Surface-Assisted Laser Desorption Ionization Mass Spectrometry(SALDI-MS) of Low-Molecular-Weight Medicines andToxic Materials Using Commercial TiO2 Nanoparticles
Tetsu Yonezawa,*1 Takashi Asano,1,2 and Masaki Matsubara1,³
1

Division of Material Science and Engineering, Faculty of Engineering, Hokkaido University,
Kita 13, Nishi 8, Kita-ku, Sapporo, Hokkaido 060-8628
2

Criminal Investigation Laboratory, Metropolitan Police Department,
2-2-1 Kasumigaseki, Chiyoda-ku, Tokyo 100-8929
E-mail: tetsu@eng.hokudai.ac.jp
Received: September 30, 2015; Accepted: November 28, 2015; Web Released: December 4, 2015

Tetsu Yonezawa
Professor Tetsu Yonezawa graduated from the Graduate School of Engineering, The University of Tokyo andreceived his Ph.D. in 1994. After spending 2 years as a post-doc in Tokyo and France, he was appointed as
an assistant professor of the Kunitake Lab., Kyushu University. After staying at Nagoya University and The
University of Tokyo as an associate professor, he has now been working at the Division of Materials Scienceand Engineering, Hokkaido University since 2008. He received the SPSJ Hitachi Chemical Award 2011,
Xingda Lectureship from Peking University in 2015 and some other awards. He has published more than 170
original papers and 20 books.

Abstract
The ability of surface-assisted laser desorption/ionization
mass spectroscopy (SALDI-MS) of low-molecular-weightcompounds using six types of commercially available TiO2
nanoparticles (NPs) was examined. In all cases, TiO2 NPs gavestrong Na+-adduct sample ion peaks. Some chemicals, whichrarely show proton-adduct sample ion peaks by MALDI-MS,
such as aspirin and barbital, could be easily detected by using
TiO2 NPs. The key factors determining the SALDI ability of
TiO2 NPs are their purity, ratio of rutile, and particle size.

³ Present address: Department of Materials and Environmental
Engineering, National Institute of Technology, Sendai College,
48 Nodayama, Medeshima-Shiote, Natori, Miyagi 981-1239

protein ricin7 and resins in adhesives using MALDI-MS8 has
been reported.
Despite these advantages, MALDI-MS is considered unsuitable for the analysis of small molecules (<500 Da), because it
uses an organic matrix and its mass spectra suﬀer from highchemical background noise due to the formation of clusters ofmatrix ions and its fragment ions.9,10 With the aim to suppress
this interfering noise, we have previously reported the use of acyclodextrin-supported organic matrix, which considerably
reduces the matrix-related signals.11
Surface-assisted laser desorption/ionization mass spectrometry (SALDI-MS) using inorganic nanoparticles or nanostructured surfaces is an organic matrix-free approach that overcomes this problem.10,12,13 The use of nanoparticles (NPs) withSALDI-MS was originally reported by Tanaka who employeda suspension of 30 nm cobalt NPs in glycerol for the mass
analysis of proteins and synthetic polymers.1 Sunner et al. usedgraphite particles as a matrix to ionize peptides and proteins.14
As another approach to matrix-free LDI-MS, Siuzdak et al.
developed a system called DIOS (desorption/ionization on
silicon), based on the use of a nanostructured surface of porous
silicon for the analysis of low-molecular-weight compounds.15
We have reported that, among the four bare metal NPs, Ag, Au,
Cu, and Pt, Pt NPs exhibited the highest SALDI-MS performance for angiotensin I because of the lower heat conductivity
and higher melting temperature of Pt.16 Bare Pt nanomaterials
with thin petals (Pt nanoﬂower) were also applied for SALDIMS.17,18 The use of semiconductor GaP NPs was reported to

346 | Bull. Chem. Soc. Jpn. 2016, 89, 346–353 | doi:10.1246/bcsj.20150337© 2016 The Chemical Society of Japan

1. Introduction
Matrix-assisted laser desorption/ionization (MALDI) is a
widely used and excellent ionization method, especially forthermally labile samples with high molecular weights.1­3
MALDI mass spectrometry (MALDI-MS) is frequently used
for the mass measurement of biopolymers and synthetic polymers. The recent rapid progress in protein chemistry has beenstimulated by MALDI-MS.4­6 MALDI-MS exhibits several
advantages such as high sensitivity to very small amounts ofsample and fast sample preparation protocols, and its use has
begun in the ﬁeld of forensic science. The detection of toxic

allow SALDI-MS with soft ionization.19 ZnO NPs20 and magnetic Fe2O3 NPs21 have also been proposed as good candidates
for SALDI-MS matrices. Kinumi et al. examined 11 types ofmetal and metal oxide powders, namely Al, Mn, Mo, Si, Sn,
SnO2, TiO2, W, WO3, Zn, and ZnO, suspended in glycerol or
liquid parraﬁn.22 Among them, TiO2 NPs proved to be the mostsuitable NPs for SALDI-MS.
TiO2 is widely used in photocatalysis, sensor technology,
catalysis support, as well as pigments. For LDI-MS, nitrogenlaser at 337 nm is frequently used and this laser light can beeﬀectively absorbed by TiO2 NPs. Furthermore, TiO2 NPs are
inexpensive and easily obtained. Its three natural polymorphs,
anatase, brookite, and rutile, show diﬀerent physical properties.
Castro et al. used TiO2 NPs in diﬀerent crystal forms,23 anddemonstrated that anatase and rutile mono-structured NPs are
more suitable for MS than anatase/rutile mixed NPs, andanatase proved to be optimal. Kawasaki et al. reported theinﬂuence of the crystalline structure of the TiO2 ﬁlm surface
on the SALDI-MS eﬃciency.24 Among the rutile, anatase, andamorphous phases, the anatase-type TiO2 gave the best results.
For example, signal/noise ratios of the mass spectra of angiotensin II obtained by rutile-, anatase-, and amorphous-TiO2
ﬁlms are 3, 28, and 9, respectively.24 TiO2 nanotube arrays
were also used as an active SALDI-MS surface25,26 and wereapplied as the sample stage for mass imaging of slices oftissues.27
As compared with MALDI-MS, SALDI-MS aﬀords advantages such as high salt tolerance, high throughput analysis, andsuitability for quantitative analyses, which make this technique
suitable for forensic science.28­35 SALDI has signiﬁcant potential as a good analytical tool for drug testing and is rapidly
advancing in the ﬁeld of forensic sciences. Its applications
include the detection of illicit drug molecules, their metabolites, and small molecules in forensic samples, including
ﬁngerprints and banknotes.

In this study, six commercially available TiO2 NPs withvarious sizes and crystalline structures were applied for the
SALDI-MS analysis of medicines and toxic materials with lowmolecular-weight (<500 Da), which are investigated in theﬁeld of forensic science. The structural characteristics of TiO2
NPs ideal for this purpose are also discussed.
2. Experimental
2.1 Chemicals and Materials.
Six types of TiO2
nanoparticles were supplied from Ishihara Sangyo Kaisha(ISK), Japan. Their properties are summarized in Table 1. PTseries NPs (PT301, PT401L, and PT401M) are rutile TiO2 andMC-series NPs (MC50, MC90, and MC150) are anatase TiO2.
The speciﬁc surface area of the NPs should be in inverse
proportion to their diameter. A small gap in the diameters canbe found, which is probably due to the diﬀerence of the measurement methods used (particle sizes are catalogue values
measured from TEM images). CHCA (α-cyano-4-hydroxycinnamic acid; organic matrix), methanol, acetonitrile (solvents), acetaminophen, testosterone, caﬀeine, diazepam, sildenaﬁl, methamidophos, aspirin (acetylacetic acid), and barbital(measurement samples) were supplied from Wako Pure Chemical (Japan) and used as received. The molecular structures andmolecular weights of the measurement samples are shownin Figure 1. Triﬂuoroacetic acid and sodium triﬂuoroacetate(Wako) were used as ionization-supporting reagents. Water was
puriﬁed by a Milli-Q system (>18 M³).
Table 1. Size and crystal structures of the TiO2 nanoparticles used for SALDI-MS measurements
Particle size
/nm
Rutile ratio/%

PT301 PT401L PT401M MC50 MC90 MC150
220
120
85
26
18
6.7
98

75

60

0.0

0.0

0.0

Figure 1. Structures and molecular weights of the medicines and toxic materials tested in this study.
Bull. Chem. Soc. Jpn. 2016, 89, 346–353 | doi:10.1246/bcsj.20150337© 2016 The Chemical Society of Japan | 347

2.2 Characterization of TiO2 NPs. In order to characterise
the TiO2 NPs used for SALDI-MS, X-ray diﬀraction investigation (Rigaku Miniﬂex II, Cu Kα) and TEM observation(Hitachi H-9500, 300 kV) were performed.
2.3 Matrix and Sample Preparation. Each type of TiO2
NPs (5 mg) was dispersed into a water/acetonitrile (1:1 (v/v),
1 cm3) mixed solvent by 5-min ultrasonication. CHCA (5 mg),the organic matrix, was also dissolved in 1 cm3 of water/acetonitrile (1:1 (v/v)) mixed solvent.
Sample solutions were prepared as follows, unless otherwise
noted. Sample molecules were dissolved in water/acetonitrile(1:1 (v/v)) or methanol at a concentration of 0.1 mg cm¹3(100 ppm).
Triﬂuoroacetic acid (TFA) and sodium triﬂuoroacetate (TFANa) were selected as ionization-supporting reagents. Aqueous
solutions of TFA and TFA-Na (0.1 wt %) were prepared.
SALDI samples were prepared using the common dried
droplet method. Brief, 1 mm3 of a matrix dispersion or solution
was deposited on a sample well (3 mmº) of a stainless steel
target plate and evaporated in air at room temperature. The
amount of TiO2 NPs is enough to obtain a uniform NP layeron the well. More TiO2 NPs may cause the contamination of
the sample chamber. In some cases, 1 mm3 of an ionizationsupporting reagent solution was deposited onto TiO2 andevaporated under vacuum. Next, 1 mm3 of a sample solution
was deposited onto the same well and evaporated in air atroom temperature. The target plate was then introduced into the
instrument.
2.4 SALDI-MS Measurement. Positive-ion mass spectra
were measured using a MALDI time-of-ﬂight (TOF) mass
spectrometer (Autoﬂex speed, Bruker Daltonics, Germany).
Measurements were performed in the positive reﬂectron modeand each spectrum was obtained by accumulating 2500 laser
shots (355 nm, 500 shots © 5 spots). In order to minimize thereduction of signal intensity, due to the consumption of sample
molecules, and the ﬂuctuation of the signal intensity, due tonon-uniform crystallization (so-called sweet spot phenomenon),random walking mode (i.e., laser irradiation positions randomly
displaced on the sample well during the pulse irradiation) was
selected for the accumulation of signals. No obvious diﬀerence
was found in the background noise of the obtained mass spectra from various TiO2 NPs. For the application of SALDI toforensic science, rapid analyses and quantitative analyses areimportant. Therefore, we have selected the positive reﬂectronmode for the rapid scanning of the samples and the random
walking mode to eliminate variability of data. The intensity of
the laser was evaluated by the laser power values, which are
an original parameter of the MALDI apparatus. Laser power(LP) is a parameter of the laser intensity but this is a control
parameter of our spectrometer. There is no unit for LP numbers.
3.1 Characterization of TiO2 NPs for SALDI-MS. In
order to compare the eﬀect of size and crystal structure of TiO2
NPs on the SALDI-MS measurements, two series were selected(PT-series and MC-series), and XRD measurement and TEM
observation were performed (Table 1; detailed XRD data in
the Supporting Information (Figures S1 and S2)). The XRD
patterns show that NPs of the PT-series contain both rutile andanatase structures. PT301 consists of 98% rutile and 2%
anatase. However, PT401M and 401L contain more anatasestructures (60% rutile and 75% rutile, respectively). On theother hand, MC-series TiO2 NPs consist of anatase phase only.
The size of the NPs was determined by TEM observation.
Their TEM images and size distributions are collected in
Supporting Information (Figure S3). PT301 (220 « 67 nm),
PT401L (120 « 46 nm), and PT401M (85 « 27 nm) shows
relatively larger particle size. On the contrary, MC-series NPs
are very small (MC50: 26 « 6.1 nm, MC90: 18 « 5.7 nm,
MC150: 6.7 « 3.9 nm). These particles did not have obvious
anisotropic structures. Broad peaks in the XRD patterns of theMC-series NPs are also an indication of their small size. In allcases, the particle sizes are relatively uniform.
3.2 Selection of the Media of TiO2 NPs and Samples, andDispersibility of TiO2 NPs. For SALDI-MS measurements,
usually NPs and samples are placed on sample wells separately.
First, a few drops of the NP dispersion are deposited on thewell and dried under reduced pressure. Next, a few drops ofthe sample solution are deposited on the same well and dried.
The process is very simple; however, a high throughput andaccuracy of mass measurements are highly required in the ﬁeldof forensic science. Thus, a water/acetonitrile mixture andmethanol were selected as media for the TiO2 NPs and for the
samples, respectively in order to minimize their evaporation
time. As shown in Figure S4 (Supporting Information), PTseries NPs were dispersed and dried uniformly on the sample
wells, whereas MC-series NPs formed some aggregates.
3.3 Eﬀect of Ionization-Supporting Reagents. The eﬀectof the ionization-supporting reagents, i.e., TFA and TFA-Na,
was evaluated. TFA and TFA-Na have been commonly used as
ionization-supporting materials in MALDI-MS and SALDI-MS
systems.28­30 However, TFA is highly toxic and is classiﬁed
as a chemical with which all contact should be avoided. Thus,
for repeated mass measurements, exclusion of TFA is very
important for the health of the operators. In order to evaluate
the eﬀect of these ionization-supporting reagents on SALDIMS measurement, TFA and TFA-Na were used with PT301.
Figure S5 (Supporting Information) illustrates the mass spectraof acetaminophen (analgesic antipyretic, 151 Da) with PT301
NPs as the inorganic matrix. In all cases, the strongest Na+adduct peak is at m/z = 174. In the absence of ionizationsupporting reagent and in the presence of TFA, the K+adduct peak (m/z = 190) can also be observed in the spectrum.
However, the peak of the protonated sample molecule (m/z =152) was not detected, even with the addition of TFA. On theother hand, an interference peak at m/z = 159, attributed to[TFA-Na + Na]+ ion, was found. However, the [M + H]+ peak
was not detected in this spectrum. Moreover, the intensity of
the Na+-adduct decreased upon addition of TFA (from 4200 to2900). Proton adduction to the analyte molecules by TFA did
not support ionization. From these results, it can be concluded
that TFA is not suitable.
The intensity of the Na+-adduct peak increased with theaddition of TFA-Na, but no obvious increase was observed(from 4200 to 6800). On the contrary, the [TFA-Na + Na]+
increased more than three times than the peak found in the
spectrum of the TFA-added sample. Using other TiO2 NPs as
inorganic matrix, no strong increase of the Na+-adduct peak348 | Bull. Chem. Soc. Jpn. 2016, 89, 346–353 | doi:10.1246/bcsj.20150337© 2016 The Chemical Society of Japan

3. Results and Discussion

could be observed. From these results, we concluded that TFANa is also not suitable as an ionization-supporting reagent.
Because considerably larger amounts of specimens should
be examined in a short period in the criminal investigationlaboratories of the police department, simpler and rapid sample
preparation and limited contamination are quite important foranalyses in forensic science. On the basis of these results, noionization-supporting reagent was used in this study.
3.4 SALDI-MS Measurements of Low-Molecular-Weight
Medicines and Toxic Materials Using Various TiO2 NPs.
Figure 2 shows the MALDI and SALDI mass spectra ofacetaminophen (151 Da) using CHCA as an organic matrix andsix types of TiO2 NPs. In the MALDI mass spectrum with
CHCA, proton-adduct peak can be observed at relatively highintensity, even at a low laser power (LP = 40). A fragment ion[M + H ¹ H2O]+ peak can be found at m/z = 134. No alkaline
metal ion-adduct peak is detected. However, the matrix-related
interfering peaks are much higher than the sample peak, whichmakes the identiﬁcation of the target molecules very diﬃcult.
On the contrary, SALDI-MS with TiO2 NPs needs a relatively
higher laser power for desorption/ionization. At LP = 60, themolecular ion peak could be observed with a low signal/noise(S/N) ratio of ca. S/N = 14­30. At LP = 80, good spectra withhigh S/N ratios can be obtained. In all SALDI-MS spectra
measured with TiO2 NPs, the strongest peak can be found at
m/z = 174, corresponding to the Na+-adduct sample ion.
Moreover, weak peaks at m/z = 190, corresponding to the K+adduct sample ion, can be observed. However, the protonadduct peak or large fragment peaks are not detected, and no
signiﬁcant matrix-related interfering peaks are found. The
largest Na+-adduct sample peak intensity was obtained with
PT401M NPs (Figure 4d), followed in order by PT401L andPT301. PT-TiO2 NPs show higher [acetaminophen ¹ Na]+
peak intensities than MC-TiO2 NPs.
Figure 3 shows the MALDI and SALDI mass spectra of
testosterone (steroid hormone, 288 Da). As discussed in theprevious section, the SALDI mass spectra with TiO2 NPs at an

LP of 70 clearly show Na+-adduct peak (m/z = 311), which
was found to be slightly lower than that of acetaminophen.
Peaks of testosterone fragment ions are not detected. The
MALDI mass spectra using CHCA as the organic matrix were
measured at a lower laser power of LP = 40, to obtain the
protonated molecular ion peak. No obvious fragment peak can
be observed either in the MALDI spectrum (A small peak at
m/z = 268 can be attributed to the contamination). PT401M
shows the highest intensity for the testosterone peak, followedby PT401L and PT301; MC-TiO2 NPs show lower peakintensities. No interfering peak can be found when PT-TiO2
NPs were used for SALDI-MS.
Figures 4 and 5 show the MALDI and SALDI mass spectra of caﬀeine (alkaloid, 194 Da) and diazepam (anxiolytic,
284 Da), respectively. In the MALDI spectra, we can ﬁnd
proton-adduct peaks. A peak at m/z = 174 in the MALDI

Figure 2. Mass spectra of acetaminophen using (a) CHCA,(b) PT301, (c) PT401L, (d) PT401M, (e) MC50, (f ) MC90,
and (g) MC150.

Figure 4. Mass spectra of caﬀeine using (a) CHCA, (b)PT301, (c) PT401L, (d) PT401M, (e) MC50, (f ) MC90,
and (g) MC150.

Bull. Chem. Soc. Jpn. 2016, 89, 346–353 | doi:10.1246/bcsj.20150337

Figure 3. Mass spectra of testosterone using (a) CHCA,(b) PT301, (c) PT401L, (d) PT401M, (e) MC50, (f ) MC90,
and (g) MC150.

© 2016 The Chemical Society of Japan | 349

Figure 5. Mass spectra of diazepam using (a) CHCA, (b)PT301, (c) PT401L, (d) PT401M, (e) MC50, (f ) MC90,
and (g) MC150.

Figure 6. Mass spectra of acetylsalicylic acid (aspirin)
using (a) CHCA, (b) PT301, (c) PT401L, (d) PT401M,(e) MC50, (f ) MC90, and (g) MC150.

spectrum of caﬀeine can be considered as a fragment peak of[M ¹ CO ¹ CH3 + Na]+. A fragment peak of [M ¹ CHO]+ ofdiazepam can be found at m/z = 255 in the MALDI spectrum.
GC-EI-MS of diazepam sometimes shows this fragment peak.
The SALDI mass spectra of the both chemicals with TiO2 NPs
show the Na+-adduct peaks (m/z = 217 and 311, respectively)
as the strongest peaks. When TiO2 NPs were used for SALDIMS, the peak intensity of caﬀeine is lower than that of
testosterone. On the other hand, the peak intensity of diazepam
is higher than that of testosterone. These ﬁndings can be
explained by the diﬀerence in acidity. Proton aﬃnities ofcaﬀeine, diazepam, and testosterone are estimated as 644
kJ mol¹1,36 966 kJ mol¹1,37 and 880 kJ mol¹1,38 respectively.
Sodium cation aﬃnities of organic molecules follow an ordersimilar to that of proton aﬃnities.39 The SALDI-MS peakintensities can be well explained by the proton aﬃnities of thecompounds. In the case of diazepam, which has a high protonaﬃnity value, proton-adduct peaks can also be observed in the
SALDI spectra. However, the strongest SALDI-MS peak was
obtained with PT401M, followed by PT401L and PT301.
Figures 6 and 7 show the MALDI and SALDI mass spectraof aspirin (acetylsalicylic acid, antipyretic and analgesic, 180
Da; Proton aﬃnity, 868 kJ mol¹1) and barbital (hypnotic, 184Da), respectively. Aspirin is the most widely used medical
compound and its overdose is a very serious problem. Barbital
was used as a sleeping aid until the 1950s, but its overdose isstill an important issue. These compounds are hardly detectedby MALDI. As shown in Figures 8a and 9a, the protonatedmolecular weight related peaks of aspirin and barbital are notobserved in the MALDI mass spectra using CHCA as theorganic matrix. Previous reports also indicated that the detection of protonated aspirin is very diﬃcult.38 On the contrary,
the Na+-adduct peaks of the both compounds (m/z = 203 and207, respectively) can be clearly observed by SALDI-MS using
TiO2 NPs. Moreover, when PT-type TiO2 NPs were used, peaks
at m/z = 225 and 207 are detected in the mass spectra of

aspirin and barbital, respectively. These peaks can be attributed to the [M + 2Na ¹ H]+ cation. Aspirin often gives the[M + 2Na ¹ H]+ signal because it can bind two alkali cations:
one between the two carbonyl oxygens of the acid and ester
groups, and the other more closely associated with the oxygensof the acid moiety.40
In both cases, no obvious fragment ion peak can beobserved, even in the SALDI-MS spectra. This is quite animportant feature of SALDI compared to MALDI. As the samelaser was used for the desorption/ionization of the organic
compounds in MALDI and SALDI, in both cases aspirin andbarbital molecules should be desorbed from the sample holder
surface. However, the mass peaks that can be used for theidentiﬁcation of these compounds could not be detected with

350 | Bull. Chem. Soc. Jpn. 2016, 89, 346–353 | doi:10.1246/bcsj.20150337© 2016 The Chemical Society of Japan

Figure 7. Mass spectra of barbitar using (a) CHCA, (b)PT301, (c) PT401L, (d) PT401M, (e) MC50, (f ) MC90,
and (g) MC150.

Figure 8. Mass spectra of sildenaﬁl using (a) CHCA, (b)PT301, (c) PT401L, (d) PT401M, (e) MC50, (f ) MC90,
and (g) MC150.

Figure 9. Mass spectra of methamidophos using (a) CHCA,(b) PT301, (c) PT401L, (d) PT401M, (e) MC50, (f ) MC90,
and (g) MC150.

MALDI, and were found only in the SALDI spectra as the
Na+-adduct ions.
Three possible explanations can be put forth for this ﬁnding.
That is: 1) the amounts of the desorbed molecules are diﬀerent in MALDI and SALDI, and thus the amounts of ionised
molecules are diﬀerent; 2) proton addition to these compounds
is more diﬃcult than alkali ion addition; and 3) alkali-adductions are more stable than the protonated ones. However, as
shown in Figure 2, the addition of an ionization-promoting
reagent (TFA or TFA-Na) did not signiﬁcantly aﬀect the
MALDI mass spectra. Thus, this phenomena can be presumably attributed to the ﬁrst argument, i.e., under our experimental conditions, the desorption ability of neutral molecules
by SALDI is better than that by MALDI. Unfortunately, proton
addition in MALDI analysis is not so eﬀective for these
Bull. Chem. Soc. Jpn. 2016, 89, 346–353 | doi:10.1246/bcsj.20150337

compounds. This high desorption ability may be ascribed tonano-dimensional microstructure eﬀect with a very highsurface area.
Mass spectra of sildenaﬁl (used to treat erectile dysfunction,
474 Da) and methamidophos (organophosphate insecticide, 141Da) were also obtained by MALDI with CHCA and by SALDI
with TiO2 NPs as shown in Figures 8 and 9, respectively.
Methamidophos has a proton aﬃnity of 897 kJ mol¹1.41 In
the case of sildenaﬁl, the MALDI spectrum showed a strong
protonated molecular ion peak at m/z = 475, and several
isotope ions using a low laser power (LP = 30). The SALDI
spectrum with PT401L shows a strong Na+-adduct peak, aswell as the protonated molecular ion peak. These signals areaccompanied by a couple of isotope ion signals. Comparing the
SALDI spectra obtained with diﬀerent PT-series TiO2 NPs, theNPs that gave stronger Na+-adduct ion peaks also gave stronger
protonated peaks. Using the MC-series TiO2 NPs, the intensitiesof the Na+-adduct and protonated ion peaks are much smaller
than those of the peaks obtained with the PT-series ones.
Methamidophos is an organophosphate insecticide used for
potatoes. However, due to its high toxicity to humans, the levelof its residues in raw agricultural products is strictly monitored.
Its use as an insecticide is prohibited in Japan. Methamidophos
can be detected as a protonated molecular ion in MALDI-MS
spectra; however, other interfering peaks with much higher
intensities can be observed. On the other hand, methamidophos
can be detected as the Na+-adduct in SALDI-MS spectra
without strong interfering or fragment ion peaks. In this case,
the MC-series NPs show slightly higher peak intensities than
the PT-series NPs.
Diazepam and sildenaﬁl show the protonated ion peaks in
the MALDI-MS spectra as well as in the SALDI-MS spectra,
especially using PT-series TiO2 NPs. In almost all cases, in
SALDI-MS, PT NPs give larger peaks than MC NPs.
Diazepam has a relatively high proton aﬃnity value; sildenaﬁlshould also have a high proton aﬃnity value because it
contains six nitrogen atoms and one SO2 group. However,
unfortunately, no relevant data could be found in the literature.
On the other hand, other compounds can be detected only as
Na+-adduct peaks by TiO2 SALDI-MS.
3.5 Eﬀect of Crystalline Structures and Sizes of TiO2 on
SALDI-MS Measurement.
Comparing the mass spectra
obtained with TiO2 NPs (Table 2), three important factors arefound to control the peak intensities of the samples in the
SALDI-MS spectra. The most important factor is purity,
because, in all cases, PT-series TiO2 NPs give higher mass
peaks than MC-series NPs. The second factor is the rutile ratio,
and the third is the size.
The SALDI eﬃciency can be determined by two parameters:
laser desorption eﬃciency and ionization eﬃciency. To date,there is no consensus regarding the mechanism underlying the
SALDI method. It is widely believed that the mechanism of
SALDI is diﬀerent from that of MALDI, and many studies
discuss the physicochemical properties required to promote
the laser desorption/ionization. Parameters that can aﬀect the
LDI eﬃciency are: (1) pulse laser-induced rapid temperature
increase, (2) laser-induced restructuring of the nanostructures,(3) high surface area (nanostructures, nanoparticles), and (4)terminal-OH groups (in the case of metal oxides) or other© 2016 The Chemical Society of Japan | 351

Table 2. [M + Na]+ intensities of the low-molecular-weight medicines and toxic materials measured by
SALDI-MS in this study
Compounds
Acetaminophen
Testosterone
CaﬀeineDiazepam
SildenaﬁlMethamidophos
AspirinBarbital

PT301
4100
3500
700
3600
21800
1200
260
70

PT401L
14800
11500
1100
12600
44100
3600
290
150

functional groups on the surface. In the cases studied herein, allanalyte molecules were detected as Na+-adduct molecular ionsby SALDI, and only a few were detected as protonated ions.
Because there is no associated proton donor on TiO2 NPs near
the excited analyte molecules, only desorption occurs, without
ionization, by pulse laser irradiation. Thus, a rapid temperature
increase of the pulse laser irradiation is crucial. Rutile-type
TiO2 has a higher UV absorption eﬃciency than anatase-type
TiO2.24 Therefore, the temperature of pure rutile TiO2 NPs
increases more rapidly than that of anatase NPs, and thedesorption eﬃciency also increased. This can be explained by agreater amount of analyte molecules desorbed from rutile TiO2
NPs by laser irradiation than that desorbed from anatase TiO2
NPs. The particle size of TiO2 NPs is also a very importantparameter for SALDI-MS sensitivity. In both series of TiO2
NPs (PT and MC), smaller NPs show higher mass peaks, andthe smaller PT NPs have higher anatase ratios than the larger
ones. Particle size also strongly aﬀects the temperature elevation, i.e., smaller particles show a more rapid increase of thetemperature than the larger ones. Thus, the desorption eﬃciency increases with the decrease of the particle sizes.
Castro et al. used anatase TiO2, rutile TiO2, and the commercially available mixture P25 for the SALDI-MS measurement of PEG200, caﬀeine, etc.23 Their results indicated that all
analytes were ionised with anatase, which is found to be moresuitable than rutile TiO2 and P25. However, in their study,
rutile TiO2 was obtained by the calcination of anatase TiO2 at
1000 °C for 6 h. Although the TEM images before and after
calcination are not presented, the particles presumably becomelarger in the process than that with the original anataseparticles.
By comparing our results with previous reports,23,24 we canconclude that the purity and rutile ratios of the TiO2 NPs, aswell as their crystal structure, strongly aﬀect the desorption ofanalyte molecules. Comparing the two series of TiO2 NPs usedin our study (PT and MC), the PT-series TiO2 NPs are bigger
than MC-series NPs, although the PT-series NPs gave stronger
mass peaks. This indicates that the rutile ratios of TiO2 NPs arequite inﬂuential on the desorption ability because of the higher
energy absorption.

PT401M
46000
24800
2600
29000
120600
3200
1100
300

MC50
3600
4100
400
5900
1300
1900
270
®

MC90
2200
10100
800
3700
3800
2300
240
®

MC150
8900
12000
4100
16400
7700
4600
1000
670

found. Sodium-adduct peaks of acetylsalicylic acid and barbital
could be clearly obtained SALDI with TiO2 NPs. MALDI canhardly detect these two compounds. Comparing with the mass
spectra obtained with these NPs, three important factors arefound to control the peak intensities of the samples. The mostimportant factor is their purity, the second is their ratio of
rutile, and the third is their particle size. This study represents
important progress in the ﬁeld of forensic science.
This work was partially supported by the Hokkaido
University (to TY) and Grant-in-Aid for Scientiﬁc Research(A) from JSPS, Japan (to TY No. 24241041).
Supporting Information
Information about X-ray diﬀraction characterization of TiO2
NPs and calculation of rutile anatase ratios. This material isavailable on http://dx.doi.org/10.1246/bcsj.20150337.
References

Six commercially available TiO2 NPs with various particle sizes, crystal structures, and purities, have been used as
SALDI-MS matrices. Using TiO2 NPs, strong sodium-adductpeaks are mainly observed but no proton-adduct peak was

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