Prediction of Chromatographic Retention Time in High Resolution Anti-Doping Screening Data Using Artificial Neural Networks

SUPPLEMENTARY INFORMATION (S.I.)

PREDICTION OF CHROMATOGRAPHIC RETENTION TIME IN HIGH RESOLUTION ANTI-DOPING SCREENING DATA USING ARTIFICIAL NEURAL NETWORKS

Thomas Miller, Alessandro Musenga, David Cowan, Leon Barron*

Analytical & Environmental Science Division King’s College London, 150 Stamford Street, London, SE1 9NH, United Kingdom.

*Corresponding author email: [email protected] ; Tel: +44 20 7848 3842; Fax: +44 20 7848 4980 Table of Contents S 1.0 Compound identifiers S3 S 2.0 Reagents S6 S 3.0 Urine sample preparation S6 S 4.0 HRMS conditions S7 S 5.0 Calculation of log octanolwater distribution coefficient S7 S 6.0 Network types and architectures S7 S 7.0 Generation of molecular descriptors S8 S 8.0 Repeatability of retention time in urine and network replicates S10 S 9.0 Substitution of experimentallyderived p Ka with predicted p Ka S15

List of Figures Figure S1. The network architecture used in (a) linear (b) PNN (c) RBF and (d) MLP models. The linear model had no hidden layers, where (b) – (d), contained hidden layers with varying number of nodes. Figure S2. The final network architecture used to predict retention time for all compounds (n=86) was a four layer MLPBP, 18:5:4:1 structure.

E P Figure S3. (a) tr vs tr using the reoptimised 18:5:4:1 multilayer perceptron (inset) using P predicted p Ka input data (trained for 2200 epochs). (b) residual errors in t r using the predicted pKa input data for all analytes (n = 86).

List of Tables Table S1 . Compounds used in the ANN with their respective SMILES strings and CAS identifier. Table S2 . Definitions of the 18 molecular descriptors used to train the ANNs.

Table S3 . Data set used when training the ANN, calculated pKa data was omitted from the training when literature cited experimental p Ka data was used. The prediction of retention time from the most optimised network is shown with the difference relative to the experimental retention time. Table S4 . The prediction of retention time for all compounds from 10 individual networks. Networks were all 18:5:4:1 MLPBP with training lasting between 20004000 epochs. Table S5 . The experimentallymeasured retention time of replicate urine samples. The analysis was performed over 3 days with urine samples 15 on the first day, 610 on the second day and 1115 on the final day. Fields marked with (–) indicate that data was not available.

S 1.0 Compound Identifiers Table S1. Compounds used in this study along with their respective SMILES strings and CAS identifiers .

Compounds SMILES CAS

Atenolol O=C(N)Cc1ccc(OCC(O)CNC(C)C)cc1 29122687

Bisoprolol O(c1ccc(cc1)COCCOC(C)C)CC(O)CNC(C)C 66722449

Carvedilol O(c4ccccc4OCCNCC(O)COc3cccc2c3c1c(cccc1)n2)C 72956093

Labetalol O=C(c1cc(ccc1O)C(O)CNC(C)CCc2ccccc2)N 36894696

Metipranolol O=C(Oc1c(c(c(OCC(O)CNC(C)C)cc1C)C)C)C 22664557

Metoprolol O(c1ccc(cc1)CCOC)CC(O)CNC(C)C 51384511

Nadolol OC(CNC(C)(C)C)COc1cccc2c1C[C@H](O)[C@H](O)C2 42200339

Timolol O[C@H](COc1nsnc1N2CCOCC2)CNC(C)(C)C 26839758

Fenoterol Oc1cc(cc(O)c1)C(O)CNC(C)Cc2ccc(O)cc2 13392182

Salmeterol OCc1cc(ccc1O)[C@H](O)CNCCCCCCOCCCCc2ccccc2 89365504

Aminoglutethimide O=C1NC(=O)CCC1(c2ccc(N)cc2)CC 125848

Clomiphene Cl/C(c1ccccc1)=C(/c2ccc(OCCN(CC)CC)cc2)c3ccccc3 911455

Tamoxifen O(c1ccc(cc1)/C(c2ccccc2)=C(\c3ccccc3)CC)CCN(C)C 10540291

Bendroflumethiazide FC(F)(F)c3c(cc1c(NC(NS1(=O)=O)Cc2ccccc2)c3)S(=O)(=O)N 73483

Bumetanide O=S(=O)(c2cc(cc(NCCCC)c2Oc1ccccc1)C(=O)O)N 28395031

Chlorothiazide O=S(=O)(c1c(Cl)cc2c(c1)S(=O)(=O)/N=C\N2)N 58946

Chlorthalidone O=S(=O)(N)c1c(Cl)ccc(c1)C2(O)c3ccccc3C(=O)N2 77361

Clopamide O=C(NN1C(CCCC1C)C)c2ccc(Cl)c(c2)S(=O)(=O)N 636544

Etacrynic acid Clc1c(C(=O)\C(=C)CC)ccc(OCC(=O)O)c1Cl 58548

Furosemide O=S(=O)(N)c1c(Cl)cc(c(C(=O)O)c1)NCc2occc2 54319

Hydrochlorothiazide O=S(=O)(c1c(Cl)cc2c(c1)S(=O)(=O)NCN2)N 58935

Indapamide O=S(=O)(N)c1c(Cl)ccc(c1)C(=O)NN3c2ccccc2CC3C 26807658

Torasemide O=S(=O)(c2c(Nc1cc(ccc1)C)ccnc2)NC(=O)NC(C)C 56211406

Triamterene C1=CC=C(C=C1)C2=NC3=C(N=C(N=C3N=C2N)N)N 396010

Xipamide CC1=C(C(=CC=C1)C)NC(=O)C2=CC(=C(C=C2O)Cl)S(=O)(=O)N 14293448

Amiphenazole C1=CC=C(C=C1)C2=C(N=C(S2)N)N 490551

Benzoylecgonine CN1[C@H]2CC[C@@H]1[C@H]([C@H](C2)OC(=O)c3ccccc3)C(=O)O 519095

Ephedrine O[C@H](c1ccccc1)[C@@H](NC)C 299423

Fenproporex N#CCCNC(Cc1ccccc1)C 16397287

Heptaminol OC(C)(C)CCCC(N)C 372667

Methamphetamine N(C(Cc1ccccc1)C)C 537462

Phendimetrazine O2C(c1ccccc1)C(N(C)CC2)C 634037

Strychnine O=C7N2c1ccccc1[C@@]64[C@@H]2[C@@H]3[C@@H](OC/C=C5\[C@@H]3C[C 57249 @@H]6N(CC4)C5)C7

Codeine CN1CC[C@]23c4c5ccc(c4O[C@H]2[C@H](C=C[C@H]3[C@H]1C5)O)OC 76573

Fentanyl O=C(CC)N(C1CCN(CC1)CCC2=CC=CC=C2)C3=CC=CC=C3 437387

Hydromorphone O=C4[C@@H]5Oc1c2c(ccc1O)C[C@H]3N(CC[C@]25[C@H]3CC4)C 466999

Morphine CN1CC[C@]23C4=C5C=CC(O)=C4O[C@H]2[C@@H](O)C=C[C@H]3[C@H]1C5 57272

Pentazocine Oc1ccc3c(c1)[C@]2([C@H]([C@H](N(CC2)C\C=C(/C)C)C3)C)C 359831

Desonide O=C\1\C=C/[C@]2(/C(=C/1)CC[C@H]3[C@H]4[C@](C[C@H](O)[C@H]23)([C@@] 638948 5(OC(O[C@@H]5C4)(C)C)C(=O)CO)C)C

Celiprolol O=C(N(CC)CC)Nc1ccc(OCC(O)CNC(C)(C)C)c(c1)C(=O)C 56980939

Formoterol O=CNc1cc(ccc1O)[C@@H](O)CN[C@H](C)Cc2ccc(OC)cc2 73573872

Terbutaline Oc1cc(cc(O)c1)C(O)CNC(C)(C)C 23031256

MDMA CC(CC1=CC2=C(C=C1)OCO2)NC 42542109

Pseudoephedrine O[C@@H](c1ccccc1)[C@@H](NC)C 90824

Carteolol O=C2Nc1cccc(OCC(O)CNC(C)(C)C)c1CC2 51781067

Oxprenolol O(c1ccccc1OC\C=C)CC(O)CNC(C)C 6452717

Probenecid O=S(=O)(N(CCC)CCC)c1ccc(C(=O)O)cc1 57669

Dichlorphenamide Clc1c(cc(cc1Cl)S(=O)(=O)N)S(=O)(=O)N 120978

Benzphetamine N(C)(Cc1ccccc1)[C@@H](C)Cc2ccccc2 156081

Fenfluramine FC(F)(F)c1cccc(c1)CC(NCC)C 458242

Mephentermine N(C(Cc1ccccc1)(C)C)C 100925

Alprenolol O(c1ccccc1C\C=C)CC(O)CNC(C)C 13655522

Pindolol CC(C)NCC(O)COc2cccc1nccc12 13523869

Propranolol CC(C)NCC(COc1cccc2c1cccc2)O 525666

Raloxifene O=C(c1c3ccc(O)cc3sc1c2ccc(O)cc2)c5ccc(OCCN4CCCCC4)cc5 84449901

Hydroflumethiazide FC(F)(F)c2c(cc1c(NCNS1(=O)=O)c2)S(=O)(=O)N 135091

Etilefrine OC(CNCC)c1cc(O)ccc1 709557

Methoxyphenamine CC(CC1=CC=CC=C1OC)NC 93301

Nikethamide O=C(N(CC)CC)c1cccnc1 59267

pmethylamphetamine NC(Cc1ccc(cc1)C)C 22683789

Oxycodone O=C4[C@@H]5Oc1c2c(ccc1OC)C[C@H]3N(CC[C@]25[C@@]3(O)CC4)C 76426

Betaxolol O(CCc1ccc(OCC(O)CNC(C)C)cc1)CC2CC2 63659187

Acetazolamide O=S(=O)(c1nnc(s1)NC(=O)C)N 59665

Amiloride Clc1nc(C(=O)\N=C(/N)N)c(nc1N)N 2016888

Polythiazide CN1C(NC2=CC(=C(C=C2S1(=O)=O)S(=O)(=O)N)Cl)CSCC(F)(F)F 346189

Amphetamine NC(C)Cc1ccccc1 300629

Sibutramine ClC1=CC=C(C2(CCC2)C(CC(C)C)N(C)C)C=C1 106650560

Clenbuterol Clc1cc(cc(Cl)c1N)C(O)CNC(C)(C)C 37148279

Modafinil O=S(C(c1ccccc1)c2ccccc2)CC(=O)N 68693118

Bambuterol O=C(Oc1cc(cc(OC(=O)N(C)C)c1)C(O)CNC(C)(C)C)N(C)C 81732469

Phenmetrazine O2C(c1ccccc1)C(NCC2)C 134496

Salbutamol OCc1cc(ccc1O)C(O)CNC(C)(C)C 18559949

Benzthiazide O=S(=O)(c1c(Cl)cc2c(c1)S(=O)(=O)/N=C(\N2)CSCc3ccccc3)N 91338

Dimethylamphetamine c1(cccc(c1C)CC(N)C)C 4075961

MDA NC(C)CC1=CC2=C(C=C1)OCO2 4764174

Buprenorphine Oc7ccc5c1c7O[C@H]3[C@]6(OC)[C@H](C[C@@]2([C@H](N(CC[C@@]123)CC4C 52485797 C4)C5)CC6)[C@@](O)(C)C(C)(C)C

Methadone CCC(=O)C(CC(C)N(C)C)(c1ccccc1)c2ccccc2 76993

Oxymorphone O=C1[C@@H]2OC3=C(O)C=CC4=C3[C@@]2([C@]5(CC1)O)CCN(C)[C@@H]5C4 76415

Acebutolol O=C(Nc1ccc(OCC(O)CNC(C)C)c(c1)C(=O)C)CCC 37517309

Dextromoramide C[C@@H](C(C1=CC=CC=C1)(C(N2CCCC2)=O)C3=CC=CC=C3)CN4CCOCC4 357562

Esmolol O=C(OC)CCc1ccc(OCC(O)CNC(C)C)cc1 103598034

Sotalol O=S(=O)(Nc1ccc(cc1)C(O)CNC(C)C)C 3930209

Pemoline O=C2\N=C(/OC2c1ccccc1)N 2152343

Toremifene ClCCC(/c1ccccc1)=C(/c2ccc(OCCN(C)C)cc2)c3ccccc3 89778267

Methylphenidate O=C(OC)C(C1CCCCN1)C2=CC=CC=C2 113451

Phentermine NC(Cc1ccccc1)(C)C 122098

S2.0 Reagents Acetonitrile, methanol (both HPLC grade), potassium dihydrogen orthophosphate and anhydrous disodium hydrogen orthophosphate were obtained from FisherScientific (Loughborough, UK). Formic acid was obtained from VWR (Leicestershire, UK). K12 βglucuronidase from E. coli was purchased from Roche (Mannheim, Germany). A total of 86 compounds deriving from the list of WADA prohibited substances screened for at the 2012 Olympic Games (London, UK) were included in this study. Stock standards were prepared at concentrations of 1 mg/mL in methanol and stored at <10 oC in the dark when not in use. Working standards were prepared from these and used to spike urine samples. These were stored in the same manner as stock standards. Analytical grade reference compounds were purchased from several different manufacturers or donated by other antidoping laboratories. These compounds included (in alphabetical order) acebutolol, acetazolamide, alprenolol, amiloride, aminoglutethimide, amiphenazole, amphetamine, atenolol, bambuterol, bendroflumethiazide, benzoylecgonine, benzphetamine, benzthiazide, betaxolol, bisoprolol, bumetanide, buprenorphine, carteolol, carvedilol, celiprolol, chlorothiazide, chlorthalidone, clenbuterol, clomiphene, clopamide, codeine, desonide, dextromoramide, dichlorphenamide, dimethylamphetamine, ephedrine, esmolol, etacrynic acid, etilefrine, fenfluramine, fenoterol, fenproporex, fentanyl, formoterol, furosemide, heptaminol, hydrochlorothiazide, hydroflumethiazide, hydromorphone, indapamide, labetalol, methylenedioxyamphetamine (MDA), methylenedioxymethamphetamine (MDMA), mephentermine, methadone, methamphetamine (MA), methoxyphenamine, methylphenidate, metipranolol, metoprolol, modafinil, morphine, nadolol, nikethamide, oxprenolol, oxycodone, oxymorphone, pemoline, pentazocine, phendimetrazine, phenmetrazine, phentermine, pindolol, pmethylamphetamine, polythiazide, probenecid, propranolol, pseudoephedrine, raloxifene, salbutamol, salmeterol, sibutramine, sotalol, strychnine, tamoxifen, terbutaline, timolol, torasemide, toremifene, triamterene and xipamide. Ultrapure water was obtained from an Elga high purity water system (Marlow, UK) with a resistivity of 18.2 M.cm. S 3.0 Urine Sample Preparation Drugfree urine samples were collected anonymously from 15 healthy volunteers and from these, 1 mL aliquots were each stored at or below 10 °C in polypropylene tubes until analysis. To acquire retention data in urine, each aliquot was then spiked with premixed concentrated solutions of all the reference compounds. To match the routine screening protocol exactly, 100 L of internal standard solution, consisting of a mixture of mefruside (10 g/mL) and d 3 epitestosterone glucuronide (1 g/mL) in methanol were also added. The addition of these compounds was required to check system performance and to assess the hydrolysis of glucuronide metabolites. Hydrolysis of glucuronide conjugates was normally achieved by addition of 1 mL of a 20 % v/v βglucuronidase working solution in phosphate buffer (0.1 M, pH 6.2) followed by incubation at 50 oC for 1 h. Following this, 2 mL of 2 % v/v formic acid was added to the samples.

S4.0 HRMS Conditions Detection was performed using fast polarity switching high resolution mass spectrometry on an Exactive instrument (Thermo Fisher Scientific, San Jose, USA), equipped with a heated electrospray ionisation (HESIII) source. The sheath gas, auxiliary gas and sweep gas flow rate settings were 70, 10 and 0 arbitrary units respectively. The capillary temperature was 250 °C; the heater temperature was 300 °C; and the positive/negative spray voltages were +4.50 kV and 3.00 kV. Nitrogen gas was used within the HESIII source and the collision cell. Enhanced resolution mode was employed at 25,000 FWHM resolution. Three events occurred during each acquisition cycle by performing a full scan both in positive and negative ionisation mode (both with disabled CID) followed by a full scan in positive ionisation mode only with CID (collision energy 30 eV). The AGC target was set to “balanced”, meaning that ~10 6 ions were collected in the Ctrap before being sent to the Orbitrap for acquisition. The scan range was m/z 1001000 for all acquisition events and the resulting scan rate was ~1.2 seconds.

S5.0 Calculation of log octanol-water distribution coefficient Calculation of the respective distribution coefficients for the Moriguchi octanolwater partition, (MlogP) coefficient and the GhoseCrippen octanolwater partition (AlogP) is shown below (S1).

(S1)

where pH was 2.44 for the samples and K ow was replaced with the respective value for the either the Moriguchi partition coefficient or GhoseCrippen partition coefficient.

S6.0 Network types and architectures Network architecture was tested and included the use of probabilistic neural (PNN) networks, radial basis function networks (RBF), linear networks and multilayer perceptrons (MLP), as shown in Figure S1 .

Figure S1.The network architecture used in (a) linear (b) PNN (c) RBF and (d) MLP models. The linear model had no hidden layers, where (b) – (d), contained hidden layers with varying number of nodes

The most accurate network architecture was a four layer MLP. This network used feedforward back propagation (BP) supervised learning. The network contained two hidden layers (1 st layer n=5, 2 nd layer n=4 nodes) and was used in the prediction of retention time for all compounds.

Figure S2. The final network architecture used to predict retention time for all compounds (n=86) was a four layer MLPBP, 18:5:4:1 structure S7.0 Generation of molecular descriptors Molecular descriptors were generated by using simplified molecular input line entry system (SMILES). Using SMILES, quantitative structureactivity relationships (QSAR) descriptors were computed by Parameter Client. Of the 18 inputs, a total of 15 were calculated using this method, the remaining descriptors were sourced from literature or calculated as outlined in S1.0. Table S2. Definitions of the 18 molecular descriptors used to train the ANNs.

Descriptor Definition pKa log of the acid dissociation constant MlogP Moriguchi octanolwater partition coefficient AlogP GhoseCrippen octanolwater partition coefficient MlogD Moriguchi octanolwater distribution coefficient AlogD GhoseCrippen octanolwater distribution coefficient NDB Number of double bonds NTB Number of triple bonds NC Number of carbon atoms NO Number of oxygen atoms NR04 Number of 4 membered rings NR05 Number of 5 membered rings NR06 Number of 6 membered rings NR07 Number of 7 membered rings NR08 Number of 8 membered rings NR09 Number of 9 membered rings UI Unsaturation index HY Hydrophilic factor NBNZ Number of benzenelike rings

Table S3. Data set used when training the ANN, calculated p Ka data was omitted from training when literaturecited experimental p Ka data was used. The prediction of retention time from the optimised network is shown with the difference relative to the experimental retention time.

(ref) E P Compound Set p Ka Calculated pKa MlogP AlogP MlogD AlogD nDB nTB nC nO nR04 nR05 nR06 nR07 nR08 nR09 UI HY nBNZ t R (min) t R (min) Difference (min) Atenolol Train 9.60 (1) 9.40 0.9250 0.6690 0.0133 -0.2427 1 0 14 3 0 0 1 0 0 0 3.00 1.99 1 1.63 1.620 0.01 Bisoprolol Train 9.50 (2) 9.40 1.5950 2.0310 0.6887 1.1247 0 0 18 4 0 0 1 0 0 0 2.81 0.28 1 4.95 5.030 -0.08 Carvedilol Train 7.90 (3) 8.20 2.1950 4.0150 1.3848 3.2048 0 0 24 4 0 1 3 0 0 2 4.46 0.83 3 6.64 6.810 -0.17 Labetalol Train 9.40 (4) 9.30 2.6740 2.3300 1.7731 1.4291 1 0 19 3 0 0 2 0 0 0 3.81 2.58 2 5.22 5.100 0.12 Metipranolol Train 9.18 (5) 9.60 2.3470 2.8580 1.4583 1.9693 1 0 17 4 0 0 1 0 0 0 3.00 0.30 1 6.02 5.620 0.40 Metoprolol Train 9.68 (1) 9.40 1.6530 1.7570 0.7371 0.8411 0 0 15 3 0 0 1 0 0 0 2.81 0.34 1 3.88 3.810 0.06 Nadolol Train 9.67 (6) 9.50 1.3580 1.1460 0.4426 0.2306 0 0 17 4 0 0 2 0 0 0 2.81 1.83 1 2.52 2.810 -0.29 Timolol Train 9.21 (7) 9.40 1.2230 1.1280 0.3326 0.2376 0 0 13 3 0 1 1 0 0 0 2.59 0.47 0 3.65 3.620 0.03 Fenoterol Train 8.50 (8) 9.10 1.6350 2.3370 0.8248 1.5268 0 0 17 4 0 0 2 0 0 0 3.70 2.69 2 2.28 2.230 0.05 Salmeterol Train 10.20 (9) 10.00 2.8720 4.2260 1.9295 3.2835 0 0 25 4 0 0 2 0 0 0 3.70 1.53 2 7.27 7.120 0.15 Aminoglutethimide Train 4.20 (10) 4.40 1.3770 1.2850 0.9361 0.8441 2 0 13 2 0 0 2 0 0 0 3.17 1.19 1 2.08 2.200 -0.12 Clomiphene Train 8.70 (11) 9.60 5.6700 6.4830 4.8091 5.6221 1 0 26 1 0 0 3 0 0 0 4.32 -0.89 3 7.49 7.680 -0.19 Tamoxifen Train 8.85 (12) 8.70 5.2010 6.3200 4.3522 5.4712 1 0 26 1 0 0 3 0 0 0 4.32 -0.92 3 7.47 7.760 -0.29 Bendroflumethiazide Train 8.40 (13) 8.60 1.5820 2.1790 0.7332 1.3302 4 0 15 4 0 0 3 0 0 0 4.09 1.88 2 7.13 7.210 -0.08 Bumetanide Train 0.70 (14) 3.20 1.7630 2.9600 1.3250 2.5220 3 0 17 5 0 0 2 0 0 0 4.00 1.82 2 7.20 7.490 -0.29 Chlorothiazide Train 6.70 (15) 6.00 0.1870 0.4500 -0.5422 -0.2792 5 0 7 4 0 0 2 0 0 0 3.59 1.54 1 1.95 1.930 0.02 Chlorthalidone Train 9.20 (16) 9.60 1.6380 1.5070 0.7481 0.6171 3 0 14 4 0 1 2 0 0 1 4.00 1.96 2 4.26 4.470 -0.21 Clopamide Train 2.90 (17) 3.60 2.0080 2.3180 1.6776 1.9876 3 0 14 3 0 0 2 0 0 0 3.32 1.16 1 4.08 4.260 -0.18 Etacrynic acid Train 3.50 (18) 2.80 3.0100 4.1520 2.6961 3.8381 3 0 13 4 0 0 1 0 0 0 3.32 -0.20 1 7.53 7.680 -0.15 Furosemide Train 3.90 (19) 3.00 0.4340 1.7140 0.0431 1.3231 3 0 12 5 0 1 1 0 0 0 3.91 2.06 1 6.75 6.650 0.10 Hydrochlorothiazide Train 7.90 (20) 9.00 -0.5470 0.0380 -1.3572 -0.7722 4 0 7 4 0 0 2 0 0 0 3.46 2.41 1 2.09 2.190 -0.10 Indapamide Train 8.30 (21) 9.30 2.5360 2.7710 1.6997 1.9347 3 0 16 3 0 1 2 0 0 1 4.00 1.08 2 6.66 6.820 -0.16 Torsemide Train 7.10 (22) 5.30 0.8600 2.1330 0.1072 1.3802 3 0 16 3 0 0 2 0 0 0 4.00 1.08 1 5.87 6.020 -0.15 Triamterene Train 6.20 (23) 6.20 2.7030 1.0170 2.0254 0.3394 0 0 12 0 0 0 3 0 0 0 4.17 3.96 1 2.92 3.000 -0.08 Xipamide Train 4.75 (24) 5.10 2.3840 2.8120 1.8577 2.2857 3 0 15 4 0 0 2 0 0 0 4.00 1.91 2 7.06 7.330 -0.27 Amiphenazole Train 8.11 (25) 5.10 2.3850 1.6830 1.5609 0.8589 0 0 9 0 0 1 1 0 0 0 3.59 2.42 1 1.94 1.840 0.10 Benzoylecgonine Train 10.14 (26) 10.80 2.1700 1.8680 1.2305 0.9285 2 0 16 4 0 1 2 1 0 0 3.17 -0.29 1 3.31 3.310 0.00 Ephedrine Train 9.60 (27) 9.40 1.6640 1.2350 0.7523 0.3233 0 0 10 1 0 0 1 0 0 0 2.81 0.51 1 1.84 2.000 -0.16 Fenproporex Train 7.23 (28) 7.90 2.1470 2.0450 1.3843 1.2823 0 1 12 0 0 0 1 0 0 0 3.00 -0.31 1 2.75 2.770 -0.02 Heptaminol Train 10.60 (29) 11.10 1.3930 0.8720 0.4311 -0.0899 0 0 8 1 0 0 0 0 0 0 0.00 1.59 0 1.46 1.290 0.17 Methamphetamine Train 9.87 (30) 10.40 2.5450 2.0660 1.6192 1.1402 0 0 10 0 0 0 1 0 0 0 2.81 -0.29 1 2.80 2.590 0.21 Phendimetrazine Train 7.60 (31) 7.50 1.8450 1.9980 1.0771 1.2301 0 0 12 1 0 0 2 0 0 0 2.81 -0.84 1 2.32 2.550 -0.23 Strychnine Train 8.26 (32) 8.40 2.9040 1.1460 2.0702 0.3122 2 0 21 2 0 2 4 1 1 6 3.17 -0.83 1 2.93 3.010 -0.08 Codeine Train 8.20 (33) 8.30 2.1690 1.6380 1.3384 0.8074 1 0 18 3 0 1 4 0 1 3 3.00 -0.35 1 1.93 2.150 -0.22 Fentanyl Train 8.40 (34) 8.90 3.7680 3.8390 2.9254 2.9964 1 0 22 1 0 0 3 0 0 0 3.81 -0.87 2 5.96 6.010 -0.05 Hydromorphone Train 8.20 (35) 8.50 1.9310 1.6570 1.1011 0.8271 1 0 17 3 0 1 4 0 1 3 3.00 -0.34 1 1.68 1.480 0.20 Morphine Train 8.21 (36) 8.20 1.9310 1.3870 1.1004 0.5564 1 0 17 3 0 1 4 0 1 3 3.00 0.28 1 1.60 1.100 0.50 Pentazocine Train 8.88 (37) 8.90 3.7770 4.3480 2.9054 3.4764 1 0 19 1 0 0 3 0 1 0 3.00 -0.43 1 5.00 5.070 -0.07 Desonide Train 12.85 (38) 12.90 1.8070 0.9620 0.7497 -0.0953 4 0 24 6 0 2 3 0 1 1 2.32 0.18 0 6.74 6.980 -0.24 Celiprolol Train 9.70 (39) 9.50 1.6960 1.5970 0.7790 0.6800 2 0 20 4 0 0 1 0 0 0 3.17 0.95 1 4.40 4.460 -0.06 Formoterol Train 7.90 (40) 8.90 1.6590 1.9290 0.8488 1.1188 1 0 19 4 0 0 2 0 0 0 3.81 1.74 2 3.92 3.940 -0.02 Terbutaline Train 8.80 (41) 9.10 1.1290 1.2540 0.2621 0.3871 0 0 12 3 0 0 1 0 0 0 2.81 2.14 1 1.45 1.440 0.01 MDMA Train 9.90 (42) 10.30 1.6700 1.8350 0.7426 0.9076 0 0 11 2 0 1 1 0 0 1 2.81 -0.24 1 2.88 2.730 0.15 Pseudoephedrine Train 9.90 (43) 9.40 1.6640 1.2350 0.7366 0.3076 0 0 10 1 0 0 1 0 0 0 2.81 0.51 1 1.85 2.000 -0.15 Carteolol Train 9.70 (44) 9.50 1.3060 1.2830 0.4139 0.3909 1 0 16 3 0 0 2 0 0 0 3.00 1.06 1 2.68 2.650 0.03 Oxprenolol Train 9.38 (3) 9.40 1.8310 2.2320 0.9301 1.3311 1 0 15 3 0 0 1 0 0 0 3.00 0.34 1 4.59 4.660 -0.07 Probenecid Train 3.40 (45) 3.70 1.9790 2.8210 1.6867 2.5287 3 0 13 4 0 0 1 0 0 0 3.32 -0.20 1 7.23 7.560 -0.33 Dichlorphenamide Train 8.30 (46) 9.00 0.1980 0.8150 -0.6383 -0.0213 4 0 6 4 0 0 1 0 0 0 3.46 2.50 1 3.98 4.010 -0.04 Benzphetamine Train 9.30 (47) 8.90 4.1430 4.1860 3.2761 3.3191 0 0 17 0 0 0 2 0 0 0 3.70 -0.93 2 5.65 5.590 0.06 Fenfluramine Train 9.57 (48) 10.20 3.7900 3.3580 2.9058 2.4738 0 0 12 0 0 0 1 0 0 0 2.81 -0.23 1 4.91 5.270 -0.36 Mephentermine Train 10.40 (49) 10.40 2.8370 2.2710 1.8847 1.3187 0 0 11 0 0 0 1 0 0 0 2.81 -0.33 1 3.51 3.140 0.37 Alprenolol Train 9.60 (50) 9.40 2.3700 2.6400 1.4567 1.7267 1 0 15 2 0 0 1 0 0 0 3.00 0.32 1 5.67 5.930 -0.26 Pindolol Train 9.25 (48) 9.50 1.3060 1.9260 0.4133 1.0333 0 0 14 2 0 1 1 0 0 1 3.46 1.14 1 2.41 2.740 -0.33 Propranolol Train 9.45 (48) 9.50 2.5340 2.5400 1.6320 1.6380 0 0 16 2 0 0 2 0 0 0 3.59 0.29 2 5.69 5.780 -0.09 Raloxifene Train 8.95 (51) 8.80 3.5080 6.4110 2.6324 5.5354 1 0 28 4 0 1 4 0 0 1 4.59 0.12 3 7.13 6.580 0.55 Hydroflumethiazide Train 8.90 (52) 8.60 -0.0770 0.3160 -0.9497 -0.5567 4 0 8 4 0 0 2 0 0 0 3.46 2.27 1 2.87 3.180 -0.31 Etilefrine Train 9.00 (53) 9.80 1.0970 0.9380 0.1769 0.0179 0 0 10 2 0 0 1 0 0 0 2.81 1.39 1 1.41 0.920 0.49 Methoxyphenamine Train 10.45 (54) 10.40 2.2240 2.0500 1.2693 1.0953 0 0 11 1 0 0 1 0 0 0 2.81 -0.28 1 3.23 3.300 -0.07 Nikethamide Train 3.50 (55) 4.00 1.0200 0.7910 0.7061 0.4771 1 0 10 1 0 0 1 0 0 0 3.00 -0.75 0 2.26 2.020 0.24 p-methylamphetamine Train 9.68 (56) 10.10 2.5450 2.1210 1.6125 1.1885 0 0 10 0 0 0 1 0 0 0 2.81 0.49 1 2.75 3.450 -0.70 Oxycodone Train 8.53 (57) 7.60 1.3810 1.0290 0.5304 0.1784 1 0 18 4 0 1 4 0 1 3 3.00 -0.33 1 2.21 2.470 -0.26 Betaxolol Verify 9.38 (3) 9.40 1.9910 2.5770 1.0901 1.6761 0 0 18 3 0 0 1 0 0 0 2.81 0.26 1 6.32 6.110 0.21 Acetazolamide Verify 7.40 (58) 7.40 -1.5830 -1.2060 -2.3582 -1.9812 3 0 4 3 0 1 0 0 0 0 3.17 1.84 0 1.73 1.780 -0.06 Amiloride Verify 8.70 (59) 7.80 0.6680 -0.8100 -0.1929 -1.6709 2 0 6 1 0 0 1 0 0 0 3.17 6.79 0 2.01 1.580 0.43 Polythiazide Verify 9.10 (60) 9.30 1.0510 2.4120 0.1404 1.5014 4 0 11 4 0 0 2 0 0 0 3.46 1.28 1 7.04 7.210 -0.17 Amphetamine Verify 9.80 (56) 9.90 2.2420 1.6350 1.3198 0.7128 0 0 9 0 0 0 1 0 0 0 2.81 0.55 1 2.26 2.370 -0.11 Sibutramine Verify 8.50 (61) 9.70 4.5150 5.1760 3.6662 4.3272 0 0 17 0 1 0 1 0 0 0 2.81 -0.88 1 7.09 7.150 -0.06 Clenbuterol Verify 9.60 (62) 9.50 2.7410 2.3710 1.8293 1.4593 0 0 12 1 0 0 1 0 0 0 2.81 2.12 1 3.05 3.770 -0.72 Modafinil Verify 19.25 (63) 14.90 2.1830 1.9460 0.9323 0.6953 2 0 15 2 0 0 2 0 0 0 3.91 0.34 2 6.17 6.010 0.16 Bambuterol Verify 9.60 (64) 9.60 1.0320 1.7270 0.1203 0.8153 2 0 18 5 0 0 1 0 0 0 3.17 0.33 1 4.24 4.590 -0.35 Phenmetrazine Verify 8.45 (65) 8.60 1.5640 1.4620 0.7183 0.6163 0 0 11 1 0 0 2 0 0 0 2.81 -0.28 1 2.04 2.510 -0.47 Salbutamol Test 9.30 (66) 9.60 1.1330 0.9160 0.2376 0.0206 0 0 13 3 0 0 1 0 0 0 2.81 2.06 1 1.54 1.450 0.09 Benzthiazide Test 9.20 (67) 9.60 2.1990 2.7750 1.3091 1.8851 5 0 15 4 0 0 3 0 0 0 4.17 1.13 2 7.57 7.020 0.54 Dimethylamphetamine Test 9.40 (68) 10.20 2.8370 2.6070 1.9361 1.7061 0 0 11 0 0 0 1 0 0 0 2.81 0.43 1 3.35 2.760 0.59 MDA Test 9.67 (69) 9.90 1.3790 1.4030 0.4636 0.4876 0 0 10 2 0 1 1 0 0 1 2.81 0.53 1 1.55 2.500 -0.95 Buprenorphine Test 8.31 (70) 8.30 3.8060 3.8980 2.9690 3.0610 0 0 29 4 0 1 6 0 1 4 2.81 0.09 1 6.80 6.550 0.25 Methadone Test 8.30 (1) 9.00 4.1030 4.3200 3.2279 3.4449 1 0 21 1 0 0 2 0 0 0 3.81 -0.90 2 6.98 7.030 -0.05 Oxymorphone Test 8.50 (71) 7.60 1.1430 0.7780 0.3150 -0.0500 1 0 17 4 0 1 4 0 1 3 3.00 0.30 1 1.52 1.210 0.31 Acebutolol Test 9.40 (72) 9.40 1.5890 1.6150 0.6881 0.7141 2 0 18 4 0 0 1 0 0 0 3.17 1.00 1 3.90 3.710 0.19 Dextromoramide Test 5.70 (73) 6.90 3.3260 3.5670 2.5809 2.8219 1 0 25 2 0 1 3 0 0 0 3.81 -0.85 2 7.14 6.970 0.17 Esmolol Test 9.50 (74) 9.40 1.8360 1.9780 0.9297 1.0717 1 0 16 4 0 0 1 0 0 0 3.00 0.33 1 4.24 4.310 -0.07 Sotalol Test 8.80 (75) 8.30 0.7090 0.9670 -0.1398 0.1182 2 0 12 3 0 0 1 0 0 0 3.17 1.25 1 1.75 1.580 0.17 Pemoline Test 10.50 (76) 0.30 1.1790 1.3360 0.2219 0.3789 2 0 9 2 0 1 1 0 0 0 3.17 0.61 1 2.65 2.700 -0.05 Toremifene Test 8.00 (77) 8.70 5.4020 6.2980 4.5851 5.4811 1 0 26 1 0 0 3 0 0 0 4.32 -0.89 3 7.45 7.680 -0.23 Methylphenidate Test 8.90 (78) 9.50 2.2660 2.1800 1.4009 1.3149 1 0 14 2 0 0 2 0 0 0 3.00 -0.31 1 3.71 3.900 -0.19 Phentermine Test 10.10 (79) 9.90 2.5450 1.8400 1.6070 0.9025 0 0 10 0 0 0 1 0 0 0 2.81 0.49 1 2.69 2.910 -0.22

S8.0 Repeatability of retention time in urine and network replicates Retention time for each compound whether derived from the ANN or urine analysis was performed in replicate to asses repeatability of the methods used. Table S3 shows the predicted retention time of each compound (n=86) across 10 individual ANNs. Table S4 shows the variation in experimental retention time for 15 replicated urine analyses.

Table S4. The prediction of retention time for all compounds from 10 individual networks. Networks were all 18:5:4:1 MLPBP with training lasting between 20004000 epochs.

P Compound tR (min)

MLP 1 MLP 2 MLP 3 MLP 4 MLP 5 MLP 6 MLP 7 MLP 8 MLP 9 MLP 10 Mean Atenolol 1.399 1.366 1.633 1.463 1.542 1.507 1.676 1.392 1.773 1.471 1.522 Bisoprolol 4.924 4.939 4.949 5.097 5.059 5.049 4.839 5.087 4.824 4.852 4.962 Carvedilol 6.554 6.558 6.644 6.596 6.777 6.880 6.719 6.490 6.807 6.771 6.680 Labetalol 5.096 5.206 5.217 5.290 5.178 5.274 5.024 5.022 5.126 4.756 5.119 Metipranolol 5.985 5.857 6.022 6.033 6.083 5.871 6.262 6.034 6.097 6.280 6.052 Metoprolol 3.870 4.069 3.875 4.103 4.174 4.023 3.749 3.892 3.792 3.741 3.929 Nadolol 2.555 2.559 2.515 2.559 2.547 2.519 2.648 2.568 2.796 2.675 2.594 Timolol 3.615 3.676 3.648 3.609 3.602 3.721 3.587 3.439 3.711 3.404 3.601 Fenoterol 2.685 2.332 2.279 2.368 2.420 2.471 2.500 2.332 2.391 2.426 2.420 Salmeterol 7.029 6.913 7.265 7.077 7.132 7.217 7.182 6.934 7.302 7.092 7.114 Aminoglutethimide 1.978 2.088 2.075 1.959 1.772 1.910 2.058 2.066 2.108 1.728 1.974 Clomiphene 7.591 7.609 7.489 7.583 7.331 7.300 7.251 7.340 7.388 7.356 7.424 Tamoxifen 7.543 7.574 7.466 7.552 7.291 7.285 7.232 7.330 7.384 7.396 7.405 Bendroflumethiazide 7.178 7.381 7.134 7.295 7.329 7.365 7.348 7.200 7.246 7.157 7.263 Bumetanide 7.453 7.693 7.203 7.556 7.338 7.426 7.349 7.298 7.317 7.298 7.393 Chlorothiazide 1.997 1.925 1.953 1.997 2.114 2.030 1.979 2.017 2.315 1.991 2.032 Chlorthalidone 4.577 4.366 4.258 4.638 4.675 4.665 4.544 4.437 4.491 4.356 4.501 Clopamide 4.285 4.320 4.084 4.448 4.479 4.255 4.375 4.251 4.338 4.196 4.303 Etacrynic acid 7.582 7.719 7.534 7.688 7.676 7.631 7.615 7.545 7.624 7.643 7.626 Furosemide 6.599 6.625 6.753 6.632 6.755 6.682 6.623 6.609 6.711 6.519 6.651 Hydrochlorothiazide 2.242 2.362 2.094 2.357 2.405 2.180 2.206 2.385 1.940 2.288 2.246 Indapamide 6.985 6.798 6.663 6.807 6.831 6.829 6.933 7.058 6.843 7.200 6.895 Torasemide 6.141 5.976 5.867 6.160 6.160 6.062 5.988 6.012 6.073 5.966 6.041 Triamterene 2.930 2.995 2.917 3.084 3.073 2.851 2.774 2.958 2.369 2.825 2.878 Xipamide 7.257 7.213 7.063 7.292 7.272 7.151 7.226 7.195 7.246 7.118 7.203 Amiphenazole 1.967 1.872 1.938 1.972 2.175 2.052 2.037 2.113 1.992 2.220 2.034 Benzoylecgonine 3.495 3.325 3.305 3.311 3.378 3.300 3.378 3.227 3.264 3.256 3.324 Ephedrine 1.741 1.891 1.837 1.893 2.022 1.883 1.828 1.765 1.977 1.832 1.867 Fenproporex 2.735 2.790 2.748 2.794 2.801 2.768 2.786 2.697 2.750 2.682 2.755 Heptaminol 1.273 1.225 1.462 1.398 1.263 1.400 1.200 1.203 1.210 1.311 1.295 Methamphetamine 2.794 2.927 2.796 2.819 2.765 2.695 2.786 2.531 2.801 2.724 2.764

S10

Phendimetrazine 2.552 2.639 2.322 2.495 2.535 2.618 2.741 2.470 2.556 2.609 2.554 Strychnine 3.066 2.793 2.926 2.954 2.913 2.873 2.947 2.950 2.966 2.873 2.926 Codeine 2.052 2.006 1.927 1.795 2.019 2.006 2.012 1.867 2.006 2.047 1.974 Fentanyl 5.805 5.991 5.963 6.061 6.101 5.995 5.962 5.833 5.897 5.943 5.955 Hydromorphone 1.870 1.922 1.676 1.747 1.883 1.894 1.824 1.795 1.882 1.806 1.830 Morphine 1.566 1.660 1.596 1.750 1.807 1.767 1.711 1.778 1.717 1.615 1.697 Pentazocine 4.876 5.130 4.995 5.138 5.071 5.055 5.098 4.931 5.083 5.051 5.043 Desonide 7.152 6.960 6.740 6.992 7.034 6.996 6.994 6.941 6.980 6.922 6.971 Celiprolol 4.505 4.481 4.396 4.588 4.618 4.573 4.485 4.492 4.378 4.452 4.497 Formoterol 3.917 3.935 3.920 3.991 3.997 3.970 3.853 3.769 4.072 3.717 3.914 Terbutaline 1.420 1.370 1.451 1.447 1.529 1.475 1.485 1.369 1.370 1.526 1.444 MDMA 3.006 2.648 2.875 2.852 2.673 2.836 2.536 2.805 2.806 2.539 2.758 Pseudoephedrine 1.754 1.922 1.849 1.913 2.048 1.899 1.863 1.779 2.003 1.845 1.888 Carteolol 2.915 2.917 2.680 2.981 2.971 2.864 2.993 2.810 2.881 2.781 2.879 Oxprenolol 4.722 4.648 4.594 4.541 4.616 4.502 4.672 4.523 4.725 4.406 4.595 Probenecid 7.318 7.474 7.234 7.440 7.350 7.452 7.367 7.129 7.211 7.107 7.308 Dichlorphenamide 4.148 3.997 3.975 4.198 4.173 4.180 4.145 4.063 4.090 4.041 4.101 Benzphetamine 5.629 5.648 5.647 5.603 5.639 5.621 5.786 5.695 5.786 5.648 5.670 Fenfluramine 4.902 5.322 4.910 5.191 5.340 5.159 5.081 5.091 5.089 4.891 5.098 Mephentermine 3.393 3.410 3.509 3.401 3.354 3.389 3.458 3.198 3.339 3.386 3.384 Alprenolol 5.681 5.759 5.674 5.695 5.568 5.783 5.697 5.427 5.791 5.336 5.641 Pindolol 2.489 2.903 2.410 2.827 2.781 2.699 2.871 2.594 2.671 2.439 2.668 Propranolol 5.426 5.802 5.690 5.815 5.837 5.743 5.816 5.796 5.808 5.741 5.747 Raloxifene 6.998 6.968 7.129 6.884 7.334 7.373 7.234 7.159 7.484 7.669 7.223 Hydroflumethiazide 2.894 2.989 2.874 2.884 2.864 2.954 3.056 2.684 2.961 2.609 2.877 Etilefrine 1.377 1.398 1.406 1.443 1.524 1.494 1.410 1.385 1.415 1.457 1.431 Methoxyphenamine 3.365 3.295 3.232 3.363 3.449 3.640 3.576 3.299 3.472 3.318 3.401 Nikethamide 2.149 2.034 2.259 2.104 1.709 2.524 2.189 2.069 2.419 1.899 2.136 pMethyl 2.827 3.031 2.746 3.044 3.038 2.642 2.846 2.991 2.785 2.871 2.882 amphetamine Oxycodone 1.841 1.898 2.211 1.785 1.760 1.775 1.600 1.583 1.675 1.566 1.769 Betaxolol 5.841 5.772 6.320 6.253 6.112 6.323 6.011 6.005 6.259 5.890 6.079 Acetazolamide 2.949 1.255 1.725 1.753 2.047 1.858 1.260 1.846 1.276 1.452 1.742 Amiloride 2.146 1.513 2.012 1.884 1.342 1.527 1.741 1.680 1.114 1.232 1.619 Polythiazide 6.908 7.563 7.044 6.906 6.853 6.915 7.103 7.089 7.393 6.771 7.055 Amphetamine 2.211 2.268 2.263 2.344 2.265 2.183 2.146 2.085 2.262 2.079 2.211 Sibutramine 7.435 7.041 7.086 7.420 7.039 6.797 7.230 7.336 7.060 6.725 7.117 Clenbuterol 3.399 3.423 3.049 3.962 3.829 3.373 3.318 3.614 3.354 3.647 3.497 Modafinil 6.030 6.203 6.168 6.023 6.168 6.698 6.790 5.676 5.282 6.125 6.116 Bambuterol 4.505 4.532 4.243 5.171 4.664 4.165 4.345 4.848 4.739 4.543 4.576 Phenmetrazine 2.266 2.291 2.042 2.240 2.249 2.412 2.395 2.210 2.376 2.180 2.266 Salbutamol 1.398 1.377 1.542 1.463 1.561 1.505 1.544 1.376 1.450 1.535 1.475

S11

Benzthiazide 7.673 7.735 7.565 7.708 7.693 7.698 7.705 7.560 7.582 7.566 7.649 Dimethyl 3.595 3.988 3.354 3.920 3.997 3.433 3.635 4.067 3.559 3.787 3.734 amphetamine MDA 1.646 1.879 1.553 1.778 1.928 1.724 1.605 1.592 1.657 1.688 1.705 Buprenorphine 6.465 6.101 6.796 5.984 5.872 5.509 5.600 6.090 6.552 6.468 6.144 Methadone 7.034 6.998 6.980 7.020 6.694 7.022 6.904 6.637 7.060 6.748 6.910 Oxymorphone 1.562 1.549 1.519 1.634 1.525 1.543 1.361 1.530 1.383 1.362 1.497 Acebutolol 3.873 4.024 3.899 3.727 3.943 3.631 3.913 3.641 4.206 3.710 3.857 Dextromoramide 6.807 5.839 7.144 6.358 6.328 6.977 6.739 7.003 6.982 6.869 6.705 Esmolol 4.562 4.459 4.241 4.372 4.579 4.106 4.582 4.388 4.312 4.427 4.403 Sotalol 1.593 1.598 1.750 1.659 1.832 1.706 1.714 1.586 2.187 1.566 1.719 Pemoline 2.169 2.104 2.651 1.949 2.706 2.809 2.385 2.449 2.852 2.255 2.433 Toremifene 7.546 7.597 7.451 7.559 7.292 7.269 7.220 7.333 7.370 7.340 7.398 Methylphenidate 3.827 3.821 3.714 3.775 4.155 4.079 4.359 3.760 3.713 3.952 3.916 Phentermine 2.595 2.765 2.693 2.844 2.813 2.465 2.642 2.645 2.593 2.669 2.672

S12

Table S5. The experimental retention time of replicate urine samples. The analysis was performed over 3 days with urine samples 15 on the first day, 610 on the second day and 1115 on the final day. Fields marked with (–) indicate that data was not available.

E tR (min)

Day 1 Day 2 Day 3 Compound 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Aminoglutethimide 2.20 2.18 2.20 2.25 2.23 2.21 2.18 2.17 2.16 2.18 2.22 2.25 2.21 2.26 2.21 Amiphenazole 1.76 1.84 1.77 1.95 1.84 1.81 1.83 1.81 1.81 1.82 1.84 1.90 1.80 1.86 1.62 Amphetamine 2.38 2.37 2.37 2.47 2.40 2.40 2.38 2.36 2.36 2.37 2.41 2.42 2.40 2.46 2.34 Bambuterol 4.60 4.59 4.60 4.62 4.59 4.54 4.58 4.58 4.58 4.57 4.58 4.52 4.60 4.55 4.56 Benzoylecgonine 3.31 3.31 3.30 3.31 3.29 3.29 3.30 3.30 3.29 3.30 3.29 3.30 3.28 3.28 3.25 Benzphetamine 5.68 5.59 5.62 5.59 5.60 5.58 5.57 5.57 5.56 5.58 5.61 5.50 5.57 5.53 5.60 Buprenorphine 6.55 6.55 6.57 6.54 6.49 6.53 6.53 6.52 6.52 6.50 6.49 6.51 6.49 6.50 Celiprolol 4.47 4.46 4.46 4.48 4.45 4.40 4.44 4.44 4.44 4.44 4.45 4.39 4.46 4.40 4.40 Clenbuterol 3.77 3.77 3.75 3.78 3.74 3.73 3.75 3.75 3.75 3.75 3.74 3.73 3.73 3.73 3.71 Codeine 2.18 2.15 2.19 2.25 2.22 2.21 2.17 2.14 2.14 2.17 2.20 2.23 2.18 2.23 2.19 Desonide 7.00 6.98 6.99 6.99 6.98 6.98 6.98 6.97 6.98 6.97 6.98 6.98 6.97 6.97 6.98 Dextromoramide 6.98 6.97 6.98 6.98 6.97 6.95 6.96 6.96 6.96 6.97 6.95 6.95 6.95 6.95 6.95 Dimethylamphetamine 2.78 2.76 2.78 2.95 2.81 2.79 2.81 2.81 2.73 2.82 2.69 2.76 2.80 2.89 2.66 Ephedrine 1.89 1.96 1.90 2.07 1.97 1.94 1.96 1.94 1.94 1.96 1.93 2.03 1.90 2.01 1.72 Esmolol 4.31 4.31 4.31 4.32 4.30 4.26 4.30 4.29 4.29 4.28 4.29 4.26 4.32 4.26 4.24 Fenfluramine 5.20 5.27 5.24 5.28 5.19 5.05 5.26 5.27 5.23 5.18 5.17 5.10 5.24 5.12 5.08 Fenoterol 2.26 2.23 2.25 2.32 2.27 2.25 2.26 2.25 2.22 2.24 2.27 2.29 2.26 2.30 2.22 Fenproporex 2.80 2.77 2.80 3.00 2.83 2.81 2.85 2.85 2.75 2.85 2.72 2.77 2.83 2.95 2.70 Fentanyl 5.99 6.01 6.04 5.99 6.02 5.94 5.97 5.96 5.96 5.95 6.17 5.84 5.94 5.94 6.00 Formoterol 3.95 3.94 3.93 3.95 3.92 3.90 3.92 3.92 3.91 3.91 3.93 3.91 3.92 3.89 3.88 Heptaminol 1.23 1.29 1.20 1.30 1.26 1.21 1.28 1.29 1.32 1.22 1.31 1.35 1.24 1.25 1.09 Labetalol 5.11 5.10 5.10 5.10 5.08 5.05 5.09 5.07 5.06 5.05 5.09 5.03 5.08 5.04 5.04 MDA 2.53 2.50 2.51 2.66 2.53 2.52 2.55 2.52 2.50 2.53 2.50 2.55 2.53 2.59 2.45 Methadone 7.05 7.03 7.04 7.03 7.03 7.02 7.02 7.02 7.02 7.02 7.01 7.00 7.00 7.01 7.02 Methamphetamine 2.63 2.59 2.61 2.80 2.61 2.60 2.64 2.61 2.59 2.64 2.59 2.64 2.65 2.72 2.56 Methoxyphenamine 3.31 3.30 3.30 3.32 3.30 3.29 3.29 3.31 3.29 3.30 3.32 3.28 3.28 3.31 3.26 Methylphenidate 3.92 3.90 3.90 3.92 3.89 3.86 3.89 3.89 3.88 3.88 3.90 3.87 3.87 3.87 3.85 Metipranolol 5.72 5.62 5.66 5.61 5.65 5.60 5.61 5.60 5.59 5.60 5.67 5.54 5.61 5.57 5.66 Metoprolol 3.84 3.81 3.82 3.84 3.80 3.77 3.80 3.80 3.80 3.81 3.82 3.78 3.80 3.78 3.75 Modafinil 6.01 6.01 6.01 6.00 6.01 5.99 5.99 5.99 5.98 5.98 6.00 6.01 5.99 5.99 5.99 Morphine 1.05 1.10 0.98 1.10 1.08 0.95 1.08 1.09 1.13 1.10 1.08 1.14 1.06 1.08 0.96 Nadolol 2.83 2.81 2.82 2.88 2.83 2.81 2.82 2.83 2.79 2.83 2.79 2.81 2.80 2.86 2.77 Nikethamide 1.99 2.02 1.99 2.03 2.00 2.00 2.00 2.01 2.02 1.99 2.05 2.10 2.05 2.06 1.99

S13

Oxprenolol 4.68 4.66 4.67 4.71 4.65 4.61 4.66 4.66 4.65 4.63 4.66 4.60 4.70 4.60 4.60 Oxycodone 2.50 2.47 2.48 2.58 2.49 2.47 2.50 2.49 2.45 2.49 2.46 2.52 2.50 2.54 2.42 Oxymorphone 1.16 1.21 1.14 1.22 1.17 1.13 1.19 1.22 1.25 1.17 1.27 1.29 1.16 1.18 1.07 Pemoline 2.68 2.70 2.66 2.69 2.69 2.67 2.68 2.69 2.67 2.67 2.69 2.70 2.69 2.69 2.64 Pentazocine 5.07 5.07 5.07 5.09 5.05 4.95 5.05 5.04 5.03 5.03 5.05 4.98 5.05 5.00 5.00 Phentermine 3.09 2.91 3.03 3.05 3.05 3.03 2.98 2.95 2.88 2.99 2.90 2.99 2.93 3.08 2.89 Pindolol 2.85 2.74 2.80 2.91 2.83 2.79 2.80 2.78 2.72 2.81 2.77 2.79 2.77 2.87 2.74 Propranolol 5.86 5.78 5.81 5.76 5.79 5.74 5.75 5.75 5.73 5.73 5.82 5.67 5.75 5.72 5.80 Salbutamol 1.40 1.48 1.39 1.47 1.44 1.42 1.47 1.47 1.48 1.39 1.46 1.52 1.41 1.44 1.22 Salmeterol 7.12 7.12 7.12 7.11 7.11 7.11 7.11 7.10 7.11 7.11 7.10 7.09 7.10 7.09 7.10 Sotalol 1.48 1.58 1.48 1.61 1.52 1.51 1.57 1.56 1.57 1.51 1.54 1.62 1.50 1.55 1.32 Terbutaline 1.35 1.44 1.34 1.42 1.37 1.36 1.42 1.43 1.42 1.32 1.42 1.46 1.36 1.38 1.18 Timolol 3.63 3.62 3.61 3.63 3.60 3.58 3.61 3.60 3.61 3.61 3.61 3.59 3.59 3.59 3.56 Toremifene 7.68 7.68 7.69 7.68 7.68 7.67 7.67 7.67 7.67 7.68 7.65 7.66 7.65 7.65 7.66 Acebutolol 3.70 3.71 3.69 3.71 3.68 3.66 3.68 3.67 3.68 3.67 3.67 3.67 3.68 3.72 3.67 Acetazolamide 1.73 1.78 1.78 1.77 1.78 1.76 1.75 1.77 1.71 1.77 1.79 1.80 1.79 1.82 1.81 Alprenolol 5.97 5.93 5.94 5.92 5.92 5.88 5.88 5.90 5.89 5.87 5.95 5.79 5.92 5.87 5.98 Amiloride 1.48 1.58 1.58 1.57 1.60 1.58 1.58 1.59 1.49 1.57 1.51 1.66 1.55 1.66 1.52 Atenolol 1.49 1.62 1.60 1.57 1.59 1.58 1.58 1.62 1.50 1.59 1.52 1.65 1.57 1.60 1.53 Bendroflumethiazide 7.21 7.21 7.21 7.21 7.20 7.20 7.19 7.20 7.19 7.19 7.21 7.20 7.20 7.21 7.21 Benzthiazide Betaxolol 6.26 6.11 6.13 6.12 6.10 6.04 6.09 6.10 6.09 6.07 6.27 6.04 6.10 6.06 6.15 Bisoprolol 5.04 5.03 5.02 5.01 5.01 4.94 5.01 5.02 5.01 4.97 5.02 4.95 5.02 4.96 4.99 Bumetanide 7.49 7.49 7.48 7.49 7.48 7.47 7.47 7.48 7.47 7.47 7.48 7.48 7.47 7.48 7.48 Carteolol 2.66 2.65 2.65 2.70 2.68 2.66 2.65 2.65 2.63 2.66 2.61 2.65 2.65 2.70 2.67 Carvedilol 6.85 6.81 6.82 6.81 6.83 6.79 6.79 6.80 6.79 6.79 6.79 6.79 6.81 6.79 6.84 Chlorothiazide 1.88 1.93 1.92 1.90 1.93 1.91 1.90 1.91 1.88 1.90 1.93 1.95 1.93 1.97 1.95 Chlorthalidone 4.45 4.47 4.45 4.46 4.45 4.42 4.44 4.44 4.44 4.41 4.44 4.46 4.46 4.47 4.47 Clomiphene 7.68 7.68 7.68 7.68 7.68 7.67 7.67 7.68 7.66 7.67 7.65 7.65 7.64 7.66 7.65 Clopamide 4.25 4.26 4.24 4.26 4.24 4.22 4.24 4.25 4.25 4.25 4.28 4.30 4.30 4.32 4.30 Dichlorphenamide 3.97 4.01 4.00 4.01 3.99 3.97 3.98 3.98 3.99 3.96 3.98 4.00 4.00 4.05 4.02 Etacrynic acid 7.68 7.68 7.69 7.68 7.68 7.68 7.67 7.67 7.67 7.67 7.67 7.67 7.67 7.69 7.68 Etilefrine 0.88 0.96 0.93 0.94 0.95 0.94 0.96 0.95 0.94 0.96 0.86 0.90 0.90 0.90 0.92 Hydrochlorothiazide 2.12 2.19 2.16 2.15 2.15 2.14 2.13 2.16 2.11 2.14 2.15 2.18 2.17 2.20 2.19 Hydroflumethiazide 3.15 3.18 3.17 3.18 3.16 3.16 3.15 3.14 3.15 3.14 3.15 3.19 3.18 3.23 3.22 Hydromorphone 1.36 1.48 1.48 1.44 1.49 1.48 1.46 1.47 1.45 1.47 1.38 1.53 1.43 1.47 1.40 Indapamide 6.82 6.82 6.82 6.82 6.82 6.80 6.80 6.81 6.80 6.80 6.82 6.81 6.81 6.82 6.82 MDMA 2.73 2.73 2.75 2.86 2.77 2.77 2.77 2.76 2.69 2.76 2.65 2.74 2.78 2.85 2.72 Phendimetrazine 2.58 2.55 2.57 2.71 2.59 2.57 2.60 2.55 2.52 2.60 2.49 2.60 2.58 2.68 2.61 Phenmetrazine 2.53 2.51 2.54 2.63 2.55 2.53 2.56 2.52 2.48 2.56 2.46 2.56 2.55 2.63 2.56 pmethylamphetamine 3.41 3.45 3.43 3.44 3.42 3.40 3.42 3.43 3.41 3.41 3.39 3.42 3.41 3.46 3.43

S14

Polythiazide Probenecid 7.56 7.56 7.55 7.56 7.55 7.54 7.54 7.55 7.54 7.54 7.55 7.54 7.54 7.55 7.56 Pseudoephedrine 1.93 2.00 1.98 2.08 2.02 2.00 2.00 1.99 1.95 2.01 1.92 2.09 1.95 2.11 1.92 Raloxifene Sibutramine 7.16 7.15 7.16 7.15 7.15 7.14 7.13 7.14 7.14 7.14 7.13 7.12 7.12 7.13 7.14 Strychnine 3.07 3.01 3.04 3.06 3.04 3.05 3.03 2.99 2.99 3.02 3.01 3.05 3.02 3.09 3.09 Tamoxifen 7.77 7.78 7.77 7.77 7.77 7.76 7.77 7.77 7.77 7.77 7.75 7.75 7.74 7.75 7.74 Torasemide 6.12 6.02 6.04 6.04 6.04 5.97 5.99 5.99 6.00 5.99 6.18 5.95 5.97 5.98 6.07 Triamterene 3.04 3.00 3.02 3.04 3.01 3.02 3.01 2.96 2.96 3.01 2.97 3.01 3.00 3.08 3.05 Xipamide 7.33 7.33 7.33 7.33 7.33 7.32 7.32 7.32 7.31 7.32 7.33 7.32 7.33 7.33 7.33

S 9.0 Substitution of experimentally-derived p Ka with predicted p Ka

The use of a separate p Ka prediction software package was investigated as a possible alternative to experimentally derived p Ka data as a molecular descriptor.

(a) 9 (b) 1.5 Training set: y = 0.9916x + 0.0884; R² = 0.9745 8 Verification set: y = 1.0548x + 0.2042; R² = 0.9965 Test set: y = 0.936x + 0.2212; R² = 0.9472 7 1

6 Training Verification 0.5 5 Test (mins)

E 4 r t 0 3

ResidualError (mins) 2 -0.5 1

0 -1 0 2 4 6 8 P Training Verification Test tr (mins) (n=61) (n=10) (n=15)

E P Figure S3. (a) tr vs tr using the reoptimised 18:5:4:1 multilayer perceptron (inset) using predicted p Ka input data P (trained for 2200 epochs). (b) residual errors in tr using the predicted p Ka input data for all analytes (n = 86).

S15

References [1] Veigl, E., Böhs, B., Mandl, A., Krametter, D., & Lindner, W. (1995). Evaluation of silica gelbased brush type chiral cation exchangers with (S)N(3, 5dinitrobenzoyl) tyrosine as chiral selector: attempt to interpret the discouraging results. Journal of Chromatography A, 694 (1), 151161. [2] Haeusler, G., Schliep, H. J., Schelling, P., Becker, K. H., Klockow, M., Minck, K. O. Enenkel, H.J., Schulze, E., Bergmann, R., Schmitges, C. –J., Seyfried, C. & Harting, J. (1985). High [beta] 1Selectivity and Favourable Pharmacokinetics as the Outstanding Properties of Bisoprolol. Journal of Cardiovascular Pharmacology , 8, S2S15. [3] PerišićJanjić, N. U., Lučić, B., Janjić, N. J., & Agbaba, D. (2003). Study of the lipophilicity and retention behavior of some betaadrenoceptor antagonists. JPC-Journal of Planar Chromatography-Modern TLC , 16 (5), 347350. [4] LlorentMartínez, E. J., Šatínský, D., & Solich, P. (2007). Fluorescence optosensing implemented with sequential injection analysis: a novel strategy for the determination of labetalol. Analytical and bioanalytical chemistry , 387 (6), 20652069. [5] Schappler, J., Guillarme, D., Rudaz, S., & Veuthey, J. L. (2008). Microemulsion electrokinetic chromatography hyphenated to atmospheric pressure photoionization mass spectrometry. Electrophoresis , 29 (1), 1119. [6] Devlin, R. G., Duchin, K. L., & Fleiss, P. M. (1981). Nadolol in human serum and breast milk. British journal of clinical pharmacology , 12 (3), 393396. [7] Kanikkannan, N., Singh, J., & Ramarao, P. (2001). In vitro transdermal iontophoretic transport of timolol maleate: effect of age and species. Journal of controlled release , 71 (1), 99105. [8] Additives. Meeting, et al. (2005). Toxicological Evaluation of Certain Veterinary Drug Residues in Food , World Health Organization, IPCS, International Programme on Chemical Safety. [9] Dougall, I. G., Harper, D., Jackson, D. M., & Leff, P. (1991). Estimation of the efficacy and affinity of the β2 ‐adrenoceptor agonist salmeterol in guinea ‐pig trachea. British journal of pharmacology , 104 (4), 10571061. [10] Menge, G., & Dubois, J. P. (1984). Determination of aminoglutethimide and N acetylaminoglutethimide in human plasma by highperformance liquid chromatography. Journal of chromatography , 310 (2), 431437. [11] Lopez, C., Nehme, R., Claude, B., Morin, P., Max, J. P., Pena, R., Pelissou, M., & Ribet, J. P. (2012). A convenient approach to simultaneous analysis of a pharmaceutical drug and its counterion by CE using dualopposite end injection and contactless conductivity detection. Chromatographia , 75 (12), 2532. [12] de Santana, D. P., Braga, R. M. C., Strattmman, R., Albuquerque, M. M., Bedor, D. C. G., Leal, L. B., & da Silva, J. A. (2008). Reversed phase HPLC determination of tamoxifen in dog plasma and its pharmacokinetics after a single oral dose administration. Química Nova , 31 (1), 47 [13] Giancotti, V. HPLCMS technique equipped with a high resolution mass analyser of new design (LTQOrbitrap). Application to pharmacological, antidoping and molecular diagnostic problems [14] Song, B., Galande, A. K., Kodukula, K., Moos, W. H., & Miller, S. M. (2011). Evaluation of the pKa values and ionization sequence of bumetanide using 1H and 13C NMR and UV spectroscopy. Drug Development Research , 72 (5), 416426.

S16

[15] Baer, J. E., Leidy, H. L., Brooks, A. V., & Beyer, K. H. (1959). The physiological disposition of chlorothiazide (Diuril) in the dog. Journal of Pharmacology and Experimental Therapeutics , 125 (4), 295302 [16] Balesteros, M. R., Faria, A. F., & de Oliveira, M. A. (2007). Determination of losartan associated with chlorthalidone or hydrochlorothiazide in capsules by capillary zone electrophoresis. Journal-Brazilian Chemical Society , 18 (3), 554 [17] Barroso, M. B., Jiménez, R. M., & Alonso, R. M. (1997). High performance liquid chromatographyelectrochemical detection applied to monitoring clopamide in human urine and pharmaceutical formulations. Journal of liquid chromatography & related technologies , 20 (4), 637650 [18] CampínsFalcó, P., HerráezHernández, R., & SevillanoCabeza, A. (1992). Sensitive determination of ethacrynic acid in urine samples by reversedphase liquid chromatography with ultraviolet detection using solidphase extraction techniques for sample cleanup. Analytica chimica acta , 270 (1), 3944 [19] Deetjen, P. (1966). Micropuncture Studies on Site and Mode of Diuretic Action of furosemide. Annals of the New York Academy of Sciences , 139 (2), 408415. [20] Dollery, C. Therapeutic Drugs , Churchill Livingstone, Edinburgh, 1999 pp. C230-234 , (137), 141. [21] Caruso, F. S., Szabadi, R. R., & Vukovich, R. A. (1983). Pharmacokinetics and clinical pharmacology of indapamide. American heart journal , 106 (1), 212220 [22] Cadwallader, A. B., De La Torre, X., Tieri, A., & Botrè, F. (2010). The abuse of diuretics as performance ‐enhancing drugs and masking agents in sport doping: pharmacology, toxicology and analysis. British journal of pharmacology , 161 (1), 116 [23] Knauf, H., Wais, U., Lübcke, R., & Albiez, G. (1976). On the Mechanism of Action of Triamterene: Effects on Transport of Na+, K+, and HVHCO ‐3‐ions. European Journal of Clinical Investigation , 6(1), 4350 [24] Hemplemann, F. W., Arzneim-Forsch., 1977, 27, 2140 [25] Esteva, A. M., Zapardiel, A., Bermejo, E., Chicharro, M., & Hernández, L. (2003). Electrochemical and spectral behaviour of 2, 4diamino5phenyltiazole determined in urine. Chemia analityczna , 48 (5), 839852. [26] Foulon, C., Menet, M. C., Manuel, N., PhamHuy, C., Galons, H., Claude, J. R., & Guyon, F. (1999). Rapid analysis of benzoylecgonine, cocaine, and cocaethylene in urine, serum, and saliva by isocratic highperformance liquid chromatography with diodearray detection. Chromatographia , 50 (1112), 721727 [27] Hasemann, P., ter Balk, M., Preu, L., & Wätzig, H. (2007). Separation of cold medicine ingredients using a precise MEKC method at elevated pH. Electrophoresis , 28 (11), 1779 1787. [28] Stevenson, G. W., & Williamson, D. (1958). Base Strengths of Cyanoamines1. Journal of the American Chemical Society , 80 (22), 59435947. [29] Morros, A., Borja, L., & Segura, J. (1985). Determination of heptaminol in plasma by thinlayer chromatography and in situ fluorimetry. Journal of pharmaceutical and biomedical analysis , 3(2), 149156 [30] Fan, Y., Feng, Y. Q., Zhang, J. T., Da, S. L., & Zhang, M. (2005). Poly (methacrylic acidethylene glycol dimethacrylate) monolith intube solid phase microextraction coupled to high performance liquid chromatography and analysis of amphetamines in urine samples. Journal of Chromatography A , 1074 (1), 916

S17

[31] Linden, C. H., Kulig, K. W., & Rumack, B. H. (1985). Amphetamines. Advanced Emergency Nursing Journal , 7(3), 1832 [32] Zhang, J., Wang, S., Chen, X., Hu, Z., & Ma, X. (2003). Capillary electrophoresis with fieldenhanced stacking for rapid and sensitive determination of strychnine and brucine. Analytical and bioanalytical chemistry , 376 (2), 210213 [33] O'Neal, C. L., Crouch, D. J., Rollins, D. E., Fatah, A., & Cheever, M. L. (1999). Correlation of saliva codeine concentrations with plasma concentrations after oral codeine administration. Journal of analytical toxicology , 23 (6), 452459 [34] Skacel, M., Knott, C., Reynolds, F., & Aps, C. (1986). Extracorporeal circuit sequestration of fentanyl and alfentanil. British journal of anaesthesia , 58 (9), 947949 [35] Goforth, H. W. (2010). HydromorphoneOROS formulation. Expert Opinion on Pharmacotherapy , 11 (7), 12071214 [36] Polard, E., Le Corre, P., Chevanne, F., & Le Verge, R. (1996). In vitro and in vivo evaluation of polylactide and polylactidecoglycolide microspheres of morphine for site specific delivery. International journal of pharmaceutics , 134 (1), 3746 [37] Ogami, S., Hayashi, S., Shibaji, T., & Umino, M. (2008). Pentazocine transport by squarewave AC iontophoresis with an adjusted duty cycle. Journal of medical and dental sciences , 55 (1), 1527 [38] Chemical Book. (2008). Retrieved December, 2012 from http://www.chemicalbook.com [39] Lipka, E., Lee, I. D., Langguth, P., SpahnLangguth, H., Mutschler, E., & Amidon, G. L. (1995). Celiprolol doublepeak occurrence and gastric motility: nonlinear mixed effects modeling of bioavailability data obtained in dogs. Journal of pharmacokinetics and biopharmaceutics , 23 (3), 267286 [40] Mukherjee, M., Pritchard, D. I., & Bosquillon, C. (2012). Evaluation of airinterfaced Calu3 cell layers for investigation of inhaled drug interactions with organic cation transporters in vitro International Journal of Pharmaceutics, 426(1), 714. [41] De Ridder, D. J., Verliefde, A. R. D., Heijman, S. G. J., Verberk, J. Q. J. C., Rietveld, L. C., Van der Aa, L., Amy, G.L., T. J., & Van Dijk, J. C. (2011). Influence of natural organic matter on equilibrium adsorption of neutral and charged pharmaceuticals onto activated carbon. Water Science & Technology , 63 (3), 416423. [42] Pichini, S. (2005). Distribution of 3, 4Methylenedioxymethamphetamine (MDMA) in non conventional matrices and its applications in clinical toxicology. [43] Chambers, E., Diehl, D., & Mazzeo, J. (2005). Simple and Fast SPEUPLCMSMS Method for an Allergy Tablet Mixture of Acids and Bases. [44] Florence, A. T. and D. Attwood (2011). Physicochemical Principles of Pharmacy , Pharmaceutical Press. [45] Van Gulpen, C., Brokerhof, A. W., Van der Kaay, M., Tjaden, U. R., & Mattie, H. (1986). Determination of benzylpenicillin and probenecid in human body fluids by high performance liquid chromatography. Journal of Chromatography B: Biomedical Sciences and Applications, 381, 365372. [46] Maren, T. H. (1995). The development of topical carbonic anhydrase inhibitors. Journal of Glaucoma, 4(1), 4962. [47] Morozowich, W., Chulski, T., Hamlin, W. E., Jones, P. M., Northam, J. I., Purmalis, A., & Wagner, J. G. (1962). Relationship between in vitro dissolution rates, solubilities, and LT50's in mice of some salts of benzphetamine and etryptamine. Journal of Pharmaceutical Sciences , 51 (10), 993996

S18

[48] Randhawa, M. A., Iqbal, A., Nasimullah, M., Akhtar, M., Yousaf, S. M., & Turner, P. (1995). HendersonHasselbalch equation is inadequate for the measurement of transmembrane diffusion of drugs and buccal drug absorption is a useful alternative. General Pharmacology: The Vascular System , 26 (4), 875879 [49] Myung, S. W., Yoon, S. H., & Kim, M. (2003). Analysis of benzene ethylamine derivatives in urine using the programmable dynamic liquidphase microextraction (LPME) device. Analyst , 128 (12), 14431446 [50] Shalaeva, M., Kenseth, J., Lombardo, F., & Bastin, A. (2008). Measurement of dissociation constants (pKa values) of organic compounds by multiplexed capillary electrophoresis using aqueous and cosolvent buffers. Journal of pharmaceutical sciences , 97 (7), 25812606 [51] Teeter, J. S., & Meyerhoff, R. D. (2002). Environmental fate and chemistry of raloxifene hydrochloride. Environmental toxicology and chemistry , 21 (4), 729736 [52] Smith, R. B., Smith, R. V., & Yakatan, G. J. (1976). Spectrofluorometric determination of hydroflumethiazide in plasma and urine. Journal of pharmaceutical sciences , 65 (8), 12081211 [53] Schwarz, M. A., Neubert, R. H., & Dongowski, G. (1996). Characterization of Interactions Between Bile Salts and Drugs by Micellar Electrokinetic Capillary Chromatography. Part I. Pharmaceutical research, 13(8), 11741180 [54] Freudenthaler, S., Meineke, I., Schreeb, K. H., Boakye, E., GundertRemy, U., & Gleiter, C. H. (1998). Influence of urine pH and urinary flow on the renal excretion of memantine. British journal of clinical pharmacology , 46 , 541546 [55] Yalkowsky, S. & Sanghvi, T. (2007). U.S Patent No. 10/548,000 [56] Twitchett, P. J., Gorvin, A. E. P., & Moffat, A. C. (1976). Highpressure liquid chromatography of drugs: II. An evaluation of a microparticulate cationexchange column. Journal of Chromatography A, 120(2), 359368 [57] Kuo, P. C., Liu, J. C., Chang, S. F., & Chien, Y. W. (1989). Invitro transdermal permeation of oxycodone:(I) effect of PH, delipidization and skin stripping. Drug Development and Industrial Pharmacy , 15 (8), 11991215 [58] Kenny, A. D. (2008). Role of carbonic anhydrase in bone: plasma acetazolamide concentrations associated with inhibition of bone loss. Pharmacology, 31(2), 97107. [59] Kaczorowski, G. J., Barros, F., Dethmers, J. K., Trumble, M. J., & Cragoe Jr, E. J. (1985). Inhibition of sodiumcalcium exchange in pituitary plasma membrane vesicles by analogs of amiloride. Biochemistry, 24(6), 13941403 [60] Hennig, U. G. G., Chatten, L. G., Moskalyk, R. E., & Ediss, C. (1981). Benzothiadiazine dissociation constants. Part I. Ultraviolet spectrophotometric pKa determinations. Analyst , 106 (1262), 557564 [61] Carvalho, J. M., da Silva, A. R., e Ricardo, A. L. D. C., Aucélio, Q., e Katia, A. L. M. A., & Leandro, C. (2012). Voltammetric determination of sibutramine in beverages and in pharmaceutical formulations. Quim. Nova , 35 (5), 988992 [62] Koole, A., Jetten, A. C., Luo, Y., Franke, J. P., & De Zeeuw, R. A. (1999). Rapid Extraction of Clenbuterol from Human and Calf Urine Using Empore™ C8 Extraction Disks. Journal of analytical toxicology , 23 (7), 632365 [63] Wang, W., Xiang, S., Zhou, X., Ji, Y., & Xiang, B. (2011). Enantiomeric Separation and Determination of the Enantiomeric Impurity of Armodafinil by Capillary Electrophoresis with Sulfobutyl Etherβcyclodextrin as Chiral Selector. Molecules, 17(1), 303314.

S19

[64] Palmarsdottir, S., Mathiasson, L., Jönsson, J. Å., & Edholm, L. E. (1997). Determination of a basic drug, bambuterol, in human plasma by capillary electrophoresis using double stacking for large volume injection and supported liquid membranes for sample pretreatment. Journal of Chromatography B: Biomedical Sciences and Applications, 688(1), 127134. [65] Vree, T. B., Muskens, A. T. J. M., & Rossum, J. V. (1969). Some physico ‐chemical properties of amphetamine and related drugs. Journal of Pharmacy and Pharmacology , 21 (11), 774775. [66] Deñola, N. L., Quiming, N. S., Saito, Y., Catabay, A. P., & Jinno, K. (2009). Sensitive Micellar Electrokinetic Chromatographic Determination of Salbutamol, Guaifenesin, and Dyphylline in Oral Formulations. Journal of Liquid Chromatography & Related Technologies®, 32(10), 14071422. [67] Neidner, W., Karsten, M., Liu, X., Swart, R., Steiner, F. & McLeod, F. (2009). Mixed Mode ReversedPhase Columns Providing a Unique Selectivity Tool in Method Scouting Studies for Pharmaceutical Analysis. Dionex. [68] Brittain, H. G. (2007). Profiles of Drug Substances, Excipients and Related Methodology: Critical Compilation of pKa Values for Pharmaceutical Substances , Elsevier Science. [69] Fan, Y., Feng, Y. Q., Zhang, J. T., Da, S. L., & Zhang, M. (2005). Poly (methacrylic acidethylene glycol dimethacrylate) monolith intube solid phase microextraction coupled to high performance liquid chromatography and analysis of amphetamines in urine samples. Journal of Chromatography A , 1074 (1), 916 [70] Avdeef, A., Barrett, D. A., Shaw, P. N., Knaggs, R. D., & Davis, S. S. (1996). Octanol, chloroform, and propylene glycol dipelargonatwater partitioning of morphine6 glucuronide and other related opiates. Journal of medicinal chemistry , 39 (22), 43774381 [71] Casy, A. F. and R. T. Parfitt (1986). Opioid Analgesics , Springer. [72] Boutroy, M. J., Bianchetti, G., Dubruc, C., Vert, P., & Morselli, P. L. (1986). To nurse when receiving acebutolol: is it dangerous for the neonate?. European journal of clinical pharmacology , 30 (6), 737739. [73] Pryanishnikova, N. T., & Raevskii, K. S. (1973). Ionization constants of some analgesics with narcotic action and their painrelieving activity. Bulletin of Experimental Biology and Medicine , 75 (3), 291293 [74] Fan, C. D., Zhao, H., & Chow, M. S. (1991). Simple and rapid highperformance liquid chromatographic assay for esmolol. Journal of Chromatography B: Biomedical Sciences and Applications , 570 (1), 217223. [75] Kärkäinen, S. (1984). Highperformance liquid chromatographic determination of sotalol in biological fluids. Journal of Chromatography B: Biomedical Sciences and Applications, 336(1), 313319. [76] Vermeulen, N. P., Teunissen, M. W., & Breimer, D. D. (1979). Pharmacokinetics of pemoline in plasma, saliva and urine following oral administration. British journal of clinical pharmacology, 8(5), 459463. [77] Af Ursin, K., Salmia, J., Niskanen, H., Kortesuo, P., Kananen, M., Kiesvaara, J., & Otsomaa, L. (2004). European Patent No. EP 1140197. Munich, Germany: European Patent Office. [78] Marchei, E., Farré, M., Pardo, R., GarciaAlgar, O., Pellegrini, M., Pacifici, R., & Pichini, S. (2010). Correlation between methylphenidate and ritalinic acid concentrations in oral fluid and plasma. Clinical chemistry , 56 (4), 585592

S20

[79] Katsu, T., Ido, K., & Kataoka, K. (2001). Acyclic neutral carrierbased polymer membrane electrode for a stimulant, phentermine. Analytical sciences , 17 (6), 745749

S21