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 octanol-water 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 experimentally-derived 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 MLP-BP, 18:5:4:1 structure. E P Figure S3. (a) tr vs tr using the re-optimised 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. S1 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 MLP-BP with training lasting between 2000-4000 epochs. Table S5 . The experimentally-measured retention time of replicate urine samples. The analysis was performed over 3 days with urine samples 1-5 on the first day, 6-10 on the second day and 11-15 on the final day. Fields marked with (–) indicate that data was not available. S2 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 29122-68-7 Bisoprolol O(c1ccc(cc1)COCCOC(C)C)CC(O)CNC(C)C 66722-44-9 Carvedilol O(c4ccccc4OCCNCC(O)COc3cccc2c3c1c(cccc1)n2)C 72956-09-3 Labetalol O=C(c1cc(ccc1O)C(O)CNC(C)CCc2ccccc2)N 36894-69-6 Metipranolol O=C(Oc1c(c(c(OCC(O)CNC(C)C)cc1C)C)C)C 22664-55-7 Metoprolol O(c1ccc(cc1)CCOC)CC(O)CNC(C)C 51384-51-1 Nadolol OC(CNC(C)(C)C)COc1cccc2c1C[C@H](O)[C@H](O)C2 42200-33-9 Timolol O[C@H](COc1nsnc1N2CCOCC2)CNC(C)(C)C 26839-75-8 Fenoterol Oc1cc(cc(O)c1)C(O)CNC(C)Cc2ccc(O)cc2 13392-18-2 Salmeterol OCc1cc(ccc1O)[C@H](O)CNCCCCCCOCCCCc2ccccc2 89365-50-4 Aminoglutethimide O=C1NC(=O)CCC1(c2ccc(N)cc2)CC 125-84-8 Clomiphene Cl/C(c1ccccc1)=C(/c2ccc(OCCN(CC)CC)cc2)c3ccccc3 911-45-5 Tamoxifen O(c1ccc(cc1)/C(c2ccccc2)=C(\c3ccccc3)CC)CCN(C)C 10540-29-1 Bendroflumethiazide FC(F)(F)c3c(cc1c(NC(NS1(=O)=O)Cc2ccccc2)c3)S(=O)(=O)N 73-48-3 Bumetanide O=S(=O)(c2cc(cc(NCCCC)c2Oc1ccccc1)C(=O)O)N 28395-03-1 Chlorothiazide O=S(=O)(c1c(Cl)cc2c(c1)S(=O)(=O)/N=C\N2)N 58-94-6 Chlorthalidone O=S(=O)(N)c1c(Cl)ccc(c1)C2(O)c3ccccc3C(=O)N2 77-36-1 Clopamide O=C(NN1C(CCCC1C)C)c2ccc(Cl)c(c2)S(=O)(=O)N 636-54-4 Etacrynic acid Clc1c(C(=O)\C(=C)CC)ccc(OCC(=O)O)c1Cl 58-54-8 Furosemide O=S(=O)(N)c1c(Cl)cc(c(C(=O)O)c1)NCc2occc2 54-31-9 Hydrochlorothiazide O=S(=O)(c1c(Cl)cc2c(c1)S(=O)(=O)NCN2)N 58-93-5 Indapamide O=S(=O)(N)c1c(Cl)ccc(c1)C(=O)NN3c2ccccc2CC3C 26807-65-8 Torasemide O=S(=O)(c2c(Nc1cc(ccc1)C)ccnc2)NC(=O)NC(C)C 56211-40-6 Triamterene C1=CC=C(C=C1)C2=NC3=C(N=C(N=C3N=C2N)N)N 396-01-0 Xipamide CC1=C(C(=CC=C1)C)NC(=O)C2=CC(=C(C=C2O)Cl)S(=O)(=O)N 14293-44-8 Amiphenazole C1=CC=C(C=C1)C2=C(N=C(S2)N)N 490-55-1 Benzoylecgonine CN1[C@H]2CC[C@@H]1[C@H]([C@H](C2)OC(=O)c3ccccc3)C(=O)O 519-09-5 Ephedrine O[C@H](c1ccccc1)[C@@H](NC)C 299-42-3 Fenproporex N#CCCNC(Cc1ccccc1)C 16397-28-7 Heptaminol OC(C)(C)CCCC(N)C 372-66-7 Methamphetamine N(C(Cc1ccccc1)C)C 537-46-2 Phendimetrazine O2C(c1ccccc1)C(N(C)CC2)C 634-03-7 Strychnine O=C7N2c1ccccc1[C@@]64[C@@H]2[C@@H]3[C@@H](OC/C=C5\[C@@H]3C[C 57-24-9 @@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 76-57-3 Fentanyl O=C(CC)N(C1CCN(CC1)CCC2=CC=CC=C2)C3=CC=CC=C3 437-38-7 Hydromorphone O=C4[C@@H]5Oc1c2c(ccc1O)C[C@H]3N(CC[C@]25[C@H]3CC4)C 466-99-9 S3 Morphine CN1CC[C@]23C4=C5C=CC(O)=C4O[C@H]2[C@@H](O)C=C[C@H]3[C@H]1C5 57-27-2 Pentazocine Oc1ccc3c(c1)[C@]2([C@H]([C@H](N(CC2)C\C=C(/C)C)C3)C)C 359-83-1 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@@] 638-94-8 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 56980-93-9 Formoterol O=CNc1cc(ccc1O)[C@@H](O)CN[C@H](C)Cc2ccc(OC)cc2 73573-87-2 Terbutaline Oc1cc(cc(O)c1)C(O)CNC(C)(C)C 23031-25-6 MDMA CC(CC1=CC2=C(C=C1)OCO2)NC 42542-10-9 Pseudoephedrine O[C@@H](c1ccccc1)[C@@H](NC)C 90-82-4 Carteolol O=C2Nc1cccc(OCC(O)CNC(C)(C)C)c1CC2 51781-06-7 Oxprenolol O(c1ccccc1OC\C=C)CC(O)CNC(C)C 6452-71-7 Probenecid O=S(=O)(N(CCC)CCC)c1ccc(C(=O)O)cc1 57-66-9 Dichlorphenamide Clc1c(cc(cc1Cl)S(=O)(=O)N)S(=O)(=O)N 120-97-8 Benzphetamine N(C)(Cc1ccccc1)[C@@H](C)Cc2ccccc2 156-08-1 Fenfluramine FC(F)(F)c1cccc(c1)CC(NCC)C 458-24-2 Mephentermine N(C(Cc1ccccc1)(C)C)C 100-92-5 Alprenolol O(c1ccccc1C\C=C)CC(O)CNC(C)C 13655-52-2 Pindolol CC(C)NCC(O)COc2cccc1nccc12 13523-86-9 Propranolol CC(C)NCC(COc1cccc2c1cccc2)O 525-66-6 Raloxifene O=C(c1c3ccc(O)cc3sc1c2ccc(O)cc2)c5ccc(OCCN4CCCCC4)cc5 84449-90-1 Hydroflumethiazide FC(F)(F)c2c(cc1c(NCNS1(=O)=O)c2)S(=O)(=O)N 135-09-1 Etilefrine OC(CNCC)c1cc(O)ccc1 709-55-7 Methoxyphenamine CC(CC1=CC=CC=C1OC)NC 93-30-1 Nikethamide O=C(N(CC)CC)c1cccnc1 59-26-7 p-methylamphetamine NC(Cc1ccc(cc1)C)C 22683-78-9 Oxycodone O=C4[C@@H]5Oc1c2c(ccc1OC)C[C@H]3N(CC[C@]25[C@@]3(O)CC4)C 76-42-6 Betaxolol O(CCc1ccc(OCC(O)CNC(C)C)cc1)CC2CC2 63659-18-7 Acetazolamide O=S(=O)(c1nnc(s1)NC(=O)C)N 59-66-5 Amiloride Clc1nc(C(=O)\N=C(/N)N)c(nc1N)N 2016-88-8 Polythiazide CN1C(NC2=CC(=C(C=C2S1(=O)=O)S(=O)(=O)N)Cl)CSCC(F)(F)F 346-18-9 Amphetamine NC(C)Cc1ccccc1 300-62-9 Sibutramine ClC1=CC=C(C2(CCC2)C(CC(C)C)N(C)C)C=C1 106650-56-0 Clenbuterol Clc1cc(cc(Cl)c1N)C(O)CNC(C)(C)C 37148-27-9 Modafinil O=S(C(c1ccccc1)c2ccccc2)CC(=O)N 68693-11-8 Bambuterol O=C(Oc1cc(cc(OC(=O)N(C)C)c1)C(O)CNC(C)(C)C)N(C)C 81732-46-9 Phenmetrazine O2C(c1ccccc1)C(NCC2)C 134-49-6 Salbutamol OCc1cc(ccc1O)C(O)CNC(C)(C)C 18559-94-9 Benzthiazide O=S(=O)(c1c(Cl)cc2c(c1)S(=O)(=O)/N=C(\N2)CSCc3ccccc3)N 91-33-8 Dimethylamphetamine c1(cccc(c1C)CC(N)C)C 4075-96-1 MDA NC(C)CC1=CC2=C(C=C1)OCO2 4764-17-4 Buprenorphine Oc7ccc5c1c7O[C@H]3[C@]6(OC)[C@H](C[C@@]2([C@H](N(CC[C@@]123)CC4C 52485-79-7 C4)C5)CC6)[C@@](O)(C)C(C)(C)C S4 Methadone CCC(=O)C(CC(C)N(C)C)(c1ccccc1)c2ccccc2 76-99-3 Oxymorphone O=C1[C@@H]2OC3=C(O)C=CC4=C3[C@@]2([C@]5(CC1)O)CCN(C)[C@@H]5C4 76-41-5 Acebutolol O=C(Nc1ccc(OCC(O)CNC(C)C)c(c1)C(=O)C)CCC 37517-30-9 Dextromoramide C[C@@H](C(C1=CC=CC=C1)(C(N2CCCC2)=O)C3=CC=CC=C3)CN4CCOCC4 357-56-2 Esmolol O=C(OC)CCc1ccc(OCC(O)CNC(C)C)cc1 103598-03-4 Sotalol O=S(=O)(Nc1ccc(cc1)C(O)CNC(C)C)C 3930-20-9 Pemoline O=C2\N=C(/OC2c1ccccc1)N 2152-34-3 Toremifene ClCCC(/c1ccccc1)=C(/c2ccc(OCCN(C)C)cc2)c3ccccc3 89778-26-7 Methylphenidate O=C(OC)C(C1CCCCN1)C2=CC=CC=C2 113-45-1 Phentermine NC(Cc1ccccc1)(C)C 122-09-8 S5 S2.0 Reagents Acetonitrile, methanol (both HPLC grade), potassium dihydrogen orthophosphate and anhydrous disodium hydrogen orthophosphate were obtained from Fisher-Scientific (Loughborough, UK).