LC and NMR Studies for Identification and Characterization of Degradation Byproducts of Olmesartan Acid, Elucidation of Their Degradation Pathway and Ecotoxicity Assessment

The discovery of various sartans, which are among the most used antihypertensive drugs in the world, is increasingly frequent not only in wastewater but also in surface water and, in some cases, even in drinking or groundwater. In this paper, the degradation pathway of olmesartan acid, one of the most used sartans, was investigated by simulating the chlorination process normally used in a wastewater treatment plant to reduce similar emerging pollutants. The structures of nine isolated degradation byproducts (DPs), eight of which were isolated for the first time, were separated via chromatography column and HPLC methods, identified by combining nuclear magnetic resonance and mass spectrometry, and justified by a proposed mechanism of formation beginning from the parent drug. Ecotoxicity tests on olmesartan acid and its nine DPs showed that 50% of the investigated byproducts inhibited the target species Aliivibrio fischeri and Raphidocelis subcapitata, causing functional decreases of 18% and 53%, respectively.


Introduction
Keeping water clean and healthy is difficult, but not impossible. The important thing is to define which substances actually constitute a danger and a risk to the health of humans and other species, to establish which ones must leave production cycles to be eliminated from the environment, and to determine which ones must be kept in concentrations below certain hazardous limits. In water, new substances, in addition to being highly persistent contaminants, carry possible health and environmental effects. Pollutants of emerging interest are considered one of the most significant environmental problems in recent years. These include perfluoroalkyl substances (PFAS) [1], cyanobacteria [2], mycotoxins [3], hormones [4], psychoactive substances [5], pesticides [6], cosmetics, and industrial additives and drugs [7]. These substances have the potential to cause adverse effects on the environment and human health, but are still largely not specifically regulated by legislation, and their effects are not yet clear [8][9][10][11][12][13][14][15]. At the local level, pollution could affect different types of substances-and also byproducts or reaction products-so much so that an aggregate and cumulative risk assessment is required that takes into account the multiplicity of exposures. The effluents of wastewater treatment plants (WWTPs), not normally designed to eliminate emerging contaminants, are one of the main sources of contamination of surface waters by micropollutants [16]. Hundreds of tons of pharmaceutical substances flow residual solvent signals (CDCl 3 , at δ H 7.27 and δ C 77.0). The proton-detected heteronuclear correlations were measured using a gradient heteronuclear single-quantum coherence (HSQC) experiment, optimized for 1 J HC = 155 Hz, and a gradient heteronuclear multiple bond coherence (HMBC) experiment, optimized for n J HC = 8 Hz. The MALDI TOF mass spectrometric analyses were performed on a Voyager-De Pro MALDI mass-spectrometer (PerSeptive Biosystems, Framingham, MA, USA).

Chlorination Experiments
A 10 −5 M OLM solution was treated for 10 min with 10% hypochlorite (molar ratio OLM/HClO 1:1 concentration, spectroscopically determined λ max 292 nm, ε 350 dm 3 /mol cm) at room temperature [34], simulating the conditions used in a typical WWTP. The experiment was conducted at pH = 10.5. The presence of OLM was quantified using a Lambda 12 UV-Vis spectrophotometer (Perkin Elmer, Waltham, MA, USA). Absorbance peaks were determined at 230 nm. The absorbance values were converted into a concentration using a calibration curve prepared from standard solutions with known OLM concentrations. The pH of the solution, measured and recorded continuously using a pH-meter, increased immediately from the initial pH of 8.0 to 10.5, and pH remained at this value during the reaction. An aliquot of the solution was taken every 15 min, quenched by sodium thiosulphate excess, filtered, dried by lyophilization, and the residue dissolved in a saturated sodium bicarbonate solution and extracted with ethyl acetate. The course of the reaction was monitored using HPLC. DP1-DP9 were isolated from the ethyl acetate extract of the aqueous solution (Scheme 1 and Figure 1) and identified by comparing their retention times with those of commercially available standard compounds, or isolated by performing preparative experiments with a solution of OLM at a concentration higher than 10 −3 M and treated with 6% hypochlorite at room temperature for 2 h. The DPs obtained were isolated using column chromatography and HPLC and were completely characterized using NMR and MS analyses.
Molecules 2021, 26, x FOR PEER REVIEW 3 of 12 was performed using an RP Gemini C18-110A preparative column (10 μm particle size, 250 × 21.2 mm i.d., Phenomenex, Bologna, Italy) with a flow rate of 7.0 mL/min. The 1 Hand 13 C-NMR spectra were recorded with an NMR spectrometer operated at 400 MHz and at 25 °C (Bruker DRX, Bruker Avance, Billica, MA, USA) and referenced in ppm to the residual solvent signals (CDCl3, at δH 7.27 and δC 77.0). The proton-detected heteronuclear correlations were measured using a gradient heteronuclear single-quantum coherence (HSQC) experiment, optimized for 1 JHC = 155 Hz, and a gradient heteronuclear multiple bond coherence (HMBC) experiment, optimized for n JHC = 8 Hz. The MALDI TOF mass spectrometric analyses were performed on a Voyager-De Pro MALDI mass-spectrometer (PerSeptive Biosystems, Framingham, MA, USA).

Chlorination Experiments
A 10 −5 M OLM solution was treated for 10 min with 10% hypochlorite (molar ratio OLM/HClO 1:1 concentration, spectroscopically determined λmax 292 nm, ε 350 dm 3 /mol cm) at room temperature [34], simulating the conditions used in a typical WWTP. The experiment was conducted at pH = 10.5. The presence of OLM was quantified using a Lambda 12 UV-Vis spectrophotometer (Perkin Elmer, Waltham, MA, USA). Absorbance peaks were determined at 230 nm. The absorbance values were converted into a concentration using a calibration curve prepared from standard solutions with known OLM concentrations. The pH of the solution, measured and recorded continuously using a pH-meter, increased immediately from the initial pH of 8.0 to 10.5, and pH remained at this value during the reaction. An aliquot of the solution was taken every 15 min, quenched by sodium thiosulphate excess, filtered, dried by lyophilization, and the residue dissolved in a saturated sodium bicarbonate solution and extracted with ethyl acetate. The course of the reaction was monitored using HPLC. DP1-DP9 were isolated from the ethyl acetate extract of the aqueous solution (Scheme 1 and Figure 1) and identified by comparing their retention times with those of commercially available standard compounds, or isolated by performing preparative experiments with a solution of OLM at a concentration higher than 10 −3 M and treated with 6% hypochlorite at room temperature for 2 h. The DPs obtained were isolated using column chromatography and HPLC and were completely characterized using NMR and MS analyses.

Chlorination Procedure and Product Isolation
Olmesartan acid (1.0 g, 2.24 mmol) was dissolved in 1.0 L of phosphate buffer (KH2PO4/K2HPO4 0.1 M) [35]. A sodium hypochlorite solution (about 6% active chlorine, molar ratio OLM/HClO 1:20; concentration spectroscopically determined at λmax of 292 nm, ε = 350 dm 3 /mol × cm) was added drop-by-drop to this solution under magnetic stirring at room temperature. The pH of phosphate buffer was adjusted at a value of 6.50 by adding a 10% H3PO4 solution, checked with a pH-meter. The reaction was stopped after 2 h with an excess of sodium thiosulphate and concentrated by lyophilization. The residue was dissolved in water and pH adjusted to 7.00, and this solution was extracted using ethyl acetate (EA). The crude EA fraction (579 mg) was chromatographed on silica gel CC, eluting with gradient of methylene chloride:methanol:acetic acid (100:0:0.5 to 70:30:0.5, v/v/v) to yield 16 fractions. The fraction EA3 (16 mg), eluted with methylene chloride/methanol/acetic acid (100:0:0.5, v/v/v), was analyzed via HPLC using a reversed-phase column and eluting with water/acetonitrile (20:80, v/v) to yield DP6 (6.7 mg). The fraction EA4 (30 mg), eluted with methylene chloride:methanol:acetic acid (98:2:0.5, v/v/v), was separated by semipreparative HPLC using a reversed-phase column Kromasil 10 μm 100 Å C18 (250 × 10 mm) and elution with a gradient of CH3COONH4 (A, pH 4.0; 10 mM) and methanol (B), starting with 70% B for 1 min and installing a gradient to obtain 100% B over 20 min at a solvent flow rate of 4 mL/min to yield DP2 (7.8 mg). The fraction EA6 (36 mg), eluted with methylene chloride:methanol:acetic acid (95:5:0.5, v/v/v), was analyzed via preparative HPLC using a reversed-phase column Gemini 10 μm C18 110 Å (250 × 21 mm) and elution with a gradient of CH3COONH4 (A, pH 4.0; 10 mM) and methanol (B), starting with 60% B for 1 min and installing a gradient to obtain 100% B over 30 min at a solvent flow rate of 7.5 mL/min to yield 5 subfractions.

Ecotoxicity Data
The acute bioluminescence test with Aliivibrio fischeri (NRRLB-11177) was carried out in accordance with the ISO 11348-3:2007 [36] standard protocol, and the algal growth inhibition test with Raphidocelis subcapitata was performed according to the ISO 8692:2012 [37] standard protocol. These organisms are established biological models and are included in most regulations for the assessment of wastewater on an end-of-pipe basis.
The inhibition of bioluminescence in the presence of the OLM and its DPs was measured after 30 min of exposure. The toxic effect values are given by the ratio of the decrease in bacterial light output emitted by the bacterium in the sample compared to the control. To provide the relevant osmotic pressure for the test organisms, the salinity concentration of the stock solution was adjusted by 2% for NaCl. The temperature during exposure was 15 • C according to the Microtox standard procedure.
The growth of algae exposed to the sample was compared with the growth of algae in a negative control. For each sample, three replicates were inoculated with 10 4 algal cells L −1 in well plates and incubated for 72 h at 23 ± 2 • C under continuous illumination. The specific growth rate of R. subcapitata in each replicate was calculated from the logarithmic increase in cell density in the interval from 0 to 72 h. R. subcapitata density was determined by an indirect procedure using a spectrophotometer (Hach Lange DR5000, Loveland, CO, USA) and a 1 cm cuvette.

Chlorination Experiments
The OLM chlorination experiments were performed by mimicking the conditions of a typical WWTP, in which a 10 −5 M solution of the drug was treated for 10 min with 10% hypochlorite (OLM:hypochlorite molar ratio of 1:1; concn.) at room temperature [38,39]. Then, the tests are repeated at much higher concentrations of the contaminant (>10 −3 M), with a much lower ratio of OLM:oxidizing agent (1:5 or 1:6), so as to ensure the degradation of the studied contaminant and the possibility of isolating sufficient quantities of degradation byproducts for the subsequent structural identification.
The course of the reaction was monitored by HPLC, and the DPs obtained were isolated by column chromatography and HPLC (Scheme 1) and completely characterized using NMR and MS analyses (see Supplementary Materials). Finally, DP1-DP9 ( Figure 1) were isolated relative percentages of 1.23, 1.35, 2.01, 1.15, 1.02, 1.15, 0.45, 0.23, and 0.15, respectively. The proposed mechanism of their formation from OLM is shown in Figure 2a,b. Except for DP9, all other DPs were isolated for the first time.

Structure Elucidation of Degradation Byproducts DP1-DP9
In the OLM treatment at the buffered pH, the concentration of DP1-DP9 was at a maximum after 2 h and in the range of 2.01 to 0.15%. The plausible mechanism of the DPs formation from OLM is shown in Figure 2a

Ecotoxicity Data
Toxicity data were summarized in Figure 3, including the effects of R. subcapitata 72 h and A. fischeri after 30 min. The analysis of toxicity data evidenced the presence of the three main groups of samples considering a threshold value for the effects statistically different from the control groups [44]: (i) no effect (−10% ≤ inhibition ≤ 10%); (ii) biostimulation effect (inhibition < −10%); and (iii) toxic effects (inhibition > 10%).

Ecotoxicity Data
Toxicity data were summarized in Figure 3, including the effects of R. subcapitata 72 h and A. fischeri after 30 min. The analysis of toxicity data evidenced the presence of the three main groups of samples considering a threshold value for the effects statistically different from the control groups [44]: (i) no effect (−10% ≤ inhibition ≤ 10%); (ii) biostimulation effect (inhibition < −10%); and (iii) toxic effects (inhibition > 10%). After 72 h of exposure, OLM presented no effect on R. subcapitata. Comparing OLM and DPs results, only DP6 showed no toxicity. Several samples presented negative growth effects (DP1, DP2, DP3, DP7, DP8, DP9), and their toxicity values ranged from After 72 h of exposure, OLM presented no effect on R. subcapitata. Comparing OLM and DPs results, only DP6 showed no toxicity. Several samples presented negative growth effects (DP1, DP2, DP3, DP7, DP8, DP9), and their toxicity values ranged from 18% to a maximum of 53%. DP4 and DP5 showed stimulatory effects. Toxicity data from A. fischeri confirmed that the parent compound OLM showed no toxic effect after 30 min of exposure.
Five of nine isolated DPs (DP3, DP4, DP5, DP6 and DP9) showed no inhibitory effect. The other DPs presented acute toxicity like DP1, DP2, DP7, and DP8 showing inhibition effects from 20% to 34%. Comparing the results of the algal growth inhibition with bacteria luminescence inhibition, it was evident that the response of A. fischeri was less sensitive, but the toxicity trends were linearly correlated (Pearson correlation, r = 0.61, moderately high correlation).

Conclusions
This paper investigated the fate of OLM following the degradation treatment by chlorination. The reaction was carried out by simulating the conditions of a typical WWTP using excess sodium hypochlorite. After the chlorination treatment, chromatographic techniques were used to isolate nine degradation byproducts, which were fully characterized by MS and NMR analyses and via comparison with a commercial standard. OLM underwent complete mineralization in about 59% of cases, and in 21% of cases was recovered as is. OLM transformed into the corresponding byproducts in 20% of cases, and about 9% of these were identified. A possible mechanism for the degradation of OLM and its degradation byproducts has been hypothesized. Half of the investigated DPs possessed anywhere from slightly to highly toxic effects on the target species Aliivibrio fischeri and Raphidocelis subcapitata; the remaining DPs presented no such effects. According to the selected battery of toxicity, the correlation of the results suggested that DPs acted very similarly in unicellular organisms. Moreover, due to the highlighted ecotoxicological effects, DPs acting on organisms of low levels of complexity could have negative effects also on the whole trophic chain (biomagnification).

Supplementary Materials:
The following are available online, Table S1: 1 H, 13 C and 2 D NMR data of Olmesartan medoximil in CD 3 OD; Table S2: 1 H, 13 C and 2 D NMR data of DP-1 in CD 3 OD; Table S3: 1 H, 13 C and 2 D NMR data of DP-2 in CD 3 OD; Table S4: 1 H, 13 C and 2 D NMR data of DP-3 in CD 3 OD; Table S5: 1 H, 13 C and 2 D NMR data of DP-4 in CD 3 OD; Table S6: 1 H, 13 C and 2 D NMR data of DP-5 in CD 3 OD; Table S7: 1 H, 13 C and 2 D NMR data of DP-6 in CD 3 OD; Table S8: 1 H, 13 C and 2 D NMR data of DP-7 in CD 3 OD; Table S9:  Funding: This research was supported by AIPRAS-Onlus (Associazione Italiana per la Promozione delle Ricerche sull'Ambiente e la Salute umana) for the grants in support of this investigation.