The research was done with partial filling micellar electrokinetic chromatography, microemulsion electrokinetic chromatography, and ultra-high performance liquid chromatography. The study focuses on determination of male and female steroids from cold and hot tap water of households in Helsinki City.
Trang 1Contents lists available at ScienceDirect
journal homepage: www.elsevier.com/locate/chroma
Heli Sirén∗, Tuomas Tavaststjerna, Marja-Liisa Riekkola
Department of Chemistry, University of Helsinki, P.O Box 55, FI-0 0 014, Helsinki 00560, Finland
a r t i c l e i n f o
Article history:
Received 13 February 2021
Revised 11 April 2021
Accepted 30 April 2021
Available online 7 May 2021
Keywords:
Steroid hormones
Tap water
Partial filling micellar electrokinetic
chromatography
Microemulsion electrokinetic capillary
chromatography
Liquid chromatography-mass spectrometry
a b s t r a c t
The research was done with partial filling micellar electrokinetic chromatography, microemulsion elec- trokinetic chromatography, and ultra-high performance liquid chromatography The study focuses on de- termination of male and female steroids from cold and hot tap water of households in Helsinki City The district ´s raw water is made run from Päijänne Lake through a water tunnel to the purification plants
in Helsinki area The effluents delivered from the plants to households as tap water were sampled and used for the study They were concentrated with solid phase extraction to exceed the detection lim- its of the three methods With partial filling method the limits were 0.50, 0.48, 0.33, and 0.50 mg/L for androsterone, testosterone, progesterone, and testosterone-glucuronide, respectively In microemul- sion method the limit values were 1.33, 1.11, and 0.40 mg/L for androsterone, testosterone, and proges- terone, respectively, and 0.83, 0.45, and 0.50 mg/L for hydrocortisone, 17- α-hydroxyprogesterone, and 17- α-methyltestosterone, respectively
In the tap water samples, progesterone concentrations represented the highest values being 0.22 and 1.18 ng/L in cold and hot water, respectively They also contained testosterone (in all samples), its glucuronide metabolite (in 25% of the samples), and androstenedione (in 75% of the samples) The ultra-high liquid chromatographic method with mass spectrometric detection was used for identification of the steroids at μg/L level
© 2021 The Authors Published by Elsevier B.V This is an open access article under the CC BY license ( http://creativecommons.org/licenses/by/4.0/)
1 Introduction
Humans and animals generate numerous natural and synthetic
steroids that are the main hormone sources e.g., in surface waters
[1–3] Concerning to steroid contamination, nearly 80% of effluent
water from wastewater treatment plants (WWTPs) contain female
hormones [ 4, 5]
Generally, purification of drinking water is processed with effi-
cient pretreatment methodologies, which are needed before water
can be delivered to humans’ households [6] Mostly, drinking wa-
ter is made of water from environment, i.e., from ground water
or surface water from lake and river systems, or from sea [7]that
is purified in water purifying plants In the present research, the
raw water is made run from Päijänne Lake through the Päijänne
Water Tunnel to the purification plants in Helsinki Raw water is
purified by precipitation of organic humus material with iron sul-
phate Furthermore, removals of the faults in smell and taste are
done with filtration through sand beds, removal of microbes with
∗ Corresponding author
E-mail address: heli.m.siren@helsinki.fi (H Sirén)
ozone gasification, organic material with carbon membrane filtra- tion, and disinfection with UV light Chlorine is added to prevent microbes ´growth in the water networks Finally, calcinated water and carbon dioxide are added [8]
When water components have not significant health effects, they are not designed to be removed totally from the effluents Therefore, several steroids are found at extremely low concentra- tions in water of various pre-treatment plants [9] Recently, it was also observed that biological processes removed steroids, but they also activated steroids to be transformed back to their steroidal precursor androstenedione Thus, the knowledge about the exis- tence of steroids in drinking water is strengthen, when raw water
is used from lake and river systems surrounded by city, agricul- tural, and industrial areas [ 10, 11]
According to literature, at many plants androgen steroids are removed from intake water using biological purification and mem- brane filtration [12–14] However, despite that in effluent water an- drogen steroids were detected [15] Recently, the female hormones
17 β-estradiol (E2) and 17 α-ethinylestradiol (EE2) were also iden- tified in tap and drinking water by GC-MS [16] Another paper showed that millimetric-size polymer ultrafilters (UF-PBSAC) used
as packed-layer membranes, removed estradiol at higher than 99% https://doi.org/10.1016/j.chroma.2021.462233
0021-9673/© 2021 The Authors Published by Elsevier B.V This is an open access article under the CC BY license ( http://creativecommons.org/licenses/by/4.0/ )
Trang 2recoveries Thus, their performance fulfilled the European Union
proposed minimum detection limit of 1 ng/L in drinking water
[17] by assuring the filter-package ´s quality for estradiol removal
from water [18] Anyhow, there are very few research papers on
determination of male steroids in local tap water supply systems of
the cities in the world [19–21] The reasons may be the published
risk assessments that the low concentrations of steroids were very
unlikely to pose risks to human health [22] Despite that, high-
quality water processing directives and regulations would be help-
ful to evaluate transparently the water quality in general [23]
In many countries, network systems for water services are out
of the coverage with regulations [ 24, 25] However, in Finland the
authorities have published results of the water quality in Helsinki
area using the purified effluent water from Päijänne Lake being
the source of drinking water The results of the laboratory data
of the year 2020 dealt the public information about exceptionally
low amounts of total organic contaminant (TOC) [26] However,
the concentrations of individual inorganic ions were more detailed
measured They were individually studied to convince the suitabil-
ity of the purified water for human use
As to steroid hormones, they are slightly water-soluble but not
volatile, which enable the preservation of their features in surface
and ground waters [ 15, 27-29] Since those water types are used in
preparation of drinking water, the processes may lead to meaning-
ful concentrations of steroids in tap water There are few recently
published papers about the studies on androgens, estrogens, and
progestogens in environmental water and in surface water, which
were used for processing of drinking water [30–32] Then, it was
assumed that the purification processes omitted the removal of
pharmaceuticals due to problems in water pre-treatment Further-
more, some groundwater plants virtually have reported that the
processes do not have any other treatment steps in the process
than aeration when producing drinking water [33–35] Anyhow,
the knowledge about the individual steroid hormones in drinking
water is important, since they have hormonal effects, such as gen-
eration of femininity among animals (estrogens), occurrence of de-
formity, and increase of birth defects [ 10, 22, 31, 35, 36] Furthermore,
clean water is the base solvent in manufacturing of commercially
products
Mainly, steroid hormones in drinking water have been de-
termined with liquid chromatography–tandem mass spectrometry
(LC-MS/MS) and gas chromatography-mass spectrometry (GC/MS)
[37–39] Despite the strength of chromatographic techniques, cap-
illary electrophoresis (CE) with UV detection has shown its usabil-
ity in separation of parent steroids from their metabolites due to
the possible utilization of CE with different kinds of methodolo-
gies and by modifying the composition of electrolyte solutions In
steroid analyses, a partial filling micellar electrokinetic capillary
chromatography (PF-MEKC) was used to enhance separation and
UV sensitivity of neutral steroid compounds in water samples from
WWTPs [ 10, 15] Then, water samples needed matrix removal and
steroid concentration before analyses
Solid phase extraction (SPE) is an excellent technique to pu-
rify high volumes of water samples and to concentrate nonpo-
lar analytes [10] Various novel configurations of SPE materials,
like magnetic solid–phase extraction [40] before GC-MS studies of
estrane, androstenedione, and progesterone hormones was used
Lately, SPE was filled with magnetite nanoparticles which were
palmitate-coated to enrich steroids and increase capacity with 100-
fold Then, limit of detection for steroids were 4 8 ng/L via SPE
when measured with GC/MS [41] The concentrates needed manip-
ulation since steroids were derivatives to improve their volatility
The concentrations of androstenedione, testosterone, and pro-
gesterone have shown to be 10 0 0-fold lower in drinking water
than in surface and ground water [28,29,42–46] According to lit-
erature, individual steroid concentrations exist at ng/L level in en-
vironmental samples [ 10, 15, 24, 28, 29, 47] The increase of toxicity of water to organisms is due to progesterone, which is permanently measured at ng/L to higher concentrations [ 48, 49] Progesterone was determined in surface water, lake and river water, tap wa- ter, and in influent and effluent water of WWTPs in various coun- tries [10,24,50–54] Its concentrations varied from 0.031 ng/L to 128.3 ng/L, being the lowest in tap water and the highest in in- fluent water to WWTPs (Supplementary data Table S1) Recently also, interlaboratory research to compare determination of ECDs in drinking water, surface water and treated wastewater in six coun- tries was organized and the results were reported [55] Solid phase extraction (SPE) was used for purification and concentration of the ECDs and finalized with situation fitting extraction techniques [ 55, 56, Supplementary data Table S2] The steroid concentrates are favorable made from small sample volumes with micro techniques [ 57, 58]
The present study was made to determine steroid hormones
in cold and hot tap water from randomly selected households in Helsinki City The analyses were done with partial filling micellar electrokinetic chromatography technique (PF-MEKC) Off-line con- centration with SPE enrichment and analyte focusing by on-line stacking were used to concentrate the steroids from ng/L (raw wa- ter level) to μg/mL concentrations (method level) The concentra- tion was mostly focused to the off-line enrichment and validation
of the capillary electrophoresis methods by the properties of the electrolytes (ionic strength, pH) and the composition of the micelle phases to obtain regionally suitable conductivity zones for focusing purposes in the capillary
Often the raw water contains humus [ 8, 24, 59, 60] Thus, some markers of the mold Botrytis cinerea were detected with PF- MEKC using UV detection and identified with UHPLC-HRMS/MS All steroids in the samples were also identified with UHPLC-HRMS/MS The observations about the mold are in correlation with those made on syntheses, with behaviour of fungal pathogen B cinerea , and with creation of the pathways written in literature [ 61, 62]
2 Materials and methods
2.1 Chemicals 2.1.1 PF-MEKC
Steroids selected for tap water studies were progesterone ( ≥98%, Sigma-Aldrich Co., Germany), androstenedione ( ≥ 98%, 63- 05-8, Sigma-Aldrich Co., Germany), testosterone ( ≥ 98%, Sigma- Aldrich Co., France), and testosterone-glucuronide (TLC: 1, STER- ALOIDS INC., USA)
2.1.2 MEEKC
The chemicals (progesterone, androstenedione, testosterone, and testosterone-glucuronide) mentioned above were used along with pregnelone ( ≥98%, Sigma-Aldrich), hydrocortisone ( ≥98%, HPLC, Sigma-Aldrich), 17- α-hydroxyprogesterone ( ≥95%, Sigma- Aldrich), fluoxymesterone (TLC: 1, STERALOIDS), 5-androsten-
3 β-ol-17-one (DHEA, 1 mg/1 mL in methanol, certified solu- tion, Sigma-Aldrich), 5 α-androstan-17 β-ol-3-one (TLC: 1, STER- ALOIDS), 11 β-hydroxytestosterone (TLC: 1, STERALOIDS), and 17 α -methyltestosterone (TLC: 1, STERALOIDS) They were studied with MEEKC as the reference for sensitivity when optimizing the CE analysis
2.1.3 LC-MS
The chemicals (progesterone, androstenedione, testosterone, and testosterone-glucuronide) mentioned above were used along with 5 β-androstan-3 α-ol-17-one glucosiduronate (TLC: 1, STER- ALOIDS), 1,3,5(10)-estratrien-3,17 β-diol 3-glucosiduronate (TLC: 1, STERALOIDS), 4-androsten-17 β-ol-3-one glucosiduronate (TLC:
Trang 31, STERALOIDS), aldosterone, 11 β,17 β-dihydroxy-9 α-fluoro-
17 α-methyl-4-androsten-3-one (9 α-fluoro-11 β-hydroxy-17 α
-methyltestosterone, fluoxy-mesterone), (TLC: 1, STERALOIDS),
4-androsten-3,11,17-trione, β-estradiol (TLC: 1, STERALOIDS),
estrone (TLC: 1, STERALOIDS), 17 α-methyltestosterone ( ≥98%,
Sigma-Aldrich), and 17 α-hydroxyprogesterone ( ≥98%, Sigma-
Aldrich) They were used for validation of the column material and
the steroid identification with liquid chromatographic methods
Methanol was used in gradient elution, since it endured better
separation between steroids, matrix, and steroid conjugates
2.1.4 Other chemicals
Ammonium acetate (AA) (98%, Sigma-Aldrich CO., Germany) for
CE electrolyte solution, ammonium hydroxide (25%, VWR Interna-
tional S.A.S, France) for pH adjustment, sodium dodecyl sulphate
(SDS) for micelle formation (99%, Oy FF-Chemicals Ab, Germany),
sodium taurocholate (STC) for SDS-STC micelles (BioXtra ≥95%
(TLC), Sigma-Aldrich Co., Germany), methanol (HPLC-MS grade,
Fisher Scientific, UK), sodium hydroxide (NaOH) for condition of
capillary and pH adjustment of the solutions [Sigma-Aldrich Co.,
Finland), and MQ-water (pH 5.8) for electrolytes, eluents, and stan-
dard solutions (Direct-Q UV Millipore, Millipore S.A., Molshheim,
France) Methanol was used as the solvent in standards and as the
marker of electroosmosis
The steroids were used as received and stored in a dark and
cold room ( + 4 °C and -20 °C) Botrydial (C 17H 26O 5, 310.38534
g/mol) standard was not available Therefore, it was identified with
the accurate mass method with non-target UHPLC-HRMS
2.2 Instruments and methods
2.2.1 LC Liquid chromatography and mass spectrometry methods in
method development
2.2.1.1 LC-UV and UHPLC-UV-ESI-MS Liquid chromatographic anal-
yses were performed with a Hewlett–Packard Series 1050 liquid
chromatograph –MSD (Palo Alto, USA) The separation was inves-
tigated on different columns (Gemini C18 column (30 × 3.00 mm,
5μm) and Gemini-NX C18 column (250 × 4.60 mm, 5μm, 110 ˚A)
from Phenomenex (Denmark) The best separation was obtained
with Gemini-NX C18 column using methanol-water (60:40, v/v)
modified by 25% ammonia-ammonium hydroxide (0.1%, v-%) as the
eluent The program was isocratic with 1 mL/min flow rate and 30
min analysis time and the detection was with UV at 247 nm Sam-
ple volume was 2 μL The MS spectra were detected at mass range
of 50-600 Da and analysed at positive mode
2.2.1.2 UHPLC-ESI-MS The liquid chromatographic analyses were
also performed with a Hewlett–Packard Series 1100 liquid chro-
matograph (Palo Alto, USA) coupled with an Esquire 30 0 0 plus ion
trap mass spectrometer (Bruker Daltonics, USA), using a Gemini-
NX C18 (30 × 3.00 mm, 5μm, Phenomenex, Denmark) column
and methanol-water (60:40, v/v) eluent containing 0.1% (v-%)
ammonia-ammonium hydroxide (25%) Electrospray ionization was
used with the following parameters: capillary voltage ±4500 V,
end plate offset ±500 V, nebulizer pressure 50 psi (nitrogen), 9
L/min of drying gas (nitrogen), and drying temperature 350 °C,
mass range 50–500 amu Androstenedione (m/z 287 Da), proges-
terone (m/z 315 Da), and testosterone (m/z 289 Da) were analysed
in positive mode with 1 μL injection volume
2.2.1.3 Tandem-MS and direct infusion A Micromass Quattro II
triple quadrupole mass spectrometer (electrospray ionization, pos-
itive and negative ESI-MS/MS) was used to find the experimental
conditions to optimize the liquid chromatographic method Direct
infusion (10 μl/min) into the mass spectrometer with the micro sy- ringe pump and scan range in MS-measurements of m/z 50–10 0 0
Da were used Parameters in the negative ionization mode were -3.2 kV, 40–48 V depending on the metabolite, and 70 °C for cap- illary voltage, cone voltage, and source temperature, respectively Parameters in the positive ionization mode were 3.5 kV, 35–50 V depending on the metabolite, and 70 °C, respectively The temper- ature of turbo ion source was set to 450 °C The optimised instru- ment settings were ion spray voltage 4500 V, nebulizer gas flow
10 L/min, curtain gas 12 L/min, collision gas 5 L/min, focusing po- tential 185 V, entrance potential 8 V, and cell exit potential 13 V The MS 2 measurements with [M – H] − (negative ionization mode, nESI) and [M + H] + (positive ionization mode, pESI) were done with 15, 25, and 35 eV collision energies Standard mix- tures at 10-26 ppm concentrations were used They were prepared
in acetonitrile-water mixture (1:1, v/v) containing 0.1 % NH 4OH (v/v), when measurements were done in nESI The solution was acetonitrile-water (1:1, v/v) containing 0.1 % HCOOH (v/v), when identification was done with positive ionization mode For the MS 2
analysis of the compounds the most intense peak in the mass spec- trum was chosen providing the mass spectra of the product ions
2.2.2 UHPLC coupled with a mass spectrometer for identification
The accurate masses of the compounds were measured with the Thermo Ultimate 30 0 0 UHPLC coupled with an Orbitrap Fu- sion TMS (Tribrid mass spectrometer) using a Kinetex C18 column (100 × 2.1 mm, 100 ˚A, Phenomenex, Denmark) A filter unit cou- pled with a precolumn was used before the analytical column Ac- curate identification was made with an orbitrap-electrospray mass spectrometer The mass spectrometer was operated in the positive ion electrospray mode (pESI) with at m/z 86.0 0–470.0 0 Da and
at EICs of the specific ion fragments The 10 μL volumes of the preconcentrated water samples were injected to the eluent at the flow rate of 0.6 mL/min (column temperature 40 °C) The parame- ters used for the mass spectrometer were as follows: spray volt- age + 30 0 0 V in pESI mode, sweep gas flow rate 0 respective ar- bitrary units (AU), sheath gas flow rate 40 AU, aux gas flow rate
12 AU, ion transfer tube temperature 350 °C, vaporizer temperature
300 °C, maximum injection time 100 ms, automated gain control (AGC) target 250 0 0 0, and S-lens RF level 60% The orbitrap reso- lution used in this work was 120 0 0 0
2.2.3 Capillary electrophoresis 2.2.3.1 PF-MEKC A Hewlett-Packard 3D capillary electrophoresis instrument (Agilent, Waldbronn, Germany) equipped with a pho- todiode array detector (190-600 nm) was used with ChemStation programmes (Agilent) for instrument running and data handling Analyses were done with PF-MEKC at +25 kV voltage, 25 °C tem- perature for 20 min time The method provided 17 μA current, which was monitored to store information about the possible in- stability of the analysis a function of time It also informs in long sequences about bubble formation (detected as current decrease to zero) Current was a good marker to detect, when the discontinu- ous solution system was stabilized to continuous one (detected as stabile current)
Fused silica capillaries (i.d 50 μm, o.d 362 μm) of 80 cm (L tot
80 cm, L eff 71.5 cm for PF-MEKC) and 60 cm (L tot 60 cm, L eff 51.5 cm for MEEKC) were from Polymicro Technologies (Phoenix,
AZ, USA) They were conditioned by sequentially flushing with 0.1
M NaOH, MQ-water, and the electrolyte solution for 20 min each
at 13.634 psi (940 mbar) When the sample was changed to an- other, the capillary was washed with water and the electrolyte so- lution for 5 min and 2 min, respectively The compounds were de- tected with UV-wavelengths of 206 nm (testosterone-glucuronide conjugate) and 247 nm (androstenedione, testosterone, and proges- terone)
Trang 42.2.3.2 MEEKC Microemulsion electrokinetic chromatography
(MEEKC) was used as the comparison method for PF-MEKC The
instrumentation and the capillary dimensions were the same as
informed above for PF-MEKC Samples were injected by pressure
of 50 mbar for 5.0 s Separation was made at + 23 kV for 30 min
The compounds were detected with UV-wavelengths of 238 nm
and 247 nm
2.2.4 Solid-phase extraction
In many cases, these steroids cannot be detected due to too
small representative sample volumes used in the analyses There-
fore, sample concentrates are needed in analyses Solid phase ex-
traction (SPE, STRATA-X C18 columns) was used for treatment of
2 L cold and hot tap water samples from the locations informed
in Ch 2.4 Before use, the sorbents for each water sample were
washed with methanol (6 mL) and MQ-water (conductivity 18 M ,
6 mL) After sorbent conditioning and introducing the sample (2
columns for 2 L), the SPE materials were dried for 30 min un-
der vacuum The adsorbed steroids were eluted with methanol
(6 mL) The eluates from the two C 18columns were combined and
evaporated to dryness under gentle nitrogen stream at 40 °C The
precipitate was reconstituted in 1 mL of methanol Methanol was
used since it was investigated to be a more quantitative eluent for
steroids than acetonitrile The control sample made of MQ-water
had an enrichment factor of 80 0 0 (2.0 0 L to 250 μL) The extract
was divided into 250 μL aliquots for the PF-MEKC and MEEKC
analyses followed by addition of 20 μL of 0.1 M NaOH into the
sample For LC-UV-MS analyses the studied sample volumes were
200 μL (in methanol-MQ-water mixture, 1:1, v/v) The enrichment
factor is 10,0 0 0 Analyses of each steroid standard and water con-
centrates were performed with five replicate injections and with
eight sequential analyses
2.3 Solutions
2.3.1 Electrolyte solutions
2.3.1.1 PF-MEKC The final micelle mixture was prepared by
adding 10 0 0 μL of 20 mM ammonium acetate (AA, pH 9.68) in
MQ-water, 440 μL of 100 mM sodium dodecyl sulphate (SDS) in
20 mM AA solution (pH 9.68) followed by addition of 50 μL of
100 mM sodium taurocholate in MQ-water, in this specific order
The concentrations of AA, SDS, and STC were 19.33 mM, 29.50 mM,
and 3.356 mM, respectively The total volume of the micelle solu-
tion was 1490 μL
In the beginning of each analysis, all solutions were sequentially
introduced into the capillary from the inlet to the outlet in the
following order: The electrolyte (ammonium acetate, AA), micelle
solution (mixture of SDS in AA and STC in MQ water), a sample
solution (a standard or a sample concentrate), and the electrolyte
(AA) Then, the micelle plug was placed after the electrolyte so-
lution used for separation and before the sample solution Before
the electric field ( + 25 kV) is switch on, the AA plug was 94.88 %,
followed by the micelle plug of 3.55% (55.7 nl), the sample plug
of 0.415% (6.46 nl), and the final electrolyte plug of 1.18 % (18.30
nl) The percentages are calculated from the total volume of the
capillary (1570 nL) [63] Furthermore, before voltage was switched
on, the analysis was forced to wait for 0.5 min to stabilize instabil-
ity due to the pressure differences ( p) between the ends of the
capillary
Originally, the PF-MEKC method was optimised for separa-
tion of pregnelone, progesterone, androstenedione, testosterone,
hydrocortisone, 17- α-hydroxyprogesterone, fluoxymesterone, 5-
androsten-3beta-ol-17-one (DHEA), 5 α-androstan-17 β-ol-3-one,
11 β-hydroxytestosterone, and 17 α-methyl-testosterone, but in this
study only for testosterone, androstenedione, progesterone, and
testosterone-glucuronide
2.3.1.2 MEEKC The microemulsion solution was made by weight- ing of 82.7 g disodium tetraborate decahydrate solution (10 mM stock solution made in water and pH adjusted to 9.2), 0.5 g hex- anol, 15 g acetonitrile, 1.2 g 1-butanol, and 0.6 g SDS The solution order was important made by weighting the components The final solution weight was 100 g It contained disodium tetraborate dec- ahydrate, hexanol, acetonitrile, 1-butanol, and SDS at 82.7, 0.5, 15, 1.2, and 0.6 w-% The MEEKC method was used for determination
of progesterone and androstenedione, but since it was not as se- lective as PF-MEKC, other steroids, corticosteroids, and phthalates could also be measured
2.3.2 Eluents in liquid chromatography
The eluents used in LC-UV-MS/MS analyses contained MQ- water and methanol The isocratic systems (Gemini NX C18) were made of methanol-water (60:40, v/v) and 0.1% (v-%) ammonia The analysis time was 30 min The eluent flow rate was 1 mL/min
In the gradient system (UHPLC-orbitrap-MS/MS) the eluents were A) 0.1% formic acid in MQ-water and B) 0.1% formic acid in methanol Gradient elution was used from 95:5 (v/v, A/B) by in- creasing B to 100% in 15 min and thereafter returning to the initial composition within 1 min, and lastly keeping the composition for
4 min to equilibrate the system The analysis time was 16 min The eluent flow rate was 0.6 mL/min
2.4 Samples, sampling, and sample storage
The household water samples were taken by the resident of the premises in the district area of Helsinki city Their locations were selected by site from the old and new city areas, but also from a city coast area and a hill The water samples were sampled on Au- gust to November in 2017 according to ISO/TC 147/SC 6 protocol [64] Within the framework of the current study, randomly chosen tap water samples from the areas mentioned below were analysed for the content of selected steroids and botrydis fungus ( B cinerea ) Cold and hot tap water (both 1 × 200 0 mL, sampled in Etu-Töölö, Kumpula (4 households and Helsinki University), Munkkiniemi, La- tokartano, Viikki, Pikku-Huopalahti, Vesala, Etelä-Haaga (2 house- holds), Kalasatama, Pasila, and Ullanlinna) They were water from households in apartment houses that were connected to water net- works with pipelines that were constructed within 15 years The exceptions were tap water from Etu-Töölö, Munkkiniemi, Etelä- Haaga, and Pasila, where the refurbishment has started or will start In addition, the exception is Kalasatama area that has re- newed pipelines The reference samples were blank water made
of MQ-water methanol (95:5, v/v) spiked with a steroid mixture
to reach 4 μg/mL steroid concentrations for the analyses In the artificial water sample the concentration was 0.5 ng/L They were pre-treated as the real samples (Ch 2.2.3)
2.5 Standard series in calibration 2.5.1 PF-MEKC and MEEKC
The stock solutions of steroids were 1100-2470 μg/mL in methanol They were diluted to 50 μg/mL solutions to prepare the working solutions Their final concentrations were 50 μg/mL for optimizing the migration, verifying electroosmosis, and adjustment
of lamp energy (sensitivity) Concentration calibration was made with solution mixtures of 0.5-6.0 μg/mL Peak areas of each steroid
in the electropherograms were calculated as a function of concen- trations
In MEEKC the concentrations in calibration were 2.0, 4.0, 6.0, 8.0, and 10.0 μg/mL
2.5.2 LC methods
The stock solutions were prepared to10 0 0 μg/mL and 200 μg/mL They were diluted to 0.05, 0.1, 0.5, 1.0, 2.0, 3.0, 4.0, 5.0, 7.0,
Trang 5and 10.0 μg/mL with methanol to make the concentration calibra-
tion and to determinate the limits of detection (LOD)
2.5.3 Mass spectrometric detection
The 10, 12, 15, 25, and 50 μg/mL concentrations were used for
validation of the MS spectra of the steroids
2.6 Method validation
Under the above-mentioned optimised conditions in the capil-
lary electrophoresis and liquid chromatography, the methods were
validated with steroid standards and with concentrated water sam-
ples
2.6.1 PF-MEKC and MEEKC
The electrophoretic mobilities (μep) were measured from the
equation μep = (L detL tot) / (t Rx V), where L det is the length of the
capillary to the detector, L totis its total length, t Rabsolute migra-
tion of the analyte to detection, and V the applied voltage during
the analyses
The limit of detection (LOD) was measured for each steroid
mixture in methanol by using the equation LOD = (height (peak))
/ (height (noise)), which should be 3 x the noise value The limit
of quantification (LOQ) was calculated as 3 x LOD
Parameters (retention factor k, efficiency N, and resolution R) of
the steroids in PF-MEKC were calculated with the Eqs.(1)–(3)
k= t a − t EOF
t EOF
1− t a
t mi
N=μe V l
2DI =μe El
R= 1
4
√
N
μ
¯
μ
(3) where t a, t EOF and t mi are migration times of an analyte, electroos-
mosis, and micelle in Eq.(1); l is the effective length of capillary,
V is the applied voltage and E the electric field in the separation,
μeis the electrophoretic mobility and D the diffusion coefficient of
a steroid in Eq (2); μ=μ2 −μ1 is the difference of the elec-
trophoretic mobilities of two steroids and μ¯ is the average of the
electrophoretic mobilities of the two steroids in Eq.(3) The mi-
celle marker was sodium taurocholate (STC), which was the other
micelle chemical in the mixture STC is a bile salts and has affinity
to SDS and it absorbs at the wavelengths sensitive for steroids
The mobility of electroosmosis is calculated from each of the
analyses by using methanol as the neutral marker Calculations
made with the equation μep = μtot − μeo, μtot = (L det L tot)/(Ut
m), and μeo = (L det L tot)/(Ut eo), where μep and μeo are the
electrophoretic mobilities of the analyte and electroosmosis, L det
is the length of the capillary to the detector, L totis the length of
the total capillary, U is the applied voltage during the analysis, and
t mand t eoare the migration times of the analyte and electroosmo-
sis, respectively
2.6.2 UHPLC-UV-MS
The method optimization was made with choosing the best
separation efficiency and the highest sensitivity for the steroids in
UV and the most selective m/z value for each of the steroids
3 Results
3.1 Optimization of the methods
Detectability of the steroid hormones was first studied exten-
sively using both UV and ESI-MS detection with liquid chromatog-
raphy Then, twelve steroids in mixtures were used to measure
separation efficiency by means of Gemini C18 and Gemini-NX C18 columns with isocratic elution ( Supplementary, Fig S1 ) The col- umn phases behaved differently: The latter one was not as good as the first one in respect of separation efficiency them all However, the system was not perfect because the steroid peaks were overly broad with low sensitivity, although at higher concentrations the better sorbent also lost resolution Despite that particularly good linearity was obtained for androstenedione, testosterone, and pro- gesterone (R 2between 0.9986-0.9996, Supplementary, Ch S3 ), even
if their movement to detection took long times, i.e., 9.800, 12.136, and 24.243 min, respectively Tandem-MS and direct infusion MS were performed to optimise ionisation and the fragment intensities ( Supplementary, Table S1 ) with nESI and pESI ( Supplementary, Figs S2–S5 ) The MS detection was promising, although the sensitivities
of the characteristic masses for androstenedione, androsterone- glucuronide, testosterone-glucuronide, and testosterone were too low for detecting them in real water samples The method lim- its of detection (MLD) for androstenedione, testosterone, and pro- gesterone were 0.181, 0.661, and 0.149 μg/mL, respectively with
UV detection Anyhow, LC-MS was only used for identification of the steroids in pre-concentrated water samples Finally, confirmed analyses of the samples were done by means of UHPLC-MS/MS to identify the steroids with accurate masses
Determination of the studied steroids in the concentrated wa- ter samples were determined with PF-MEKC, which was opti- mised with standards ( Experimental Ch 2.1, 2.5, and 2.6 ) Due to the structural and small polarity differences of the steroids they did not interact interdependently with micelles in the binary mi- cellar liquid (sodium dodecyl sulphate (SDS) and sodium tauro- cholate (STC)) zone before the separation electrolyte solution An- drostenedione is the lightest of the steroids, why it migrated as the first compound to detection Testosterone is a slightly heav- ier than androstenedione, but the linear geometry due to the OH- group allows interaction with the micelles and migration as the second to detection On the contrary, progesterone has the highest mass of the studied steroids due to its side-chain CO-CH 3, which branches the structure, with the ketone group out of the nonpo- lar part of the molecule Thus, progesterone can intensively inter- act with the binary micelles and migrate as the last steroid before the micelles Only testosterone-glucuronide (the anionic glucoside conjugate with pKa 3.30, migrated near electroosmosis showing the least interaction with micelles compared to testosterone (pKa 19.04–19.09) and progesterone (pKa 18.92) The method optimiza- tion showed that all steroid glucuronide conjugates migrated be- fore the parent steroid The final PF-MEKC method for tap water measurements was validated with androstenedione, testosterone, and progesterone standard mixtures Table1informs that the sys- tem was efficient (N, calculated with N = 5.54 (t m/w h) 2for each of the studied steroids In addition, their resolution (R between peaks
1 and 2; R = 2( t 2−t 1)/(( w 1+ w 2)) was quantitative, with no over- lapping with each other, matrix compounds, or the micelles The advantage of the PF-MEKC method was also that it was repeatable regarding to the separation of the endogenous steroid hormones and testosterone-glucuronide ( Table2) The correctness
of the obtained results was verified by the relative standard de- viations of the absolute migration times of individual steroids, their electrophoretic mobilities, and the mobility of electroosmosis, which were 4.0–6.3% (RSD 1.4–3.6% in a mixture), 2.8–6.7% (RSD 4.7–7.5% in a mixture), and 3.2–4.1% (RSD 1.6–10% in a mixture), respectively ( Table 2) At applied voltage of + 25 kV the relative standard deviation of the current from run-to-run was 5.4%
In general, the correlation coefficients for all steroid compounds were higher than 0.99 (R 2) ( Table3) The method limits of detec- tion (MLD, S/N 3) and method limits of quantification (MLQ, 3 x MLD) were from 0.33 to 0.61 μg/mL and from 0.99 to 1.84 μg/mL, respectively This meant that the steroid concentrations in the
Trang 6Table 1
Validation data of selectivity parameters in PF-MEKC
Steroid Retention factor (k) ∗ Efficiency (N) ∗ [x 10 5 ] Resolution (R) ∗
∗ Average values ∗∗ Testosterone-glucuronide could not been used in the study, since it could not
be detected at 247 nm
Table 2
Validation data of sensitivity parameters in PF-MEKC and MEEKC
Steroid PF-MEKC Migration ∗ time [min] RSD [%] Mobility ∗ [m 2 V −1 s −1 ] RSD [%] Repeated analyses series ∗∗
Steroid MEEKC Migration ∗ time [min] RSD [%] Mobility ∗ [m 2 V −1 s −1 ] RSD [%] Repeated analyses series ∗∗
∗∗ The 6 series were done with a standard mixture randomly chosen days during one month
Table 3
Calibration data of the steroids with PF-MEKC and MEEKC Optimised parameters of the steroids in PF-MEKC and in MEEKC under the conditions explained in Experi- mental
Compound
Concentra-tion range in PF-MEKC
Concentration calibration equation ∗∗ Correlation (R 2 )
-max 0.70)
1.84 (min 1.59 -max 2.10)
y = 0.293x + 0.053 0.996
0.42-max 0.53)
1.48 (min 1.26 -max 1.71)
y = 0.341x + 0.077 0.997
0.27-max 0.39)
0.99 (min 0.81 -max 1.17)
y = 0.570x + 0.080 0.993 Testosterone-
glucuronide
0.43-max 0.81)
1.72 (min 1.29 -max 2.43)
y = 0.394x + 0.005 0.991
range in MEEKC [μg/mL]
MLD [μg/mL] ∗∗ MLQ [μg/mL] ∗∗) Concentration
calibration equation ∗∗ )
Correlation (R 2 )
17- α-hydroxy-
progesterone
17- α-methyl-
testosterone
∗ No standard available; identification was made with UHPLC-ESI-orbitrap-MS and tandem-MS with accurate mass method ( less than 2; five decimals)
∗∗ Average values
water samples (at ng/L level) needed enhancement to fulfil the
method-related quantification ranges in PF-MEKC-UV, because the
lowest amount in concentration calibration was 0.5 mg/L with UV
at 254 nm The performance of the technique was studied by mea-
suring six standard series by detecting the accuracy of the calibra-
tion range during a day and during each day in two months The
results showed stable system throughout the work
The usability of PF-MEKC method was compared with data
obtained microemulsion electrokinetic capillary chromatography
(MEEKC) MEEKC was chosen as the reference method since the
buffer itself does not absorb as much as the fully micellar MEKC
buffer modified with SDS However, the measurements showed
that the MEEKC buffer could not give as low MDL values as those
in PF-MEKC As seen in Table3they were 1.33, 1.11, and 0.40 mg/L for androsterone, testosterone, and progesterone, respectively The advantage of MEEKC was the fast analyses, since even progesterone (the last compound) could migrate within 8.5 min ( Fig 1) Thus, the PF method could be applied for sensitive, selective, and accu- rate determination of steroids in tap water
3.2 Screening of steroid hormones with capillary electrophoresis
Steroid concentrations are extremely low in tap water [ 21, 60] Therefore, the water samples needed concentration enhancement with SPE for steroid detection at the limits of the PF-MEKC method [ 10, 15, 24, 65, 66]
Trang 7Fig 1 Capillary electrophoresis separation of steroid standards in methanol
(A) A PF-MEKC-UV profile Peaks (1) testosterone glucuronide, (2) fluoxymes-
terone, (3) androstenedione, (4) testosterone, (5) 17 α-hydroxyprogesterone, (6)
17 α-methyltestosterone, and (7) progesterone Concentration of all is 3 μg/mL
(B) A MEEKC-UV profile Peaks: (1) hydrocortisone, (2) androstenedione, (3) 17-
α-hydroxyprogesterone, (4) testosterone, (5) 17- α-methyltestosterone, (6) proges-
terone Detection at 247 nm Conditions as explained in Experimental Current 13
μA Concentration 20.0 μg/mL
The off-line concentration factor (enrichment factor, F) made
with SPE was 80 0 0 (2.0 0 L water enriched to 250 μL concentrated
sample.) Thus, by using SPE the ng/L concentrations could be con-
centrated to μg/mL (mg/L) level., which was the method detec-
tion level in CE with UV detection The enrichment factor in on-
line concentration was not measured, but it was detected from
very narrow speak shapes of the non-ionic and non-polar steroids
Therefore, the recoveries of the analytes at 5.0 μg/mL depended on
their detectability According to the present studies, the yields from
water were 33.8% and 64.4% for the parent steroids and testos-
terone glucuronide, respectively However, when the solvent was
water-methanol mixture (1:1, v/v), the yields for all compounds
were 80–90%
The matrix effect calculated from the analyte peak area in pres-
ence of matrix (after SPE) was divided by the analyte peak area
(standard) without matrix [ 67, 68] gave the average values between
1.3 and 2.9 for androstenedione, testosterone, and progesterone
in PF-MEKC Non-spiked MQ-water was also extracted and back-
ground area at the migration range of the target analytes were
subtracted from the concentrated sample profiles of the target an-
alytes when calculating recovery In the present work, the enrich-
ment factor was 80 0 0 (meaning 2.0 0 L water sample concentrated
to 250 μL), which was chosen, since 250 μL volumes for four times
made repetitions were needed The MEEKC used as the reference
was an important screening method to verify the PF-MEKC pro-
files due to hydrocortisone, 17- α-hydroxyprogesterone, and 17- α
-methyltestosterone, since in MEEKC the interaction of 17- α-func-
tionalised steroids had considerable different mobility than their
parent steroids Because the sensitivity was better in PF-MEKC-UV
than in MEEKC-UV, the partially filled system was selected for the
capillary electrophoresis study
Method development with LC was studied with twelve dif-
ferent steroids and glucuronide metabolites using UV and MS
detectors The efficiency of isocratic elution with the 30 mm
column used was quite good, although androstenedione could
not be separated from estrones, 17 α-methyltestosterone, and
17 α-hydroxyprogesterone However, by using a longer column (250 mm) hydrocortisone, 11- β-hydroxytestosterone, fluoxymes- terone, androstenedione, testosterone, 17 α-hydroxyprogesterone,
17 α-methyl-testosterone, and progesterone were separated Again, the MEEKC method was used to receive reference to the estrones However, then high concentrations of the steroids were needed, which lead to overlapping of the glucuronide conjugates of estrone, estradiol, and estratriol Thus, more LC studies were done, but with direct infusion of the standards to MS and with UHPLC-orbitrap-
MS The UHPLC studies showed that the concentrated samples en- abled detection of testosterone-glucuronide, androstenedione, an- drosterone, testosterone, estrogens, and progesterone, although the method was not used for quantification However, the traditional LC-ESI-MS was practical for detecting of the steroid fragmenta- tion of steroids and their identification using direct infusion with nESI and pESI modes Mostly, the steroids were identified with pESI mode, except detection of the glucuronide conjugates with both nESI and pESI modes Optimisation with high sensitivity was needed in tandem-MS by modifying the selective collision voltage ( Table4)
3.3 Determination of the concentrates of purified water samples
Steroids in the tap water samples were prepared as con- centrates before the PF-MEKC Any of the concentrates of puri- fied water samples was steroid-free The PF-MEKC electrophero- grams of the concentrates of purified water samples showed peaks corresponding to testosterone glucuronide, androstenedione, testosterone, and progesterone The analytes were identified using UHPLC-ESI-orbitrap-MS/MS to prove their existence in the concen- trates To convince the steroids, the concentrated samples were spiked with 3 μg/mL steroid standards They were detected by the increase of their peak areas compared to those detected for the concentrate samples The steroids were also identified with elec- trophoretic mobility using methanol as the electroosmosis marker Similar procedure was performed with the MEEKC method, but the SPE concentrated samples spiked concentrations were 8.6 and 20 μg/mL, since the MLD values were higher with MEEKC-UV method The PF-MEKC method allowed excellent relation between absorp- tion and concentration compared with the equal correlation in MEEKC was only satisfactory ( Table3)
The electropherograms of concentrated water samples justified that the steroids in cold and hot water samples were androstene- dione, testosterone, and progesterone ( Fig.2) All the samples con- tained progesterone Its concentration was higher than those of androstenedione and testosterone Progesterone concentrations in cold and hot tap water from Helsinki households ranged between 0.012-0.330 ng/L and 0.054-0.765 ng/L, respectively
Fig.3shows the variation of progesterone concentration in the
16 locations studied in Helsinki City According to literature on
LC studies, progesterone was found to be 0.003–0.5 ng/L in wa- ter samples [ 50, 54] Earlier, similar quantities at the range of 0.01– 0.33 ng/L were also measured with PF-MEKC [15] In the present study, the samples contained progesterone, testosterone (in 100%
of samples), its glucuronide metabolite (in 25% of samples), and androstenedione (in 75% of the samples) The MEEKC method gave similar information about the steroids in cold and hot water sam- ples In addition to the analytes discovered, in MEEK the additional steroids were 17- α-hydroxyprogesterone and hydrocortisone, but also diethyl phthalate Their concentrations were 0.006–0.056 ng/L The results showed that the concentrations of progesterone were the highest in the western and the central districts of Helsinki City ( Table 5) The reasons for that could not be discovered, but the ages of the water pipelines influence the steroid concentration The regional differences may also be the reason, since materials of the pipelines (steel, polymer coated, other), flow rate of water, and
Trang 8Table 4
Mass fragments used for identification of some steroids in LC-ESI-MS
Confirmation ions m/z [Da]
(% = normalised intensities
of the fragments) Solvent
Ionization mode negative (-) positive ( + )
464 (20%)
465 (5%)
Acetonitrile-water (1:1, v/v) cont 0.1% ammonium hydroxide
MS(-) collision 25 eV
463 (100%) Ions 75, 85, 113, 403 (low abundance)
MS 2 (-) collision 25 eV
466 (30%)
MS(-) collision 25 eV
465 (20%) Ions 75 (98%),
85 (100%),
113 (50%)
MS 2 (-) collision 35 eV
288 (30%)
328 (40%)
329 (5%)
MS( + ) collision 20 eV
97 (100%)
109 (70%)
123 (10%)
MS 2 ( + ) collision 25 eV
290 (20%)
330 (20%)
MS( + ) collision 20 eV
289 (100%)
271 (3%)
253 (4%)
97 (85%)
109 (50%)
MS 2 ( + ) collision 15 eV
Table 5
Concentrations of progesterone, testosterone, and androstenedione in cold and hot tap water ex- tracts determined with the PF-MEKC The water samples were from Latokartano, Pikku-Huopalahti, Vesala, Etelä-Haaga, Kalasatama, Pasila, Kumpula (1-5), Ullanlinna, Etu-Töölö, Munkkiniemi, and Vi- ikki Conditions are as explained in Experimental Testosterone-glucuronide was detected, but it was not quantified, since the amount was near its MLQ value
Progesterone Testosterone Androstenedione Steroids
Pikku-Huopalahti 0.033 0.056 0.001 0.003 nd 0.008 0.034 0.059
Etelä-Haaga 1 0.033 0.077 0.007 0.008 nd 0.006 0.040 0.091 Kalasatama 0.006 0.018 0.001 0.009 0.001 0.008 0.007 0.035 Pasila 0.018 0.026 0.002 0.008 0.001 0.005 0.021 0.039 Etelä-Haaga 2 0.006 0.011 0.007 0.008 nd 0.005 0.013 0.024
16 extracts Average values from 4 repetitions Three replicate samples (á 250 μL) from the concentrates of 1
mL (nd = not determined)
network of households’ connections with the water pipeline have
disparity
The steroid results showed that the concentration of proges-
terone in tap water from Ullanlinna was moderately higher in
hot water than in cold water (0.134 and 0.563 ng/L, respectively)
( Table 5) Similar effect was noticed in water samples of La-
tokartano, Pikku-Huopalahti, and Etelä-Haaga In addition, elevated
amounts of other steroids were found in Ullanlinna compared to
water in Kumpula ( Table 5) Testosterone and androstenedione
amounts were 30% lower in Kumpula (hill area) than in Ullanlinna
(seaside) Overall, in seaside area the steroid concentrations were higher than in the central area and new constructed suburban ar- eas
Visual perception about the hot water samples from Ullanlinna and Etelä-Haaga showed before SPE treatment that they were red- dish in colour The filtrated precipitation and the soluble mold were identified with UHPLC-ESI-orbitrap-MS(MS 2) The phytotoxic sesquiterpene metabolite botrydial from Botrytis cinerea ( Supple- mentary reference S11 ) mold was identified in all concentrates of purified water Identification of botrydial made with SCAN( +) MS
Trang 9Fig 2 Electropherograms as examples of hot tap water samples analysed with PF-
MECK-UV The concentrate samples are from (A) Vesala and (B) Pasila, and (C)
the reference water Compounds: (1) androstenedione, (2) testosterone, (3) proges-
terone, and (4) botrydial The compounds were identified by spiking Detection at
the UV wavelength 247 nm Separation conditions are explained in Experimental
and MS 2 modes gave accurate molar mass of 311.18533 Da for
C 17H 27O 5 ( 0.008250 ppm) with the fragment of 170.15390 Da
(80%) The MS 2 results to confirm botrydial from the isolation
ion 311.18454 Da (C 17H 27O 5, -2.4 4 430 ppm) gave the fragments
293.17419 Da (100%), 265.17931 Da (50%), 255.12222 Da (20%), and
237.11170 Da (10%)
Both cold and hot tap water contained botrydial as high as 861–
3900% of the biologically grown mold with respect to a drilled well
water Except for the purified water all samples from Vesala area
very rich in mold growth being at 2320–2460% level The lowest
botrydial amounts (9% in cold water and 23% in hot water) were
detected in the new built suburban area Viikki
4 Discussion
The results showed that all water samples pretreated for con-
centrates of purified water from Päijänne Lake contained proges-
terone Its high concentration in tap water might be caused by
the endogenic progesterone but also contraceptive pharmaceuticals
since steroids need specific removal from effluents of purification
plants The water samples can be contaminated with mold which
may grow in the water pipes The observation is supported by lit-
Fig 3 Progesterone concentrations in tap water samples determined with the
PF-MEKC technique The concentrated water samples were from Latokartano, Pikku-Huopalahti, Vesala, Etelä-Haaga, Kalasatama, Pasila, Kumpula, Ullanlinna, Munkkiniemi, and Viikki Conditions are as explained in Experimental
erature that describes about discoveries of filamentous fungi iden- tified from 14 drinking water systems in Norway [59] The paper informs that mold is detected in cold water However, in our study, the concentrations of botrydial metabolite of B cinerea were higher
in hot water than in cold water samples That was supported the knowledge that only a small number of mold can grow at tem- peratures of 4 °C or below ( Supplementary reference S12 ) Steroids belonging to oil compounds can resist B cinerea , since mold cause biodegradation of natural materials and may grow on dead organic matter everywhere in nature Thus, tap water may have an odd odor and flavor problems
5 Conclusions
Capillary electrophoresis showed capability in studies of male and female steroids in concentrates of water which was made run from Päijänne Lake and delivered from purification plants to Helsinki City Partial filled micellar electrokinetic chromatography with UV detection applied for SPE treated concentrates of purified water was sensitive and selective for infinite small steroid concen- trations The methods used detected also microbial pollutants in drinking water Our results prove that by applying UV detection at specific wavelength for steroids, the steroid identification can be conducted
Statement of human and animal rights
In this project, humans or animals have not been used to test the water samples We have neither had any sensory evaluator boards The persons in households gave the permission to sam- ple the tap water when they wanted The households also have received results of their own water samples analysed by the in- strumentation informed in methods
Declaration of Competing Interest
The authors declare that they do not have competing financial interest concerning the project They do not have any conflicts ei- ther
CRediT authorship contribution statement Heli Sirén: Methodology, Investigation, Data curation, Writing review & editing Tuomas Tavaststjerna: Writing review & edit- ing, Investigation, Data curation Marja-Liisa Riekkola: Supervi- sion
Trang 10The Helsinki City Foundation (Grant 2017) is acknowledged for
the financial support The authors acknowledge I Rekola, A Puo-
lakka, M Tilli, and L Guricza for their assistance in laboratory
Supplementary materials
Supplementary material associated with this article can be
found, in the online version, at doi: 10.1016/j.chroma.2021.462233
References
[1] A.C Naldi , P.B Fayad , S Sauve , M Prevost , Analysis of steroid hormones and
their conjugated forms in water and urine by on-line solid-phase extraction
coupled to liquid chromatography tandem mass spectrometry, Chem Central
J 1 (2016) 1–17
[2] J.K Leet , S Sassman , J.J Amberg , A.W Olmstead , L.S Lee , G.T Ankley ,
M.S Sepulveda , Environmental hormones and their impacts on sex differen-
tiation in fathead minnows, Aquat Toxicol 158 (2015) 98–107
[3] C.P Silva , M Otero , V Esteves , Processes for the elimination of estrogenic
steroid hormones from water: a review, Env Pollut 165 (2012) 38–58
[4] A.C Johnson , H.-R Aerni , A Gerritsen , M Gibert , W Giger , K Hylland , M Jür-
gens , T Nakari , A Pickering , M.J.F Suter , A Svenson , F.E Wettstein , Compar-
ing steroid estrogen, and nonylphenol content across a range of European
sewage plants with different treatment and management practices, Water Res
39 (2005) 47–58
[5] Z.H Liu , Y Kanjo , S Mizutani , Removal mechanisms for endocrine disrupting
compounds (EDCs) in wastewater treatment — physical means, biodegradation,
and chemical advanced oxidation: a review, Sci Total Env 407 (2009) 731–748
[6] S.Y Wee , A.Z Aris , Endocrine disrupting compounds in drinking water supply
system and human health risk implication, Env Inter 106 (2017) 207–233
[7] https://www.cdc.gov/healthywater/drinking/public/water _ sources.html
[8] https://yle.fi/uutiset/3-8731424
[9] M Ferrey , Wastewater Treatment Plant Endocrine Disrupting Chemical Moni-
toring Study, Report, Minnesota Pollution Control Agency, 2011
[10] H Sirén , S El Fellah , Androgens, oestrogens, and progesterone concentrations
in wastewater purification processes measured with capillary electrophoresis,
Env Sci Poll Res 24 (2017) 16765–16785
[11] M.J Benotti , R.A Trenholm , B.J Vanderford , J.C Holady , B.D Stanford , S.A Sny-
der , Pharmaceuticals and endocrine disrupting compounds in U.S drinking wa-
ter, Environ Sci Technol 43 (2009) 597–603
[12] M.O Barbosa , N.F.F Moreira , A.R Ribeiro , M.F.R Pereira , A.M.T Silva , Occur-
rence and removal of organic micropollutants: an overview of the watch list
of EU Decision 2015/495, Water Res 94 (2016) 257–279
[13] D Balabani ˇc, D Hermosilla, A Blanco, N Merayo, A Krivograd Klemen ˇci ˇc, The
possibility of removal of endocrine disrupters from paper mill waste waters
using anaerobic and aerobic biological treatment, membrane bioreactor, ultra-
filtration, reverse osmosis and advanced oxidation processes, Environmental
Toxicology III, www.witpress.com , ISSN 1743-3541 (on-line); WIT Transactions
on Ecology and the Environment, Vol 132, © 2010 WIT Press; Environmental
Toxicology III, pp 33-44 doi:10.2495/ETOX10 0 041
[14] P Rajasulochana , V Preethy , Comparison on efficiency of various techniques in
treatment of waste and sewage water – a comprehensive review, Resource-Ef-
fici Technol 2 (2016) 175–184
[15] H Sirén , S El Fellah , Steroids contents in waters of wastewater purification
plants: determination with partial-filling micellar electrokinetic capillary chro-
matography and UV detection, Int J Env Anal Chem 96 (2016) 1003–1021
[16] R Vallejo-Rodríguez , P.B Sánchez-Torres , A López-López , E León-Becerril ,
M Murillo-Tovar , Detection of steroids in tap and drinking water using an op-
timized analytical method by gas chromatography–mass spectrometry, Expo
Health 10 (2018) 189–199
[17] Proposal for a Directive of the European Parliament and of the Council on
the quality of water intended for human consumption (recast) COM (2017)
753 final 2017/0332 (COD) p 18 https://ec.europa.eu/environment/water/
water-drink/pdf/revised _ drinking _ water _ directive.pdf
[18] M Tagliavini , P.G Weidler , C Njel , J Pohl , D Richter , B Böhringer , A.I Schäfer ,
Polymer-based spherical activated carbon – ultrafiltration (UF-PBSAC) for the
adsorption of steroid hormones from water: material characteristics and pro-
cess configuration, Water Res 185 (2020) 116249
[19] C Chia-Yang , W Tzu-Yao , W Gen-Shuh , C Hui-Wen , L Ying-Hsuan ,
L Guang-Wen , Determining estrogenic steroids in Taipei waters and removal
in drinking water treatment using high-flow solid-phase extraction and liq-
uid chromatography/tandem mass spectrometry, Sci Total Environ 378 (2007)
352–365
[20] E Magi , C Scapolla , M Di Carro , C Liscio , Determination of endocrine-disrupt-
ing compounds in drinking waters by fast liquid chromatography-tandem mass
spectrometry, J Mass Spectrom 45 (2010) 1003–1011
[21] Y Yu , L Wu , A.C Chang , Seasonal variation of endocrine disrupting com-
pounds, pharmaceuticals and personal care products in wastewater treatment
plants, Sci Total Env 442 (2013) 310–316
[22] C.J Houtman , J Kroesbergen , K Lekkerkerker-Teunissen , J.P van der Hoek , Hu-
man health risk assessment of the mixture of pharmaceuticals in Dutch drink-
ing water and its sources based on frequent monitoring data, Sci Total Env
496 (2014) 54–62 [23] H.Fr Schröder , W Gebhardt , M Thevis , Anabolic, doping, and lifestyle drugs, and selected metabolites in wastewater—detection, quantification, and be- haviour monitored by high-resolution MS and MS n before and after sewage treatment, Anal Bioanal Chem 398 (2010) 1207–1229
[24] S El Fellah , G Duporte , H Siren , Steroid hormones, inorganic ions and botry- dial in drinking water Determination with capillary electrophoresis and liq- uid chromatography-orbitrap high resolution mass spectrometry, Microchem
J 133 (2017) 126–136 [25] R Mäkinen, Drinking water quality and network materials in Finland Sum- mary Report (2008)
[26] Finnish accreditation service, T225, (EN/ISO/IEC 17025, Quality of water 1.1.- 31.12.2020
[27] A Arditsoglou , D Voutsa , Partitioning of endocrine disrupting compounds in inland waters and wastewaters discharged into the coastal area of Thessa- loniki, Northern Greece, Environ Sci Pollut Res 17 (2010) 529–538 [28] E Vulliet , C Cren-Olivé, Screening of pharmaceuticals and hormones at the regional scale, in surface and groundwaters intended to human consumption, Env Poll 159 (2011) 2929–2934
[29] S.T Glassmeyer , E.T Furlong , D.W Kolpin , A.L Batta , R Benson , J.S Boone ,
O Conerly , M.J Donohue , D.N King , M.S Kostich , H.E Masha , S.L Pfaller , K.M Schenck , J.E Simmons , E.A Varughese , S.J Vesper , E.N Villegas , V.S Wil- son , Nationwide reconnaissance of contaminants of emerging concern in source and treated drinking waters of the United States, Pharmaceuticals Sci Total Environ 581-582 (2017) 909–922
[30] V.L Cunningham , C Perino , V.J D’Aco , A Hartmann , R Bechter , Human health risk assessment of carbamazepine in surface waters of North America and Eu- rope, Regul Toxicol Pharmacol 56 (2010) 343–351
[31] A.C Johnson , M.D Juergens , R.J Williams , K Kuemmerer , A Kortenkamp , J.P Sumpter , Do cytotoxic chemotherapy drugs discharged into rivers pose a risk to the environment and human health? An overview and UK case study,
J Hydrol 348 (2008) 167–175 [32] E Vulliet , C Cren-Olivé, Screening of pharmaceuticals and hormones at the regional scale, in surface and groundwaters intended to human consumption, Environ Pollut 159 (2011) 2929–2934
[33] M.J Benotti , B.D Stanford , E.C Wert , S.A Snyder , Evaluation of a photocat- alytic reactor membrane pilot system for the removal of pharmaceuticals and endocrine disrupting compounds from water, Water Res 43 (2009) 1513–1522 [34] K Lekkerkerker-Teunissen , E.T Chekol , S.K Maeng , K Ghebremichael , C.J Hout- man , A.R.D Verliefde , J.Q.J.C Verberk , G Amy , R van Egmond , Pharmaceutical removal during managed aquifer recharge with pretreatment by advanced ox- idation, Water Sci Technol Water Supply 12 (2012) 755–767
[35] C.J Houtman , J Kroesbergen , K Lekkerkerker-Teunissen , J.P van der Hoek , Hu- man health risk assessment of the mixture of pharmaceuticals in Dutch drink- ing water and its sources based on frequent monitoring data, Sci Total Env
496 (2014) 54–62 [36] E Magi , C.M Scapolla , M Di Carro , C Liscio , Determination of endocrine-dis- rupting compounds in drinking waters by fast liquid chromatography–tandem mass spectrometry, J Mass Spectr 45 (2010) 1003–1011
[37] L Barreiros , J.F Queiroz , L.M Magalhaes , A.M.T Silva , M.A Segundo , Analy- sis of 17 β-estradiol and 17- α-ethinylestradiol in biological and environmental matrices-a review, Microchem J 126 (2016) 243–262
[38] R Vallejo-Rodríguez , P.B Sánchez-Torres , A López-López , E León-Becerril ,
M Murillo-Tovar , Detection of steroids in tap and drinking water using an op- timized analytical method by gas chromatography–mass spectrometry, Expo- sure Health (2017) 1–11
[39] H.M Kuch , K Ballschmiter , Determination of endocrine-disrupting phenolic compounds and estrogens in surface and drinking water by HRGC −(NCI) −MS
in the picogram per liter range, Environ Sci Technol 35 (2001) 3201–3206 [40] I Vasconcelos , C Fernandes , Magnetic solid phase extraction for determination
of drugs in biological matrices, TrAC Trends Anal Chem 89 (2017) 41–52 [41] R.A Pérez , B Albero , J.L Tadeo , E Molero , C Sánchez-Brunete , Analysis of steroid hormones in water using palmitate coated magnetite nanoparticles solid-phase extraction and gas chromatography–tandem mass spectrometry, Chromatographia 77 (2014) 837–843
[42] M Velicu , R Suri , Presence of steroid hormones and antibiotics in surface wa- ter of agricultural, suburban and mixed-use areas, Env Monit Ass 154 (2009) 349–359
[43] E Aydin , I Talinli , Analysis, occurrence and fate of commonly used pharmaceu- ticals and hormones in the Buyukcekmece Watershed, Turkey, Chemosphere
90 (2013) 2004–2012 [44] H Chang , S Wu , J Hu , M Asami , S Kunikane , Trace analysis of androgens and progestogens in environmental waters by ultra-performance liquid chromatog- raphy-electrospray tandem mass spectrometry, J Chrom A 1195 (2008) 44–
51 [45] H.T.T Thuy , T.D Nguyen , The potential environmental risks of pharmaceuticals
in Vietnamese aquatic systems: case study of antibiotics and synthetic hor- mones, Env Sci Pollut Res 20 (2013) 8132–8140
[46] P.D Scott , M Bartkow , S.J Blockwell , H.M Coleman , S.J Khan , R Lim , J.A Mc- Donald , H Nice , D Nugegoda , V Pettigrove , L.A Tremblay , M.S.J Warne , F.D.L Leusch , An assessment of endocrine activity in Australian rivers using chemical and in vitro analyses, Env Sci Pollut Res 21 (2014) 12951–12967 [47] T.Y Fang , S.M Praveena , C de Burbure , A.Z Aris , S.N Ismail , I Rasdi ,Analytical techniques for steroid estrogens in water samples - a review, Chemosphere 165 (2016) 358–368