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Accepted Manuscript: Measurement
Accepted Manuscript: Measurement
PII: S0263-2241(18)30286-0
DOI: https://doi.org/10.1016/j.measurement.2018.04.011
Reference: MEASUR 5412
Please cite this article as: E.O. Er, B. Özbek, S. Bakırdere, A sensitive and selective analytical method for the
simultaneous determination of sildenafil and tadalafil in water, energy drinks and sewage sludge matrices by LC-
QTOF-MS/MS, Measurement (2018), doi: https://doi.org/10.1016/j.measurement.2018.04.011
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A sensitive and selective analytical method for the simultaneous determination of
sildenafil and tadalafil in water, energy drinks and sewage sludge matrices by LC-
QTOF-MS/MS
1
Yıldız Technical University, Chemical Engineering Department, 34349 İstanbul, TURKEY
2
Yıldız Technical University, Department of Chemistry, 34349 İstanbul, TURKEY
quadrupole-time-of-flight.
Highlights
• A fast and direct injection method was developed for sildenafil and tadalafil
determination.
• The developed method was applied to energy drink, tap water and sewage sludge
samples.
• 17 different energy drinks were determined for sildenafil and tadalafil contents.
1
Abstract
Given that the adulteration of dietary products with unauthorized content of erectile
dysfunction drugs has increased in the world, public health is threatened worldwide. The
illegal use of these drugs poses serious health risks to consumers and the environment. The
order to control the abuse of erectile dysfunction drugs. Therefore, a sensitive and selective
method was developed for the simultaneous determination of sildenafil and tadalafil using
was performed with an electrospray ionization mass spectrometer through the targeted
MS/MS mode within 4.0 minutes. Calibration plots for both analytes were linear with
correlation coefficients greater than 0.9992 within the range of 0.50 – 2000 ng/g. LOD/LOQ
values were calculated as 0.16/0.53 and 0.28/0.93 ng/g for sildenafil and tadalafil,
respectively. The validated assay was successfully applied to energy drink, tap water and
different brands of energy drinks and the results were compared with the written ingredients
on the bottles. The tap water and sewage sludge samples were also analyzed and the recovery
Keywords: Energy drinks, erectile dysfunction drugs, LC-QTOF-MS/MS, sewage sludge, tap
water
2
1. Introduction
Sildenafil and tadalafil are active pharmaceutical ingredients of erectile dysfunction (ED)
drugs which are marketed all over the world [1]. They act as phosphodiesterase-5 (PDE-5)
inhibitors which break down cyclic guanosine monophosphate (cGMP). The mechanism leads
increased blood flow and induces smooth muscle relaxation [2]. Besides erectile dysfunction
treatment, PDE-5 inhibitors are also used for heart failure and cardiac dysfunction [3].
Despite the clinical efficacy of these drugs, significant adverse effects including headaches,
back pain, facial flushing, dyspepsia, dizziness, rash and visual disorder have been reported
[4]. Besides that, the cardiovascular risk factor for ED drugs is still controversial [4].
threatens public health. Even though the approved active ingredients are presented with their
side effects, toxicity and maximum dose, other significant parameters such as purity, quality
and efficacy could make the products risky for environmental and human health [5]. These
risks come up with the presence of chemical contaminants in the drug ingredients such as
pesticides, heavy metals, mycotoxins or the interaction of these supplements with prescription
drugs [6]. It is well known that illegal addition of the some ingredients in dietary supplements
with unknown properties and upper dosage limits (100 mg/day for sildenafil [7] and 20
mg/day for tadalafil [8]) have potential risks to public health [5]. In order to overcome these
issues, the continuous and regular screening of ED drugs in dietary supplements should be
performed since many reports have been published confirming ED drugs presence in these
supplements [14].
On the other hand, the contamination of ED drugs in aquatic environment is a global problem
around the world, it is crucial to determine the contaminant levels in environmental samples
for assessing the environmental risks to take precautionary actions for safety of the water
cycle [9]. As sildenafil and tadalafil were reported to exhibit high stability when subjected to
3
a wide range of environmental conditions for 45 days, the detection of their levels in
environmental samples is required [10]. Therefore the detection of ED drugs at very small
There are numerous reports on the quantification of sildenafil, tadalafil and sometimes their
plasma, hair and waste water samples. Techniques such as high performance liquid
18], ion mobility spectrometry (IMS) [19], electrochemical detection (ECD) [20], liquid
mass spectrometry (LC-MS/MS) [23-28] have been reported. Several extraction methods for
sample preparation were reported in most studies including solid phase extraction (SPE) [29],
liquid-liquid extraction [23] and dispersive liquid-liquid microextraction [30]. All of these
methods include several steps for sample preparation which are time-consuming. Therefore, a
direct injection method was used in this study and to the best of our knowledge, a direct
spectrometry (QTOF-MS) has not been reported for simultaneous determination of sildenafil
and tadalafil in energy drink, tap water and sewage sludge samples.
The aim of this work was to develop a simple and reliable method for simultaneous
determination of sildenafil and tadalafil in energy drinks, tap water and sewage sludge. As an
innovative tool, LC-QTOF-MS/MS method has been applied due to its high sensitivity,
reproducibility, lower detection limits and high resolution. The system provides accurate
4
2. Materials and Methods
All chemicals used throughout this study were of high-purity grade. In all dilutions and
standard preparations, ultra-pure water generated from a Milli-Q instrument was used.
Acetonitrile (ACN), ammonium formate and formic acid were purchased from Merck.
Sildenafil was obtained from SMS Pharmaceuticals while tadalafil was obtained from
Zhejiang Huahai Pharmaceutical Co. Stock solutions of analytes were prepared from their
pure compounds as 1000 µg/g stock solutions (mass based) stored at +4 °C. Mixtures of
standard solutions were prepared by dilution of the stock solutions and prepared daily when
2.2 Instrumentation
An Agilent HPLC (1260 Binary pump, 1260 HiP ALS auto sampler, 1260 HiP Degasser and
1260 TCC column oven) coupled with an Agilent 6530 ESI-QTOF-MS detector was used in
C18, 3.0 × 150 mm, 2.7 µm) analytical column equipped with a guard column (5.0 × 2.1 mm).
Agilent MassHunter 7.0 Qualitative Analysis and MassHunter Forensic and Toxicology
Library Software were used to process spectra for the accurate mass data of molecular ions.
The software provides a list of possible formula by using a CHNO algorithm by comparing
theoretical with measured isotopic pattern and an internal library confirms the molecular
throughout this study can be found in Table 1. Zopiclone was used as internal standard (IS) at
a concentration of 100 ng/g. It was spiked to calibration standards and real samples before
5
analysis to eliminate instrumental errors due to injection volume variations, changes in MS
Seventeen energy drink samples were analyzed for their sildenafil and tadalafil contents.
A-D samples were purchased from a petrol station while E–R were purchased from
supermarkets in Istanbul, Turkey. According to the statements given on the bottle of the
samples, the origin of the A-K samples, M-N samples, O, P and R samples were Turkey,
USA, Canada, United States and Israel, respectively. All of the brands were analyzed in three
replicates from the same manufacturing batch. None of the brands were specified as
For the determination of sildenafil and tadalafil in sewage sludge, a sample was taken from
the aeration tank of the Atakoy Advanced Biological Treatment Plant of Istanbul Water and
Sewerage Administration (İSKİ). Tap water was sampled from Esenler district of Istanbul,
Turkey. All samples were diluted with 50/50 (v/v) ACN/water solution and filtered with a
The gradient elution method described in Table 1 gave a proper chromatographic separation.
The retention times were reproducible for sequential runs. The highest resolution of analytes
in a short period of time was obtained by using the gradient elution of ACN to 50/50 (v/v)
formic acid buffer at pH 3.5/ACN at a flow rate of 0.40 mL/min. The retention times of IS,
sildenafil and tadalafil were obtained at 1.79, 2.18 and 3.43 minutes, respectively. Under the
6
optimum conditions, sildenafil, tadalafil and IS were separated in only 4.0 minutes and one
analysis was completed within 10 minutes to wash the column from residues and prepare it
Chromatograms revealed completely separated peaks and no interference peak was observed.
Although complete separation is not needed for the tandem mass spectrometry applications, it
generally enhances detectability and reduces suppression effects [32]. The conditions for the
The spectrometric acquisition was performed in targeted MS/MS mode for compound
selection to achieve maximum sensitivity and accuracy. Targeted MS/MS scanning mode
presents high selectivity with the selection of precursor ion with unit-mass resolution
followed by the fragmentation of the precursor ion in the collision cell [33]. The MS
acquisition parameters including dry gas temperature, drying gas flow, nebulizer gas flow,
capillary voltage, fragmentor voltage and collision energy were optimized to enhance the
sensitivity. In the optimization part, all the system parameters having the effect on S/N ratio
for the analyte signal have been optimized to get lower detection limits. The optimization
steps were given in Table 2 while optimum MS parameters were given in Table 1.
7
Table 1. Operating parameters of the LC-QTOF-MS method
Parameter Value
Column temperature 40 °C
Injection volume 20 µL
0.0 100 0
2.00 100 0
4.00 0 100
5.50 0 100
7.00 100 0
8.00 100 0
8
Fragmentor voltage 150 V
Table 2. Optimization steps for MS acqusition parameters (Maximum reponses are indicated
in bold)
Response (Count-per-second)
Parameter Steps
Sildenafil Tadalafil
250 655484 111984
Drying gas
300 1750962 222130
temperature (˚C)
350 2471781 340770
40 3920409 392139
Nebulizer gas
50 3737187 367774
flow (psig)
60 3674407 384527
8 2356051 323806
Drying gas flow
10 3345074 392139
(l/min)
13 3920409 394883
3.5 2778941 331405
Capillary Voltage
4.0 3920409 450775
(kV)
4.5 2953509 380998
150 3862918 450775
Fragmentor
200 3819338 340770
voltage (V)
250 2471781 81056
10 60176 351611
Collision Energy 20 328136 540044
(V) 30 1148241 85785
40 1638611 4220
Figures 1 and 2 show the full scan spectrums of sildenafil, tadalafil and IS, and the proposed
fragmentation behavior for the observed ion of the structures. The identification of the
structures were easily achieved with the combination of chromatogram and mass spectrum
data which exhibits protonated molecular ion and its fragment ions with high resolution (<5
ppm mass accuracy). The precursor ions and product ions were determined after a series of
collision energies and selected according to optimum abundance of fragment ions. The
quantification of analytes were processed by selecting the most intensive fragment ions as
9
quantifier and the second highest transitions were selected as qualifier to confirm the
Figure 1. Full scan product ion mass spectra of A) zopiclone (IS) at a CID of 20 V B)
10
Figure 2. Proposed fragmentations of A) sildenafil and B) tadalafil structure by ESI+ analysis
Table 3. Quantifier/qualifier ions for each compounds and analytical performance of LC-
QTOF-MS/MS system
Sildenafil Tadalafil IS
R2 0.9993 0.9992
11
3.3 Analytical figures of merit
The method performance was evaluated in terms of linearity, %recovery, limits of detection
(LOD) and limits of quantification (LOQ). The calibration curve was established between
0.50-2000 ng/g in 50/50 (v/v) ACN/water solution to investigate linearity. The ratio values in
calibration curve were calculated as peak area of analytes dividing by the peak area of internal
standard in the same chromatogram. The calibration plots of sildenafil and tadalafil were
given in Figure 3 and the extracted ion chromatograms (EIC) of mixed standard solutions of
12
Linearity was investigated in the range of 0.50-2000 ng/g where the correlation coefficients
(R2) were 0.9993 and 0.9992 for sildenafil and tadalafil, respectively. The typical equations
were y = 0.003x-0.0278 for sildenafil and y = 0.0011x-0.0211 for tadalafil. LOD and LOQ
values were calculated using six replicate analysis of the lowest concentration in calibration
plots (1.0 ng/g) that provides signal to noise ratio more than five for the quantifier ion of each
analyte. The values were correctly confirmed with the ratio of qualifier ion. The following
The LOD/LOQ values were 0.16/0.53 and 0.28/0.93 ng/g for sildenafil and tadalafil,
Figure 4. Extracted ion chromatograms of mixed standard solutions of sildenafil and tadalafil
13
The method selectivity was examined with three different matrices to check the presence of
any component which could affect the detectability of target analytes. The extracted ion
chromatograms in Figure 5 clearly showed that there was no interfering components at the
retention times of target analytes in the matrices. The constancy of retention times were
evaluated and ranged from 0.05% to 0.54%. Furthermore, mass tolerance limit was also
considered as an important parameter for selectivity. High mass resolutions only with 5 ppm
mass tolerance for both precursor and product ions distinguished analytes from other matrix
14
3.4 Quantitative determination of sildenafil and tadalafil in energy drinks
drinks were performed simultaneously in a short run period. Although energy drinks have
very complex matrices, direct injection was applied to reduce the sample treatment steps and
analysis period. Quantitative determination of sildenafil and tadalafil in the samples were
performed by taking the averages of three replicates of the same manufacturing batch. The
results obtained for sildenafil and tadalafil ingredients of the samples were given in Table 4.
The relative standard deviations were calculated for Brands A, B, C and D as 6.21, 3.22, 2.13
and 3.79 %, respectively. The results obtained for Brands A, B and C were 1520 ± 94, 825 ±
27 and 187 ± 4 µg/g as sildenafil, respectively. Tadalafil was not detected in Brands A, B and
C. Tadalafil was the only species detected in Brand D at a concentration of 39.1 ± 1.5 µg/g.
detected, mg
There was no sildenafil and tadalafil detected in the rest of the thirteen brands of energy
drinks and this suggested they were not present or below the detection limits. Brand E was
used for spiking experiments to determine the recoveries of sildenafil and tadalafil in energy
drink matrices. The percent recoveries were obtained by adding the target analytes to obtain
15
250 ng/g spiked samples (four replicates). Recoveries were calculated from the regression
equation of the calibration plot of target analytes. The percent recoveries for sildenafil and
tadalafil were obtained as 101.69 ± 5.92 and 96.55 ± 0.23, respectively. The chromatogram
presented in Figure 6-A indicates good separation of analytes and internal standard with
smooth peak shapes. The high recoveries indicate that the target analytes were not affected by
3.5 Determination of sildenafil and tadalafil in sewage sludge and tap water
Qualitative and quantitative determination method with high accuracy and precision was
applied to sewage sludge and tap water samples. Sildenafil and tadalafil content of sewage
sludge and tap water were analyzed before recovery studies. Neither sildenafil nor tadalafil
was detected in the samples within detection limits. The recovery studies were performed by
spiking experiments to show the suitability of the developed method. For this purpose, proper
amounts of analytes were added to sewage sludge and tap water samples to obtain 250 ng/g
spiked samples. The chromatograms of spiked samples for sewage sludge and tap water
matrices were presented in Figure 6-B and 6-C. It is clear that resolution of the analytes was
high enough for quantification even though high resolution is not needed in LC-QTOF-
MS/MS system. The percent recoveries of sewage sludge matrix for sildenafil and tadalafil
were obtained as 97.65 ± 3.62 and 107.71 ± 3.91, respectively, while these values in tap water
matrix were obtained as 103.61 ± 2.42 and 103.42 ± 2.54 for sildenafil and tadalafil,
respectively. It is the good indication that water matrices could be analyzed for their sildenafil
16
Figure 6. Spiking experiments for A) energy drink, Brand E B) sewage sludge and C) tap
water.
4. Conclusions
In the present study, an analytical method was developed for qualitative and quantitative
determination of sildenafil and tadalafil in three matrices namely; energy drinks, sewage
sludge and tap water samples. All the system parameters in chromatographic separation and
detection steps were optimized to get high sensitivities for both analytes. The method was
applied through the direct injection of sample followed by liquid chromatography tandem
17
mass spectrometry with a quadrupole mass analyzer. The method provided high sensitivity,
accuracy and precision with very low detection limits. The developed method presents lower
limits of detection/quantification and offers fast and direct analysis if compared to other
methods published in literature [26, 34, 35]. Spiking experiments were performed in all
matrices. There was no peak shift observed for analytes and the recoveries showed the
reliability of the developed method for the matrices analyzed. Amounts of sildenafil and
tadalafil in seventeen different energy drinks were determined under the optimum conditions.
Four samples were found to contain high amounts of sildenafil and tadalafil, which have
potential risks to human health. All the results obtained show that the developed method can
be applied for the simultaneous determination of the analytes in the matrices analyzed in a
Acknowledgements
This work was supported by Research Fund of the Yildiz Technical University. Project
Number: FDK-2017-3186. The authors thank Science and Technology Application and
Conflicts of Interest
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