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1 Cite this: DOI: 10.1039/c0xx00000x 2 3 www.rsc.org/xxxxxx 4 5 ARTICLE TYPE 6 7 Molecular modelling and synthesis of polymer for the extraction of 8 9 and from human 10 11 Iuna Tsyrulneva,*a Olga Zaporozhets,a Elena Piletskab and Sergey Piletskyb 12 13 5 Received (in XXX, XXX) Xth XXXXXXXXX 20XX, Accepted Xth XXXXXXXXX 20XX 14 DOI: 10.1039/b000000x 15 16 A new solid phase extraction resin with high affinity towards amiloride and triamterene was developed with the assistance of 17 computational modelling. Itaconic acid was selected as the best functional monomer capable of forming strong complexes with amiloride 18 and triamterene. Conditions for selective solid phase extraction of the from human urine and for their separation using custom- 19 10 designed adsorbent were optimised. It was shown that this adsorbent provided high level of purification of the diuretics from human 20 urine, which made possible their quantification using spectrophotometry.

21 Manuscript 22 1. Introduction amiloride and triamterene is complicated due to their similar 23 Amiloride (3,5-diamino-6-chloro-N-(diaminomethylene)- molecular structures and chemical properties. 24 15 -2-carboxamide) and triamterene (6-phenylpteridine- Commercial adsorbents (nylon membranes, C18 discs, etc.) do 25 2,4,7-triamine) (Figure 1) are -sparing diuretics, not always provide necessary selectivity towards the amiloride 26 commonly used for the treatment of and congestive 55 and triamterene. Besides, in most cases the reduction of the 27 . The mechanism of these diuretics action involves matrix effect has only been achieved by 100x dilution of urine, 28 blocking the epithelial channel and inhibiting sodium which also leads to the loss of sensitivity. In recent years, new 29 20 reabsorption (1). This promotes increasing of the amount of salt technique for selective pre-concentration of samples based on the 30 and water removed from kidneys and thus, decreases blood application of molecularly imprinted polymers (MIPs), was Accepted 31 volume. The main characteristic of these diuretics is that even in 60 developed as an alternative to the use of generic adsorbents. MIPs 32 small doses these drugs cause high-volume urine are widely used for extraction and purification of a number of 33 decreasing further their concentration and making difficult to biological and chemical substances: e.g. drugs, amino acids, 34 25 identify and quantify them. Thus, highly sensitive methods are pollutants, etc. (15-20). The application of MIPs for extraction of 35 required for the determination of these diuretics. drugs from human and calf urine has already been studied (21, 36 Amiloride and triamterene are usually used in conjunction with 65 22). Due to their selectivity and recognition properties MIPs are 37 or loop diuretics as it helps to correct potassium often called ‗synthetic receptors‘. Compared to natural receptors, 38 imbalances caused by these drugs. Due to its potassium-sparing MIPs are characterised by higher physical stability, strength, 30 properties amiloride and triamterene can cause and resistance to high temperatures and pressure, and inertness

39 Methods 40 acidosis. Wrong prescription and excessive abuse of these towards acids, bases, ion metals and organic solvents. However, 41 diuretics can lead to negative side effects. Besides, the presence 70 the important complication caused by the use of MIPs in 42 of amiloride and triamterene in urine is a proof of doping analytical application is possible leaching of the template 43 consumption (2). International Olympic Committee and World molecule from the cross-linked polymer. One of the common 44 35 Anti-Doping Agency (WADA) established the requirement for approaches to avoid this problem involves the imprinting of the 45 anti-doping laboratories to control the presence of these template analogue (23). Nevertheless, it requires a chemical 46 compounds in urine at the level less or equal to minimum 75 modification of the target, and might lead to reduced affinity and -1 47 required performance limit, which is 0.2 µg mL (3). specificity of the polymeric material. A promising approach, 48 The widely used methods for the determination of amiloride ―virtual imprinting‖, was proposed by Breton et al. (24). In this 40 and triamterene are high performance liquid and gas case polymer composition is designed using computational 49 Analytical 50 chromatography with mass-spectroscopic detection. These methods but polymer is made in the absence of the template. 51 methods provide the necessary selectivity and sensitivity (4-7). 80 52 The other methods for determination of triamterene and amiloride 53 include spectrophotometry and fluorimetry (8-14). However, 54 45 direct analysis of urine samples is complicated because complex 55 urine matrix interferes with the determination of analytes. Thus, a) b) 56 preliminary liquid-liquid or solid phase extraction (SPE) is necessary. Besides, only few articles propose selective 57 Fig. 1 Structures of amiloride (pKa=8.7) (a) and triamterene (pKa=6.2) 58 determination of amiloride in the presence of triamterene and (b). 59 50 their separation (11, 12). Simultaneous determination of 60 This journal is © The Royal Society of Chemistry [year] [journal], [year], [vol], 00–00 | 1 Analytical Methods Page 2 of 6

1 The apparent specificity in this case originates from the careful wavelength of 420 nm, of triamterene - at an excitation 2 selection of appropriate monomers and from random creation of wavelength of 360 nm and emission wavelength of 440 nm. 3 affinity binding sites in the polymer. Spectrophotometric measurements were performed on 4 Design of the polymers with predicted properties for the spectrophotometer SpectroQuest-2800 UV-vis spectrophotometer 5 5 extraction of diuretics from aqueous solutions and biological 60 (Unico, USA). The calibration curves for amiloride and 6 liquids hasn‘t attracted much attention. Recently polymers for triamterene were built at an absorbance wavelength of 360 nm 7 extraction of (25, 26), (26) and and 370 nm, respectively. pH measurements were performed 8 hydrochlorthiazide (27, 28) were proposed. using pH-meter PH150MI (IT, Russia) equipped with a glass 9 The aim of the work was to design a new sorbent with electrode. The SPE process was assisted by a peristaltic pump 10 10 predicted high affinity towards amiloride and triamterene and to 65 Mityflex 913 (Anko, USA). 11 develop simple, sensitive and inexpensive method of SPE of 2.4. Molecular modelling 12 these diuretics from aqueous solutions and human urine. 13 Molecular modeling was undertaken using a workstation from 14 Research Machines running the CentOS 5 GNU/Linux operating 15 2. Materials and methods system, configured with a 3.2GHz core 2 duo processor, 4 GB 16 70 memory and running the SYBYL 7.3 software suite (Tripos Inc., 15 2.1. Ethics statement 17 St. Louis, Missouri, USA). The LEAPFROG algorithm was 18 Research comprising study of urine effect was conducted in applied to screen the library of functional monomers for their 19 Ukraine. Urine samples were provided by 5 laboratory possible interactions with the template, resulting in a table 20 employees, who gave their verbal informed consent to have the ranking the monomers according to their binding scores (in kcal -1 21 samples used for research purposes. All samples were treated 75 mol ). The library contained 20 functional monomers which are Manuscript 22 20 anonymously. commonly used in molecular imprinting and possessed 23 polymerisable residues and residues able to interact with a 2.2. Materials 24 template through ionic and bonds, van der Waals‘ and 25 Standard solutions of 6 drugs (amiloride, dipole–dipole interactions (29). The charges for each atom were 26 , bumetanide, chlorthiazide, furosemide, 80 calculated using Powell method in combination with Gasteiger- 27 triamterene) (SIGMA, St. Louis, MO, USA) were prepared by Huckel charges and Tripos Force Field. The same method was 28 25 dissolving 1 mg of the corresponding diuretic in 10 mL of applied to refine the structures of the monomers using the 29 methanol (MeOH). Methanol stock solutions of diuretics were gradient minimisation method (the minimisation is stopped when 30 stored in a tightly closed container in a cool and dry place. energy gradient is lower than 0.001 kcal mol-1). The functional Accepted 31 Working solutions were obtained by taking an appropriate 85 monomer which demonstrated the highest binding energy 32 volume of standard solution, evaporating MeOH in a nitrogen towards the amiloride and triamterene was selected for the 33 30 stream and diluting it with water. Standard aqueous solution was polymer preparation. freshly prepared daily. 34 2.5. Polymer synthesis 35 Itaconic acid (IA), ethyleneglycol dimethacrylate (EGDMA), 36 2,2-azobis(isobutyronitrile) (AIBN), dimethylformamide (DMF), General procedure for preparation of custom-designed polymers 37 HPLC grade MeOH and acetonitrile (MeCN) were purchased 90 was following: 6.5 g of monomer IA and 26.0 g of cross-linker 38 35 from Merck (Darmstadt, Germany). All chemicals were used EGDMA were dissolved in DMF. The initiator AIBN (0.33 g, 1% without further purification. Sodium phosphate dibasic, sodium of total solid weight of monomer and cross-linker) was then

39 Methods 40 phosphate monobasic, sodium carbonate, sodium hydrogen added. The solutions were deoxygenated by purging with 41 carbonate, formic acid (FA) and hydrochloric acid (HCl) (all nitrogen for 10 min and then thermally polymerised in oil bath at 42 Merck, Darmstadt, Germany) were of analytical reagent grade. 95 80 ºC for 20 hours. The resultant polymer monolith was crushed, 43 40 Negative urine samples were obtained from healthy volunteers ground using Ultracentrifuge Mill (Retsch, UK) and sieved. The 44 who did not consume studied diuretics. Samples were stored in particle size fraction 45-125 μM was collected. Fine particles 45 polypropylene bottles at a temperature of -20 ºC. Before were removed by repeated sedimentation in MeOH twice and left 46 conducting the experiment samples were defrozen and to dry for 2 hours. 1-mL SPE tubes were packed with 25, 50 and 47 centrifuged. 100 100 mg of polymer and used for the optimisation of SPE protocol and for the measurement of the polymer‘s capacity. 48 45 2.3. Instrumentation

49 2.6. Evaluation of binding Analytical High performance liquid chromatography (HPLC) was performed 50 - 51 on Agilent1200 liquid chromatograph system equipped with Aqueous solutions of amiloride and triamterene (1 mL, 2 μg mL 1) were added to 50 mg of polymer particles. Samples were 52 fluorescent detector (FD). The column was kept in column oven 105 shaken in water bath for 6 hours and centrifuged for 10 min at 53 at the temperature 25 ºC. The chromatographic separation was 3000 rpm. The concentration of free analyte was determined 54 50 performed using a Luna 5 μm C18(2) (150 x 4.6 mm) column using spectrophotometer and HPLC-FD set-up. The amount of 55 (Phenomenex, USA). Instrumental parameters of amiloride and triamterene bound to the polymer was calculated 56 chromatographic determination were following: flow rate - 0.5 -1 by subtracting the equilibrium amount of analyte from the initial 57 mL min , volume of sample injected - 20 μL, mobile phase – 70/30 (v/v) MeOH/water. The detection of amiloride was 110 amount of analysed diuretic. 58 55 performed at an excitation wavelength of 285 nm and emission 59 60 2 | Journal Name, [year], [vol], 00–00 This journal is © The Royal Society of Chemistry [year] Page 3 of 6 Analytical Methods

1 2.7. Optimisation of the SPE protocol in aqueous solution pH 4.5. Then, 1 mL of spiked urine was filtered through 2 solutions polymer. Cleaning step included washing with 2 mL of 50% 3 60 MeOH, 1 mL of 0.1 M HCl, 2 mL of MeOH and with 1 mL of 4 In order to find the most favorable pH range for the adsorption of phosphate buffer, pH 7.5. Triamterene was eluted by 1-mL 5 amiloride and triamterene, the following experiment was aliquot of MeOH acidified with 0.1 M hydrochloric acid (10%, 6 5 conducted: SPE cartridges were packed with 50 mg of polymer -1 v/v). Every fraction was analysed by UV-vis spectrophotometer 7 and loaded with 1 mL of amiloride or triamterene (2 μg mL ) in and compared with blank solution. 8 0.1 M phosphate buffer, pH 5.0, 6.0 and 7.0, which corresponds to the pH range of normal human urine (5.5-7.0). The adsorption 9 65 2.9. Testing specificity using different groups of diuretics of amiloride and triamterene was evaluated using 10 Several diuretics, such as furosemide, bumetanide, 10 spectrophotometer. 11 , chlorthiazide and bendroflumethiazide, were 12 The elution of amiloride and triamterene from the polymer was optimised. MeCN, MeOH, DMF, MeOH acidified with 0.1 M tested in order to estimate the selectivity of the designed polymer. 13 1 mL of analyte solution containing 0.5 μg of amiloride or 14 HCl or 5% FA and MeCN acidified with 5% FA, were tested. In order to evaluate the effectiveness of a regeneration 70 triamterene and 0.5, 5.0 or 50 μg of each functional analogue was 15 filtered through cartridges. The cartridges were washed with 2 16 15 procedure the cartridge was loaded and washed as it was described above. Regeneration of the cartridge was performed by mL of MeOH. Amiloride or triamterene were eluted with 1 mL of 17 MeOH acidified with 0.1 M HCl (10%, v/v). Fractions were 18 washing the polymer with MeOH with subsequent treatment with the mixture of MeOH and 0.1 M HCl in order to remove any analysed using UV-vis spectrophotometer. 19 traces of diuretics. The cartridges were reconditioned using 20 75 20 MeOH and water. 3. Results and Discussion

21 Manuscript 50-mg amounts of the polymer were packed into empty 1-mL 22 3.1. Study of interactions between monomer and cartridges between two polyethylene frits. For the determination templates 23 of amiloride the cartridges were conditioned with 2 mL of MeOH 24 The selection of functional monomer is very important because it and 2 mL of 0.1 M phosphate buffer solution, pH 6.0. 1-mL 25 provides the basis for the strong interactions and therefore, 25 aliquots of amiloride solutions in 0.1 M phosphate buffer 26 80 desirable high binding. The computational modelling was solution, pH 6.0, of known concentrations (10-500 ng mL-1) were 27 performed for protonated amiloride and triamterene. The charged filtered through polymer cartridges. forms of monomers were used in the virtual screening in order to 28 For the determination of triamterene the cartridges were generate the molecular complexes with highest binding. 29 conditioned with 2 mL of MeOH and 2 mL of 0.1 M phosphate 30 Accordingly to the molecular modelling study, IA (Figure 2) was 30 buffer solution, pH 4.5. 1-mL aliquots of triamterene solutions in Accepted 85 found as the best monomer for the imprinting of amiloride and 31 0.1 M phosphate buffer solution, pH 4.5, of known triamterene because it demonstrated the highest binding energy, 32 concentrations (2-200 ng mL-1) were filtered through polymer when compared to other monomers (Tables 1, 2). It would be 33 cartridges. expected that under acidic conditions IA, which possesses two 34 The cartridge was washed with 2 mL of MeOH. Amiloride or 35 carboxyl groups, will demonstrate strong ionic interactions with 35 triamterene were eluted using 1 mL of MeOH/HCl 90/10 (v/v). 90 positively charged diuretic molecules (Figure 3). 36 Fractions were collected, dried under a nitrogen stream, dissolved It was found that under acidic conditions carboxyl group of IA 37 in 100 μL of mobile phase (MeOH/H O 70/30) and analysed 2 is an excellent hydrogen bond donor and acceptor. As it can be 38 using HPLC-FD set-up. Every fraction was analysed by seen from Figure 2, at pH≤2.8 IA molecule is neutral and does 39 spectrophotometer without any additional pre-treatment and Methods not form complexes with positively charged amiloride and 40 40 compared to blank solutions (elution solvent without drugs). 41 95 triamterene. At pH≥5.5 negatively charged dianion of IA interacts 42 2.8. Testing specificity using human urine with charged amine groups of triamterene and amiloride by 43 Human urine was diluted in 2 times by phosphate buffer with pH electrostatic mechanism of binding and thus, forming stable complexes. 44 4.5 or 6.0 and spiked with known concentration (10-200 and 25- -1 The synthesis of custom-designed polymer was performed by 45 500 ng mL ) of triamterene or amiloride, respectively. Each 100 free-radical polymerisation of IA (20%, w/w) in the presence of 46 45 cartridge containing 50 mg of IA-based polymer was treated in a 47 different way for amiloride and triamterene. large excess of cross-linker (80%, w/w), as it was described in the 48 For SPE of amiloride polymer cartridge was conditioned with Experimental section.

49 2 mL of MeOH and 2 mL of 0.1 M phosphate buffer solution pH Analytical

50 6.0. Then, 1 mL of spiked urine was filtered through polymer. 105 51 50 Cleaning step included washing with 2 mL of the mixture of

52 MeOH with water (1:1), 1 mL of 0.1 M HCl, 2 mL of MeOH and 53 with 1 mL of 0.05 M carbonate buffer, pH 10.0. Amiloride was 54 eluted by 1-mL aliquot of MeOH acidified with 0.1 M

55 hydrochloric acid (10%, v/v). Every fraction was analysed using 110 56 55 spectrophotometer and compared with blank solution.

57 For SPE of triamterene polymer cartridge was conditioned Fig. 2 Structure of IA (pK =3.85, 5.45). 58 with 2 mL of MeOH and 2 mL of 0.1 M phosphate buffer a

59 60 This journal is © The Royal Society of Chemistry [year] Journal Name, [year], [vol], 00–00 | 3 Analytical Methods Page 4 of 6

1 Table 1 Binding energies (kcal mol-1) of the molecular complexes Table 2 Binding energies (kcal mol-1) of the molecular complexes 2 between amiloride and functional monomers between triamterene and functional monomers 3 4 Monomer Binding energy, kcal mol-1 Monomer Binding energy, kcal mol-1 5 Itaconic acid -43.1 Itaconic acid -36.0 6 2-Acrylamido-2-methyl-1- Acrylamide -35.2 7 -41.2 propanesulfonic acid Methacrylic acid -31.3 8 Urocanic acid -39.7 N,N‘-methylene-bis-acrylamide -28.8 9 Trifluoroacetic acid -37.1 Urocanic acid -26.8 10 1-vinylimidazole -32.6 11 MeOH and target analytes were eluted with 1 mL of MeOH 12 3.2. Optimisation of SPE conditions 55 acidified by 0.1 M HCl (10% v/v). High binding capacity of the 13 Different solvents were tested in order to optimise loading, polymer (up to 40 g kg-1) allowed the 500-times pre- 14 5 washing and elution conditions. It was observed that the type of concentrating the diuretics from aqueous solutions. The 15 interaction between templates and monomer depended on the possibility of re-using cartridges packed with IA polymer was 16 nature of the medium. In MeOH electrostatic interactions assessed in the experiments in optimising washing and elution 17 between amino group of the diuretics and carboxylic groups of IA 60 protocols. It was observed that no degradation of the polymer was 18 are reduced by interaction with polar solvent molecules. It is observed after 30 cycles of regeneration and re-use. 19 10 known that electrostatic interactions play the main role in the So, based on described above experiments, the optimal 20 formation of molecular complexes in aprotic solvent. protocol for selective and accurate SPE of amiloride and

21 Hydrophobic interactions in aqueous solutions contribute to the triamterene from aqueous solutions was following. The cartridge Manuscript 22 non-specific interactions. 65 was conditioned with 0.1 M phosphate buffer solution, pH 6.0 23 It was found that water influenced the increase of hydrophobic and 4.5 for determination of amiloride or triamterene, 24 15 interactions between template and the polymer. As a result the respectively. 1-mL aliquots of amiloride or triamterene in 0.1 M 25 polymer demonstrated high affinity towards amiloride and phosphate buffer solution pH 6.0 and 4.5, respectively, were 26 triamterene with a 99% recovery in aqueous solutions. Thus, filtered through polymer cartridges. The cartridge was washed 27 water was selected as a loading solvent. 70 with 2 mL of MeOH. Amiloride or triamterene were eluted using 28 Buffer solutions of amiloride and triamterene at pH 5.0, 6.0 1 mL of MeOH acidified with 0.1 M HCl (10% v/v). 29 20 and 7.0 were prepared in order to estimate the adsorption of the 30 corresponding diuretics. It was observed that both analytes were Accepted 31 completely adsorbed under the specified conditions. Their 32 retention at pH 5.0 and 6.0 was caused by formation of 75 33 complexes diuretic-monomer involving electrostatic forces and 34 25 weak non-specific interactions. At pH 7.0 neutral form of 35 triamterene contributed to non-specific retention by forming 36 hydrophobic bonds. 37 It was considered that polar properties of MeOH will prevent 80 38 formation of hydrogen bonds between analyte and polymer 30 matrix. Therefore, MeOH was chosen as a washing solvent due to

39 Methods 40 its ability to decrease effectively the strength of hydrophobic 41 bonds and eliminate non-specific interactions. 42 Figure 4 demonstrates that MeOH containing 10% 85 43 hydrochloric acid is an optimal solvent for elution of amiloride 44 35 and triamterene. It appeared, that in acidic medium negatively 45 charged IA transforms in a neutral molecule. Inability of 46 methanol acidified with 5% formic acid to cause the break of 47 bonds can be the evidence of strong binding between template 90 48 and monomer. Other tested solvents, like MeCN and DMF, were 40 not suitable as eluents because of low solubility of diuretics. 49 Analytical 50 The amount of the polymer per cartridge was also optimised. 51 The recoveries of diuretics from cartridges packed with 25, 50 52 and 100 mg of polymer were tested. The highest recovery was 95 53 observed for cartridges packed with 50-100 mg. The recovery of 54 45 diuretics at 25 mg of polymer was less than 60%. Quantity of the 55 polymer equal to 50 mg was chosen for the practical extraction of 56 the target analytes from solution. In our experiments 500 mL of solution spiked with 2 μg of amiloride or triamterene were 57 100 Fig. 3 Molecular complexes between triamterene (a) and amiloride (b) 58 filtered through polymeric adsorbent. Polymer was washed with and IA. 59 50 60 4 | Journal Name, [year], [vol], 00–00 This journal is © The Royal Society of Chemistry [year] Page 5 of 6 Analytical Methods

1 molecules of triamterene and amiloride were uncharged, it 2 allowed them to be bound by IA-based polymer mostly by 3 hydrophobic forces. It was only possible to remove the target 4 35 analytes by using the mixture of MeOH with HCl. 5 Designed polymer was characterised by high recoveries of 6 diuretics (Table 3). Apparently, the weak interactions between the 7 components of urine matrix and diuretics could be destroyed by 8 combined treatment with HCl and MeOH. 9 40 The results presented in Table 3 demonstrate that under 10 optimal conditions quantitative recovery and elution of amiloride 11 and triamterene were achieved. It was also established that 12 preliminary clean-up of the matrix was effective as it allowed 13 removing completely the interference with the components of 14 45 urine, which greatly compromised the determination of amiloride 15 and triamterene. 16 Fig. 4 Selection of solvent for elution of amiloride ( ) and triamterene 17 ( ). 3.4. Testing the specificity using different groups of diuretics 18 19 3.3. Evaluation of SPE for urine samples Acidic diuretics (furosemide and bumetanide) and thiazide 20 50 diuretics (bendroflumethiazide, chlorthiazide and 5 The possibility to apply custom-designed adsorbent for the 21 hydrochlorothiazide) (Figure 5) were chosen for the evaluation of Manuscript 22 recovery of amiloride and triamterene from human urine samples the selectivity of adsorbent in SPE experiments. It was found that 23 was tested. These diuretics are supposed to form ion associates the interfering substances were extracted from aqueous solutions 24 with kynurenine, xanthurenic and folic acids (30) which results in during the loading step. It is known that under optimal conditions, 25 disability to detect them without preliminary clean-up of the 55 which were used in the experiment (pH 4.5 and 6.0), are 26 10 matrix. neutral and acidic diuretics are negatively charged. Their 27 In order to remove the interfering components of the urine extraction by negatively charged polymer can be explained by 28 matrix, washing of the polymers with acids, MeCN, MeOH and involvement of mostly hydrophobic and, partially, electrostatic 29 their mixtures with hydrochloric and formic acids was performed. forces. It was expected that MeOH will be effective solvent for 30 Aqueous MeOH was effective in the removal of salts, very polar 60 the removal of non-specifically bound compounds. In contrast, 31 15 components of urine and urobilin. It was found that the strength under these conditions the analytes of interest (triamterene and Accepted 32 of 5% formic acid was not sufficient to break the bonds between amiloride) are positively charged. They are bound with charged 33 IA and interfering substances. At the same time, 0.1 M HCl was carboxylic groups of IA mostly by electrostatic forces. Thus, 34 effective in neutralising all organic acids. It was observed that target analytes can only be eluted from the polymer using MeCN demonstrated no recovery of diuretics and slight recovery 35 65 acidified MeOH, which allows their separation from the 36 20 of urine components, which can be the evidence of its inability to interfering compounds and, therefore, their purification. 37 dissolve these substances. MeOH demonstrated high level of 38 recovery of urine components. So, combined treatment with HCl and MeOH gave the possibility to elute components of urine,

39 Methods 40 presumably derivatives of isoindole, which are characterised with 41 25 strong absorbance intensity in the area where diuretics absorb light and allowed to clean-up urine matrix. Since in the buffer 42 a) b) 43 44 Table 3 The results of spectrophotometric detection of amiloride and triamterene after SPE from human urine (n=3). 45 30 46 Diuretic Added, Found, Recovery, c) d) 47 ng mL-1 ng mL-1 % 48 Amiloride 25.0 24.9±0.1 99.4

49 Analytical 50.0 51.0±0.3 102.0 50 100.0 98.9±0.1 98.9 51 250.0 250.0±0.6 100.0 52 500.0 495±1 99.0 53 Triamterene 10.0 10.10±0.05 101.0 e) 54 20.0 19.9±0.1 99.4 55 70 Fig. 5 Structures of bendroflumethiazide (pKa=8.5) (a), bumetanide 50.0 49.5±0.1 99.0 56 (pKa=3.6) (b), hydrochlorothiazide (pKa=7.9, 9.2) (c), chlorthiazide 100.0 100.2±0.1 100.2 57 (pKa=6.7, 9.5) (d) and furosemide (pKa=3.9) (e). 200.0 198.0±0.2 99.0 58

59 60 This journal is © The Royal Society of Chemistry [year] Journal Name, [year], [vol], 00–00 | 5 Analytical Methods Page 6 of 6

1 2 0.8 3 3 4 Notes and references 5 0.6 aTaras Shevchenko National University of Kyiv, 62A Volodymyrska St., 6 Kyiv, Ukraine 01033. [email protected]. +380679452988; b 7 1 45 Department of Chemistry, College of Science and Engineering, 0.4 2 University of Leicester, LE1 7RH, UK. 8 1a 2a 9 Absorbance 1 R. Ventura, J. Segura, J. Chromatogr. B, 1996, 687, 127. 10 0.2 2 WADA, The world anti-doping code—the 2009 prohibited 11 50 list: international standard, World Anti-Doping Agency, 12 Montreal, 2009. 3 WADA, Minimum required performance limits for detection 13 0.0 250 300 350 400 of prohibited substances (technical document TD2012MRPL), 14 Wavelength, nm World Anti-Doping Agency, Montreal, 2012. 15 55 4 P. Van Eenoo, F.T. Delbeke, Chromatographia, 2004, 59, 39. 16 5 C. Brunelli, C. Bicchi, A. Di Stilo, A. Salomone, M. Vincenti, 17 Fig. 6 Absorbance spectra of standard solutions of triamterene (1, black J. Sep. Sci., 2006, 29, 2765. 6 R. Ventura, M. Roig, N. Monfort, P. Saez, R. Berges, J. solid line) and amiloride (2, red solid line); their mixture (3, blue solid 18 Segura, Eur. J. Mass Spectrom., 2008, 14, 191. line); triamterene, which was eluted with MeOH (1a, black dotted line); 19 60 7 O. Zaporozhets, I. Tsyrulneva, M. Ischenko, Am. J. Anal. 20 5 amiloride, which was eluted with MeOH/HCl (2a, red dotted line). Chem., 2012, 3, 320. 8 M. I. Toral, S. Pope, S. Quintanilla, P. Richter, Int. J. Pharm. ,

21 Manuscript 3.5. Testing of the aqueous solution contained both 2002, 249, 117. 22 diuretics 9 M. Kartal, N. Erk, J. Pharm. Biomed. Anal., 1999, 19, 477. 23 65 It was shown that selective elution of amiloride and triamterene 10 C. M. Peralta, L. P. Fernández, A. N. Masi, Microchem. J., 24 2011, 98, 39. from polymer surface could be achieved, which is based on the 25 11 I. Tsyrulneva, O. Zaporozhets, Pharm. Pharm., 2013, 4, 520. 10 difference in their chemical properties. At pH 7.5 molecule of 26 12 J. A. M. Pulgarı́n, A. A. Molina, P. F. López, Anal. Chim. amiloride is charged, while under the same conditions triamterene Acta, 2001, 449, 179. 27 is neutral. 70 13 G. A. Ibañez, G. M. Escandar, A. Espinosa Mansilla, A. Muñoz de la Peña, Anal. Chim. Acta, 2005, 538, 77. 28 The protocol of separation and quantification of each 14 M. L. Luis, J. M. G. Fraga, A. I. Jiménez , F. Jiménez, O. 29 individual diuretic involved conditioning of the polymer with 30 Hernández, J. J. Arias, Talanta, 2004, 62, 307. 15 MeOH and 0.1 M phosphate buffer pH 7.5, and filtration of 15 A. Zander, P. Findlay, T. Renner, B. Sellergren, A. Swietlow, Accepted 31 aqueous solution spiked with amiloride and triamterene through 75 Anal. Chem., 1998, 70, 3304. 32 adsorbent, followed by washing with 0.1 M phosphate buffer, pH 16 P. Martin, I. D. Wilson, D. E. Morgan, G. R. Jones, K. Jones, Anal. Commun., 1997, 34, 45. 33 7.5, in order to obtain required difference in the charges of the 34 17 W. M. Mullett, E. P. C. Lai, Anal. Chem., 1998, 70, 3636. tested diuretics and quantitative elution of triamterene with 18 S. Scorrano, L. Mergola, R. Del Sole, G. Vasapollo, Int. J. 35 20 MeOH (Figure 6). Amiloride is eluted by MeOH acidified with 80 Mol. Sci., 2011, 12, 1735. 36 0.1 M HCl (Figure 6). 19 A. Bossi, F. Bonini, A. P. F. Turner, S. A. Piletsky, Biosens. 37 Bioelectron., 2007, 22, 1131. 38 20 V. Pichon, F. Chapuis-Hugon, Anal. Chim. Acta, 2008, 622, Conclusions 48–61.

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