Sampling and sample preparation

Samples of influent and permeate from the MBR were continuously collected as 24-h-composite samples by a peristaltic pump over the course of ten days. Since permeate samples were taken with a delay of two days to match them with the corresponding influent samples according to the HRT, the sampling period represents an actual time series of eight days. Grab samples of the concentrate with mixed-liquor suspended solids were collected three times during the study. All samples were taken in clean glass bottles rinsed with ultra pure water, heptane and acetone and subsequently heated overnight at > 240 °C. Samples were kept in the dark and transported on ice. Extraction was carried out within six hours of sampling.

Regarding acidic compounds and ICMs, the influence of direct acidification during sampling (by providing acid in the sampling bottles beforehand) and acidification in the laboratory after sampling before extraction was investigated (data not shown). For the majority of analytes, the different procedures yielded results which differed only slightly (< 10%), with no clear preference for one method, so subsequently samples were acidified after collection.

The liquid phase of the concentrate was separated from the MLSS by centrifugation directly after arrival at the laboratory. Samples were centrifuged for 60 min with 3500 r/min in a temperature-controlled centrifuge (Rotanta 460R, Hettich Lab Technology, Tuttlingen, Germany).

Samples of influent and supernatant of the concentrate were passed through binder-free glass fibre filters (MN 85/70 BF, Machery-Nagel, Düren, Germany; average retention capacity: 0.6 µm;

diameter: 55 mm) which were conditioned prior to filtration by sequentially soaking them in heptane, acetone and methanol for 15 min each, rinsing with Milli-Q water twice and storing in Milli-Q water overnight before use.

Before extraction, samples were separated into aliquots for the determination of neutral analytes and the analysis of acidic drugs, bezafibrate and ICMs. The latter aliquots were acidified with H2SO4 to a pH of 2.3–2.8, while the native samples of the first aliquot were controlled to have a pH of 7–8. All samples were augmented with internal standards before SPE (see below).

3.2.5ANALYTICAL PROCEDURES

The analytical protocols used in this study followed in large parts procedures previously described by Hirsch et al., 2000, Hummel et al., 2006, Magdeburg et al., 2014 (antibiotics, acidic compounds: NSAIDs and bezafibrate, psycho-active compounds, ICMs), Schulz et al., 2008 and Kormos et al., 2009 (TP of iopromide).

3.2.5.1SOLID PHASE EXTRACTION AND CLEAN-UP

NEUTRALANALYTES: ANTIBIOTICS, PSYCHO-ACTIVECOMPOUNDS, OPS. Neutral analytes were extracted with Oasis HLB cartridges (500 mg, 6 mL, Waters, Milfort, U.S.). Prior to extraction, the cartridges were conditioned with 1 x 5 mL heptane, 1 x 5 mL acetone, 2 x 5 mL methanol and 3 x5 mL Milli-Q water (Figure 3-3). Samples were passed through the cartridges with a flow rate of approximately 5 mL/min. Following SPE, the HLB material was dried completely under a steady nitrogen stream for approximately 60–90 min. Elution was accomplished with 5 x 2 mL acetone. Extracts were evaporated to approximately 100 µL by a gentle nitrogen stream before the vials were rinsed with 300 µL of methanol, followed by a second reduction to 100 µL and a final addition of 400 µL Milli-Q water resulting in a final sample volume of 500 µL. Samples were kept at 4 °C in the dark until LC-MS/MS analysis.

ACIDICANALYTES: NSAIDSANDBEZAFIBRATE. For the extraction of acidic compounds, Oasis MCX cartridges (500 mg, 6 mL, Waters, Milfort, U.S.) were used. The procedure was in principle as described above except for the final dilution, which was carried out with formic acid (Figure 3-3).

ICMSAND TP. ICMs and their TP were extracted with Isolute ENV⁺ cartridges (200 mg; 3 mL, Biotage, Uppsala, Sweden) following the scheme depicted in Figure 3-3.

Since the SPE procedure for ICMs is highly sensitive to matrix material in the water sample, after conditioning the MCX cartridges were mounted on top of the ENV⁺ cartridges and the water extraction was performed in one step to shield the ENV⁺ cartridges from matrix material (Figure 3-4).

3.2.5.2LC-MS/MS-ANALYSIS

For chromatographic separation, an Agilent 1200 Series (Agilent Technologies, Waldbronn, Germany) liquid chromatographic system equipped with membrane degasser, binary high-pressure gradient pump, autosampler and column thermostat was used. The detection was carried out on a tandem mass spectrometer (Applied Biosystems/MDS Sciex 4000 Q Trap Qq-LIT-MS; Applied Biosystems, Langen, Germany) using multiple reaction monitoring (MRM). For the analysis, electrospray ionization (ESI) was used except for organophosphorus compounds, which were analysed by atmospheric pressure chemical ionization (APCI). The specific modes are detailed below. For each target compound two MRM transitions were monitored for quantification (transition 1) and confirmation (transition 2). Instrument control, peak detection

Antibiotics, psycho-active

compounds, OPs NSAIDs and bezafibrate ICMs

Figure 3-3: Overview of the SPE procedures for antibiotics, psycho-active compounds, OPs (left), NSAIDs and lipid regulators (middle) and ICMs (right).

ACIDICANALYTES: NSAIDSANDBEZAFIBRATE. Chromatography was performed with a Zorbax Eclipse XDB-C8 (150 x 4.6 mm; 5 µm) equipped with a Zorbax XDB-C8 guard column (4.6 x 12.5 mm, 5 µm, both purchased from Agilent Technologies (Waldbronn, Germany). The solvents used were acetonitrile (A) and 10 mM formic acid (B). Separation started with 60% A changing to 5% A within 6 min, was kept isocratic for 8 min before it was returned to the starting conditions within 1 min and was held isocratic for the final 5 min of the chromatographic run. Flow rate was set to 400 µL/min, injection volume was 15 µL. Mass spectrometry was carried out with ESI in negative mode with the following conditions: CAD:

5 psi; curtain gas: 30 psi; ion source gas 1: 45 psi; ion source gas 2: 50 psi; source temperature:

650 °C, entrance potential (EP): -10 V. Other parameters are shown in Table 3-1.

ANTIBIOTICS. Chromatography was carried out with a Chromolith^ Performance RP-18e column (100 x 4.6 mm, 130 Å, 2 µm) preceded by a Chromolith^ RP-18e (5 x 4.6mm) guard column, both supplied by Merck, Darmstadt, Germany. Mobile phase A consisted of a 10 mM ammonium formiate buffer adjusted to pH 4 with formic acid. Acetonitrile served as mobile phase B.

LC-MS/MS-analysis Conditioning of Oasis MCX cartridge (500 mg;

6 mL ): 1 x 5 mL heptane, 1 x 5 mL acetone, 2 x

Table 3-1: Target compounds and the corresponding ESI-MS/MS parameters for the analysis of acidic

Figure 3-4: SPE installation for acidic analytes and ICMs.

Chromatography started with 0% B, held for 10 min, followed by an increase of B to 26% in 5 min, further increase to 38% within 2 min and held isocratic for 6 min before increasing B to 100% within 6 min, holding isocratic for 4 min before returning to initial conditions within 2 min which were held for 4 min. The flow rate was 400 µL/min. Injection volume was 5 µL; the temperature of the column oven was set to 25 °C.

Electrospray ionization (ESI) was executed in positive mode. Conditions for ESI were set as followed: collision gas: 5 psi; curtain gas: 35 psi; ion source gas 1: 45 psi; ion source gas 2:

40 psi; source temperature: 650 °C; entrance potential: 10 V; ionspray voltage: 5.5 kV. Further details of the MS/MS analysis are listed in Table 3-2.

Table 3-2: Target compounds and the corresponding ESI-MS/MS parameters for the analysis of antibiotics (RT: retention time; DP: declustering potential; CE: collision energy; CXP: collision cell exit potential).

Compound CAS

ICMSANDTRANSFORMATIONPRODUCTSOFIOPROMIDE. A Chromolith^ Performance RP-18e (100 x 4.6 mm, 130 Å, 2 µm) column preceded by a Chromolith^ RP-18e (5 x 4.6 mm) guard column (both supplied by Merck, Darmstadt, Germany) was used for chromatographic separation. Mobile phase A was produced by mixing 980 mL Milli-Q water (additioned with 20 mmol/L NH3 and adjusted to pH 5.7 with acetic acid) with 20 mL acetonitrile. Mobile phase B was produced by mixing 40% of mobile phase A with 60% acetonitrile. The gradient started with 100% A for six minutes, changed to 20% A within further 6 min and was held for 3 min before returning to 100% A within 0.5 min and held isocratic at these conditions for the final 4.5 min. Flow rate was 600 µL/min, injection volume was 25 µL and the temperature of the column oven was adjusted to 30 °C.

Detection was performed with electrospray ionization (ESI) in positive mode (collision gas:

medium; curtain gas: 30 psi; ion source gas 1 and 2: 40 psi; source temperature: 600 °C;

entrance potential: 10 V; see Table 3-3).

The transformation products of iopromide were analysed according to Schulz et al., 2008, where all details of the procedure are described.

Table 3-3: Target compounds and the corresponding ESI-MS/MS parameters for the analysis of ICMs (RT:

retention time; DP: declustering potential; CE: collision energy; CXP: collision cell exit potential).

Compound CAS

PSYCHO-ACTIVECOMPOUNDS. Chromatographic separation was accomplished with a Synergi Polar-RP 80 Å column (150 x 3 mm, 4 µm) with a SecurityGuard column (Polar-RP, 4 mm × 3 mm) (both purchased from Phenomenex, Aschaffenburg, Germany). Two mobile phases were used: (A) 10 mM ammonium formiate buffer adjusted to pH 4 with formic acid and

within 13 min to 80% B, which was kept for 7 min before returning to starting conditions within 1 min. Starting conditions were held for 10 min for re-equilibration. The flow rate was 500 µL/min, injection volume was 25 µL, and the temperature of the column oven was kept at 25 °C. Electrospray ionization (ESI) was executed in positive mode with the following parameters: collision gas: 6 psi; curtain gas: 25 psi; ion source gas 1 and ion source gas 2: 40 psi;

source temperature: 450 °C; entrance potential: 10 V; ionspray voltage: 5.5 kV. Further details of the MS/MS analysis are shown in Table 3-4.

Table 3-4: Target compounds and the corresponding ESI-MS/MS parameters for the analysis of psycho-active compounds (RT: retention time; DP: declustering potential; CE: collision energy; CXP: collision cell

OPS. Chromatographic separation was achieved with a Synergi Polar-RP 80 Å column (150 x 3 mm, 4 µm) with a SecurityGuard guard column (Polar-RP, 4 mm × 3 mm) (both purchased from Phenomenex, Aschaffenburg, Germany). The used mobile phases were Milli-Q water (A) and MeOH (B). Chromatography started with 64% B, was increased to 76% B within 0.5 min and further increased to 100% B within 15.5 min, held isocratic for 4 min and returned to initial conditions with 64% B within 0.1 min and then was kept isocratic for 4.9 min. The flow rate was set to 400 µL/min, injection volume was 10 µL, and the temperature of the column oven was 25 °C. Mass-spectrometric analysis was carried out in positive mode, utilizing atmospheric pressure chemical ionization (APCI), using the following parameters: collision gas, medium; curtain gas: 30 psi, nebulizer current, 3 µA; ion source gas 1: 60 psi; source temperature: 500 °C; entrance potential: 10 V. Declustering potential was 80 V and collision cell exit potential 10 V. Further details are given in Table 3-5.

Table 3-5: Target compounds and the corresponding APCI-MS/MS parameters for the analysis of OPs (RT:

retention time; DP: Declustering potential; CE: Collision energy; CXP: Collision cell exit potential).

for all analytes: DP [V]:80, CXP [V]: 10; ¹ Precursor ion/product ion;