Analysis of some pharmaceuticals in municipal wastewater of Almadinah Almunawarah

2.1 Chemicals and reagents

The following chemicals and reagents were used: reagent water (Milli-Q, 18.2 MΩ cm, Elix10, Millipore, USA), ethylenediaminetetraacetic acid disodium salt dehydrate (Na₂ETDA, Sigma, ACS reagent, 99.0–101.0%), ascorbic acid (Sigma, ultra grade, >99.0%), formic acid (Fischer Scientific, analytical reagent grade, 98%), ammonium hydroxide solution (Sigma–Aldrich, ACS reagent, NH₃ content 28–30%), methanol (Sigma–Aldrich, Chromasolv grade, 99.9% min), acetonitrile (ACN, Fisher Scientific, HPLC Gradient grade), dimethyldichlorosilane (DMDCS, 5% in toluene), and toluene (GC grade, 99.5%, Panreac, Barcelona-Spain). The drugs of interest were: acetaminophen (98%), carbamazepine, diclofenac sodium salt, erythromycin ethyl succinate, norfluoxetine (>97%), ranitidine hydrochloride, scopolamine N-butyl bromide (hyosine-N-bytyl bromide, >99%), and trimethoprim (>98.5%) which were purchased from Sigma–Aldrich, ibuprofen (>99%), metronidazole (99.9%), sulfamethoxazole (99.9%), and warfarin sodium salt (99.9%) from Fluka, and atenolol, captopril, cephalexin, chlorpheniramine maleate, metformin hydrochloride (99.8%), and simvastatin (98.3%) were obtained as a gift from Al-Jazeera Pharmaceutical Industries, Riyadh, Saudi Arabia. Stock solutions of these drugs (∼2000 μg mL⁻¹) were prepared as follows: approx. 20.0 mg of each drug was accurately weighed in a glass test tube and dissolved in 10 mL water or MeOH depending on analyte solubility. In order to minimize drugs' degradation, the test tubes containing the stock solutions were wrapped with aluminum foil and stored refrigerated at 4 °C. Working solutions of single analytes as well as mixtures of analytes (10.0 ng mL⁻¹ each) were prepared in methanol as required from the stock solutions and stored in a similar way as for the stock solutions.

Three types of HPLC mobile phases were prepared: formic acid (A1, 0.3% (v/v) in water); ammonium hydroxide (A2, 20 mM in water); and acetonitrile: methanol (B, 2:1).

2.2 Samples collection and pre-treatment

Wastewater samples were collected from the only STP in Almadinah Almunawarah. The current total capacity of this plant is 300,000 m³ day⁻¹. In addition to domestic sewage, the plant sometimes receives partially-treated industrial and medical wastewater. The plant undertakes tertiary treatment. Most of the treated wastewater is discharged into the nearby Al-Khlail wadi (valley) and used mainly for irrigation, and about 15% of this water is transported in trucks and used for watering of green areas and trees in public parks and streets of Almadinah Almunawarah.

Wastewater samples have been collected biweekly for a period of 4 months both from the inlet of the plant after initial screening (influents) and from the outlet after chlorination (tertiary treated, effluents). Composite samples were collected for 24 h using a portable water sampler (WS750 dual bottle sampler from Global Water, CA, USA) at a rate of 100 mL h⁻¹.

The procedure described by Batt et al. (2008) for the analysis of pharmaceuticals in waste and surface water was adopted for this work with modifications. Batt et al. procedure was developed and applied for the analysis of pharmaceuticals in wastewater and surface water samples obtained from New Mexico and East Fork River in Cincinnati, Ohio, respectively. The pH of the samples was measured upon arrival to the laboratory and found to be close to neutral values (pH 7.2–7.6) and therefore the samples were used without any pH adjustment. The samples were then filtered using a vacuum filter funnel (porosity 25–50 μm, Aldrich). To each 500 mL of filtered samples, a 2 mL solution containing Na₂ETDA (5.00 g L⁻¹, used as a metal chelating agent) and ascorbic acid (25.0 mg L⁻¹, used to remove any chlorine residues that may be present in the samples) was added before extraction.

Analytes in the samples were extracted at the same day of collection using Oasis MCX cartridges (mixed mode, 150 mg from Waters, Milford, MA, USA). The material inside these cartridges is made of hydrophobic-lipophilic balanced copolymer that can retain acidic, basic, and neutral analytes. A drug behaves as an acid, base, or neutral in solution based on the functional group(s) it processes and on the pH of the solution. In this context, “acidic and neutral” analytes described in this work are those pharmaceuticals that are eluted off the SPE Oasis MCX cartridge with ACN, whereas basic analytes are those that are eluted by ACN in 5% ammonium hydroxide. The reason for dividing the analytes into 2 groups (“acidic and neutrals” and basics) was to enhance the sensitivity of the MS detector and to avoid any complexity during analysis.

Before extraction, each SPE cartridge was conditioned with ACN (6 mL) followed by reagent water (6 mL). Samples prepared as described above were passed through reconditioned cartridges at a rate of 3–5 mL min⁻¹ with the aid of a vacuum pump. Each cartridge was then slowly rinsed with a 2 mL solution of formic acid (2%) and allowed to dry under vacuum.

Acidic and neutral analytes in each sample were first eluted with ACN (2 × 4 mL) into a small glass tube using a vacuum manifold (20 positions from Waters, Milford, MA, USA). Basic analytes retained on the cartridge material were then eluted by ACN solution (2 × 4 mL) containing ammonium hydroxide (5%) into a separate glass tube. Each eluate was then concentrated to dryness with the help of a TurboVap LV Concentration Evaporator Workstation (Caliper Life Sciences, Runcorn, UK) at 40 °C under a gentle stream of N₂. The contents of the first tube were reconstituted with ACN in water (0.50 mL, 20 + 80), whereas the eluate in the other tube was reconstituted with MeOH in water (0.50 mL, 20 + 80). The first fraction is termed from now on as “acidics and neutrals,” whereas the second one as “basics.” Reconstituted samples were transferred to glass vials and analyzed by LC–MS. This procedure has been first tried using solutions of mixed standards as described in the section 2.3.2 below.

2.3 Samples analysis

3.1 UV analysis

According to the UV spectra of all analytes (not shown here) an optimal wavelength of 210–215 nm can be used for all analytes when using the A2–B (1:1) mobile phase (210 nm was used). On the other hand, the use of the A1–B (1:1) mobile phase resulted in a different spectral pattern and a different optimal wavelength was obtained for most of the analytes. Additionally the following compounds showed no absorption signals in the investigated wavelength range: captopril, erythromycin ethyl succinate, and hyosine-N-butyl bromide. As a consequence multiple wavelengths have been used to monitor the analytes: 225, 230, 240, and 270 nm; each of them has been used to monitor a sub group of the analytes.

3.2 HPLC-DAD analysis

Experiments conducted as described in Section 2.2 above showed that the analytes eluted as basics were: atenolol, ranitidine, simvastatin, and trimethoprim. In contrast, erythromycin ethyl succinate showed no HPLC-DAD signal, but confirmed by LC–MS to elute in the basics group. The rest of the analytes was eluted in the acidics and neutrals fraction.

3.3 LC–MS/MS Analysis

Several solid phase extraction cartridges were used in the literature for the extraction of pharmaceuticals from wastewater including Oasis HLB, Oasis MCX, Oasis WCX, Strata-X, Supelco C8, Supelco C18, Varian Focus, and Merck LiChrolut-EN. The Oasis MCX, which was employed in this work, has been shown to provide high overall recoveries. (Batt et al., 2008; Lacey et al., 2008).

Extracted MRM chromatogram, mass spectrum, and product ion spectrum for each analyte that has been detected in the wastewater in concentrations greater than the LOD are shown in Figs. 1–5. Out of the 19 targeted drugs, 5 pharmaceuticals have been found both in raw (influents) and treated (effluents) wastewater samples. The concentrations of these pharmaceuticals are shown in Tables 4 and 5. Although most pharmaceuticals have high solubility in water and hence remain soluble in the aqueous phase, some drugs have lower solubility and remain insoluble as the solid material in wastewater. Since the samples were filtered before extraction, the reported concentrations in this work represent only the water-soluble fraction of the analytes.

Close

Figure 1 Extracted MRM chromatogram (1a), mass spectrum (1b), and product ion spectrum (1c) for acetaminophen.

Export to PPT

Close

Figure 2 Extracted MRM chromatogram (2a), mass spectrum (2b), and product ion spectrum (2c) for atenolol.

Export to PPT

Close

Figure 3 Extracted MRM chromatogram (3a), mass spectrum (3b), and product ion spectrum (3c) for cephalexin.

Export to PPT

Close

Figure 4 Extracted MRM chromatogram (4a), mass spectrum (4b), and product ion spectrum (4c) for metformin.

Export to PPT

Close

Figure 5 Extracted MRM chromatogram (5a), mass spectrum (5b), and product ion spectrum (5c) for norfluoxetine.

Export to PPT

The detected drugs have been found in most of the samples as shown in Tables 4 and 5. As expected, the concentrations of individual pharmaceuticals in the wastewater influents were found to be low with an average concentration of 2.04–38.9 ng mL⁻¹. The highest detected concentration was for acetaminophen (99.6 ng mL⁻¹), followed by metformin (31.2 ng mL⁻¹), norfluoxetine (10.4 ng mL⁻¹), atenolol (4.03 ng mL⁻¹), and cephalexin (3.23 ng mL⁻¹). Interestingly, the amounts of these pharmaceuticals in the treated wastewater (effluents) did not differ much from those found in the raw wastewater (influents), with a concentration range of 0.545–31.2 ng mL⁻¹. The highest concentration detected was for acetaminophen (90.5 ng mL⁻¹), followed by norfluoxetine (11.7 ng mL⁻¹), cephalexin (2.83 ng mL⁻¹), metformin (4.51 ng mL⁻¹), and atenolol (2.01 ng mL⁻¹).

The findings of this study is consistent with those of many reports in the literature, where low concentrations of pharmaceuticals have been found in municipal wastewater (Bartelt-Hunt et al., 2009; Batt et al., 2008; Gracia-Lor et al., 2010; Miège et al., 2009; Radjenovic et al., 2009; Rosal et al., 2010). For example, Rosal et al. reported the occurrence of around 70 pharmaceuticals and PPCPs in influents of municipal wastewater with some compounds in the ng mL⁻¹ concentration range (e.g. paraxanthine, caffeine, and acetaminophen), whereas the rest were in the ng L⁻¹ range (Rosal et al., 2010). In another study by Gracia-Lor et al., 13 out of 20 investigated drugs were detected in the influents of urban wastewater samples with salicylic acid having the highest concentration (276.7 ng mL⁻¹) (Gracia-Lor et al., 2010).

On the other hand, effluents of urban wastewater and receiving waters were reported also to contain many pharmaceuticals at low concentrations. (Bartelt-Hunt et al., 2009; Gracia-Lor et al., 2010; Joss et al., 2008; Lacey et al., 2008; Santos et al., 2007; Soliman et al., 2007; Spongberg and Witter, 2008; Ternes, 1998; Zhou et al., 2009) Clearly, this indicates that most of the current wastewater treatment practices are inefficient in completely removing such contaminants. For instance, 5 pharmaceuticals (i.e. propranolol, sulfamethoxazole, carbamazepine, indomethacine and diclofenac) were found in all wastewater and the receiving surface water samples in England with carbamazepine having the highest levels (2.336 ng mL⁻¹). The reported removal efficiencies for these compounds from the wastewater were in the range of 43–92%.(Zhou et al., 2009) In another study, 5 out of 6 drugs (diclofenac, ibuprofen, ketoprofen, naproxen, carbamazepine, and caffeine) have been detected in both influent and effluent samples from 4 STPs in Seville–Spain in the ng mL⁻¹ concentration range. The reported removal rates for these drugs were between 6% and 98%. (Santos et al., 2007) Similarly, 5 out of 10 pharmaceuticals detected in the influents of 3 STPs in Duplin-Ireland have been found in the wastewater effluents with concentrations of <1 ng mL⁻¹ for most samples (Lacey et al., 2008).

The removal efficiency of pharmaceuticals from wastewater is actually dependent on several factors including the climatic conditions, the type of wastewater treatment and its operational conditions (e.g. temperature, redox conditions, solids and hydraulic retention time) as well as the age of the activated sludge used in the plant, but the main factor is reported to be the physico-chemical nature of most pharmaceuticals, which is the acidity and high solubility in water with very low solid–liquid partition. These factors, especially the last one lead to a very poor sorption of these compounds onto sludge and hence leaving them soluble in the aqueous phase (Gracia-Lor et al., 2012; Verlicchi et al., 2012).

It is also worth noting here that some of the pharmaceuticals that were detected in the wastewater were also found in the sludge of the wastewater treatment plans. This is due to the low solubility of such drugs and therefore they remain insoluble and appear in the sludge (Gao et al., 2012; Jelic et al., 2011).

When considering a single pharmaceutical at low concentrations such as those reported in this investigation and other work, it may be assumed that not many health risks can be associated with long term exposure to such a drug. But the health risks associated with exposure to a large number of pharmaceuticals, their metabolites, and transformation products, even at low concentrations, cannot be ignored.