1. Results and Discussion
Detection and measurements of trace pharmaceuticals in real wastewater samples with an application to anti-ulcer agents, omeprazole, esomeprazole and anti-platelet agent clopidogrel, using sensitive UPLC-MS methodology
Kazi Mohsin1, Leda G. Bousiakou2, George A. Karikas3
1 Kayyali Chair for Pharmaceutical Industries, College of Pharmacy, King Saud University, Riyadh, Saudi Arabia, 2 Department of Physics and Astronomy, King Saud University, Riyadh, Saudi Arabia, 3 Department of Medical Laboratories Technology, Technological Educational Institute of Athens, Athens, Greece.
Objectives
Results and Discussion
The aim is to develop a rapid, accurate and sensitive analytical method for the simultaneous quantification and chemical characterization of the most frequently found pharmaceuticals in real wastewater samples from the Riyadh South Tertiary, in Saudi Arabia.
Table 1: UPLC/MS optimized conditions for six selected pharmaceuticals and hormones. ES-electrospray ionization; monoisotopic molecular weight; Q, quantification
Introduction
Compound
Therapeutic group
Polarity (ES) MW
Q Transition
Cone (V)
Capillary
Retention time(min)
Atorvastatin
Anti-cholesterol & lipid regulator -
558.64
557.51 40 3
8.32
Clopidogrel
Anti-platelet agent +
321.82
322.34 25
7.87
Omeprazole
Anti-ulcer agent
345.1
346.3 20
6.41
Esomeprazole
Anti- ulcer agent
345.42
346.38
9.86
Diphenhydramine
Antihistamine (Sedative)
255
256.43 15
2.8
5.52
Oestrogen
Steroid hormone
270
269.39 80
6.84
Pharmaceuticals coming from a wide spectrum of therapeutic classes are used in human medicine worldwide. After administration of the active pharmaceutical compounds, some drugs are metabolised, while others remain intact before being excreted. Although the pharmaceutical residues in the water cycle are at low concentrations (below therapeutic doses), the continuous release of these pharmaceuticals and hormones into the environment could have adverse effects to many living organisms for their entire lifetime .
A number of analytical methods for determining pharmaceuticals in aqueous environmental samples using LC/MS have been previously reviewed. Sensitive, selective and reproducible methods are of major importance in providing realistic data regarding their presence both in surface waters and wastewater. Thus, there is extensive interest in this area in improving the current analytic methods.
The chlorination effect on the treatment of waste water (Fig shows omeprazole conc. Below)
Methods
Table 2: Concentrations of the target pharmaceuticals detected wastewater samples. Data represented as mean only, n=3
Fig 2: Recoveries (>90%, RSD<10%, n=5) for the pharmaceuticals & hormone from wastewater
An ultra-performance liquid chromatography–tandem mass spectrometry (UPLC–MS) method has been developed for the targeted analytes including analgesic and anti-inflammatory agents, anti-cholesterol statins and two antibiotics of same chemical groups. Solvent extraction technique was applied for the quantitative analysis of targeted analytes in wastewater samples. The method was validated in considering the following parameters of linearity, recovery, precision, limits of detection (LOD) and limits of quantification (LOQ). Linear range of the current method was established by analysis of six standard calibration solutions to cover the concentration range of 1ppb-1ppm (1µg/L-1mg/L).
Compound
Raw wastewater (µg/ml)
Before chlorination (µg/ml)
After chlorination (µg/ml)
Omeprazole
0.048
0.012
0.001
Esomeprazole
0.069
0.036
0.002
Clopidogrel
0.033
0.015
Fig 4: UPLC/MS chromatograms of omeprazole detected in waste water from inlet (raw), before- after chlorination in Riyadh tertiary south plant
All compounds were satisfactorily determined by single injection with a chromatographic run time of less than 10 minutes. The process efficiency (combination of the matrix effect and the extraction process recovery) for selected compounds was evaluated in sewage wastewater (before & after chlorination) samples. The method was successfully validated in wastewater (WW) at low concentration levels, as expected for pharmaceuticals in these matrices (0.1, 0.5 and 1μg/L in WW). Apart from few exceptions, the instrumental limits of detection varied between 1 and 10ppb. The limits of quantification were estimated from sample chromatograms at the lowest spiked levels tested and normally were 100ng/L for WW. The developed method was applied to the analysis of 60 WW samples and confirmed the presence of anti-ulcer agents, omeprazole, esomeprazole and anti-platelet agent clopidogrel above the chromatographic detection limit (0.0001ug/ml) before and after chlorination. Measurements of influent sewage characteristics from the same treatment WW plant are also studied before and after chlorination. The overall BOD, COD and TSS have been significantly reduced by the chlorination process.
Fig 1: Schematic flow diagram of the Riyadh South Tertiary Plant (Capacity = 200,000m3/d)
Chlorination had a substantial effect on the waste water sample analysis collected from NWC, south plant in Riyadh, Saudi Arabia. The presence of anti- ulcer agent omeprazole was significant at the influent (raw wastewater) as well as before chlorination (i.e., Fig a, b conc & 0.012µg/mL). On the other hand, the omeprazole conc. was substantially decreased to 10 fold after chlorination process (Fig 4c, conc µg/mL).
Fig 3: UPLC/MS chromatograms and spectra of the pharmaceuticals that detected for positive mode in effluent waste water sample. (A, A1) omeprazole, (B, B1) esomeprazol, and (C, C1) clopidogrel
Conclusions
Anti-ulcer agents, omeprazole, esomeprazole and anti-platelet agent clopidogrel were detected in this work among the most detected pharmaceuticals in wastewater samples. Antidepressant agent diphenhydramine, cholesterol regulator atorvastatin and the female hormone oestrogen were not detected in any of the three sampling points.
ACKNOWLEDGEMENT
This research has been co-funded by the European Union (European Social Fund) and Greek national resources under the framework of the “Archimedes III: Funding of Research Groups in TEI of Athens” project of the “Education & Lifelong Learning” Operational Programme.
Reference
Gracia-Lor et al. J Chromatogr A. 2010;1217(5):622-32
Bousiakou L et al. Pharmakeftiki 25, I, 2013