Capillary electrophoresis and liquid chromatography for determining steroids in concentrates of purified water from Päijänne Lake

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.

Journal of Chromatography A 1649 (2021) 462233 Contents lists available at ScienceDirect Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma Capillary electrophoresis and liquid chromatography for determining steroids in concentrates of purified water from Päijänne Lake Heli Sirén∗, Tuomas Tavaststjerna, Marja-Liisa Riekkola Department of Chemistry, University of Helsinki, P.O Box 55, FI-00014, 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 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 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 The districts´ 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 limits 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 microemulsion method the limit values were 1.33, 1.11, and 0.40 mg/L for androsterone, testosterone, and progesterone, 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/) 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 efficient pretreatment methodologies, which are needed before water can be delivered to humans’ households [6] Mostly, drinking water 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 sulphate 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 filtration, and disinfection with UV light Chlorine is added to prevent ´ microbesgrowth 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 concentrations 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 existence of steroids in drinking water is strengthen, when raw water is used from lake and river systems surrounded by city, agricultural, and industrial areas [10,11] According to literature, at many plants androgen steroids are removed from intake water using biological purification and membrane filtration [12–14] However, despite that in effluent water androgen steroids were detected [15] Recently, the female hormones 17β -estradiol (E2) and 17α -ethinylestradiol (EE2) were also identified 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/) H Sirén, T Tavaststjerna and M.-L Riekkola Journal of Chromatography A 1649 (2021) 462233 recoveries Thus, their performance fulfilled the European Union proposed minimum detection limit of ng/L in drinking water [17] by assuring the filter-packages´ 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, highquality water processing directives and regulations would be helpful 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 suitability 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 meaningful 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 Furthermore, some groundwater plants virtually have reported that the processes 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 generation of femininity among animals (estrogens), occurrence of deformity, 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 determined with liquid chromatography–tandem mass spectrometry (LC-MS/MS) and gas chromatography-mass spectrometry (GC/MS) [37–39] Despite the strength of chromatographic techniques, capillary electrophoresis (CE) with UV detection has shown its usability in separation of parent steroids from their metabolites due to the possible utilization of CE with different kinds of methodologies 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 purify high volumes of water samples and to concentrate nonpolar 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 100fold Then, limit of detection for steroids were - ng/L via SPE when measured with GC/MS [41] The concentrates needed manipulation since steroids were derivatives to improve their volatility The concentrations of androstenedione, testosterone, and progesterone have shown to be 10 0-fold lower in drinking water than in surface and ground water [28,29,42–46] According to literature, 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 water, and in influent and effluent water of WWTPs in various countries [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 influent 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 countries 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 concentration with SPE enrichment and analyte focusing by on-line stacking were used to concentrate the steroids from ng/L (raw water level) to μg/mL concentrations (method level) The concentration 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 PFMEKC 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] 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%, 6305-8, Sigma-Aldrich Co., Germany), testosterone (≥ 98%, SigmaAldrich Co., France), and testosterone-glucuronide (TLC: 1, STERALOIDS 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%, SigmaAldrich), fluoxymesterone (TLC: 1, STERALOIDS), 5-androsten3β -ol-17-one (DHEA, mg/1 mL in methanol, certified solution, Sigma-Aldrich), 5α -androstan-17β -ol-3-one (TLC: 1, STERALOIDS), 11β -hydroxytestosterone (TLC: 1, STERALOIDS), and 17α 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 5β -androstan-3α -ol-17-one glucosiduronate (TLC: 1, STERALOIDS), 1,3,5(10)-estratrien-3,17β -diol 3-glucosiduronate (TLC: 1, STERALOIDS), 4-androsten-17β -ol-3-one glucosiduronate (TLC: H Sirén, T Tavaststjerna and M.-L Riekkola Journal of Chromatography A 1649 (2021) 462233 1, STERALOIDS), aldosterone, 11β ,17β -dihydroxy-9α -fluoro17α -methyl-4-androsten-3-one (9α -fluoro-11β -hydroxy-17α methyltestosterone, fluoxy-mesterone), (TLC: 1, STERALOIDS), 4-androsten-3,11,17-trione, β -estradiol (TLC: 1, STERALOIDS), estrone (TLC: 1, STERALOIDS), 17α -methyltestosterone (≥98%, Sigma-Aldrich), and 17α -hydroxyprogesterone (≥98%, SigmaAldrich) 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 infusion (10 μl/min) into the mass spectrometer with the micro syringe pump and scan range in MS-measurements of m/z 50–10 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 capillary 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 temperature of turbo ion source was set to 450°C The optimised instrument settings were ion spray voltage 4500 V, nebulizer gas flow 10 L/min, curtain gas 12 L/min, collision gas L/min, focusing potential 185 V, entrance potential V, and cell exit potential 13 V The MS2 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 mixtures at 10-26 ppm concentrations were used They were prepared in acetonitrile-water mixture (1:1, v/v) containing 0.1 % NH4 OH (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 MS2 analysis of the compounds the most intense peak in the mass spectrum was chosen providing the mass spectra of the product ions 2.1.4 Other chemicals Ammonium acetate (AA) (98%, Sigma-Aldrich CO., Germany) for CE electrolyte solution, ammonium hydroxide (25%, VWR International 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 standard 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 (C17 H26 O5 , 310.38534 g/mol) standard was not available Therefore, it was identified with the accurate mass method with non-target UHPLC-HRMS 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 UHPLC coupled with an Orbitrap Fusion TMS (Tribrid mass spectrometer) using a Kinetex C18 column ˚ Phenomenex, Denmark) A filter unit cou(100 × 2.1 mm, 100 A, pled with a precolumn was used before the analytical column Accurate 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 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 parameters used for the mass spectrometer were as follows: spray voltage + 30 0 V in pESI mode, sweep gas flow rate respective arbitrary 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, and S-lens RF level 60% The orbitrap resolution used in this work was 120 0 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 analyses were performed with a Hewlett–Packard Series 1050 liquid chromatograph –MSD (Palo Alto, USA) The separation was investigated on different columns (Gemini C18 column (30 × 3.00 mm, ˚ 5μm) and Gemini-NX C18 column (250 × 4.60 mm, 5μm, 110A) 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 mL/min flow rate and 30 analysis time and the detection was with UV at 247 nm Sample volume was μL The MS spectra were detected at mass range of 50-600 Da and analysed at positive mode 2.2.3 Capillary electrophoresis 2.2.3.1 PF-MEKC A Hewlett-Packard 3D capillary electrophoresis instrument (Agilent, Waldbronn, Germany) equipped with a photodiode 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 temperature for 20 time The method provided 17 μA current, which was monitored to store information about the possible instability 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 discontinuous 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 (Ltot 80 cm, Leff 71.5 cm for PF-MEKC) and 60 cm (Ltot 60 cm, Leff 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 each at 13.634 psi (940 mbar) When the sample was changed to another, the capillary was washed with water and the electrolyte solution for and min, respectively The compounds were detected with UV-wavelengths of 206 nm (testosterone-glucuronide conjugate) and 247 nm (androstenedione, testosterone, and progesterone) 2.2.1.2 UHPLC-ESI-MS The liquid chromatographic analyses were also performed with a Hewlett–Packard Series 1100 liquid chromatograph (Palo Alto, USA) coupled with an Esquire 30 0 plus ion trap mass spectrometer (Bruker Daltonics, USA), using a GeminiNX 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), L/min of drying gas (nitrogen), and drying temperature 350°C, mass range 50–500 amu Androstenedione (m/z 287 Da), progesterone (m/z 315 Da), and testosterone (m/z 289 Da) were analysed in positive mode with μL injection volume 2.2.1.3 Tandem-MS and direct infusion A Micromass Quattro II triple quadrupole mass spectrometer (electrospray ionization, positive and negative ESI-MS/MS) was used to find the experimental conditions to optimize the liquid chromatographic method Direct H Sirén, T Tavaststjerna and M.-L Riekkola Journal of Chromatography A 1649 (2021) 462233 2.2.3.2 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 The compounds were detected with UV-wavelengths of 238 nm and 247 nm 2.3.1.2 MEEKC The microemulsion solution was made by weighting of 82.7 g disodium tetraborate decahydrate solution (10 mM stock solution made in water and pH adjusted to 9.2), 0.5 g hexanol, 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 decahydrate, 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 selective as PF-MEKC, other steroids, corticosteroids, and phthalates could also be measured 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 Therefore, sample concentrates are needed in analyses Solid phase extraction (SPE, STRATA-X C18 columns) was used for treatment of 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 , mL) After sorbent conditioning and introducing the sample (2 columns for L), the SPE materials were dried for 30 under vacuum The adsorbed steroids were eluted with methanol (6 mL) The eluates from the two C18 columns were combined and evaporated to dryness under gentle nitrogen stream at 40°C The precipitate was reconstituted in 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 (2.0 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 Analyses of each steroid standard and water concentrates were performed with five replicate injections and with eight sequential analyses 2.3.2 Eluents in liquid chromatography The eluents used in LC-UV-MS/MS analyses contained MQwater 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 The eluent flow rate was 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 increasing B to 100% in 15 and thereafter returning to the initial composition within min, and lastly keeping the composition for to equilibrate the system The analysis time was 16 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 August to November in 2017 according to ISO/TC 147/SC 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 × 20 0 mL, sampled in Etu-Tưưlư, Kumpula (4 households and Helsinki University), Munkkiniemi, Latokartano, Viikki, Pikku-Huopalahti, Vesala, Etelä-Haaga (2 households), Kalasatama, Pasila, and Ullanlinna) They were water from households in apartment houses that were connected to water networks with pipelines that were constructed within 15 years The exceptions were tap water from Etu-Töölö, Munkkiniemi, EteläHaaga, and Pasila, where the refurbishment has started or will start In addition, the exception is Kalasatama area that has renewed pipelines The reference samples were blank water made of MQ-water - methanol (95:5, v/v) spiked with a steroid mixture to reach 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) 2.3 Solutions 2.3.1 Electrolyte solutions 2.3.1.1 PF-MEKC The final micelle mixture was prepared by adding 10 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 solution 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 solution 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 to stabilize instability due to the pressure differences ( p) between the ends of the capillary Originally, the PF-MEKC method was optimised for separation of pregnelone, progesterone, androstenedione, testosterone, hydrocortisone, 17-α -hydroxyprogesterone, fluoxymesterone, 5androsten-3beta-ol-17-one (DHEA), 5α -androstan-17β -ol-3-one, 11β -hydroxytestosterone, and 17α -methyl-testosterone, but in this study only for testosterone, androstenedione, progesterone, and testosterone-glucuronide 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 concentrations 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 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, H Sirén, T Tavaststjerna and M.-L Riekkola Journal of Chromatography A 1649 (2021) 462233 and 10.0 μg/mL with methanol to make the concentration calibration and to determinate the limits of detection (LOD) separation efficiency by means of Gemini C18 and Gemini-NX C18 columns with isocratic elution (Supplementary, Fig S1) The column 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 progesterone (R2 between 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, androsteroneglucuronide, testosterone-glucuronide, and testosterone were too low for detecting them in real water samples The method limits of detection (MLD) for androstenedione, testosterone, and progesterone 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 water samples were determined with PF-MEKC, which was optimised 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 micellar liquid (sodium dodecyl sulphate (SDS) and sodium taurocholate (STC)) zone before the separation electrolyte solution Androstenedione is the lightest of the steroids, why it migrated as the first compound to detection Testosterone is a slightly heavier than androstenedione, but the linear geometry due to the OHgroup 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-CH3 , which branches the structure, with the ketone group out of the nonpolar part of the molecule Thus, progesterone can intensively interact 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 optimization showed that all steroid glucuronide conjugates migrated before the parent steroid The final PF-MEKC method for tap water measurements was validated with androstenedione, testosterone, and progesterone standard mixtures Table informs that the system was efficient (N, calculated with N = 5.54 (tm /wh )2 for each of the studied steroids In addition, their resolution (R between peaks and 2; R = 2(t2 −t1 )/((w1 +w2 )) was quantitative, with no overlapping 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 (Table 2) The correctness of the obtained results was verified by the relative standard deviations 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 (R2 ) (Table 3) The method limits of detection (MLD, S/N 3) and method limits of quantification (MLQ, 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 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 capillary electrophoresis and liquid chromatography, the methods were validated with steroid standards and with concentrated water samples 2.6.1 PF-MEKC and MEEKC The electrophoretic mobilities (μep ) were measured from the equation μep = (Ldet Ltot ) / (tR x V), where Ldet is the length of the capillary to the detector, Ltot is its total length, tR absolute migration 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 x the noise value The limit of quantification (LOQ) was calculated as 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= N= R= ta − tEOF tEOF − μe V l 2DI 1√ N = ta tmi μe El 2D μ μ¯ (1) (2) (3) where ta , tEOF and tmi are migration times of an analyte, electroosmosis, 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, μe is the electrophoretic mobility and D the diffusion coefficient of a steroid in Eq (2); μ = μ2 − μ1 is the difference of the electrophoretic mobilities of two steroids and μ ¯ is the average of the electrophoretic mobilities of the two steroids in Eq (3) The micelle 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 = (Ldet Ltot )/(Ut m ), and μ eo = (L det L tot )/(Ut eo ), where μ ep and μ eo are the electrophoretic mobilities of the analyte and electroosmosis, L det is the length of the capillary to the detector, L tot is the length of the total capillary, U is the applied voltage during the analysis, and tm and teo are the migration times of the analyte and electroosmosis, 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 Results 3.1 Optimization of the methods Detectability of the steroid hormones was first studied extensively using both UV and ESI-MS detection with liquid chromatography Then, twelve steroids in mixtures were used to measure H Sirén, T Tavaststjerna and M.-L Riekkola Journal of Chromatography A 1649 (2021) 462233 Table Validation data of selectivity parameters in PF-MEKC Steroid Retention factor (k)∗ Efficiency (N)∗ [x 105 ] Resolution (R)∗ Androstenedione (And) Testosterone (Testo) Progesterone (Prog) 1.57 3.01 3.49 9.37 9.76 9.84 114 (And-Testo) 26 (Testo-Prog) not measured ∗ Average values∗∗ Testosterone-glucuronide could not been used in the study, since it could not be detected at 247 nm Table Validation data of sensitivity parameters in PF-MEKC and MEEKC Steroid PF-MEKC Migration∗ time [min] RSD [%] Mobility∗ [m2 V−1 s−1 ] RSD [%] Repeated analyses series∗∗ Androstenedione Testosterone Progesterone Testosterone-glucuronide Electroosmosis 9.422 10.909 11.276 7.231 5.648 6.3 5.2 5.8 4.0 3.2 -2.705 × 10−8 -3.256 × 10−8 -3.370 × 10−8 -1.478 × 10−8 6.752 × 10−8 2.8 6.1 6.7 5.3 4.1 6 6 24 Steroid MEEKC Migration∗ time [min] RSD [%] Mobility∗ [m2 V−1 s−1 ] RSD [%] Repeated analyses series∗∗ Hydrocortisone Androstenedione 17-α -hydroxy progesterone Testosterone 17-α -methyl testosterone Progesterone Electroosmosis 5.271 6.453 6.887 7.014 7.203 8.347 4.178 6.0 7.4 7.3 9.7 11.9 10.8 4.1 -4.248 × 10−8 -3.470 × 10−8 -3.251 × 10−8 -3.192 × 10−8 -3.109 × 10−8 -2.683 × 10−8 3.216 × 10−8 2.9 3.5 4.0 7.9 8.5 9.5 3.6 6 6 6 20 ∗∗ The series were done with a standard mixture randomly chosen days during one month Table 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 Experimental Compound Concentra-tion range in PF-MEKC [μg/mL] Androstene-dione 0.5-6.0 ∗ 0.996 y=0.341x + 0.077 0.997 y=0.570x + 0.080 0.993 y=0.394x + 0.005 0.991 Correlation (R2 ) 0.884 0.880 0.931 Concentra-tion range in MEEKC [μg/mL] 2.0-10.0 2.0-10.0 2.0-10.0 0.50 (min 0.48 -max 0.70) 0.48 (min 0.42-max 0.53) 0.33 (min 0.27-max 0.39) 0.50 (min 0.43-max 0.81) MLD [μg/mL]∗ ∗ 1.84 (min 1.59 -max 2.10) 1.48 (min 1.26 -max 1.71) 0.99 (min 0.81 -max 1.17) 1.72 (min 1.29 -max 2.43) MLQ [μg/mL] ∗ ∗ ) 0.83 1.33 0.45 2.50 4.00 1.35 Concentration calibration equation∗∗ ) y=0.621x + 0.707 y=1.493x - 0.471 y=1.355x - 0.310 2.0-10.0 2.0-10.0 1.11 0.50 3.32 1.51 y=0.935x + 0.937 y=1.161x + 1.464 0.837 0.517 2.0-10-0 0.40 1.19 y=1.646x - 1.043 0.906 Progesterone 0.5-6.0 Testosteroneglucuronide Compound 0.5-6.0 ∗∗ y=0.293x + 0.053 MLQ [μg/mL] 0.5-6.0 ∗ Correlation (R2 ) MLD [μg/mL] Botrydial Testosterone Hydro-cortisone Androstene-dione 17-α -hydroxyprogesterone Testosterone 17-α -methyltestosterone Progesterone Concentration calibration equation∗∗ No standard available; identification was made with UHPLC-ESI-orbitrap-MS and tandem-MS with accurate mass method ( 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 measuring six standard series by detecting the accuracy of the calibration 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 less than 2; five decimals) in PF-MEKC As seen in Table they 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 (Fig 1) Thus, the PF method could be applied for sensitive, selective, and accurate 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] H Sirén, T Tavaststjerna and M.-L Riekkola Journal of Chromatography A 1649 (2021) 462233 17α -hydroxyprogesterone However, by using a longer column (250 mm) hydrocortisone, 11-β -hydroxytestosterone, fluoxymesterone, androstenedione, testosterone, 17α -hydroxyprogesterone, 17α -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-orbitrapMS The UHPLC studies showed that the concentrated samples enabled detection of testosterone-glucuronide, androstenedione, androsterone, 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 fragmentation 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 (Table 4) 3.3 Determination of the concentrates of purified water samples Fig Capillary electrophoresis separation of steroid standards in methanol (A) A PF-MEKC-UV profile Peaks (1) testosterone glucuronide, (2) fluoxymesterone, (3) androstenedione, (4) testosterone, (5) 17α -hydroxyprogesterone, (6) 17α -methyltestosterone, and (7) progesterone Concentration of all is μg/mL (B) A MEEKC-UV profile Peaks: (1) hydrocortisone, (2) androstenedione, (3) 17α -hydroxyprogesterone, (4) testosterone, (5) 17-α -methyltestosterone, (6) progesterone Detection at 247 nm Conditions as explained in Experimental Current 13 μA Concentration 20.0 μg/mL Steroids in the tap water samples were prepared as concentrates before the PF-MEKC Any of the concentrates of purified water samples was steroid-free The PF-MEKC electropherograms 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 concentrates To convince the steroids, the concentrated samples were spiked with 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 electrophoretic 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 absorption and concentration compared with the equal correlation in MEEKC was only satisfactory (Table 3) The electropherograms of concentrated water samples justified that the steroids in cold and hot water samples were androstenedione, testosterone, and progesterone (Fig 2) All the samples contained 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 shows 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 water 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 samples 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 The off-line concentration factor (enrichment factor, F) made with SPE was 80 0 (2.0 L water enriched to 250 μL concentrated sample.) Thus, by using SPE the ng/L concentrations could be concentrated to μg/mL (mg/L) level., which was the method detection level in CE with UV detection The enrichment factor in online 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 testosterone 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 presence 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 background area at the migration range of the target analytes were subtracted from the concentrated sample profiles of the target analytes when calculating recovery In the present work, the enrichment factor was 80 0 (meaning 2.0 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 profiles due to hydrocortisone, 17-α -hydroxyprogesterone, and 17-α methyltestosterone, since in MEEKC the interaction of 17-α - functionalised 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 different 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, 17α -methyltestosterone, and H Sirén, T Tavaststjerna and M.-L Riekkola Journal of Chromatography A 1649 (2021) 462233 Table Mass fragments used for identification of some steroids in LC-ESI-MS Compound Concentration [μg/mL] Testosterone glucuronide 10 Androsterone glucuronide Androstenedione Testosterone Confirmation ions m/z [Da] (% = normalised intensities of the fragments) 463 (100%) 464 (20%) 465 (5%) 463 (100%) Ions 75, 85, 113, 403 (low abundance) 465 (100%) 466 (30%) 15 Ionization mode negative (-) positive (+) Solvent Acetonitrile-water (1:1, v/v) cont 0.1% ammonium hydroxide MS2 (-) collision 25 eV MS(-) collision 25 eV MS2 (-) collision 35 eV 465 (20%) Ions 75 (98%), 85 (100%), 113 (50%) 287 (100%) 288 (30%) 328 (40%) 329 (5%) 97 (100%) 109 (70%) 123 (10%) 289 (100%) 290 (20%) 330 (20%) 289 (100%) 271 (3%) 253 (4%) 97 (85%) 109 (50%) 12 12 MS(-) collision 25 eV MS(+) collision 20 eV MS2 (+) collision 25 eV MS(+) collision 20 eV MS2 (+) collision 15 eV Table Concentrations of progesterone, testosterone, and androstenedione in cold and hot tap water extracts 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 Viikki 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 Water sources Cold [ng/L] Hot Cold [ng/L] Hot Cold [ng/L] Hot Cold [ng/L] Steroids Hot Latokartano Pikku-Huopalahti Vesala Etelä-Haaga Kalasatama Pasila Etelä-Haaga Kumpula Kumpula Kumpula Kumpula Kumpula Ullanlinna Etu-Töölö Munkkiniemi Viikki 16 extracts 0.013 0.033 nd 0.033 0.006 0.018 0.006 0.022 0.027 0.018 0.020 0.029 0.016 0.029 0.015 0.027 0.036 0.056 0.015 0.077 0.018 0.026 0.011 0.028 0.037 0.023 0.022 0.036 0.060 0.043 0.019 0.027 0.001 0.001 0.001 0.007 0.001 0.002 0.007 nd nd nd nd nd 0.003 nd nd nd 0.002 0.003 0.002 0.008 0.009 0.008 0.008 nd nd nd nd nd 0.005 nd 0.007 0.008 nd nd nd nd 0.001 0.001 nd nd nd nd nd nd nd nd nd nd nd 0.008 nd 0.006 0.008 0.005 0.005 nd nd nd nd nd nd nd nd nd 0.014 0.034 0.001 0.040 0.007 0.021 0.013 0.022 0.027 0.018 0.020 0.029 0.019 0.029 0.015 0.027 0.038 0.059 0.017 0.091 0.035 0.039 0.024 0.028 0.037 0.023 0.022 0.036 0.065 0.043 0.026 0.035 Average values from repetitions Three replicate samples (á 250 μL) from the concentrates of mL (nd = not determined) network of households’ connections with the water pipeline have disparity The steroid results showed that the concentration of progesterone 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 Latokartano, 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 areas Visual perception about the hot water samples from Ullanlinna and Etelä-Haaga showed before SPE treatment that they were reddish in colour The filtrated precipitation and the soluble mold were identified with UHPLC-ESI-orbitrap-MS(MS2 ) The phytotoxic sesquiterpene metabolite botrydial from Botrytis cinerea (Supplementary reference S11) mold was identified in all concentrates of purified water Identification of botrydial made with SCAN(+) MS H Sirén, T Tavaststjerna and M.-L Riekkola Journal of Chromatography A 1649 (2021) 462233 Fig Progesterone concentrations in PF-MEKC technique The concentrated Pikku-Huopalahti, Vesala, Etelä-Haaga, Munkkiniemi, and Viikki Conditions are tap water samples determined with the water samples were from Latokartano, Kalasatama, Pasila, Kumpula, Ullanlinna, as explained in Experimental erature that describes about discoveries of filamentous fungi identified 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 temperatures 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 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 concentrations 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 Fig Electropherograms as examples of hot tap water samples analysed with PFMECK-UV The concentrate samples are from (A) Vesala and (B) Pasila, and (C) the reference water Compounds: (1) androstenedione, (2) testosterone, (3) progesterone, and (4) botrydial The compounds were identified by spiking Detection at the UV wavelength 247 nm Separation conditions are explained in Experimental and MS2 modes gave accurate molar mass of 311.18533 Da for C17 H27 O5 ( 0.008250 ppm) with the fragment of 170.15390 Da (80%) The MS2 results to confirm botrydial from the isolation ion 311.18454 Da (C17 H27 O5 , -2.44430 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 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 sample the tap water when they wanted The households also have received results of their own water samples analysed by the instrumentation informed in methods Declaration of Competing Interest The authors declare that they not have competing financial interest concerning the project They not have any conflicts either Discussion The results showed that all water samples pretreated for concentrates of purified water from Päijänne Lake contained progesterone 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- CRediT authorship contribution statement Heli Sirén: Methodology, Investigation, Data curation, Writing review & editing Tuomas Tavaststjerna: Writing - review & editing, Investigation, Data curation Marja-Liisa Riekkola: Supervision H Sirén, T Tavaststjerna and M.-L Riekkola Journal of Chromatography A 1649 (2021) 462233 Acknowledgments [23] The Helsinki City Foundation (Grant 2017) is acknowledged for the financial support The authors acknowledge I Rekola, A Puolakka, M Tilli, and L Guricza for their assistance in laboratory [24] Supplementary materials 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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. .. 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 meaningful concentrations of steroids in tap water. .. Progesterone was determined in surface water, lake and river water, tap water, and in influent and effluent water of WWTPs in various countries [10,24,50–54] Its concentrations varied from 0.031 ng/L to