Table 1.
Published multiple-class analytical methods for endocrine disrupting chemicals in human urine
| Analyte | Urine volume | Enzyme Type | Sample preparation | Sample extraction | Instrumental method | LOD (ng/mL) | Reference |
|---|---|---|---|---|---|---|---|
| Total: 35 analytes 16 BRPs, 12 OH-PAHs, 5 OH-PBDEs, TCS, and TBBPA |
2.0 mL | β-glucuronidase/arylsulfatas e enzyme (Helix pomatia, β-glucuronidase activity ≈ 100,000 U/mL; sulfatase activity ≈ 47,500 U/mL) | enzyme amount: 10 μL (i.e., 50 U/100 μL urine); buffer: 1 mL of 1 M NaOAc (pH 5.5); incubation time: overnight at 37 °C | Oasis HLB SPE cartridge (500 mg, 6 mL) |
Single injection: Instrument: LC-MS/MS; LC column: Poroshell 120 EC-C18 (100 mm × 4.6 mm, 2.7 μm); Mobile phase: 5 mM NH4Ac in water and ACN; Run time: 27 min; Injection volume: 10 μL; |
0.008–0.161 | [10] |
| Total: 21 analytes 7 BPs, 7 parabens, 5 benzophenones, TCC, and TCS |
1.0 mL | β-glucuronidase enzyme (Type HP-2 from Helix pomatia, β-glucuronidase activity ≥ 100,000 U/mL; sulfatase activity ≈ 7500 U/mL) | enzyme amount: 2.0 μL (i.e., 20 U/100 μL urine); buffer: 0.1 mL of 1 M NH4Ac (≈ 6.0); incubation time: 24 h at 37 °C | vortex-assisted dispersive liquid-liquid microextraction |
Single injection: Instrument: LC-MS/MS; LC column: Brownlee Aq C18 column (100 mm × 4.6 mm, 5 μm); Mobile phase: water and MeOH; Run time: 12 min; Injection volume: 10 μL; |
0.01–0.20 | [9] |
| Total: 19 analytes 5 BADGEs, 5 benzophenones, 7 parabens, TCC, and TCS |
0.5 mL | β-glucuronidase/arylsulfatas e enzyme (Helix pomatia, β-glucuronidase activity ≈ 145,700 U/mL; sulfatase activity ~ 887 U/mL) | enzyme amount: 0.15 μL (i.e., 4.4 U/100 μL urine); buffer: 0.3 mL of 1 M NH4Ac (no pH adjustments); incubation time: 12 h at 37 °C; | liquid-liquid extraction with EtAc |
Two injections: Instrument: LC-MS/MS; LC column: Betasil C18 (2.1 mm × 100 mm, 5 μm); 1st injection for 5 BADGEs: Mobile phase: water and MeOH; Run time: 30 min; Injection volume: 10 μL; 2nd injection for remaining analytes: Mobile phase: 0.1% FA in water and MeOH; Run time: 30 min; Injection volume: 10 μL; |
0.06–0.6 | [11] |
| Total: 21 analytes 7 BPs, 7 parabens, 5 benzophenones, TCS, and TCS |
5.0 mL | β-glucuronidase enzyme (type HP-2 from Helix pomatia, β-glucuronidase activity ≈ 197,000 U/mL) | enzyme amount: 20 U/100 μL urine; buffer: not provided; incubation time: overnight at 37 °C; | air-assisted liquid-liquid microextraction |
Single injection: Instrument: LC-MS/MS; LC column: Atlantis T3 dC18 column (75 mm × 2.1 mm, 3.0 μm); Mobile phase: water and MeOH; Run time: 10 min; Injection volume: 10 μL; |
0.01–0.30 | [12] |
| Total: 12 analytes 7 PhMs, 4 parabens, and BP-3 |
3.0 mL | β-glucuronidase enzyme (Type HP-2 from Helix pomatia, β-glucuronidase activity ≥ 200,000 U/mL) | enzyme amount: 25 pL (i.e., 167 U/100 μL urine); buffer: 0.75 mL of 1 M NaOAc (pH 4.5); incubation time: overnight at 37 °C; | Bond Elut Certify SPE cartridge (130 mg, 10 mL) |
Single injection: Instrument: LC-MS/MS; LC column: Kinetex Phenyl-Hexyl column (2.1 mm × 100 mm, 1.7 μm); Mobile phase: 0.1% HAc in both water and ACN; Run time: 20 min; Injection volume: 5 μL; |
0.09–0.37 | [13] |
| Total: 23 analytes 8 BPs and 15 OH-PAHs |
0.5 mL | β-glucuronidase enzyme (from E. coli K12, β-glucuronidase activity ≈ 140 U/mL | enzyme amount: 20 μL (i.e., 0.6 U/100 μL urine); buffer: 1 mL of 0.5 M NH4Ac (pH 6.0); incubation time: 14 h at 37 °C; | liquid-liquid extraction with mixture of EtAc:pentane:toluene (5:4:1, v:v) |
Two injections: Instrument: LC-MS/MS; 1st injection for 8 BPs: LC column: Betasil C18 (2.1 mm × 100 mm, 5 μm); Mobile phase: 0.1% NH4OH in water and MeOH; Run time: 20 min; Injection volume: 10 μL; 2nd injection for 15 OH-PAHs: LC column: Agilent Eclipse Plus C18 column (100 mm × 4.6 mm, 3.5 μm) Mobile phase: water and MeOH; Run time: 20 min; Injection volume: 2 μL; |
0.003–0.36 | [14] |
| Total: 19 analytes 14 PhMs and 5 BPs |
0.05 mL | β-glucuronidase enzyme (from E. coli K12, β-glucuronidase activity ≈ 200 U/mL | enzyme amount: 0.5 μL (i.e., 0.2 U/100 μL urine); buffer: 0.025 mL of 1 M NH4Ac (pH 6.5); incubation time: 1.5 h at 37 °C; | Online Strata X SPE cartridge (20 mm × 2.0 mm, 25 μm) |
Single injection: Instrument: LC-MS/MS; LC column: Synergi MAX-RP column (150 mm × 3.0 mm; 4 μm); Mobile phase: 0.05% HAc in both water and ACN; Run time: 13 min; Injection volume: 50 μL; |
0.005–0.39 | [15] |
| Total: 6 analytes 4 PhMs, BPA, and 4-n-NP |
0.9 mL | β-glucuronidase enzyme (from abalone, β-glucuronidase activity > 100,000 U/mL, arylsulfatase activity < 20,000 U/mL) | enzyme amount: 0.5 U/100 μL urine; buffer: 0.25 mL of 1 mM NH4Ac (no pH adjustment); incubation time: 2 h at 37 °C; | ABS Elut NEXUS SPE cartridges (60 mg, 3 mL) |
Two injections: Instrument: LC-MS/MS; LC column: Acquity UPLC BEH Phenyl column (100 mm × 2.1 mm, 1.7 μm); 1st injection for PhMs: Mobile phase: 0.1% FA in water and MeOH; Run time: 22 min; Injection volume: 5 μL; 2nd injection for BPA and 4-n-NP: Mobile phase: 0.05% NH4OH in water and MeOH; Run time: 14 min; Injection volume: 5 μL; |
0.3–0.5 | [16] |
| Total: 260 analytes pesticides |
0.1 mL | no enzymatic deconjugation step | Quick, Easy, Cheap, Effective, Rugged, and Safe (QuEChERS) method |
Single injection: Instrument: LC-MS/MS; LC column: Kinetex C18 column (100 mm × 2.1 mm, 2.6 μm); Mobile phase: 5 mM AF and 0.1% FA in both water and MeOH; Run time: 15 min; Injection volume: 4 μL; |
10 | [17] | |
| Total: 13 analytes pesticides |
1.0 mL | acidic hydrolysis using 5 mL of HCl (50%) and heating in a 90 °C water bath for 45 min for deconjugation | Liquid-liquid extraction with hexane followed by derivatization with N-tert-butyldimethylsilyl-N-methyltrifluoroacetamide |
Single injection: Instrument: GC-MS; GC column: Restek RTX-35 column (30 m × 0.25 mm, 0.25 μm); Run time: 30 min; Injection volume: 1 μL; |
0.025–0.10 | [18] | |
| Total: 10 analytes pesticides |
1.0 mL | β-glucuronidase enzyme (type H-1 from Helix pomatia with a specific activity of ~500 U/mg) | enzyme amount: 56 units/100 μL urine; buffer: 0.75 mL of 0.2 M NaOAc; incubation time: > 6 h at 37 °C; | Oasis HLB SPE cartridge (30 mg, 1 mL) |
Single injection: Instrument: LC-MS/MS; LC column: Betasil C18 column (100 mm × 2.1 mm; 3 μm); Mobile phase: 0.1% HAc in water and ACN; Run time: 20 min; Injection volume: 30 μL; |
0.1–0.6 | [19] |
| Total: 121 analytes 45 plasticizers and metabolites, 34 environmental phenols, 31 pesticides, 11 OH-PAHs; |
0.5 mL | β-glucuronidase/arylsulfatas e enzyme (Helix pomatia, β-glucuronidase activity ≈ 100,000 U/mL; sulfatase activity ≈ 47,500 U/mL) | enzyme amount: 20 μL (i.e., 400 units/100 μL urine); buffer: 0.5 mL of 1 M NH4Ac (pH 5.5); incubation time: overnight at 37 °C | ABS Elut NEXUS SPE cartridge (60 mg, 3 mL) |
Two injections: Instrument: LC-MS/MS; 1st injection for 43 analytes: LC column: Ultra AQ C18 column (2.1 mm × 100 mm, 3 μm); Mobile phase: 0.1% HAc in both water and MeOH; Run time: 12 min; Injection volume: 5 μL; 2nd injection for 78 analytes: LC column: Betasil C18 column (100 mm × 2.1 mm, 5 μm) Mobile phase: water and ACN; Run time: 9 min; Injection volume: 5 μL; |
≤ 0.1 for 101 analytes 0.1–1.0 for 18 analytes ≥ 1.0 for 2 analytes |
the present study |
OH-PAHs: monohydroxylated polycyclic aromatic hydrocarbons; BRPs: brominated phenols; OH-PBDEs: hydroxyl polybrominated diphenyl ethers; TCC: triclocarban; TCS: triclosan; TBBPA: tetrabromobisphenol A; BPs: bisphenols; BADGEs: bisphenol A diglycidyl ethers; PhMs: phthalate metabolites; BPA: 4,4’-(1-methylethylidene)bisphenol; NH4Ac: ammonium acetate; FA: formic acid; HAc: acetate acid; NH4OH: ammonium hydroxide; AF: ammonium formate; ACN: acetonitrile; MeOH: methanol; EtAc: ethyl acetate; NaOAc: sodium acetate; LOD: limit of detection.