Simple and rapid method for monitoring pharmaceuticals in wastewater
Suominen, Mikko (2013)
Suominen, Mikko
2013
Ympäristö- ja energiatekniikan koulutusohjelma
Luonnontieteiden tiedekunta - Faculty of Natural Sciences
This publication is copyrighted. You may download, display and print it for Your own personal use. Commercial use is prohibited.
Hyväksymispäivämäärä
2013-06-05
Julkaisun pysyvä osoite on
https://urn.fi/URN:NBN:fi:tty-201306131213
https://urn.fi/URN:NBN:fi:tty-201306131213
Tiivistelmä
Thousands of tons of pharmacologically active ingredients are used annually. The compounds end up into the environment either directly or from wastewater treatment plants. Also pharmaceutical factories generate point source emissions. Pharmaceuticals in the environment have adverse and potentially unidentified effects and elimination of pharmaceutical emissions at point sources is needed. To support this work, reliable analytical methods capable of measuring pharmaceuticals in environmental matrices are needed.
The aim of this Master’s thesis was to develop an HPLC-UV method for the measurement of acetyl salicylic acid (ASA), ciprofloxacin (CPX), paracetamol (PCM), sulfamethoxazole (SMX), diclofenac (DIC) and erythromycin (ERY) from wastewater. In order to detect ERY derivatization was needed. Pretreatment of samples was optimized in terms of sample pH. SPE recoveries and repeatabilities were determined and also breakthrough of analytes was investigated. The aim was to achieve quantification limits of 0.05 mg/l.
Two separate methods were developed. A separate method for the derivatized ERY was needed because the derivatization product was extremely hydrophobic. The HPLC method for ASA, CPX, DIC, PCM and SMX used a 250 mm x 4.6 mm x 5 μm C18 column, a gradient using 1 % acetic acid, 0.2 % triethylamine : ACN as mobile phases, a flow rate of 1 ml/min. ASA was detected at 275 nm and the rest of the compounds at 265 nm.
To retain ASA, SMX, PCM and DIC from wastewater C18 SPE sorbents were used and sample pH adjusted to 2. For CPX strong cation exchange sorbents were used. ASA, DIC and SMX didn’t show analyte breakthrough up to 200 ml but for PCM analyte breakthrough occurred after 50 ml. Recoveries were 88.3 ± 3.6 % for ASA, 107.4 ± 1.1 % for SMX and 86.9 ± 8.5 % for DIC using 100 ml sample volumes and 84.7 ± 4.6 % using 50 ml sample volume. Recovery of CPX was 77.8 ± 3.7 % using 100 ml sample volume.
Taking sample enrichment during SPE pretreatment into account, method detection limits were 0.037 mg/l for PCM, 0.043 mg/l for ASA, 0.003 mg/l for SMX, 0.009 mg/l for DIC and 0.048 mg/l for CPX using 1 ml as the final HPLC sample volume. Therefore quantification at the 0.05 mg/l level could be done.
Derivatization of ERY was carried out by evaporating the sample solvent and reacting the residue with FMOC-Cl and phosphate buffer (pH 8.25) at 60 oC for 15 minutes. A 50 mm x 4.6 mm x 5 μm C8 column, isocratic elution with ACN:Milli-Q water 80:20 (v:v), a flow rate of 2 ml/min and a detection wavelength of 265 nm were used. Linearity of the HPLC method was fair (R2 = 0.927) and instrumental quantification limit was 9.6 μg of ERY. ERY was extracted from wastewater at pH 10 using C18 SPE sorbents. The mean recovery was 82.7 % ± 36.5 %. Breakthrough of ERY wasn’t investigated because of poor derivatization repeatability. Taking sample enrichment into account, quantification of ERY at the 0.05 mg/l level could be achieved by extracting approximately 230 ml of the sample.
The aim of this Master’s thesis was to develop an HPLC-UV method for the measurement of acetyl salicylic acid (ASA), ciprofloxacin (CPX), paracetamol (PCM), sulfamethoxazole (SMX), diclofenac (DIC) and erythromycin (ERY) from wastewater. In order to detect ERY derivatization was needed. Pretreatment of samples was optimized in terms of sample pH. SPE recoveries and repeatabilities were determined and also breakthrough of analytes was investigated. The aim was to achieve quantification limits of 0.05 mg/l.
Two separate methods were developed. A separate method for the derivatized ERY was needed because the derivatization product was extremely hydrophobic. The HPLC method for ASA, CPX, DIC, PCM and SMX used a 250 mm x 4.6 mm x 5 μm C18 column, a gradient using 1 % acetic acid, 0.2 % triethylamine : ACN as mobile phases, a flow rate of 1 ml/min. ASA was detected at 275 nm and the rest of the compounds at 265 nm.
To retain ASA, SMX, PCM and DIC from wastewater C18 SPE sorbents were used and sample pH adjusted to 2. For CPX strong cation exchange sorbents were used. ASA, DIC and SMX didn’t show analyte breakthrough up to 200 ml but for PCM analyte breakthrough occurred after 50 ml. Recoveries were 88.3 ± 3.6 % for ASA, 107.4 ± 1.1 % for SMX and 86.9 ± 8.5 % for DIC using 100 ml sample volumes and 84.7 ± 4.6 % using 50 ml sample volume. Recovery of CPX was 77.8 ± 3.7 % using 100 ml sample volume.
Taking sample enrichment during SPE pretreatment into account, method detection limits were 0.037 mg/l for PCM, 0.043 mg/l for ASA, 0.003 mg/l for SMX, 0.009 mg/l for DIC and 0.048 mg/l for CPX using 1 ml as the final HPLC sample volume. Therefore quantification at the 0.05 mg/l level could be done.
Derivatization of ERY was carried out by evaporating the sample solvent and reacting the residue with FMOC-Cl and phosphate buffer (pH 8.25) at 60 oC for 15 minutes. A 50 mm x 4.6 mm x 5 μm C8 column, isocratic elution with ACN:Milli-Q water 80:20 (v:v), a flow rate of 2 ml/min and a detection wavelength of 265 nm were used. Linearity of the HPLC method was fair (R2 = 0.927) and instrumental quantification limit was 9.6 μg of ERY. ERY was extracted from wastewater at pH 10 using C18 SPE sorbents. The mean recovery was 82.7 % ± 36.5 %. Breakthrough of ERY wasn’t investigated because of poor derivatization repeatability. Taking sample enrichment into account, quantification of ERY at the 0.05 mg/l level could be achieved by extracting approximately 230 ml of the sample.