Direct nano-electrospray

royalsocietypublishing.org/journal/rsos
ionization tandem mass
spectrometry for the
Research
quantification and
Cite this article: Amer S, Zarad W, El-Gendy H, identification of
Abdel-Salam R, Hadad G, Masujima T, Emara S.
2019 Direct nano-electrospray ionization tandem
mass spectrometry for the quantification and
metronidazole in its dosage
identification of metronidazole in its dosage form
and human urine. R. Soc. open sci. 6: 191336.
form and human urine
http://dx.doi.org/10.1098/rsos.191336

Received: 5 August 2019

Sara Amer1,2, Walaa Zarad1, Heba El-Gendy1,
Accepted: 4 October 2019 Randa Abdel-Salam3, Ghada Hadad3,
Subject Category: Tsutomu Masujima2,† and Samy Emara1
Chemistry
1
Faculty of Pharmacy, Misr International University, Km 28 Ismailia Road, Cairo 11865, Egypt
2
Subject Areas: Quantitative Biology Center (QBiC), RIKEN, 6-2-3 Furuedai, Suita, Osaka 565-0874, Japan
3
analytical chemistry Pharmaceutical Analytical Chemistry Department, Faculty of Pharmacy, University of Suez
Canal, Ismailia 41522, Egypt
Keywords: SA, 0000-0002-7381-5361
direct mass spectrometry, nanospray electrospray
ionization, nanospray capillary, metronidazole, A rapid, sensitive and direct nano-electrospray ionization-tandem
mass spectrometry (NS-ESI-MS/MS) method, using an offline
human urine, 1, 3, 6- polytyrosine
nanospray (NS) capillary, has been developed and validated for
the analysis of metronidazole (MTZ). A mixture of 2 µl MTZ
Author for correspondence:
sample solution prepared in an ionization solvent consisting of
Sara Amer
methanol : water : formic acid in a ratio of 80 : 20 : 0.3, together
e-mail: sara.m.amer@hotmail.com with 2 µl of an internal standard (IS), 1,3,6-polytyrosine, is
loaded into the back of the NS capillary. The NS capillary was
fitted into the ion source at a distance of 3 mm between the NS
tip and MS orifice. The sample is then analysed and acquired a
† sustainable signal that allowed for data compilation across
Deceased 3 March 2017.
This article has been edited by the Royal Society various data points for MTZ identification and quantification.
The quantification relied on the ratio of the [M + H]+ peaks
of Chemistry, including the commissioning, peer
of MTZ and IS with m/z values of 172.0717 and 182.0812,
review process and editorial aspects up to the respectively, while the identification relied on the MS/MS of the
point of acceptance. precursor ions [M + H]+ of both compounds and their fragments
at 128.05 for MTZ and 165.1 and 136.07 for the IS. The NS-ESI-
MS/MS method was accurate and precise for the quantification
of MTZ over the concentration range from 2.5 to 25 000 ng ml−1.

© 2019 The Authors. Published by the Royal Society under the terms of the Creative
Commons Attribution License http://creativecommons.org/licenses/by/4.0/, which permits
unrestricted use, provided the original author and source are credited.
 The applicability of the method was confirmed by MTZ analysis in its pharmaceutical dosage 2
form and detection of the analyte in clinical human urine samples without any sample

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treatment procedure.

1. Introduction
Metronidazole (1-(2-hydroxyethyl)-2-methyl-5-nitromidazole) (MTZ) is one of the essential drugs used to
treat protozoal infections of Trichomonas vaginalis and anaerobic microbial infections [1]. MTZ is also used
to treat infections such as giardia in pets [2]. It is the most widely used nitroimidazole, and is well
tolerated at doses of less than or equal to 2 g d−1. It is an essential drug in the Egyptian market due
to the high level of pollution and other factors that contribute to a high number of cases involving

R. Soc. open sci. 6: 191336

diarrhoea and intestinal infections [3].
Several methods have been reported for the determination of MTZ from its individual or combined
formulations with other active ingredients using spectrophotometry [4–7], electrochemistry [8–11],
capillary electrophoresis [12–14], gas chromatography (GC) [15–18], high-performance liquid
chromatography (HPLC) coupled with ultraviolet [19–25] and mass spectrometric detector methods
[26–34].
The ability for separation has allowed LC to be the first choice in method selection for a wide
range of drugs. Perhaps the greatest advantage in using LC as a separation technique in coupling
with MS as a detector (LC-MS) is the ability to identify multiple compounds in unresolved
chromatographic peaks.
However, chromatographic methods have their drawbacks. All previous published LC-MS methods
clarified that mobile phase composition and type of stationary phase have to be investigated to
understand the occurrence of both analyte and internal standard (IS) peaks. This usually involved
prolonged chromatography run time. Also, carry-over is one of the most common problems in
developing the LC-MS method and the sources of this problem range from hardware devices and the
selection of appropriate rinse solvents to challenges with chromatography. Choosing interface and
mass spectrometer settings is another issue that should be considered. All these have been considered
due to the desire to set a shorter timeline in drug discovery and development which led to the need
for rapid and sensitive approaches for drug evaluation in dosage forms, as well as monitoring in
biological matrices for further study.
The nanospray (NS) capillary, produced by Humanix (Hiroshima, Japan), devised a new coupling
technique with MS. Masujima’s group has been using NS capillaries in the field they have coined as
‘live single cell MS’, in which they trap single cells for analysis in order to identify many bioactive
compounds [35–42]. However, with continuous improvement, these NS capillaries can stand out as a
promising analytical tool for pharmaceutical quality control applications. The current study uses
these NS capillaries as a replacement for LC-MS interface systems and directly sprays the sample into
the mass spectrometer. Their unique shape and minute tip bore size allow for optimum electrospray
and small droplet size, leading to more ion formation. Also, the instrument’s unique set-up under
ambient conditions can be applied on a wide variety of thermo-sensitive drugs. In the present study,
we did not use the LC interface and its associated problems. The use of disposable NS capillaries
reduces the potential for contamination, carry-over effect. In addition, the small volume approach
allows for the introduction of less matrix components into the MS system, thus extending the useful
life of the equipment, in contrast with traditional LC methods. This study explores the use of
these NS capillaries in the development and validation of a direct NS-ESI-MS/MS method to
evaluate the analysis of MTZ in tablet dosage forms in 30 s, with the intention of possible
applications for various routine pharmaceutical developments. To the best of our knowledge, it is the
first study of a drug analysis using direct NS-ESI-MS/MS. Sample solutions were loaded into the NS
capillaries and then analysed using this technique, acquiring sustainable signal that allowed for
swift data compilation across various data points for compound identification and quantification.
The detection of MTZ in clinical urine samples is still required and will reveal the applicability of the
direct NS-ESI-MS/MS design to significantly reduce sample run time, drug detection limit and may
also have an impact on the qualitative and quantitative analysis of drugs in biological fluids.
The applicability of the proposed direct NS-ESI-MS/MS method was assessed by analysing random
urine samples collected from patients in treatment with MTZ.
2. Material and methods 3

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2.1. Chemical and materials
HPLC grade methanol (Tokyo Chemical Industry Co., LTD, Japan) was used. Formic acid (Sigma Aldrich,
Germany) was of reagent grade (greater than or equal to 95%). 1,3,6- polytyrosine solution (Thermo Fisher
Scientific) was used as IS. MTZ was a gift from Sanofi-Aventis (Egypt). Flagyl tablets (Batch no. 5EG102)
were manufactured by Sanofi-Aventis. Each tablet contains 500 mg MTZ. Ultrapure water (Organo,
Puric-ω, Tokyo, Japan) was prepared daily and used throughout the experiment.

2.2. Standard solutions and calibration

An equivalent amount of MTZ powder was dissolved in methanol and used as a stock solution of
5 mg ml−1. From the stock solution, serial dilutions were prepared to cover the concentration range of

R. Soc. open sci. 6: 191336

0.25–2500 µg ml−1 of working solutions using the same solvent. All stock and working solutions were
kept at −20°C. The calibration curve of MTZ was prepared by diluting the above working solutions
100 times with methanol to obtain a series of calibration standard solutions ranging from 2.5 to
25 000 ng ml−1. Each concentration was injected in replicates of three.

2.3. Instrumentation
Full MS analysis was carried out on a high-resolution mass spectrometer (OrbitrapVelos Pro, Thermo
Fisher Scientific). The Velos Pro mass spectrometer was equipped with an offline NS ion source
(Thermo Fisher Scientific). The NS ion source was positioned on a manual XYZ stage, and the XYZ
stage was placed in front of the mass spectrometer inlet. Once the NS capillary was mounted on the
ion source, the XYZ stage was then used to move the NS capillary to the optimum position. To aid in
this, a video camera was positioned between the NS capillary tip and the inlet of the MS instrument,
allowing for the user to observe and calculate the distance between the two. MS/MS analysis was
done in the ion trap mode of the instrument. Helium gas (99.999% purity) was used as the collision
gas. Collision energy was set between 20 and 25 eV in collision-induced dissociation (CID) mode. The
ionization solvent used consisted of 80 : 20 : 0.3 ratio between methanol : water : formic acid. Solutions
to be analysed were loaded into NS capillaries of 1 µm tip pore size (Humanix, Hiroshima, Japan)
using pipette tips (Eppendorf, GELoader tips). The spray voltage applied was 1000 V. Full MS scans
were monitored in the following ranges: 150–380, 150–212, 165–190 and alternating scans between
170–174 and 180–184, each for 30 s. All MS/MS spectra were recorded using an average across 30 s of
scans. Ion trap calibration was done once at the very beginning of the experiment in the positive
mode using the positive ion mode calibration solution, which was prepared as stated in the Thermo
Scientific LTQ Velos Pro Manual. Positive mode Fourier transform (FT) manual calibration was done
every morning with the NS ionization source using commercially available polytyrosine solution.

2.4. Dosage form analysis

According to the label, Flagyl contains 500 mg of MTZ per tablet (Sanofi-Aventis Egypt). A total of 10
tablets were weighed and then pulverized and homogenized. An accurately weighed portion of the
powder equivalent to 250 mg of MTZ was transferred to a 50 ml volumetric flask and then dissolved in
about 30 ml of methanol using a shaker for 30 min. After extraction, the obtained solution was
quantitatively diluted to volume with methanol. The solution was then filtered; the first portion of the
filtrate was discarded and the remainder was used as stock sample solution. A known volume of sample
stock solution was diluted quantitatively with methanol to obtain a concentration of 500 ng ml−1 MTZ.

2.5. Urine analysis

The development of a convenient direct MS tool to detect MTZ in urine samples is still required.
Consequently, the developed direct NS-ESI-MS/MS method was further applied to detect MTZ in
random human urine samples with high sensitivity, selectivity and without any sample pretreatment
or separation procedures. Urine samples were collected from volunteers after the oral administration
of a single dose of MTZ in some cases. In other cases, urine samples were collected after the
administration of MTZ and other prescribed drugs. Urine samples were loaded into the NS capillaries 4
and processed as mentioned under instrumentation.

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2.6. Selectivity
Five blank preparations (ionization solvent) were prepared and injected to determine the selectivity of the
method. The degree of selectivity was determined by the presence or absence of interference m/z peaks at
the m/z values of MTZ and IS.

2.7. Data analysis

Data acquisition was performed using Thermo Xcalibur software and raw data were analysed on Microsoft
Excel. Detected peaks were estimated by the exact mass value of ±5 ppm tolerance, and annotated by
KEGG database (http://www.kegg.jp/kegg/) and HMDB database (http://www.hmdb.ca/). MS/MS

R. Soc. open sci. 6: 191336

spectra were compared against MetFrag (https://msbi.ipb-halle.de/MetFrag/).

2.8. Assay validation

Three different concentration levels (10, 100 and 10 000 ng ml−1) of MTZ were prepared to determine
intra- and inter-day precision and the accuracy of the proposed method against the calibration curve.
Intra-assay precision was assessed as the relative standard deviation (RSD) of the mean MTZ/IS ratio
obtained on the same day. Inter-assay precision was calculated using the RSD of the mean MTZ/IS
ratio on five consecutive days. Accuracy was determined as the per cent of the relative errors (REs) of
the mean measured concentrations using the following equation:
 
mean measured concentration – nominal concentration
RE% =
100:
nominal concentration

3. Results and discussion

3.1. NS offline ionization advantages
The NS capillaries are minute, hollow, borosilicate glass tubes 28 mm long and, depending on the selected
size, have a tip bore size ranging from 1 to 10 µm wide. These tips are platinum coated, allowing
for electrical conductivity [43]. Samples are loaded into the NS capillary from the back side using a
gel-loading pipette. After mounting the capillary onto the ion source, electric voltage is applied to start
the spray, producing an electric field between the NS capillary and MS inlet. Under this influence, ions
of similar polarity, as the applied voltage, are repelled and migrate towards the NS tip.
At the moment of electric voltage application, ions of similar charge to that of the applied voltage are
repelled and migrate towards the NS outlet. In the current study, the positive charge of the applied
voltage repels the ions of similar charge, causing them to migrate towards the NS outlet. A Taylor
cone is then formed due to the equilibrium between the electrostatic force and the surface tension of
the sample liquid. While the electrostatic force pulls the liquid from the capillary towards the MS
inlet, the surface tension runs in the opposite direction. Immediately after, a fine jet of the solution is
released from the tip of the Taylor cone, which then easily disperses into small droplets. These
droplets are composed of equidistant multicharges and many analyte molecules. A decreased size
of these droplets initially occurs due to solvent evaporation, until repulsion forces (Coulomb forces) of
the like charges overcome the surface tension of the said droplet. At this point, the Rayleigh limit is
said to be reached and Coulomb fission occurs. This process is repeated until singular charged analyte
molecules are produced, which the MS inlet enters. This process is illustrated in figure 1.
Due to the inherently low flow rates achieved solely by electrostatic and capillary forces, nano-ESI has
several advantages over traditional ESI techniques with higher flow rates. A lower flow rate solution is
known to reduce the size of primarily produced charged droplets, and the generated droplets by an NS
tip are about 100 times smaller than those of conventional ESI sources [44], allowing for a more efficient
use of the total sample with minimum loss of analyte material in comparison with conventional ESI
techniques. Therefore, initial droplets need fewer droplet fission events and less solvent evaporation
prior to the release of a gas-phase ion. Analytically, the developed direct NS-ESI-MS/MS method is
 5
MS capillary
nanospray tip
inlet

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at 250˚ C
+
– – + +
(a) + + 3 mm distance
+ + + –
ambient conditions mass analysis
– – + +
+ –

power supply
+ve –ve

(b) (c) (d) (e) (f)

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+ +
- + + - + + + +
+ + + +
- + + ++ - + + + + ++
+ ++++ +
+ + + +
+ + + + +
+ + + ++ + + + + + +
- - ++ ++ - - + ++ ++ + +++ + + + +
+ + +
+

Taylor cone
Rayleigh limit

Figure 1. (a) The overall framework of NS ionization. (b) Upon application of positive voltage, ions that carry positive charge are
repelled and migrate to the NS tip. (c) The positive ions continue to accumulate until a Taylor cone is produced. (d ) The jet stream
produced from the Taylor cone produces comparatively large primary multicharged droplets. (e) Desolvation decreases the size of the
primary droplets, pushing the like charges closer and closer until the Rayleigh limit is reached, in which repulsion forces surpass
surface tension and ( f ) Coulomb fission occurs. This process is then repeated until singularly charged analyte molecules are formed,
which are analysed by the mass spectrometer.

unique, in that it achieves a quantifiable signal without the need for separation, thereby removing the
downsides of method coupling.

3.2. Method optimization

3.2.1. Voltage
For MTZ determination, preliminary experiments were carried out on a voltage range of 800–1300 V. It
was observed that the applied low voltage caused considerable instability in the analyte signal, whereas
the voltage greater than 1000 V led to the loss of the MS signal. An adequate voltage application of
1000 V, resulting in excellent sensitivity and stability of the analyte signal, was selected for further study.

3.2.2. MS inlet temperature

Different inlet temperatures were tested to promote thermal declustering, if needed. For this experiment,
a range of 200–400°C was evaluated in increments of 50. Little difference was shown between the results,
indicating minimal ion clustering of the sample, so a default of 250°C was set.

3.2.3. Distance to MS inlet

Experimentation was done on a singular concentration at different distances between the NS capillary tip
and MS inlet ranging 1–4 mm, measured by the scaled XYZ stage and video camera in order to find the
position that attained the highest ion count. The best results were achieved at a distance of 3 mm, which
was kept constant throughout the present study.

3.2.4. Ionization solvent, composition and ratio

The effect of ionization solvent for offline ionization of MTZ was done with different ratios between
methanol and ultrapure water, with the addition of 3% formic acid. A 100 ng ml−1 solution of standard
 9 6
8

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7

MTZ/IS ratio
6
5
4
3
2
1
0
50 60 70 80 90 100
methanol % v/v
Figure 2. Dependence of the signal intensity of MTZ on the methanol ratio of the ionization solvent.

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MTZ IS
(a) 40 000

35 000
30 000
signal intensity

25 000
20 000
15 000
10 000
5000
0
(b) 100 000

80 000
signal intensity

60 000

40 000

20 000

(c) 200 000 28 000 000

180 000 27 000 000
160 000
MTZ signal intensity

26 000 000
IS signal intensity

140 000
120 000 25 000 000
100 000 24 000 000
80 000 23 000 000
60 000
22 000 000
40 000
20 000 21 000 000
0 20 000 000
time interval (30 s)
Figure 3. Comparison between IS and MTZ signal at (a) 10 ng ml−1, (b) 100 ng ml−1 and (c) 10 000 ng ml−1 over a 30 s interval.

MTZ was prepared and injected with different solvent ratios, ranging from 50–100%, in replicates of 3.
Average [M + H]+ signal intensities of MTZ to IS were analysed and summarized in figure 2. As
shown, the maximum ionization of MTZ was observed at 80% methanol, as indicated by the signal
 (a) RT: 1.50–2.00 7
NL:
100 2.06E6
1.63 1.96 m/z=
1.77 1.91

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1.58 172.07 – 172.07 F:
90 1.69 1.83 FTMS + p NSI
Full ms
[165.00-190.00]
MS 1ug_1
80

70
relative abundance

60

50

40

30

20

10

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0
1.50 1.55 1.60 1.65 1.70 1.75 1.80 1.85 1.90 1.95 2.00
time (min)

1ug_1#1-88 RT: 1.53-1.99 AV: 18 NL: 1.76E6

(b) F: FTMS + p NSI Full ms [165.00-190.00]
172.0718
100

90

80

70
relative abundance

60

50

40

30

20

10

0
172.060 172.065 172.070 172.075 172.080 172.085 172.090
m/z

Figure 4. Screenshot of XCalibur platform depicting TIC (a) of selected MTZ m/z peak (b).

ratio of analyte to IS. Accordingly, the optimum ionization solvent for MTZ was evaluated to be 80 : 20 :
0.3 (v/v) methanol : water : formic acid.

3.2.5. Internal standard selection

The application of an IS was the key in turning direct MS from a qualitative to a quantitative tool. Most, if
not all, methods choose their IS to either be a compound of very similar chemical structure or the labelled
isotope of the compound to be analysed. This is to ensure both the drug and IS undergo the same
conditions, and if there is a loss, both are lost with the same ratio. For these reasons, deuterium-
labelled isotopes are considered the optimum choice for mass spectrometric analysis as they are
almost identical to the tested compound except that 4–6 hydrogen atoms are replaced with deuterium
[45,46]. But labelled isotopes are much more expensive than their unlabelled counterparts, and some
may require special storage conditions in order to maintain their integrity.
By contrast, the current method does not require a chemically related IS, because there is no
pretreatment step nor chromatographic separation in which there is any chance of loss of sample. The
only requirement in the choice of an IS is that it be of adequate, stable and similar ionization
efficiencies. For this reason, the selected IS was the same solution used for FT calibration-1,3,6-
polytyrosine solution. This solution has excellent ionization, and contains three major m/z peaks,
allowing for the quantification at any m/z range. This also gives us the advantage of quantifying
several compounds within the same run. This undergoing experiment used the peak of 1-tyrosine as
 100ng_3 # 1-84 RT:1.53-1.99 AV: 17NL: 1.82E5
(a) F: FTMS + p NSI Full ms [165.00-190.00] 8
169.1224
100

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95
90
85
80
75
70
relative abundance

65
60 173.1536
55
177.1638
50
45
40
35 172.0718
30
175.1329
25 167.1068 170.1177
182.0812
20 171.1381
15

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178.1672
10 179.1067 181.1224 183.6012
5 174.1571 180.1747 185.0121
187.1481
166.0358 168.6145 176.1363 189.0699
0
166 168 170 172 174 176 178 180 182 184 186 188 190
m/z
172_1 #1-80RT:0.28-0.52 AV: 9NL:7.44E4
(b) F: FTMS + c NSI Full ms2 172.07@cid25.00 [50.00-200.00]
128.05
100
95
90
85
80
75
70
relative abundance

65
60 172.07
55
50
45
40
35
30
25
20
15
–CH3CH0
10
5
0
100 105 110 115 120 125 130 135 140 145 150 155 160 165 170 175 180 185 190 195 200
m/z
182_1 # 1-34RT:0.20-0.46 AV:10NL:4.53E3
(c) F: FTMS -p NSI Full ms2 182.02@cid25.00 [50.00-190.00]
100 165.10
95
90
85
80
75
70 182.08
relative abundance

65
60
55
50
45 136.07
40
35
30
25
20 –NH3
15
10 –(H2O+CO)
5
0
100 105 110 115 120 125 130 135 140 145 150 155 160 165 170 175 180 185 190
m/z

Figure 5. (a) Full mass spectrum depicting both m/z = 172.0717 of MTZ and m/z = 182.0795 of IS. (b) MS/MS spectrum of MTZ at
CID = 25 eV. (c) MS/MS spectrum of 1-tyrosine at CID = 25 eV.

the IS, as it is the closest in m/z value to that of MTZ. To ensure our hypothesis, a comparison between
MTZ and IS signal was assessed at three different concentration levels, and the results are shown in
figure 3. This graphical depiction illustrates that there is a clear correlation between the ionization
Table 1. Summary of calibration results of MTZ over three consecutive days by direct NS-ESI-MS/MS method. 9

calibration range (2.5–25 000 ng ml−1)

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day 1 day 2 day 3 average

slope 0.0678 0.0603 0.0657 0.0646

intercept −0.4294 −0.0507 0.4611 −0.0063
2
R 0.9982 0.9959 0.9977 0.9973

efficiencies of the two signals, leading to the conclusion that both signals of the analyte and IS are
continuously affected at the same ratio.

3.2.6. Sample loading

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An aliquot 2 µl sample solution, together with 2 µl of an IS, is loaded into the back of the NS capillary. It
is important to note that any handling of an NS capillary must be carefully done using forceps. Doing so
will prevent any damage to the outer coat, prevent contamination by the human hand as well as greatly
decrease the chance of breaking the NS capillary tip. After the solvent has been added, the NS capillary is
to be centrifuged for a couple of seconds. This step is critical in removing the air gap at the front end of
the NS capillary and moving the sample to the very tip, for maximum spray results. This way, the NS
capillary will be ready to be mounted within the offline ion source.

3.3. Data extraction and interpretation

The data were extracted from XCalibur to Microsoft Excel in the following steps: (i) two active windows
in XCalibur should be open—one depicting the total ion chromatogram (TIC) of the run, depicted in
figure 4a and the other representing the average mass spectrum of the run, depicted in figure 4b.
(ii) Activate the mass spectrum window by clicking on the pin icon on the top right corner. Zoom in
on m/z = 172.0717 within the mass spectrum. (iii) Activate the TIC window by clicking on its
respective pin icon. Then select the m/z from 172.07 to 172.074. (Note: a very narrow range was
chosen due to the fact that as concentration increased, the base peak width increased. Also, as a
limitation of XCalibur, it is not possible to only choose one exact m/z. If one does so, the programme
automatically calculates the TIC of this m/z ± 1.) A stable signal should appear in the TIC window.
This indicates that the ionization of MTZ is stable and constant throughout the run. (iv) Right click
the TIC window and choose ‘Export@Clipboard (Chromatogram)’. Then, open an empty Microsoft
Excel file. Choose the first cell and paste the contents. The TIC window will be pasted and numerical
points in a XY format, X as time and Y as peak intensity. (v) Below the column of the MTZ intensity,
calculate the area under the curve (AUC) of the TIC using the following equation:

¼ Sum product ððA2:A100  A1:A99)
ðB2:B100 þ B1:B99ÞÞ
0:5

This equation represents the trapezoid rule, which divides the graph into multiple trapezoids and
calculates the summation of all trapezoids formed. Note: A1 and B1 represent the cell position that
contains the first XY point; A2 and B2 represent the position of the second XY point; A99 and B99
represent the position of the second to last XY point; and A100 and B100 represent the position of the
last XY point. (vi) Repeat the previous steps on the same run, but on the peak of the IS at m/z range
of 82.079–182.084. Paste the results next to the results of MTZ and calculate the AUC. (vii) Finally,
calculate the ratio of MTZ/IS as AUCMTZ/AUCIS.

3.4. Quantification and confirmation

Full MS spectra were used for quantification in a range containing both m/z values of [M + H]+ of MTZ
(172.0717) and IS (182.0812), both seen in the spectrum of figure 5a. MS/MS analysis was performed for
confirmation of both drug and IS and were compared to the accepted spectral patterns of the respective
compounds. As illustrated in figure 5b, the precursor of ion [M + H]+ at m/z = 172.1 gave way to 2-methyl-
5-nitromidazole after the complete removal of 1N side chain at m/z = 128.05. Figure 5c depicts the MS/MS
Table 2. Precision and accuracy validation of MTZ by direct NS-ESI-MS/MS method. 10

10 ng ml−1

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concentration
run no. day 1 day 2 day 3 day 4 day 5

1 113.79 101.09 108.62 92.36 102.87

2 106.73 111.73 97.12 99.69 106.08
3 108.63 95.99 96.26 111.11 120.34
4 112.07 94.31 107.05 110.60 111.31
5 96.65 117.04 106.22 103.62 107.12
intra-day results
mean 107.57 104.03 103.05 103.48 109.54

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s.d. % 6.70 9.95 5.88 7.85 6.74
RSD % 6.23 9.56 5.70 7.59 6.15
RE % 7.57 6.03 3.05 3.48 9.54
inter-day results
mean 105.54
s.d. % 2.86
RSD % 2.71
RE % 5.94

concentration 100 ng ml−1

run no. day 1 day 2 day 3 day 4 day 5

1 115.60 117.03 106.02 103.24 110.28

2 111.70 112.82 97.96 100.40 107.57
3 120.43 111.75 102.09 96.49 106.61
4 116.52 110.06 107.07 105.73 114.13
5 113.65 108.55 112.45 112.23 116.95
intra-day results
mean 115.58 112.04 105.12 103.62 111.11
s.d. % 3.28 3.22 5.45 5.91 4.38
RSD % 2.84 2.88 5.18 5.70 3.94
RE % 15.58 12.04 5.12 3.62 11.11
inter-day results
mean 109.49
s.d. % 4.99
RSD % 4.56
RE % 9.49

concentration 10 000 ng ml−1

run no. day 1 day 2 day 3 day 4 day 5

1 95.55 105.28 99.16 94.41 110.28

2 101.37 107.32 101.22 103.53 111.13
3 91.20 104.56 99.72 99.56 111.46
4 96.05 106.84 103.11 94.78 105.87
5 105.74 109.25 107.49 98.37 107.45
(Continued.)
Table 2. (Continued.) 11

10 000 ng ml−1

royalsocietypublishing.org/journal/rsos
concentration
run no. day 1 day 2 day 3 day 4 day 5

mean 97.98 102.14 98.13 109.23 106.65

s.d. % 5.64 1.84 3.35 3.75 2.45
RSD % 5.75 1.72 3.28 3.82 2.25
RE % −2.01 6.65 2.14 −1.86 9.23
mean 102.83
s.d. % 5.04
RSD % 4.90

R. Soc. open sci. 6: 191336

RE % 2.83

Table 3. Determination of MTZ in commercial formulations through 500 ng ml−1 sample solution by direct NS-ESI-MS/MS
method.

run no. 1 2 3 4 5 average

concentration found (ng ml−1) 518.21 545.45 538.33 510.16 490.65 520.56
recovery % 103.64 109.09 107.67 102.03 98.13 104.11
RSD % 2.58 6.43 5.42 1.44 1.32 3.44
RE % 3.64 9.09 7.67 2.03 −1.87 4.11

spectrum of 1-tyrosine in which the precursor ion, [M + H]+ at m/z = 182.1 is dissociated into several
fragments including the loss of NH3 at m/z = 165.1, and the loss of H2O + CO at m/z = 136.07.

3.5. Validation
3.5.1. Linearity
The effect of m/z range was examined in order to achieve the most accurate and precise linear equation.
The method compared the AUC of the MTZ/IS ratio versus concentration over four m/z ranges: 150–380,
150–212, 165–190 and alternating scans between m/z ranges 170–174 and 180–184, each run for 30 s.
Regression equations were compared and the R 2 value (R 2) of each range was found to be 0.979,
0.990, 0.999 and 0.993, respectively. These results indicate that a smaller range that contains both
analyte peak and IS peak gives more accurate results. We inferred also that too small a range may
have too low an ion count to be reliable, and that both analyte and IS should be present in the same
scan to undergo the exact same instrument conditions. Therefore, the range from m/z 165 to 190 was
chosen as the best range to be used for quantification. Once m/z range was established, day-by-day
calibration curve over the concentration range from 2.5 to 25 000 ng ml−1 was done on 3 different days
in order to observe the degree of change in regression. Over 3 days, R 2 was calculated to be 0.9977,
0.9959 and 0.9982, respectively (n = 3) (table 1).

3.5.2. Limit of detection and limit of quantitation

The limit of detection (LOD) and limit of quantification (LOQ) were determined according to the ICH
guidelines for validation of analytical procedures [47] and were found to be 0.5 ng ml−1 and
2.5 ng ml−1, respectively.

3.5.3. Accuracy and precision

Intra- and inter-day precisions and accuracies of the three different concentration levels are summarized
in table 2. Daily recovery per cent ranged from 91.21 to 120.44%. Intra-day RSD% ranged from 1.72 to
 (a) YBlank_1 #2-75 RT: 0.53-0.98 AV: 17 NL: 2.90E6 12
F: FTMS + p NSI Full ms [150.00-212.00]
202.0474
100

royalsocietypublishing.org/journal/rsos
95
90
85
80
75
70
65
60
55
50
45
40
35
191.1044 197.1148
30
25
20

R. Soc. open sci. 6: 191336

15 180.0656 203.0513
10 152.0221 158.9221 170.0925 174.8960
164.9301 207.0781
5 162.1126 192.1077 198.1182
176.0108 185.0423 189.0471 209.1146
0
155 160 165 170 175 180 185 190 195 200 205 210
m/z
(b) 1ug_1 #1-87 RT: 0.52-0.99 AV: 18 NL: 7.45E5
F: FTMS + p NSI Full ms [150.00-212.00] 172.0717
100
95
90
85
197.1147
80
75
70
65
60
55
199.1343
50
45
40 151.1117
201.1636
35
30
173.1535
25 160.9910 185.1191 191.1641
20 169.1223 175.1328
158.1539
15 157.1223
177.1637
10 162.9736 195.1743 208.1399
153.1274
5 167.1067 202.1669
0
155 160 165 170 175 180 185 190 195 200 205 210
m/z
(c) Y8hrs_1 #1-89 RT: 0.52-0.98 AV: 18 NL: 2.30E6
F: FTMS + p NSI Full ms [150.00-212.00]
202.0475
100
95
90
85
80 170.0925
75
70
65
60
55 197.1148
203.0522
50
45 172.0718
40 156.0768
35
30
191.1043
25
180.0656
20
15 166.0725
157.0609 189.0470
10 152.0319 174.8960
158.9221 185.0425
5 178.0588 182.0579 198.1182
0
155 160 165 170 175 180 185 190 195 200 205 210
m/z

Figure 6. Depicting spectrum of (a) drug-free human urine sample, (b) drug-free urine spiked with 50 ng ml−1 MTZ and (c) clinical
urine sample after oral administration of MTZ.
 sampleb_3 #2-238 RT: 0.02-6.30 AV: 23 NL: 2.83E6
F: FTMS + c NSI Full ms [50.00-1500.00]
114.0661
100 sampleb_1 #2-222 RT: 1.81-2.08 AV: 11 NL: 1.49E6
sampleb_1 #1-222 RT: 0.61-0.90 AV: 12 NL: 4.23E5
F: FTMS + c NSI Full ms [300.00-349.99] 346.0394
F: FTMS + c NSI Full ms [100.00-149.99] 100
121.0716
95 95
28 vitamin B3 omeprazole
90
26 85 346.1209
90 24
m/z = 123.0549 80
m/z = 346.1209
75
22
70
85 20 65
60
18
55
80 16 50
14 45
40 cefotaxime
75 12 128.0450 35
10 30 m/z = 456.0642
8 25
70 20
6
15 348.1027
123.0549 127.0497
4 120.0649 10
65 126.0521 344.0191
5 347.0133
2 345.0116
0
344.0 344.5 345.0 345.5 346.0 346.5 347.0 347.5 348.0 348.5
456.0642
60 119 120 121 122 123 124 125 126 127 128 m/z
m/z

55
50
45 149.0233
lactulose Na+ adduct
40
m/z = 365.1054
35
271.1880
30 MTZ
m/z = 172.0718 365.1054
25 414.0536

20
249.2060
15 191.1643 231.1203
121.0719 279.1590
10 478.0460
158.1540 333.1883
133.1011 172.0718 375.1987 425.1870
5 301.1409 355.0697 396.0428
494.0199
0
100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500
m/z

Figure 7. A mass spectrum of a clinical urine sample taken from a patient after oral ingestion of a number of drugs, including vitamin B3 (m/z = 123.0549), MTZ (m/z = 172.0718), omeprazole (m/z = 346.1209), lactulose
(m/z = 365.1064 Na+ adduct) and cefotaxime (m/z = 456.0642).
13

R. Soc. open sci. 6: 191336 royalsocietypublishing.org/journal/rsos

9.56 and RE% ranged from 1.86 to 11.11% over the three different concentrations. Inter-day RSD% ranged 14
from 2.71 to 4.90% and RE% ranged from 2.83 to 9.49%.
There is no interference m/z peak at both m/z values of MTZ and the IS from the five different

royalsocietypublishing.org/journal/rsos
injections of the blank (ionization solvent), hence the method is selective. A high degree of selectivity
was also examined in the validity of the developed nano-ESI-MS/MS method to determine MTZ in
tablets as there was no interference arising from the commonly used additives. Experimental results
after analysis of a 500 ng ml−1 preparation offered good recovery of 104.11% with 3.44% RSD, making
the present technique promising for quality control studies, as documented in table 3. When using the
ionization solvent for preparation of MTZ standards and samples, with the aid of an IS, the matrix
effects are not taken into account for validation of the method because they do not affect
reproducibility or linearity of MTZ.

3.5.4. Detection of MTZ in human urine

R. Soc. open sci. 6: 191336

When MTZ is prescribed for the treatment of trichomoniasis, it is unusual to discontinue treatment;
however, it is possible that the patient may not take the medicine, and the doctor may wonder
whether the medication has been properly absorbed or not. MTZ is also believed to reduce the desire
for alcohol in alcoholism [48]. Here, patient cooperation may be questionable, and a question arises as
to whether or not the drug is taken. Therefore, it may seem useful to determine whether MTZ has
been taken and whether it has been absorbed or not. Several LC-MS methods can easily study the
presence of MTZ in blood or in urine, but these cannot be carried out without sample pre-treatment
procedures [49,50]. In the absence of a blood sample, the simple and quick detection of MTZ in urine
is of practical interest upon applying the direct MS technique. Thus, the developed direct NS-ESI-MS/
MS method was applied to detect the analyte in random human clinical urine samples after oral
administration of the MTZ or MTZ with other co-administered prescribed drugs. The obtained results
were compared with the medical records of the patients to verify the reliability of the method.
Figure 6 reports the results obtained by analysing the drug-free human urine sample (figure 6a),
spiked drug-free urine sample (figure 6b) and real sample containing MTZ alone (figure 6c). Figure 7
reports the result obtained by analysing the real sample containing MTZ with other co-administered
drugs (vitamin B3, omeprazole, lactulose and cefotaxime). The results showed that direct detection of
MTZ is feasible despite the presence of other matrix interfering compounds or in the presence of
potentially co-administered drugs.

4. Conclusion
A rapid, sensitive and selective direct NS-ESI-MS/MS technique has been developed and validated to
quantify MTZ in tablet dosage forms. The direct NS-ESI-MS/MS system developed in the present
study can provide an alternative to conventional chromatographic techniques and be very promising
to various applications, based on the very low sample volume and short runtime. The direct NS-ESI-
MS/MS measurement exhibits a strong ionization technique and provides high resolution, which can
provide easy-to-interpret mass spectra for MTZ and other pharmaceuticals. The developed method
was also designed for continuous detection of drug intake of patients via screening of the drug in
urine samples. Urine samples collected from volunteers were analysed directly without any extraction
process. The obtained results were in agreement with the medical records of the volunteers which, in
turn, demonstrated the reliability of the method. The results also reveal that direct detection of MTZ
in human urine is feasible with direct NS-ESI-MS/MS despite the presence of matrix interfering
compounds or other co-administered drugs. Thus, the developed method can be used in clinical
practices to detect MTZ or other pharmaceuticals in urine samples in a short time.

Ethics. All volunteers gave informed consent prior to donating urine.

Data accessibility. The datasets supporting this article have been uploaded in the Dryad Digital Repository: https://dx.
doi.org/10.5061/dryad.778340g [51].
Authors’ contributions. G.H. and S.E. were involved in conceptualization; T.M. contributed to methodology; W.Z. and
H.E.-G. contributed to software; S.A., W.Z. and H.E.-G. validated; S.A. contributed to formal Analysis.; W.Z.,
H.E.-G. and R.A. contributed to investigation; W.Z. and H.E.-G. took care of resources; S.A., W.Z. and H.E.-G.
contributed to data duration; writing—original draft preparation was done by S.A.; writing—review and editing
were done by W.Z., H.E.-G. and S.E.; S.E. supervised.
Competing interests. We declare we have no competing interests.
Funding. We received no funding for this study.
Acknowledgements. S.A. acknowledges the support of RIKEN, Quantitative Biology Center—Japan, for the Internship
programme and she is grateful to all technical staffs of the Laboratory for Single Cell Mass Spectrometry for their
extensive help during her internship at RIKEN.
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