Accepted Manuscript

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

Elif Öztürk Er, Belma Özbek, Sezgin Bakırdere

PII: S0263-2241(18)30286-0
DOI: https://doi.org/10.1016/j.measurement.2018.04.011
Reference: MEASUR 5412

To appear in: Measurement

Received Date: 7 August 2017

Revised Date: 4 April 2018
Accepted Date: 5 April 2018

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

Elif Öztürk Er1, Belma Özbek1, Sezgin Bakırdere2*

1
Yıldız Technical University, Chemical Engineering Department, 34349 İstanbul, TURKEY
2
Yıldız Technical University, Department of Chemistry, 34349 İstanbul, TURKEY

Corresponding Author: Sezgin Bakırdere, Yıldız Technical University, Department of

Chemistry, Davutpaşa Campus, Esenler, 34349, İstanbul, Turkey. Email:

bsezgin23@yahoo.com, Fax: +90 212 3834134

Abbreviations: ECD, electrochemical detection; ED, erectile dysfunction; EIC, extracted

ion chromatogram; IMS, ion mobility spectrometry; PDE-5, phosphodiesterase-5; QTOF,

quadrupole-time-of-flight.

Highlights

• A fast and direct injection method was developed for sildenafil and tadalafil

determination.

• The method presented high accuracy and precision.

• 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.

4 of 17 energy drinks found to contain high amounts of sildenafil and tadalafil.

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

contents of dietary supplements and environmental samples should be monitored carefully in

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

LC-QTOF-MS/MS. The chromatographic separation was successfully achieved. The analysis

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

sewage sludge samples. The quantitative measurements were performed on seventeen

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

values were calculated for sildenafil and tadalafil.

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].

Furthermore, the illegal marketing of ED drugs in ‘generic’ form or as ‘natural supplement’

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

levels become crucial for analytical and toxicological laboratories [11].

There are numerous reports on the quantification of sildenafil, tadalafil and sometimes their

metabolites in pharmaceuticals, dietary supplements, bulk powders, human blood, urine,

plasma, hair and waste water samples. Techniques such as high performance liquid

chromatography (HPLC) [10, 12-15], gas chromatography-mass spectrometry (GC-MS) [16-

18], ion mobility spectrometry (IMS) [19], electrochemical detection (ECD) [20], liquid

chromatography-mass spectrometry (LC-MS) [21, 22] and liquid chromatography-tandem

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

injection method by liquid chromatography (LC) coupled to quadrupole time-of-flight mass

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

identification, confirmation and quantification of components in samples using quadrupole

and collision cell combined with electrospray ionization (ESI) [31].

4
2. Materials and Methods

2.1 Chemicals and reagents

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

analysis was conducted.

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

both the qualitative and quantitative determination of sildenafil and tadalafil.

Chromatographic separation of analytes were performed by an Agilent Poroshell 120 (EC-

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

formula with fragment ions. Instrumental parameters of LC-QTOF-MS/MS systems used

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

measurements and ionization differences during analysis.

2.3 Sample preparation

Seventeen energy drink samples were analyzed for their sildenafil and tadalafil contents.

Samples were given the arbitrary names A, B, C, D, E, F, G, H, I, J, K, L, M, N, O, P and R.

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

containing sildenafil and tadalafil ingredients on the bottles.

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

0.45 µm syringe filter before analysis.

3. Results and Discussions

3.1 Chromatographic separation

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

for the next run.

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

developed chromatographic separation of the analytes can be seen in Table 1.

3.2 Mass spectrometry

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

LC Mobile phase A: 50/50 (v/v) ACN/H2O at pH 3.5 buffer

(Ammonium formate/formic acid)

B: ACN with 0.10% (v/v) formic acid

Flow rate 0.40 mL/min

Column type Poroshell (3.0 x 150 mm, 2.7 µm) EC C18

Column temperature 40 °C

Injection volume 20 µL

Gradient elution Time, min A (%) B (%)

0.0 100 0

2.00 100 0

4.00 0 100

5.50 0 100

7.00 100 0

8.00 100 0

Q-TOF MS Drying gas temperature 350 °C

Nebulizer gas flow 40 psig

Capillary voltage 4.0 kV

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

consistency of quantifier/qualifier ratio between calibration standards and unknown samples.

The selected quantifier and qualifier ions were presented in Table 3.

Figure 1. Full scan product ion mass spectra of A) zopiclone (IS) at a CID of 20 V B)

sildenafil at a CID of 40 V and C) tadalafil at a CID of 20 V

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

Precursor ion [M+H]+ 475.2107 390.1429 389.1096

Quantifier/qualifier ion 58.0657/ 100.0993 268.1071/135.0435 245.0217/217.0270

LOD/LOQ, ng/g 0.16/0.53 0.28/0.93

Linear range, ng/g 0.50-2000 0.50-2000

Linear equation y = 0.003x – 0.0278 y = 0.0011 -0.0211

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

sildenafil, tadalafil and IS at 0.50-2000 ng/g concentrations were shown in Figure 4.

Figure 3. Calibration plots of sildenafil and tadalafil

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

formulas were applied in the calculations;

LOD = 3 x Standard deviation/slope

LOQ = 10 x Standard deviation/slope

The LOD/LOQ values were 0.16/0.53 and 0.28/0.93 ng/g for sildenafil and tadalafil,

respectively. The analytical performance of the method was presented in Table 3.

Figure 4. Extracted ion chromatograms of mixed standard solutions of sildenafil and tadalafil

between 0.50 - 2000 ng/g concentration range (100 ng/g IS spiked)

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

components with a high level of confidence.

Figure 5. Extracted ion chromatograms of sildenafil, tadalafil and IS in blank matrices

14
3.4 Quantitative determination of sildenafil and tadalafil in energy drinks

Qualitative and quantitative determination of sildenafil and tadalafil in seventeen energy

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.

Table 4. Quantitative determination of sildenafil and tadalafil in energy drinks

Amount in one bottle

Sildenafil, µ g/g Tadalafil, µ g/g for the analyte(s)

detected, mg

Brand A 1520 ± 94 Not detected 228 ± 14

Brand B 825 ± 27 Not detected 124 ± 4

Brand C 187 ± 4 Not detected 28.0 ± 0.6

Brand D Not detected 39.1 ± 1.5 5.87 ± 0.22

Brand E to R Not detected Not detected --

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

the complex matrix components.

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

and tadalafil contents with high accuracy.

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

short period of time in routine analysis laboratories.

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

Research Center of Yıldız Technical University for instrumental analysis support.

Conflicts of Interest

The authors declare no conflict of interest.

18
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