Detection of melamine in milk products by surface desorption atmospheric pressure chemical ionization mass spectrometry.
ABSTRACT Without any sample pretreatment, trace amounts of melamine in various milk products were rapidly detected noting the characteristic fragments (i.e., m/z 110, 85, and 60) in the MS/MS spectrum of protonated melamine molecules (m/z 127) recorded by using surface desorption atmospheric pressure chemical ionization mass spectrometry. Signal responses of the most abundant ionic fragment (m/z 85) of protonated melamine were well correlated with the amounts of melaime in milk products, showing a dynamic range about 5 orders of magnitude. The limit of detection (LOD) was found to be 3.4 x 10(-15) g/mm(2) (S/N = 3) for the detection of pure melamine deposited on the paper surface, which was much lower than that for detection of melamine in powdered milk (1.6 x 10(-11) g/mm(2), S/N = 3) or liquid milk (1.3 x 10(-12) g/mm(2), S/N = 3). The significant difference in LOD was ascribed to the relatively strong molecular interactions between melamine and the matrix such as proteins in the milk products. As demonstrated using desorption electrospray ionization (DESI) for melamine detection, weakening the molecular interaction between analytes and proteins is proposed as a general strategy to improve the sensitivity of ambient mass spectrometry for direct detection of analytes bound in protein matrixes. The relative standard deviation (RSD) and the recovery of this method were found to be 5.2 approximately 11.9% and 87 approximately 113%, respectively, for the detection of melamine in milk products. A single sample analysis was completed within a few seconds, providing a particularly convenient way to rapidly screen melamine presence in milk products.
- SourceAvailable from: PubMed Central[show abstract] [hide abstract]
ABSTRACT: A straight-forward analytical strategy called internal extractive electrospray ionization mass spectrometry (iEESI-MS), which combines solvent extraction of chemicals inside a bulk sample with in situ electrospray ionization mass spectrometry, has been established to directly characterize the interior of a bulk sample with molecular specificity. The method allows both qualitative and quantitative analysis of analytes distributed in a 3-dimensional volume (e.g., 1 ~ 100 mm(3)) of various synthetic and biological matrices (e.g., chewing gum, leaves, fruits, roots, pork, lung tissues) without either mashing the sample or matrix separation. Using different extraction solvents, online chromatographic separation of chemicals inside the sample volume was observed during iEESI-MS analysis. The presented method is featured by the high speed of analysis, high sensitivity, low sample consumption and minimal sample preparation and/or degradation, offering unique possibilities for advanced applications in plant science, clinical diagnosis, catalyst studies, and materials science.Scientific Reports 08/2013; 3:2495. · 2.93 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Plasma-based ambient mass spectrometry is emerging as a frontier technology for direct analysis of sample that employs low-energy plasma as the ionization reagent. The versatile sources of ambient mass spectrometry (MS) can be classified according to the plasma formation approaches; namely, corona discharge, glow discharge, dielectric barrier discharge, and microwave-induced discharge. These techniques allow pretreatment-free detection of samples, ranging from biological materials (e.g., flies, bacteria, plants, tissues, peptides, metabolites, and lipids) to pharmaceuticals, food-stuffs, polymers, chemical warfare reagents, and daily-use chemicals. In most cases, plasma-based ambient MS performs well as a qualitative tool and as an analyzer for semi-quantitation. Herein, we provide an overview of the key concepts, mechanisms, and applications of plasma-based ambient MS techniques, and discuss the challenges and outlook. © 2013 Wiley Periodicals, Inc. Mass Spec Rev.Mass Spectrometry Reviews 12/2013; · 7.74 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: The Kjeldahl method and four classic spectrophotometric methods (Biuret, Lowry, Bradford and Markwell) were applied to evaluate the protein content of samples of UHT whole milk deliberately adulterated with melamine, ammonium sulphate or urea, which can be used to defraud milk protein and whey contents. Compared with the Kjeldahl method, the response of the spectrophotometric methods was unaffected by the addition of the nitrogen compounds to milk or whey. The methods of Bradford and Markwell were most robust and did not exhibit interference subject to composition. However, the simultaneous interpretation of results obtained using these methods with those obtained using the Kjeldahl method indicated the addition of nitrogen-rich compounds to milk and/or whey. Therefore, this work suggests a combination of results of Kjeldahl and spectrophotometric methods should be used to screen for milk adulteration by these compounds.Food Chemistry 12/2013; 141(4):3649-55. · 3.33 Impact Factor
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Detection of Melamine in Milk Products by Surface Desorption
Atmospheric Pressure Chemical Ionization Mass Spectrometry
Shuiping Yang, Jianhua Ding, Jian Zheng, Bin Hu, Jianqiang
Li, Huanwen Chen, Zhiquan Zhou, and Xiaolin Qiao
Anal. Chem., 2009, 81 (7), 2426-2436• DOI: 10.1021/ac900063u • Publication Date (Web): 05 March 2009
Downloaded from http://pubs.acs.org on March 31, 2009
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Detection of Melamine in Milk Products by Surface
Desorption Atmospheric Pressure Chemical
Ionization Mass Spectrometry
Shuiping Yang,†Jianhua Ding,†,‡Jian Zheng,§Bin Hu,†Jianqiang Li,†Huanwen Chen,*,†,‡
Zhiquan Zhou,|and Xiaolin Qiao*,|
Department of Applied Chemistry, East China Institute of Technology, Fuzhou, 344000 P. R. China, College of
Chemistry, Jilin University, Changchun, 130023 P. R. China, Beijing Centre for Physical and Chemical Analysis,
Beijing 100089, and College of Information Science and Engineering, Harbin Institute of Technology at Weihai,
Weihai 264000 P. R. China
Without any sample pretreatment, trace amounts of
melamine in various milk products were rapidly detected
noting the characteristic fragments (i.e., m/z 110, 85, and
60) in the MS/MS spectrum of protonated melamine
molecules (m/z 127) recorded by using surface desorp-
tion atmospheric pressure chemical ionization mass
spectrometry. Signal responses of the most abundant ionic
fragment (m/z 85) of protonated melamine were well
correlated with the amounts of melaime in milk products,
showing a dynamic range about 5 orders of magnitude.
The limit of detection (LOD) was found to be 3.4 × 10-15
g/mm2(S/N ) 3) for the detection of pure melamine
deposited on the paper surface, which was much lower
than that for detection of melamine in powdered milk
(1.6 × 10-11g/mm2, S/N ) 3) or liquid milk (1.3 ×
10-12g/mm2, S/N ) 3). The significant difference in
LOD was ascribed to the relatively strong molecular
interactions between melamine and the matrix such
as proteins in the milk products. As demonstrated
using desorption electrospray ionization (DESI) for
melamine detection, weakening the molecular interac-
tion between analytes and proteins is proposed as a
general strategy to improve the sensitivity of ambient
mass spectrometry for direct detection of analytes
bound in protein matrixes. The relative standard
deviation (RSD) and the recovery of this method were
found to be 5.2∼11.9% and 87∼113%, respectively,
for the detection of melamine in milk products. A single
sample analysis was completed within a few seconds,
providing a particularly convenient way to rapidly
screen melamine presence in milk products.
Melamine (1,3,5-triazine-2,4,6-triamine, MW 126) is a basic
organic compound, which has been widely used in polymer resins
or as raw material in chemical industry. Because of its high
nitrogen level (66% nitrogen by mass), melamine gives the
analytical characteristics of protein molecules when the Kjeldahl
method is used for protein analysis. Thus, melamine can be used
to adulterate protein-rich diets by unethical manufacturers. For
example, melamine was found in pet food and blamed for killing
thousands of cats and dogs in the U.S. in 2007.1Recently, high
levels of melamine were reported in pet food, wheat gluten, and
various dairy products.2-4A safety limit of melamine ingestion
has been officially set to be 1 ppm for infant formula.5,6However,
melamine concentrations in the adulterated milk products were
as high as ∼3300 ppm,7posing extreme danger to consumers. In
* Corresponding author. Dr. Huanwen Chen. Fax: (+)86-794-8258320. Phone:
(+)86-794-8258703. E-mail: email@example.com.
†East China Institute of Technology.
§Beijing Centre for Physical and Chemical Analysis.
|Harbin Institute of Technology at Weihai.
(1) Brown, C. A.; Jeong, K.-S.; Poppenga, R. H.; Puschner, B.; Miller, D. M.;
Ellis, A. E.; Kang, K.-I.; Sum, S.; Cistola, A. M.; Brown, S. A. J. Vet. Diagn.
Invest. 2007, 19, 525–531.
Anal. Chem. 2009, 81, 2426–2436
10.1021/ac900063u CCC: $40.75 2009 American Chemical Society
Analytical Chemistry, Vol. 81, No. 7, April 1, 2009
Published on Web 03/05/2009
September 2008, milk products tainted by melamine induced renal
failure and even death in pets and several infants in China.
Therefore, fast, unambiguous detection of trace amounts of
melamine in food products is of paramount importance.
Food is a typical sample of complex matrixes. Melamine in
food can be detected by either mass spectrometry (MS),8-17
chromatography,8-11,15,18-20or enzyme-linked immunosorbent
assay (ELISA)21when the matrixes are cleaned up using separa-
tion techniques such as gas chromatography (GC),18,20,22liquid
chromatography (LC),8-11,13,15,16and capillary electrophoresis
(CE).23Mass spectrometry-based methods are preferable for
melamine detection because of the high sensitivity and high
specificity. However, because of the complicated matrixes, exten-
sive multiple-step sample pretreatments (e.g., extractions, pre-
concentration, derivatization, etc.) which take tens of minutes or
even hours are normally required by conventional mass spec-
trometry-based methods. For example, the U.S. Food and Drug
administration (FDA) has published a method for screening
melamine in pet food using gas chromatography/mass spectrom-
etry (GC/MS),18which takes about 3 h for a single run due to
the tedious sample pretreatment. Undoubtedly, these methods are
not ideally suitable for rapid detection of melamine in a large
number of milk products.
Because of the capability of high throughput analysis, ambient
mass spectrometry is of increasing interest for analysis of samples
with complex matrixes. Surface desorption atmospheric pressure
chemical ionization mass spectrometry (DAPCI-MS) has been
used for direct analysis of various samples24-28with neither
sample pretreatment nor chemical contamination. With the use
of a low sheath gas flow or even without sheath gas, DAPCI can
be used for direct analysis of powdered samples, sticky liquids,
and biological tissues without a notable loss of sensitivity. Thus,
DAPCI-MS is selected as a representative ambient ionization
technique for fast screening of melamine in various milk products.
Experimental data show that DAPCI-MS serves as a sensitive,
rapid analytical tool for specific detection of melamine in both
powdered milk and liquid milk, without any sample pretreatment.
MATERIALS AND METHODS
All the milk samples were bought in local super markets and
were directly used without further treatment. Each measurement
was performed with 20 mg of powdered milk or 20 µL of undiluted
milk suspension, which was deposited on a piece of clean filter
paper to form a sample spot about 1 cm2. Melamine was added
into water and then sonicated for 30 min until no crystal was
visible to prepare the stock solution (3000 ppm). Nanograms
of melamine were deposited on a filter paper surface and then
dried in air before DAPCI-MS analysis. Chemicals such as
melamine (A.R. grade), methanol (A.R. grade), acetic acid (A.R.
grade) were bought from Chinese Chemical Reagent Co. Ltd.
(Shanghai, China). All chemicals were directly used without
any pretreatment other than dissolution and dilution with
deionized water when it was necessary.
Commercial powdered/liquid milk products containing no
melamine were used as blank powdered/liquid milk samples. Pure
melamine fine powder was precisely added into blank powdered
milk samples (1 + 9, w:w) to make a mixture containing melamine
of high concentration. The mixture was then carefully ground and
stirred to allow the even distribution of melamine in the whole
mixture. The same procedure was followed when a portion of the
mixture was diluted with blank powdered milk to make a mixture
containing melamine of lower concentrations (e.g., 1%). This
procedure was repeated several times to make a series of dilute
melamine milk powder standards. A drop of water (20 µL) was
dripped onto a piece of filter paper to form a wet area (∼1 cm2),
which was saturated by pure water but no water oozing out.
The wet side of the filter paper was pressed onto the dry
powdered milk sample, resulting in an even distribution of a
thin layer (about 1 mm in thickness) of the dry milk powders
(15∼20 mg) on the wet surface of the filter paper. The milk
strip was immediately placed on a sample holder under the
discharging needle of the DAPCI source for direct desorption/
ionization, with a distance of 2-3 mm between the tip of the
needle and the sample surface. Care was taken to ensure that
the center of the sample area was right under the discharging
needle. No powdered milk was blown off by the sheath gas of
the DAPCI source. A certain amount of pure melamine was
directly added into undiluted milk to form a heterogeneous
liquid mixture, which was then sonicated for 30 min to prepare
a standard liquid milk sample. The liquid milk sample was
deposited on a piece of filter paper to form a sample spot
around 1 cm2, which was then directly analyzed without drying
the sample or any other treatment. Note that all the liquid milk
standards were readily analyzed within 3 min after the
Experiments were carried out using a commercial linear ion
trap mass spectrometer (LTQ-XL, Finnigan, San Jose, CA) installed
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Analytical Chemistry, Vol. 81, No. 7, April 1, 2009
with a surface desorption chemical ionization source. The principle
and experimental setup for the DAPCI source were previously
described elsewhere.26-28Briefly, the DAPCI source (schemati-
cally shown in Figure 1) and the LTQ mass spectrometer were
set to work in positive/negative ion detection mode. A gentle
nitrogen sheath gas flow (0.15∼0.2 MPa, 250∼450 mL/min) was
used to bring saturated water vapor as the chemical reagent for
the production of primary ions in the DAPCI source. The gas flow
tube was heated to 450 °C, and the ionization region of the DAPCI
source was maintained at 150∼175 °C, ensuring that the water
droplets brought by the nitrogen gas flow were completely
vaporized before ionization. This maximizes the ionization
efficiency by facilitating the release of melamine from the sample
and results in a stable signal level. The DAPCI source assembly
was coaxially coupled to the LTQ mass spectrometer, allowing a
15 mm distance between the discharge tip and the instrument
inlet. The milk samples, either as powders or as liquid suspension,
were directly supplied to the DAPCI source using a paper surface
placed at 2-3 mm from the discharge tip. The sample spot was
located midway between the ion entrance of the LTQ instrument
and the DAPCI discharge needle. The angle (R) formed between
the discharge needle and the sample holder was 30∼45°, and the
angle (?) formed by the ion entrance capillary with respect to
the sample holder was 20∼25 °. The primary reagent ions,
accelerated by the electric field, impacted the sample surface for
desorption/ionization, so that protonated melamine ions (m/z 127)
were created at ambient pressure and then introduced through
the ion guide system into the LTQ mass analyzer for mass
analysis. The corona discharge voltage was ±4.5 kV with a
discharge current about 1-2 mA. The temperature of the heated
capillary of the LTQ instrument was maintained at 150 °C. The
default voltages for conversion dynode and detectors were used,
but the voltages for the ion guide system (including voltages for
the heated capillary, tube lenses, etc.) were optimized using the
signal (m/z 127) of authentic melamine (∼5 ng) on a paper
surface. Further optimization was not performed.
All the MS mass spectra were recorded with an average
duration time of 0.1 min and were background subtracted.
Collision-induced dissociation (CID) was performed with 20-35%
collision energy (CE) to the precursor ions of interest, which were
isolated with a mass window width of 1 m/z unit. MS/MS spectra
could be collected with a recording time more than 0.1 min if
necessary. Compounds of interest were identified using MS and
CID data matching of the unknown compounds against authentic
RESULTS AND DISCUSSION
Direct Analysis of Powdered Milk. Ambient Ionization
Techniques for Direct Analysis of Powdered Samples. Many ambient
mass spectrometry techniques including desorption electrospray
ionization (DESI),29-32direct analysis in real time (DART),17,25,33
atmospheric pressure glow discharge (APGD),35,36atmospheric
solids analysis probe (ASAP),37,38and neutral desorption extractive
electrospray ionization (ND-EESI)39-43are available for fast
detection of analytes on various solid surfaces with minimal sample
pretreatment. Nonfat powdered milk is a manufactured dairy
product composed of very fine particles. Typically, the average
amounts of major nutrients in the unreconstituted milk are (by
weight) 36% protein, 52% carbohydrates (predominantly lactose),
1.3% calcium, and 1.8% potassium.44Milk powders contain all 20
standard amino acids and are high in soluble vitamins and
minerals.44Therefore, powdered milk is a representative case of
complicated biological samples, which challenge modern analytical
tools for rapid analysis due to the matrixes and fine size of
particles. For instance, milk powders cannot be directly supplied
for fast analysis even using the well-established DESI technique,
because the relatively strong sheath gas flow of an open-air DESI
source30,45-50blows the powders away from the right position,
resulting in serious contamination in the source region. Certainly,
DESI and other techniques requiring high-pressure sheath gas
can be used to analyze powdered samples after the samples are
processed to form a solid surface (e.g., a tablet). However, the
sample preparation steps decrease the analysis speed.
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Figure 1. Schematic illustration of the heated DAPCI source for
melamine detection. The schematics are not proportionally scaled.
Analytical Chemistry, Vol. 81, No. 7, April 1, 2009
Surface desorption atmospheric pressure chemical ionization
mass spectrometry has been successfully applied to rapid detec-
tion of powdered samples such as amino acids and glucose using
ambient moisture for primary ion production.26,28In autumn, the
relative humidity of the local air is usually lower than 45%, which
is not sufficient for primary ion production in DAPCI. To ensure
sufficient reagent (i.e., water) in the nitrogen sheath gas, saturated
water vapor was carried into the DAPCI source by the gentle
nitrogen gas flow flushing through an acetic acid aqueous solution
(250 mL, 1%, v:v).
Characteristic Fragmentation of Protonated Melamine. Under
the experimental conditions, the DAPCI-MS spectrum of authentic
melamine (1 ng) deposited on a filter paper surface was recorded,
showing a predominant peak at m/z 127 (shown in Figure 2a).
Upon CID (25% CE, 30 ms), the precursor ions (m/z 127)
generated ions of m/z 110, 85, and 60 as the major fragments
(shown in Figure 2b), by the loss of NH3, NH2CN, and C2HN3,
respectively. In the MS/MS spectrum of the deuterium labeled
melamine ions (m/z 134), the precursor ions produced major
fragments of m/z 114, 90, and 66 (shown in the Figure 2c),
which were ascribed to the loss of ND3, ND2CN, and C2DN3from
the parent ions, respectively. The fragmentation pattern was
identical to that observed using the protonated melamine
molecules (m/z 127), validating the fragmentation pathways
of the precursor ions. These data suggest the fragment of m/z
60 or 66 observed from the protonated melamine (m/z 127) or
the isotope labeled molecule (m/z 134) contains 6 hydrogen
or deuterium atoms. Its possible structure is proposed in Figure
2b. Interestingly, under the experimental conditions, the ionic
fragment of m/z 68, which was observed as a major fragment in
was almost undetectable. This difference was probably caused by
the different CID conditions. For example, m/z 68 showed up
when the CID experiments were performed with either high
collision energy (e.g., 35%) or high source/heated capillary
temperature (e.g., 300 °C). In the MS3experiment, the parent
ions of m/z 85 (m/z 90 for the deuterium labeled ions) yielded
a predominant signal at m/z 68 (m/z 70), probably by the loss
of NH3(ND3). This confirmed that the fragment of m/z 68
observed in the MS/MS spectrum was mainly derived from
the fragment of m/z 85. As m/z 68 increased to high
abundance, the intensity of m/z 85 drastically decreased. This
is also in agreement with previous studies.9-11However, it is
beneficial to maximize the abundance of the fragment of m/z 85
for sensitive quantification of melamine in our experiments. In
the MS/MS spectrum of protonated melamine, the signal intensity
ratio of m/z 109 to m/z 110 varied with CID conditions (i.e.,
collision energy, CID duration time). Preliminary data show that
Figure 2. Mass spectra of melamine recorded by DAPCI-MS: (a) mass spectrum of authentic melamine (1 ng) on filter paper surface; (b)
MS/MS spectrum of protonated melamine (m/z 127); (c) MS/MS spectrum of protonated deuterium melamine (m/z 134); (d) MS/MS/MS spectrum
of the ionic fragments (m/z 85) produced from protonated melamine (m/z 127); (e) MS/MS/MS spectrum of the ionic fragments (m/z 90) produced
from protonated deuterium melamine (m/z 134); (f) mass spectrum of powdered milk on a filter paper surface, the signal detected at m/z 127
yielded the same MS/MS spectrum as that of protonated authentic melamine (shown in Figure 2b).
Analytical Chemistry, Vol. 81, No. 7, April 1, 2009