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High-throughput trace melamine analysis in complex mixtures
w
Guangming Huang,
a
Zheng Ouyang*
b
and R. Graham Cooks*
a
Received (in Cambridge, UK) 15th October 2008, Accepted 11th November 2008First published as an Advance Article on the web 1st December 2008
DOI: 10.1039/b818059hAmbient ionization using a low-temperature plasma (LTP)probe combined with tandem mass spectrometry (MS/MS)allows detection and quantitation of melamine in milk powder,whole milk and other products at levels down to low ppb inanalysis times of a few tens of seconds.
In September 2008, deliberate contamination of dairy and otherproducts with the synthetic chemical melamine resulted in amajor outbreak of renal disease (urolithiasis) and associateddeaths in infants. The addition of melamine to dairy products isa fraudulent activity designed to produce an incorrectly highreading in the measurement of protein content based on totalnitrogen (the Kjeldahl nitrogen determination method
1
). Thissituation has created an immediate need for analytical methodsthat have the following properties: (1) high sensitivity, (2)specificity of compound identification, (3) rapid detection (highsample throughput), (4) matrix independence, (5) simple,rugged, and reliable instrumentation, and (6) quantitativeaccuracy. No existing method is completely adequate as seenby the performance of a variety of methods explored recentlyin connection with earlier related incidents, for example,that involving pet food contamination by melamine whichoccurred in March of 2007. Amongst the methods are (i) liquidchromatography/tandem mass spectrometry (LC/MS/MS)
2–6
which meets all the requirements except speed of analysisand the instrumentation characteristics of item (5); (ii) directanalysis in real time (DART) mass spectrometry
7
which showspromising performance although detection limits need signifi-cant improvement; (iii) gas chromatography/mass spectrometry(GC/MS)
8–10
which isnot a high-throughput method; (iv) liquidchromatography/ultraviolet detection (LC/UV)
9,11
which hassimilar drawbacks and (v) capillary electrophoresis/massspectrometry
12
which remains to be adequately evaluated. Theconsensus seems to be that the combination of liquid chromato-graphy and tandem mass spectrometry is currently the best wayof achieving these objectives even though the sample pretreat-ment procedures mean that high-throughput analysis is notpossible.In this communication we report on the use of a variant on anew ionization method based on the use of a low-temperatureplasma (LTP)
13
for determining traces of melamine in complexsamples in the ambient environment without any prior samplepreparation. LTP ionization is among a number of newambient ionization methods
14–20
that include desorptionelectrospray ionization (DESI),
14–16
DART,
21
electrospraylaser desorption ionization,
22–24
desorption atmospheric pre-ssure chemical ionization,
25
flowing afterglow-atmosphericpressure glow discharge,
26
plasma-assisted desorption ioniza-tion,
27
dielectric barrier discharge ionization
28
and glowdischarge ionization.
29
These methods, introduced over thepast few years, allow the direct mass spectrometric analysis of compounds present in solid samples and appear to haveconsiderable practical value. In the case of the LTP experi-ment,
13,30
a plasma is generated using a small handheldprobe and it is directed onto the sample by a slow stream(0.5 L min
À
1
) of helium. Suction of the MS vacuum systempulls the resulting gas phase ions into the mass spectrometer.In the variant used here, ionization is thermally assisted byheating the sample.The experiment employs tandem massspectrometry (MS/MS)to provide the necessary chemical specificity, while ruggednessand simplicity are the result of implementation on a simple iontrap mass spectrometer, making the assay efficient and relativelyinexpensive. Melamine is ionized directly in the ambientenvironment without prior preparation of the samples, allowingthe experiment to be done in a high-throughput fashion, at a rateof two samples per minute. The linear dynamic range is morethan two orders of magnitude and a variety of complex mixturescan be assayed, apparently without interference. The experi-ments were done by exposing the solid melamine-containingmaterial at approximately 170
1
C directly to the LTP generatedin a discharge barrier desorption ionization event. Liquids suchas milk evaporated as they were heated but the residueswere simply examined as they were, without prior samplepreparation.Fig. 1 displays the mass spectrum of melamine and theproduct ion MS/MS spectrum of the protonated molecule(
m
/
z
127). The principal fragment ions,
m
/
z
110 and
m
/
z
85,are associated with loss of ammonia and cyanamide, respec-tively. Note that in Fig. 1a, protonated melamine and itscharacteristic fragment ion
m
/
z
85 are observed, indicating thatLTP produces both the intact protonated molecule and itsfragment when desorption is thermally assisted.
13
Fig. 1d and eshow the characteristic increase in ‘‘chemical noise’’ seen forthe mass spectrum taken for a complex mixture and therelative insensitivity of the MS/MS data to the matrix.
31,32
Improvements in speed and detection limit were achieved byoptimizing the temperature and by restricting the MS/MSexperiment to single reaction monitoring (SRM). The SRMconditions were optimized using the following values: isolationwindow, 1.5; activation energy, 45;
Q
, 0.4; activation time, 75 ms.
a
Department of Chemistry and Center for Analytical InstrumentationDevelopment, 560 Oval Drive, Purdue University, West Lafayette,IN 47907, USA. E-mail: cooks@purdue.edu;Fax: +1 (0)765 494 9421; Tel: +1 (0)765 494 5263
b
Weldon School of Biomedical Engineering, Purdue University,206 South Intramural Drive, West Lafayette, IN 47907, USA.E-mail: ouyang@purdue.edu; Fax: +1 (0)765 496 1912;Tel: +1 (0)765 494 2214
w
Electronic supplementary information (ESI) available: Experimentalsection and Fig. S1–S5. See DOI: 10.1039/b818059h
556
|
Chem. Commun.,
2009, 556–558This journal is
c
The Royal Society of Chemistry 2009
COMMUNICATION www.rsc.org/chemcomm
|
ChemComm
 
To examine pure melamine, 5
m
L aliquots of an aqueoussolution containing added melamine were deposited ontothe surface of a glass slide over an area of 2 mm
Â
2 mm.The sample dried rapidly at the temperature used (170
1
C) andthe analytical curve (Fig. 2a) shows a linear response of theSRM intensity
vs.
melamine concentration over the range of 12.5–5000 pg with an experimentally estimated detection limitof 5 pg. The relative standard deviation (RSD) varies from5.8% to 10.2% for five replicates at different melamine con-centrations within the linear response range. Similarly, formelamine spiked into milk powder, 10
m
L of an aqueoussolution containing melamine and 10 mg milk powder wasdeposited into an area of 4 mm
Â
4 mm. Direct analysis wasconducted once the solution had dried. The analyticalcurve (Fig. 2b) displayed a linear range from 10–2500 ng g
À
1
(100 pg–25 ng melamine spiked into 10 mg milk powder) witha detection limit of 6 ng g
À
1
(60 pg melamine in 10 mg milkpowder). The RSD varied from 5.2% to 9.8% at differentmelamine concentrations within the linear range. This perfor-mance is on the same order as the LC/MS/MS method where1.7 ng g
À
1
has been reported for a tissue sample
6
and the limitof detection (LOD) is much lower than the value of 50 ng g
À
1
in a complex matrix given in the Food and Drug Administra-tion (FDA)’s Interim Melamine and Analogues Safety/RiskAssessment report (May 24, 2007).
33
Fig. 2c shows the tandem mass spectrum recorded usingSRM for 10 mg milk powder spiked with 0 g (control), 0.1 ngand 1 ng melamine to reach the final concentrations of 0, 10and 100 ng g
À
1
. At the concentration of 10 ng g
À
1
, a significantpeak can be observed for melamine in milk powder. A detailedsummary of the analytical performance of the present methodfor samples in different matrices, including liquid milk, is givenin Table 1. Precisions were in the 5–10% RSD range, indica-tive of the quality of the measurement and the fact that thesamples were homogeneously mixed.The LTP method not only allows direct and rapid analysisat high sensitivity, it also allows high throughput. This isshown in Fig. 3 for the case of eight milk powder samples(10 mg each) spiked with 50 ng melamine. The whole experi-ment took less than 3 minutes, around 25 seconds per sample,of which the measurement time is only a small fraction. Thehigh-throughput feature together with the lack of samplepretreatment is extremely useful when large numbers of samples need to be screened. It can also be seen (inset to
Fig. 1
(a) LTP mass spectrum and (b) MS/MS product ion spectrumof protonated melamine,
m
/
z
127, recorded using 50 ng of pure solidmelamine. Corresponding mass spectra for a sample consisting of (c) 0 ng and (d) 20 ng melamine spiked into 10 mg milk powder.(e) MS/MS product ion spectrum of the protonated molecule from(d) used for high specificity melamine confirmation.
Fig. 2
SRM MS/MS intensity of the characteristic fragment (
m
/
z
85)from the protonated molecule (
m
/
z
127)
versus
amount of (a) puremelamine in water (5
m
L) on a glass slide and (b) melamine spiked in10 mg milk powder. (c) SRM data for 10 mg milk powder spiked with0, 0.1 and 1 ng melamine.
Table 1
Analytical performance of melamine detection in different matricesMatrix Linear range
c
(absolute amount) LOD (absolute amount)Water
a
2.5–1000 ng mL
À
1
(12.5 pg–5 ng) 1 ng mL
À
1
(5 pg)Liquid milk
a
30–5000 ng mL
À
1
(150 pg–25 ng ) 15 ng mL
À
1
(75 pg)Synthetic urine
a
20–5000 ng mL
À
1
(100 pg–25 ng ) 10 ng mL
À
1
(50 pg)Milk powder
b
10–2500 ng g
À
1
(100 pg–25 ng) 6 ng g
À
1
(60 pg)Soy milk powder
b
20–3000 ng g
À
1
(200 pg–30 ng) 10 ng g
À
1
(100 pg)
a
Melamine spiked in 5
m
L matrix.
b
Melamine spiked in 10 mg matrix.
c
RSD values vary from 4.1% to 12.7% for five replicates at each melamineconcentration within the linear range.
Fig. 3
High-throughput data for eight control samples (0.5 to3.0 min) and eight 50 ng 10 mg
À
1
milk powder samples (3.5 to6.5 min) using single reaction monitoring at 170
1
C. Inset (left)temperature effect on LTP signal for melamine; inset (right) photo-graph of the eight samples on a glass slide.
This journal is
c
The Royal Society of Chemistry 2009
Chem. Commun.,
2009, 556–558
|
557
of 00

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