Electronic Supplementary Material for
Direct Analysis of Large Living Organism by Megavolt
Electrostatic Ionization Mass Spectrometry
Kwan-Ming Ng, Ho-Wai Tang, Sin-Heng Man, Pui-Yuk Mak, Yi-Ching Choi,
Melody Yee-Man Wong
Department of Chemistry, and State Key Laboratory of Synthetic Chemistry,
The University of Hong Kong, Hong Kong, China
Correspondence to: Kwan-Ming Ng; e-mail: kwanmng@hku.hk
Experimental Procedures and Materials
Chemicals, Solvent and Others
The explosives (2,4-ditnitrotoluene at >99.9% purity and 2,4,6-trinitrophenol at >99.0 % purity) and
cocaine (> 97 % purity) standards were purchased from Sigma Aldrich (St. Louis, MO, USA). Toluene
(AR grade) was purchased from VWR International (Radnor, PA, USA). Acetonitrile (HPLC grade) was
purchased from Scharlab S.L. (Sentmenat, Barcelona, Spain). Water (HPLC grade) was purchased from
RCI Labscan (Pathumwan, Bangkok, Thailand). Formic acid (ACS grade, > 98 % purity) was purchased
from Sigma Aldrich.
Red wine (Vintage 2009, Reserve Shiraz, Jacob’s Creek, South Australia, Australia), mint flavored
chewing gum (Airwaves, Wrigley, IL, USA) and painkiller tablet (each contain 500 mg acetaminophen,
Panadol, GlaxoSmithKline, County Waterford, Ireland) were purchased from a local supermarket.
Mass Spectrometry
All mass spectrometric measurements were performed using a hybrid quadrupole-time-of-flight (Q-TOF)
tandem mass spectrometer (Q-TOF Premier, Waters Micromass, Manchester, UK). The original ion
extraction cone in the ion source was replaced by an extended ion source, which extended the mass
spectrometer inlet away from the ion source block for easier sampling. The extended ion source was
electrically grounded to prevent the accumulation of electrostatic charge. In addition, to prevent possible
electrostatic damage, the mass spectrometer was shielded from electrical charges using several
electrically grounded metal plates. Instrumental control and data acquisition were performed using
MassLynx 4.1 system software (Waters Corporation, Milford, MA, USA).
The experimental settings of the mass spectrometer were as follows. Ions collected from the inlet would
pass through an ion source block which was set at 80 °C. No pneumatic assistance, including cone gas or
nebulising gas, was used. The sample cone voltage was set at 30 V. In TOF-MS experiment, the ions
were guided through an argon-filled collision cell with collision energy of 2 eV for better ion
transmission. For tandem mass spectrometry experiment, ions of interest would be selected by a
quadrupole mass filter and then guided into the collision cell with collision energy from 2 – 14 eV
(laboratory frame), depending on the extent of fragmentation of the respective analyte ion (except for
[Toluene - H]+ which required a higher collision energy of 35 eV). The ions were then mass analyzed by
a reflectron TOF mass analyzer and detected by a microchannel plate detector set at 2.1 kV. The mass
resolving power of the TOF mass analyzer was tuned to ~6,500 (at m/z 304.1, Full-width at Half
Maximum, FWHM). For the general TOF-MS experiments, the mass accuracy of the Q-TOF mass
spectrometer was calibrated to be within 20 ppm (at m/z 304.1468) with an external calibration. For
accurate mass measurement of volatile compounds in human breath gas, TOF-MS experiment was
performed immediately after mass calibration, the mass accuracy was within 6 ppm (at m/z 90.9772). The
vacuum pressure of the source, collision cell and TOF mass analyzer regions were 1.68, 6.22 × 10-3, 8.73
× 10-7 mbar respectively. In general, each TOF MS spectrum was a combination of 30 – 60 scan. The
scan time for each scan was 1 s and the interscan time was 0.1 s. The acquisition mass range was m/z 10
– 1,000.
Electrostatic Generators
The positive (+0.2 megavolt) and negative (-0.4 megavolt) electrostatic potential were generated using
two Van de Graaff generators (Science First, Yulee, FL, USA). The Van de Graaff generators used in all
our experiments were the same type, but the model adopted for the positive ion mode study is different
from that adopted for the negative ion mode study. The difference between the two models is size of the
domes (that used for the accumulation of the electrostatic charge). Both domes (for the positive and
negative electrostatic generators) are spherical in shape, but their diameters and circumferences are
different. The circumferences of the domes used for the Van de Graaff generators generating positive
electrostatic potential (+0.2 megavolt) and negative electrostatic potential (-0.4 megavolt) were 56.5 cm
and 100.8 cm respectively. The diameters of the domes used for the Van de Graaff generators generating
positive electrostatic potential (+ 0.2 megavolt) and negative electrostatic potential (-0.4 megavolt) were
approximate 18.0 cm and 32.1 cm, respectively.
Direct Analysis of Explosives on Latex Glove
A laboratory latex glove was worn on a hand of a healthy individual, and then a small amount (< 1 mg) of
the explosive compounds was deposited on the fingertip region of the glove. The individual was
electrically floated to megavolt potential by putting one of his hand on the metal ball of the Van de Graaff
generator for the gradual accumulation of charge for about 2 seconds, then the gloved hand of the
individual was moved in front of (~ 8 cm away from) the extended inlet of the mass spectrometer. The
Van de Graaff generator (with negative potential at 0.4 MV) was switched on only when the individual
had put his hand on the metal ball. Ions of the explosives were readily detected upon the electrostatic
charging of the individual. The individual was isolated from the surrounding to avoid charge leakage. A
diagram showing the experimental setup is depicted in Figure 1.
Direct Analysis of Explosives and Flammable Solvent on Cloth/ Tissue Paper
Explosive powder/ a drop of toluene was deposited on a piece of cotton cloth/ tissue paper. The cloth/
paper was held by an individual, and then put in front of the mass spectrometer inlet after the individual
was electrostatically charged by a Van de Graaff generator. Negative electrostatic potential was applied
for the detection of explosives, and positive electrostatic potential was applied for the detection of toluene.
Direct Analysis of Cocaine on Bare Hand
The experimental procedure was essentially similar to that of the analysis of explosives on a glove, except
that cocaine (< 0.5 mg) was deposited directly on the fingertip of an individual, and also a Van de Graaff
generator which generated a positive electrostatic potential (0.2 MV) was employed.
Direct Analysis of Acetaminophen in Painkiller Tablet
The experimental procedure was essentially similar to that of the analysis of cocaine on bare hand, except
that a painkiller tablet instead of powder was directly held by an individual, with the addition of 200 μL
of an aqueous solution (containing 50% acetonitrile and 0.1 % formic acid) onto the tablet.
Direct Analysis of Human Breath after Drinking Red Wine and Chewing Mint Flavored Gum
For the analysis of human breath after drinking red wine, a healthy individual first drank a cup of red
wine. After 3 minutes, breath gas of the individual was directed to the extended inlet via a glass tube (~
20 cm long), and he was then electrostatically charged by a Van de Graaff generator generating a positive
electrostatic potential (0.2 MV). The breath gas analysis of the individual after chewing the mint-flavored
gum (for 5 minutes) was performed with the same procedure.
Direct Analysis of Normal Breath of Individuals
For the direct analysis normal breath of four healthy individuals, the procedure was essentially the same
as that in the analysis of breath after drinking red wine, except that the breath of the individuals can be
directed to the extended inlet of the mass spectrometer, without the intake of any special food or drink
prior to analysis.
Supplementary Figures
(d) Blank glove without the
explosive compound 2,4,6-TNPh
(a) Blank glove without the
explosive compound 2,4-DNT
Relative Intensity (%)
Relative Intensity (%)
227.2
100
100
50
157.1
0
150
199.2
171.1
160
170
180
190
m/z
200
210
227.2
228.2
227
50
213.2
228
220.1
229
241.2
228.2
0
210
220
230
240
m/z
250
[2,4,6-TNPh – H][2,4-DNT – H]
181.0
50
199.2
157.1
0
150
171.1
160
170
180
190
m/z
200
210
[2,4-DNT – H]-
Relative Intensity (%)
88.0
50
80
0
50
[2,4-DNT – H – NO2]135.0
116.0
[2,4-DNT – H – NO]151.0
160
120
100
m/z
150
200
227.2 228.0
228.2
227
50
228
229
241.2
213.2
220.1
0
210
100 (f)
Relative Intensity (%)
181.0
(c)
100
228.0
227.2
(e)
100
Relative Intensity (%)
(b)
100
Relative Intensity (%)
-
220
230
m/z
240
[2,4,6-TNPh – H – NO]198.0
[2,4,6-TNPh
– H – NO2]182.0
227.2
250
227.2
228.0
[2,4,6-TNPh - H]228.0
50
180
0
50
210
100
150
m/z
X10
240
200
250
Figure S1. Direct detection of explosives on latex glove by megavolt electrostatic ionization mass spectrometry,
while the individual was electrostatically charged up to 0.4 megavolt potential at negative polarity. Mass spectra
showing the background of a blank latex glove in the absence of explosive compounds (a) 2,4-DNT and (d) 2,4,6TNPh. Mass spectra showing the detection of (b) [2,4-DNT - H]- at m/z 181.0; and (e) [2,4,6-TNPh - H]- at m/z 228.0
from latex glove containing the explosives, both in form of deprotonated ions, and their respective MS/MS spectra in
(c) and (f). The collision energy used in the MS/MS experiment for [2,4-DNT - H]- and [2,4,6-TNPh - H]- were 2 and
13 eV respectively (laboratory frame). (2,4-DNT: 2,4-Dinitrotoluene; 2,4,6-TNPh: 2,4,6,-Trinitrophenol)
(a) Blank cloth without the
explosive compound 2,4-DNT
(c) Blank cloth without the explosive
compound 2,4,6-TNPh
50
100
157.1
Relative Intensity (%)
Relative Intensity (%)
100
171.1
199.2
0
150
160
170
180
190
200
210
227.2
227.2
228.2
50
227
220.1
213.2
0
210
228.2
220
230
m/z
181.0
Relative Intensity (%)
Relative Intensity (%)
0
150
199.2
160
171.1
170
180
m/z
241.2
240
250
[2,4,6-TNPh – H]-
100 (d)
50
157.1
229
m/z
[2,4-DNT – H]100 (b)
228
190
200
210
227.2
227.2
213.2
228.0
220
228.2
227
220.1
50
0
210
228.0
230
228
241.2
240
229
250
m/z
Figure S2. Direct detection of explosives on cloth by megavolt electrostatic ionization mass spectrometry, where an
electrostatic potential at 0.4 megavolt at negative polarity was applied on the cloth. Mass spectra showing the
background of a blank cloth in the absence of (a) 2,4-DNT; and (c) 2,4,6-TNPh. Mass spectra showing the detection
of (b) [2,4-DNT - H]- at m/z 181.0; and (d) [2,4,6-TNPh - H]- at m/z 228.0 from cloth containing the explosives. Both
explosives were detected in form of deprotonated ions, (2,4-DNT: 2,4-Dinitrotoluene; 2,4,6-TNPh: 2,4,6,Trinitrophenol)
(a)
Relative Intensity (%)
100
[(Men)2 + H – Men – H2O]+
139.1
[(Men)2 + H – Men]+
157.2
[(Men)2 + H – C10H18]+
175.2
[(Men)2 + H – H2O]+
295.3
50
100
0
[Men + H + Men]+
313.3
50
200
100
150
300
200
250
300
m/z
(b)
Relative Intensity (%)
100
350
[Men + H + Bg]+
263.2
[Bg + H]+
107.1
– [menthol]
- (C10H20O)
50
0
50
100
150
200
250
300
m/z
Figure S3. Characterization of menthol-related ions in the breath of a healthy individual
after chewing mint flavored gum. (a) MS/MS spectrum of protonated menthol dimer
([Men + H + Men]+) at m/z 313.3, showing the detection of several menthol-related
fragment ions, such as [(Men)2 + H – H2O]+, [(Men)2 + H – C10H18]+, [(Men)2 + H – Men]+,
and [(Men)2 + H – Men – H2O]+. (b) MS/MS spectrum of protonated adduct of menthol
with background molecule ([Men + H + Bg]+) at m/z 263.2, showing the neutral loss of a
menthol molecule. [Bg + H]+ at m/z 107 is the protonated ion of the background
molecule. The collision energy used in the MS/MS experiment for [Men + H + Men]+ and
[Men + H + Bg]+ were 12 and 10 eV respectively (laboratory frame). (Men: Menthol)
[DiEtBu + H – C2H5OH]+
129.0
Relative Intensity (%)
100
50
[DiEtBu + H – C2H5OH – H2O]+
101.0
0
50
100
m/z
*
[DiEtBu + H]+
175.1
150
200
Figure S4. Characterization of ions of red wine volatile organic compounds in the breath of
a healthy individual after drinking red wine (50ml). MS/MS of protonated diethyl
butanedioate [DiEtBu + H]+ at m/z 175.1 detected in the breath after drinking red wine.
Neutral loss of ethanol (C2H5OH) and subsequent loss of water (H2O) was observed,
which conformed to the structure of the [DiEtBu + H] + precursor ion. Diethyl butanedioate
is a reported volatile component found in red wine. * the ion peak at m/z 149 is the
fragment ion of an interference ion at m/z 177.0. The collision energy used in the MS/MS
experiment for [DiEtBu + H]+ was 5 eV. (DiEtBu: Diethyl butanedioate).
Relative Intensity (%)
100
(e)
163.1
151.1
169.1
50
163.0
155.1
0
150
158.1
155
167.1
160
163.1
100
165
Relative Intensity (%)
(a)
170
50
163.0
0
162.8
162.9
(b)
151.1
169.1
163.0
155.1
0
150
158.1
155
167.1
160
165
170
50
0
162.8
162.9
50
155.1
158.1
155
167.1
160
165
Relative Intensity (%)
Relative Intensity (%)
163.0
163.3
170
163.2
163.3
163.2
163.3
163.0
50
0
162.8
162.9
m/z
163.0
163.1
m/z
(d)
(h)
151.1
163.0
163.1
169.1
50
155.1
155
158.1
167.1
160
165
163.0
100
170
Relative Intensity (%)
Relative Intensity (%)
163.2
163.1
100
163.1
0
150
163.1
(g)
151.1
169.1
100
163.0
m/z
(c)
0
150
163.3
163.0
m/z
100
163.2
163.1
100
50
163.1
(f)
163.1
Relative Intensity (%)
Relative Intensity (%)
100
163.0
m/z
m/z
163.1
50
0
162.8
m/z
162.9
163.0
163.1
m/z
Figure S5. Chemical profiling of the breath of four healthy individuals electrically floated at 0.2 megavolt at
positive polarity. Mass spectra of selected m/z range from m/z 150 to 170 of the breath profile of (a) volunteer 1,
(b) volunteer 2, (c) volunteer 3, and (d) volunteer 4, which show that the breath profile in the selected m/z range
for the 4 individuals were generally similar. However, for the ions at m/z 163.0 and 163.1, their relative
abundance among the breath profile of the 4 individuals were quite different ((e) volunteer 1, (f) volunteer 2, (g)
volunteer 3, and (h) volunteer 4), suggesting the megavolt electrostatic ionization MS technique may help reveal
the difference among the breath profile of different individuals.
100
217.2
50
211.1
0
210
215.1
213.1
212
219.2
214
216
218
220
214.1
(e)
50
213.1
0
212.5
213.5
100
217.2
50
213.1 215.1
219.2
0
210
212
214
216
218
220
m/z
213.1
214.1
(c)
215.1
211.1
219.2
0
210
212
214
214.1
213.1
0
212.5
213.5
216
218
220
217.2
0
210
213.1
212
215.1
219.2
214
216
m/z
218
220
214.5
213.5
(h)
214.1
217.15
0
216.9
m/z
217.1
217.3
(k)
217.16
50
217.09
0
216.9
100
213.2
213.5
217.08
217.1
217.3
m/z
50
0
212.5
217.3
50
100
214.1
0
212.5
217.1
m/z
50
100
50
211.1
(j)
m/z
Relative Intensity (%)
Relative Intensity (%)
214.1
(d)
214.5
(g) 213.1
m/z
100
0
216.9
100
50
100
217.2
50
217.08
m/z
Relative Intensity (%)
Relative Intensity (%)
100
(f)
Relative Intensity (%)
211.1
50
217.15
m/z
Relative Intensity (%)
214.1
Relative Intensity (%)
Relative Intensity (%)
(b)
214.5
(i)
m/z
m/z
100
100
Relative Intensity (%)
214.1
Relative Intensity (%)
(a)
Relative Intensity (%)
Relative Intensity (%)
100
214.5
(l)
50
0
216.9
217.15
217.09
217.1
217.3
m/z
Figure S6. Chemical profiling of the breath of four healthy individuals electrically floated at 0.2 megavolt at
positive polarity: Mass spectra of selected m/z range from m/z 210 to 220 of the breath profile of (a) volunteer 1;
(b) volunteer 2; (c) volunteer 3; and (d) volunteer 4, which show that the breath profile in the selected m/z range
for the four individuals were generally similar. However, for the ions at m/z 213.2, it has a significantly higher
relative abundance in the breath of (g) volunteer 3 than that of (e) volunteer 1, ( f) volunteer 2 and (h) volunteer
4. In addition, difference in the breath profile among the four individuals was also observed in the relative
abundance of ions at m/z 217.09 and m/z 217.15, where the relative abundance of ions at m/z 217.15 was 2 – 4
times of that of ions at m/z 217.09 for (i) volunteer 1, (k) volunteer 3 and (l) volunteer 4. But for the breath profile
of (j) volunteer 2, the relative abundance of ions at /z 217.15 was almost the same as that of ions at m/z 217.09.