peak at rn/z 117.0883 with a 40% relative intensity represents an ion
formed by the loss of water from the protonated molecule. The structure
of DPG is shown with A. The base peak in the DEGEspectrum is a fragment ion at m/z 73.0670. The protonated molecule peak also at m/z
135.0995 has a 52% relative intensity. There is a peak at m/z 89.0586
with a 50% relative intensity representing a fragment ion of the protonated molecule. A mass spectral peak at rn/z 117.0883 with a relative intensity of 15% representsan ion formed by the lossof water from the protonated molecule. The structure of DEGE is shown with 8.
3
|
i\
.....
Figure 4. Spectrum of DEGE in the presence of D20 (A). The peak
cluster beginning at nominal m/z 135 is enlarged in order to see the
presence of a peak representing [M + H] § and two consecutive H/D exchanges. Spectrum of DPG in the presence of D20 (8). The peak cluster
beginning at nominal m/z 135 is enlarged in order to see the presence
of a peak representing [M + H] § and three consecutive H/D exchanges.
All of these mass spectral data are presented in profile mode.
309
Journal of Analytical Toxicology, Vol. 31, July/August 2007
A
"~
....:'.-
~4
'
i
I
I
, ,LII
i
i
I
i~
ii
llr/&.15/
luM
m.zt~l
I
,
Figure 5. Mass spectraof uncontaminated (A) and contaminated dog food
(B). Mass spectral peaks endogenousto the dog food are marked with asterisks. The base peak in spectrum A is at m/z 145.0520. The base peak
in spectrum B is at m/z 127.0715, consistentwith the exact massof a protonated molecule of melamine. Notice that all the endogenous mass
spectral peaks in the contaminated dog food have a much lower relative
intensity to the protonated molecule of melamine in the mass spectrum
of the contaminated dog food. A mass spectral peak at m/z | 27.0395 is
present in the mass spectrum of the uncontaminated dog food; it is also
visible in the inserted profile display of the melamine protonated
molecule peak but does not affect the peak assigment.
!,%
a
121071]
T" iT
F4*.-ClbN~I'
o5.o~04
H-ii 4-.
Im9HI*
noted that the mass spectrum of both the contaminated and
the uncontaminated dog food exhibited a peak with nominal
rn/z 127. However, the accurate mass for this peak in the spectrum of the uncontaminated dog food was determined to be
127.0395 Da. Using the "Elemental Compositions" program in
the MS Tools software with an error limit of 2 mmu and elemental constraints of C 1/20 H 0/50 O 0/10 N 0/10, the only resuiting formula was [C6H603+ H]. The protonated molecule of
this C6H603species and the protonated molecule of melamine
can be partially separated with the resolving power of the AccuTOF as seen in the insert in Figure 5B. This means that the
centroided value of the peak with nominal rn/z 127 represents
only the protonated molecule of melamine in the contaminated dog food.
Further, the isotope-peak intensities associated with the ion
with nominal m/z 127 in the mass spectrum of the contaminated dog food were consistent with the elemental composition
of [melamine + H]§ C3HTN6.In order to verify that the base
peak represented [melamine + H]§ a search in the NIST05
Mass Spectral Database was performed using the elemental
composition C3H6N6. The search resulted in one spectrum,
1,3,5-triazine-2,4,6-triamine commonly known as melamine.
The third degree of analyte analysis, in-source CAD,was carried
out (Figure 6) for both the contaminated pet food sample and
an authentic sample of melamine. The stability of the triazine,
a conjugated-ring structure, is evident in its fragmentation
behavior as the base peak of both spectra represents the protonated molecule, even at relatively high fragmentation energy
(60 V). A mass spectral peak at nominal m/z 85 is present in
each spectrum representing a logical loss of a molecule of
cyanamide, N=C-NH2, from the protonated molecule; this [M
- 42] § peak was not present until the in-source CAD voltage
was increased to 60 V to induce fragmentation. The loss of
cyanamide from the protonated molecule is a result of chargeinduced fragmentation. Scheme 1 shows a possible mechanism by which the fragment ion of nominal m/z 85 is produced.
It should be noted that the measured accurate mass-tocharge ratio value for the protonated molecule of melamine in
m . H-OH .,r
rn/z 127
m,'=
Figure 6. Mass spectrum of pure melamine acquired using in-source
CAD with Orifice-1 operated at 60 V (A). The basepeak at m/z 127.0713
representsthe protonated molecule. The peak at m/z 85.0504 with a 54%
relative intensity representsa fragment ion consistent with the loss of a
molecule of cyanamide. The structure of a protonated molecule of
melamine is also shown. Mass spectrum of melamine in the contaminated
dog food acquired using in-source CAD with Orifice-1 operated at 60 V
(B). The base peak at m/z 127.0723 representsthe protonated molecule.
The peak at m/z 85.0495 with 38% intensity relative to the basepeak represents a fragment ion consistent with the loss of a molecule of
cyanamide. ]he peak at nominal m/z 114 is consistent with creatinine, a
degradation product of creatine. This unrelated ion with nominal rn/z 114
is representedin the mass spectrum acquired by in-source CAD because,
unlike MS-MS, it is not possible to isolate a precursor ion of a specific
mass-to-charge ratio value prior to insource CAD.
310
H,/N"~,. C ~ N'~,, C /
I
N",,, H
H//N"....C ~ / N ' ~ C / N ' ~ . , H
II
-
H=-"i~'-.
/
/ ~c/~
I(
N.~/.
~1
/ \
/
~
Z
m/z
Scheme
I
NH~
85
1. Fragmentation mechanism of protonated molecule.
Journal of AnalyticalToxicology,Vol. 31, July/August2007
A
o
f
l
essary, a sample of the pet food could be dried before the H/D
exchange analysis. As can be seen from Figure 7, the amount
of melamine present in the contaminated pet food was well
above the 100-ppm level resulting in the exchange of all seven
hydrogen atoms.
Conclusions
B
Figure 7. Mass spectra presented in the profile mode of standard
melamine in the presence of deuterium oxide and the structure of protonated molecule of melamine with all active hydrogen atoms exchanged
for deuterium (A) and melamine detected in contaminated pet food in the
presence of deuterium oxide (B). The peak representing the protonated
molecule in each spectrum is labeled with [M + HI§ The mass spectral
peaks due to H/D exchange are labeled with the number of hydrogen
atoms exchanged with deuterium. Because of the peak intensity relative
to the intensityof the previous nominal mass-to-charge ratio value peak,
it is easy to distinguish a peak representing H/D exchange from the 13C
isotopepeak.
the spectrum obtained by in-source CAD for the standard was
127.0713 and in the contaminated dog food was 127.0723
(Figure 6). This 1 mmu range in these two measurements represents the range of variation for all measurements.
The H/D exchange analysis was performed on the standard
melamine and the contaminated pet food. Figure 7 shows the
spectrum of each in the presence of deuterium oxide. A cluster
of peaks representing the protonated molecule and seven H/D
exchanges are present in each spectrum as predicted by the active hydrogens in melamine's structure. This provided the
fourth degree of confirmation and results in an unambiguous
identification.
The LOD results for the third and fourth degrees of confirmation are summarized in Table I. The LOD of H/D exchange
was found to be 100 ppm where all seven hydrogen atoms exchanged for deuterium. At 75 ppm, only five hydrogen atoms
exchanged for deuterium. However, melamine could be detected at 1 ppm with three degrees of confirmation: accurate
mass measurement, isotope peak intensities, and CAD fragmentation.
As the concentration of the melamine decreases in the standards in Table I, the number of sites undergoing H/D exchange
is diminished. This can be explained by the fact that the standards were prepared in a methanol/water solution. The water
in the standards would definitely compete for the deuterium resuiting in fewer exchanges with the melamine at lower concentrations. The standards were prepared using water because
this provided a matrix that was similar to the pet food. The pet
food did contain water. If confirmation below 100 ppm was nec-
The use of a DARTion source coupled with high-resolution
mass spectral analysis yields exact mass measurement and accurate isotope-peak intensities to detect and identify melamine
in contaminated pet food. The presence of melamine in the pet
food is confirmed by in-source CADfragmentation with LOD of
1 ppm. To eliminate any ambiguity due to the presence of
many other compounds present in the complex pet food matrix, H/D exchange is used as a fourth degree of identification
confirmation at LOD 100 ppm without the need for extensive
sample extraction, derivatization, or further analysis by
GC-MS.
The limitations of this method involve the lack of an ability
to have the H/D exchange confirmation below 100 ppm in wet
food, the inability to select precursor ions for the in-source
CAD confirmation of melamine, and the possible interference
by materials that have the same nominal mass as melamine but
cannot be separated from the ions representing the melamine
because of the resolving power of the instrument. However,
even in the case of unresolved contaminants, their presence
can be confirmed by analysis of uncontaminated pet food and
the probability of those contaminants having the same number
of active hydrogen atoms is low. Although no effort was made
to establish a quantitation scheme, a method could easily and
quickly be developed because of analyses performed on other
compounds in dog food.
Acknowledgments
The authors gratefully acknowledge the financial support of
ITT AdvancedScience and Engineering, Reston, VA,and the assistance provided by the Pacific Mass Spectrometry Facility in
the Chemistry Department at the University of the Pacific.
DART is a registered trademark of JEOL USA, Peabody, MA.
LTQ-FTis a trademark of Thermo Fisher, Waltham, MA.ALPO
is a registered trademark of Soci~t~ des Produits Nestl~ S~.,
Vevey, Switzerland.
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