Table S3: Sample Preparation, Instrumental Analysis, Validation Parameters and Application

Table S3: Sample Preparation, Instrumental Analysis, Validation Parameters and Application

Table S3: Sample preparation, instrumental analysis, validation parameters and application of sent standard mixture for laboratory coded 3

COMPOUND / SAMPLE PREP / INTRUM ANALYSIS / VALIDATION PARAM / Application of sent mixture
Sample preparation
(sample pre-treatment: pH adjustment, filter pore size, material and trade name, extraction method and conditions, additional clean-up, derivatisation, internal standard…) / Instrumental analysis
(operational parameters: separation and detection condition,…) / Validationparameters
(sensitivity, accuracy, recovery, reproducibility, repeatability,…) / a. Standard mixture was used for calibration
b. Standard mixture was used for check-up
c.Standard mixture was not used
CP /
  1. Filtration through 0.45 µm cellulose acetate membranes (Advantec MFS. Inc, CA, USA)
  2. Transferred 250mL×3 into 3 polypropylene bottles
  3. pH adjustment to 2 using H2SO4 (1/3-acid/water), 200 and 250µL were required for surface and wastewater
  4. Addition of isotopically labelled:20µL caffeine-d9 2.5ppm was added to each bottle as surrogate.
  5. Concentration by double SPE: Sample volume, 250 mL; WAX cartridge (6cc, 150mg, particle size 30µm, Waters Corp., USA) on top, HLB cartridge(6cc, 200mg, particle size 30µm, Waters Corp., USA)
  6. Conditioning:
  1. WAX: 4mL of 0.1% NH4OH/methanol followed by 4mL methanol and finally 4ml H2O
  2. HLB: 10mL MeOH followed by 10mL H2O
  1. Load samples to cartridges at equal rate, dry by air flow for 20 min.
  2. Washing:
  3. WAX: 4mL of 25mM aqueous sodium acetate pH 4
  4. HLB: 5mL aqueous acetic acid pH 2
  5. Elution: 4mL MeOH for WAX, 4mL MeOH for HLB
  6. Extract was dried under gentle N2 gas stream to dryness.
  7. Reconstitute to 0.5mL using solution MeOH/H2O-70/30 v/v.
  8. Add 30µL 4-NP 2.0ppm as internal standard.
/ -Analysis by LC-MS/MS
-Equipment: UPLC Dionex 3000 coupling with AB Sciex 5500 QTrap (Toronto, Canada).
-LC separation on a reversed-phase column C18 column (3.5µm, 40mm × 2.1mm ID, Higgins Analytical, CA, USA).
-Ultrapure water (A) and acetonitrile (B), both containing 0.1% of formic acid, were employed as mobile phase (flow-rate 0.3 mL min-1). LC gradient: 0–0.3 min, 40% B; 2 min, 100% B; 2-2.2 min, 100% B; 2.2–3 min, 40% B.
-MS/MS conditions: positive ESI; SRM quantification transition, 261.1 > 139.9; SRM confirmation transition, 261.1 > 232.9; collision energy, 29 eV and 23 eV, respectively; declustering potential, 191 V / -Quantification by the isotope dilution method with calibration curves prepared in HPLC-water
-Linearity: determination coefficient r2 = 0.99
-Sensitivity: method limit of quantification (s/n10) = 1.5 ngL-1. Accuracy: relative recovery (calculated with respect to the IS, in standard solution, n=5) = 114±20%
-Repeatability: standard deviation (RSD, n=5) = 8 %. / a
ETO /
  1. Filtration through 0.45 µm cellulose acetate membranes (Advantec MFS. Inc, CA, USA)
  2. Transferred 250mL×3 into 3 polypropylene bottles
  3. pH adjustment to 2 using H2SO4 (1/3-acid/water), 200 and 250µL were required for surface and wastewater
  4. Addition of isotopically labelled: 20µL caffeine-d9 2.5ppm was added to each bottle as surrogate.
  5. Concentration by double SPE: Sample volume, 250 mL; WAX cartridge (6cc, 150mg, particle size 30µm, Waters Corp., USA) on top, HLB cartridge (6cc, 200mg, particle size 30µm, Waters Corp., USA)
  6. Conditioning:
  1. WAX: 4ml of 0.1% NH4OH/methanol followed by 4ml methanol and finally 4mL H2O
  2. HLB: 10mLMeOH followed by 10mL H2O
  1. Load samples to cartridges at equal rate, dry by air flow for 20 min.
  2. Washing:
  3. WAX: 4ml of 25mM aqueous sodium acetate pH 4
  4. HLB: 5ml aqueous acetic acid pH 2
  5. Elution: 4mLMeOH for WAX, 4mLMeOH for HLB
  6. Extract was dried under gentle N2 gas stream to dryness.
  7. Reconstitute to 0.5mL using solution MeOH/H2O-70/30 v/v.
  8. Add 30µL 4-NP 2.0ppm as internal standard.
/ -Analysis by LC-MS/MS
-Equipment: UPLC Dionex 3000 coupling with AB Sciex 5500 QTrap (Toronto, Canada).
-LC separation on a reversed-phase column C18 column (3.5µm, 40mm × 2.1mm ID, Higgins Analytical, CA, USA).
-Ultrapure water (A) and acetonitrile (B), both containing 0.1% of formic acid, were employed as mobile phase (flow-rate 0.3 mL min-1). LC gradient: 0–0.3 min, 40% B; 2 min, 100% B; 2-2.2 min, 100% B; 2.2–3 min, 40% B.
-MS/MS conditions: positive ESI; SRM quantification transition, 589.1>229; SRM confirmation transition, 589.1>185; collision energy, 19 eV and 59eV, respectively; declustering potential, 91 V / -Quantification by the isotope dilution method with calibration curves prepared in HPLC-water
-Linearity: determination coefficient r2 = 0.99
-Sensitivity: method limit of quantification (s/n >10) = 2 ngL-1. Accuracy: relative recovery (calculated with respect to the IS, in standard solution, n=5) = 109±16%
-Repeatability: relative standard deviation (RSD, n=5) 10 %. / a
IF /
  1. Filtration through 0.45 µm cellulose acetate membranes (Advantec MFS. Inc, CA, USA)
  2. Transferred 250mL×3 into 3 polypropylene bottles
  3. pH adjustment to 2 using H2SO4 (1/3-acid/water), 200 and 250µL were required for surface and wastewater
  4. Addition of isotopically labelled: 20µL caffeine-d9 2.5ppm was added to each bottle as surrogate.
  5. Concentration by double SPE: Sample volume, 250 mL; WAX cartridge (6cc, 150mg, particle size 30µm, Waters Corp., USA) on top, HLB cartridge (6cc, 200mg, particle size 30µm, Waters Corp., USA)
  6. Conditioning:
  7. WAX: 4mL of 0.1% NH4OH/methanol followed by 4mL methanol and finally 4mL H2O
  8. HLB: 10mLMeOH followed by 10mL H2O
  9. Load samples to cartridges at equal rate, dry by air flow for 20 min.
  10. Washing:
  11. WAX: 4ml of 25mM aqueous sodium acetate pH 4
  12. HLB: 5ml aqueous acetic acid pH 2
  13. Elution: 4ml MeOH for WAX, 4mLMeOH for HLB
  14. Extract was dried under gentle N2 gas stream to dryness.
  15. Reconstitute to 0.5mL using solution MeOH/H2O-70/30 v/v.
  16. Add 30µL 4-NP 2.0ppm as internal standard.
/ -Analysis by LC-MS/MS
-Equipment: UPLC Dionex 3000 coupling with AB Sciex 5500 QTrap (Toronto, Canada).
-LC separation on a reversed-phase column C18 column (3.5µm, 40mm × 2.1mm ID, Higgins Analytical, CA, USA).
-Ultrapure water (A) and acetonitrile (B), both containing 0.1% of formic acid, were employed as mobile phase (flow-rate 0.3 mL min-1). LC gradient: 0–0.3 min, 40% B; 2 min, 100% B; 2-2.2 min, 100% B; 2.2–3 min, 40% B.
-MS/MS conditions: positive ESI; SRM quantification transition, 261>92; SRM confirmation transition, 261>153.9; collision energy, 31 eV and 31eV, respectively; declustering potential, 46 V / -Quantification by the isotope dilution method with calibration curves prepared in HPLC-water
-Linearity: determination coefficient r2 = 0.99
-Sensitivity: method limit of quantification (s/n >10) = 1.5 ngL-1. Accuracy: relative recovery (calculated with respect to the IS, in standard solution, n=5) = 114±20%
-Repeatability: standard deviation (RSD, n=5) = 4 %. / a
GEM /
  1. Filtration through 0.45 µm cellulose acetate membranes (Advantec MFS. Inc, CA, USA)
  2. Transferred 250mL×3 into 3 polypropylene bottles
  3. pH adjustment to 2 using H2SO4 (1/3-acid/water), 200 and 250µL were required for surface and wastewater
  4. Addition of isotopically labelled: 20µL caffeine-d9 2.5ppm was added to each bottle as surrogate.
  5. Concentration by double SPE: Sample volume, 250 mL; WAX cartridge (6cc, 150mg, particle size 30µm, Waters Corp., USA) on top, HLB cartridge (6cc, 200mg, particle size 30µm, Waters Corp., USA)
  6. Conditioning:
  7. WAX: 4mL of 0.1% NH4OH/methanol followed by 4mL methanol and finally 4mL H2O
  8. HLB: 10mLMeOH followed by 10mL H2O
  9. Load samples to cartridges at equal rate, dry by air flow for 20 min.
  10. Washing:
  11. WAX: 4ml of 25mM aqueous sodium acetate pH 4
  12. HLB: 5mL aqueous acetic acid pH 2
  13. Elution: 4mLMeOH for WAX, 4mLMeOH for HLB
  14. Extract was dried under gentle N2 gas stream to dryness.
  15. Reconstitute to 0.5mL using solution MeOH/H2O-70/30 v/v.
  16. Add 30µL 4-NP 2.0ppm as internal standard.
/ -Analysis by LC-MS/MS
-Equipment: UPLC Dionex 3000 coupling with AB Sciex 5500 QTrap (Toronto, Canada).
-LC separation on a reversed-phase column C18 column (3.5µm, 40mm × 2.1mm ID, Higgins Analytical, CA, USA).
-Ultrapure water (A) and acetonitrile (B), both containing 0.1% of formic acid, were employed as mobile phase (flow-rate 0.3 mL min-1). LC gradient: 0–0.3 min, 40% B; 2 min, 100% B; 2-2.2 min, 100% B; 2.2–3 min, 40% B.
-MS/MS conditions: positive ESI; SRM quantification transition, 264>112; SRM confirmation transition, 264>94.9; collision energy, 23 eV and 57eV, respectively; declustering potential, 146 V. / -Quantification by the isotope dilution method with calibration curves prepared in HPLC-water
-Linearity: determination coefficient r2 = 0.99
-Sensitivity: method limit of quantification (s/n >10) = 1.5 ng L-1. Accuracy: relative recovery (calculated with respect to the IS, in standard solution, n=5) = 105±18%
-Repeatability: standard deviation (RSD, n=5) = 8 %. / a
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