The determination of drugs in waste-water and soil samples by liquid chromatography and mass spectrometry.

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Final Report

Version I

The determination of drugs in waste-water and soil samples by

liquid chromatography and mass spectrometry.

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Final Report

The determination of drugs in waste-water and soil samples by liquid chromatography and mass spectrometry.

Version 1 Group:

Avans University of Applied Sciences. Forensic Laboratory Investigation.

Academie voor Technologie, Gezondheid en Milieu. Forensisch Laboratorium Onderzoek.

Author:

M.H.F. Graumans mhf.graumans@student.avans.nl

Supervisors:

Dr. L. Barron leon.barron@kcl.ac.uk

Drs. A.L.B.M. Biemans albm.biemans@avans.nl

Period:

From: 01-02-2012 until: 30-06-2012. Location:

Department of Forensic Science and Drug Monitoring, Analytical & Environmental Science Division, School of Biomedical Sciences ,King’s College London.

Franklin Wilkins building, Stamford Street 150, London, United Kingdom.

Forensisch Laboratorium Onderzoek, Avans Hogeschool, Lovensdijksstraat 61-63, Breda, The Netherlands.

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Acknowledgements

This final report is written during a graduating internship period at the Department of Biomedical Sciences, Forensic Science & Drug Monitoring at King’s College London. The following report will contain a literature study and laboratory results who are gained during the interpretation and examination of complex matrices. The work for this project is done by a student of the study Forensic Laboratory Science of Avans University Breda, the Netherlands. To write this final report I owe my gratitude to: Leon Barron for the great help, inspiration and providing information that is needed during the literature and laboratory investigation. I also want to thank the Mass Spectrometry Facility of King’s College for the additional information and use of their instruments. Thanks to King’s college in general for the great hospitality, possibility for a graduating project, instrument purchase and the great lab facilities. I also want to thank John Cassella for the possibility to take soil samples at the Staffordshire University and Ad Biemans for guidance from my home institution.

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Abstract

In the field of forensic science, the investigation of complex matrices for the determination of drug analytes by the multidiscipline environmental forensics involves a lot of different approaches such as; analytical chemistry, geochemistry, (eco) toxicology, statistics, chemical engineering and atmospheric chemistry. As with pharmaceuticals, also illicit drugs and metabolites of these compounds are eliminated through waste water [1]. Unfortunately these products are a rising problem as contaminants for the environment. The Department of Forensic Science & Drug Monitoring wants to optimise a research method that is possible for the identifying of drugs in complex matrices[2].

Water samples are collected in the London Thames in the vicinity of Europe’s biggest water treating plant nearby Beckton. The water samples are examined with the analytical MS/MS method to determine possible drugs. In order to prepare the water samples for LC-ESI-MS/MS determination, a method is optimised with the analytical methods such as liquid chromatography (LC)-UV detection and solid phase extraction. Preliminary results have shown, that with (LC)-UV detection drugs analytes can be detected in the μg/l range. During the solid phase extraction with a mixed mode cartridge, recovery percentages for different analytes lie between 13 to 102 % for 29 different pharmaceuticals. Also a separation method for soil is prepared by using an IKA Turax® _{homogenizing and grinding system.}

Results with the LC-ESI-MS/MS has revealed that it is possible to determine drugs in water and sediment originating from the Thames. Now an optimised method has been established, it is important that a more extensive study is made during a longer period of time. With an extensive study, more analytes in different matrices can be determined in ng/L to μg/l concentrations. Also the interdisciplinary use of the validated method will make it possible to affect different forensic approaches.

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1. Introduction

The name drugs, is a collective name for pharmaceuticals and stimulants. Stimulants are normally better described as; substances which stimulate the brain as a result of mental and physical impact. The intended use of drugs are for therapeutic effect, to lighten severe pain, especially for human or veterinary health reasons. Mainly opiates such as morphine and diamorphine (heroin) are clinically used to lighten pain, but some persons find the effect of opiates pleasurable and take it for non-therapeutic purposes, which better can be described as the abuse of drugs. The categorisation of drugs are based on their chemical structure and specific biological activity. Pharmaceutically active compounds or active pharmaceutical ingredients (APIs) are very complex molecules. The biological, physicochemical properties and functionalities of these complex molecules are all different, because they are developed and used for their biological activity. Many pharmaceutical drugs are currently abused were once they were promoted as helpful substances by the food and pharmaceutical industries. Examples of misused pharmaceutical products are substances as; heroin, cocaine and amphetamine. Especially the chemical molecules of the pharmaceutical compounds, have molecular weights from 200 to 1000 Dalton1, these APIs are small molecules. Pharmaceuticals are a diverse group of chemicals that include prescription, non-prescription medications and veterinary drugs, so the classifying can be easily done in two classes, licit and illicit drugs. In paragraph 2, Table 2.1 will give an impression in which classes the drugs can be subdivided [1][2].

1.1 Licit- and illicit drugs

Drugs are ingrained in the modern society, mainly alcohol and nicotine are a good example of widely accepted drugs. The term ‘licit drug’ is therefore mainly used when the use of it is accepted by law and as long a doctor prescribes them. The drugs that a doctor normally will prescribe are; antihypertensive, cardiovascular, hypnotics, sedatives, antipsychotic, analgesics, anti- inflammatory and antimicrobial agents. Illicit drugs are substances that are classified as illegal by law but also the trading of prescribed drugs is illegal. The most common abused drugs are; amphetamine, methamphetamine, MDMA, heroin, cocaine and cannabis. Not only these drugs are the drugs of abuse, there are many more and the most of them are normally prescribed by a doctor for therapeutic treatment. The active pharmaceutical ingredients APIs normally exist as solid forms. These solid forms are salts that can form polymorphs. These APIs can form more than ten different polymorphs. A polymorphs of a drug will have an identical chemical composition, but with very different properties. These polymorphs will normally include differences in bioavailability, solubility, chemical, physical stability and dissolution rate. All these factors will involve the metabolism of animals and humans, but also the impact into the environment when they come directly into it. Since the introduction of medical treatment there has always been excretion of APIs into the environment. These APIs will affect organisms and also humans who will live in an ecosystem, different publicised research articles highlighted identified drugs in the environment [2][3][4].

1.2 Drugs in the environment

To understand how drugs and other chemicals come into the environment, the biological aspect about the ecosystem is the basic knowledge how an environment is build up. An ecosystem is a community of living organisms (humans, animals and plants), non-living matter (soil, water, air) and environmental factors. Together this community forms an interaction system that is in

1

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equilibrium with each other for the existence of life. As long the human do not disturb or influences the important life cycle, an ecosystem will be in balance. Often human actions affect our environment, this can affect the ecosystem. The consumption of pharmaceuticals and drugs of abuse are different for each country. Many pharmaceuticals will only be given on prescription, while there are also countries where it possible to buy the pharmaceutical without any prescription. These differences between countries, makes it impossible to measure the use of drugs. It is understandable that the pathways of input of pharmaceuticals and drugs into the environment is accepted as a normal case. All active compounds may enter the environment by different routes. The biggest input of drugs into the environment will be come from metabolites and transformation products after excretion of human beings and animals. As described in the research report of the Environmental Protection Agency [5], the biggest source of environmental exposure has been recognised from municipal sewage. The entering of pharmaceuticals in the environment is much higher from municipal waste sources than the manufacturing of industry. The several pathways of input of pharmaceuticals and drugs into the environment are showed in figure 1.

Normally drug products are used for therapeutic effects, so the APIs can interact in the treated animal or human body. During the metabolite process in a treated body, molecules resulting changes of the originally chemical structure. Many drugs undergo a structural change in bodies of humans and animals, by enzymes in the liver and microorganisms in the bowel. The metabolism is different for each drug, some drugs are less metabolised before they will be excreted against drugs that are largely metabolised. After excretion of these metabolite products, they will come into the environment where biotic and non biotic processes will create transformation products. These transformation products will be created in technical facilities such as sewage and drinking water treatment plants. Other molecules can also be formed into the environment after excretion of parent compounds. An example of transformation processes are hydrolysis and photo oxidation. Many parent compounds of drugs will be bio-transformed by organisms that are present in soil such as bacteria and fungi. Biotransformation and biodegradation will do the same as metabolism, changing the chemical structure of the active molecules. This degradation will result in physical-chemical change, so that the molecule will be less toxic or enhance the water solubility [3][4].

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1.2.1. River water.

Water is the main source that is essential for life on earth. Roughly the surface of the earth is for 70% covered by water. The hydrologic cycle, shown in figure 2 is a cycle that is the basic principle for the circulation of water in the environment. The use of water for life is formulated as an easy ecosystem: drinking water → waste water → receiving water. A river is a major source of water that is used for multipurpose, water supply, shipping, fishing and irrigation works. Often along a river a lot of agriculture, urban development and industry is found. Normally rivers will flow water from higher areas to the lower parts at the earth’s surface. Most of the rivers are oceanic and will end in the sea. The so called continental rivers are ending in lakes, swamps or desserts. Continental rivers are periodic, unlike oceanic rivers that are often permanent water drains. Rivers are divided into

Figure 2: Hydrologic cycle is the influence of water on the life of on earth. The hydrologic cycle is essential in the form of different types of water that can occur[6].

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three areas: upper, middle and lower reaches. In the upper area erosion prevails, in the lower course is a lot of sedimentation, while in the middle area the river is winding with lateral erosion and accumulation of debris. The course of the rivers is strongly influenced by the geological structure. In areas with complex geological structure the river will follow usually not the shortest way to the sea, but are forced to move more or less long distances in a different direction. The amount of water that a river will drain is depending on the watershed, soil and climatic conditions. As well when the water level becomes higher, the flow rate decrease as result in sedimentation of sludge. The amount of sludge carried by the rivers, is often enormous and will give the river water often a brown- yellowish colour, which depends on the origin of the geological area. A winding river often clashes against the bank, that result into a hollow bank, whereby the water level rises and the flow decreases. Lateral erosion and accumulation ensure an increasing of meanders [7][8][9].

1.2.1.1.Thames River

The River Thames is ±336 km (210 miles) long and starts from its source in Gloucestershire. From Gloucestershire the river runs through the interior of England to the North Sea. The river and its tributaries are located in an area of 16,133 km2 that is called Thames basin. Thames basin is an heavily urbanised area, that includes the Medway towns2 and cities such as; Oxford, Reading, Slough, Luton and London. Figure 3 shows the Thames Basin, with the river Thames and its tributaries. The main river in the basin is the River Thames, that is subdivided in the non-tidal and tidal area. The non-tidal part is ±235 km (147 miles) long and is separated into the tidal Thames at Teddington Lock. Teddington Lock is on the western suburbs of London, and consist of three locks. The tidal part of the Thames is ±158

km (99

miles)long and supports one of the most ecologically diverse estuaries in England and Wales. The Thames estuary is the area where the river meets the North Sea in Eastern England, the water mainly in the London area is salty, because freshwater is mixed with the water of the North Sea. The water level in the tidal area falls by 7 meters (23 ft) twice a day and differs on the time of year.

2_{Medway Towns: The Medway Towns are the Towns Chatham, Gillingham, Rainham and Rochester, who}

are clustered around the estuary of the river Medway which is located in the Thames basin [15].

Figure 3: The Thames basin area, with the tributaries (light blue) and the Thames River (dark blue), trough the interior of England[10].

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The flow-out takes between 6 to 9 hours and the flow-in of the North Sea 4 to 5 hours. To protect London against flooding from the sea, nearby Woolwich the Thames Barrier is built. Especially the Thames river is an important water source that is providing two-thirds of London’s drinking water. The company ThamesWater®

is United Kingdom’s biggest sewerage company who serving 14 million costumers while using the water sources in Thames basin area [11][12][13][14].

1.2.2 Drugs in water

Activity of rivers and streams are one of the major interest for environmental (geo)scientists. This fluvial activity are common used as dumping ground or hiding places for materials from waste. Runoff from the surface will involves the cycling system, because water is exposed to contaminants which will be released in the environment. One of these pollutants, are the drugs that will come in the aquatic system. Water is an important medium for the transporting of pollutants in the environment. Several studies have shown that pharmaceutical substances could be measured in wastewater of medical care units and sewage treatment plantings. The concentrations of drugs in surface water and effluent from sewage treatment plants have concentrations in the ng/l to µg/l range [16]. As shown in figure 1 these compounds come up in the water by several factors. Compounds are disposed in a variety of forms and can end up in water by runoff of, farms, landfill leachate and sewage treatment. An easy example how drugs are released in water, is the drugs applied in veterinary medicine for therapeutic purposes. When the excreted manure with substances of drugs(metabolites) will be spread out on the fields, the substances will end up into the soil. After a heavy rainfall, the water will solve with the drug substances and the water with the drugs will be discharged to the groundwater. That the drugs substances will be discharged to the groundwater is, because water naturally moves down toward the lowest point, see also figure 2. The investigation of pharmaceuticals in the environment is currently intensively increased in the work field of environmental sciences. Pharmaceuticals are been around for several decades in the environment but are now brought to attention. This attention is important because the toxicity of pharmaceuticals could be hazardous to earthly organisms. As described in the literature [17], wastewater treatment plants have been identified as sources of pharmaceuticals entering into the environment. The removal of pharmaceuticals are very low, the compounds that are not removed will be released into the effluent streams of a WWTP (wastewater treatment plant). Several studies have shown not only licit drugs in the environment are detectable, but also a selected group of illicit drugs are. In the literature [18], a 1-year study have determinate drugs and their metabolites in wastewater plants of the city Florence (Italy). Samples were taken from the right and left banks of the Arno river and sampled during a period of July 2006-2007. The reported concentrations are in ng/L of the substances cocaine, benzoylecgonine(cocaine metabolite) and morphine(heroin metabolite). The results of the investigation in Florence has highlighted that the use of illicit drugs can be determined in WWTP analysis in a specific area. A similar research for illicit drugs in waste water treatment plants is carried out in the Paris area (France) [19], to determine cocaine, cenzolecgonine (major metabolite of cocaine), amphetamine, 3,4-methylenedioxymethamphetamine (MDMA) and buprenorphine. Effluent water samples are taken from four different WWTP in the area of Paris. The results have showed that Amphetamine is rarely detected and other compounds such as cocaine and benzolecgonine were measured in the water samples. Especially interesting results are also derived in the European Union in order of a project of European Monitoring Centre for Drugs and Drug Addiction (EMCDDA). Several rivers and lakes in Italy were investigated, including the Thames River in England [20]. The Thames was sampled along different areas such as, Oxfordshire (at New Bridge and Shillinford Bridge, 170 and 120 km above the tidal Thames) and mainly in the

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London area (Chiswick Bridge, House of Parliament and Tilbury). The sample areas where downstream whereby Tilbury was 20km from the WWTP Beckton. The results are determined for the drugs; benzoylecgonine, cocaine, amphetamine, methamphetamine, MDA, MDMA, morphine and THC-COOH. The concentrations in the upper area Shillinford Bridge and New Bridge (0,13 to 6 ng/L) are lower in comparison with the results downstream in the London area (0,41 to 42 ng/L). Similarly to other investigations, results reported for therapeutic pharmaceuticals, appears widespread in the environment [3][4][21].

1.2.2.1 Water solubility of drugs

Pharmaceutical compounds are derived from different chemical molecular structures. The typically forms are composed of several interlinked aromatic cores and multiple substituents3 that containing atoms as N, P, O, S, and halogens. As described in paragraph 1.2.2 different substances are found in effluent of sewage treatment plants, surface water, groundwater and in drinking water. When an active compound is biodegradable, the concentration in effluents will be lower. Polar compounds such as pharmaceuticals may be more likely to remain in aqueous media, and are difficult to eliminate. The less polar compounds adsorb easily onto sludge particles during the waste water treatment [22]. An example of how the solubility of a drug is created is described for the drug ketamine [23]. Ketamine is rapidly metabolised in the body by phase I reaction to the active metabolite norketamine and inactive metabolite hydroxyl-norketamine. This metabolism process takes place in the liver by the enzyme cytochrome P450. The metabolism of ketamine into norketamine is done by an oxidation (demethylating) which forms norketamine. The norketamine is followed by a hydroxylation process that yields hydroxy-norketamine. The phase II biotransformation reaction includes glucuronidation conjugation with glutathione and amino acids. The conjugation with glutathione and amino acids will increase the solubility of hydroxyl-norketamine and will forms dehydro-norketamine that is a hydrophilic metabolite. As also with other molecules, the electronegative atoms and aromatic groups makes a drug molecule high polarisable in water. The polar functional groups of the pharmaceuticals will interact in water via polar interactions, whereby hydrogen bonding can be formed [3][24]

1.2.3.Wastewater Treatment.

Wastewater treatment plants (WWTPs) receive water from different sources, as well from agricultural, industrial, household and community sewage. During the treatment primary and secondary treatment involving the cleaning process. Whereby wastewater is prepared for reintroduction into the environment. Wastewater normally consist components such as bacteria, organic matter, inorganic species and pollutants. During the pre-treatment process, large objects such as; glass, roots, and rocks are removed. The pre-treated water is than pumped into storage reservoirs for the primary treatment, where debris, solid, sandy and soil material will settle-out. To remove much as possible, chemical coagulant4 will be added to the water. Fine suspended material will bind together to form flocs, so it can be removed during the settlement. Sunlight will break down some bacteria and organic material, but in order to remove the dissolved organic matter, secondary treatment is needed. With main approaches at the secondary treatment, more organic compounds, including drugs, can be degraded by microbes or adsorption with more coagulant. Treatment systems for attaining secondary treatment are oxidation pond systems, fixed films and suspended films. Most secondary treatment is based on

3

Substituent: The replacing of hydrogen atoms in the molecule, examples of a substituent are; ethyl, methyl and propyl groups.

4

Chemical coagulant: Chemical coagulant is a chemical used in the water treatment. Normally iron chloride (FeCl3) is used, to incorporated particles into flocs [x]

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aerobic- but particularly anaerobic cleaning systems who are designed to treat industrial wastes, such as wastes from pharmaceutical plants or hospitals. An oxidation pond, or also called lagoon, is a system where wastewater will degrade. Aeration of the water with microflora will enhance the biotic phase. The surface water layer in a lagoon will be aerobic and the bottom layer is the anaerobic environment that includes sludge deposits. With the use of fixed filter systems the contamination of the effluent is separated. The slow filters are based on biological action, where the biological growth of bacteria and other micro-organisms is supported. Micro-organisms will breakdown organic compounds during the slow filtration through sand filters. The essential part of filtration is to reduce turbidity, removing particles that are larger than a particular including microorganisms and total suspended solids. Suspended-filter systems consists extended aeration systems and activated sludge systems. Activated sludge include a diverse range of the added micro-organisms, that consume the biodegradable organic substrates in wastewater. A lot of flocs (clump) of microorganisms form together a mass of microbes, what is described as the activated sludge. Activated sludge occurs when suspended microorganisms break down organic pollutants by reducing the biochemical oxygen demand (BOD). Activated sludge is the primary route for the removal of the dissolved druganalytes and is a so called ‘concentration point’ for bioactive compounds, who are not fully biodegraded yet. Solids retention time is an important factor for the biotransformation by microorganisms, a longer period of sludge treatment will cause better biotransformation and transforming abilities of the present druganalytes. Most sludge is transformed into biosolids for landfill or agriculture, with conventional treatment or enhance treatment. Enhance treatment will reduce volume by thermal drying and with conventional treatment the sludge is maturated for several days with anaerobic digestion at a temperature of 35 oC. When the prepared biosolid is used as landfill or manure, some pharmaceuticals with insufficient biotransformation will be released into the environment [5] [21][25][26].

1.2.4 Drugs in soil.

The entering points of drugs into soils are various, with the possible pathways; dust, biosolids and as well runoff from manure. Manure and biosolids(sediment) are the main sources of drugs release into soil. Biosolids is sewage sludge that is generated from wastewater treatment plants (paragraph 1.2.3.). The sewage sludge can be used for the utilise of agriculture, landfill ore soil conditioner. Manure and as well biosolids are both used as source of nutrients. The basic nutrient compounds are; phosphorus, nitrogen and organic matter (humus). The main goal of spreading out biosolids or manure is to enhance the physical properties of soil and stimulate plant growth. By the spreading of these materials, any present drugs may interact with the soil. In the literature [27] is described, that pharmaceuticals are determined in sludge from WWTPs. This research shows that the concentration of pharmaceuticals in solid waste samples can be much higher than in aqueous samples. Several factors will involve the sorption of drugs into soils, especially the sorption potential can affect. That the concentration of drugs in solid waste sometimes can be higher than in soil samples is because the mobility through sludge is more difficult. The interaction of pharmaceuticals with soil particles is the major influence of mobility. Parameters such as cationexchange, cation bridging and hydrogen bonding are involving the mobility of pharmaceuticals through sludge, sediment and soil.

In the published article of; Environmental, Toxicology and Chemistry [29], has described the interaction and adsorption of drugs into soil. Sorption will affect the transport of pharmaceuticals though the soil before they come into the ground or surface water. Pharmaceuticals that are adsorbed by soil are less biologically available for biodegradation. Many pharamaceuticals that are released by manure are ionisable compounds, with pKa values

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within the pH range of natural soils. These natural pH range make it possible that compounds are found in the environment as negative, neutral, zwitterionic, and positively charged species. Most soil particles are negatively charged (anions), so there can take place cationexchange. The interaction of adsorption of the pharmaceutical compounds will electrostatically attracted with cations(positively charged ions) to the negative charged soil particle. The polar functional groups of the pharmaceuticals will interact adsorption via polar interactions, hereby hydrogen bondings are formed with minerals and organic fractions of the soil particle[21][28][30].

1.2. Analytical methods

During this project several techniques of analytical science are used to identify and determine amounts of drugs in waste-water and soil samples.

1.2.1 Solid phase extraction

Solid phase extraction (SPE), is a widely used technique for the cleaning of samples before chromatographic analysis. SPE separations are carried out on special cartridges. These cartridges are available in different sizes and packing materials that is required for the extraction. As shown in figure 5 the solid phase is placed in a small cartridge, where the sample will be added and forced through under vacuum. The basic idea of SPE is based on the interaction of hydrophobic organic functional groups to a solid surface. This solid surface exists of powdered particles (sorbent material). Several powdered particles exist and has its own chemical interaction. Sorbent material can interact with the solid phase by Van der Waals forces, polar, non-polar and electrostatic attraction. With the development of a wide range of more polar phases and more essentially, ionic phases, the SPE technique evolved to provide a simple and rapid technique for the fractionation of drugs from complex matrices. Especially mixed mode particles such as hydrophilic lipophilic balanced (HLB) cartridges are universal. The polymeric reversed-phase sorbent that is made from a specific range of two monomers (hydrophilic N-vinylpyrrolidone and lipophilic divinylbenzene) will provide extraction of a wide range of acidic, basic and neutral compounds. HLB cartridges will have a higher recovery than the traditional C18 silica based sorbents. The packing material in a reversed phase SPE cartridge is composed with silica that is modified with aryl and alkyl functional groups. An alkyl group is explained as –CH2- and the aryl group are aromatic ring structures (example: phenyl)[31][32][33].

Earlier studies have determined drugs in waste-water, soil and sludge samples while using solid phase extraction. Solid phase extraction is a widely used analytical extraction method for the separation of analytes in complex matrices. Studies as [17][28][29]have optimised analytical methods for the detection of pharmaceuticals in complex matrices. During their study they have used different SPE cartridges which are optimised for the separation of the analytes. For each sorbent is the output compared with the highest taken acceptable recovery percentage >70%.

Figure 4: A schematic view how a SPE cartridge is constructed [1].

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During their investigation there is concluded that a 6ml Waters Oasis HLB cartridges gave the best recovery with 15 of the 21 target compounds yielding for sludge samples. In the study of [17]is a Strata X SPE cartridge elected as the best extraction method for waste-water samples. It is clear that the use of the right cartridge will depend on several factors. The choice for a cartridge depends on the sorbent material and the interaction that will give the highest average recovery. Also the matrix with the investigation analyte, which has to be extracted, is important how the sorbent particles will react[21].

1.2.2 High performance liquid chromatography

The basic idea of chromatography is the separation of mixtures of compounds, into their individual components. The separation is based on interaction of the compound with the mobile phase and stationary phase. This project focussed on the use of high performance liquid chromatography and therefore the key concepts for this method are described in this paragraph. Figure 6 illustrates a modern HPLC instrument that consists a; solvent reservoir, degasser, pump, injector port, column, detector and data processing system.

Figure 5: A block diagram of a modern HPLC instrument [16].

The solvent reservoir of a HPLC system delivers the mobile phase. The mobile phase is a liquid mixture that is made up of water and an organic solvent. In reversed phase HPLC organic solvents such as acetonitrile or methanol are used. A degasser is used to remove air from the system The pump is essential for a HPLC system to force the mobile phase through the column under high pressure. Different pumps are available for a HPLC system to drive more than 1 mobile phase. Binary and quaternary pumps are used for complex gradient elution. A binary pump will drive two mobile phases at the same time, and the quaternary pump is able to drive up to four different mobile phases at the same time. The pump will drive the mobile phase under high pressure, with flow rates from 0,1 to 10ml/min. The injecting system of a HPLC instrument is via a rotating valve system that can be used manual or automatically. The injecting system has two positions, a “Load” and “Inject”. In the load position the sample is pushed into a fixed volume loop, and stands in connection with the waste flask. When the injector is in load position, the mobile phase will flow through the column without the injected sample. Once the injector is filled with the sample, the injector is turned in inject position. The inject position is in direct connection with the pump and the column. The injected sample will be taken by the flow of the mobile phase, and now the sample can undergo the chromatographic separation. The

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column, contains the stationary phase, and is packed with absorbent particles. Columns can vary in dimension and packing particle size, the most used particles are micro porous or diffusive particles, permeable to solvent. The mobile phase moves around the particles which can consist of polymeric silica or carbon with a bonded phase for analyte interaction. The separation with reversed phase performance liquid chromatography (RP-HPLC) on the stationary phase (column) is non-polar and the mobile phase is polar. When a component of the investigation sample also is non-polar, it will have affinity with the non-polar particles in the stationary phase. The detector of a HPLC can vary, but the most used detector is UV/VIS detector. The ultraviolet-visible (UV-VIS) detector has a high sensitivity to measure amounts of UV absorbing analyte and absorbing analytes with suitable chromophores that absorb in the visible region of the light spectrum. The detector type is chosen for the application of the type of chromatography that is needed for the analyte type that will be separated. A more confirmation technique that can be used, for the separation and measuring of components is a mass spectrometer [1][31][32].

1.2.3 High pressure liquid chromatography- mass spectrometry

High pressure liquid mass spectrometry, also known as liquid chromatography-mass spectrometry (LC-MS), is an extremely resourceful instrumental technique. The instrumentation of LC-MS consists of a HPLC that is expanded via a suitable boundary to a mass spectrometer. During the LC-MS chromatography, liquid chromatography is used for the separation of a sample. The separated analyte will then be introduced into the mass spectrometer as a single compound. Figure 6 will give the basic steps how the analyte is introduced into the mass spectrometer for analysis.

When an analyte enters the mass spectrometer the source will ionisate the analyte. The ionisation can be achieved in different ways. The most used techniques in a LC-MS system, is electrospray ionisation (ESI). Other techniques that also can be used are atmospheric pressure chemical ionisation (APCI) and atmospheric pressure photo ionisation (APPI),but the use of electrospray ionisation in LC-MS is the most common, to analyse drugs, toxicological- and environmental samples. Electrospray ionisation will spray the mobile phase with analytes eluting from the LC system into a chamber at atmospheric pressure, where solvent and contaminates are evaporated, and the target compounds are ionised in an interface, see figure 7 The ionisation is done in presence of a

Sample Introduction

Ionisation Analyser Amplification & detection

Output

Figure 6: Block diagram how a mass spectrometer works [13].

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strong electrostatic field and a heated drying gas (example for used gas is nitrogen (N2)). The drying gas will evaporate solvent and the pressure of the electrostatic field will cause separation of the analyte molecules. While the solvent is evaporated the charge concentration of the resulted droplets are increased. The gas phase of the ions arise when ions will pass through a capillary chamber that is defined as the mass chamber where the ions will be analysed by analyser of the mass spectrometer. The separated ions will be focused onto a detector where the ions are converted to a measurable electrical current. The electrical current is a signal in the form of different peaks showing the particular ions. The series of peaks that are generated representing the mass over charge value (m/z). There is measured in m/z because the separation in mass spectrometer is on basis of mass and charge. To measure more accurate, tandem mass spectrometry is used. Tandam mass spectrometry is an MS/MS analyser where a quadrupole is placed in three order (triple quadropole). When the mixture of ions enters the first quadropole Q1, where just the precursor ion will pass to the second stage Q2. The second quadropole is the collision cell where all ions will collide with the collision gas (N2 or Ar). During this collision product ions (daughter ions) are formed, and passes straight on to the third quadruple Q3. At Q3 only the specific product ions are allowed to the detectorwhere the detection of molecular weight differences between daughter ions and the original molecular ion is measured. An example of tandem mass spectrum can be given by a pure sample of caffeine. The precursor ion [M+H]+ (m/z 195)is selected by quadrupole Q1 and dissociated in Q2. Then all fragments with the mass of > m/z 138 were analysed at Q3, to give the full mass spectrum. The chromatogram of caffeine will show signals, where the sum of m/z 57 + 138 will form 195 that is selected by Q1. The identification for caffeine will be noted for m/z transition as (195>138)[1][5][31][32][35].

1.3. Overall objective

Similar research studies have shown that legal an illegal drugs are detectable in water and solid samples. The majority applied chemical methods are the use of gas chromatography or liquid chromatography. During the research investigation in the published article[36] the analytical methods; SPE and gas chromatography-mass spectrometry (GC-MS) are applied for the detection of anti-inflammatory drugs(ibuprofen, naproxen, ketoprofen and diclofenac) in wastewater and as well in the published article of [37], non polar drugs are determined in sewage water. Recent studies [17],[19] and [28], have shown the use of liquid chromatography mass spectrometry (LC-MS). The use of these techniques is based on reversed phase retention mechanisms. To develop a more general approach for the determination of a broad range of drugs compounds in complex matrices, the combination, and optimisation of analytical methods is important. This report will outline the design of an analytical method which is capable for the determination of a rage of drugs in complex matrices, where waste water is the target sample. The laboratory research will have overlap with various disciplines in forensic science, but the field of environmental forensics is the central part of this investigation. During the laboratory investigation drugs and related substances are determinate with analytical research methods. To investigate the complex samples it is important that they are first purified by solid phase extraction. After purification the samples are analysed by High Performance Liquid Chromatography and the accurate method Liquid Chromatography-Mass Spectrometry/Mass Spectrometry. The interpretation of the amount of the confirmed substances is carried out by using spiked water samples, as well the recovery percentage of the SPE cartridge is reported.

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2. Experiment

2.1 Reagents

Analytical grades with a purity of ≥97 %; amitriptyline hydrocloride, antipyrine, atenolol, bezafibrate, clofibric acid, diclofenac sodium salt, flurbiprofen, gemfibrozil, ibuprofen sodium salt, indomethacin, ketoprofen, meclofenamic acid sodium salt, mefenamic acid, (±)-metoprolol (±) tartrate salt, nifedipine, nortriptyline hydrochloride, propanolol hydrochloride, ranitidine hydrochloride, salicylic acid, sulfamethazine sodium salt, sulfamethoxazole, sulfaphenazole, tamoxifen, tramadol hydrochloride and warfarin were all obtained from Sigma-Aldrich

[Steinheim, Germany]. Diazepam standard is purchased under license from Sigma-Aldrich [Poole, United Kingdom], and the analytes; caffeine, triclosan and trimethoprim with a purity of ≥95 % are from Fluka [Buchs, Switzerland].

Used solvents were all HPLC grade [Fisher Scientific, Walthams,USA], ultra high purity water that is used for the preparation of mobile phase, extraction solvents and standards is obtained from a Synergy® System by Millipore with resistivity 18,2 mΩ.cm at 25oC [Millipore, Bedford, USA]. Ammonium acetate is that is used as buffer in solutions is ordered from [BDH Hipersulv Murarrie, Austrialia], and pH adjustment solvent is diluted acetic acid by [BdH AnalaR Murarrie, Austrialia].

Stock solutions with a concentration of 1000 ppm [1000 mg/L] are prepared for each druganalyte in ultra high purity water or methanol HPLC grade. All prepared standards are stored in a fridge at ±4 oC. Two internal standards were prepared with a concentration of 10 ppm (10 mg/L )by solving 100 µL of each 1000 ppm analyte into a 10 ml flask. Table 2.1 shows the drug analytes that are studied during the project. The table will focus on the molecule structure, class of the drug, m/z transition logP and pKa values.

Name and Class:

LogPref pKaref Structure: m/z transition

Amitriptyline Antidepressant 4.62 9.42 (278>233) Antipyrine (phenazone) Analgesic 0.17 1.45 (189)

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Atenolol Beta blocker 0.16 9.20 (267>190) Bezafibrate Antilipemic 4.25 3.60 (363>316) Caffeine Stimulant licit -0.07 14.00 (195>138) Clofibric acid Hyperlipidaemic 2.58 3.46 (213>127) Negative mode electrospray ionisation. Diazepam Benzodiazepine/ sedative 2.86 3.39 (285>257)

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Diclofenac Analgesics and Anti-inflammatory drugs 4.15 4.40 (294>250) Negative mode electrospray ionisation. Flurbiprofen Analgesics and Anti-inflammatory drugs licit 4.16 4.33 - Gemfibrozil Hyperlipidaemic - - (249>121) Negative mode electrospray ionisation. Ibuprofen Analgesics and Anti-inflammatory drugs - - (205>162) Negative mode electrospray ionisation. Indomethacin Analgesics and Anti-inflammatory drugs

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Ketoprofen Analgesics and Anti-inflammatory drugs 3.12 4.45 (255>209) Meclofenamic Acid Analgesics - - (294>258) Mefenamic Acid Analgesics and Anti-inflammatory drugs - - (194>138) Metropolol Beta blocker 1.88 9.31 (268>116) Nifidipine Beta blocker 3.17 1.00 (345)

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Nortriptyline Antidepressant 4.39 9.70 (264>233) Propranolol Beta blocker 3.48 9.49 (260>116) Ranitidine Histamine H2-receptor antagonist 0.15 8.20 (315>270) Salicylic Acid Analgesic metabolite 2.36 3.50 (137>93) Negative mode electrospray ionisation.

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Sulphametazine Antibiotic 0.89 7.40 (279>156) Sulfamethoxazole Antibiotic 0.89 5.60 (254>156) Sulfaphenazole Cardiovascular - - -

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Tamoxifen Anti- cancer drug

6.58 8.85 (372>327) Tramadol Analgesic 2.51 9.19 (264) Triclosan Antimicrobial 4.53 8.10 (287>289) Negative mode electrospray ionisation. Trimethoprim Antibiotic 0.91 6.60 (291>123)

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Warfarin Anticoagulant 2.60 5.05 (307>161) Negative mode electrospray ionisation.

2.1.1 High Performance Liquid Chromatography

The high performance liquid chromatograph(HPLC) system that is used during the method validation, was an Agilent HP1100[Santa Clara, California, USA]. The Agilent HP1100 consist a quaternary pump with as well in the system present degasser, a programmable injector and autosampler. The HPLC is controlled by the software Agilent Chemstation [Rev. A. 10.02 [1757] 1990-2003] on a Hewlett- Packard Vectrave computer. Measurements were carried out by an UV diode array detector, at the wavelengths 220, 254 and 270nm. All analysis are performed on a Waters Sunfire 150 x 2.1mm with a particle size of 3,5µm octadecylisilica analytical column. The used flow was set to 0,2ml/min, with an injection volume of 20µL. The two mobile phases A en B, are programmed with gradient conditions. Table 2.2 shows the gradient program that is used during the separation. All runs are re-equilibrated with a post time of 15 minutes. The two used mobile phases are prepared by dissolving of 10 mM of ammonium acetate (CH3COONH4) in 1L high purity water with acetonnitrile (MeCN). Mobile phase A is prepared in a 90:10 ratio and B is created by dissolving in a 20:80 ratio. Both solutions are filtered under high pressure suction through a Buchner flask, while using an 0.2 µm micro-pore filter. During the filtration, particulate matter will be removed, so they have no effect on the HPLC. Both mobile phase bottles are labelled and degassed in an ultrasonic bath during 15 to 20 minutes.

Table 2.2: High performance liquid chromatography conditions. HPLC: Column: Agilent HP1100 Waters Sunfire 150 x 2.1mm; 3,5µm Flowrate: 0,2ml/min Post Time: 15min Gradient: Wavelengths: 220nm 254nm 270nm Time (min)

Mobile phase A Mobile phase B 0,00 5,00 28,00 55,00 100% 100% 50% 0% 0% 0% 50% 100%

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2.1.2 Solid Phase Extraction

To calculate the recovery percentage of a Waters Oasis 200mg HLB cartridge, n≥6 samples of internal standard 1 and n≥6 samples of internal standard 2 were prepared with concentration of 1 mg/L in a 1 L volumetric flask. Also samples are prepared in a 1 L volumetric flask with adjusted pH, to stabilise the different pKa values of the druganalytes, 10 mM ammonium acetate is added. All samples are made up to the mark with high purity water in volumetric flask. pH values are adjusted to ±5,5 by adding drop-wise on a magnetic stirrer 1 M of Acetic Acid. All solutions are transferred into pre-washed eluens bottles, and loaded on a conditioned SPE cartridge. It is important to condition the solid phase cartridge. By filling the cartridge with methanol, a water-miscible condition is prepared. Because the sample will be handled in a more polar phase it is important to remove the excess methanol with a least 5 proper rinses of high purity water. The water will create an aqueous condition, which is equal to the sample matrices. During the conditioning of the cartridge little vacuum is required to achieve a good flow. In combination with a Phenomenex SPE Vacuum manifold [Phenomenex Benelux, the Netherlands], the vacuum is set on -50 to -70 kPa, with a required flow of 1 mL/minute. All samples are reconstituted after drying by a stream of with 1 ml of mobile phase A.

2.2 Sample collection

(Waste)-water samples are collected from the river Thames in the Greater-London area in the vicinity of the wastewater treatment plant Beckton. The WWTP of Beckton treats 2,8000 million litres of sewage per day from a population of 3,5 million people, in an area of 300 km2, in the North and East London site. The vital role of the WTTP is to treat sewage and protect the environment. Figure 8 gives an overview, of this area. The official sample site was at Linton Mead on the south side of the Thames river. The samples are taken at 11 May 2012 at 8.30h during the flow-out of flood (04:59h; 7,04 m) to eb (11.07h; 0,90 m)5. During the sampling three eluens bottles of ±1,0 L water are taken and transported to the laboratory. (Control) soil samples are taken in Stoke-on-Trent on different depths, placed in plastic vials and frozen at -18 o

C. The sample taking was in cooperation with the Staffordshire University.

2.2.1. Sample preparation river water

The river water samples require additional preparation before they are loaded for analytical methods. After sampling, the river samples were directly filtrated by high suction filtration trough a Buchner flask, using an 0.2 µm micro-pore filter. The spiked samples with the concentrations of 1 ppb (1 µg/L) and 10 ppm (10 µg/L) are prepared in a 1 L volumetric flask. The flask is proper cleaned by rinsing with pure methanol and two to three rinses of high purity water. After cleaning the volumetric flask, internal standard mix 1 and 2 are added in the concentrations 1 and 10 µg/L. To stabilise the different pKa values of the druganalytes, 10 mM ammonium acetate is added. An appropriate concentration is prepared by filling, the volumetric flask up to the mark with yellowish/clear filtrated Thames water. To start with the SPE extraction both spiked solutions are adjusted to pH ±5,5 by adding drops of 1M Acetic Acid. 1 L of the filtrated Thames water is used to prepare a neat sample, as well from the spiked samples the pH is adjusted to ±5,5. All the three prepared samples are loaded on labelled Waters Oasis 200mg HLB cartridge with 6ml content, in combination with a Phenomenex SPE Vacuum manifold. First the SPE cartridges were conditioned, by filling the cartridges with methanol. Because the sample is handled in an more polar aqueous phase, the methanol is removed with a least 5 proper rinses of high purity water. The water will create an aqueous condition, that is equal to the sample matrices. The vacuum is settled between -50 to -70 kPa, with a flow of

5

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1ml/min. After all samples were loaded, the cartridges are washed with a proper rinse of high purity water. All extracts are eluted in 10ml 50:50 ethylacetate/acetone mixture and collected in a glass tube.The collected extracts were placed under stream of air, until the glass tubes were completely dry, all samples are reconstituted in 1 ml mobile phase A and vortexed for at least 5 minutes. All reconstituted samples are transferred in a labelled chromatography injection vial.

2.2.2. Sample preparation soil and sediment

After filtration of the river water samples, the used filters were placed in a glass beaker with high purity water. The residue that is retained on the filter, is removed with a spatula by scraping careful in the water. The solution is then transferred in a Falcontube, that is placed in a centrifuge for at least 5 minutes at 4000 rcf, a formed brown residue is located at the bottom of the tube and the supernatant (water) is poured off. The sediment pellet is concentrated on a clock glass, and air dried overnight. A control soil sample is placed on a clean clock glass and incubated in an oven for at least 2 hours at 150 oC, the baked soil is grinded in a mortar, and transferred to a clean labelled tube, 1,0 g of the homogenised dried soil sample is weighed into an IKA® Turrax [IKA® Werke, Germenay] vial, and solved in 5ml of 50% methanol:water solution. As well the dried sediment is weighed and transferred into a Turrax vial, and filled with 5 ml of 50% methanol:water solution. Another control sample is prepared and spiked with internal

Figure 8: This picture shows an overview where the water samples are taken. The general picture shows the Greater London Area with the Thames estuary that meets the North Sea. The Yellow and Red mark shows the sample point Linton Mead, what is in the vicinity of WWTP Beckton (Orange Mark).

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standard mix 1 and 2. All extraction vials are placed separately on the IKA® Ultra Turrax Tube Drive control, and grinded for 2 minutes at 5000 rpm. To remove the methanol solution from the soil samples, suction filtration under pressure is used. A Millipore Glass Fiber Filters, Filtertype AP25 0,8 to 8,0 µm filter [Billerica, MA, USA] is placed directly on the bottom of a syringe that is connected with a Phenomenex SPE Vacuum manifold. All samples are filtrated into a clear solution, and placed in a glass tubes. 3ml of extract is transferred in a 50 mL volumetric flask and made up to the mark with high purity water. All samples are loaded on a conditioned Waters Oasis 200mg HLB cartridge and reconstituted into an LC-injectionvial. Figure 9 shows the schematic overview how the extraction method for sediment and soil samples is carried out.

2.3 Liquid-Chromatography- electro spray ionisation- tandem mass

spectrometry (LC-ESI-MS/MS)

During the liquid chromatography, an Agilent HP1100 apparatus is used. The LC, contains a quaternary pump, degasser, programmable injector and autosampler. Samples are separated on the Agilent HP1100 and with electrospray ionisation in positive and negative mode introduced on the Micromass Quatro-LC. As well on the HPLC method, a Waters Sunfire 150 x 2.1mm with a particle size of 3,5µm octadecylisilica analytical column is used [Waters Cooperation, Milford, MA, USA]. All prepared samples during the validation process as well the real samples from the Thames water as the sediment and control soil are injected with volume of 10 µL. The flow rate was set to 0,2 ml/min with a gradient program for mobile phase A and B. Instrument control of the LC-ESI-MS/MS is carried out using a Netfinity 3000 computer. Data analysis is used with the software program Masslynx V.3,5.

1ug/L propranolol in 0,1% formateacetate 50% Methanol is injected with an full Hamilton syringe on a Harvard apparatus, for cleaning the line. Analyte determination and confirmation is carried out by using the library catalogue, where during a previously study all analytes already were investigated by direct infusion. All base peaks ions are noted in table 2.1.

Figure 9: A schematic view how the, dried soil/sediment is placed in a Turrax vial, that will be filled with 50% methanol. After grinding, all liquid is filtrated with high suction filtration, and solved into a volumetric flask. After SPE extraction the sample is reconstituted into an LC-injectionvial.

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2.4 Method validation

To design a method that is useful for the detection of drugs in (waste-)water, a method validation is required. All standards are first separately injected with a concentration of 10 mg/L, for the retention time determination with the use of high performance liquid chromatography. Also the recovery percentage for the Waters Oasis 200 mg, 6 ml HLB cartridge is calculated with adjusted and inappropriate pH, for n=6 samples with a concentration of 1 mg/L. As well the linearity in a concentration range of 0,1 to 30 µg/L internal standard 1 and 2 are analysed in duplicate. Correlation coefficients (r2) greater than 0,95 are used for each analyte with n≥ 6 data points. A repeatability study is carried out for the instrumental retention time precision, by injecting internal standard 1 and 2 for n=10 with a concentration of 10 mg/L. The limit of detection (LOD) is calculated as a concentration corresponding to a signal-to-noise ratio of 10:1. Limit of detection is determined by the lowest observable concentration that give a signal to noise ratio at 3:1.

3. Results and Discussion

3.1 High performance liquid chromatography

High performance liquid chromatography is used for druganalytes determination, all used drug analytes are injected individual and determined on their expected retention times. Table 3.1 gives an overview of the repeatability of internal standard 1 and 2. The retention time is given by the repeatability of n=10 injections with a concentration of 10 mg/L. Also the correlation is given, and drawn up by a calibration curve with at least n≥6 data points in the range of 0,1 to 30 µg/L.

Table 3.1: Data overview of repeatability, and calibration curve during HPLC method. Analyte: tr (min)A RSD (%) n=10B Instrument RSD (%) PrecisionC R2 n≥6 High purity waterD Atenolol Caffeine Antipyrine Metoprolol Ketoprofen Tramadol Benzafibrate Propanolol Indomethacin Meclofenamic Acid Nortriptyline Nifedipine Diazepam Tamoxifen 5,2 9,4 19,5 23,7 24,4 25,8 26,2 30,1 30,7 32,5 36,4 38,4 39,4 46,5 1,5 1,4 0,2 <0,1 <0,1 0,1 <0,1 <0,1 0,1 0,1 0,1 <0,1 <0,1 <0,1 15,7/16,0 8,8/8,9 6,8/9,2 9,8/10,6 8,4/9,4 6,8/8,3 10,7/11,4 12,9/11,4 20,4/21,4 52,2/53,8 19,5/21,7 12,1/24,2 7,4/8,4 13,9/15,2 0,9834 0,9997 0,9998 0,9913 0,9920 0,9956 0,9953 0,9910 0,9957 <0,9500 0,9821 0,9849 0,9904 <0,9500

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A

:Mean retention time of n=10 injections of 10 mg/L Individual druganalyte.

B_{:Relative Standard Deviation of mean retention time.} C

:Relative Standard Deviation of n=6

D

: Correlation coefficient for n≥ 6 data points in the range of 0,1 to 30 µg/L.

3.2 SPE cartridge recovery

The experimental pKa values, are obtained externally from the research article [38]. Recovery percentages are calculated for the various druganalytes by using a Waters Oasis® HLB 30µL 6cc/200mg cartridge. The already established SPE method which is described in [5], is during the laboratory research modified for further analysis to extract a broad range of druganalytes in (waste-) water. The original method for the use of Waters Oasis HLB sorbent describes the application for acidic, basic and neutral pharmaceuticals, based on the hydrophobic, hydrophilic and π-π interactions. Results in table 3.2 are obtained for pH adjustment and non-optimised pH. During the recovery investigation is became clear that the recovery for 29 different drug analytes is possible and that optimised pH, has an overall recovery for 14 compounds that is higher than >70 %. The druganalytes; meclofenamic acid, salicylic acid, sulfamethazine and triclosan are harder to extract and have shown to have lower recovery rates than <25 %.

Salicylic Acid Sulfaphenazole Sulfamethazine Ramtidine Clofibric Acid Thrimethoprim Metoprolol Warfarin Flurbriprofen Ibuprofen Diclofenac Mefenamic Acid Amitriptyline Gemfibrozil Triclosan 5,6 15,9 16,5 19,5 21,4 22,7 24,2 25,5 29,3 29,6 30,8 31,6 38,9 39,9 45,7 1,4 0,5 0,4 0,1 0,1 0,1 0,9 0,1 0,1 <0,1 0,1 0,1 <0,1 0,1 <0,1 14,1/14,6 16,1/13,4 10,4/11,0 1,9/3,1 4,3/4,7 2,8/3,1 9,9/8,6 6,4/44,6 2,2/34,8 4,1/31,6 14,6/27,2 44,6/10,2 23,1/18,3 35,9/35,0 22,3/20,2 0,9957 0,9982 0,9673 0,9916 0,9990 0,9962 0,9980 0,9907 0,9954 0,9846 0,9802 0,9541 <0,9500 0,9941 0,9519

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Table 3.2: Recovery percentage (%) for different druganalytes with Waters Oasis® HLB 30µL 6cc/200mg cartridge.

*n= 5

3.3 Liquid-Chromatography- electro spray ionisation- tandem mass

spectrometry (LC-ESI-MS/MS)

For the identification of the druganalytes in internal standard 1 and 2, a mix of both internal standards of 5 ppm is injected. Separations of the analytes are obtained with two sequential positive and negative ESI-MS/MS runs. During a previous study many druganalytes were infused by direct infusion. Each analyte in the concentration of 10 mg/L was injected and optimised in positive and negative modes. The base peak ions in this research project are compared with the saved analytes on the acquisition program in the Masslynx V.3,5 software. Running of the samples on ESI-MS/MS mode required more sensitivity for all target compounds, figure 10 shows the separation of the target compounds in positive mode and the negative mode is drawn up in figure 11. Table 3.3 gives a comprehensive overview of the data that is obtained for the developed method in high purity water.

Analyte: pKaRef. Recovery %

No adjusted pH % RSD n=6 Recovery % Adjusted pH 5,5 RSD % n=6 Atenolol Caffeine Amitipyrine Metoprolol Ketoprofen Tramadol Bezafibrate Propanolol Indomethacin Meclofenamic Acid Nortriptyline Nifedipine Diazepam Tamoxifen Salicylic Acid Sulfaphenazole Cimetidine Sulfamethazine Ranitidine Clofibric Acid Thrimethoprim Warfarin Flurbriprofen Ibuprofen Diclofenac Mefenamic Acid Amitriptyline Gemfibrozil Triclosan 9.20 14.00 9.42 9.31 4.45 9.19 3.60 9.49 4.50 - 9.70 1.00 3.46 8.85 3.50 - 6.80 7.40 8.20 3.46 6.60 5.05 4.33 - 4.40 - 9.42 - 8.10 56,0 79,7* 99,8 115,8 85,9 61,2 76 98,1 43,7 48,2 87,2 70,9 83,0 >120 14,6 67,6 - 0 97,9 86,5 89,2 70,7 74,8 89,9 52,8 >120 >120 106,4 11,5 32,6 49,2* 12,8 22,5 18,6 41,7 9,9 44,2 39,9 46,9 196,5 55,5 11,5 68,8 4,8 13,9 - 0 6,0 3,9 2,3 5,2 8,3 1,6 10,4 14,7 11,3 5,4 8,9 59,8 87,7 97,5 70,7 90,3 56,2 80,2 92,8 37,4 20,9 34,6 55,5 92,1 39,6 13,4 67,3 68,4* 15,0 92,3 68,8 85,7 61,9 102,5 64,8 45,2 92,5 66,9 73,8 18,3 16,1 8,9 9,2 10,6 9,4 8,3 11,4 11,4 21,4 53,8 21,7 24,2 8,4 15,2 14,6 13,4 11,0* 66,8 3,1 4,7 3,1 44,6 34,8 31,3 27,2 10,2 18,3 35,1 20,2

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Figure 10: Chromatogram of internal standard 1+2 with a concentration of 5 mg/L in positive mode.

Figure 11: Chromatogram of internal standard 1+2 with a concentration of 5 mg/L in negative mode. 5.438 7,6 10,017 15,231 16,503 20,317 21,59 23,755 26,426 27,952 30,241 34,056 35,964 37,236 0 1000000 2000000 3000000 4000000 5000000 6000000 7000000 0 10 20 30 40 50 60

Intens

ity

(arb)

Retention Time (min)

Internal Standard 1+2 ESI

+

_(5ppm)

5.438: Atenolol (267>190) 7.600: Sulfamethoxazole (254>156) 10.017: Caffeine (195>138) 15.231: Ranitidine (315>270) 16.503: Sulfamethazine (279>156) 20.317: Thrimethoprim (291>123) 21.590: Metoprolol (268>116) 23.755: Ketoprofen (255>209) 26,426: Bezafibrate (363>316) 27.952: Propanolol (260>116) 30.241: Indomethacin (358>174) 34.056: Nortriptyline (264>233) 35.964: Amitriptyline (278>233) 37.236: Diazepam (285>257) 5,874 20,373 26,355 29,532 38,307 0 1000000 2000000 3000000 4000000 5000000 6000000 7000000 0 10 20 30 40 50 60

Int

ens

ity

(

ar

b)

Retention Time (min)

Internal Standard1+2 ESI

-

_(5ppm)

5.874: Salysilic Acid (137>93) 20.373: Clofibric Acid (213>127) 26.355: Warfarin (307>161) 29.532: Diclofenac (294<250) 38.307: Gemfibrozil (249>121)

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Table 3.3: Comprehensive overview of the data obtained for the established method in high purity water. Analyte: Positive mode (ESI+)MS/MS tr (min)A RSD (%)B n=6 Recovery %C Adjusted pH 5,5

LOD in High purityD water (ng/L)

LOQ in High purityE water (ng/L) R2 n≥6 HighF purity water Sulfamethoxazole Atenolol Caffeine Ranitidine Sulfamethazine Thrimethoprim Metoprolol Ketoprofen Bezafibrate Propranolol Indomethacin Nortriptilyine Diazepam 5,2 6,2 10,0 15,0 17,7 21,0 22,5 23,2 25,0 29,2 30,0 35,1 37,23 1,3 0,8 0,6 0,5 0,4 <0,1 <0,1 <0,1 0,3 <0,2 <0,1 <0,1 <0,1 95,4 41,0 103,1 >120 5,00 >120 42,3 >120 75,0 50,1 >120 101,4 >120 22,0 86,7 112,2 236,8 41,3 89,2 58,8 22,1 39,4 5,1 29,0 138,2 55,3 73,4 289,1 374,1 789,4 137,7 297,2 195,7 73,5 131,2 51,2 26,5 460,7 184,4 0,989 0,9735 0,9717 <0,95 0,9947* 0,972 0,9962 0,9709 0,9836* <0,95 0,959 0,9713 0,9924 Negative mode (ESI-)MS/MS

tr (min) LOD in High purity

water (ng/L)

LOQ in High purity water (ng/L) Salicylic Acid Cloforbic Acid Triclosan Warfarin Diclofenac Gemfibrozil 5,5 19,9 24,6 24,9 28,9 38,3 - - - - - - - - - - - - 124,1 16,9 361,1 101,8 43,7 70,4 413,7 56,3 1203,7 339,2 145,6 234,6 - - - - - - A:Mean retention time of n=6 injections of 1 µg/L internal standard.

B:Relative Standard Deviation of mean retention time. C:Recovery percentage calculated with MS/MS method D:Limit of Detection calculated for internal standard at 10 mg/L. E: Limit of Quantitation calculated for internal standard at 10 mg/L F: Correlation coefficient for n≥ 6 data points in the range of 0,1 to 30 µg/L.

The Limit of Detection (LOD) and Limit of Quantitation (LOQ) were calculated, to indicate the selectivity of the used method. With the Limit of Quantitation the lowest concentration for a druganalytes with MS/MS detection is calculated. The results would suggest that the LOD in high purity water for the 19 analytes is between 5,1 and 138,2 ng/L. Where propranolol shows the lowest concentration, that will response above the noise level for this MS/MS method, and will also be the most sensitive detectable analyte. Especially caffeine, ranitidine nortriptyline, salicylic acid, triclosan and warfarin show less sensitivity. Also, LOQ for 14 analytes are quite high, and lay above 100 ng/L. Considering that the method is developed for the detection of analytes in complex matrices, this method needs to be optimised, in order to detect at trace levels. As well in comparing with the calibration curve design, the analytes, bezafibrate and sulfmethazine, could not be quantified at the concentrations 0,1; 0,25; 0,5 and 1,0 µg/L. Recovery percentage obtained for sulfamethoxazole was not detected with the HPLC method. Other recovery percentages that were obtained are in agreement that with LC-MS/MS is more accurate measured. Notable values are obtained for the analytes metoprolol (HPLC recovery 70 %, LC-MS/MS 42,3 %), propranolol (HPLC recovery 92 %, LC-MS/MS 50 %), indomethacin HPLC recovery 37 %, LC-MS/MS >120%) and nortriptyline (HPLC recovery 34 %, LC-MS/MS 101 %). Recoveries are not calculated for the negative mode ions. Considering that the developed method for the SPE extraction is used for a wide variety of drug analytes with pKa values ranging from 3, 46 to 14,0 it is important to compare more mixed mode particles that will react with Van der Waals forces, polar, non-polar and electrostatic attraction for the application of general screening of compounds at trace levels.

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3.3.1. Thames river water

The developed method was applied to the identification of druganalytes in sediment and river water samples from the London Thames river. Several peaks are identified by their base peak ions. The Thames water samples were screened in duplicate with tandem mass spectrometry(MS/MS) and displayed in figure 12. MS/MS data for the injection of 10 mg/l standards of atenolol, caffeine, thrimethoprim, metoprolol and propranolol in high purity water are given in figure 13.

Figure 12: MS/MS chromatogram of a neat Thames sample.

Figure 13: MS/MS comparison chromatogram with the analytes. 6,201 10,017 20,954 22,607 29,22 36,725 0 50000 100000 150000 200000 250000 0 10 20 30 40 50 60 Int ensit y ( ar b )

Retention Time (Min)

Neat Thames Sample

Atenolol (267>190) 6,201 Caffeine (195>138) 10,017 Trimethoprim (291>123) 20,954 Metoprolol (268>116) 22,607 Propranolol (260>116) 29,22 0 2000000 4000000 6000000 8000000 10000000 12000000 0 10 20 30 40 50 60 In te n si ty (ar b )

Retention Time (min)

The determination of drugs in waste-water and soil samples by liquid chromatography and mass spectrometry.

Final Report

Version I