3.1. Derivatization
Derivatization is the most common and flexible choice for optimal chromatographic separation and MS measurement of analytes. The high polarity and thermal instability, and low molecular weight, of hormones can result in their easy adsorption on the chromatographic column or their decomposition at the injector port. This requires derivatizing them to increase their volatility and decrease their polarity —and hence dipole–dipole interactions— prior to injection into GC–MS for their determination at trace (subnanogram-per-liter) levels, something that can be easily and rapidly accomplished by trimethylsilylation. In fact, this silylation treatment ensures completeness of reaction —and obtainment of a single main product— and conversion of all hydroxyl groups in the hormones (Kumirska et al. 2015; Ben Sghaier et al. 2017), all with adequate sensitivity and selectivity. The high volatility, low thermal degradation, fast reaction with compounds containing hydroxyl groups, and good solubility in common organic solvents of N,O-bis(trimethylsilyl)trifluoroacetamide led us to choose it as derivatization reagent, in combination with the catalyst trimethylchlorosilane for optimal derivatization of synthetic and natural hormones spanning a wide range of polarity (Hernando et al. 2004). Based on previous reports, BSTFA containing 1% TMCS is an excellent derivatization reagent for estrogens, androgens and progestogens (Zuo et al. 2007; Azzouz et al. 2010; Suri et al. 2012; Albero et al. 2013; Aftafa et al. 2014; Kumirska et al. 2015; Ben Sghaier et al. 2017).
Microwave irradiation has been found to reduce the time needed to derivatize analytes from several hours to a few minutes, especially with hormones (Zuo et al. 2007; Söderholm et al. 2010; Azzouz and Ballesteros 2012), provided the optimum conditions as regards solvent, irradiation time and microwave power are established. Using an effective solvent for the sample and derivatization products is important with a view to reducing the amount of silylating reagent needed and preventing hydrolysis of the products by exposure to moisture (Bowden et al. 2009). In this work, volumes of 50 µL of individual solutions of the reagents were added to 100 µL of a solution containing a 1 µg L− 1 concentration of each analyte in ethyl acetate, acetonitrile, acetone, petroleum ether or n-hexane to identify the most suitable solvent. As can be seen from Fig. 2, the best results were obtained with petroleum ether, acetonitrile and ethyl acetate, the first being selected on the grounds of compatibility with the stationary phase of the chromatographic column —reportedly, the BSTFA–TMCS mixture shortens column lifetime—, sensitivity and stability (Albero et al. 2013). The influence of the volumes of solvent and derivatizing reagent was examined over the range 25–100 µL and the best results were found to be provided by 35 µL of petroleum ether and 70 µL of BSTFA + 1% TMCS.
The influence of the microwave irradiation conditions was examined by comparing the relative responses of all compounds with those obtained by using a water bath. For this purpose, a volume of 100 mL of aqueous sample containing a 100 ng L− 1 concentration of each analyte was fed into the continuous system as described in Sect. 2.4. Then, the extract was evaporated to dryness under a gentle nitrogen stream and the dry residue reconstituted with 70 µL of a mixture of BSTFA + 1% TMCS and 35 µL of petroleum ether for further derivatization in the microwave oven at a variable power (100–400 W) and irradiation time (1–5 min). Using a power greater than 250 W was found to detract from the analytical signals, possibly as a result of degradation of the analytes, and so did using a power of 200 W or a time longer than 4 min. By contrast, an irradiation power below 200 W resulted in poor derivatization. A microwave setting of 200 W and an irradiation time of 4 min were thus selected. By contrast, conventional thermal derivatization required heating at 60°C for 25 min. Using the microwave oven for silylation thus reduced the reaction time from 25 min to only 4 min, which can be especially interesting for the fast derivatization of multiclass mixtures of hormones. In any case, progesterone and androstenedione could not be derivatized because they contained no hydroxyl groups for binding to Si(CH3)3 groups in the reagent, so they had to be determined underivatized.
3.2. Solid phase extraction variables
Selecting an appropriate sorbent for SPE was one of the most important tasks with a view to maximizing sorption efficiency and optimizing sample treatment. Optimization tests involved passing 100 mL of an aqueous standard solution containing a 100 ng L− 1 concentration of each analyte at pH 7, adjusted with dilute NaOH or HCl, at 5 mL min− 1 through the sorbent column in triplicate. The sorbents included polymers (Amberlite XAD-2, Oasis HLB and LiChrolut EN), polar materials (Silica Gel and Florisil), reversed phase silica sorbents containing octadecyl groups (nonpolar) and Isolute NH2, all of which were used in amounts of 80 mg to pack sorbent columns. The sorption efficiency of the sorbent materials was assessed by comparing the amounts of analytes present in 1 mL fractions of the aqueous solutions before (100% recovery) and after passage through the sorbent column (unsorbed analyte). Both fractions were collected in glass vials and evaporated to dryness under a gentle nitrogen stream for derivatization with 70 µL of BSTFA + 1% TMCS mixture and 35 µL of petroleum ether that was placed in a household microwave oven at 200 W for 4 min. Finally, 1 µL aliquots of the treated solutions were injected into the GC–MS system for analysis. As can be seen from Table 2, the highest sorption efficiency, close to 100%, for the analytes as a whole was achieved with Oasis HLB. By contrast, the efficiency of the other polymeric sorbents (LiChrolut EN, Amberlites XAD-2) and RP-C18 never exceeded 44 % on average, that of Florisil and Isolute NH2 was lower than 18.2%, and that of the polar sorbent (silica gel) even lower (6.6 % only).
Table 2
The sorption efficiency (%) of the hormones on different sorbent materials.
Compound | Silica gel | LiChrolut EN | Oasis-HLB | Florisil | Amberlite XAD-2 | RP-C18 | IsoluteNH2 |
Hexestrol | 30.0 | 55.0 | 98 | 18 | 22 | 26,5 | 60 |
Diethylstilbestrol | 25,4 | 61.0 | 100 | 14 | 25,4 | 32 | 54,2 |
Dihydrotestosterone | 5.0 | 33.0 | 97 | 20 | 35 | 41 | 1 |
Estrone | 3,5 | 46,7 | 95 | 19,9 | 30,4 | 38 | 45,7 |
Androstenedione | 3 | 28.0 | 95 | 16 | 30 | 37 | 0 |
Norethindrone | 0 | 25,3 | 97 | 23 | 14,3 | 32,6 | 0 |
17β-estradiol | 5,8 | 41,6 | 100 | 9,7 | 27,6 | 28,7 | 0 |
Testosterone | 2.0 | 46.0 | 98 | 12 | 33 | 39 | 0 |
17α-ethinyl estradiol | 11.0 | 58,9 | 100 | 27,8 | 46,6 | 36,9 | 76 |
Pregnenolone | 0 | 22.0 | 97 | 19,5 | 35 | 11 | 0 |
Levorgestrel | 0 | 28,7 | 95 | 17,7 | 32,9 | 18,1 | 0 |
Progesterone | 0 | 80,7 | 97 | 30,5 | 75,9 | 13,8 | 0 |
Estriol | 0 | 39,4 | 99 | 3,48 | 17,4 | 34,3 | 0 |
Sorption efficiency average | 6.6 | 43.6 | 97.5 | 17.8 | 32.7 | 29.9 | 18.2 |
The organic solvents used as eluents differed in polarity and included methanol, ethanol, acetone, ethyl acetate and acetonitrile. Acetone resulted in the highest chromatographic peaks by effect of its increased eluting efficiency, the other solvents being roughly 1.5 times less efficient. Consequently, we chose to use acetone as eluent for all hormone classes.
The influence of the amount of Oasis-HLB was investigated by using various columns containing 20–120 mg of sorbent. For this purpose, a series of calibration graphs were run for each hormone and column by passing 100 mL of aqueous standard solutions containing variable concentrations (5–500 ng L− 1) of each analyte and subsequently eluting the columns with 500 µL of acetone. Analytical signals increased with increasing amount of sorbent up to 80 mg and decreased above 90 mg owing to the need for a higher volume of eluent to ensure complete elution of the hormones. A working column packed with 80 mg of Oasis-HLB was thus used in subsequent tests.
Then, the influence of eluent volumes over the range 50–800 µL was investigated by using loops of variable length in the injection valve (IV2 in Fig. 1). The desorption efficiency increased with increasing injected volume up to 600 µL, above which the signals for all analytes leveled off. The absence of carryover was confirmed by a second injection of 600 µL of acetone. A single injection of 600 µL of acetone therefore sufficed for complete elution of all hormones and that was the volume adopted as optimal.
Flow rates over the range 1–6 mL min− 1 for the sample during the preconcentration step and for air (eluent carrier) during elution had no effect on analyte sorption or elution efficiency. In fact, peaks areas remained constant throughout, which led us to choose 5 mL min− 1 for both the sample and air in order to ensure a high throughput. An identical air flow rate was used to dry the sorbent column before elution.
The sample pH strongly influenced the efficiency with which the hormones were retained by the sorbent. Tests conducted over the pH range 3.0–9.0 by passing 100 mL of an aqueous standard solution containing a 100 ng L− 1 concentration of each hormone adjusted with dilute HCl or NaOH revealed that the best results were obtained at 6.5–7.5, so neutral pH (7.0) was selected for subsequent tests. By contrast, the ionic strength of the water samples, adjusted with potassium chloride, had no effect on the analytical signal up to 1.5 M.
The breakthrough volume is important in that it is directly related to preconcentration factors, so it influences limits of detection and quantification —and hence sensitivity. The impact of this variable was assessed by passing volumes of 10–300 mL of aqueous solutions containing a 100 ng L− 1 concentration of each analyte at pH 7 through the SPE unit. Volumes up to 200 mL resulted in no loss of analytes from the column and maximized the sorption efficiency for all analytes (∼100%); higher volumes, however, decreased the sorption efficiency by effect of the sorbent being overloaded and/or the sample matrix being coeluted with the analytes. A sample volume of 100 mL was therefore selected that provided a concentration factor of 167 with 600 µL of solvent.
3.3. Analytical performance
The proposed SPE–GC–MS method, depicted in Fig. 1, was used under the above-described optimum conditions to determine thirteen natural and synthetic hormones in various types of water to assess analyte detectability, linearity range, precision and recovery. Table 1 shows the resulting figures of merit. Validation tests involved spiking 100 mL samples of ultrapure water with a few microliters of standard solutions containing all the hormones at concentrations over the range 0.04–800 ng L− 1 that were treated as described in Sect. 2.4 to construct calibration curves. Each calibration solution additionally contained a 500 ng L− 1 concentration of internal standard (triphenylphosphate). The equations for the standard curves were obtained by plotting analyte-to-internal standard peak area ratios against analyte concentrations. Linearity was excellent for all analytes (correlation coefficients were all higher than 0.9981). Limits of detection (LODs), defined as the analyte concentrations providing chromatographic peaks equal to three times the regression standard deviation, Sy/x, divided by the slope of the calibration graph, ranged from 0.01 to 0.30 ng L− 1. LODs were also calculated as the lowest concentrations providing chromatographic signals three times higher than background noise. Tests on real samples aimed at determining LODs provided results similar to those for distilled water. The lower limit of the linear range was taken to be the limit of quantification (LOQ) and calculated as 3.3 × LOD.
The precision of the proposed method was measured in terms of reproducibility and repeatability by calculating the within- and between-day relative standard deviation (RSD), respectively, for 12 individual standard mixtures of ultrapure water containing three different concentrations of each hormone (5, 50 or 200 ng L− 1). Analyses were performed on the same day (within-day precision) or on 7 different days (between-day precision). As can be seen from Table 1, precision was quite acceptable; thus, RSD values ranged from 3.0 to 5.5% for repeatability (within-day precision) and 4.5 to 7% for reproducibility (between-day precision). The good precision obtained can be ascribed to the use of automated SPE and an internal standard to correct chromatographic errors in terms of relative area (analyte-to-internal standard peak area ratio).
Accuracy in terms of standard deviation was checked by assessing analyte recovery from various types of water (drinking, mineral, river, swimming pool, well and waste) that were spiked with three different concentrations of each hormone (5, 50 or 200 ng L− 1) for analysis in triplicate (n = 3). Because many of the water samples studied contained some hormone, recoveries were calculated by subtracting previously quantified endogenous compounds from total contents. As can be seen from Table 3, all hormones were accurately identified; also, average recoveries were acceptable (92–103%), which testifies to the applicability of the proposed method to any type of water sample —a result, no doubt, of the highly efficient SPE unit used to pretreat samples.
Table 3
Average recoveries of hormones spiked to water samplesa.
Hormones | Tap | Mineral | Swimming Pool | Well | River | Waste water |
5 ng L− 1 | 50 ng L− 1 | 5 ng L− 1 | 50 ng L− 1 | 5 ng L− 1 | 50 ng L− 1 | 5 ng L− 1 | 50 ng L− 1 | 5 ng L− 1 | 50 ng L− 1 | 5 ng L− 1 | 50 ng L− 1 |
Estrogens | | | | | | | | | | | | |
Hexestrol | 101 ± 5 | 96 ± 4 | 95 ± 4 | 103 ± 5 | 95 ± 5 | 101 ± 4 | 101 ± 4 | 98 ± 5 | 99 ± 5 | 101 ± 4 | 98 ± 5 | 100 ± 4 |
Diethylstilbestrol | 92 ± 4 | 99 ± 4 | 100 ± 6 | 95 ± 4 | 92 ± 4 | 96 ± 4 | 96 ± 4 | 102 ± 4 | 99 ± 5 | 100 ± 5 | 101 ± 5 | 97 ± 4 |
Estrone | 99 ± 4 | 97 ± 4 | 94 ± 6 | 103 ± 5 | 95 ± 4 | 99 ± 6 | 96 ± 4 | 98 ± 5 | 101 ± 4 | 98 ± 5 | 102 ± 4 | 95 ± 5 |
17β-estradiol | 96 ± 4 | 101 ± 6 | 97 ± 6 | 94 ± 5 | 99 ± 6 | 101 ± 5 | 100 ± 4 | 99 ± 5 | 101 ± 5 | 101 ± 4 | 96 ± 5 | 102 ± 5 |
17α-ethinylestradiol | 101 ± 5 | 92 ± 5 | 99 ± 5 | 92 ± 4 | 97 ± 6 | 93 ± 3 | 101 ± 4 | 101 ± 4 | 98 ± 4 | 99 ± 5 | 97 ± 6 | 102 ± 4 |
Estriol | 99 ± 6 | 100 ± 5 | 97 ± 4 | 93 ± 4 | 103 ± 7 | 97 ± 5 | 101 ± 6 | 96 ± 5 | 102 ± 5 | 100 ± 5 | 102 ± 4 | 100 ± 5 |
Androgens | | | | | | | | | | | | |
Testosterone | 100 ± 4 | 98 ± 4 | 99 ± 6 | 95 ± 6 | 100 ± 6 | 95 ± 4 | 100 ± 4 | 102 ± 5 | 97 ± 4 | 96 ± 4 | 101 ± 3 | 96 ± 4 |
Dihydrotestosterone | 101 ± 4 | 102 ± 6 | 98 ± 4 | 100 ± 5 | 97 ± 4 | 96 ± 4 | 97 ± 4 | 92 ± 4 | 96 ± 4 | 101 ± 5 | 98 ± 4 | 100 ± 5 |
Androstenedione | 99 ± 5 | 96 ± 4 | 100 ± 5 | 102 ± 4 | 101 ± 5 | 93 ± 4 | 92 ± 4 | 94 ± 4 | 98 ± 4 | 99 ± 4 | 99 ± 4 | 97 ± 4 |
Progestogens | | | | | | | | | | | | |
Progesterone | 101 ± 5 | 97 ± 6 | 98 ± 5 | 95 ± 4 | 100 ± 4 | 94 ± 6 | 98 ± 6 | 97 ± 4 | 97 ± 4 | 100 ± 4 | 99 ± 4 | 98 ± 5 |
Norethindrone | 102 ± 5 | 95 ± 4 | 102 ± 5 | 92 ± 4 | 98 ± 4 | 100 ± 5 | 93 ± 6 | 102 ± 5 | 100 ± 5 | 101 ± 6 | 99 ± 6 | 102 ± 5 |
Levorgestrel | 102 ± 5 | 93 ± 4 | 102 ± 5 | 97 ± 4 | 96 ± 5 | 95 ± 4 | 99 ± 4 | 97 ± 4 | 102 ± 5 | 99 ± 4 | 99 ± 5 | 100 ± 5 |
Others | | | | | | | | | | | | |
Pregnenolone | 98 ± 4 | 103 ± 5 | 99 ± 5 | 94 ± 5 | 101 ± 5 | 100 ± 5 | 99 ± 4 | 94 ± 5 | 99 ± 4 | 95 ± 4 | 100 ± 4 | 97 ± 5 |
a 80 mg of HLB Oasis sorbent, sample adjusted at pH 7 for all hormones; volume, 100 mL (n = 3, ± SD). |
3.4. Determination of hormones in water
The practical use of the proposed SPE–GC–MS method for determining hormones was assessed by applying it to various types of real waters samples (tap, mineral, well, swimming pool, pond, river and waste) from Spain and Morocco. Volumes of 100 mL of the different types of samples were analyzed by using the method in triplicate. All samples were passed through a 0.45 µm membrane filter and adjusted to pH 7 prior to insertion into the continuous SPE system. As can be seen from Table 4, none of the hormones was present in tap or mineral water. Also, norethindrone, levorgestrel, estriol, pregnenolone, diethylstilbestrol and androstenedione were detected in none of the samples. On the other hand, estrone was the natural estrogen most frequently detected in all other types of water (well, pond, swimming pool and river), but especially in river water (concentrations of 13–83 ng L− 1), which are similar to those of coated South Florida surface water, where it was encountered at levels up to 79.5 ng L− 1 by Ng et al. (2021). One other hormone found in many samples was dihydrotestosterone (28–64 ng L− 1). Also, testosterone was found in pond and river water, at concentrations from 6 to 24 ng L− 1. Ben Sghaier et al. (2017) found testosterone at levels from 5.4 to 6.5 ng L− 1 in river water (Table 5). By contrast, other hormones such as 17β-estradiol, hexestrol, 17 α-ethinylestradiol and progesterone were found at lower levels (3.0–12 ng L− 1) but still similar to those in surface waters reported by other authors (Ben Sghaier et al. 2017; Zhou et al. 2020). By way of example, Fig. 3 shows the chromatogram of a swimming pool water analyzed by the proposed method.
Table 4
Hormones found in water samples (± SD, ng L− 1, n = 3) using the proposed SPE-GC-MS method.
Sample | Hexestrol | Estrone | 17β-estradiol | 17α-ethinylestradiol | Testosterone | Dihydrotestosterone | Progesterone |
Tap 1 | nqa | nq | nq | nq | nq | nq | nq |
Tap 2 | nq | nq | nq | nq | nq | nq | nq |
Mineral 1 | nq | nq | nq | nq | nq | nq | nq |
Mineral 2 | nq | nq | nq | nq | nq | nq | nq |
Well 1 | nq | 14 ± 1 | nq | nq | nq | nq | nq |
Well 2 | nq | 27 ± 1 | 3.3 ± 0.2 | nq | nq | nq | nq |
Swimming pool 1 | 5.1 ± 0.3 | 15 ± 1 | nq | nq | nq | 28 ± 1 | nq |
Swimming pool 2 | 3.0 ± 0.2 | 43 ± 2 | nq | 4.0 ± 0.2 | nq | 31 ± 2 | 13 ± 1 |
Ponq 1 | nq | 21 ± 1 | nq | nq | nq | 38 ± 2 | nq |
Ponq 2 | nq | 25 ± 1 | 3.6 ± 0.2 | nq | 6.0 ± 0.3 | 28 ± 1 | nq |
River 1 | nq | 13 ± 1 | nq | nq | 17 ± 1 | 42 ± 2 | nq |
River 2 | nq | 83 ± 4 | nq | nq | 24 ± 1 | 64 ± 3 | nq |
Waste 1 | 10 ± 1 | 110 ± 10 | 12 ± 1 | 12 ± 0.1 | 31 ± 2 | 76 ± 4 | 24 ± 1 |
Waste 2 | 14 ± 1 | 93 ± 4 | 22 ± 1 | 7.0 ± 0.4 | 57 ± 3 | 45 ± 2 | 16 ± 1 |
a nq: not quantified |
Table 5
Comparison of the proposed method with existing alternatives for the determination of hormones in aqueous samples
Analytes | Samples | Pretreatment methoda | Analytical techniquea | Analytical featuresa | Hormone concentrations in real samples | Reference |
Estrogens (and pharmaceuticals) | Surface and waste water | SPE- derivatization (BSTFA + 1% TMCS) | GC–MS GC–ECD | LOD: 2.1–4.2 ng L− 1 RSD < 17.5% Recovery: 58–107% | 8–120 ng L− 1 | Migowska et al. 2012 |
Estrogens | River water | SPME | LC–MS/MS | LOD: 0.012–0.693 ng L− 1 RSD < 10% Recovery: 87– 98% | 0.10–7.53 ng L− 1 | Aftafa et al. 2014 |
Estrogens and progestogens | Underground, estuary, sea and waste water | BAµE | LC–DAD | LOD: 50–100 ng L− 1 RSD < 14 % Recovery: 87–102 % | 300–4300 ng L− 1 | Almeida and Nogueira 2015 |
Estrogens, androgens, progestogens (and phenols) | River water | ASE–GPC–SPE-derivatization (MSTFA) | GC–MS | LOD: 0.3–0.8 ng L− 1 RSD < 10 % Recovery: 60–95% | 1.2–15.9 ng L− 1 | Huang et al. 2015 |
Estrogens, androgens, progestogens (and other EDCs) | River water | SPE- derivatization (BSTFA + 1% TMCS) | GC–MS | LOD: 0.33–3.33 ng L− 1 Recovery: 52–71% | 5.4–116 ng L− 1 | Ben Sghaier et al. 2017 |
Androgens, estrogens, corticosteroids and progestogens | Sea and fresh tap water | SPE | UHPLC– HRMS | LOD: 0.06–10 ng L− 1 RSD < 10.5 % Recovery: 95–109% | 0.26–39 ng L− 1 | Huysman et al. 2017 |
Natural and synthetic progestogens | Surface and waste water | SPE | LC–APCI/APPI–HRPS | LOQ: 0.02–0.87 ng L− 1 RSD < 33 % Recovery: 60–140% | 0.14–110 ng L− 1 | Golovko et al. 2018 |
Natural and synthetic progestogens | River and sewage effluents | SPE | LC–MS/MS | LOD: 0.008–0.12 ng L− 1 RSD < 17% Recovery: 43–116% | 0.04–38 ng L− 1 | Shen et al. 2018 |
Androgens, estrogens, corticosteroids and progestogens | River and waste water | SPE | LC–MS/MS | LOD: 0.01–40 ng L− 1 RSD: 1–13% Recovery: 56–126% | 1.0–220 ng L− 1 | Zhang et al. 2018 |
Androgens, estrogens, progestogens (and bisphenol A) | Tap, surface and waste water | SPE | UHPLC–MS/MS | LOD: 0.05–1.0 ng L− 1 RSD: 1.3–19 % Recovery: 70–130% | 0.80–790 ng L− 1 | Goeury et al. 2019 |
Androgens, estrogens and progestogens | Surface and waste water | SPE | LC–MS/MS | LOD: 1.9–44 ng L− 1 Recovery: 42–144% | 1.9–384 ng L− 1 | González et al. 2020 |
Androgens, estrogens, progestogens (and other EDCs) | Swimming pool water | SPE | LC-MS | LOD: 0.02–0.28 ng L− 1 RSD < 13.5 % Recovery: 72–118% | 0.02–78.8 ng L− 1 | Zhou et al. 2020 |
Natural and synthetic estrogens (and other emerging pollutants) | Surface water (river and canal) | SPE | LC-HRMS | LOD: 0.2–10.5 ng L− 1 RSD < 20 % Recovery: 96–101% | 5.1–285 ng L− 1 | Ng et al. 2021 |
Androgens, estrogens, progestogens and pregnenolone | Drinking, mineral, river, swimming pool, well and waste water | SPE- derivatization (BSTFA + 1% TMCS) | GC–MS | LOD: 0.01– 0.30 ng L− 1 RSD: 3.0–7.0% Recovery: 92–103% | 3.0– 104 ng L− 1 | This work |
aASE–GPC: accelerated solvent extraction–automated gel permeation chromatography. BAµE: Bar adsorptive microextraction. BSTFA: N,O-bis(trimethylsilyl)trifluoroacetamide. GC–ECD: Gas chromatography with electron capture detection. GC–MS: Gas chromatography–mass spectrometry. GC–MS/MS: Gas chromatography–tandem mass spectrometry. LC–MS/MS: High performance liquid chromatography–tandem mass spectrometry. LC–DAD: High–performance liquid chromatography–diode array detection. LC–APCI/APPI–HRPS: Liquid chromatography tandem atmospheric pressure chemical ionization/atmospheric pressure photoionization with hybrid quadrupole/orbital trap mass spectrometry operated in the high resolution product scan mode. LC–HRMS: Liquid chromatography–high resolution mass spectrometry. LC–MS/MS: Liquid chromatography–tandem mass spectrometry. LOD: Limit of detection. LOQ: Limit of quantification. MSTFA: N-methyl-N-(trimethylsilyl)trifluoroacetamide. ND: Not detected. RSD: Relative standard deviation. SPE: Solid–phase extraction. SPME: Solid–phase microextraction. TMCS: Trimethylchlorosilane. UHPLC– HRMS: Ultrahigh performance liquid chromatography– high resolution mass spectrometry. UHPLC–MS/MS: Ultra–high performance liquid chromatography–tandem mass spectrometry. UWWTPs: Urban wastewater treatment plants |
The waste water samples contained various estrogens (hexestrol, estrone, 17β-estradiol and 17α-ethinylestradiol) at concentrations from 7.0 to 110 ng L− 1. These results are consistent with previously reported values (Table 5) such as those of Migowska et al. (2012) and Goeury et al. (2019). The androgen concentrations in waste water were highest for dihydrotestosterone (45–76 ng L− 1), followed by testosterone (31–57 ng L− 1). Interestingly, these concentrations are lower than those found in Chinese waste water (201 ng L− 1 for dihydrotestosterone and 53.3 ng L− 1 for testosterone; Yu et al. 2019) but higher than those in waste water from Argentina (33 ng L− 1 for dihydrotestosterone and 16 ng L− 1 for testosterone; Gonzalez et al. 2020). Progesterone, at 16–24 ng L− 1, was the only progestogen detected in waste water, these levels being considerably lower than those reported by Golovko et al. (2018): 0.11–110 ng L− 1. By way of example, Fig. 3 shows the chromatograms of a swimming pool water and a waste water analyzed by the proposed method.