Analytical and Bioanalytical Chemistry Electronic Supplementary Material Multi-residue enantiomeric analysis of human and veterinary pharmaceuticals and their metabolites in environmental samples by chiral liquid chromatography coupled with tandem mass spectrometry detection Dolores Camacho-Muñoz, Barbara Kasprzyk-Hordern 1
Table S1 Published enantioselective methods that employed AGP CSP and LC-MS/MS for the determination of cpacs in environmental matrices Compound Environmental matrix Sample treatment Analytical determination Method quantification limits Ibuprofen 1 Influent wastewater (1 ml) Effluent wastewater and river water (25 ml) SPE (Oasis HLB) LC-MS/MS (TQD) Chiral-AGP column (1 x 4 mm, 5 µm particle size) 1mM NH 4 OAc/ACN 98:2, v/v, (ph 7.);.2 ml/min 8-27 ng L -1 in wastewater 3-8 ng L -1 in river water Clotrimazole, econazole, miconazole, tebuconazole, propiconazole 2 Wastewater (1-2 ml) Sludge.1 g USE-SPE (Oasis HLB) UHPLC-MS/MS (TQD) Chiral-AGP column (1 x 4 mm, 5 µm particle size) 1mM NH 4 OAc/ACN 9:1, v/v, (ph 7.);.3 ml/min.3-1 ng L -1 in wastewater 3-29 ng g -1 in sewage sludge Ketoconazole 2 Deaminated metoprolol 3 Fluoxetine and norfluoxetine 4 Effluent wastewater (5 ml) Influent (2 ml) and effluent (5 ml) wastewater Chloramphenicol Soil (2 g) isomers 5 Plant samples (1 g) SPE (Oasis HLB) SPE (Evolute CX- 5 cartridges) Shaking- LLE Chiral-HSA column (1 x 2 mm, 5 µm particle size) LC-MS/MS (TQD) Chiral-AGP column (1 x 2 mm, 5 µm particle size) 1mM NH 4 OAc/MeOH 95:5: v/v, (ph 5.);.22 ml/min LC-MS/MS (TQD) Chiral-AGP column (1 x 2 mm, 5 µm particle size) 1mM NH 4 OAc/ACN 97:3, v/v, (ph 4.4);.22 ml/min LC-MS/MS (TQD) Chiral-AGP column (15 x 2 mm, 5 µm particle size) 1mM NH 4 OAc/ACN 98:2, v/v, (ph 4.);.4 ml/min 4.8 ng L -1 in effluent wastewater 3.6-4.3 ng L -1 in influent wastewater.9-1. ng L -1 in effluent wastewater 2-5 ng g -1 TQD: Triple quadrupole detector; USE: Ultrasonic solvent extraction; LLE: liquid-liquid extraction; 1 Wang Z, Huang Q, Yu Y, Wang C, Ou W, Peng X (213) Environ Geochem Health 35:683 91; 2 Huang Q, Zhang K, Wang Z, Wang C, Peng X (212) Anal Bioanal Chem 43:1751 176; 3 Barclay VKH, Tyrefors NL, Johansson IM, Pettersson CE (212) J Chromatogr A 1269:28 217; 4 Barclay VKH, Tyrefors NL, Johansson M, Pettersson CE (212) J Chromatogr A 1227: 15 114; 5 Berendsen BJA, Zuidema T, de Jong J, Stolker LAM, Nielen MWF (211) Anal Chim Acta 7:78 85. 2
Table S2 Structures, molecular weighs, CAS numbers and physicochemical properties of the studied PACs Compound Structure CAS pk a Log K ow Solubility Uses (mg L -1 ) Anti-inflammatory drugs S-(+)-O-Desmethylnaproxen (M) C 13H 12O 3 5279-1-4 4.34 b 2.54 a 2296 a Metabolite 6α-Methylprednisolone C 22H 3O 5 83-43-2 12.5 a 1.99 a 12 a Human/Veterinary Antibacterial drugs Amoxicillin C 16H 19N 3O 5S 26787-78- 2.44 a.61 a 3433 a Human/Veterinary Ampicillin 3H2O C 16H 19N 3O 4S 3H 2O 7177-48-2 2.44 a 1.35 a 11 a Human/Veterinary 3
Table S2(Continued) Compound Structure CAS pk a Log K ow Solubility Uses (mg L -1 ) Danofloxacin C 19H 2FN 3O 3 112398-8- 6.22, 9.43 d.39 d 65.6 d Veterinary Penicillin G sodium salt C 16H 17N 2NaO 4S 69-57-8 2.45 a 1.67 a 21 a Human/Veterinary Penicillin V potassium salt C 16H 17N 2O 5SK 132-98-9 2.44 a 1.88 a 65 a Human/Veterinary Ceftiofur C 19H 17N 5O 7S 3 837-57-6 2.63 a 2.5 a 4 e Veterinary 4
Table S2(Continued) Compound Structure CAS pk a Log K ow Solubility Uses (mg L -1 ) Doxycycline hyclate C 23H 29ClN 2O 9 2439-14-5 3.5, 7.7, 9.5 f -3.4 b 63 b Human/Veterinary Erythromycin C 37H 67NO 13 114-7-8 8.88 a 3.6 a 1437 a Human/Veterinary Minocycline HCl C 23H 28ClN 3O 7 13614-98-7 8.25 b.5 g 59.32 g Human Oxytetracycline HCl C 22H 25ClN 2O 9 258-46- 3.27 a -.9 a 313 a Human/Veterinary 5
Table S2(Continued) Compound Structure CAS pk a Log K ow Solubility Uses (mg L -1 ) Tetracycline HCl C 22H 25ClN 2O 8 64-75-5 3.3 a -1.3 a 231.1 a Human/Veterinary 1R,2R-(-)-Chloramphenicol base C 9H 12N 2O 4 716-61- n.a. -.32 a 26 a Metabolite 1R,2S-(-)-Florfenicol C 12H 14Cl 2FNO 4S 73231-34-2 1.7 a -.12 a 132 h Veterinary Antifungal drug (+)-Griseofulvin C 17H 17ClO 6 126-7-8 >9 i 2.18 i 8.64 i Veterinary 6
Table S3 Optimized MRM conditions for the analysis of the target analytes by LC-MS/MS Compound CV/CE MRM 1 (quantification) CV/CE MRM 2 (confirmation) Mode Amoxicillin 28/27 366.1 > 28.1 28/17 366.1 > 349.4 ESI + Ampicillin 44/1 35.3> 16. 44/28 35.3 > 16.5 ESI + Ceftiofur 5/2 524.> 241. 5/5 524.> 125. ESI + 1R,2R-(-)-Chloramphenicol base 3/15 213.> 164.5 3/15 213.> 132. ESI + Danofloxacin 65/2 358.1 > 34. 38/38 358.1 > 255.2 ESI + S-(+)-O-Desmethylnaproxen 2/1 215.> 17.5 ESI - Doxycycline hyclate 4/4 445.3 > 428.7 8/34 445.3 >154. ESI + Erythromycin 12/32 734.7 > 158.5 2/6 734.7 > 115.9 ESI + 1R,2S-(-)-Florfenicol 25/1 356.1 > 335.9 25/2 356.1 > 185. ESI + (+)-Griseofulvin 45/25 353.2 > 69. 45/23 353.2 > 165. ESI + 6α-Methylprednisolone 48/25 375.> 161. 48/2 375.> 357. ESI + Minocycline 44/36 458.3 > 441.6 ESI + Oxytetracycline 4/18 461.2 > 426.2 4/4 461.2 > 21.4 ESI + Penicillin G 48/2 335.2 > 16.1 48/2 335.2> 176. ESI + Penicillin V 54/4 351.2 > 114.2 54/4 351.2 > 16.1 ESI + Tetracycline 32/28 445.3 > 41.2 32/3 445.3 > 427. ESI + Table S4 Linearity and range, enantiomeric fraction, instrumental and method detection and quantification limits Compound Linearity range (µg L -1 ) R 2 IDL S/N (µg L -1 ) IQL S/N (µg L -1 ) Surface water (n=3) MDL calc (ng L -1 ) MQL calc (ng L -1 ) Effluent wastewater (n=3) MDL calc (ng L -1 ) MQL calc (ng L -1 ) Amoxicillin a 16.3-196.995 4.9 16.32 13.97 45.58 1.95 35.74 Ampicillin a 1.64-395.998.49 1.64.81 2.64.62 2.3 Ceftiofur a 1.64-59.999.49 1.64 29.41 96.28 - - 1R,2R-(-)-Chloramphenicol base b 1.68-88.999.51 1.68 1.89 6.35 2.24 7.54 S-(+)-O-Desmethylnaproxen c 34.6-624.995 1.4 34.63 9.54 33.5 19.44 67.31 1R,2S-(-)-Florfenicol c 3.32-1.987 1. 3.32 - - - - Griseofulvin d 17.1-1.99 5.13 17.8 5.1 17.41 1.5 35.89 6α-Methylprednisolone d 298-895.997 89.44 297.84 7.55 262.64 164.61 612.83 Penicillin G a 16.4-592.997 4.93 16.42 3.67 12.4 - - Penicillin V a 174-416.992 51.95 173.99 131.58 455.25 - - IS used: a Ketoprofen-d3, b Ifosfamide-d4, c Naproxen-d3, d Tetramisole-d5, : Enantiomeric fraction (x: mean, σ: standard deviation; Absolute shown in brackets); IDL S/N: Instrumental detection limit based on signal to noise approach; IQL S/N: Instrumental quantification limit based on signal to noise approach; MDL calc: Method detection limit; MQL calc: Method quantification limit 7
Table S5 Inter- and intra-day precision (RSD ), retention time and resolution of the studied PACs Intra-day RSD (n=3) 1 (μg L -1 ) 5 (μg L -1 ) 1 (μg L -1 ) 3 (μg L -1 ) 1 T (μg L -1 R (min) R S D1 D2 D3 D1 D2 D3 D1 D2 D3 D1 D2 D3 ) Inter-day RSD (n=9) Amoxicillin 4. - 9.8 16.9 16..4 2.7 6.6 7.7 2.1 1.5 1.2.9 2. 14.4 17.3 7.8 5.2 Ampicillin 4.2-8.1 1.3 6.7 2.3 5. 2. 7. 4.9 5.5.7 2. 2.3 14.9 16.8 12.2 4.7 Ceftiofur 7. - 5.4 8.8 6.2 4.3 9.2 5.4 1.5 6. 2.8.3 1.1 1.8 1.1 24.2 6.7 4.8 1R,2R-(-)-Chloramphenicol base 18.6-7.3 9.4 3.8 6.4 3.1 4. 2.9 3.6 3.2 1.8.7 1.2 14.7 2.8 18.4 19.2 S-(+)-O-Desmethylnaproxen 4.8-2.8 14.6 2.3 13. 4.5 4.6 2.6 3.9 6.5 2.8 3.3 3.1 33.5 26.7 12. 11. 1R,2S-(-)-Florfenicol 13.7-5. 17.7 18.3 6.7 3.5 7.3 7.6 4.2 2.4 2.6 2.7 1.2 26.5 17.1 9.7 8.5 Griseofulvin 76.3-6.1 15. 2.2 2.3 6.1 2.1 4.1 2.7 1.5 1.3 1.6 1.6 31.5 19.7 15.2 7.8 6α-Methylprednisolone 42.1 - - - - - - - 2.6 12.6 3.5 11.2 1.3 5.3 - - 6.2 36.6 Penicillin G 4.2-21.7 1.6 15.7 1.2 6.8 7.4.7 5.5 5.3 21.3 2.9 3.2 16. 14.5 6.1 15. Penicillin V 9.7 - - - - - - - 9.5 12.8 1.2 9.3 6. 5.2 - - 1.9 16. T R: Retention time; R S: Resolution 5 (μg L -1 ) 1 (μg L -1 ) 3 (μg L -1 ) 8
Table S6 Recovery (), intra-day precision (RSD ), enantiomeric fraction and matrix effect () in surface water and effluent wastewater Surface water (n=3) Effluent wastewater (n=3) Recovery Recovery ME Recovery Recovery ME Compound () a () b () () c () d () Amoxicillin 35.8 ± 21.8 - -5.7 91.3 ± 25. - -5.7 Ampicillin 62.1 ± 29.5 - -21.3 162.1 ± 22.7 - -21.3 Ceftiofur 1.7 ± 92.7 - -77. - - -77. 1R,2R-(-)-Chloramphenicol base 26.5 ± 9. 13.5 ± 3.8-51.1 44.6 ± 3.5 19.7 ± 15.2-51.1 S-(+)-O-Desmethylnaproxen 14.8 ± 1.2-14.3 12.9 ± 14. - 14.3 1R,2S-(-)-Florfenicol - - 12.4 - - 12.4 Griseofulvin 98.1 ± 1. - 53.6 95.2 ± 5.1-53.6 6α-Methylprednisolone 113.4 ± 5.7 - - 97.2 ± 3. - - Penicillin G 136.4 ± n.a. - -11.6 - - -11.6 Penicillin V 38. ± n.a. - - - - - ME: Matrix effect; a Concentration level:.6 µg L -1 ; b Concentration level:.1 µg L -1 ; c Concentration level: 1.2 µg L -1 ; d Concentration level:.2 µg L -1 9
Conditioning and equilibration 4 ml methanol + 2 ml deionized water (ph 7.5) Sample load 5 ml filtered river water 25 ml filtered effluent wastewater 2 ml deionized water Wash 2 ml deionized water (5 NH 4 OH) Elution 1 4 x 1 ml methanol Elution 2 2 x 1 ml methanol (2 CH 2 O 2 ) HLB HLB HLB MAX HLB MAX Evaporation, reconstitution and injection on HPLC-MS/MS MAX MAX MAX Combine eluates Fig. S1 Flow chart 1 1 1 Amoxicillin 1 5. 1. 15. 2. 25. 3. 35. 4. 45. 5. 55. 6. 65. 7. 75. 8. 85. 9. 95. 1. 1 5. 1. 15. 2. 25. 3. 35. 4. 45. 5. 55. 6. 65. 7. 75. 8. 85. 9. 95. 1. 15. 11. 1 5. 1. 15. 2. 25. 3. 35. 4. 45. 5. 55. 6. 65. 7. 75. 8. 85. 9. 95. 1. 15. 11. Ampicillin 1 5. 1. 15. 2. 25. 3. 35. 4. 45. 5. 55. 6. 65. 7. 75. 8. 85. 9. 95. 1. 1 5. 1. 15. 2. 25. 3. 35. 4. 45. 5. 55. 6. 65. 7. 75. 8. 85. 9. 95. 1. 15. 11. 1 5. 1. 15. 2. 25. 3. 35. 4. 45. 5. 55. 6. 65. 7. 75. 8. 85. 9. 95. 1. 15. 11. S-(+)-O-Desmethyl naproxen 1 5. 1. 15. 2. 25. 3. 35. 4. 45. 5. 55. 6. 65. 7. 75. 8. 85. 9. 95. 1. 1 5. 1. 15. 2. 25. 3. 35. 4. 45. 5. 55. 6. 65. 7. 75. 8. 85. 9. 95. 1. 15. 11. 1 5. 1. 15. 2. 25. 3. 35. 4. 45. 5. 55. 6. 65. 7. 75. 8. 85. 9. 95. 1. 15. 11. Penicillin G 5. 1. 15. 2. 25. 3. 35. 4. 45. 5. 55. 6. 65. 7. 75. 8. 85. 9. 95. 1. 15. 11. 1 1 1 Ceftiofur 1 5. 1. 15. 2. 25. 3. 35. 4. 45. 5. 55. 6. 65. 7. 75. 8. 85. 9. 95. 1. 1 5. 1. 15. 2. 25. 3. 35. 4. 45. 5. 55. 6. 65. 7. 75. 8. 85. 9. 95. 1. 15. 11. 1 5. 1. 15. 2. 25. 3. 35. 4. 45. 5. 55. 6. 65. 7. 75. 8. 85. 9. 95. 1. 15. 11. Penicillin V 1 5. 1. 15. 2. 25. 3. 35. 4. 45. 5. 55. 6. 65. 7. 75. 8. 85. 9. 95. 1. 1 5. 1. 15. 2. 25. 3. 35. 4. 45. 5. 55. 6. 65. 7. 75. 8. 85. 9. 95. 1. 15. 11. 1 5. 1. 15. 2. 25. 3. 35. 4. 45. 5. 55. 6. 65. 7. 75. 8. 85. 9. 95. 1. 15. 11. Florfenicol 5. 1. 15. 2. 25. 3. 35. 4. 45. 5. 55. 6. 65. 7. 75. 8. 85. 9. 95. 1. 5. 1. 15. 2. 25. 3. 35. 4. 45. 5. 55. 6. 65. 7. 75. 8. 85. 9. 95. 1. 15. 11. 5. 1. 15. 2. 25. 3. 35. 4. 45. 5. 55. 6. 65. 7. 75. 8. 85. 9. 95. 1. 15. 11. 1 1 1 Chloramphenicol base 5. 1. 15. 2. 25. 3. 35. 4. 45. 5. 55. 6. 65. 7. 75. 8. 85. 9. 95. 1. 5. 1. 15. 2. 25. 3. 35. 4. 45. 5. 55. 6. 65. 7. 75. 8. 85. 9. 95. 1. 15. 11. 5. 1. 15. 2. 25. 3. 35. 4. 45. 5. 55. 6. 65. 7. 75. 8. 85. 9. 95. 1. 15. 11. 1 1 1 6α-Methylprednisolone 5. 1. 15. 2. 25. 3. 35. 4. 45. 5. 55. 6. 65. 7. 75. 8. 85. 9. 95. 1. 5. 1. 15. 2. 25. 3. 35. 4. 45. 5. 55. 6. 65. 7. 75. 8. 85. 9. 95. 1. 5. 1. 15. 2. 25. 3. 35. 4. 45. 5. 55. 6. 65. 7. 75. 8. 85. 9. 95. 1. 15. 11. 1 1 1 (+)-Griseofulvin 5. 1. 15. 2. 25. 3. 35. 4. 45. 5. 55. 6. 65. 7. 75. 8. 85. 9. 95. 1. 5. 1. 15. 2. 25. 3. 35. 4. 45. 5. 55. 6. 65. 7. 75. 8. 85. 9. 95. 1. 15. 11. 1 1 1 Praziquantel-d11 E1 E2 E1 E2 E1 E2 5. 1. 15. 2. 25. 3. 35. 4. 45. 5. 55. 6. 65. 7. 75. 8. 85. 9. 95. 1. 5. 1. 15. 2. 25. 3. 35. 4. 45. 5. 55. 6. 65. 7. 75. 8. 85. 9. 95. 1. 15. 11. 5. 1. 15. 2. 25. 3. 35. 4. 45. 5. 55. 6. 65. 7. 75. 8. 85. 9. 95. 1. 15. 11. 1 1 1 Praziquantel E1 E2 E1 E2 E1 E2 5. 1. 15. 2. 25. 3. 35. 4. 45. 5. 55. 6. 65. 7. 75. 8. 85. 9. 95. 1. 2 4 6 8 1 5. 1. 15. 2. 25. 3. 35. 4. 45. 5. 55. 6. 65. 7. 75. 8. 85. 9. 95. 1. 15. 11. 5. 1. 15. 2. 25. 3. 35. 4. 45. 5. 55. 6. 65. 7. 75. 8. 85. 9. 95. 1. 15. 11. 2 4 6 8 1 2 4 6 8 1 (min) (min) (min) Fig. S2 Chromatogram of PACs sold as enantiomerically pure (except praziquantel) in standard solution (left) and surface water (center) and effluent wastewater (right) spiked at 3 μg L -1 of each enantiomer 1
12 1 Recovery () 8 6 4 2 Pharmacologically active compound HLB (ph 7.5) HLB (ph 2.) MAX (ph 7.5) HLB-MAX (ph 7.5) MCX (ph 2.) Fig. S3 Optimization of SPE conditions 1. Enantiomeric fraction.8.5.3. Pharmaceutically active compound HLB (ph 7.5) HLB (ph 2.) MAX (ph 7.5) HLB-MAX (ph 7.5) MCX (ph 2.) Fig. S4 Enantiomeric fraction of cpacs in different SPE conditions 11
1..8 Aminorex 1..8 Naproxen.6.6.4.4.2.2. 1 2 3 4. 1 2 3 4 1. Tetramisole 1. Ketoprofen.8.8.6.6.4.4.2.2. 1 2 3 4. 1 2 3 4 1. 3-N-Dechloroethylifosfamide 1. Praziquantel.8.8.6.6.4.4.2.2. 1 2 3 4. 1 2 3 4 1. Ifosfamide 1. Ibuprofen.8.8.6.6.4.4.2.2. 1 2 3 4. 1 2 3 4 1..8.6 1,11-Dihydro-1-hydroxy carbamazepine 1..8.6 Dihydroketoprofen E1+E2 E3 E4.4.4.2.2. 1 2 3 4. 1 2 3 4 1..8 Fexofenadine 1..8 Chloramphenicol.6.6.4.4.2.2. 1 2 3 4. 2 4 6 8 Concentration (µg L -1 ) Concentration (µg L -1 ) Fig. S5 Enantiomeric fraction of cpacs in standard solutions prepared at different concentrations 12