Rotating multitip micropillar array electrospray ionization-mass spectrometry for rapid analysis and high-throughput screening

Article abstract of DOI:10.1016/j.ijms.2011.11.013

The applications of rapid screening benzodiazepines from urine and determining synthetic reaction products and kinetics semi-quantitatively using a microfabricated rotating multitip electrospray ionization (ESI) platform for mass spectrometry (MS) is presented. The ESI tips are based on lidless micropillar array ESI (μPESI) sources where the transfer of liquid is based on capillary forces without external pumping. A single silicon platform contains 60 separate μPESI tips which can be individually used by rotating the whole platform by six degrees each time from tip to tip using a computer control. The rotating multitip μPESI platform with an ion trap mass spectrometer was successfully demonstrated in rapid identification and monitoring of intermediates and final products in chemical synthesis within 10 min. The system was also applied to high-throughput screening of benzodiazepines from urine samples. Urine samples were extracted with solid-phase extraction (SPE) using C₁₈ phase ZipTip pipettes, enabling the use as small sample volumes as 50 μL of the urine sample. Therefore the whole sample treatment and the analysis with the rotating multitip μPESI-MS took only 5 min per sample.

Full text of DOI:10.1016/j.ijms.2011.11.013

International Journal of Mass Spectrometry 310 (2012) 65–71
Contents lists available at SciVerse ScienceDirect
International Journal of Mass Spectrometry
journal homepage: www.elsevier.com/locate/ijms
Rotating multitip micropillar array electrospray ionization-mass spectrometry
for rapid analysis and high-throughput screening
Teemu Nissiläa,b, Nina Backmanb, Marjo Kolmonenc,d, Antti Leinonend, Alexandros Kiriazisb,
Jari Yli-Kauhaluomab, Lauri Sainiemib,e, Risto Kostiainenb, Sami Franssilae, Raimo A. Ketolaa,∗
a Centre for Drug Research, Faculty of Pharmacy, University of Helsinki, Finland
b Division of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Helsinki, Finland
c Hjelt Institute, Department of Forensic Medicine, University of Helsinki, Finland
d Doping Control Laboratory, United Medix Laboratories Ltd., Helsinki, Finland
e Department of Materials Science and Engineering, Aalto University School of Technology, Espoo, Finland
a r t i c l e i n f o
a b s t r a c t
Article history:
The applications of rapid screening benzodiazepines from urine and determining synthetic reaction prod-
ucts and kinetics semi-quantitatively using a microfabricated rotating multitip electrospray ionization
(ESI) platform for mass spectrometry (MS) is presented. The ESI tips are based on lidless micropillar array
ESI (␮PESI) sources where the transfer of liquid is based on capillary forces without external pumping.
A single silicon platform contains 60 separate ␮PESI tips which can be individually used by rotating the
whole platform by six degrees each time from tip to tip using a computer control. The rotating multitip
␮PESI platform with an ion trap mass spectrometer was successfully demonstrated in rapid identification
and monitoring of intermediates and final products in chemical synthesis within 10 min. The system was
also applied to high-throughput screening of benzodiazepines from urine samples. Urine samples were
extracted with solid-phase extraction (SPE) using C18 phase ZipTipTM pipettes, enabling the use as small
sample volumes as 50 ␮L of the urine sample. Therefore the whole sample treatment and the analysis
with the rotating multitip ␮PESI-MS took only 5 min per sample.
Received 4 November 2011
Received in revised form
22 November 2011
Accepted 22 November 2011
Available online 2 December 2011
Keywords:
Electrospray ionization
Multitip ion source
Mass spectrometry
Rapid analysis
Micropillar
Screening
© 2011 Elsevier B.V. All rights reserved.
Reaction monitoring
1. Introduction
contains an array of nanoelectrospray needles etched in a sil-
icon wafer. Typical flow rates used in systems range from 20
A current trend in analytical chemistry has been miniaturization
and microfabrication of analytical instruments and integration of
different kind of functionalities on the same microchip. One impor-
tant research field in analytical chemistry has been miniaturization
of ion sources for mass spectrometry [1,2]. For example, various
miniaturized electrospray ionization (ESI) sources have been devel-
oped from glass [3,4], silicon [5], and different types of polymers
[6,7]. The miniaturized ion sources can increase ionization effi-
ciency and thereby the sensitivity of measurements, and minimize
the use of organic solvents [8]. Due to low manufacturing costs, the
microchip ion sources can be replaced, when their performance is
not any more acceptable in terms of sensitivity and stability, or they
can be disposable if needed.
to 300 nL/min. It has three operation modes, namely chip-based
infusion, LC–MS fraction collection, and liquid extraction surface
analysis (LESATM) [10]. These modes can be used for example, in
rapid analysis of proteins [11] and lipids [12], drug and metabolite
analysis [13,14], and surface analysis of tissues [15]. Other multi-
array ion source systems are, for example, a multichannel device
with an array of ESI tips [16] chemically etched fused-silica ESI
emitter for improved sensitivity [17], an array of enclosed ESI tips
made from an SU-8 polymer [18], and a micromachined silicon-
based ultrasonic ejector array [19]. A rotating ion source platform
has been previously designed, using a polycarbonate microdevice
with radial closed channels extending to the circumference of the
disk where an opening at the rim acted as an ESI tip [20]. Another
microfabricated ion source platform suitable for automation, a
rotating sample surface for desorption electrospray ionization MS
(DESI/MS), was previously published [21]. In this system suitable
surface spots for DESI were printed on the edge of a CD disc (totally
50 spots), and after introduction of a sample to the sampling spot,
DESI/MS was applied to a rapid analysis of the sample. In this way
a rapid quantitative determination of caffeine in two diet sport
To speed up analysis cycle time and to diminish contamination
risks, multi ion sources have been recently developed, NanomateTM
being the most common of them [9]. The Nanomate system
∗
Corresponding author. Tel.: +358 9 19159194.
E-mail address: raimo.ketola@helsinki.fi (R.A. Ketola).
1387-3806/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.ijms.2011.11.013

66
T. Nissilä et al. / International Journal of Mass Spectrometry 310 (2012) 65–71
Fig. 1. Chemical structures of the benzodiazepines studied.
drinks using isotopically labeled caffeine as an internal standard
was demonstrated. The total analysis time of seven calibration
standards and two sport drink samples (45 separate analyses) was
7.5 min. A similar rotating multinozzle emitter array for nanoelec-
trospray ionization was recently fabricated from silicon [22]. The
platform consisted of 96 identical 10-nozzle emitters in a circular
array on a 3-in. silicon chip. For sampling the inlets on the silicon
platform were connected to 96 silica capillaries via the PTFE tubing,
thus enabling high-throughput measurements.
platform was combined to ion trap and time-of-flight (TOF) mass
spectrometers.
2. Experimental
2.1. Chemicals and samples
HPLC-grade acetonitrile, methanol, and formic acid were
obtained from Sigma (St. Louis, Mo, USA). Water was purified
with Milli-Q purification system (Millipore, Molsheim, France). A
solvent used for flushing the microchip before measurements con-
sisted of 95% acetonitrile or methanol and 1% formic acid in water.
1 mL of 100 ␮M samples of benzodiazepines in methanol, namely
nordiazepam, oxazepam, temazepam, ␣-OH-alprazolam, and ␣-
OH-midazolam were obtained from United Medix Laboratories Ltd.
(Helsinki, Finland). The structures of the compounds are shown
in Fig. 1. Pooled urine and authentic positive urine samples, ana-
lyzed previously with immunological and GC–MS methods, were
obtained from United Medix Laboratories Ltd. Spiked urine sam-
ples were prepared to contain all benzodiazepines with a cut-off
concentration of 200 ng/mL in pooled urine.
We have previously shown that an open silicon micropillar array
channel can induce a spontaneous sample transfer to a sharp tip
where an electrospray plume is formed [23,24]. The open channel
enables very easy sampling of liquid samples onto the microchip
and a rapid transfer of the liquid to the tip of the microchip for
ESI. The open micropillar array channel made of SU-8 polymer can
also be used as a microreactor for protein digestion studies [25].
When a silicon based ␮PESI microchip was covered with titanium
dioxide (TiO2) it could be used for performing of photocatalytic
reactions and mimicking phase I metabolism with an UV lamp, and
a direct analysis of reaction products with MS [26]. The ␮PESI chip
has also been covered with a glass lid to enable liquid chromato-
graphic separation using the micropillar array as a stationary phase
combined with a monolithically integrated ␮PESI tip for MS detec-
tion [27]. The ␮PESI system has also been fabricated in a rotating
multitip format [28]. In this format a silicon wafer has 60 individ-
ual ESI tips at the periphery of a circular microchip platform. It was
shown that a computer-controlled turning from one tip to another
one improves repeatability from tip-to-tip because positioning the
microchip in front of the mass spectrometer was much easier and
more repeatable. In addition, the analysis times were shorter and
contamination was minimized as an unused tip was possible to use
with every single sample.
2.2. The rotating multitip ꢀPESI microchip
The basic analytical properties and fabrication process of the
rotating multitip ␮PESI microchips have been previously published
[28]. Briefly, the 60 tip microchips are fabricated on a silicon wafer.
The fabrication process uses two mask levels: nested masks of
silicon dioxide (SiO2) and aluminum (Al). The SiO2 mask defines
the microreactor chamber and the fluidic channel embedded with
micropillars while the aluminum mask defines the sharp thru-
wafer etched ESI tip. The ESI tip, the sampling spot, and the flow
channel are created with deep reactive ion etching (DRIE). Both
the microreactor and the flow channel contain micropillars (diam-
eter 60 ␮m, space between the pillars 15 ␮m, height 22 ␮m, Fig. 2c)
to support spontaneous sample flow towards the electrospray tip.
There are 60 tips on one platform and the angles between the tips
are 6 degrees (Fig. 2a and b).
The aim of the study was to evaluate the performance of the
rotating multitip ␮PESI source in rapid analyses. The feasibility
of the multitip-␮PESI was studied in the rapid follow-up analysis
of synthesis processes and in high-throughput screening analysis
of abused drugs in urine samples using a quick sample prepa-
ration method of ZipTipTM solid-phase extraction (SPE) [29,30]
prior to the measurement with ␮PESI/MS. The multitip ␮PESI

T. Nissilä et al. / International Journal of Mass Spectrometry 310 (2012) 65–71
67
Fig. 2. (a) A rotating multitip-␮PESI platform containing 60 individual tips combined with MS. (b) Three individual ␮PESI tips on a rotating multitip platform. Width of the
pillar array channel is 1 mm. (c) A scanning electron micrograph of a micropillar array of the chip. (d) A single ␮PESI chip with a ZipTip solid-phase extraction pipette.
2.3. Mass spectrometry
(A, Fig. 3) was studied. The purity of starting heptafulvene was
checked with NMR before reaction and no impurities were found.
Reaction was carried out in a round-bottomed flask placed in an
acetone–ice bath (−15 ◦C). Heptafulvene A (6.00 mg, 1.0 equiv.) was
dissolved in dichloromethane (2.0 mL), producing a 10 mM reac-
tion solution of m-chloroperoxybenzoic acid (m-CPBA, Aldrich, 77%,
11.0 mg, 2.5 equiv.) to initiate the oxidation reaction. From the mag-
netically stirred reaction mixture, a 10-␮L aliquot was taken and
diluted with 1 mL of 0 ◦C (ice cold) 95% methanol/1% formic acid/4%
water by shaking by hands for a few seconds. 1.5 ␮L of the diluted
sample was introduced to the ␮PESI tip for immediate analysis of
remaining starting materials, reaction intermediates and products.
The whole sampling procedure took about 10–15 s. The first four
samples were taken with a rate of one sample in 1 min and the
last one was taken after 10 min of adding the oxidizing agent to a
reaction mixture.
Waters Q-TOF Micro (Waters, UK), Bruker MicroTOF (Bruker
Daltonics, Germany), and Agilent 6330 Ion trap (Agilent Technolo-
gies, Santa Clara, CA, US) were used in MS experiments. The Q-TOF
and MicroTOF, which were used in the drug screening, were cali-
brated using a sodium formate solution in external calibration. The
microchip positioned in front of the cone of a mass spectrometer
to the distance of 5 mm (Fig. 2a). Nitrogen produced by a high-
purity nitrogen generator was used as a cone gas (flow 40 L/h, cone
temperature 80 ◦C). A platinum electrode connected the high volt-
age source of the MS to the microchip. The high voltage of 3.5 kV
was used in experiments for electrospray ionization in a positive
ion mode. A mass range of m/z 80 to 800 was monitored. With
Agilent 6330 ion trap mass spectrometer, which was used for reac-
tion monitoring, the microchip was positioned to a 5-mm distance
from the MS entrance cone. Ultra scan and positive ion modes were
used and drying gas temperature was set to 150 ◦C with a flow
rate of 2.0 L/min. No nebulizer gas was used. A multitip ion source
chip was attached to a rotating platform (Thorlabs CR1-Z7/M, Thor-
labs Sweden AB, Gothenburg, Sweden). The rotating platform was
controlled by Thorlabs computer software APT motor controller.
Each sample was pipetted onto an unused tip, thus minimizing
cross-contamination between samples. After measurements the
whole ␮PESI microchip was cleaned with a Piranha solution (3:1
mixture of ammonium hydroxide (NH4OH) and hydrogen peroxide
(H2O2) in an ultrasonic bath, whereafter it was rinsed with deion-
ized water and methanol and let to dry in room temperature. After
this cleaning procedure the microchip was ready to be reused.
2.5. Urine sample pretreatment
A
spiked pooled urine sample was prepared to contain
nordiazepam, oxazepam, temazepam, ␣-OH-alprazolam, and
␣-OH-midazolam with concentration of 200 ng/mL of each com-
pound. Solid-phase extraction of urine sample was done with 10-␮L
C18-ZipTipTM pipettes (Millipore, Molsheim, France) (Fig. 2d). First,
the tips were conditioned three times with 10 ␮L of acetonitrile
and three times with 10 ␮L of 10% aqueous acetonitrile keeping
the tip wet the whole time. After conditioning, the tip was loaded
five times with 10 ␮L of the sample, washed ten times with 10 ␮L
of 10% aqueous acetonitrile, and after the last washing step the tip
was let dry. Elution of the sample directly to the microchip was
made with 2 ␮L of 95% methanol/1% formic acid/4% water.
2.4. Organic synthesis and sampling
2 mL of authentic positive benzodiazepine urine samples were
enzymatically hydrolyzed using 500 ␮L of 800 mM sodium phos-
phate buffer (pH 7.0) with 20 ␮L of ␤-glucuronidase (E. coli,
Roche Diagnostics GmbH, Mannheim, Germany). The samples were
The synthesis of tropones from heptafulvenes has been pre-
viously described [31]. Briefly, the oxidative cleavage of the
semicyclic carbon–carbon double bond of the starting heptafulvene

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T. Nissilä et al. / International Journal of Mass Spectrometry 310 (2012) 65–71
Fig. 3. A synthesis reaction pathway and structures of the intermediates [31] and final reaction products, showing their molecular weights and the m/z values of ionized
molecules.
vortexed and incubated in 55 ◦C water bath for 30 min. After that
the same ZipTip-solid phase extraction method was used for pre-
treatment as with spiked urine samples.
and trioxygenated byproducts of the starting compound A, respec-
tively, and the ion at m/z 173 is protonated m-CPBA. The ion at
m/z 321 may represent two compounds, namely the protonated
molecule of an oxidation product of A and a tropylium cation. The
ion at m/z 515 is a sodium adduct of the intermediate D. The 37Cl iso-
tope ion at m/z 517 confirmed the presence of one chlorine atom in
the molecule. The ion at m/z 391 was formed also in the experiment
without the starting material A and its origin remained unclear,
even with MS/MS data obtained. The ion at m/z 286 is a fragment
ion from D, produced by the loss of m-CPBA and HF, as evidenced
in MS/MS measurements (Table 2). The identity of product D has
not been previously confirmed with any other method due to its
relatively rapid reactivity to form a product E. The structures of the
products were confirmed by MS/MS spectra (Table 1). All synthesis
products showed an ion [M+H–HF]+ confirming that the products
contained at least one fluorine atom (in-CF3 group) and are derived
from the starting material A. The intermediates B/C showed a loss of
formaldehyde, producing an ion at m/z 291 ([M+H–CH2O]+) which
confirmed that these intermediates contained at least one oxy-
gen close to the isopropyl group. The intermediate D showed a
loss of both m-chlorobenzoic acid and m-CPBA, confirming that
the starting material A has reacted with m-CPBA. The final prod-
uct E showed a specific loss of carbon monoxide (CO), producing
a six-membered ring and confirming the existence of a ketone
group in the molecule. An increase and/or a decrease of the starting
material and reaction products as a function of time are shown in
Fig. 4b. The analytical cycle was fast enough for the measurement
of relative abundances of each reaction product. Interestingly, the
multitip-␮PESI-MS measurement of the pure starting compound A
showed also mono- and dioxygenated products (ions at m/z 321 (B)
and 337), even without using any oxidant. The starting compound
A was known to be highly reactive, thus it could be oxygenated
3. Results and discussion
3.1. Rapid analysis of reaction products from organic synthesis
The multitip-␮PESI with an ion trap MS was applied to monitor-
ing of reaction products from a synthesis of tricyclic tropones from
heptafulvenes which has been previously published but the struc-
tures of the intermediate products have not been confirmed yet
[31]. The synthesis is rather rapid, as the whole synthesis is com-
pleted in 10 min. However, despite being carried out as a “one-pot”
process, it consists of several chemical reaction steps, thus yielding
intermediate products whose structures have not been confirmed
so far with any analytical methods. Therefore, the new multitip-
␮PESI chip was found to be a suitable choice for the analysis as it
provides rapid analyses, hence kinetics could also be determined
from the analytical results. Due to high concentration of the start-
ing material, and hence of the reaction products, a liquid sample
from the reaction vessel was first diluted with methanol by 100-fold
prior to the analysis to avoid overloading of the analytical instru-
ment and especially ES ionization. Dilution was made with a chilled
solvent to prevent the reaction from proceeding further before the
analysis.
The mass spectra obtained from the experiments supported the
postulated synthesis route (Fig. 3a) [31]. As an example, a mass
spectrum of synthesis products measured at 2.5 min after start-
ing the reaction is shown in Fig. 4a. The starting compound A was
observed at m/z 305, intermediates at m/z 321 and 515 and the final
product at m/z 279. The ions at m/z 337 and 353 are dioxygenated

T. Nissilä et al. / International Journal of Mass Spectrometry 310 (2012) 65–71
69
already in the solvent by residue oxygen or during ES ionization
[32,33]. Another advantage of the new analytical method is the
very low amount of the starting compound needed for the anal-
ysis (150 pmol ∼ 50 ng), therefore the synthesis could be done in a
microscale, for example using a microreactor on a chip integrated
with an on-chip ESI source. The results obtained in this experiment
show that the multitip-␮PESI-MS can be used for easy, convenient,
and rapid monitoring of organic reactions.
3.2. High-throughput screening of drugs
Screening of drugs from urine samples is commonly performed
with immunological methods, but the drawback of those methods
is that they can give false positive or negative results because of
the lack of the specificity or sensitivity. Gas chromatography–mass
spectrometry (GC/MS) [34,35] and liquid chromatography–mass
spectrometry (LC/MS) [36,37] are more selective and sensitive
methods for screening, but they are still relatively slow for high-
throughput screening analysis. Therefore, the feasibility of the
multitip-␮PESI platform combined with a TOF/MS instrument was
evaluated in this study for screening of selected benzodiazepines
from urine samples. For sample treatment a rapid ZipTip based
method was chosen to take advantage of the fast analytical method.
The purpose was also to minimize the total volume of urine used,
and it was noticed that 50 ␮L of urine sample was enough to achieve
sensitivity level required for detection of drugs at cut-off concen-
trations (200 ng/mL) used in immunological screening methods.
The analytes were eluted with 2 ␮L of 95% methanol with 1% of
formic acid which was directly deposited on the ␮PESI sampling
spot and subsequently analyzed with MS. Temazepam, oxazepam,
nordiazepam, ␣-OH-midazolam, and ␣-OH-alprazolam are com-
monly used target compounds in the screening of benzodiazepines
in urine samples, and therefore they were selected for the ZipTip-
multitip-␮PESI-TOF/MS analysis [38].
The compatibility of the ZipTip-multitip-␮PESI-TOF/MS for
screening of benzodiazepines was demonstrated with an anal-
ysis of spiked urine samples and 12 authentic urine samples,
previously detected as positive samples with immunological and
GC/MS methods. In the same analysis sequence also standard
solutions at cut-off concentration levels (200 ng/mL) and negative
control samples (unspiked urine samples) were analyzed. Fig. 5a
shows a mass spectrum of a spiked urine sample measured with
ZipTip-multitip-␮PESI-TOF/MS. The spiked urine sample contained
benzodiazepines, namely nordiazepam, oxazepam, temazepam,
␣-OH-alprazolam, and ␣-OH-midazolam with a concentration of
200 ng/mL. All benzodiazepines were detected with adequate sig-
nal intensity for positive identification when a mass spectrum of
a reagent blank was subtracted from the mass spectrum of the
sample. Fig. 5b shows a typical mass spectrum measured with
the ZipTip-multitip-␮PESI-TOF instrument from an authentic urine
sample that contained oxazepam (a protonated molecule at m/z
287) and ␣- or/and 4-OH-midazolam (protonated molecule at m/z
342). The ions at m/z 289 and 344 were concluded to be protonated
oxazepam and ␣- or/and 4-OH-midazolam with a 37Cl isotope,
respectively. The sample was previously analyzed with GC/MS and
the same compounds were detected, showing the capability of
ZipTip-multitip-␮PESI-TOF method for high-throughput screening
of benzodiazepines from urine samples. The sample treatment with
ZipTip extraction was effective as the signals from matrix compo-
nents were minimal, as shown in the mass spectrum of a blank
urine samples (Fig. 5c). The blank urine sample contained some
matrix compounds, but the ions detected did not interfere with the
analysis of benzodiazepines as they had clearly different accurate
masses from those of the analytes. The urine samples were also
analyzed directly with multitip-␮PESI-TOF/MS without the ZipTip
extraction but as assumed, the matrix components interfered with
Fig. 4. (a) A mass spectrum of synthesis products measured at 2.5 min after start-
ing the reaction. (b) Relative intensities of the starting compound and the reaction
products by a function of time measured by the multitip-␮PESI platform with ion
trap MS. The RSDs of three replicate experiments are also shown.
Table 1
MS/MS data of the starting compound and reaction products measured by multitip-
␮PESI platform with ion trap MS.
Compound
Precursor ion, m/z
Product ions in MS/MS (m/z) and their
structures
A
305, [M+H]+
303 [M+H–H2]+
289 [M+H–CH4]+
285 [M+H–HF]+
283 [M+H–H2–HF]+
269 [M+H–CH4–HF]+
319 [M+H–H2]+
B/C
321, [M+H]+/M+
301 [M+H–HF]+
299 [M+H–H2–HF]+
291 [M+H–CH2O]+
283 [M+H–HF–H2O]+
279 [M+H–H2–2HF]+
495 [M+Na–HF]+
D
E
515, [M+Na]+
279, [M+H]+
475 [M+Na–2HF]+
359 [M+Na-m-chlorobenzoic acid]+
339 [M+Na-m-chlorobenzoic acid–HF]+
286 [M+Na-m-CPBA–HF]+
259 [M+H–HF]+
251 [M+H–CO]+
239 [M+H–2HF]+
231 [M+H–CO–HF]+
211 [M+H–CO–2HF]+

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T. Nissilä et al. / International Journal of Mass Spectrometry 310 (2012) 65–71
Table 2
Analytical results of benzodiazepines analyzed with quantitative GC/MS and
screening ZipTip-multitip-␮PESI-TOF/MS methods. Compounds with concentra-
tions below the cut-off value (200 ng/mL) of the ZipTip-multitip-␮PESI-TOF/MS are
in parenthesis.
Sample
GC/MS, ng/mL
␮PESI-TOF/MS, with 20 mDa
window
1
2
␣-OH-alprazolam, 2494
Oxazepam, 396
␣- and/or 4-OH-alprazolam
Desmethyldiazepam
(desmethyldiazepam, 155)
(temazepam, 193)
3
4
5
␣-OH-alprazolam, 462
Oxazepam, 206
(desmethyldiazepam, 32)
(temazepam, 26)
␣-OH-midazolam, overload
Desmethyldiazepam, 7499
Temazepam, 4601
␣- and/or 4-OH-alprazolam
␣- and/or 4-OH-midazolam
Desmethyldiazepam
Temazepam
Oxazepam, 4409
␣-OH-midazolam, 1486
(desmethyldiazepam, 164)
(oxazepam, 152)
␣- and/or 4-OH-midazolam
Desmethyldiazepam
(temazepam, 25)
6
7
Oxazepam, 10000
Oxazepam
␣- and/or 4-OH-midazolam
Temazepam
␣-OH-midazolam, 164
Temazepam, 67207
Oxazepam, 20292
Oxazepam
Desmethyldiazepam, 3150
Desmethyldiazepam, 455
Oxazepam, 1052
Temazepam, 551
Oxazepam, 397
Desmethyldiazepam
Desmethyldiazepam
8
9
Desmethyldiazepam
(desmethyldiazepam, 57)
(␣-OH-alprazolam, 171)
(temazepam, 33)
10
11
12
Oxazepam, 33810
Oxazepam
␣-OH-midazolam, 946
(␣-OH-alprazolam, 189)
Temazepam, 4065
Desmethyldiazepam, 220
Oxazepam, 2377
␣- and/or 4-OH-midazolam
␣- and/or 4-OH-alprazolam
Temazepam
Desmethyldiazepam
Oxazepam
Desmethyldiazepam, 244
Oxazepam, 312
Desmethyldiazepam
Oxazepam
Temazepam, 1801
Temazepam
not detected in other five samples, due to either ion suppres-
sion caused by matrix components or other benzodiazepines, low
recovery of oxazepam during the extraction (not likely as it can
easily been detected from spiked samples) or decomposition of
oxazepam during thaw and freeze cycles as the urine samples could
not be analyzed with the ZipTip-multitip-␮PESI-TOF/MS method
at the same time as with the other methods. However, all samples
were detected as positives with the ZipTip-multitip-␮PESI-TOF/MS
method and in routine analysis all positive screening results would
be confirmed with a quantitative analytical method, such as GC/MS
or LC–MS/MS techniques.
Fig. 5. (a) A mass spectrum of a spiked urine sample containing nordiazepam (m/z
271), oxazepam (m/z 287), temazepam (m/z 301), ␣-OH-alprazolam (m/z 325), and
␣-OH-midazolam (m/z 342) with a concentration of 200 ng/mL, (b) a mass spectrum
of an authentic positive urine sample containing oxazepam (m/z 287) and ␣- and/or
4-OH-midazolam (m/z 342), and (c) a mass spectrum of a blank urine sample. All
samples were analyzed with ZipTip-multitip-␮PESI-TOF/MS. Background (a mass
spectrum of a reagent blank) was subtracted from the mass spectra A and B.
The whole analytical procedure (SPE and ␮PESI-TOF/MS mea-
surement) was rapid, as the total analysis time was approximately
5 min per sample. In addition, the consumption of samples and
reagents was minimal, as only 50 ␮L of the urine sample was
needed and less than 200 ␮L of solvents were used in SPE per
sample. This new approach could be used in the future as a new
alternative for conventional screening methods.
the ionization and detection of the analytes which were not visible
in the mass spectra measured (data not shown).
All authentic urine samples containing benzodiazepines or their
conjugates were detected as positive with the ZipTip-multitip-
␮PESI-TOF/MS method using a detection window of 20 mDa for
identification (Table 2). In some urine samples the concentrations
of individual benzodiazepines determined by GC/MS were actu-
ally below the cut-off values, therefore it was expected that these
compounds would not necessarily be detected by ZipTip-multitip-
␮PESI-TOF/MS method. No false positives were observed from
negative control samples with the method. The results obtained
were in a good agreement with those obtained with the GC/MS
method, except in the case of oxazepam and one sample (no. 8)
with temazepam. Oxazepam was detected in five samples but was
4. Conclusions
Parallelization of the ␮PESI tips to a rotating multitip plat-
form combined with MS was shown to be a promising method
for high-throughput screening in qualitative and semi-quantitative
analyses. The speed and easiness of the rotating multitip ␮PESI
platform with ion trap MS proved to be adequate for rapid mon-
itoring of reaction products in an organic synthesis. The amount

T. Nissilä et al. / International Journal of Mass Spectrometry 310 (2012) 65–71
71
of the starting compound needed for the analysis was at a pmol
range, which enables utilization of microreactors for the synthesis,
still providing high sensitivity in the analysis. The rotating multitip
␮PESI/MS combined with ZipTipTM solid-phase extraction method
proved to be efficient approach for specific screening of benzodi-
azepines from urine, being rapid, sensitive, and selective. The next
step in the development is to automate both the ZipTipTM SPE and
␮PESI/MS analysis which would reduce the total analysis time from
the current five min per sample to approximately one min per sam-
ple. This should also further enhance sensitivity and reproducibility
of the measurements.
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Acknowledgements
We gratefully acknowledge The Academy of Finland (Projects
129633, 138674, and 108276), The Finnish Funding Agency for
Technology and Innovation (TEKES) (project 40399/08), Walter och
Lisi Wahl Stiftelse för Naturvetenskaplig Forskning, the European
Commission (Contract No. LSHB-CT-2004-503467), and Graduate
School in Pharmaceutical Research for financial support of this
work.
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