Development of a novel passive sampler based on a combination of membrane assisted solvent extraction and molecularly imprinted polymer for monitoring of selected pharmaceuticals in surface waters Sinegugu Khulu A thesis submitted to the Faculty of Science, University of the Witwatersrand in fulfilment of the requirements for the degree of Doctor of Philosophy July 2022 i DECLARATION I declare that this dissertation is my own, unaided work. It is being submitted for the degree of Doctor of Philosophy at the University of the Witwatersrand, Johannesburg. It has not been submitted before for any degree or examination at any university Sinegugu Khulu 26th day of July 2022 at Johannesburg ii PUBLICATIONS AND MANUSCRIPTS This thesis is based on the following papers in which the candidate was the principal author: Synthesis, characterization and application of a molecularly imprinted polymer as an adsorbent for solid phase extraction of selected pharmaceuticals from water samples Sinegugu Khulu, Somandla Ncube, Tebogo Kgame, Elizabeth Mavhunga, Luke Chimuka Published in Polym. Bull. 79, 1287–1307 (2022). https://doi.org/10.1007/s00289-021-03553-9 Multivariate optimization of a two-way technique for extraction of pharmaceuticals in surface water using a combination of membrane assisted solvent extraction and a molecularly imprinted polymer Sinegugu Khulu, Somandla Ncube, Yannick Nuapia, Lawrence Mzukisi Madikizela, Hlanganani Tutu, Heidi Richards, Kuria Ndungu, Elizabeth Mavhunga, Luke Chimuka Published in Chemosphere, Volume 286 (2022), 131973, ISSN 0045-6535, https://doi.org/10.1016/j.chemosphere.2021.131973 Development and application of a membrane assisted solvent extraction- molecularly imprinted polymer based passive sampler for monitoring of selected pharmaceuticals in surface water Sinegugu Khulu, Somandla Ncube, Yannick Nuapia, Lawrence Mzukisi Madikizela, Elizabeth Mavhunga, Luke Chimuka Manuscript under review in Water Research, Manuscript number: WR70110 https://doi.org/10.1007/s00289-021-03553-9 https://doi.org/10.1016/j.chemosphere.2021.131973 iii CANDIDATE CONTRIBUTION TO ARTICLES Synthesis, characterization and application of a molecularly imprinted polymer as an adsorbent for solid phase extraction of selected pharmaceuticals from water samples Sinegugu Khulu, Somandla Ncube, Tebogo Kgame, Elizabeth Mavhunga, Luke Chimuka Candidate contribution: Principal author, involved in idea conceptualization, performed the experiments and wrote the manuscript. Tebogo Kgame was an honours student who assisted with experimentation. Other co-authors helped with analysis of results and manuscript editing. Multivariate optimization of a two-way technique for extraction of pharmaceuticals in surface water using a combination of membrane assisted solvent extraction and a molecularly imprinted polymer Sinegugu Khulu, Somandla Ncube, Yannick Nuapia, Lawrence Mzukisi Madikizela, Hlanganani Tutu, Heidi Richards, Kuria Ndungu, Elizabeth Mavhunga, Luke Chimuka Candidate contribution: Principal author, involved in conceptualizing the idea, performed the experiments and wrote the manuscript. Co-authors helped with analysis of results and manuscript editing. Development and application of a membrane assisted solvent extraction- molecularly imprinted polymer based passive sampler for monitoring of selected pharmaceuticals in surface water Sinegugu Khulu, Somandla Ncube, Yannick Nuapia, Lawrence Mzukisi Madikizela, Elizabeth Mavhunga, Luke Chimuka Candidate contribution: Principal author, involved in idea conceptualization, performed the experiments and writing of the manuscript. Co-authors helped with analysis of results and manuscript editing. iv CONFERENCE OUTPUTS Synthesis and application of a selective molecularly imprinted polymer in solid-phase extraction of emerging pollutants from dam water (ChromSA postgraduate student workshop, September 2019). Multivariate optimization of a two-way technique for extraction of pharmaceuticals in surface water using a combination of membrane assisted solvent extraction and a molecularly imprinted polymer (ChromSA postgraduate student workshop, October 2021) – Awarded 3rd Prize for oral presentation. v OTHER RESEARCH OUTPUTS Lawrence Mzukisi Madikizela, Cornelius Rimayi, Sinegugu Khulu, Somandla Ncube, Luke Chimuka, Pharmaceuticals and personal care products Chapter 10 in the book titled: Emerging Freshwater Pollutants, published in Elsevier, 2022, 171-190, ISBN 9780128228500, https://doi.org/10.1016/B978-0-12-822850-0.00009-0 https://doi.org/10.1016/B978-0-12-822850-0.00009-0 vi ABSTRACT Pharmaceuticals are an important group of persistent emerging pollutants due to being continuously detected in the aquatic environment. Pharmaceuticals are compounds whose therapeutic effects enhance the health of individuals. However, these compounds have the potential to enter the environment as part of effluents from wastewater treatment plants while other sources include improper disposal of expired and unused medication and human excreta. Although these pollutants exist in trace levels (µg L-1 to ng L-1) in environmental samples, they have gained a lot of attention in the science community owing to the perceived detrimental effects on human health and ecotoxicological effects in aquatic life. As such, determination of pharmaceuticals in the environment has become an essential component in environmental monitoring studies. Various analytical techniques have been reported mainly grab sampling followed by solid phase extraction being the most reported. However, the challenge has been that these pharmaceuticals exist in trace levels which requires extremely sensitive approaches. At the same time, they exist as mixtures which requires the need for analytical methods which allows for multi-residue analysis at a time. One of the obvious choices of sampling is grab sampling that involves instantaneous collection of samples. The main challenge of such is that this requires large amounts of samples as well as potential to miss the episodic events of pollution. In this regard, recent studies now advocate for passive sampler-based approaches where a sorbent is deployed in the environment over a period of time. This allows for estimation of time-weighted average (TWA) concentrations which caters for episodic events of pollution. In this regard, the purpose of the current study was to develop an alternative passive sampling technique based on a combination of a molecularly imprinted polymer (MIP) sorbent and membrane assisted solvent extraction (MASE) for the determination of pharmaceuticals belonging to five different classes in surface water. The approach was to synthesize a smart polymer using molecular imprinting technology, place it inside a semi-permeable polypropylene membrane and finally place it in a protective chamber. The chamber and its content, now referred to as the passive sampler was then deployed vii under optimized conditions for monitoring of the five model pharmaceuticals belonging to different groups. The first part of the work involved synthesizing a smart polymer, the MIP for efficient and selective extraction of pharmaceuticals belonging to different groups. This was done by selecting an appropriate template for molecular imprinting process. Cavity tuning experiments which involved the utilization of all the target pharmaceuticals whether as single or multi-template were conducted. The venlafaxine imprinted polymer was successfully selected based on its cross-selectivity for the selected pharmaceuticals. The synthesized polymer attained maximum matrix-matched adsorption capacities ranging from 206 to 418 ng mg-1 for individual pharmaceuticals within 80 min. Batch adsorption and kinetic studies indicated that the binding of the selected pharmaceuticals on the MIP particles resulted in multiple interactions through chemisorption. An analytical method for determination of the target pharmaceuticals was successfully developed using liquid chromatography-mass spectrometry (LC-MS), giving detection limits ranging from 0.03 to 0.31 ng mL-1 and quantification limits ranging from 0.12 - 3.81 ng mL-1 for individual pharmaceuticals. The venlafaxine imprinted polymer was further applied as a selective sorbent for solid phase extraction of an antiretroviral (nevirapine), an antidepressant (venlafaxine), a muscle relaxant (methocarbamol), an anticonvulsant (carbamazepine) and a cardiac stimulant (etilefrine) in dam water samples, yielding recoveries ranging from 43 - 69%. This preliminary data indicated that MIP cross selectivity can be an essential and attractive approach in the monitoring of organic pollutants belonging to different classes in environmental water bodies. This work, presented as Paper 1 in the thesis was published in Polymer Bulletin Journal. The synthesized venlafaxine imprinted smart polymer was then used in combination with a membrane assisted solvent extraction technique, referred to as MASE-MIP for the extraction of these compounds in complex environmental water samples. The MASE-MIP combination utilized the cross-selectivity of the synthesized venlafaxine viii MIP whilst preventing co-extraction of larger molecules to yield cleaner extracts and increased selectivity. After efficient extraction, the sample extracts were analyzed using liquid chromatography-quadrupole time-of-flight mass spectrometry (LC- qTOF/MS). The MASE-MIP was optimized for various significant experimental parameters such as the influence of the sample salt content, the stirring rate, the stirring time and the amount of MIP using a central composite design. Optimum extraction conditions for a sample volume of 18 mL were found to be 5 g of salt content, a stirring rate of 400 rpm, an extraction time of 60 min and 50 mg of MIP, yielding good extraction efficiencies ranging from 38 – 91% for individual pharmaceuticals. The optimized MASE-MIP-LC-qTOF/MS method yielded detection limits in the range of 0.09 to 0.20 ng mL-1 and quantification limits ranging from 0.31 to 0.69 ng mL-1 for individual pharmaceuticals. Furthermore, the optimized extraction method was applied in environmental monitoring of selected pharmaceuticals in two important rivers in South Africa. All selected model compounds were detected in the water samples at concentrations ranging from 0.19 to 2.48 ng mL-1. This illustrated emphasis of a need to continuously monitor the presence of these compounds in environmental waters. The monitoring could be done through the proposed analytical method which has proven to be precise and accurate. This work, presented as Paper 2 in this thesis has been published in Chemosphere journal. The developed MASE-MIP technique was further used in combination with the passive sampling technique to form a MASE-MIP based passive sampler for extraction and monitoring of selected pharmaceuticals in environmental water bodies. This technique utilized the cross-selectivity of the synthesized MIP, the size exclusion and protective membrane and allowed for preconcentration of the target pharmaceuticals into the green receiver solvent (ionic liquid). The passive sampler approach was based on allowing the targeted compounds to diffuse selectively in an integrative manner through the polypropylene membrane which housed an ionic liquid as a green receiving solvent and a MIP. Upon successful diffusion, the analytes were selectively adsorbed by a MIP. The technique was optimized for parameters such as effects of biofouling, ix deployment time and solvent type for the receiver phase. Furthermore, the passive sampler was calibrated in laboratory-based experiments to obtain sampling rates (Rs) for each target pharmaceutical with the view to attain estimated time weighted average (TWA) concentrations of the targeted pharmaceuticals in environmental waters. The optimum matrix-matched sampling rates obtained ranged from 0.0007 - 0.0018 L d-1 for individual pharmaceuticals, whilst the method detection and quantification limits ranged from 2.45 - 3.26 ng L-1 and 8.06 - 10.81 ng L-1, respectively. Upon deployment in a dam situated in a highly populated township in South Africa, only etilefrine and methocarbamol were detected and quantified at maximum TWA concentrations of 12.88 and 72.29 ng L-1, respectively. This work is well presented as Paper 3 in this thesis. Paper 3 is a manuscript under review in Water Research journal. The MASE-MIP based passive sampler showed proven ability to selectively extract targeted pharmaceuticals prior to their determination using LC-qTOF/MS. In this case, the presented experimental procedures allow for detection of trace level environmental concentrations of the targeted pharmaceuticals, making them suitable alternative analytical methods that can be utilized for monitoring of these compounds in environmental water bodies. x DEDICATION This work is dedicated to my dearest daughter Zibusiso: Thank you for your patience, your love and for all the nights you spent without me. All these kept me going during the most challenging parts of this journey. I hope this accomplishment is enough inspiration and proof that you can do anything you put your mind to. To my parents, my pillars of strength: Thank you for believing in me without ceasing. Thank you for all your prayers for they have kept me this far. Ngibonga angiphezi boKhulu KaBayeni! xi ACKNOWLEDGEMENTS Oh, what a journey this has been! So, to God - Our Father, thank you for taking me this far, thank you for being my strength when I was weak. All the glory belongs to you! I would like to express my deepest gratitude to my supervisors Prof. Luke Chimuka and Dr. Somandla Ncube for their dedication, guidance, support and patience throughout my studies. Thank you for always finding time even after office hours to share your academic and life experiences with me. Thank you for grooming and shaping me into the person I have become academically. I would also like to express my sincere gratitude to Prof. Lawrence Madikizela and Dr. Yannick Nuapia for their guidance, support and for sharing their knowledge and skills in the field of Environmental Analytical Chemistry. To Prof. Madikizela, thank you for also proofreading this work. To my co-authors, Prof. Hlanganani Tutu, Dr. Kuria Ndungu, Dr. Heidi Richards and Tebogo Kgame, thank you so much for your contributions. A Special thanks to Prof. Elizabeth Mavhunga for her contribution, mentorship and unwavering support. Thank you for always taking me to writing retreats with your students, it went a long way. I thank the Wits School of Education for their financial support; knowing my tuition fees were covered kept me focused throughout this journey. To the Environmental Analytical Chemistry Technician (Mokgaetjie “Ses Andy” Monyai) and the Chromatography Technicians (Dr. Eric Laka, Thapelo “Thaps” Mbhele and Refilwe Moepya), thank you for always assisting when I get stuck and for providing a healthy research environment. To the Environmental Analytical Chemistry Research Group (special thanks to Kgomotso Maiphetlho and Thapelo Ramalepe), thank you for your support. Lastly, a very big thank you goes to my family (my mother, my father, my daughter, my sister -Nompumelelo Nhleko, my cousin and prayer partner Musawenkosi Mchunu), my friends (Nompumelelo Mncube, Nqobile Mncube, Pinkie Ntola, Faith xii Zondi) and every other person who supported and encouraged me throughout this journey, it really went long a way! xiii TABLE OF CONTENTS DECLARATION ........................................................................................................... i PUBLICATIONS AND MANUSCRIPTS ................................................................... ii CANDIDATE CONTRIBUTION TO ARTICLES ..................................................... iii CONFERENCE OUTPUTS ......................................................................................... iv OTHER RESEARCH OUTPUTS ................................................................................. v ABSTRACT ................................................................................................................. vi DEDICATION .............................................................................................................. x ACKNOWLEDGEMENTS ......................................................................................... xi LIST OF FIGURES .................................................................................................. xviii LIST OF TABLES ..................................................................................................... xix LIST OF ABBREVIATIONS ..................................................................................... xx CHAPTER ONE ........................................................................................................... 1 1. INTRODUCTION ............................................................................................. 2 CHAPTER TWO ........................................................................................................... 6 2 LITERATURE REVIEW................................................................................... 7 2.1 Sources of pharmaceutical compounds in the aquatic environment ......................... 7 2.2 Techniques for extraction of pharmaceuticals .......................................................... 8 2.2.1 MIPs as adsorbents for SPE .............................................................................. 9 2.2.2 Membrane based extraction techniques .......................................................... 10 2.3 Sampling techniques for monitoring pharmaceuticals in water .............................. 12 2.3.1 Grab sampling ................................................................................................. 12 2.3.2 Passive sampling ............................................................................................. 12 xiv 2.4 Occurrence of the selected pharmaceuticals in the environment ............................ 16 REFERENCES ............................................................................................................ 17 CHAPTER 3 ................................................................................................................ 34 3 AIMS AND OBJECTIVES ............................................................................. 35 3.1 Aim of the study ...................................................................................................... 35 3.2 Objectives ............................................................................................................... 35 3.3 Hypothesis............................................................................................................... 35 3.4 Motivation ............................................................................................................... 35 3.5 Overall Approach .................................................................................................... 36 CHAPTER 4 ................................................................................................................ 38 MANUSCRIPTS AND PUBLICATIONS ................................................................. 38 4 PAPER 1 .......................................................................................................... 39 Abstract ....................................................................................................................... 40 4.1 Introduction ............................................................................................................. 42 4.2 Materials and Methods ............................................................................................ 45 4.2.1 Chemicals and reagents ................................................................................... 45 4.2.2 Instrumentation and apparatus ........................................................................ 45 4.2.3 Synthesis of a molecularly imprinted polymer ............................................... 46 4.2.4 Molecularly imprinted polymer selection ....................................................... 47 4.2.5 Batch adsorption studies ................................................................................. 47 4.2.6 Effect of sample pH and elution solvent ......................................................... 50 4.2.7 Application of the MIP in spiked dam water samples .................................... 50 xv 4.3 Results and discussions ........................................................................................... 51 4.3.1 Synthesis and characterization of the best MIP .............................................. 51 4.3.2 Effect of sample pH ........................................................................................ 55 4.3.3 Batch adsorption ............................................................................................. 56 4.3.4 Effect of elution solvent .................................................................................. 60 4.3.5 Applicability of the synthesized polymer in environmental water samples .... 61 4.4 Conclusions ............................................................................................................. 63 Funding ........................................................................................................................ 63 Conflict of interests ..................................................................................................... 63 Availability of data ...................................................................................................... 63 Code Availability ........................................................................................................ 63 REFERENCES ............................................................................................................ 64 4 PAPER 2 .......................................................................................................... 71 4.1 Introduction ............................................................................................................. 74 4.2 Materials and Methods ............................................................................................ 76 4.2.1 Chemicals and reagents ................................................................................... 76 4.2.2 Synthesis of the molecularly imprinted polymer ............................................ 76 4.2.3 Instrumentation and apparatus ........................................................................ 77 4.2.4 MASE preparation .......................................................................................... 78 4.2.5 General MASE-MIP extraction procedure ...................................................... 78 4.2.6 Optimization of extraction parameters ............................................................ 79 xvi 4.2.7 Validation and application of the MASE-MIP technique ............................... 80 4.2.8 Sampling and description of study area .......................................................... 80 4.3 Results and discussions ........................................................................................... 83 4.3.1 Optimization of extraction conditions ............................................................. 83 4.3.2 Method validation and application .................................................................. 90 4.3.3 Application of the developed analytical methods in monitoring of selected pharmaceuticals in surface water ....................................................................................... 93 4.4 Conclusions ............................................................................................................. 97 Funding ........................................................................................................................ 97 REFERENCES .................................................................................................................. 98 4 PAPER 3 ........................................................................................................ 113 4.1 Introduction ........................................................................................................... 116 4.2 Materials and Methods .......................................................................................... 120 4.2.1 Chemicals and reagents ................................................................................. 120 4.2.2 Synthesis of a molecularly imprinted polymer ............................................. 120 4.2.3 Instrumentation and apparatus ...................................................................... 121 4.2.4 Development of a MASE-MIP based passive sampler ................................. 122 4.2.5 Optimization of extraction parameters .......................................................... 124 4.2.6 Study site and field deployment of passive samplers .................................... 126 4.2.7 MASE-MIP extraction of water samples ...................................................... 127 4.3 Results and discussions ......................................................................................... 128 xvii 4.3.1 Optimization of extraction conditions ........................................................... 128 4.3.2 Validation and application of a passive sampling device in environmental monitoring 135 4.4 Conclusions ........................................................................................................... 137 Funding ...................................................................................................................... 138 REFERENCES ................................................................................................................ 138 5 CONCLUSIONS AND RECOMMENDATIONS FOR FUTURE WORK .. 145 Appendix A ............................................................................................................... 148 xviii LIST OF FIGURES Figure 1 Representation of the three accumulation regimes governed by exposure time of a passive sampler…………………….……………………………………………14 xix LIST OF TABLES Table 1 Physicochemical properties and molecular structures of the selected model pharmaceuticals………………………………………………………………….........5 Table 2 Environmental concentrations of the selected pharmaceuticals……………………..……………………………………………….17 xx LIST OF ABBREVIATIONS DES Deep eutectic solvent DVB Divinylbenzene HF-LPE Hollow fiber liquid phase extraction HF-LPME Hollow-fibre liquid-phase microextraction HLB Hydrophilic lipophilic balance IL Ionic liquid LLE Liquid-liquid extraction MASE-MIP membrane assisted solvent extraction-molecularly imprinted polymer MIP Molecularly imprinted polymer PAH polycyclic aromatic hydrocarbon POCIS polar organic chemical integrative sampler SA South Africa SBSE Stir-bar sorptive extraction SPE Solid phase extraction SPME Solid phase microextraction WWTPs Wastewater treatment plants 1 CHAPTER ONE This chapter gives a general introduction to the study and gives information on the selected model pharmaceuticals which includes their physiochemical properties and health effects. 2 1. INTRODUCTION Droughts accompanied with the deteriorating water quality have become the main threats to South Africa’s ability to supply sufficient quality water in order to meet its demands and to ensure environmental sustainability [1]. The largest threat to quality water supply is due to water pollution through various ways. The unavoidable wide usages of pharmaceuticals has seen them being frequently detected in the environmental waters and this has raised a lot of attention amongst the science community and the general public at large [2–4]. Subsequently, pharmaceuticals have been identified as part of the emerging water pollutants owing to their negative health effects in biota (especially fish) as well as increased microbial drug resistance in these species [5–8]. Growth in industries and human settlements have been identified as the contributors in the pollution of water in both rural and urban areas in Africa and across the globe [9]. This is due to inaccessibility of modern ablution facilities particularly in most African regions as well as improper disposal of unused or expired medications which in turn contribute to the direct contamination of surface water bodies [10]. Although this is the case, pharmaceuticals are primarily introduced into the environment through treated wastewater treatment plants (WWTPs) effluents [11–14]. WWTPs receive very large quantities of pharmaceuticals via human excreta due to their high excretion rates from the human body [10]. Most pharmaceuticals are highly polar and soluble in water which result in their limited elimination during the wastewater treatments processes and then enter the environment [15–17]. In WWTPs, some pharmaceuticals undergo incomplete degradation whilst some of the degradation products can be reversed to their active biological forms through de-conjugation [4, 18]. Due to this, it becomes important to monitor the amount of pharmaceuticals that are discharged into the aquatic environment. 3 The selected pharmaceuticals for the study belong to five different therapeutic groups and as a result, they possess different therapeutic and physicochemical properties. The pharmaceuticals were, an antiretroviral (nevirapine), an antidepressant (venlafaxine), a muscle relaxant (methocarbamol), an anticonvulsant (carbamazepine) and a cardiac stimulant (etilefrine). Venlafaxine is a widely used antidepressant whilst methocarbamol is used as a muscle relaxant drug for the treatment of symptoms of musculoskeletal disorders such as painful spasm and dislocations [19, 20]. Nevirapine is commonly used to prevent mother to child transmission of HIV and treating the HIV-1 infection, whilst carbamazepine and etilefrine are widely used as a basic treatment of epilepsy and hypertension, respectively [21, 22]. Physicochemical properties and molecular structures of the studied compounds are summarized in Table 1. As shown in Table 1, these compounds have n-octanol-water partition coefficient logarithms (logKow) ranging from 0.1 – 2.9, dissociation constants (pKa) ranging from 9.1 – 15.96 and high water solubilities which increases their chances of escaping the WWTP process and in turn end up in the environment. Amongst the investigated pharmaceuticals, venlafaxine and methocarbamol have been linked with neurobehavioral disorders in aquatic animals with reports of neuroendocrine disruption resulting to reduced aggressive nest defense and distracted feeding patterns in exposed fish [20, 23]. In a study conducted in Spain, venlafaxine was found to encourage acute deadly phytotoxicity of polystichum setiferum plant [24]. A different target pharmaceutical, carbamazepine has shown severe negative impact on benthic organisms [22]. For example, in a study conducted by Oetken et al, the results obtained revealed that this compound poses a potential threat on the survival of Chironomus riparius and other related aquatic insects [25] [21]. The need for sensitive monitoring techniques for monitoring of pharmaceuticals in aqueous medium has led to calls for better methods that can detect them in trace levels where they also exist as mixtures in complex samples. Traditionally, solid phase 4 extraction of samples that were collected via grab sampling has been the approach of choice. However, this approach is limited for applications that require detection of analytes that exist in parts per trillion, hence the need for advancement of the existing techniques. In this work, a passive sampler based on membrane assisted solvent extraction and a molecularly imprinted polymer combination was developed and applied in the monitoring of the selected model pharmaceuticals. The passive sampler was deployed in the Orlando Dam, a dam situated in the heart of an overly populated Soweto township in South Africa. The idea was to develop a method which can analyze more than one class of pharmaceuticals at a time and determine their environmental TWA concentrations. This was accomplished by synthesizing a MIP with an affinity for all target compounds (Paper 1). The cross-selectivity of the MIP was then used in combination with the semi-permeable polypropylene membrane bag for selective extraction of these compounds in environmental water bodies with high sample matrix (Paper 2). The selective MASE-MIP combination was then used in the development of a passive sampler for monitoring of the selected pharmaceuticals over a period of 14 days. The passive sampler was aimed at pre-concentrating the trace environmental concentrations to quantifiable levels, as well as to understand the extent of environmental pollution by estimation of TWA concentrations for individual compounds (Paper 3). It is the first time that the MASE-MIP combination is applied as a passive sampler in the monitoring of pharmaceutical in the environments. Furthermore, it was the first time that MASE-MIP combination utilized a greener solvent (ionic liquid) in the receiver phase. 5 Table 1 Physicochemical properties and molecular structures of the selected model pharmaceuticals Compound Structure Therapeutic Class Molecular weight (g.mol-1) LogKow pKa Water Solubility (mg L-1) Acid Base Carbamazepine Anticonvulsant 236.27 2.45 15.96 -3.8 1.05 x 102 Etilefrine Cardiac stimulant 181.23 0.1 9.1 9.73 17.7 Methocarbamol Muscle relaxant 241.24 0.6 13.6 -3.4 267 Nevirapine Antiretroviral 266.3 2.5 10.37 5.06 1.38 x 104 Venlafaxine Antidepressant 277.4 2,9 14.42 8.91 7.2 x 103 6 CHAPTER TWO This chapter discusses the sources of pharmaceuticals in the environment, the different extraction and sampling techniques for the analysis of the selected pharmaceuticals in the environment, with more emphasis on MIPs as SPE sorbents and their combination with the MASE technique. Occurrence of the selected pharmaceuticals in the environment is also reviewed. 7 2 LITERATURE REVIEW 2.1 Sources of pharmaceutical compounds in the aquatic environment Detection of pharmaceuticals in African environmental water bodies and globally is due to various sources, ranging from effluents (WWTP, nursing homes or hospitals, pharmaceutical factories) to improper disposals of medication [4, 11, 16, 26, 27], with the main source of their detection being WWTP [14, 28]. A study conducted in South Africa revealed that WWTP receives variable amounts of pharmaceutical at different times of the day [29]. This could be attributed to the frequency at which the compound is used and the rate at which it is excreted from the body. A number of studies have been conducted globally to monitor the ability of numerous selected WWTPs for the removal of the selected model pharmaceuticals during the water treatment process [5, 21, 30]. For example, in Kenya, a study aimed at studying the ability of WWTP to remove pharmaceuticals revealed that removal efficacies of nevirapine ranged from 11 to 49% [23]. In a study conducted in South Africa, nevirapine was reported to be resistant to degradation [21]. On a separate South African study, nevirapine also showed removal efficacy of 22.5% during the WWTP process [31], whilst a study in Greece showed negative removal efficacies of carbamazepine in WWTP [30]. All these studies report inefficiencies of various WWTPs to remove these pharmaceuticals in water, which introduces these compounds into surface water bodies. It was also discovered that although WWTPs are said to be the main source of pharmaceuticals in the environment, medical waste effluents were also found to have a great potential [11]. For example, in most African countries, there is scarcity of proper waste management such as incinerators for medical and pharmaceutical factories [32, 33] which results in their waste being released into the environment untreated. This was observed in studies conducted in Nigeria and Zimbabwe where there was no pre- treatment of medical waste performed, thereby resulting in these wastes being disposed together with municipality wastes into the environment [32, 33]. 8 2.2 Techniques for extraction of pharmaceuticals Nowadays, high end developed and selective analytical instrumentation such as chromatographic techniques coupled with mass spectrometry are available for determination of pharmaceuticals [34, 35]. The identification of the pharmaceutical compounds through the m/z ions provides selectivity as it detects and identify any co- eluting compounds as separate responses. However, samples cannot be directly injected into the system without any pre-treatment as that could negatively affect the accuracy and precision of results [35] and also damage different components of the techniques e.g. the column. In this regard, sample treatment and clean-up steps are considered as crucial steps prior to chromatographic determination of pharmaceuticals in complex samples such as environmental water bodies [34, 36]. The primary objective of sample treatment is the reduction of matrix effects and preconcentration of analytes (trace enrichment) by separating the analytes from the main sample matrix [35, 37]. There is a variety of existing pre-concentration and extraction techniques applied in the analysis of pharmaceuticals. These include liquid-liquid extraction (LLE), solid phase extraction (SPE), hollow fiber liquid phase extraction (HFLPE), stir-bar sorptive extraction (SBSE), solid-phase microextraction (SPME) etc. [38–41]. LLE as a traditional sample preparation technique has several drawbacks which include consumption of large quantities of organic solvents, not easy to automate as well as being time consuming [34]. The other mentioned alternative sample preparation techniques have an advantage of utilizing less amounts organic solvents compared to LLE. However, they lack selectivity when it comes to analysis of pharmaceuticals in environmental water samples since these coexist with other organic compounds in environmental water bodies. Although there is a number of SPE sorbents available commercially for environmental applications, such as reversed- phase C18, Oasis hydrophilic lipophilic balance (HLB), anionic-exchange (Oasis MAX), carbon nanotubes and styrene divinylbenzene (DVB) [42]; some of them still lack selectivity towards targeted compounds and are designed for single use [39]. Moreover, for trace analysis, sample preparation may be prolonged 9 especially with large sample volumes used in trying to preconcentrate the sample into quantifiable levels [43]. In some cases, even after carefully optimizing the SPE extraction method, the analyte signals still get suppressed due to the presence of matrix effects (co-elution of matrix components with analytes) [44]. The SPE technique was further improved by automating the technique and further coupling it to the chromatographic determination system. This did away with the technique being laborious and resulted in improved detection limits. However, this possesses a high potential of analytes passing through the column (mostly polar compounds) where used preconcentration volumes are high [43]. This then raised a need for more selective sorbents for SPE. Over the recent years, researchers have been working hard to develop suitable and selective sorbents for SPE applications. As such, there has been high interest in the application of molecularly imprinted polymers (MIPs) as smart adsorbents for SPE in order to increase selectivity by providing specific interactions between the polymer and the target compound [37, 45, 46]. 2.2.1 MIPs as adsorbents for SPE MIPs are artificial materials which have synthetically generated recognition sites that can specifically bind with target compounds or closely related compounds [44, 47, 48]. These materials are synthesized through an interaction of functional monomers, the target molecule and a cross-linking agent leading to a three-dimensional network polymer [44, 49–51]. Monomers are chosen based on their ability to interact with the functional groups present in the template molecule [44]. Once polymerization has been established, the template molecules are removed with an aid of relevant solvents to create binding sites complementary to target analytes through shape, size and functional groups [44, 47]. MIPs have added advantages of being robust, stable and resistant over a wide range of temperature, pH and solvents (thermal and chemical stability), easy to prepare and can be stored over a long period of time without losing its binding affinity towards targeted compounds [47, 48]. The gained interest into MIPs as adsorbents for SPE is due to their high selectivity towards the targeted compounds [52–54] and their reusability [55, 56]. Other advantages of MIPs over traditional SPE 10 sorbents are that they are not affected by changes in the matrix composition resulting into better and higher recoveries and improved detection limits [57]. MIPs were mostly introduced to target hydrophilic compounds. There has been advances in the application of MIPs as SPE adsorbents where compounds belonging to different groups are analysed through the phenomenon referred to as cross-selectivity [57, 58]. A few studies have utilized this phenomenon for example, quite recently, Wang et al. analyzed 20 compounds consisting of sulfonamides, tetracyclines and fluoroquinolones using a single MIP [59], whilst Herrero-Hernández et al. used a single MIP for analysis of two classes (3 phenoxy acid herbicides and 4 phenols) [58]. The advantage of this is that multiple analytes can be analyzed by a single MIP whilst retaining good selectivity towards targeted compounds. In this work also, in Paper 1 this phenomenon was observed where pharmaceutical compounds belonging to five different classes were extracted through a single MIP. 2.2.2 Membrane based extraction techniques There has been a growing interest in the utilization of membranes for microextraction of pollutants from aqueous environmental samples [60, 61]. These techniques are based on equilibrium partitioning of analytes from the aqueous sample phase into the receiver phase containing the extraction solvent where the analytes are preferentially enriched [62]. Liquid-phase microextraction (LPME) was first introduced as a technique which created a barrier between the aqueous sample and the receiver solvent to avoid the mixing of the two phases [63, 64]. This technique showed high selectivity and high reduction of matrix effects [60, 65]. This is a three-phase system where the analyte is first extracted into an organic solvent that impregnates the walls of the membrane, and then back extracted into an aqueous acceptor solution adjusted to the adequate pH [65, 66]. To minimize the extraction steps, Quintana et al, utilized hollow-fibre liquid-phase microextraction (HF-LPME) for enrichment and clean up in a single step in the analysis of pharmaceuticals in wastewater samples [67]. HF-LPME used very small volumes of about 10 µL which was very difficult to manipulate. For that reason, a technique based on a microporous polypropylene bag was introduced and combined with a number of 11 applications with an aid of gas chromatography determination [63, 64, 66, 68]. The membrane bag can house up to 1 mL of organic solvent where analytes are extracted through the membrane bag which also acts as protective barrier which limits coextraction of matrix in the sample [35, 62]. Recent studies have shown that using a microporous membrane to prevent interferences from accessing the MIP is an attractive approach to dealing with matrix effect [35]. This approach is known as membrane assisted solvent extraction-molecularly imprinted polymer (MASE-MIP). The approach has been reported to reduce matrix effects by preventing high molecular weight interfering compounds and particulate matter from clogging the MIP cavities, yield cleaner extracts and provide increased selectivity [34, 35, 37, 69]. Typically, its set-up consists of a polypropylene membrane bag (4 cm long, 6 mm internal diameter, 0.22 mm pore size and 160 mm thickness), filled up to 1 mL organic solvent and up to 100 mg MIP particles in the receiver phase. Target compounds diffuse through the membrane bag into a receiver phase containing the organic solvent and the MIP. There is a number of parameters which influences the movement of analytes from the aqueous donor phase into the organic receiver phase such as sample salt content, stirring rate, temperature, contact time, mass of the MIP etc. [60, 63, 68]. To attain acceptable analyte recoveries and improved selectivity, the extraction method needs to be properly optimized. This technique works quite well in samples where matrix components are high because in such instances, MIPs tend to lose their recognition abilities after a few extractions [35]. This is especially for MIPs where interaction of the target template and the monomer is through hydrogen bonding; these interactions tend to be weak and are prone to interferences [34]. The technique has been successfully applied in the extraction of polycyclic aromatic hydrocarbons (PAHs) in wastewater [69] and in the extraction of triazines from aqueous extracts of cow peas and maize baby corn [70], yielding high enrichment factors, high selectivity and low limits of detection and quantification. 12 Paper 2 of this work studies the extraction of the five selected model pharmaceuticals from environmental water samples. It was observed that this phenomenon resulted in improved analyte recoveries, improved limits of detection and quantification as a result of improved selectivity and reduced matrix effects. 2.3 Sampling techniques for monitoring pharmaceuticals in water 2.3.1 Grab sampling Normally, pharmaceuticals are present in very low concentrations in aquatic environment (µg L-1 – ng L-1 or sometimes pg L-1 ) [27, 71, 72]. Most of the monitoring programmes utilize grab sampling of specific amounts of water at a specific time. However, this sampling method has drawbacks in monitoring the presence of pollutants as it only provides pollutant concentrations at the time of sampling, which might not be a true reflection of the extent of pollution since pollutant concentrations vary over time [73, 74]. For example, wastewater effluent plants receive varying amounts of pollutants in a day [29], which subsequently end up in the environment at variable concentrations. This then result in episodic events of pollution being missed [74]. Automatic sampling systems which can frequently sample a number of water samples at specific times can be a solution to this, however, this is costly and mostly impractical as it would be laborious [73]. In addition, pre-concentrating environmental pollutants to quantifiable concentration levels can be expensive and tedious as large sample volumes are required which is accompanied by several sample preparation steps [75– 77]. Furthermore, grab sampling does not offer data on the bioavailable fraction of the pollutants [78]. 2.3.2 Passive sampling Passive sampling technique is based on unprompted flow of analytes from the sample phase to the receiver phase of the passive sampler as a result of different analyte concentration between the two phases [73, 79]. This process continues until equilibrium is reached. Passive samplers offer enhanced sensitivity to analytes through pre-concentration over a longer period of time and, reduce the use of organic solvents 13 in sample preparation [79]. In some cases, passive samplers also offer increased selectivity towards target compounds depending on the trapping material used in the receiver phase [73]. The acceptor phase can be a porous adsorbent, a solvent or a chemical reagent [80]. Passive sampling devices allow for time integrated or time weighted average (TWA) concentrations which are collected in situ without having to alter the bulk of sampling medium [81]. This sampling method considers variations in pollutant concentrations and in turn increases the capabilities of the device to detecting and quantifying contaminants at trace levels, which is very difficult with grab sampling [71, 78, 82, 83]. Passive samplers offer more practical approaches to sampling, like ease of operation, less labouring, being very cost effective nonmechanical devices which does not need for energy sources in their operation [73, 76, 82]. An ideal sampler would normally consist of an acceptor phase and a membrane which separates the acceptor phase and the aqueous sample. The acceptor phase is selected based on its ability to accept or extract target compounds from the water body [84]. During the deployment period, the accumulation of analytes onto the sampler sorbent can be described in three regimes; the first one is the linear regime, the second is the curvilinear regime and the third one is the equilibrium regime [85] as presented in Fig. 1. In the time-integrative, the uptake of analytes of the receiver phase is said to be linear meaning that the concentration in the receiver will increase, whilst the concentration of analytes in the sample medium phase decrease. The second one refers to curvilinear, whereas in the third phase the movement of analyte is stationary. This is the phase at which the extraction between the receiver and sample phases is at equilibrium. In this regard, kinetic passive samplers therefore have an advantage of adsorbing contaminants from episodic events where concentrations of the pollutants are variable [73, 79, 81]. There are different types of passive samplers depending on the kind of analytes. There are passive samplers suited for organic compounds such as the polar organic chemical integrative sampler (POCIS), Chemcatcher, etc.) and those suitable for metals such as the one based on diffusive gradients in thin-films technique (DGT) and those based on polymer inclusion membrane (PIM). 14 Figure 1. Representation of the three accumulation regimes governed by exposure time of a passive sampler [85] 2.3.2.1 Chemcatcher The Chemcatcher passive sampler uses a diffusion-limiting membrane and a solid- phase receiving phase. The receiver phase is be placed and sealed in an inert plastic housing. The accumulation and selectivity rates are controlled by the choice of the diffusion-limiting membrane and that of the solid-phase receiving phase [79]. There is a number of different designs available depending on the combination of the sorbent and the diffusion-limiting membrane. One example of a used design is for non-polar organic compounds with logKow values higher than 4. This sampler uses the commercially available C18 empore disk as a receiving phase and low-density polyethylene diffusion-limiting membrane [73]. In one study, Vrana et al. calibrated the Chemcatcher sampler for measurements of time-weighted average concentrations of hydrophobic micropollutants, such as polyaromatic hydrocarbons and organochlorine pesticides in water [86]. 15 2.3.2.2 Polar organic chemical integrative sampler The polar organic chemical integrative sampler (POCIS) is used for monitoring of hydrophilic contaminants, like pesticides, pharmaceutical drugs, etc. At the moment, POCIS is the extensively used and commercially available passive sampler in environmental monitoring of pharmaceuticals [85, 87]. It consists of a sorptive receiver phase normally covered by microporous polyethersulphone membrane and the type of the receiving phase material can be changed to suit the type of targeted analytes [73]. In recent times, it has been observed that the POCIS is very effective in the qualitative analysis of pharmaceuticals but has drawbacks when it comes to quantitative analysis of these compounds. For these reasons, the POCIS has been considered a semi- quantitative tool for monitoring of pharmaceuticals [76, 88, 89]. The drawbacks are caused by its vulnerability to physicochemical parameters like the sample conductivity, dissolved organic matter, pH, biofouling, water flow and temperature [90–92]. For these reasons, the accumulation of pharmaceuticals onto the sorbent material is not clearly understood. Subsequently, the accumulation rates, widely known as sampling rates (Rs) which are obtained from calibrating the sampler in the laboratory may not be applicable for field application with the view to estimate the bioavailable concentration in the field water bodies [93]. There is currently few studies that have resorted to in situ calibrations of the POCIS, with the view to minimize the doubts in the estimation of the accumulated concentration [83, 84, 94]. This is an indication that more research is needed in the search for alternative passive samplers for monitoring of the bioavailable concentrations of pharmaceuticals in environmental waters. It is for these reasons that in the present work, a novel passive sampler based on a combination of membrane assisted solvent extraction and molecularly imprinted polymer (MASE-MIP) was designed and optimized for monitoring of the selected model compounds in aquatic environments. The sampler also investigated the possibilities of using green and stable solvents such as the deep eutectic solvents (DESs) and liquids (ILs) in the receiver phase. In recent years, DESs which share few properties with ILs have gained a lot of interests in the various fields of analytical 16 chemistry e.g. sample preparation, micro extraction, mass spectrometry etc. [95, 96]. Paper 3 of this work explains the optimization in detail. This brings the novelty into this approach since DESs and ILs have not been applied as a receiver in the MASE- MIP, even more as a receiver phase component in passive sampling. DESs have mostly been applied in extraction of pharmaceuticals using LLE based extraction techniques, where the extraction solvent would be in contact with the sample medium [97]. The tested IL (aliquat 336) has been applied in a variety of applications in the analysis of pharmaceuticals. This includes its use as functional monomer in the synthesis of a MIP for selective extraction of abacavir from polluted water [98]. Another application was in the synthesis of polymer inclusion membranes for extraction of antibiotics from water samples [99, 100]. In another study, aliquat 336 was used as a carrier for a technique based on emulsion liquid membrane in the extraction of acetaminophen in aqueous solutions [101]. Quite recently, it was utilized in the modification of waste- tyre derived activated carbon for the extraction of non-steroidal drugs in wastewater [102] 2.4 Occurrence of the selected pharmaceuticals in the environment Occurrence of pharmaceuticals in the environment is still on going as researchers are trying to understand their toxicity and fate. Table 2 summarizes some of the work that has been done in the monitoring of the selected pharmaceuticals around the globe. The data presented on Table 2 projects nevirapine as being the predominant one. Carbamazepine was detected at range of 0.10 – 1.65 ng mL-1 in the Umngeni River, South Africa and in the Nairobi Basin, Kenya, whilst it was quantified at 0.42 ng mL-1 in ground water in California, United States [23, 103, 104]. In a South African study, methocarbamol was detected at a maximum concentration of 0.096 ng mL-1 [105], whilst in a study in the US, its accumulation on the exposed fish was 0.023 ng mL-1 [104]. Amongst all the selected pharmaceuticals, etilefrine is the least monitored globally. In a study by Rimayi and co-workers, etilefrine was not detected in all the sampling site [105], whilst in another one also by Rimayi, it was detected in some sites [106]. However, concentrations are not available as that was a screening study. Overall, 17 it can be noted that these compounds are present in the environment, and this raises a need for their frequent monitoring. Findings on their environmental monitoring for this work are well presented in Paper 2 and Paper 3. Table 2 Environmental concentrations of the selected pharmaceuticals Pharmaceutical compound Matrix Concentration (ng mL-1) References Carbamazepine South Africa Kenya United State Surface water Surface water groundwater 0.19 – 1.65 0.10 0.42 [107] [23] [104] Etilefrine South Africa Surface water d [106] methocarbamol South Africa United State Surface water Exposed fish Up to 0.096 0.023 [105] [20] Nevirapine South Africa South Africa Kenya Surface water Surface water Surface water 0.11 – 0.23 0.13 – 1.48 4.86 [108] [109] [110] venlafaxine Soth Africa Poland Surface water Surface water Surface water Up to 0.026 Up to 0.026 Up to 0.250 [105] [111] [112] d- detected REFERENCES 1. Olaniran AO, Naicker K (2014) Assessment of physico-chemical qualities and heavy metal concentrations of Umgeni and Umdloti Rivers in Durban , South Africa. Environ Monit Assess 186:2629–2639. https://doi.org/10.1007/s10661- 013-3566-8 2. 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A brief summary of the overall approach is also presented. 35 3 AIMS AND OBJECTIVES 3.1 Aim of the study The main aim of the study was to develop a MASE-MIP-based passive sampler for determination of a variety of pharmaceuticals from environmental surface water samples. 3.2 Objectives • To synthesize and characterize a MIP that can selectively bind to etilefrine, methocarbamol, venlafaxine, carbamazepine and nevirapine. • To develop and optimize a MASE-MIP technique for effective extraction of targeted pharmaceuticals from environmental water samples • To design and optimize MASE-MIP based passive sampler for the determination of target analytes • To apply the developed technique in the analysis of target pharmaceuticals 3.3 Hypothesis The developed MASE-MIP-based passive will offer an effective and practical approach for monitoring and measuring of the bioavailable concentrations of various pharmaceuticals in surface water. 3.4 Motivation Pharmaceuticals are key contributors to the pollution of surface water pollution in aquatic environments which includes rivers, dams and lakes to name a few. South Africa is still slacking behind in understanding the extent of water pollution and the assessments of risks associated with pharmaceuticals in its aquatic environments. For this to be achieved, reliable analytical methods for monitoring of pharmaceuticals should be developed and verified. Most of the available methods and techniques for risk assessment of pharmaceuticals in the aquatic environment involve determining the total concentration. Novel techniques that measure the bioavailable fraction are still limited. Passive samplers are an ideal approach for sensitive monitoring and measuring of the bioavailable concentration of pharmaceuticals in environmental surface waters. 36 As such, in this work a novel MASE-MIP-based passive sampler was successfully designed and investigated as part of the search for effective and practical analytical methods for analysis of the bioavailable fraction of pharmaceuticals in the environment. This encompassed a technical aspect of the designing of the passive sampler followed by laboratory optimization and lastly the application of the sampler in surface waters. 3.5 Overall Approach The first step in this work was to synthesize a MIP for selective and efficient extraction of model pharmaceuticals belonging to five different therapeutic classes. Extensive MIP cavity tuning experiments were performed for the selection of a polymer with an affinity for all the five model pharmaceuticals. The selected MIP was characterized for stability, parameters related to polymer pores and the functional groups present on the polymer surface. Batch adsorption studies were also conducted with the view to understand the binding of compounds on the MIP cavities. The polymer was further applied to environmental samples using spiked dam water samples. In this case the performance of MIP was investigated. This first part of the research was then presented in Paper 1 which is already published in Polymer Bulletin Journal. The selected polymer was then used in the development of a two-way technique based on MASE-MIP. A central composite design was applied to optimize several parameters for this technique, and its performance reported as percentage recovery, limit of detection and quantification and relative standard deviation values. The optimized MASE-MIP technique was then applied in the extraction of the model compounds from surface water samples of two of the major South African Rivers. This second part of the research yielded Paper 2 which is already published in the Chemosphere journal. In the third part of the work a passive sampling device was developed and combined with the MASE-MIP technique to form a MASE-MIP based passive sampler. This sampler was then optimized for extraction of the target pharmaceuticals in environmental water bodies. This was followed by deployment of these samplers in Orlando Dam, a dam situated in the heart of Soweto township (South Africa), for 37 environmental monitoring of the selected pharmaceutical compounds. This part of the work produced Paper 3 which is a manuscript under review in Water Research Journal. 38 CHAPTER 4 MANUSCRIPTS AND PUBLICATIONS This chapter presents two published articles and one manuscript for consideration in the examination of this thesis. The design and reference formatting of the articles and the manuscript were formatted to suit the requirements of this thesis. On all the papers, tables and figures have been placed next to where they appear in the text in order to meet the requirements for the submission of this thesis. Also, the page numbers for published articles and the manuscript have been formatted to cater for submission requirements of this dissertation. Highlights submitted to each journal are not included in this dissertation. Supplementary document for paper 2 is included in this dissertation. Paper 1 and 2 have been published while Paper 3 is a manuscript under review. 39 4 PAPER 1 This Paper is entitled “Synthesis, characterization and application of a molecularly imprinted polymer as an adsorbent for solid phase extraction of selected pharmaceuticals from water samples” has already been published in Polymer Bulletin. This article presents extensive MIP-cavity tuning experiments and batch adsorption studies which led to the selection of a venlafaxine imprinted polymer with high affinity for all five model pharmaceuticals belonging to different classes. Polym. Bull. (2022), (IF = 2.870) https://doi.org/10.1007/s00289-021-03553-9 Sinegugu Khulu – Principal Author Somandla Ncube – Co-Supervisor Tebogo Kgame – BSc honours student Elizabeth Mavhunga – Co-author Luke Chimuka – Main Supervisor https://doi.org/10.1007/s00289-021-03553-9 40 Synthesis, characterization and application of a molecularly imprinted polymer as an adsorbent for solid phase extraction of selected pharmaceuticals from water samples Sinegugu Khulu a,b, Somandla Ncube c, Tebogo Kgame a, Elizabeth Mavhunga b, Luke Chimuka a* a Molecular Sciences Institute, University of Witwatersrand, Private Bag X3, Johannesburg, 2050, South Africa b School of Education, University of Witwatersrand, Private Bag X3, Johannesburg, 2050, South Africa c Department of Chemistry, University of South Africa, Private X6, Florida, 1710, South Africa * Luke.Chimuka@wits.ac.za Abstract Most pollutant compounds exist as mixtures in the environment. In this regard, one of the 12 principles of green analytical chemistry emphasize the need for methods that allow for analysis of multiple compounds versus those that analyze a single analyte at a time. In this work, we present a molecularly imprinted polymer (MIP) synthesized for the selective and efficient extraction of selected pharmaceuticals belonging to five different classes namely: an antiretroviral (nevirapine), an antidepressant (venlafaxine), a muscle relaxant (methocarbamol), an anticonvulsant (carbamazepine) and a cardiac stimulant (etilefrine) from surface water samples. Cavity tuning experiments using the target pharmaceuticals as a single or multi-template were conducted and the venlafaxine imprinted polymer was successfully selected for the study based on its high selectivity towards targeted pharmaceuticals. Batch adsorption and kinetic studies showed that adsorption of the selected pharmaceuticals onto the particles of the polymer followed a Freundlich adsorption isotherm as well as a pseudo second order adsorption model. This indicated heterogeneity of the binding surface 41 energies on the MIP resulting in multiple interactions through chemisorption. An analytical method for quantification of the compounds using liquid chromatography- mass spectrometry (LC-MS) was successfully developed, with detection limits ranging from 0.03 to 0.31 ng mL-1 and quantification limits in the 0.12 - 3.81 ng mL-1 range. The imprinted polymer was then evaluated as a selective adsorption sorbent for solid phase extraction (SPE) of the selected pharmaceuticals in dam water samples followed by LC-MS analysis, giving recoveries ranging from 43 - 69%. Keywords: Molecularly imprinted polymer; pharmaceuticals; nevirapine; carbamazepine; venlafaxine 42 4.1 Introduction In the modern society, the detection of pharmaceutical drugs and their residues in surface waters has raised concern due to their potential negative impact on the human health if consumed unknowingly from contaminated food and water sources [1, 2]. The widespread usage of pharmaceuticals has seen them primarily entering ground water and surface water through wastewater treatment plant (WWTP) effluents [3–6]. WWTPs receive high quantities of pharmaceuticals via human urinary and faecal excretion, and improper disposal of expired medication from nursing homes, hospitals and domestic effluents as well as untreated effluents from the pharmaceutical industry [5, 7–9]. Literature suggests that the presence of pharmaceuticals in the environmental water is due to them surviving wastewater treatment processes [10]. Pharmaceutical drugs are water soluble and have a high polarity which allows them to escape wastewater treatment processes easily [6], others undergo incomplete degradation while some degradation products could even be returned to their biologically active forms through de-conjugation [3, 10, 11]. Although pharmaceuticals are present in the aquatic environment in relatively small concentrations (µg - ng L-1) [1, 3, 12], their residues can cause ecotoxic effects, hormonal disruption, and drug resistance [13]. Sample preparation prior to chromatographic determination and clean-up steps are of great importance in the analysis of pharmaceuticals from environmental water bodies [14, 15]. There is a number of existing extraction and pre-concentration techniques used in sample preparation including solid-phase extraction, hollow fibre liquid-phase extraction, stir-bar sorptive extraction etc. [16–20]. However, these lack selectivity as pharmaceuticals normally co-exist with other organic compounds in environmental water systems. Over the years there has been a great interest in the application of molecularly imprinted polymers (MIPs) as sorbents for solid phase extraction technique to increase analyte selectivity in the presence of very complex samples [2, 21–23]. The interest in MIPs as adsorbents is due to their great selectivity towards target compounds based on shape, size and functional groups[24–27] and the fact that they can be re-used [5, 28–31]. 43 Most studies in environmental monitoring have mainly focused on synthesizing MIPs that target a single pharmaceutical or a group of pharmaceuticals belonging to the same class. In the environment pharmaceuticals do not exist in isolation but as mixtures of different classes some of which have similar physico-chemical properties. Principle no. 8 of the 12 principles of green analytical chemistry emphasizes the need for methods that allow for analysis of multiple compounds versus those that analyze a single analyte at a time [32]. MIPs have been shown to bear synthetic recognition sites which can specifically bind to a target molecule as well as other closely related molecules [33, 34]. Ncube et al., 2019 observes that this phenomenon, referred to as cross selectivity, can be utilized when there is need to analyze multiple analytes belonging to different classes in complex samples [23]. Studies have been mentioned where a single MIP has been applied in analysis of different classes of environmental pollutants. For example, Herrero-Hernández et al. used a single MIP for analysis of two classes (3 phenoxyacid herbicides and 4 phenols) [35], while recently Wang et al. analyzed 20 compounds consisting of sulphonamides, tetracyclines, and fluoroquinolones using a single MIP [36]. As such, the objective of this present work was to develop a single MIP that can be used to target five different classes of pharmaceuticals from environmental water sources. In this regard, various cavity tuning experiments were done to identify the imprinting template that could produce cavities on a single MIP with a high affinity for all targeted classes of pharmaceuticals. The model compounds included an antiretroviral (nevirapine), an antidepressant (venlafaxine), a muscle relaxant (methocarbamol), an anticonvulsant (carbamazepine), and a cardiac stimulant (etilefrine). Table 1 shows the physicochemical properties and molecular structures of the model pharmaceuticals. The synthesized MIP was applied as an adsorbent for solid phase extraction of the target compounds in dam water samples. 44 Table 1 Physicochemical prope